Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Molecular mechanism of Alzheimer's disease pathogenesis in Down syndrome Sun, Xiulian 2006

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2007-268056.pdf [ 17.98MB ]
Metadata
JSON: 831-1.0100546.json
JSON-LD: 831-1.0100546-ld.json
RDF/XML (Pretty): 831-1.0100546-rdf.xml
RDF/JSON: 831-1.0100546-rdf.json
Turtle: 831-1.0100546-turtle.txt
N-Triples: 831-1.0100546-rdf-ntriples.txt
Original Record: 831-1.0100546-source.json
Full Text
831-1.0100546-fulltext.txt
Citation
831-1.0100546.ris

Full Text

M O L E C U L A R MECHANISM OF ALZHEIMER'S DISEASE PATHOGENESIS IN DOWN SYNDROME by XIULIAN SUN B. Medicine, Nankai University, 1999 M . Medicine, Nankai University, 2001 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Neuroscience) T H E UNIVERSITY OF BRITISH COLUMBIA December 2006 © Xiulian Sun, 2006 ABSTRACT Alzheimer's disease (AD) is the most common neurodegenerative disease leading to dementia. Neuritic plaques and neurofibrillary tangles are the two hallmarks of A D neuropathology. The molecular mechanism underlying A D pathogenesis remains unknown. It is believed that deposition of amyloid P (A(3) protein in the brain plays a pivotal role in A D pathogenesis. Ap\ the central component of neuritic plaques, is derived from (3-amyloid precursor protein (APP) by sequential cleavages by (3- and y-secretase. Nearly all individuals with Down Syndrome (DS) show characteristic A D pathological changes after their 30s. The molecular mechanism by which A D pathogenesis develops in DS patients is poorly defined. BACE1 is the major p-secretase in vivo. BACE2 is the homolog of BACE1 and located on chromososme 21. In this study, we cloned and functionally characterized BACE2 gene promoter. Our studies show that the BACE2 gene promoter has a higher activity in non-neuronal cells while BACE1 promoter has a higher activity in neuronal cells. Although both can be activated by SP1, the transcription of BACE1 and BACE2 are distinctly regulated. Even though they are homologous in amino acid sequence, BACE1 and BACE2 cleave APP at distinct sites, leading to their opposing functions in Ap production. N-terminal sequencing of BACE2 cleavage product shows that the cleavage site of BACE2 in APP is located between the 19th and 20th amino acid of A(3. Thus, BACE2 is identified as a novel 0-secretase. Overexpression of BACE2 drastically decreases Ap production in cells, whereas overexpression of BACE1 greatly increases Ap production. We and others have shown that Ap is elevated in brains of DS ii patients. Our study further shows that p-secretase activity is abnormally increased. Further study reveals that BACE1 protein levels are markedly increased in DS fetal brain tissues. Time-lapse live imaging, cell fractionation, and pulse-chase experiments show that B ACE1 accumulates abnormally in the Golgi of DS cells. These data demonstrate that abnormal BACE1 accumulation leads to elevated pVsecretase activity and subsequent A(3 deposition in DS patients. Our results provide a novel molecular mechanism by which A D develops in DS and suggest that inhibiting BACE1 or potentiating BACE2 would benefit AD patients. iii T A B L E O F C O N T E N T S ABSTRACT ii T A B L E OF CONTENTS iv LIST OF TABLES x LIST OF FIGURES ••• • xi LIST OF ABBREVIATIONS xiii ACKNOWLEDGEMENTS xxi CO-AUTHORSHIP STATEMENTS xxiii C H A P T E R 1: General introduction to Alzheimer's disease 1 1.1. Overview of Alzheimer's disease 2 1.1.1. Early stories of Alzheimer's disease 2 1.1.3. Epidemiology of Alzheimer's disease 3 1.1.3. Risk factors predisposing to Alzheimer's disease 5 1.1.3.1. Age 7 1.1.3.2. Gender and education 7 1.1.3.3. Genetic factors 8 1.1.3.4. Medical history and treatment 8 1.1.3.5. Vascular factors 10 1.1.3.6. Health habits and diet 14 1.1.4. Current Diagnosis of Alzheimer's disease 15 1.1.4.1. Clinical diagnosis 15 iv V 1.1.4.2. Pathological diagnosis 16 1.1.5. Current treatment for Alzheimer's disease 17 1.2. Pathogenesis of Alzheimer's disease 20 1.2.1. Genetics of Alzheimer's disease 20 1.2.1.1. Familial mutations in APP 20 1.2.1.2. Familial mutations in presenilin 1 and 2 23 1.2.1.3. ApoE4 polymorphisms in the late-onset Alzheimer's disease... 25 1.2.2 Neuritic plaques and amyloid P protein generation 26 1.2.2.1. P-amyloid precursor protein (APP) 27 1.2.2.2. P-site cleaving enzyme (p-secretase, BACE1) 32 1.2.2.3. a-site cleaving enzyme (cc-secretase) 36 1.2.2.4. y-site cleaving enzyme (y-secretase) 37 1.2.2.5. Ap clearance..; 42 1.2.2.6. AP immunization 45 1.2.3. Neurofibrillary tangles and tauopathy 47 1.2.4. Transgenic mouse models of Alzheimer's disease 54 1.2.4.1. APP transgenic mice 54 1.2.4.2. Presenilin and multiple gene transgenic mice 55 1.2.5. The Ap cascade hypothesis of Alzheimer's disease 56 1.2.6. Down Syndrome, a valuable model of Alzheimer's disease 59 1.3. Objectives and hypotheses 60 1.4. References 61 v CHAPTER 2 : The human BACE2 gene has distinct transcriptional regulation from BACE1 gene 102 2.1. Introduction 103 2.1.1. Overview of eukaryotic gene promoters 103 2.1.2. Identification of the BACE2 gene and its tissue-specific expression 105 2.2. Materials and methods 107 2.2.1. Cell culture 107 2.2.2. Molecular cloning of BACE2 promoter and construction of chimeric luciferase reporter plasmids 108 2.2.3. Primer extension I l l 2.2.4. Transfection 112 2.2.5. Promoter assay 112 2.3. Results : 113 2.3.1. Cloning the human BACE2 gene promoter and computer analysis for putative transcription factors 113 2.3.2. Primer extension to identify the transcription starts sites 113 2.3.3. Identification of BACE2 promoter and its transcriptional activity 115 2.3.4. SP1 can regulate BACE2 gene expression 118 2.3.5. Comparative sequence analysis of BACE1 and BACE2 genes 121 2.3.6. Distinct transcriptional activation of BACE1 and BACE2 genes in neuronal and non-neuronal cell lines 122 2.4. Discussion 124 vi 2.5. References 128 C H A P T E R 3: BACE2, as a novel APP 9-secretase, is not responsible for the pathogenesis of Alzheimer's disease in Down Syndrome 131 3.1. Introduction 132 3.2. Materials and methods 135 3.2.1. Plasmid construction 135 3.2.2. Transient and stable transfections 136 3.2.3. Immunoblotting and immunoprecipitation 137 3.2.4. N-terminal sequencing 141 3.2.5. APP23 mice and genotyping 142 3.2.6. Primary neuronal culture 143 3.2.7. Lentivirus generation and infection 144 3.2.8. Ap40/42 sandwich ELISA assay 144 3.3. Results 145 3.3.1. BACE2 and BACE1 distinctly regulate APP processing and Ap generation 145 3.3.2. N-terminal sequencing analysis shows that BACE2 cleaves APP at a novel 9-site 149 3.3.3. Overexpression of BACE2 markedly reduced Ap generation in primary neurons from AD transgenic mice 152 3.3.4. BACE2 transcription is elevated in DS patients 153 3.3.5. BACE2 protein levels remain unchanged in DS patients 155 vii 3.4. Discussion 156 3.5. References ••••• 161 C H P A T E R 4: Increased B A C E 1 Maturation contributes to Alzheimer's Disease pathogenesis in Down Syndrome 168 4.1. Introduction 169 4.2. Materials and methods 172 4.2.1. Cell cultures, plasmids and transfections 172 4.2.2. Western blots 173 4.2.3. Immunoprecipitation 174 4.2.4. A|340/42 sandwich ELISA assay 174 4.2.5. Quantitative RT-PC and real time PCR 174 4.2.6. Karyotyping of DS cell lines 175 4.2.7. Time-lapse live cell image analysis 176 4.2.8. Cell fractionation 176 4.2.9. Pulse-chase assay 177 4.2.10. Tunicamycin treatment 177 4.3. Results 178 4.3.1. Markedly elevated (3-secretase activity in DS brains 178 4.3.2. Increased BACE1 total and glycosylated protein levels in DS brains.... 180 4.3.3. BACE1 transcription was unchanged in DS 182 4.3.4. Time-lapse analysis of BACE1 trafficking in DS cell lines 184 viii 4.3.5. Abnormal BACE1 protein trafficking and accumulation in the Golgi of DS cells •. 185 4.4. Discussion 187 4.5. References 189 C H A P T E R 5: General discussion... 195 5.1. Molecular rationale of B A C E 1 as a primary drug target 197 5.2. Molecular rationale ofBACE2 as a novel drug target 200 5.3. Furture directions 202 5.4. Conclusions 204 5.5. References 205 ix LIST O F T A B L E S Table 2.1. Putative Transcription Factor Binding Sites In The Human BACE1 and BACE2 Genes LIST O F FIGURES Figure 1.1. Prevalence of Alzheimer's disease as a Function of Age in Men and Women 5 Figure 1.2. Distribution of A D severity in different age groups 6 Figure 1.3. Schematic diagrams showing APP mutations linked to early-onset Alzheimer's diseases 23 Figure 1.4. Schematic diagrams showing mutations in presenilin 1 linked to early-onset Alzheimer's disease 24 Figure 1.5. APP processing by a-, (3-, and y-secretase 29 Figure 1.6. Intracellular trafficking of APP. 30 Figure 1.7. Pathways of microglia activation in Alzheimer's disease 47 Figure 1.8. Tau gene structure, pre-mRNA alternative splicing in CNS and protein isoforms translated from alternative tau mRNAs 50 Figure 1.9. The bar code of tauopathies and their classification 53 Figure 1.10. The sequence of pathogenic events leading to A D proposed by the A(3 cascade hypothesis 57 Figure 2.1. The eukaryotic transcriptional machinery 104 Figure 2.2. Core promoter elements 105 Figure 2.3. The human BACE2 gene promoter sequence 115 Figure 2.4. Functional analysis of the BACE2 gene promoter 118 Figure 2.5. Potentiation of BACE2 Promoter Activity by SP1 120 xi Figure 2.6. Comparative sequence and promoter activity analysis of the human BACE1 and BACE2 gene 123 Figure 3.1. Regulation of APP processing and A(3 production by BACE1 and BACE2 149 Figure 3.2. N-terminal sequencing of the APP C-terminal cleaved by BACE2 showed that BACE2 cleaves APP at a novel 0 site 151 Figure 3.3: Overexpression of BACE2 by lentivirus decreased A(3 production in primary transgenic neurons 153 Figure 3.4. Increased BACE2 mRNA levels in DS patients 154 Figure 3.5. Protein level of BACE2 remains unchanged in DS 156 Figure 4.1. Marked elevation of Ap and C99, the major P-secretase product in the DS brains 180 Figure 4.2. Significant increase in total and mature BACE1 protein levels in the DS brains 182 Figure 4.3. BACE1 transcription was unchanged in DS patients whereas APP mRNA was increased by about 1.5 times 183 Figure 4.4. Time-lapse analysis of BACE1 trafficking in DS cell lines: 185 Figure 4.5. Abnormal trafficking and accumulation of mature BACE1 protein in DS 287 Figure 5.1. BACE2, as a novel APP 9-secretase, is not responsible for A D pathogenesis in DS 201 Figure 5.2. Increased BACE1 maturation contributes to A D pathogenesis in DS 203 xii LIST O F ABBREVIATIONS Ach acetylcholine AchEIs acetylcholinesterase inhibitors AD Alzheimer's disease A D A M a disintegrin and metalloprotease AEBSF 4- (2-Aminoethyl) benzenesulphonyl fluoride AICD APP intracellular cytopasmic domain A M V avian myeloblastosis virus ANF atrial naturetic peptide A N O V A analysis of variance API activator protein-1 APLP-1 (3 amyloid precursor-like protein-1 APLP-2 (3 amyloid precursor-like protein-2 APP amyloid precursor protein ATP adenosine triphosphate BACE1 Beta-Site amyloid precursor protein (APP)-cleaving enzyme 1 BACE2 Beta-Site amyloid precursor protein (APP)-cleaving enzyme 2 BBB blood-brain barrier B C A bicinchoninic acid bME (3-mercaptoethanol bp base pair xiii BRE TFIIB recognition element BRI3 brain-specific type II membrane protein C/EBP CCAAT/Enhancer Binding Protein CA1 Cornu ammonis CD44 cluster of differentiation 44 cdk cyclin dependent kinase cDNA complementary deoxyribonucleic acid CIAP alkaline phosphatase CK1 casein kinase 1 CL-4B cross-linked 4% agarose beads CNS central nervous system CREB cyclic A M P / C a + + response element binding protein CSF cerebrospinal fluid CTF C-terminal fragment DCC deleted in colorectal carcinoma D C E Downstream Core Element D M E M Dulbecco's modified Eagle's medium DNA deoxyribonucleic acid DPE Downstream Promoter Element DRAP Down region Aspartyl protease DS Down Syndrome DTT dithiothreitol xiv DUB deubiquitinating enzyme E l ubiquitin activating enzyme E2s ubiquitin conjugating enzymes E3s ubiquitin ligating enzyme E C L enhanced chemiluminescent E D T A ethylene diamine tetraacetic acid EGFP enhanced green fluorescent protein ELISA Enzyme-Linked Immunosorbent Assay ER Endoplasmic Reticulum ERGIC ER Golgi Intermediate Compartment FBS fetal bovine serum F D A food and drug administration FTD frontotemporal dementia with parkinsonism G A B A Gamma-aminobutyric acid GATA1 G A T A binding protein 1 GFP green fluorescent protein G P X glutathione peroxidase GR glucocoticoid receptor Grb2 growth factor receptor-bound protein 2 GSK-3 Glycogen synthase kinase 3 GTFs general transcription factors HB homogenization buffer X V H D L high density lipoprotein HEK293 human embryonic kidney 293 cell line HEPES N- (2-hydroxyethyl)-piperazine-N'-2-ethanesulf0nic acid HIV human immunodeficiency virus HMG-CoA hydroxymethylglutaryl coenzyme A HSF-1 Heat shock factor protein 1 IAPP Islet amyloid peptide IC50 the half maximal inhibitory concentration IDE insulin degrading enzyme JIP c-jun N-terminal kinase (JNK) interacting proteins (JIP) JNK c-jun N-terminal kinase K bp kilo base pair Kcnb2 potassium voltage-gated channel, Shab-related subfamily, member 2 Ki enzyme inhibition constant KO knockout LB Luria broth L E F Lymphoid enhancing factor L D L low density lipoprotein L F lipofectamine LRP lipoprotein receptor-related protein M bp mega base pair M A F v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian) xvi M A P microtubule-associated protein M A R K microtubule-affinity-regulating-kinase MCI mild cognitive impairment MDC9 Metalloproteinase-like, disintegrin-like, and cysteine-rich protein-9 MJD Machado-Joseph disease mM micromolar mM millimolar M M S E Mini-Mental State Examination mRNA messenger ribonucleic acid M T E Motif Ten Element MZF-1 myeloid zinc finger 1 N2A neuro-2a NEP neutral endopeptidase NF1 nuclear factor 1 NF-kB nuclear factor K B NMDA A/-methyl-D-aspartic acid NRSE neuronal restrictive silencer element NRSF neuronal restrictive silencer factor NSAIDs non-steroid anti-inflammatory agents NTF N-terminal fragment Oct-1 Octamer-binding transcription factor 1 OD optical density xvii O T U Otubain protease p75NTR p75 neurotrophin receptor PBS phosphate buffered saline PCR polymerase chain reaction PDGF platelet derived growth factor PH potential of hydrogen PKA protein kinase A PLSCR1 phospholipid scramblase 1 Pol II RNA polymerase II PSGL-1 P-selectin glycoprotein ligand 1 PVDF PolyVinylidine DiFluoride R A G E advanced glycation end products RAP receptor associated protein RE-1 repressor element-1 REST RE-1 silencing transcription factor RIPA radioimmunoprecipitation Assay R L U relative luciferase units RMSD root-mean-square deviation RNA ribonucleic acid RT-PCR reverse transcriptase polymerase chain reaction SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate - polyacrylamide gel electrophoresis xviii She Src homology 2 domain containing siPvNA small interference RNA SOD superoxide dismutase SP1 specificity protein 1 T A C E tumor necrosis factor a converting enzyme TAFs TBP-associated factors T B E Tris/Borate/EDTA T C F T cell factor TBP T A T A binding protein T E M E D tetramethylethylenediamine TF transcription factor T G N trans-Golgi network thy-1 thymus cell antigen 1 Tip60 Tat interacting protein TMP21 21 kDa transmembrane trafficking protein T N F a tumor necrosis factor a TSS transcription start site U C H ubiquitin C-terminal hydrolase USF upstream stimulatory factor USP ubiquitin-specific protease UTR untranslated region WHIMS Women's Health Initiative Memory Study xix WT wild type WT-1 Wilms tumor homolog xx A C K N O W L E D G E M E N T S I would like to acknowledge my supervisor and mentor Dr. Weihong Song for his guidance, advice and enthusiasm. Dr. Song offered me incredible possibilities and freedom to explore in science. I am particularly grateful for his encouragement, kindness and patience, both personally and professionally. I would like to thank my supervisory committee members Dr. Max Cynader, Dr. Anthony Phillips, and Dr. William Jia for their helpful insights and invaluable time. I am impressed and encouraged by their dedication, curiosity, and passion in pursuit of science. I would like to give special thanks to Dr. Hong Qing, Dr. Weihui Zhou and Dr. Yigang Tong for their patience and instructions. I thank Andrea Human and Kelley Bromley for proofreading this thesis. I would also love to acknowledge all my colleagues in Dr. Song's lab, including Guiqiong He, Alan Chen, Michel Christensen, Fang Cai, Bin Chen, Ke Wang, Shengcai Wei, Yingcheng Wang, Jane Wang, and Oliver Holmes. It is a great pleasure to work with them. In addition, I would love to thank all the friends I met in Vancouver, including Wei, Ming, Haizhong, Jian, Triny and lots of others. They are making my graduate . studies in U B C a pleasant memory forever, t am fortunate to share with them moments of pleasure and passion, as well as melancholy and uncertainty. xxi I would love to appreciate all my family members. I would like to thank my parents, brother and sisters for their unwavering support and encouragement. And last but not least, I will thank my dear love, Qichen Li , for his unconditional love and support during my studies. xxii C O - A U T H O R S H I P S T A T E M E N T S For the work described in Chapter 2, "The human BACE2 gene has distinct transcriptional regulation from BACE1 gene", I developed the experimental approach together with the senior author. I performed most of the bench-work related to BACE2 gene promoter assay and BACE2 function. The co-first author Yingcheng Wang did most of the BACE2 promoter constructs cloning and primer extension in Fig 2.4.1 did all the data analysis and wrote the first manuscript for this paper. The senior author supervised the work and wrote the final draft for publication. For the work described in Chapter 3, "BACE2, as a novel APP 0-secretase, is not responsible for the pathogenesis of Alzheimer's Disease in Down Syndrome", I developed the experimental approach together with the senior author and performed a majority of the bench-work. Coauthor Guiqiong He helped me to do the Ap ELISA in Fig 3.3 and I did all the data analysis. I wrote the first manuscript and the senior author attended the revisions required for final publication. For the work described in Chapter 4, "Increased BACE1 Maturation contributes to Alzheimer's Disease pathogenesis in Down Syndrome", I performed most of the bench-work. Coauthor Yigang Tong performed the RT-PCR for BACE1 and APP in Fig 4.3. He also helped me to do the C99 western blot and AP ELISA in Figure 4.1. The other coauthors Hong Qing and Chia-Hsiung Chen assisted me in preparing materials. I wrote the first draft for the paper. The senior author supervised the work and wrote the final draft for publication. xxiii C H A P T E R 1: General Inrtroduction to Alzheimer's Disease 1 1.1. Overview of Alzheimer's disease 1.1.1. Early stories of Alzheimer's disease On Nov 4, 1906 in Tubingen, Dr. Alois Alzheimer gave a historic lecture that made his name known at the 37th Conference of South-West German Psychiatrists. In the lecture, Dr. Alzheimer presented the case of patient Auguste D, who was a 51-year-old woman showing progressive cognitive impairment, disorientation and psychosocial incompetence (Alzheimer, 1906). Four and a half years after initial institutionalization, patient Auguste D died and her brain and records were sent to Dr. Alzheimer to examine. At necropsy, Dr. Alzheimer described the neuropathological changes in Auguste D, including brain atrophy, prominent neuronal loss and two distinct types of lesions visible by Bielschowsky silver preparation (Alzheimer, 1907). He described an abnormal change in the brain: "Dispersed over the entire cortex, and in large numbers especially in the superior layers, miliary foci could be found which represented the sites of deposition of a peculiar substance in the cerebral cortex". The "military foci" were later termed as amyloid plaques. He also noted peculiar changes in the neurofibrils: "In the center of an otherwise almost normal cell there stands out one or several fibrils due to their striking thickness and peculiar impregnability. Then they accumulated forming dense bundles and eventually the nucleus and cytoplasm disappeared and only a tangled bundle of fibrils left". The "tangled bundle of fibrils" was later known as neurofibrillary tangles. Several other cases resembling the clinical and neuropathological changes described by Dr. Alzheimer were reported by Dr. Fischer, Dr.Perisini, Dr. Bonfiglio and Dr. Kraepelin around a similar time (Bonfiglio, 1908b; Fischer, 1907b; Kraepelin, 1910; Perusini, 1910). In 1910, Dr. Emil Kraepelin suggested this senile dementia characterized by 2 plaques and neurofibrillary tangles to be named "Alzheimer's disease" (AD) (Kraepelin, 1910). It was not until 1927 that Dr. Divry identified that amyloid is the major component of plaques (Divry and Florkin, 1927a). And Dr.Scholz (1938) found that amyloid is also deposited in the cerebral vessels in A D brains (Scholz, 1938). The paired helical neurofilaments in neurofibrillary tangles were observed by electron microscopy in the 1960s (Kidd, 1963; Terry, 1963). In 1982, neurons of the nucleus basalis of Meynert were found to undergo a profound (greater than 75%) and selective degeneration in AD brains (Whitehouse et al., 1982). Nearly three decades after the identification of amyloid in neuritic plaques, the amyloid protein was purified and sequenced from fibrils of cerebrovascular amyloidosis (Glenner and Wong, 1984a; Glenner and Wong, 1984b) and from plaque cores of both AD and aged DS patients (Masters et al., 1985). Three years after the identification of amyloid protein, the (3-amyloid precursor protein (APP) gene was identified and mapped to chromosome 21 (Goldgaber et al., 1987; Kang et al., 1987; Robakis et al., 1987; Tanzi et al., 1987). Meanwhile, phosphorylated tau was also discovered from neurofibrillary tangles in A D brain by its antigenic activity (Grundke-Iqbal et al., 1986a; Grundke-Iqbal et al., 1986b; Hiara et al., 1986; Kosik et al., 1986; Nukina and Hiara, 1986). 1.1.2. Epidemiology of Alzheimer's disease AD is the most common form of irreversible dementia, accounting for two thirds of cases, with vascular dementia accounting for much of the remains. In Canada, there are about 435,000 patients over 65 affected by A D and related dementias in 2006 (Group, 2000), with an economic cost of approximately 5.5 billion per year (Ostbye and Crosse, 3 1994). A D is the 8th leading cause of death in the United States and 5th leading cause of death among the elderly according to National Center for Health Statistics (2002). The incidence rate of AD is approximately 1% annually between the ages of 65 to 70 years and 8-10% annually above age 85, with incidence then declining after age 93 (Kawas et al., 2000; Miech et al., 2002). The incidence rate is slightly higher for women and for African Americans and Caribbean Hispanics (Andersen et al., 1999; Perkins et al., 1997) (Figure 1.1). The lifetime risk of AD or another dementia varies from 12-19% for women and 6-10% for men over the age of 65 based on data from the Framingham study (Seshadri et al., 1997). Among elderly people of 65 years old, A D has a prevalence of approximately 6-10% and doubles every five years to reach approximately 30-50% at age 85. There were an estimated 4.5 million A D patients in 2005, which is expected to increase to 11.3 to 16 million by 2050 as the U S population ages. Medicare for beneficiaries with A D in the U.S is expected to increase by 75%, from 91 billion in 2005 to 160 billion in 2010. Unless there was a breakthrough in the prevention and treatment of AD, the cost would bankrupt the medicare system (Lewin, 2004). Figure 1.1: Prevalence of Alzheimer's disease as a Function of Age in Men and Women. The prevalence of A D increases with age. Women have a higher prevalence than men. Among elderly people of 65 years old. Adapted from Nussbaum, R. L. et al. N Engl J Med 2003;348:1356-1364. 4 S 5004 S «Q0»J < i f u W0-20W 1«H Women / / «5-&S W-'ji ?&I?B «As4 S$.»S* 91^94 s95 Age [pr] A D has a long preclinical phase, followed by mild cognitive impairment (MCI) in which the person has an isolated memory impairment, and, finally, dementia. MCI is defined as the presence of consistent memory impairment without decrements in other areas of cognition or function. Stringent definitions of MCI, such as performance on memory tests that is more than one standard deviation below age- and education-adjusted norms, discriminate MCI from normal aging. Using this definition, the conversion rate of those with MCI to AD is as much as 15% per year or 50-70% over 4 to 5 years, to the approximately 1 % per-year conversion rate in age-matched controls (Sadock, 2004). A D is a progressive and irreversible neurodegenerative disease leading to dementia. The duration of disease varies from 2 to 20 years with an average of 3-4 years (Ostbye et al., 1999; Wolfson et al., 2001). In general, patients lose approximately 3 5 points on the M M S E per year (Fig 1.2). The decrement can be relatively gradual in the mild stage but takes a more dramatic turn in the moderate stage (Sadock, 2004). Figure 1.2: Distribution of A D severity in different age groups. Prevalence of severe (Mini-Mental State Examination score, <=9), moderate (Mini-Mental State Examination score, 10-17), and mild (Mini-Mental State Examination score, >=18) Alzheimer disease, in each of 3 age groups. Adapted from Hebert, L. E. et al. Arch Neurol 2003;60:1119-1122. 1.1.3. Risk factors predisposing to Alzheimer's disease A majority of A D cases is sporadic, which accounts for more than 95% of A D population. There are at least four well-defined risk factors for AD: aging, familial association, DS and having ApoE4 allele. Some epidemiological studies have disclosed other risk factors including lower education, female gender, stroke, head injury, atherosclerosis, hypertension, diabetes, high homocysteine levels, high L D L cholesterol, smoking, etc. In contrast, use of non-steroid anti-inflammatory agents (NSAIDs), hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, moderate alcohol intake, and strong social support are protective against A D (Tierney, 2006). 6 1.1.3.1. Age Age is the greatest risk factor for A D (Fig 1.1, 1.2). In Canada, A D has a prevalence of approximately 5% over age 65. The prevalence is 1% between age 65-74, 7% between age 75-84 and 25% above age 85. Age may contribute to AD pathogenesis by mechanisms involving oxidative stress, hypoperfusion, accumulation of misfolded proteins, or other causes. As the postmitotic tissue with a high energy demand, the brain is particularly susceptible to oxidative stress. Although the brain is only 2% of our body weight, it consumes approximately 20% of the body's resting oxygen (Raichle, 2001). Oxidized DNA, lipids and protein products are increased in A D patients compared to controls (Floyd and Hensley, 2002; Markesbery and Carney, 1999; Mhatre et al., 2004). Also some antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPX), soluble vitamin C, A and E are decreased in MCI and A D patients (Mecocci, 2004). Oxidative stress may be a common pathophysiological pathway involved in many neurodegenerative diseases. But how oxidative stress selectively damages particular populations of neurons to induce different forms of disease remains unknown. 1.1.3.2. Gender and education Women have a higher prevalence of AD than men (Figure 1.1), a difference that could be due to higher incidence or longer survival after developing the disease. The odds ratio for women to develop A D relative to men is 1.56 (Gao et al., 1998). Several studies have shown that less educated individuals have a higher incidence of A D or cognitive decline (Ott et al., 1995; Stern et al., 1994). One possible explanation is that educated 7 people may be able to compensate for any cognitive decline such that the diagnosis of A D is delayed. 1.1.3.3. Genetic factors A family history of dementia is an important risk factor. Meta-analysis has shown that the risk for AD is 3.5 times higher in individuals that have at least one first-degree relative with dementia (van Dijk et al., 1991). There is a stronger family history in early-onset A D compared to late-onset AD. Some families show an autosomal dominant pattern of inheritance for early-onset AD. Mutations in APP, presenilin 1 (PS1) or presenilin 2 (PS2) can cause A D (refer to chapter 1.2.1). People with DS show A D pathological changes after their middle age (refer to chapter 1.2.6). ApoE4 polymorphism can alter the risk for late-onset A D (refer to chapter 1.2.1). Linkage analysis has also revealed other loci associated with late-onset A D (refer to chapter 1.2.1). 1.1.3.4. Medical history and treatments A history of head trauma with loss of consciousness can increase the risk of developing A D in males by approximately 1.8-2.5 times (Fleminger et al., 2003; Mortimer et al., 1991; van Duijn et al., 1992); however, a history of head trauma did not affect the risk of developing A D in females (Fleminger et al., 2003; van Duijn et al., 1992). It was also reported that A(3 immunoactivity can be detected in multiple cortical regions in 30% of people that have had severe head injury (Roberts et al., 1994). The observation in clinic-based case-control studies that a history of rheumatoid arthritis was inversely associated with A D suggested an association of anti-inflammatory 8 drugs with A D risk (McGeer et al., 1996). Several incidence and case-control studies consistently show that the long-term use of NSAIDs is protective against A D (in t' Veld et al., 2001; McGeer and McGeer, 2006). Postmortem studies show inflammatory changes surrounding neuritic plaques, suggesting possible involvement of inflammatory factors in disease development; however, it remains unknown if inflammation is a causative factor or compensatory effect of the pathological changes of AD. Though several studies have shown that NSAIDs can selectively decrease A(342 production (Eriksen et al., 2003; Weggen et al., 2001), randomized clinical trials failed to demonstrate NSAIDs as preventive to A D (Aisen et al., 2003; Reines et al., 2004; Thai et al., 2005). Animal studies have shown that estrogen has multiple neuroprotective effects, including increased cerebral blood flow, stimulation of cholinergic activity, prevention of neural trophy, and reversal of glucocorticoid damage. Some early observational studies suggest that long-term hormone replacement therapy may attenuate cognitive decline in postmenopausal women; however, the Women's Health Initiative Memory Study (WHIMS), a randomized controlled clinical trial showed that for women aged 65 years or older, hormone therapy did not reduce incidence of dementia or MCI and actually had an adverse effect on cognition, which was greater among women with lower cognitive function at initiation of treatment (Espeland et al., 2004; Shumaker et al., 2004). WHIMS was discontinued earlier than planned because it showed that hormone therapy increased risks for coronary heart disease outcomes, stroke, venous thromboembolism, and breast 9 cancer, although risks for hip fracture and colon cancer were decreased(Rossouw et al., 2002) . It is a general consensus that depression may be a prodromal feature of A D , but it is debated if depression is also a risk factor. A recent meta-analysis showed that depression predisposed to AD with an odds ratio of 2.03 and the interval of depression was positively associated with the risk of AD, suggesting that depression may be an independent risk factor of AD (Ownby et al., 2006). One possible explanation is that depression leads to hippocampal damage through excessive glucocorticoid secretion. Indeed, hippocampal atrophy and the severity of cognitive impairment in A D patients were correlated with dysfuntion of the hypothalamic-pituitary-adrenal axis (Pomara et al., 2003) . There is also in vivo and in vitro evidence showing that prolonged exposure to corticosteroids is associated with neuronal apoptosis in hippocampus and explicit memory impairment (de Quervain et al., 2003; Newcomer et al., 1998; Newcomer et al., 1999; Reagan and McEwen, 1997). 1.1.3.5. Vascular factors Epidemiological evidence has shown that vascular risk factors are involved in the development of AD. The large overlap in risk factors, pathology and clinical symptoms have challenged the current consideration of A D and vascular dementia as two discrete disorders. Besides the neuritic plaques and neurofibrillary tangles, cerebral amyloid angiopathy and microangiopathy are particularly common in AD. Cerebral amyloid angiopathy is a risk factor for cerebrovascular disorders such as cerebral haemorrhage 10 and is a feature of some early-onset familial A D (FAD). Incidence of cerebrovascular lesions is higher in AD than in age-matched controls (Esiri et al., 1999; Heyman et al., 1998; Jellinger and Attems, 2003; Jellinger and Attems, 2005). White matter hyperintensities on magnetic resonance imaging were frequentiy found to be associated with AD. And recent studies have shown that a history of stroke can increase A D prevalence by approximately twofold among elderly patients (Altieri et al., 2004; Schneider et al., 2003; Snowdon et al., 1997; Vermeer et al., 2003; White et al., 2002), with the risk being highest when stroke is concomitant with atherosclerotic vascular risk factors (Honig et al., 2005; Honig et al , 2003; Roher et al., 2003). Furthermore, patients with stroke or cerebral infarction show poorer cognitive performance and greater severity of clinical dementia (Heyman et al., 1998). A postmortem study of people without dementia found more senile plaques in those with coronary diseases and higher neurofibrillary tangle counts in the brains of those with previous hypertension (Sparks et al., 1990; Sparks et al., 1995). Reduced cerebral perfusion is also a common vascular component among AD risk factors (de la Torre, 2002; de la Torre, 2004). Several prospective studies show that hypertension and atherosclerosis can increase the risk for A D (Skoog and Gustafson, 2002; Skoog and Gustafson, 2003). A longitudinal population study showed that high blood pressure preceded A D by 10-15 years (Skoog et al., 1996). In the Honolulu-Asia aging study, a high systolic or diastolic blood pressure preceded A D and vascular dementia by 20-26 years, with the risk being highest among those who were never treated for hypertension (Freitag et al., 2006; Launer et al., 2000; Petrovitch et al., 2000). Further evidence indicating that hypertension 11 increases risk of developing A D came from the Finnish study and Rotterdam study (in't Veld et al., 2001; Kivipelto et al , 2001a; Kivipelto et al., 2001b; Ruitenberg et al., 2001). Some risk factors, such as diabetes and oxidative stress, are shared between hypertension and A D and may account for the increased risk. Hypertension also leads to atherosclerosis and arteriosclerosis and a major risk factor for stroke and ischemic white matter lesions, all of which have been shown to be risk factors for AD. Several prospective studies also show that having diabetes doubles the risk for developing A D (Taubes, 2003). Interestingly, diabetes and A D are linked not only epidemiologically, but may also share some pathophysiological pathways. Both type 2 diabetes mellitus and A D are associated with senescence and localized amyloid deposition that may interfere with normal cellular functioning. Neurodegeneration is also a prominent feature of type 2 diabetes, manifested by peripheral and autonomic neuropathy. Other complications of diabetes include macroangiopathy and microangiopathy. Macroangiopathy, atherosclerosis in large vessels, leads to coronary heart disease and stroke, accounting for the majority of diabetes mortality and also being risk factors for AD. Microangiopathy leads to diabetic nephropathy and eventually renal failure. Amyloid angiopathy is an important pathological change in brains of A D patients. During embryogenesis, pancreas development is regulated by the notochord in parallel with neural development and utilizes some transcription factors also critical for neural development (Cleaver and Krieg, 2001). Regarding the molecular pathogenesis, islet amyloid deposition exists in up to 96 % of type 2 diabetes examined by autopsy (Clark et al., 1995). Islet amyloid peptide (IAPP), a soluble 37 amino acid peptide, was isolated from amyloid deposits in the pancreas. Overexpression of human IAPP in transgenic 12 mice leads to pancreatic amyloid deposition and diabetes (Verchere et al., 1996). Beta-site amyloid precursor protein (APP)-cleaving enzyme l (BACEl) , an important enzyme involved in A P generation, is also highly expressed in the pancreas; however, its functions there remain unkown. Insulin degrading enzyme (DDE) can degrade both insulin and A(3, but has a higher affinity for insulin, suggesting that insulin may compete with Ap to promote Ap deposition in cases of hyperinsulinemia. Interestingly, IDE levels were decreased by approximately 50% in A D patients carrying ApoE4 alleles and IDE deficient mice developed both AD and diabetes (Cook et al., 2003; Farris et al., 2003). High plasma homocysteine levels have been shown to increase risk for A D in both case-control and prospective studies (Morris, 2003). Hyperhomocysteinemia is also a risk factor of vascular disease (Selhub et al., 1995), which may account for the higher risk for AD; however, one prospective study showed that hyperhomocyteinemia may be an independent risk factor for A D (Seshadri et al., 2002), with a stronger association in autopsy confirmed A D cases (Clarke et al., 1998). Some earlier epidemiological studies show that high cholesterol in mid-life is a risk factor for AD (Kivipelto et al., 2001b; Lesser et al., 2001) and that statins, a family of cholesterol reducing drugs, are protective against A D (Jick et al., 2000; Wolozin et al., 2000; Yaffe et al., 2002). However, later prospective studies failed to find an association of total and/or H D L cholesterol levels with incidence of A D in the Honolulu-Asia aging study and Framingham Study (Kalmijn et al., 2000; Tan et al., 2003). Some studies even found the opposite relationship between high cholesterol level and A D (Mielke et al., 13 2005; Reitz et al., 2004; Romas et al., 1999). Similarly, three large prospective studies failed to find a reduced risk of A D with statin use (Group, 2002; L i et al., 2004; Shepherd et al., 2002). 1.1.3.6. Heal th habits and diet Light to moderate alcohol intake is beneficial to the elderly by reducing mortality from coronary heart diseases as well as cerebrovascular diseases. Several studies have shown that moderate alcohol consumption is protective against A D (Huang et al., 2002; Luchsinger et al., 2004). In contrast, heavy alcohol consumption is a risk factor of dementia, probably due to the neurotoxic effects of alcohol (Harper and Kril, 1990; Saunders et al., 1991). Though earlier population based case-control studies showed that smoking was inversely related to A D (van Duijn and Hofman, 1991), several later cohort follow-up studies showed smoking is actually a risk factor for A D (Juan et al., 2004; Merchant et al , 1999; Ott et al., 1998; Wang et al., 1999). Earlier pathological studies show increased aluminum concentration in A D brain, suggesting that aluminum is a risk factor for AD. Epidemiological studies showed high aluminum concentration in drinking water was associated with A D with a relative risk of 2.14 (Rondeau et al., 2000). A recent study also showed higher aluminum and lower magnesium and phosphorus concentrations were associated with A D (Andrasi et al., 2005). Some studieshave shown certain dietary factors may be associated with AD. Fish 14 and cereal consumption may protect against AD, while high dietary fat and caloric intake, as well as vitamin deficiencies in B6, B12 and folate, and antioxidants such as vitamin E and C, may increase A D risk (Capurso et al., 1997; Solfrizzi et al , 2003). 1.1.4. Current Diagnosis of Alzheimer's disease 1.1.4.1. Clinical diagnosis A D is a clinical-pathological syndrome in which clinical dementia is due to AD-type neuropathological lesions (Hyman, 1997). Clinical diagnosis of dementia is achieved by taking a careful history, clinical examinations and selected tests. A clear social and clinical history is important to differentiate dementia due to A D from other causes such as HIV, Parkinson's and Huntington's disease. Selected lab tests and imaging are used to rule out correctable or contributory causes of dementia. The neurological examination emphasizes assessment of mental status. The most common test is the Marshall Folstein's Mini-Mental State Examination (MMSE), a 30-point scale that covers: orientation (10 points); immediate and delayed recall (6 points); naming and repeating (3 points); concentration (or concentration and calculation, for example, spelling a word backwards or doing serial subtraction) (5 points); following a three-step command (3 points); and following a written command, copying a figure, and writing a sentence (3 points)(Sadock, 2004; Tierney, 2006). The most commonly used criteria in A D diagnosis are those of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) published in 1984(McKhann et al., 1984). According to NINCDS-ADRDA, "possible A D " represents those with not fully consistent development of dementia 15 symptoms or when a secondary dementing disorder is also present. A diagnosis of "probable AD" requires: 1) dementia, established by clinical examination and documented by the M M S E or a similar test and confirmed by neuropsychological tests, 2) deficits in two more areas of cognition, 3) progressive worsening of memory and other cognitive functions, 4) no disturbance of consciousness, 5) onset between ages 40 and 90, most often after age 65, and 6) no systemic disorders or other brain diseases that could account for the progressive deficits in memory and cognition. Using these criteria, the accuracy of the clinical diagnosis in the best of hands, as determined by subsequent pathological confirmation, approaches 90% (Blacker et al., 1994). The "definitive diagnosis of A D " depends on finding typical neuritic amyloid plaques and neurofibrillary tangles on microscopic examination of brain tissue. 1.1.4.2. Pathological diagnosis The minimum criteria for neuropathological diagnosis of A D requires that: 1) there are substantial numbers of plaques widely distributed throughout many parts of the cerebral cortex with at least 10/mm2 in some areas, 2) there is a high concentration of neurofibrillary tangles in the hippocampus and adjacent structures, especially hippocampal field CA1 and the entorhinal and perirhinal cortices, with an average tangle density in the entorhinal cortex of at least 5/mm2 which is much higher in layers II and IV, and 3) there are indications of cytoskeletal changes in the neocortex as well as in the hippocampal formation. Three types of senile plaques are visible under microscope. The "classical" or "mature" ones have both an amyloid center and a neuritic crown. A(3 is the major component in the amyloid center. The crown mainly consists of dystrophic neurites 16 that are labeled with anti-NFT and anti-tau markers. The "burned out" or "hypermature" plaques have a dense core of amyloid surrounded by reactive astrocytes but no dystrophic neurites. The "diffuse" or "immature" plaques contain a loose accumulation of amyloid that can be revealed by A(3 immunostaining. Senile plaques are widely distributed in neocortex including primary sensory and motor areas and association cortex as well as limbic areas. In addition, there must also be at least some tangles and neuropil threads in the neocortex. These are most prominent in cortical regions near the hippocampus and other limbic areas such as the inferior temporal cortex. With increasing severity of dementia in the later stages of AD, there is an increase in tangle density. This is especially true in the vulnerable areas in and around the hippocampus, but is also seen in the neocortex. Although the total number of plaques does not increase substantially, the proportion of neuritic and cored plaques does increase with increasing dementia severity (Price, 1997). 1.1.5. Current treatment for Alzheimer's disease In general there are three types of treatment for A D dementia: 1) treatment of cognitive symptoms mainly by replacement strategies that focus on neurochemical deficits in A D , 2) treatment of A D risk factors to slow down the course of the disease or correct reversible causes, 3) treatment of associated symptoms or behaviors that complicate dementia (Sadock, 2004). Unfortunately, there has been no cure for A D that can stop disease progress, partly due to its unknown pathogenesis. 17 Numerous neurochemical abnormalities have been demonstrated by autopsy or by functional neuroimaging in A D patients. As the disease progresses to later stages, these abnormalities include deficits in acetylcholine (Ach), norepinephrine, serotonin, dopamine, G A B A , glutamate, corticotrophin-releasing factor and somatostatin. Deficits in the cholinergic system have been described most robustly. The activity of choline acetyl transferase, the final step in Ach synthesis, is substantially reduced in AD. This is particularly evident in the nucleus basalis of Meynert and some neocortical regions. This neurochemical abnormality has led to the development of acetylcholinesterase inhibitors (AChEIs) as the most often prescribed medication for mild to moderate AD. AChEIs can slow the breakdown of Ach in the synapse and thereby increase the effective Ach available. The drugs currently available include tacrine (Cognex), donepezil (Aricept), rivastigmine (Exelon), and galantamine (Reminyl). These medications can generally improve clinical symptoms of A D for 6-12 months. After that, the patients will usually continue to decline with a similar rate as the untreated cases. Glutamate and N M D A receptor overactivation has also been implicated in A D (Bossy-Wetzel et al., 2004; Hardingham and Bading, 2002). Excitotoxicity, related to an excess release of glutamate, can lead to calcium influx and oxidative and nitrosative stress in neurons, resulting in neuronal death in A D and other neurodegenerative diseases including vascular dementia and Parkinson's disease (Lipton, 2005; Lipton, 2006). This concept has led to Memantine as a newly FDA approved drug for AD. Memantine is an uncompetitive N M D A receptor antagonist, which is well tolerated and efficacious in 18 treating mild to severe A D in clinical trials (Reisberg et al., 2003; Reisberg et al., 2006; Wilcock, 2003). Other agents that have been evaluated include ergoloid mesylates (Hydergine), antioxidants, NSAIDs, HMG-CoA reductase inhibitors (collectively known as statins), and estrogen replacement. Hydergine, the brand name for a specific combination of four dihydro derivatives of ergotoxine, also referred to as ergoloid mesylates, was introduced to clinical medicine in 1949. It is a mild vasodilator and was used as a treatment for peripheral vascular disease, hypertension, and angina pectoris. Currently, it is used almost exclusively for treating patients with either dementia, or age-related cognitive symptoms. In the United States, the FDA has approved hydergine for idiopathic decline in mental capacity and a recent meta-analysis showed that hydergine has significant treatment effects in patients with dementia compared to placebo (Olin, 2004). Vitamin E is a fat-soluble antioxidant and can readily pass the blood brain barrier. Vitamin E can neutralize the toxic effect of peroxide and free radicals and is thought to inhibit the process of lipid peroxidation that can damage the polyunsaturated fatty acids essential to cell membrane integrity. It has been proposed that oxidative stress and free radical damage are associated with neuronal death in A D (Arlt et al., 2002; Marcus et al., 1998). Vitamin E was shown to be neuroprotective in vitro and was able to delay deterioration in APP transgenic mice in vivo (Sung et al., 2004). Some studies have shown decreased vitamin E in A D patients (Jimenez-Jimenez et al., 1997). And several studies have shown that supplementation with vitamin E, or vitamin E in combination 19 with vitamin C can delay functional deterioration in AD patients (Sano et al., 1997), but had no effect in Parkinson's disease or Huntington's disease (Group, 1993). The Cochrane systemic review concluded that there was not sufficient evidence to support that vitamin E is effective against A D (Tabet, 2005). More controlled clinical trials are needed to evaluate the efficacy of vitamin E in AD. Inflammation and microglia activation characterize many neurodegenerative diseases including A D (Eikelenboom et al., 2002). Epidemiological studies have suggested that long-term use of NSAIDs can protect against A D (Etminan et al., 2003). Although several studies have shown that NSAIDs can selectively decrease Ap42 production (Eriksen et al., 2003; Weggen et al., 2001), randomized clinical trials have failed to provide evidence of slowing down disease course (Aisen et al., 2003; Reines et al., 2004; Thai et al., 2005). Similarly, although epidemiological studies have shown that statins can protect against AD (Wolozin et al., 2000), other prospective studies did not obtain consistent results (Group, 2002; Li et al., 2004; Shepherd et al , 2002; Zandi et al., 2005). Multicenter clinical trials also did not support estrogen replacement as a treatment for A D (Mulnard et al., 2000; Shaywitz and Shaywitz, 2000). 1.2. Pathogenesis of Alzheimer's disease 1.2.1. Genetics of Alzheimer's disease 1.2.1.1. Familial mutations in APP gene It has been known for decades that A D can cluster in families and can be inherited in an autosomal dominant fashion. Early onset cases occurring before ages 60 to 65 20 comprise approximately 6-7% of all A D cases, among which about 7% of cases are familial with an autosomal dominant inheritance pattern and high penetrance (Nussbaum and Ellis, 2003). Except for the earlier age of onset, phenotypic analysis has shown that AD is highly similar in both histopathological and clinical manifestations in familial and sporadic AD. Although familial cases are rare, the importance extends far beyond its frequency, as they have allowed researchers to identify some of the critical pathogenetic pathways of AD. Cerebral amyloid angiopathy is most commonly associated with normal aging, AD, DS, and cerebral hemorrhage. Amyloid deposits in patients with hereditary cerebral hemorrhage with amyloidosis of Dutch type are antigenically related to and homologous in sequence with Ap in A D (van Duinen et al., 1987). An E22Q (E693Q in APP751) mutation in APP was identified in a Dutch family with severe amyloid angiopathy and early death in afflicted patients by their fifth and sixth decade of life (Levy et al., 1990; Van Broeckhoven et al , 1990). Another D23N (D694N in APP751) mutation in APP was identified from an Iowa family with autosomal dominant dementia beginning in their sixth or seventh decade of life (Grabowski et al., 2001). The Dutch and Iowa mutations result in loss of negative charge at Ap position 22 and 23 and increase the fibrillogenesis and pathogenecity of Ap (Van Nostrand et al., 2001). In a Flemish family that had presenile dementia and cerebral haemorrhage due to cerebral angiopathy, an A692Q mutation was identified in the APP gene (Hendriks et al., 1992). In affected members of two families with early-onset AD, Goate et al (1991) identified a heterozygous mutation in the APP gene (V717I) (Goate et al., 1991). The APP V717I mutation, termed the 21 London mutation, was also identified in many other families (Karlinsky et al., 1992; Raux et al., 2005). London mutations (V717I), together with other mutations occurring around the y-secretase site such as V717F, V717G, and T714I, can increase the ratio of Ap42 to Ap40 (De Jonghe et al , 2001; Kumar-Singh et al., 2000; Moechars et al., 1999; Suzuki et al , 1994). A(342, has two additional hydrophobic amino acids, alanine and isoleucine, at its C-terminus, and can form insoluble fibrils more readily and rapidly than Ap40 (Suzuki et al., 1994). Mullan et al (1992) later identified a KM670/671NL double mutation in a Swedish family (Mullan et al., 1992). The Swedish mutation, occurring close to the p-secretase cleavage site, can increase cleavage at the P-site. Triplication of APP may contribute to the early-onset of A D in DS. A recent report by Rovelet-Lecrux et al showed that duplication of the APP locus is associated with autosomal dominant early-onset A D with severe cerebral angiopathy in several families (Rovelet-Lecrux et al., 2006). Thus far, 30 mutations have been identified in the APP gene in 80 families, which accounts for approximately 13% of total early onset F A D cases (Fig 1.3. see the complete list at http://www.molgen.ua.ac.be/ADMutations). Figure 1.3: Schematic diagram showing A P P mutations linked to early-onset A D . Blue colored amino acids indicate AP40 and AP42 (light blue circles). Gray colored letters indicate the transmembrane domain of APP. Three digit numbers refer to positions of the known APP mutations linked to A D according to APP770 isoform. Red letters indicate the mutated amino acids. Scissors show the known a-, p-, y-secretase sites respectively. (See the detailed list on http://www.molgen.ua.ac.be/ADMutations) 22 1.2.1.2. Familial mutations in presenilin 1 and 2 genes The notion that FAD is genetically heterogeneous has led to intensive searching for loci other than APP, which account for FAD in many families with no link to chromosome 21. Linkage analysis identified a locus on chromosome 14q24.3 linking to some non-Volga German families (Schellenberg et al., 1992). Further linkage analysis and positional cloning identified the gene presenilin 1 on chromosome 14, several missense mutations of which were found to cosegregate with early-onset FAD (Sherrington et al., 1995). Shortly after presenilin J was identified, linkage analysis in Volga German kindred mapped an FAD locus at Chrlq31-43 and a gene homologous to 23 presenilin 1, called presenilin 2 (Levy-Lahad et al., 1995a; Levy-Lahad et al., 1995b). Later intensive genetic surveys identified approximately 150 mutations in presenilin 1 and 10 mutations in presenilin 2 that account for 61% and 3% of early-onset A D respectively. Unlike the cluster of APP mutations around the [3- or y-secretase cleavage sites, mutations in presenilin spread along the whole sequence (Figure 1.4). Mutations in presenilin 1 and 2 lead to selective increases in A(342, the highly amyloidogenic form of AP (Scheuner et a l , 1996). Mutations in presenilin 1 are associated with the earliest and most aggressive form of A D , commonly occurring before the age of 50 and leading to their demise in their 60s (Selkoe, 2001a). Figure 1.4: Schematic diagrams showing mutations in presenilin 1 linked to early-onset AD. Red letters indicate the identified mutations in presenilin 1. Mutations in presenilin 1 spread along the whole sequence. Adapted from http://www.molgen.ua.ac.be/ 24 1.2.1.3. ApoE4 polymorphisms in late-onset Alzheimer's disease Early-onset FAD predisposed by APP and presenilin mutations only accounts for approximately 1% of total A D cases. Studies on late-onset FAD have failed to find an association with APP or presenilin 1 and 2; however, further genetic studies have provided evidence for linkage with chromosome 19 (Pericak-Vance et al., 1991), where ApoE locus was identified (Corder et al., 1993; Saunders et al., 1993; Strittmatter et al., 1993a; Strittmatter et al., 1993b). ApoE4 gene dosage has an effect on the risk of developing AD, the age of onset, the accumulation of neuritic plaques in the brain, and the reduction of choline acetyltransferase. Three alleles of ApoE, epsilon 2, 3 and 4, are located on chromosome 19ql3. Three isoforms of ApoE, encoded by codons 112 and 158, have been shown to modify the risk of late-onset AD. At codon 112/158, ApoE2, E3 and E4 contain cysteine/cysteine, cysteine/arginine, and arginine/arginine, respectively(Rall et al., 1982; Weisgraber et al., 1981). The three isoforms have 0, 1+, and 2+ charges to account for electrophoretic differences. Compared to the more common ApoE epsilon 3 that is present in approximately 80% of Mediterranean population, the ApoE epsilon 4 allele that is present in 30% of Americans increases the risk of A D while the ApoE epsilon 2 with a 10% prevalence decreases the risk for A D (Corbo and Scacchi, 1999; Farrer et al., 1997). The risk for A D increases from 20-90% and mean age of onset decreases from 84 to 68 years with increasing number of ApoE epsilon 4 alleles. Homozygosity for APOE epsilon 4 is virtually sufficient to cause A D by age 80 (Corder et al., 1993). ApoE is shown to bind to Af3 both in vitro and in A D patients (Strittmatter et al , 1993a; Strittmatter et al , 1993b). In transgenic mice, ApoE 25 epsilon 4 is shown to increase the deposition but not its production of A(342 (Holtzman et al., 2000a; Holtzman et al., 2000b). A recent study among Swedish twins indicates that 48% of late-onset A D risk factors are attributable to genetic factors (Pedersen et al., 2004). The risk attributable to the ApoE4 allele is approximately 20-29% (Slooter et al., 1998), with the effect decreasing with age. Further genome-wide linkage studies performed in extended families and affected sibling pairs suggest that multiple genes are associated with sporadic AD. These linkage and association studies show highly consistent association with specific gene regions such as the loci on chromosome 12, 10, 9, and 6 (Kamboh, 2004). 1.2.2. Neuritic plaques and P-amyloid generation Originally described as "miliary foci" by Dr. Alzheimer, neuritic plaques are one of the two neuropathological hallmarks of AD, with neurofibrillary tangles being the other (Alzheimer, 1907). Dr. Fischer gave a detailed description of neuritic plaques observed in a large number of brains affected with senile dementia (Fischer, 1907a): "The plaques were scattered, in part also in small groups, in the cerebral cortex, and most densely in the layer of the small pyramidal cells; to a lesser extent they were found in the close vicinity of smallest vessels, the capillaries. With the Hematoxylin-Eosin stain, the plaques show a characteristic structure that differs with their size. The smallest of the foci are of a roundish spherical form, with a diameter of 10-20um. The corpuscle consists predominantly of opaque 26 clots. The next larger plaques have a central part of granular or clot-like nature, which are connected with a ring-shaped halo of a 15-25um diameter. The next-largest plaques have a size of 60-80um, more rarely from 100-120um. The center consists of different shades of opaque-bluish colored clots, in which oil immersion revealed a tangled network of rigid, dark-colored vessels. Around the ring, the tissue is more brighdy colored with a filamentous structure and an obvious radial disposition. The smallest plaques are always cell-free and more numerous than the larger ones; in some cases we observed a shrunken nucleus surrounded by residual cytoplasm, which had an appearance, in part, of glial, and in part of neuronal origin and appeared to represent a gradual atrophy. These cell residues are never found in the center but can be found exclusively in the periphery of the small focus. " Lately amyloid was identified as the major component of neuritic plaques (Divry and Florkin, 1927b). Identification of A(3 from these plaques and further studies concerning genetics of FAD have led to the (3 amyloid hypothesis of A D pathogenesis. 1.2.2.1. (3-amyloid Precursor Protein (APP) The purification and sequencing of the A|3 protein from amyloid plaques allowed the creation of oligonucleotide probes to isolate the APP cDNA. The APP gene was cloned by cDNA screening and mapped to chromosome 21 by in situ hybridization (Goldgaber et al., 1987; Kang et al , 1987; Robakis et al., 1987; Tanzi et al , 1987). The APP gene contains 19 exons spanning approximately 300kb in 21q21.3 (location chr21: 27 26,174,733-26,465,003), and has several isoforms resulting from alternative splicing of exons 1-13, 13a and 14-18, among which APP770, APP751 and APP695 are predominant isoforms. APP770 (isoform a) is the longest transcript containing exons 1-18 (mRNA sequence NM_000484, protein sequence NP_000475). APP751 (isoform b) is similar to APP770 but lacks exon 8 (mRNA sequence NM_201413, protein sequence NP_958816). APP695 (isoform c) contains exons 1-6 and 9-18 (mRNA sequence NM_201414, protein sequence NP_958817). Exon 7, which is missed in APP695, encodes a serine protease inhibitor domain called the Kunitz domain. The Kunitz domain renders APP also known as protease nexin n, which was first identified as a potent antichymotrypsin inhibitor (Van Nostrand et al., 1989). APP695 is the predominant form in neuronal tissues, whereas APP751 is the predominant variant elsewhere. The A(3 is encoded by exons 16 and 17. APP is a type I transmembrane protein and undergoes posttranslational proteolytic processing by a-, (3-, and y-secretase. In normal conditions, the predominant pathway involves a-secretase cleavage between the 16th and 17th residue of Ap and precludes Ap production, a-secretase cleavage of APP generates APP C-terminal C83 and secretory APP N-terminal sAPPcc. C83 is further processed by y-secretase to generate p3 fragment and C57 (APPy). Ap production is initiated by P-secretase cleavage. Cleavage by P-secretase generates the secreted sAPPP and APP C-terminal C99. y-secretase further cleaves C99 at two sites to generate Ap40 or AP42, with AP40 being predominant (Figure 1.5). Gervais et al (1999) showed that the APP cytoplasmic tail, 31 amino acids of the C-terminus, can be cleaved by caspase-3 during apoptosis, resulting in elevated Ap 28 production (Gervais et al., 1999). Whereas another study showed cleavage by caspase-3 results in the loss of APP endocytosis signal YENP and is independent of the amyloidogenic pathway (Soriano et al., 2001). Figure 1 . 5 : APP processing by a-, pV, and y-secretase. C83 is generated by a-secretase cleavage. The subsequent cleavage by y-secretase generated p3, which is non-amyloidogenic. C99 is generated from cleavage of [3-secretase. And subsequent y-secretase cleavage of C99 generates Af3. a C 8 3 APP a -c leavage, non-amyloidogenic pathway p-cleavage, amyloidogenic fpathway C 9 9 AB P Newly synthesized APP matures through the constitutive secretory pathway from the endoplasmic reticulum (ER) to the plasma membrane (Figure 1.6). APP undergoes several post-translational modifications including N- and O-glycosylation, phosphorylation, and tyrosine sulfuration. It is estimated that only 10% of APP can reach the plasma membrane in cultured cells where APP is processed primarily by a-secretase 29 (Sisodia, 1992). The endocytosis motif located at the C-terminus of APP (YENPTY) renders APP efficiently internalized to late endosomes, where a fraction of APP is recycled back to the membrane and a fraction is degraded in lysosomes (Koo and Squazzo, 1994). Approximately 70% of APP is internalized within minutes. Mutation of the endocytosis motif can decrease the internalization of APP and subsequently reduce Ap generation (Perez et al., 1999). Several cytosolic adaptors with phosphotyrosine-binding domains, including Fe65, She, Grb2, Mintl, Mint2, Mint3, XI1 Figure 1.6: Intracellular trafficking of APP. Nascent APP molecules mature through the constitutive secretory pathway. Once APP reaches the cell surface, it is rapidly internalized and subsequently trafficked through endocytic and recycling compartments back to the cell surface or degraded in the lysosome. Non-amyloidogenic processing mainly occurs at the cell surface where a-secretases are present. Amyloidogenic processing involves transit through the endocytic organelles where APP encounters p- and y-secretases. (Adapted from Vetrivel and Thinakaran Afewro/ogy.2006; 66: S69-S73 ). Plasma membrane ERGIC 30 and c-jun N-terminal kinase (JNK) interacting proteins (JIP) can bind to the APP cytoplasmic tail near the YENPTY motif and regulate APP trafficking and processing (King and Scott Turner, 2004). BACE1 is predominandy localized in the late Golgi/trans Golgi network (TGN) and endosomes and cleaves APP during endocytic/recycling steps. AP is mainly generated in the T G N where the y-secretase complex is enriched. It is estimated that only 6% of y-secretase complex components are located on the cell surface, whereas the majority is localized at the ER Golgi Intermediate Compartment (ERGIC), Golgi/TGN and late endosomes (Chyung et al., 2005). Other physiological functions of APP are largely unknown. APP is an evolutionarily conserved protein and has two paralogues, amyloid precursor-like protein 1 (APLP1) and APLP2, both of which lack the Ap domain. Knockout of APP or either of APLP1 or APLP2 only results in subtle neurological deficits (von Koch et al., 1997); however, the APP/APLP2 and APLP1/APLP2 double knockout mice show early postnatal lethality (Heber et al., 2000; Wang et al., 2005), which suggests that APP may have functions other than Ap generation. Some studies suggest that APP may function as a G-protein coupled receptor, or function in cell adhesion, synaptic transmission and plasticity (Turner et al., 2003). Some studies also show that the APP intracellular domain (AICD) can interact with Fe65 and Tip60 to function in a nuclear signaling pathway (Cao and Sudhof, 2001). 31 1.2.2.2. P-site cleaving enzyme (P-secretase, BACE1) A P is generated from APP by p- and y-secretase cleavage. The identity of these secretases has long been highlights of A D research. Cleavage site characteristics suggest aspartyl protease as the candidate protease. About 100 years after its initial description by Dr. Alzheimer, five groups nearly simultaneously identified BACE1 as the P-secretase by employing different experimental approaches including expression cloning, genomic search and protein purification (Hussain et al., 1999; Lin et al., 2000; Sinha et al , 1999; Vassar et al., 1999; Yan et al., 1999). BACE1 matches all characteristics of p-secretase including tissue distribution, subcellular localization, PH optimum and substrate sequence preference. But the identity of y-secretase is still unkown. BACE1 is expressed in all tissues, with the highest expression in the pancreas and also high levels in the brain (Yan et al., 1999). BACE1 mRNA is preferentially detected in neurons of all brain regions but not in glial cells (Marcinkiewicz and Seidah, 2000; Vassar et al., 1999; Yan et al., 1999). BACE1 is located on chromosome Ilq23.2-q23.3 and has four splice variants: 1-510 (NM_012104), 1-476 (NM_138972), 1-457 (NM_138971), 1-432 (NM_138973 ) (Bodendorf et al., 2001; Ehehalt et al., 2002; Murphy et al., 2001; Tanahashi and Tabira, 2001; Zohar et al., 2003). All the four isoforms are expressed in the brain and pancreas (Ehehalt et al., 2002). BACE1 full length 1-510 is endoglycosylase-H resistant. BACE457 and BACE476 are sensitive to endoglycosylase-H treatment, suggesting their localization in the ER. BACE457 and BACE476 have a reduced P-secretase activity (Bodendorf et al., 2001; Ehehalt et al., 32 2002; Tanahashi and Tabira, 2001). BACE1 gene is tightly regulated in transcriptional level and can be potentiated by SP1 and oxidative stress (Christensen et al., 2004; Tong et al., 2005). BACE1 gene expression is relatively low in normal conditions probably due to its weak promoter activity and effect of its uAUGs on leaky scanning and reinitiation during translation (Li et al., 2006; Zhou and Song, 2006). BACE1 is a type I membrane-bound aspartyl protease of 501 amino acids. BACE1 polypeptide has two D (T/S) G motifs that form conserved active sites of aspartyl proteases. Majority df BACE1 is located in the Golgi and endosomes, with a small amount observed in the ER and lysosomes (Vassar et al., 1999). BACE1 undergoes a complex set of post-translational modifications during its maturation. It is synthesized as a precursor protein containing a 21 amino acid signal peptide followed by an N-terminal propeptide domain that is removed at residue E46 by furin or furin-like convertases in T G N (Benjannet et al., 2001; Bennett et al., 2000a; Capell et al., 2000b; Creemers et al., 2001). The prodomain is required for efficient exit from ER but barely plays a strong inhibitory role in p-site cleavage (Benjannet et al., 2001). There are four predicted N-glycosylation sites at Asn 153, -172, -223 and -354 in BACE1, and the p-secretase activity is dependent on the extent of N-glycosylation (Capell et al., 2000b; Charlwood et al., 2001; Haniu et al., 2000; Huse and Doms, 2000). BACE1 has six cysteine residues that form three disulfide bonds connecting C216 to C420, C278 to C443, and C330 to C380 (Fischer et al , 2002; Haniu et al., 2000). This cysteine bridge formation is critical for maturation but not essential for APP processing (Fischer et al., 2002). BACE1 is palmitoylated at three Cys 478, -482, and -485 residues within its transmembrane 33 /cytosolic tail and is sulfurated at mature N-glycosylated moieties (Benjannet et al., 2001). Palmitoylation seems to suppress BACE1 shedding but doesn't significantly affect p-secretase activity (Benjannet et al., 2001). The cytoplasmic domain and its phosphorylation play important roles in BACE1 maturation and its intracellular trafficking through the T G N and endosomal system (Capell et al., 2000b; Haniu et al., 2000; Walter et al., 2001). BACE1 can be phosphorylated at S498 by CK-1 kinase and the phophorylation only occurs after full maturation (Walter et al., 2001). A recent study shows that BACE1 is ubiquitinated and degraded via ubiquitin-proteasome pathway, with a half-life of approximately 9 hours (Qing et al., 2004). Inhibition of ubiquitin-proteasome pathway by specific proteasome inhibitors can markedly enhance the generation of C99 and Ap (Qing et al , 2004). BACE1 is also processed between Leu 2 2 8 and Ala to generate stable N- and C-terminal fragments that remain covalendy associated via a disulfide bond (Huse et al., 2003). BACE1 dimmerizes prior to its full maturation and pro-peptide cleavage and homodimerization of BACE1 may facilitate the binding and cleavage of its substrates (Schmechel et al., 2004; Westmeyer et al., 2004). APP is the best-characterized BACE1 substrate. BACE1 cleaves APP695 isoform at a major site between Met 5 9 6 and Asp 5 9 7 ( E V K M * D A E ) to produce the C99 fragment. BACE1 also cleaves at a minor site between Tyr-10 and Glu-11 of A(3 to release a lower level of C89 fragment. Double mutation of Lys 5 9 5 -Met 5 9 6 (KM) to Asn 5 9 5 -Leu 5 9 6 (NL) in APP increases P-secretase activity and results in early onset of A D in a Swedish family (Cai et al., 2001; Citron et al, 1992). Mutation of M596V reduces the p-site cleavage of APP (Citron et al., 1995). P-secretase has the specificity for a negative charge at PI' and 34 a hydrophobic residue at PI. This specificity is novel for an aspartic protease, since most other superfamily members show a preference for hydrophobic residues at PI' (Arnold et al., 1997). Aspartic proteases are a family of endopeptidases whose catalytic activity depends on the aspartic residue. The pepsin family comprises of cathepsin D, cathepsin E, chymosin, gastricsin, pepsin A, pepsin F, rennin, BACE1 and BACE2, all of which have sequence homology. They are bilobed molecules with an aspartic residue (D T/S G) in each lobe and the active-site cleft located between the lobes (Dunn, 2002). Mutation of either aspartic acid renders the enzyme inactive (Bennett et al., 2000a; Hussain et al., 1999). BACE1 can also process APLPs (Li and Sudhof, 2004; Pastorino et al., 2004). Other substrates of BACE1 includ sialyltransferase ST6Gal I and P-selectin glycoprotein ligand 1 (PSGL-1) (Kitazume et al , 2005; Kitazume et al., 2003; Lichtenthaler et al , 2003). In the year of 2001, several groups developed BACE1 knockout mice by disruption of either or both of the active aspartic protease motifs (Cai et al., 2001; Luo et al , 2001; Roberds et al., 2001). BACE1 deficient mice are healthy and have no discernable phenotype. Interestingly, p-secretase activity is abolished in brains and cultured neurons of BACE1 knockout mice (Cai et al., 2001; Luo et al., 2001). No Ap or C99 can be detected in brains of BACE1 knockout mice crossed with APP transgenic mice (Roberds et al., 2001). BACE1 deficiency can also rescue memory deficit in APP transgenic mice, correlated with a reduction in Ap levels (Ohno et al., 2004). There is no BACE2 compensatory changes found in BACE1 knockout mice (Luo et al., 2003). Studies on BACE1 knockout mice provide strong evidence indicating that BACE1 is the 35 P-secretase in vivo. Further, inhibition of BACE1 as a treatment for A D may be free of mechanism-based side effects. Double transgenic mice that overexpress both APP and BACE1 have increased amyloid depostition (Willem et al., 2004); however, another study shows that there is an increase of amyloid deposition in modest BACE1 overexpression and decrease of amyloid deposition in high BACE1 overexpression mice (Lee et al., 2005). 1.2.2.3. a-site cleaving enzyme (a-secretase) a-secretase cleavage at site between Lysl6 and Leu 17 of A|3 is constitutive and predominant in normal condition. The non-amyloidogenic pathway of a-secretase cleavage not only precludes A P formation but also generates sAPPa that has neuroprotective and memory enhancing effect (Furukawa et al., 1996; Mattson et al., 1999; Meziane et al., 1998). a-secretase activity in the cells can be enhanced by activators of protein kinase C (PKC), such as phorbol esters, agonists of metabotropic glutamate receptors and muscarinic M l and M3 acetylcholine receptors (Buxbaum et al., 1993; Lee et al., 1995; Nitsch et al., 1992). Enhancement of a-site cleavage is also concomitant with reduction in P-secretase processing, suggesting that augmentation of a -secretase may be beneficial in treating A D (Lee et al., 1995). It is suggested that a -secretase cleavage is dependent on the a-helical secondary structure and on a distance of 12-13 residues from the membrane, rather than on a primary sequence motif (Sisodia, 1992). The identity of a-secretase remains elusive. Inhibition of sAPPa secretion by a zinc-chelating agent has led to the interest in zinc-dependent metalloproteases (Esler and Wolfe, 2001). Three potential candidates have been identified: the tumor necrosis factor 36 a (TNF-a) converting enzyme (TACE), a disintegrin and metalloprotease-10 (ADAM 10) and metalloproteinase-like, disintegrin-like, and cysteine-rich protein-9 (MDC9) (Black et al., 1997; Buxbaum et al., 1998b; Koike et al., 1999; Lammich et al., 1999; Moss et al., 1997). T A C E can also process a large number of receptors including TNF-a, P75 TNF receptor, L-selectin adhesion molecules, T G F - a and Notch (Brou et al., 2000). Adverse side effects are implicated with upregulation of a-secretase as a therapeutic strategy, due to their functional redundancy and the fact that every candidate has alternative substrates. 1.2.2.4. y-site cleaving enzyme (y-secretase) A P is generated by y-secretase cleaveage of APP C99 at y-site. The y-secretase cleaves the hydrophobic integral membrane domain of its substrates, resulting in the release of extracellular and intracellular fragments. The intramembrane proteolytic activity renders y-secretase components difficult to identify. Linkage analysis studies identified presenilin 1 and presenilin 2 as causative genes involved in early onset F A D (Sherrington et al., 1995). Presenilin 1 is a 467 amino acid protein encoded by a gene located on chromosome 14q24.3. Drosophila with presenilin loss-of-function exhibits a lethal Notch-like phenotype (Struhl and Greenwald, 1999; Ye et al., 1999). Further evidence shows that presenilin is required for proteolytic release and nuclear translocation of Notch (De Strooper et al., 1999; Song et al , 1999). Presenilin is an essential component of y-secretase (De Strooper et al., 1999; De Strooper et al., 1998; Li et al., 2000a; Song et al., 1999); however, it remains controversial if presenilin contains the catalytic center of y-secretase. Most of autosomal mutations leading to early onset F A D reside in PS1 and PS2. PS1 has 6 to 8 transmembrane (TM) domains (Doan et al , 37 1996; Li and Greenwald, 1996; Li and Greenwald, 1998; Nakai et al , 1999). The endoproteolysis between TM6 and TM7 generates two stable fragments, N-terminal fragment (NTF) and C-terminal fragment (CTF), which associate with other components of y-secretase and constitute the active form of presenilin (Thinakaran et al., 1996). Mutation of either of two aspartyl residues, embedded in T M domains 6 and 7, inhibits endoproteolysis and leads to presenilin loss-of-function (Ray et al., 1999; Steiner et al., 1999; Wolfe et al., 1999). Regions flanking the aspartate residues are evolutionarily conserved (Steiner et al., 2000). Another evidence comes from the photoaffinity labelling of PS1 (and PS2) by potent y-secretase inhibitors that are designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease (Li et al., 2000b). Both of Ap and NICD production are abolished in PS1/PS2 double knockout cells (Zhang et al., 2000). Though PS1 FAD mutant transgenic mice lack obvious phenotype, PS1 FAD mutant overexpression facilitates the AP accumulation and plaque formation in APP mutant transgenic mice. Although all the above evidence supports that presenilin constitutes the active sites of y-secretase, there are some experimental data that are difficult to reconcile with this conclusion. For instance, when only Asp257 is mutated, Notch processing by the y-secretase is inhibited, whereas APP processing continues; however, mutation of Asp385 abolishes processing in both Notch and APP (Capell et al , 2000a; Kim et al., 2001). There are more than 150 mutations spreading along PS1 gene, all of which have similar effects on Notch and APP processing. PS1 is most abundant in the ER, theERGIC, cis-Golgi network and some transport vesicles (Annaert et al., 1999); however, most of presenilin-dependent y-secretase cleavage of 38 APP occurs in late compartments (Perez et al., 1999). This spatial paradox also does not support that presenilin constitutes the active sites of y-secretase. Biochemical studies show that y-secretase activity is associated with a high molecular weight complex of 250-1000 kDa in size, which encourages the searching for presenilin cofactors that constitute the y-secretase complex. Nicastrin is the first member identified by immunoextraction with the antibody against PS1 (Yu et al., 2000). Nicastrin is a glycosylated 130 kDa protein that can bind to both presenilin NTF and CTF and thus stabilize presenilin. Exit of nicastrin from the ER to cell surface requires presenilin (Edbauer et al., 2002b). Another component of the y-secretase complex, Aph-1 was identified by a screening for the gene mutations leading to "anterior pharynx-defective phenotype", a Notch receptor (glp-1) homolog deficient phenotype in C.elegans (Goutte et al., 2002). Aph-1 is a 30 kDa protein with several transmembrane domains. Aph-1 is also required for proper trafficking of nicastrin from the ER to cell surface. Another screening of presenilin enhancers in partial presenilin deficient C.elegans strains identified Pen-2 and Aph-1, being associated with presenilin and nicastrin (Francis et al., 2002). Disruption of either Aph-1 or Pen-2 also exhibits the Notch pathway phenotype. Pen-2 is a small hairpin like membrane protein with a molecular weight of 12 kDa. By coexpression presenilin, nicastrin, Aph-1 and Pen-2 in a yeast Saccharomyces cerevisiae lacking endogenous y-secretase activity, Edbauer et al (2003) shows that the four components are necessary and sufficient to constitute the active y-secretase (Edbauer et al., 2003). Aph-1 can stabilize presenilin holoprotein, whereas Pen-2 is required for presenilin endoproteolysis to confer the y-secretase activity (Takasugi et al., 2003). Pen-2 39 is also required to stabilize the presenilin NTF and CTF heterodimer after the endoproteolysis (Prokop et al., 2004). Aph-1 interacts with immature nicastrin to form an early intermediate, upon which presenilin C-terminus binds to form a subcomplex of Aph-1, nicastrin and presenilin (Kaether et al., 2004; LaVoie et al., 2003; Niimura et al., 2005). By interacting with the transmembrane domain 4 of presenilin-1, Pen-2 is recruited into y-secretase complex and confers y-secretase activity (Kim and Sisodia, 2005; Watanabe et al., 2005). y-secretase cleaves APP at a major Af340 and a minor A(342 site. Mutations in PS1 and PS2 are found to be able to shift the preferred y-cleavage site from A(340 to A(342 (Scheuner et al., 1996); however, how the presenilin mutations shift the cleavage site remains unknown, y-secretase can also process APP at a e-site that resembles the S3 cleavage site in Notch to release APP intracellular cytopasmic domain (AICD) (Edbauer et al., 2002a; Gu et al , 2001; Kimberly et al., 2001; Weidemann et al., 2002). AICD can be stabilized by Fe65 and translocate into the nucleus in a Notch-like manner (Kimberly et al., 2001). Unlike y-cleavage, e-cleavage can be affected by PH, with more AICDe49 cleavage in plasma membrane and more AICDe51 at endosomes and at lower PH (Fukumori et al , 2006). Substrates of y-secretase are a subset of integral membrane proteins that also include Notch, APLP-1, APLP-2, N/E-cadherin, ErbB-4, lipoprotein receptor-related protein (LRP), Delta, Jagged, deleted in colorectal carcinoma (DCC), p75 neurotrophin receptor (p75NTR), syndecan-1, Nectin-l-a and cluster of differentiation 44 (CD44) 40 (Koo and Kopan, 2004). How the y-secretase is regulated to process this variable subset of substrates is largely unknown. Several proteins such as calsenilin, P-catenin, glycogen synthase kinase 3-a (GSK-3a), and 21 kDa transmembrane trafficking protein (TMP21) are shown to be able to interact with presenilin and regulate the y-secretase activity. Calsenilin, identified by yeast two-hybrid system using the last 103 amino acids of PS2 as the bait, is shown to be able to regulate the proteolysis of PS2 (Buxbaum et al., 1998a). Calsenilin, interacting with presenilin C-terminal, can also modulate calcium store and contribute to apoptosis (Jo et al., 2001; Lilliehook et al., 2002). Overexpression of calsenilin can enhance A P generation and mediate A p induced apoptosis (Jo et al., 2001; Jo et al., 2004; Lilliehook et al., 2003). P-catenin was identified by a yeast two-hybrid system using 263-407 amino acids of PS1 (Yu et al., 1998; Zhou et al., 1997). P-catenin is an important mediator of Wnt signaling pathway. In the absence of Wnt signaling, p-catenin is phosphorylated by C K - l a and GSK-3p and subsequently degraded by ubiquitin-proteasome pathway (Amit et al., 2002; Kitagawa et al., 1999). Activation of Wnt signaling by binding to its cell surface receptor stabilizes P-catenin and promotes its translocation into the nucleus and association with lymphoid enhancing factor/T-cell factor (LEF/TCF) transcription factors to activate its target genes' transcription (Lustig and Behrens, 2003). Furthermore, p-catenin can bind to E-cadherin at residues 833-862 to form adherens junction complexes at the cell-cell adhesion sites (Nelson and Nusse, 2004). Presenilin binds to E-cadherin at amino acids 760-771, the juxtamembrane region of E-cadherin, while p-catenin binds to E-cadherin at the membrane distal region (Baki et al., 2001). E-cadherin stabilizes the formation of PS1, E-cadherin and P-catenin protein complexes, in which PS1 promotes the phosphorylation and ubiquitination of p-catenin 41 and negatively regulates Wnt signaling pathway (Kang et al., 1999; Kang et al , 2002; Serban et al., 2005). GSK-3P and microtubule associated protein tau are shown to interact with PS1 at residues 250-298, interaction with which is proposed to promote the phosphorylation of tau by GSK-3p (Lucas et al., 2001; Takashima et al., 1998); however, a recent study shows that GSK-3 inhibitor lithium chloride can reduce Ap production and the underlying mechanism is mediated by GSK-3oc (Phiel et al., 2003). 1.2.2.5. Ap clearance The accumulation of Ap in the brain is determined by the rate of Ap generation versus clearance. There are two ways to remove Ap from the brain, proteolytic degradation and receptor-mediated transport from the brain. Toxic molecules including AP in the brain are cleared out through blood-brain barrier (BBB) via production and turnover of cerebrospinal fluid (CSF), and from BBB into bloodstream (Silverberg et al., 2003). Two receptors, the low-density lipoprotein receptor-related protein (LRP) and the receptor for advanced glycation end products (RAGE) are involved in receptor-mediated flux of Ap across BBB (Silverberg et al., 2003). Firstly recognized as a large endocytic receptor key to metabolism of cholesterol and ApoE containing lipoproteins, LRP is a multifunctional protein involved in endocytosis, cargo transport, and subcellular trafficking, with pivotal functions in fundamental cellular signaling pathways (Herz and Marschang, 2003). The ligand binding domains (cluster I-IV) are located on the heavy chain of LRP and can bind to variable proteins including APP, ApoE, a2-macroglobulin, tissue plasminogen activator, 42 plasminogen activator inhibitor-1, factor VIII and lactoferrin (Zlokovic, 2004). LRP regulates A(3 clearance via transporting A(3 from brain to blood through B B B (Zlokovic, 2004). LRP antagonists are shown to specifically reduce efflux of A(3 from the brain by up to 90% (Shibata et al., 2000). A(3 load is doubled in mice when LRP function is disrupted by knocking out receptor associated protein (RAP), the critical LRP chaperone (Van Uden et al., 2002). Studies show that A(3 directly interacts with clusters II and IV of LRP, which mediate binding of A p with brain capillary, AP endocytosis and transcytosis across mouse B B B . LRP has a higher affinity with Ap40 than AP42 and Dutch or Iowa mutant A p . High concentration (>luM) of A p can also promote proteasome-dependent LRP degradation in endothelium (Deane et al., 2004). Furthely, genetic polymorphism of LRP gene is shown to be associated with late-onset A D (Hollenbach et al., 1998; Kang et al., 1997; Lambert et al., 1998a; Wavrant-DeVrieze et al., 1999). While LRP is the major protein mediating AP efflux, R A G E is the major player in mediating AP influx into the brain (Deane et al., 2003). R A G E is a multifunctional protein belonging to the immunoglobulin superfamily and binds to a large set of different proteins including A p . R A G E is expressed in neurons, microglial cells and astrocytes in normal human brain (Li et al., 1998). R A G E expression is increased in A D patients, particulary in neurons close to neuritic plaques and in cells of AP containing vessels (Lue et al., 2001; Yan et al., 1996). Interaction of A p with R A G E in neurons elicits an N F - K B -dependent release of macrophage colony-stimulating factor and cellular oxidative stress (Du Yan et al., 1997). R A G E is suggested to be the cell surface receptor for A p on neurons (Du Yan et al., 1997). 43 Another way to clear A(3 out of the brain is through proteolytic cleavage by some endopeptidases including insulin degrading enzyme (IDE) or insulysin, and neprilysin. Other candidate A^-degrading proteases include plamin, tPA/uPA, endothelium enzyme -1 and matrix metallproteinase (Selkoe, 2001b). The substrates of IDE are small peptides of diverse sequence including Ap\ amylin, insulin, glucagon, atrial naturetic peptide (ANF), APP C-terminal and TGF-cc. IDE probably recognizes a conformation that is prone to conversion to a J3-pleated sheet structure (Bennett et al., 2000b). As a majority of IDE is located at cytoplasm as a soluble form, IDE is proposed to degrade the soluble pool of A(3 (Vekrellis et al., 2000). IDE has a preference to insulin, and therefore the increase of insulin will inhibit IDE mediated degradation of other substrates including Af3. One study shows that IDE levels are reduced by approximately 50% in patients carrying ApoE4 allele compared to controls, suggesting that reduced IDE level and the interaction of ApoE4 with IDE may be risk factors for A D (Cook et al., 2003). Disruption of IDE in mice shows elevated Af3 accumulation, hyperinsulinemia and glucose intolerance (Farris et al., 2003; Miller et al., 2003). Mild overexpression of IDE can diminish A(3 plaque formation by 50% in APP transgenic mice (Leissring et al., 2003). Another A(3 cleaving enzyme is neprilysin, also known as neutral endopeptidase (NEP) or membrane metalloendopeptidase (MME). Neprilysin is a type II membrane glycoprotein that can hydrolyze circulating peptides including Af3, enkephalin, cholecystokinin, neuropeptide Y, substance P, glucagon, neurotensin, oxytocin, and bradykinin. Neprilysin is able to degrade insoluble AP oligomers, degradation of which is also decreased by neprilysin inhibitors (Iwata et al., 2001). Furthermore, overexpression 44 of neprilysin is able to decrease A(3 accumulation in mice (Farris et al., 2003). It is shown that somatostain, a neprilysin modulator, is reduced with aging, correlated with reduction in neprilysin and accumulation of Af5 in late-onset A D (Iwata et al., 2002; Saito et al , 2005). 1.2.2.6. Ap immunization The innate immune response is associated with A3 deposition in brains of AD patients (Monsonego and Weiner, 2003) (Fig 1.7). Ap is the toxic molecule that provokes activation of microglia and astrocytes and subsequent cascade of innate immune response, including activation of complements, expression of chemokines, secretion of proinflammatory cytokines such as interleukin-ip and TNF-a, and secretion of nitric oxide (El Khoury et al., 1996; Husemann et al., 2001; Monsonego et al., 2003; Wyss-Coray and Mucke, 2002). Results from both active and passive Ap immunizations in A D transgenic mice have been of support to the involvement of immune response in AD. Parenteral immunization of APP transgenic mice with synthetic Ap in complete Freund's adjuvant can markedly decrease the number and density of AP deposits in the brain, with concomitant improvements in neuritic dystrophy and gliosis (Vehmas et al., 2001). Passive administration of AP antibodies reveals similar neuropathological improvements, suggesting that AP antibody plays a major role in active immunization (Bard et al., 2000; Dodart et al., 2002). Further positive results are obtained from mucosal Ap immunization (intranasal) to APP transgenic mice (Weiner et al., 2000). AP lowering effects of AP immunization lead to clinical trials in A D patients. Although the Phase I clinical trial failed to reveal any severe complications, a subsequent Phase II trial was discontinued 45 shortly after its initiation when 18 of 298 treated patients developed central nervous system (CNS) inflammatory reactions (Nicoll et al., 2003; Orgogozo et al., 2003; Senior, 2002). Subsequent evaluation of those immunized A D patients who developed effective titers of Ap antibody did show slower cognitive decline (Hock et al., 2003): Consistently, postmortem studies showed that plaque burden was decreased in the neocortex in brains of immunized patients compared to non-immunized controls (Nicoll et al., 2003). The future immunotherapy approach is still promising with improvement in immunization strategy and more knowledge of the basic mechanism of immune response to Ap. Figure 1.7. Pathways of microglia activation in Alzheimer's diseases. Microglia are bone marrow-derived cells that acquire ramified morphology in the intact CNS. In response to A8 deposition in AD, microglial cells are activated and differentiate into phagocytic cells (CDllb + ) (left), which induce a proinflammatory environment and secrete IL-16, TNF-7, NO, free radicals, chemokines, and activate complement. The NO secreted by CD1 lb + cells may enhance T cell apoptosis in the CNS. A second pathway for microglial cells is to differentiate into APCs (right), which are induced in the presence of GM-CSF and/or IFN-7 secreted by microglia, astrocytes, or other immune cells (T cells, macrophages) that infiltrate the CNS. As a result, microglia cells differentiate to dendritic-like cells that then may function as APCs for both T H I and TH2 cells. These cells also may migrate from the CNS to secondary lymph nodes and induce T cell activation. In AD, this pathway could suppress the toxic innate immune response and can be enhanced by TH2 immunization. Adapted from Monsonego, A., and Weiner, H. L. (2003). Science 302, 834-838. 46 Ail deposition 1.2.3. Neurofibrillary tangles and tauopathy Originally described as " the tangled bundle of fibrils", the neurofibrillary tangle is the other neuropathological hallmark in AD brains (Alzheimer, 1907). Dr. Bonfiglio had a detailed description of these neurofibrillary tangles in 1908 (Bonfiglio, 1908a): " The neurofibrillary alteration is widespread in the whole cortical grey matter, but affects mainly the layers of the large and small pyramidal cells; in the temporal lobe, where they are most frequent, they affect almost 1/3 of the neurons. The alteration begins in a circumscribed region of the cell body. In a cell which appears normal, a group of neurofibrils stand out for their unique thickness and intense staining. As the alteration proceeds, all the neurofibrils one after the other thicken and merge in dense bundles which progressively occur only at the periphery of the cells, where they twine in multiple crisscrossed swirls: thus they assume various shapes: claw-like forms, pyramids, ovals, halfmoons, etc. At this 47 stage the stain no longer reveals the cell protoplasm; however, the nucleus of the neuron persists and often seems pyknotic. Thereafter, however, even the nucleus disappears, and to disclose the site of the original neuron, only the tangles of dense fibrillary bundles remain which are sometimes in contact with a neuroglial nucleus, easily recognized by its features. " Electron microscopy reveals that neurofibrillary tangles are composed of paired helical neurofilaments, the high insolubility of which renders themeasy to purify (Kidd, 1963; Terry, 1963). Antibodies raised against purified paired helical neurofilaments can recognize microtubule-associated protein tau, and vice versa (Grundke-Iqbal et al., 1986a; Kosik et al., 1986; Nukina and Hiara, 1986). Furthermore, it is found that tau protein in neurofibrillary tangles is abnormally hyperphosphorylated (Grundke-Iqbal et al., 1986b; Diara et al., 1986). The cytoskeletal network in eukaryotic cells, composed of microtubules, actin and intermediate filaments, is involved in determining cell architecture, intracellular transport, modulation of surface receptors, mitosis, cell motility and differentiation. Cytoskeletafdynamics and organization depend on protein self-associations and interactions with regulatory elements such as microtubule-associated proteins (MAPs). Tau is a member of MAP family that also includes MAP-1 A, MAP-IB, MAP-1C, MAP-2, MAP-2C and MAP-4 (Maccioni and Cambiazo, 1995). Tau is localized in neurons, preferentially in axonal compartments, although nonneuronal cells usually have trace amounts (Sergeant et al., 2005). Tau gene spans more than 1 lOkb in chromosome 17q21 48 and contains 16 exons (Andreadis, 2005). Exon 4A and 8 are skipped in human brain and are specific in peripheral tissues. Exon 1,4, 5, 7, 9, 11, 12 and 13 are constitutively spliced, while exon 2, 3 and 10 are alternatively spliced, which gives rise to 6 isoforms in human brain ranging from 352 to 441 amino acids (Figure 1.8). The tau isoforms differ from each other by the presence of 3- or 4-repeat region in the C-terminal and the presence of one or two inserts at the N-terminal region. Encoded by exon 2 and 3, the two 29 amino acid sequences give different lengths to the N-terminal of tau. These two inserts are highly acidic, followed by a basic proline-rich domain. The N-terminal part of tau is referred to as projection domain since it projects from the microtubule surface where it may interact with other cytoskeletal or membrane proteins (Brandt et al., 1995). The C-terminal repetitive region of tau is referred to as microtubule assembly domain. The physiological function of tau includes microtubule assemble, neurite outgrowth and axonal transport. Figurel.8. Tau gene structure, pre-mRNA alternative splicing in the CNS and protein isoforms translated from alternative tau mRNAs. (A) The human tau gene, located on chromosome 17 at position q21, spans over 110 kb. It is composed of 16 exons numbered from -1 to 14. The start codon is located in exon 1 and two codons are described: one in the intron between exons 13 and 14 and the second in exon 14. The 3' ending of exon 14 is not completely characterized in human. The initiation of transcription is indicated by +1. (B) Schematic representation of Tau mRNAs. In the CNS, exons 4A, 6 and 8 are constitutively skipped. Exons -1 and 14 correspond to the 5' and 3' untranslated regions of Tau mRNA, respectively. Two cleavage-polyadenylation sites are described: one in intron 13/14 and one in exon 14. The polyadenylated Tau mRNA including exon 14 is less represented in human. In the CNS, the alternative splicing of exons 2, 3 and 10 generates six tau proteins. Inclusion of exon 49 3 is associated with that of exon 2, whereas exon 2 can be included alone. (C) The six human brain tau isoforms are represented as they are resolved by polyacrylamide gel electrophoresis. The tau isoform lacking the alternative exons 2, 3 and 10 is the only tau isoform to be expressed in fetal CNS (Sergeant et al., 2005). A Gene +1 ! 2 3 4 4a 5 6 7. ,8 9 10 • • • • w m m m — i -in m to so Start codon Ml U12 13 70 SO 90 T Stop cottons B mRNA 1112 13 14 lj In) /rV-HU tn) Adult CNS tau protein isoforms Foetal CNS tau protein isoforms E2E3 i one X I X 352 Alternative exons included 2+3+10 2-3 2+10 2 10 0 There are 80 putative serine or threonine phophorylation sites and 5 tyrosine sites on the longest tau isoform, among which there are at least 30 phophorylation sites being experimentally verified, ln vitro, tau is a substrate for many protein kinases; however, the number of protein kinases that actually phosphorylate tau in vivo is probably much lower. GSK-3P, cdk5, protein kinase A (PKA) and microtubule-affinity-regulating-kinase 50 (MARK) are shown to be able to phosphorylate tau in vivo (Johnson and Stoothoff, 2004). Phosphorylation of tau at specific sites can regulate tau's function (Johnson and Stoothoff, 2004). For example, phosphorylation of KXGS motifs within the microtubule-binding repeats strongly reduces binding of tau to microtubules in vitro and in vivo (Biernat et al., 1993; Biernat and Mandelkow, 1999). Furthermore, phosphorylation of tau can be regulated by O-glycosylation (Liu et al., 2004). In pathological conditions, tau forms insoluble polymers in brains of A D and other tauopathies. Phosphorylation-dependent antibodies such as AT100, AP422, 988, PHF-27 or T G / M C antibodies only detect tau in paired helical filaments, suggesting that aberrant phosphorylation is a key feature of tau isolated from brains of A D patients and other individuals with tauphothies (Sergeant et al., 2005). Tauopathy features a subset of neurodegenerative diseases, in which phosphorylation and content of tau isoforms are differrent. Five classes of tauopathies have been defined depending on the type of tau aggregates that constitute the "Bar Code" for neurodegenerative disorders (Figure 1.9) (Sergeant et al., 2005). Most mutations in tau are associated with frontotemporal dementia with parkinsonism (FTD) (Goedert et al., 1998). 58 mutations in 113 families have been identified in tau gene on chromosome 17, with 40 mutations being pathogenic. Depending on their functional effects, mutations on tau proteins may be divided into two groups: mutations affecting the alternative splicing of exon 10 and leading to changes in the proportion of 4R- and 3R-tau isoforms, and mutations modifying tau interactions with microtubules (See complete list http://www.molgen.ua.ac.be/ADMutations/default.cfm?MT=0&ML=0&Page=Home). 51 Figure 1.9. The bar code of tauopathies and their classification. ( A ) Human brain tissue from patients affected by different tauopathies, which are separated by polyacrylamide gel electrophoresis, and pathological tau proteins revealed by Western blotting using phospho-dependent tau antibodies (e.g., AD2 monoclonal antibody recognizing the phosphorylated Ser396/404 of tau, numbering according to the longest tau isoform). Four different electrophoretic patterns of pathological tau proteins are illustrated. These are composed of pathological tau bands at 60, 64, 69 and 74 kDa, which correspond to pathological tau that are found in aggregates. Type I of aggregate is characterized by the presence of the four pathological tau components, whereas types II and Ul include two major pathological tau components at 64 and 69 kDa, or 60 and 64 kDa, respectively. Finally, the fourth aggregate type, characterized by a strong pathological tau component at 60, 64 and 69 kDa components, is often observed depending on the severity of the affected region analyzed. These four main patterns of pathological tau thus represent a bar code of tauopathies. (B) Neurological disorders for which a pathological tau pattern has been defined are classified according to the "bar code". Five classes are defined including a unique or multiple neurological disorders. The four aggregate types are detailed as well as the pathological tau pattern identified. Class 0 includes a unique neurological disorder characterized by the loss of expression of tau protein and lacking distinctive neuropathological features, which is Frontal dementia of non-AD, non-Pick type (Sergeant et al., 2005). 52 Bar code classification of Tauopathies Aggregates Type! Type II type III type IV B Tauopathies: 5 Classes ClaSS 0 F r o n t a l lube dementia n u n - A O , non-Pick LOSS of t a t l protein expression • C e r e b r a l aging ( individuals aged over 75 vears) • A lzhe imer ' s disease (sporadic a n d familial} • A L S / p a r U n u m i s m - d e m e n C i a complex of G u a m P I 1 ' ^ r ^ i n s o n w i t h dementia o f f Juadeloopc *~ l a s s l , Niemann-Pfck disease type C Aggregates types • Postencephalitic p a r k i n s o n i s m • F a m i l i a l B r i t i s h dementia Dementia pugilistica I > | V I Class 2 Class 3 • Down's svndrome • F l tH' - l? ' Tan 6(1,64 and 69 •Cor t i cobasa l degenerat ion • A rgv roph i l i c gra in demet ia •Progressive supranuclear patsv • F T D P - 1 7 Type II Tau doublet: Tau 64 and 69 •Pick's disease •FTDP-17 Type III Tau doublet: Tau 60 and 64 Class 4 * Myotonic dystrophy of type f and 11 Type IV Tau 60 The R406W mutation in tau gene causes a clinical picture closely resembling AD but not FTD (Rademakers et al., 2003). Tauopathy usually coexists with neuritic plaques in AD; however, how these two pathologies are related is unkown, although it is speculated that AP deposition precedes abnormal tau aggregates (Hardy and Selkoe, 2002). The tau P301L mutation transgenic mice develop motor and behavioral deficits, as well as age- and gene-dose-dependent neurofibrillary tangles in their brains (Lewis et al., 53 2000). Injection of Ap42 into brains of P301L mutant tau transgenic mice causes 5-fold increase in the number of neurofibrillary tangles in cell bodies within the amygdala, from where neurons project to injection sites, suggesting that Ap42 fibrils can accelerate neurofibrillary tangle formation in vivo (Gotz et al., 2001). Furthermore, crossing the mutant APP transgenic mice Tg2576 with mutant tau transgenic mice JNPL3 reveals accelerated formation of neurofibrillary tangles while A p deposition remains the same, suggesting that either APP or A p influences the formation of neurofibrillary tangles (Lewis et al., 2001). A recent study shows that A P forms stable complexes with soluble tau and prior phosphorylation of tau inhibits complex formation (Guo et al., 2006). It is also shown that alpha-synuclein induces fibrillization of tau and that coincubation of tau and alpha-synuclein synergistically promotes fibrillization of both proteins (Giasson et al., 2003), suggesting that an interaction between alpha-synuclein and tau drives the formation of pathological inclusions in neurodegenerative diseases. 1.2.4. Transgenic mouse models of Alzheimer's disease 1.2.4.1. APP transgenic mice Transgenic mice expressing human pathogenic gene mutations have been critical for biomedical research and treatment development. Soon after the identification of APP and presenilin mutations in F A D families, several approaches have been used to generate APP and presenilin transgenic mouse models. There are dozens of mouse strains developed with various APP and presenilin mutations (complete list referred to http://www.alzforum.org/res/com/tra/default.asp). Mice expressing wild type human APP under APP promoter in Y A C (Lamb et al., 1993) or driven by platelet derived growth 54 factor-P (PDGF-P) promoter can not reveal any plaque formation in their brains (Mucke et al., 2000); however, various degrees of plaque deposition and memory deficit are observed in transgenic mice overexpressing human APP Swedish, London, Arctic, Dutch and Indiana mutant genes. The degree of pathological changes and memory deficit are generally correlated with APP gene expression levels due to the usage of several neuronal specific transgene promoters, such as human or mouse thy-1 promoter, hamster prion promoter and PDGF-P promoter. Tg2576 mice overexpressing human APP695 Swedish mutant under hamster prion promoter show correlative appearance in behavioral, biochemical and pathological abnormalities reminiscent of AD (Hsiao et al., 1996). APP23 mice developed by Sturchler-pierrat et al are among the best-studied AD mouse models (Sturchler-Pierrat et al., 1997). Human APP Swedish mutant is overexpressed about 7 times of endogenous APP level by mouse thy-1.2 promoter in APP23 mice. Ap plaques can be observed in APP23 mice as early as 6 months and progressively with age. The memory deficit can be observed from 3 months of age and declines with age (Lalonde et al , 2005; Van Dam et al., 2003). 1.2.4.2. Presenilin and multiple gene transgenic mice Transgenic mice with various PS1/PS2 mutations can not reveal A D pathological changes; however, the APP and PS1/PS2 double transgenic mice show accelerated amyloid plaque deposition and memory impairment (Borchelt et al., 1997; Dewachter et al., 2000; Richards et al., 2003; Siman et al., 2000). Mice bearing various human tau mutations show neuronal loss and paired helical neurofibrillary tangles. A recent report shows that inducible mutant tau transgenic mice develop progressive age-related 55 neurofibrillary tangles, neuronal loss and behavioral impairments. Turnoff of the tau transgene stabilizes memory deficit and neuronal loss whereas neurofibrillary tangles continue to accumulate, suggesting the tauopathy is not sufficient to induce memory deficit and neuronal loss (Santacruz et al., 2005). Tau and APP double crossed mice develop amyloid deposition at a similar age as mice with APP alone, but exhibits substantially enhanced neurofibrillary tangles (Lewis et al., 2001). Modest expression of human BACE1 in APP transgenic mice accelerates AR deposition whereas high BACE1 expression reduces amyloid formation (Lee et al., 2005). Crossing APP transgenic mice with ADAM10 transgenic mice reduces AR plaques, whereas crossing with dominant inactive mutant ADAM10 mice enhances the number and size of amyloid plaques (Postina et al., 2004). 1.2.5. The AR cascade hypothesis of Alzheimer's disease The past two decades of A D research has resulted in a partial understanding of the pathogenesis of AD. Despite the remaining debate, a general hypothesis is developed to provide a potential explanation for the etiology of the disease, for the development of the characteristic neuropathology and initiation of clinical symptoms associated with AD. The central point lies in generation and aggregation of Aft protein to form senile plaques, and selective neuronal loss in hippocampus and cerebral cortex, the brain areas vital for learning and memory. It is therefore referred to as AR cascade hypothesis (Figure 1.10) (Hardy and Selkoe, 2002; Selkoe, 2001a; Tanzi and Bertram, 2005). Validity of this hypothesis is supported by genetic studies in F A D pedigrees, pathologic and behavioral 56 changes in transgenic mice bearing FAD or ApoE mutations, AR neurotoxicity studies, and anti-amyloid studies in transgenic mice and in clinical trials. Figure 1.10: The sequence of pathogenic events leading to A D proposed by the AR cascade hypothesis. The curved violet arrow indicates that AR oligomers may directly injure the synapses and neurites of brain neurons, in addition to activating microglia and astrocytes. (Adapted from Hardy and Selkoe, 2002, Science;297:353-6.) Missense mutations in APP, PS1, or PS2 genes Increased A[342 production and accumulation Ap42 oligomerization and deposition as diffuse plaques Subtle effects of Ap oligomers on synapses Microglial and astrocytic activation (complement factors, cytokines, etc.) v . Progressive synaptic and neuritic injury Altered neuronal ionic homeostasis; oxidative injury Altered kinase/phosphatase activities > tangles Widespread neuronal/neuritic dysfunction and cell death with transmitter deficits Dementia 57 Several controversies still remain although most researchers accept the Aft cascade hypothesis. One concern is that the number of neuritic plaques in the brain does not correlate well with the degree of cognitive impairment experienced by A D patients. It is noted that the memory deficit and severity of A D correlates better with the soluble Aft level (not visible to immunohistochemistry) than the counts of amyloid plaques (McLean et al , 1999). Some recent reports suggest that oligomeric form of Aft is more toxic than the insoluble fibrils (De Felice et al., 2004; Hardy and Selkoe, 2002; Kayed et al., 2003; Lambert et al., 1998b). Another concern is that the APP bearing transgenic mice undergoing progressive Aft deposition can not show prominent neuronal loss or neurofibrillary tangles. One explanation is that there may be species difference in neuronal vulnerability between human and rodents. Another explanation is that Aft deposition precedes formation of neurofibrillary tangles and the mice may not be able to live long enough to develop tangles. APP transgenic mice crossed with the tau transgenic mice show substantial neurofibrillary tangles, implying that the amyloid plaque formation precedes the formation of neurofibrillary tangles (Lewis et al., 2001). Furthermore, injection of AR42 fibrils into brains of P301L mutant tau transgenic mice causes five-fold increase in the numbers of neurofibrillary tangles (Gotz et al., 2001). Studies of A D patients with DS also show that Aft deposition predates neurofibrillary tangle formation (Mann et al., 1986). Several questions remain elusive regarding the Aft cascade hypothesis. As the hypothesis is derived mainly from genetic studies in FAD, how the Aft deposition occurs in sporadic A D patients is largely unknown. Also the mechanism by which the pathological changes, such as AR deposition causes selective neuronal death and memory deficit, remains elusive. 58 1.2.6. Down Syndrome, a valuable model of Alzheimer's disease Firstly described by Dr. John Langdon Down in 1866, DS is the most common genetic cause of mental retardation, affecting approximately one in 800 to 1,000 babies (Down, 1866). DS, also called trisomy 21, is caused by an extra copy of chromosome 21 (Jacobs et al., 1959; Lejeune et al., 1959). Besides mental retardation, DS patients often have a characteristic face, and many other typical features including microcephaly and short stature. Many persons affected by DS can survive to adulthood, but the aging process seems to be accelerated. At autopsy, individuals with DS inevitably develop characteristic AD neuropathological abnormalities including neuritic plaques and neurofibrillary tangles following their middle age (Wisniewski et al., 1985). The soluble AP can be detected in brains of DS patients as early as 21 gestational weeks of age and amyloid plaques can be seen in subjects as young as 8 years of age. By the age of 30, Ap deposition can be found in up to 50% of brain specimens from individuals with DS. DS is a valuable model system for understanding A D pathogenesis (Busciglio et al , 2002; Gyure et al., 2001; Lott and Head, 2001). The A D pathogenesis in DS remains unknown. Some genes triplicated on chromosome 21 are proposed to contribute to DS pathogenesis as well as the development of A D in DS. Chromosome 21 contains about 367 genes, including APP and BACE2. The extra copy of APP is proposed to contribute to A D pathogenesis in DS patients; however, an APP expression study in development and aging of DS shows that APP is detected in fetuses and infants, but the immunoreactivity disappears from childhood to young adulthood and reappears from late adulthood, correlating with the 59 appearance of senile plaques (Arai et al., 1997). The disappearance of APP irnmunoreactivity in childhood and early adulthood cannot explain the continuous accumulation of A(3 in DS patients. Furthermore, the onset of AD varies greatly in DS patients, which cannot be explained by the 99% duplication of APP gene due to trisomy of chromosome 21. So we propose that some other genes also contribute to A D pathogenesis in DS patients. C99, the P-secretase cleavage product of APP, and A[3 are markedly increased in DS patients, suggesting increased p-secretase activity besides APP duplication (Busciglio et al., 2002). As BACE2 is the homolog of BACE1 and it is located in the Down Syndrome Critical Region, the extra copy of BACE2 may therefore play a role in abnormal processing of APP in DS. 1.3. Hypothesis and objectives Hypothesis: Abnormal P-secretase is involved in pathogenesis of Alzheimer's disease in Down Syndrome A i m l : To investigate the molecular mechanism oiBACE2 gene transcriptional regulation. Aim2: To define the function of B A C E 2 in APP processing, and its role in A D pathogenesis in DS. Aim3: To study the role of B A C E 1 in A D pathogenesis in DS. 60 1.4. References Aisen, P. S., Schafer, K. A., Grundman, M . , Pfeiffer, E. , Sano, M . , Davis, K. L. , Farlow, M . R., Jin, S., Thomas, R. G., and Thai, L. J. (2003). Effects of rofecoxib or naproxen vs placebo on Alzheimer disease progression: a randomized controlled trial. Jama 289, 2819-2826. Altieri, M . , Di Piero, V., Pasquini, M . , Gasparini, M . , Vanacore, N., Vicenzini, E . , and Lenzi, G. L. (2004). Delayed poststroke dementia: a 4-year follow-up study. Neurology 62, 2193-2197. Alzheimer, A. (1906). Uber einen eigenartigen schweren Erkrankungsprozefi der Hirnrinde. Neurologisches Centralblatt 23, 1129-1136. Alzheimer, A. (1907). Uber eine eigenartige Erkrankung der Hirnrinde. Allgemeine Zeitschrift fur Psychiatrie und Psychisch-Gerichtiiche Medizin 64, 146-148. Amit, S., Hatzubai, A., Birman, Y., Andersen, J. S., Ben-Shushan, E. , Mann, M . , Ben-Neriah, Y., and Alkalay, I. (2002). Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev 16, 1066-1076. Andersen, K., Launer, L. J., Dewey, M . E . , Letenneur, L. , Ott, A., Copeland, J. R., Dartigues, J. F., Kragh-Sorensen, P., Baldereschi, M . , Brayne, C., et al. (1999). Gender differences in the incidence of A D and vascular dementia: The E U R O D E M Studies. E U R O D E M Incidence Research Group. Neurology 53, 1992-1997. Andrasi, E. , Pali, N., Molnar, Z., and Kosel, S. (2005). Brain aluminum, magnesium and phosphorus contents of control and Alzheimer-diseased patients. J Alzheimers Dis 7, 273-284. Andreadis, A. (2005). Tau gene alternative splicing: expression patterns, regulation and modulation of function in normal brain and neurodegenerative diseases. Biochim Biophys Acta 1739, 91-103. Annaert, W. G., Levesque, L. , Craessaerts, K., Dierinck, I., Snellings, G., Westaway, D., George-Hyslop, P. S., Cordell, B., Fraser, P., and De Strooper, B. (1999). Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons. J Cell Biol 147, 277-294. 61 Arai, Y., Suzuki, A., Mizuguchi, M . , and Takashima, S. (1997). Developmental and aging changes in the expression of amyloid precursor protein in Down syndrome brains. Brain Dev 19, 290-294. Arlt, S., Beisiegel, U., and Kontush, A. (2002). Lipid peroxidation in neurodegeneration: new insights into Alzheimer's disease. Curr Opin Lipidol 13, 289-294. Arnold, D., Keilholz, W., Schild, H. , Dumrese, T., Stevanovic, S., and Rammensee, H. G. (1997). Substrate specificity of cathepsins D and E determined by N-terminal and C-terminal sequencing of peptide pools. Eur J Biochem 249, 171-179. Baki, L. , Marambaud, P., Efthimiopoulos, S., Georgakopoulos, A., Wen, P., Cui, W., Shioi, J., Koo, E . , Ozawa, M . , Friedrich, V. L. , Jr., and Robakis, N. K. (2001). Presenilin-1 binds cytoplasmic epithelial cadherin, inhibits cadherin/pl20 association, and regulates stability and function of the cadherin/catenin adhesion complex. Proc Natl Acad Sci U S A 98, 2381-2386. Bard, F., Cannon, C , Barbour, R., Burke, R. L. , Games, D., Grajeda, FL, Guido, T., Hu, K., Huang, J., Johnson-Wood, K., et al. (2000). Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 6, 916-919. Benjannet, S., Elagoz, A., Wickham, L. , Mamarbachi, M . , Munzer, J. S., Basak, A., Lazure, C , Cromlish, J. A., Sisodia, S., Checler, F., et al. (2001). Post-translational processing of beta-secretase (beta-amyloid-converting enzyme) and its ectodomain shedding. The pro- and transmembrane/cytosolic domains affect its cellular activity and amyloid-beta production. J Biol Chem 276, 10879-10887. Bennett, B. D., Denis, P., Haniu, M . , Teplow, D. B., Kahn, S., Louis, J. C , Citron, M . , and Vassar, R. (2000a). A furin-like convertase mediates propeptide cleavage of B A C E , the Alzheimer's beta -secretase. J Biol Chem 275, 37712-37717. Bennett, R. G., Duckworth, W. C , and Hamel, F. G. (2000b). Degradation of amylin by insulin-degrading enzyme. J Biol Chem 275, 36621-36625. Biernat, J., Gustke, N., Drewes, G., Mandelkow, E. M . , and Mandelkow, E: (1993). Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding. Neuron 11, 153-163. 62 Biernat, J., and Mandelkow, E. M . (1999). The development of cell processes induced by tau protein requires phosphorylation of serine 262 and 356 in the repeat domain and is inhibited by phosphorylation in the proline-rich domains. Mol Biol Cell 10,121-1 AO. Black, R. A., Rauch, C. T., Kozlosky, C. J., Peschon, J. J., Slack, J. L. , Wolfson, M . F., Castner, B. J., Stocking, K. L. , Reddy, P., Srinivasan, S., et al. (1997). A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 385, 729-733. Blacker, D., Albert, M . S., Bassett, S. S., Go, R. C , Harrell, L. E. , and Folstein, M . F. (1994). Reliability and validity of NINCDS-ADRDA criteria for Alzheimer's disease. The National Institute of Mental Health Genetics Initiative. Arch Neurol 51,1198-1204. Bodendorf, U., Fischer, F., Bodian, D., Multhaup, G., and Paganetti, P. (2001). A splice variant of beta-secretase deficient in the amyloidogenic processing of the amyloid precursor protein. J Biol Chem 276, 12019-12023. Bonfiglio, F. (1908a). Di speciali reperti in un caso di probabile sifilide cerebrale. Riv Sper Fremiatria 34,196-206. Bonfiglio, F. (1908b). Di speciali reperti in un caso di probabile sifilide cerebrale. Riv Sper Fremiatria 34, 196-206. Borchelt, D. R., Ratovitski, T., van Lare, J., Lee, M . K., Gonzales, V. , Jenkins, N. A., Copeland, N. G., Price, D. L. , and Sisodia, S. S. (1997). Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron 79, 939-945. Bossy-Wetzel, E . , Schwarzenbacher, R., and Lipton, S. A. (2004). Molecular pathways to neurodegeneration. Nat Med 10 Suppl, S2-9. Brandt, R., Leger, J., and Lee, G. (1995). Interaction of tau with the neural plasma membrane mediated by tau's amino-terminal projection domain. J Cell Biol 131, 1327-1340. Brou, C , Logeat, F., Gupta, N., Bessia, C , LeBail, O., Doedens, J. R., Cumano, A., Roux, P., Black, R. A., and Israel, A. (2000). A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease T A C E . Mol Cell 5, 207-216. 63 Busciglio, J., Pelsman, A., Wong, C , Pigino, G., Yuan, M . , Mori, H. , and Yankner, B. A. (2002). Altered metabolism of the amyloid beta precursor protein is associated with mitochondrial dysfunction in Down's syndrome. Neuron 33, 677-688. Buxbaum, J. D., Choi, E. K., Luo, Y., Lilliehook, C , Crowley, A. C , Merriam, D. E. , and Wasco, W. (1998a). Calsenilin: a calcium-binding protein that interacts with the presenilins and regulates the levels of a presenilin fragment. Nat Med 4, 1177-1181. Buxbaum, J. D., Koo, E. H. , and Greengard, P. (1993). Protein phosphorylation inhibits production of Alzheimer amyloid beta/A4 peptide. Proc Natl Acad Sci U S A 90, 9195-9198. Buxbaum, J. D., Liu, K. N., Luo, Y., Slack, J. L . , Stocking, K. L. , Peschon, J. J., Johnson, R. S., Castner, B. J., Cerretti, D. P., and Black, R. A. (1998b). Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem 273, 27765-27767'. Cai, H. , Wang, Y., McCarthy, D., Wen, H., Borchelt, D. R., Price, D. L. , and Wong, P. C. (2001). BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci 4, 233-234. Cao, X., and Sudhof, T. C. (2001). A transcriptionally [correction of transcriptively] active complex of APP with Fe65 and histone acetyltransferase Tip60. Science 293,115-120. Capell, A., Steiner, H., Romig, H., Keck, S., Baader, M . , Grim, M . G., Baumeister, R., and Haass, C. (2000a). Presenilin-1 differentially facilitates endoproteolysis of the beta-amyloid precursor protein and Notch. Nat Cell Biol 2, 205-211. Capell, A., Steiner, H., Willem, M . , Kaiser, H. , Meyer, C , Walter, J., Lammich, S., Multhaup, G., and Haass, C. (2000b). Maturation and pro-peptide cleavage of beta-secretase. J Biol Chem 275, 30849-30854. Capurso, A., Solfrizzi, V., Panza, F., Torres, F., Mastroianni, F., Grassi, A., Del Parigi, A., Capurso, C , Pirozzi, M . R., Centonze, S., and Misciagna, G. (1997). Dietary patterns and cognitive functions in elderly subjects. Aging (Milano) 9, 45-47. Charlwood, J., Dingwall, C , Matico, R., Hussain, I., Johanson, K., Moore, S., Powell, D. J., Skehel, J. M . , Ratcliffe, S., Clarke, B., et al. (2001). Characterization of the 64 glycosylation profiles of Alzheimer's beta -secretase protein Asp-2 expressed in a variety of cell lines. J Biol Chem 276,16739-16748. Christensen, M . A., Zhou, W., Qing, H., Lehman, A., Philipsen, S., and Song, W. (2004). Transcriptional regulation of BACE1, the beta-amyloid precursor protein beta-secretase, by Spl. Mol Cell Biol 24, 865-874. Chyung, J. H. , Raper, D. M . , and Selkoe, D. J. (2005). Gamma-secretase exists on the plasma membrane as an intact complex that accepts substrates and effects intramembrane cleavage. J Biol Chem 280, 4383-4392. Citron, M . , Oltersdorf, T., Haass, C , McConlogue, L. , Hung, A. Y., Seubert, P., Vigo-Pelfrey, C , Lieberburg, I., and Selkoe, D. J. (1992). Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production. Nature 360, 672-67r4. Citron, M . , Teplow, D. B., and Selkoe, D. J. (1995). Generation of amyloid beta protein from its precursor is sequence specific. Neuron 14, 661-670. Clark, A., de Koning, E. J., Hattersley, A. T., Hansen, B. C , Yajnik, C. S., and Poulton, J. (1995). Pancreatic pathology in non-insulin dependent diabetes (NIDDM). Diabetes Res Clin Pract 28 Suppl, S39-47. Clarke, R., Smith, A. D., Jobst, K. A., Refsum, H., Sutton, L. , and Ueland, P. M . (1998). Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol 55, 1449-1455. Cleaver, O., and Krieg, P. A. (2001). Notochord patterning of the endoderm. Dev Biol 234, 1-12. Cook, D. G., Leverenz, J. B., McMillan, P. J., Kulstad, J. J., Ericksen, S., Roth, R. A., Schellenberg, G. D., Jin, L. W., Kovacina, K. S., and Craft, S. (2003). Reduced hippocampal insulin-degrading enzyme in late-onset Alzheimer's disease is associated with the apolipoprotein E-epsilon4 allele. Am J Pathol 162, 313-319. Corbo, R. M . , and Scacchi, R. (1999). Apolipoprotein E (APOE) allele distribution in the world. Is APOE*4 a 'thrifty' allele? Ann Hum Genet 63 (Pt 4), 301-310. 65 Corder, E. EL, Saunders, A. M , Strittmatter, W. J., Schmechel, D. E. , Gaskell, P. C , Small, G. W., Roses, A. D., Haines, J. L. , and Pericak-Vance, M . A. (1993). Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science 261, 921-923. Creemers, J. W., Ines Dominguez, D., Plets, E. , Serneels, L. , Taylor, N. A. , Multhaup, G., Craessaerts, K., Annaert, W., and De Strooper, B. (2001). Processing of beta-secretase by furin and other members of the proprotein convertase family. J Biol Chem 276, 4211-4217. De Felice, F. G., Vieira, M . N., Saraiva, L. M . , Figueroa-Villar, J. D., Garcia-Abreu, J., Liu, R., Chang, L. , Klein, W. L. , and Ferreira, S. T. (2004). Targeting the neurotoxic species in Alzheimer's disease: inhibitors of Abeta oligomerization. Faseb J 18, 1366-1372. De Jonghe, C , Esselens, C , Kumar-Singh, S., Craessaerts, K., Serneels, S., Checler, F., Annaert, W., Van Broeckhoven, C , and De Strooper, B. (2001). Pathogenic APP mutations near the gamma-secretase cleavage site differentially affect Abeta secretion and APP C-terminal fragment stability. Hum Mol Genet 10, 1665-1671. de la Torre, J. C. (2002). Alzheimer disease as a vascular disorder: nosological evidence. Stroked, 1152-1162. de la Torre, J. C. (2004). Is Alzheimer's disease a neurodegenerative or a vascular disorder? Data, dogma, and dialectics. Lancet Neurol 3, 184-190. de Quervain, D. J., Henke, K., Aerni, A., Treyer, V., McGaugh, J. L . , Berthold, T., Nitsch, R. M . , Buck, A., Roozendaal, B., and Hock, C. (2003). Glucocorticoid-induced impairment of declarative memory retrieval is associated with reduced blood flow in the medial temporal lobe. Eur J Neurosci 17, 1296-1302. De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Murmn, J. S., Schroeter, E. H. , Schrijvers, V. , Wolfe, M . S., Ray, W. J., et al. (1999). A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398, 518-522. De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H. , Guhde, G., Annaert, W., Von Figura, K., and Van Leuven, F. (1998). Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391, 387-390. 66 Deane, R., Du Yan, S., Submamaryan, R. K., LaRue, B., Jovanovic, S., Hogg, E. , Welch, D., Manness, L. , Lin, C , Yu, J., et al. (2003). R A G E mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain. Nat Med 9, 907-913. Deane, R., Wu, Z., Sagare, A., Davis, J., Du Yan, S., Hamm, K., Xu, F., Parisi, M . , LaRue, B., Hu, H. W., et al. (2004). LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms. Neuron 43, 333-344. Dewachter, I., Van Dorpe, J., Smeijers, L. , Gilis, M . , Kuiperi, C., Laenen, I., Caluwaerts, N., Moechars, D., Checler, F., Vanderstichele, H., and Van Leuven, F. (2000). Aging increased amyloid peptide and caused amyloid plaques in brain of old APP/V717I transgenic mice by a different mechanism than mutant presenilin 1. J Neurosci 20, 6452-6458. Divry, P., and Florkin, M . (1927a). Sur les proprietes optiques de l'amyloi'de. CR Societe de Biologie (Paris) 97, 1808-1810. Divry, P., and Florkin, M . (1927b). Sur les proprietes optiques de ramyloi'de. CR Societe de Biologie (Paris) 97, 1808-1810. Doan, A., Thinakaran, G., Borchelt, D. R., Slunt, H. H. , Ratovitsky, T., Podlisny, M . , Selkoe, D. J., Seeger, M . , Gandy, S. E . , Price, D. L. , and Sisodia, S. S. (1996). Protein topology of presenilin 1. Neuron 17,1023-1030. Dodart, J. C , Bales, K. R., Gannon, K. S., Greene, S. J., DeMattos, R. B., Mathis, C , DeLong, C. A., Wu, S., Wu, X., Holtzman, D. M . , and Paul, S. M . (2002). Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model. Nat Neurosci 5, 452-457. Down, J. L. H. (1866). Observations on an ethnic classification of idiots. London Hospital Reports 3, 259-262. Du Yan, S., Zhu, H. , Fu, J., Yan, S. F., Roher, A., Tourtellotte, W. W., Rajavashisth, T., Chen, X., Godman, G. C , Stern, D., and Schmidt, A. M . (1997). Amyloid-beta peptide-receptor for advanced glycation endproduct interaction elicits neuronal expression of macrophage-colony stimulating factor: a proinflammatory pathway in Alzheimer disease Proc Nad Acad Sci U S A 94, 5296-5301. Dunn, B. M . (2002). Structure and mechanism of the pepsin-like family of aspartic peptidases. Chem Rev 102, 4431-4458. 67 Edbauer, D., Willem, M . , Lammich, S., Steiner, H., and Haass, C. (2002a). Insulin-degrading enzyme rapidly removes the beta-amyloid precursor protein intracellular domain (AICD). J Biol Chem 277, 13389-13393. Edbauer, D., Winkler, E. , Haass, C , and Steiner, H. (2002b). Presenilin and nicastrin regulate each other and determine amyloid beta-peptide production via complex formation. Proc Natl Acad Sci U S A 99, 8666-8671. Edbauer, D., Winkler, E. , Regula, J. T., Pesold, B., Steiner, H. , and Haass, C. (2003). Reconstitution of gamma-secretase activity. Nat Cell Biol 5, 486-488. Ehehalt, R., Michel, B., De Pietri Tonelli, D., Zacchetti, D., Simons, K., and Keller, P. (2002). Splice variants of the beta-site APP-cleaving enzyme BACE1 in human brain and pancreas. Biochem Biophys Res Commun 293, 30-37. Eikelenboom, P., Bate, C , Van Gool, W. A., Hoozemans, J. J., Rozemuller, J. M . , Veerhuis, R., and Williams, A. (2002). Neuroinflammation in Alzheimer's disease and prion disease. Glia 40, 232-239. El Khoury, J., Hickman, S. E. , Thomas, C. A., Cao, L. , Silverstein, S. C , and Loike, J. D. (1996). Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils. Nature 382, 716-719. Eriksen, J. L . , Sagi, S. A., Smith, T. E . , Weggen, S., Das, P., McLendon, D. C , Ozols, V. V., Jessing, K. W., Zavitz, K. H., Koo, E. H. , and Golde, T. E . (2003). NSAIDs and enantiomers of flurbiprofen target gamma-secretase and lower Abeta 42 in vivo. J Clin Invest 112, 440-449. Esiri, M . M . , Nagy, Z., Smith, M . Z., Barnetson, L. , and Smith, A. D. (1999). Cerebrovascular disease and threshold for dementia in the early stages of Alzheimer's disease. Lancet 354, 919-920. Esler, W. P., and Wolfe, M . S. (2001). A portrait of Alzheimer secretases-new features and familiar faces. Science 293,1449-1454. Espeland, M . A., Rapp, S. R., Shumaker, S. A., Brunner, R., Manson, J. E . , Sherwin, B. B., Hsia, J., Margolis, K. L. , Hogan, P. E. , Wallace, R., et al. (2004). Conjugated equine estrogens and global cognitive function in postmenopausal women: Women's Health Initiative Memory Study. Jama 291, 2959-2968. 68 Etminan, M . , Gill, S., and Samii, A. (2003). Effect of non-steroidal anti-inflammatory drugs on risk of Alzheimer's disease: systematic review and meta-analysis of observational studies. Bmj 327, 128. Farrer, L. A., Cupples, L. A., Haines, J. L. , Hyman, B., Kukull, W. A., Mayeux, R., Myers, R. H. , Pericak-Vance, M . A., Risch, N., and van Duijn, C. M . (1997). Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium. Jama 278, 1349-1356. Farris, W., Mansourian, S., Chang, Y., Lindsley, L. , Eckman, E. A., Frosch, M . P., Eckman, C. B., Tanzi, R. E. , Selkoe, D. J., and Guenette, S. (2003). Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci U S A 100, 4162-4167. Fischer, F., Molinari, M . , Bodendorf, U., and Paganetti, P. (2002). The disulphide bonds in the catalytic domain of B A C E are critical but not essential for amyloid precursor protein processing activity. J Neurochem 80, 1079-1088. Fischer, O. (1907a). Miliare Nekrosen mit drusigen Wucherungen der Neurofibrillen, eine regelmaBige Veranderung der Hirnrinde bei seniler Demenz. Monatsschr Psychiatr Neurol 22, 361-372. Fischer, O. (1907b). Miliare Nekrosen mit drusigen Wucherungen der Neurofibrillen, eine regelmaBige Veranderung der Hirnrinde bei seniler Demenz. Monatsschr Psychiatr Neurol 22, 361-372. Fleminger, S., Oliver, D. L. , Lovestone, S., Rabe-Hesketh, S., and Giora, A. (2003). Head injury as a risk factor for Alzheimer's disease: the evidence 10 years on; a partial replication. J Neurol Neurosurg Psychiatry 74, 857-862. Floyd, R. A., and Hensley, K. (2002). Oxidative stress in brain aging. Implications for therapeutics of neurodegenerative diseases. Neurobiol Aging 23, 795-807. Francis, R., McGrath, G., Zhang, J., Ruddy, D. A., Sym, M . , Apfeld, J., Nicoll, M . , Maxwell, M . , Hai, B., Ellis, M . C , et al. (2002). aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of betaAPP, and presenilin protein accumulation. Dev Cell 3, 85-97. 69 Freitag, M . FL, Peila, R., Masaki, K., Petrovitch, H. , Ross, G. W., White, L. R., and Launer, L. J. (2006). Midlife pulse pressure and incidence of dementia: the Honolulu-Asia Aging Study. Stroke 37, 33-37. Fukumori, A., Okochi, M . , Tagami, S., Jiang, J., Itoh, N., Nakayama, T., Yanagida, K., Ishizuka-Katsura, Y., Morihara, T., Kamino, K., etai. (2006). Presenilin-dependent gamma-secretase on plasma membrane and endosomes is functionally distinct. Biochemistry 45, 4907-4914. Furukawa, K., Sopher, B. L. , Rydel, R. E. , Begley, J. G., Pham, D. G., Martin, G. M . , Fox, M . , and Mattson, M . P. (1996). Increased activity-regulating and neuroprotective efficacy of alpha-secretase-derived secreted amyloid precursor protein conferred by a C-terminal heparin-binding domain. J Neurochem 67, 1882-1896. Gao, S., Hendrie, H. C., Hall, K. S., and Hui, S. (1998). The relationships between age, sex, and the incidence of dementia and Alzheimer disease: a meta-analysis. Arch Gen Psychiatry 55, 809-815. Gervais, F. G., Xu, D., Robertson, G. S., Vaillancourt, J. P., Zhu, Y., Huang, J., LeBlanc, A., Smith, D., Rigby, M . , Shearman, M . S., et al. (1999). Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloidogenic A beta peptide formation. Cell 97, 395-406. Giasson, B. I., Forman, M . S., Higuchi, M . , Golbe, L. I., Graves, C. L. , Kotzbauer, P. T., Trojanowski, J. Q., and Lee, V. M . (2003). Initiation and synergistic fibrillization of tau and alpha-synuclein. Science 300, 636-640. Glenner, G. G., and Wong, C. W. (1984a). Alzheimer's disease and Down's syndrome: sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res Commun 122, 1131-1135. Glenner, G. G., and Wong, C. W. (1984b). Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120, 885-890. Goate, A., Chartier-Harlin, M . C , Mullan, M . , Brown, J., Crawford, F., Fidani, L. , Giuffra, L. , Haynes, A., Irving, N., James, L. , and et al. (1991). Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature 349, 704-706. 70 Goedert, M . , Crowther, R. A., and Spillantini, M . G. (1998). Tau mutations cause frontotemporal dementias. Neuron 21, 955-958. Goldgaber, D., Lerman, M . I., McBride, O. W., Saffiotti, U., and Gajdusek, D. C. (1987). Characterization and chromosomal localization of a cDNA encoding brain amyloid of Alzheimer's disease. Science 235, 877-880. Gotz, J., Chen, F., van Dorpe, J., and Nitsch, R. M . (2001). Formation of neurofibrillary tangles in P3011 tau transgenic mice induced by Abeta 42 fibrils. Science 293, 1491-1495. Goutte, C , Tsunozaki, M . , Hale, V. A., and Priess, J. R. (2002). APH-1 is a multipass membrane protein essential for the Notch signaling pathway in Caenorhabditis elegans embryos. Proc Natl Acad Sci U S A 99, 775-779. Grabowski, T. J., Cho, H. S., Vonsattel, J. P., Rebeck, G. W., and Greenberg, S. M: (2001). Novel amyloid precursor protein mutation in an Iowa family with dementia and severe cerebral amyloid angiopathy. Ann Neurol 49, 697-705. Group, C. S. o. H. a. A. W. (2000). The incidence of dementia in Canada. The Canadian Study of Health and Aging Working Group. Neurology 55, 66-73. Group, H. P. S. C. (2002). MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled trial. Lancet 360, 7-22. Group, T. P. s. S. (1993). Effects of tocopherol and deprenyl on the progression of disability in early Parkinson's disease. The Parkinson Study Group. N Engl J Med 328, 176-183. Grundke-Iqbal, I., Iqbal, K., Quinlan, M . , Tung, Y. C , Zaidi, M . S., and Wisniewski, H. M . (1986a). Microtubule-associated protein tau. A component of Alzheimer paired helical filaments. J Biol Chem 261, 6084-6089. Grundke-Iqbal, I., Iqbal, K., Tung, Y. C , Quinlan, M . , Wisniewski, H. M . , and Binder, L. I. (1986b). Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc Nad Acad Sci U S A 83, 4913-4917. 71 Gu, Y., Misonou, H., Sato, T., Dohmae, N., Takio, K., and Diara, Y. (2001). Distinct intramembrane cleavage of the beta-amyloid precursor protein family resembling gamma-secretase-like cleavage of Notch. J Biol Chem 276, 35235-35238. Guo, J. P., Arai, T., Miklossy, J., and McGeer, P. L. (2006). Abeta and tau form soluble complexes that may promote self aggregation of both into the insoluble forms observed in Alzheimer's disease. Proc Natl Acad Sci U S A 103,1953-1958. Gyure, K. A., Durham, R., Stewart, W. F., Smialek, J. E. , and Troncoso, J. C. (2001). Intraneuronal abeta-amyloid precedes development of amyloid plaques in Down syndrome. Arch Pathol Lab Med 125, 489-492. Haniu, M . , Denis, P., Young, Y., Mendiaz, E. A., Fuller, J., Hui, J. O., Bennett, B. D., Kahn, S., Ross, S., Burgess, T., et al. (2000). Characterization of Alzheimer's beta -secretase protein B A C E . A pepsin family member with unusual properties. J Biol Chem 275,21099-21106. Hardingham, G. E. , and Bading, H. (2002). Coupling of extrasynaptic N M D A receptors to a CREB shut-off pathway is developmentally regulated. Biochim Biophys Acta 1600, 148-153. Hardy, J., and Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353-356. Harper, C. G., and Kril, J. J. (1990). Neuropathology of alcoholism. Alcohol Alcohol 25, 207-216. Heber, S., Herms, J., Gajic, V., Hainfellner, J., Aguzzi, A., Rulicke, T., von Kretzschmar, H. , von Koch, C , Sisodia, S., Tremml, P., et al. (2000). Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members. J Neurosci 20, 7951-7963. Hendriks, L. , van Duijn, C. M . , Cras, P., Cruts, M . , Van Hul, W., van Harskamp, F., Warren, A., Mclnnis, M . G., Antonarakis, S. E . , Martin, J. J., and et al. (1992). Presenile dementia and cerebral haemorrhage linked to a mutation at codon 692 of the beta-amyloid precursor protein gene. Nat Genet 1, 218-221. Herz, J., and Marschang, P. (2003). Coaxing the L D L receptor family into the fold. Cell 112, 289-292. 72 Heyman, A., Fillenbaum, G. G., Welsh-Bohmer, K. A., Gearing, M . , Mirra, S. S., Mohs, R. C., Peterson, B. L. , and Pieper, C. F. (1998). Cerebral infarcts in patients with autopsy-proven Alzheimer's disease: CERAD, part XVUI. Consortium to Establish a Registry for Alzheimer's Disease. Neurology 57, 159-162. Hock, C , Konietzko, U., Streffer, J. R., Tracy, J., Signorell, A., Muller-Tillmanns, B., Lemke, U., Henke, K., Moritz, E . , Garcia, E., etal. (2003). Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease. Neuron 38, 547-554. Hollenbach, E. , Ackermann, S., Hyman, B. T., and Rebeck, G. W. (1998). Confirmation of an association between a polymorphism in exon 3 of the low-density lipoprotein receptor-related protein gene and Alzheimer's disease. Neurology 50, 1905-1907. Holtzman, D. M . , Bales, K. R., Tenkova, T., Fagan, A. M . , Parsadanian, M . , Sartorius, L. J., Mackey, B., Olney, J., McKeel, D., Wozniak, D., and Paul, S. M . (2000a). Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer's disease. Proc Nad Acad Sci U S A 97, 2892-2897. Holtzman, D. M . , Fagan, A. M . , Mackey, B., Tenkova, T., Sartorius, L. , Paul, S. M . , Bales, K., Ashe, K. H., Irizarry, M . C , and Hyman, B. T. (2000b). Apolipoprotein E facilitates neuritic and cerebrovascular plaque formation in an Alzheimer's disease model. Ann Neurol 47,139-1 Al. Honig, L. S., Kukull, W., and Mayeux, R. (2005). Atherosclerosis and AD: analysis of data from the US National Alzheimer's Coordinating Center. Neurology 64, 494-500. Honig, L. S., Tang, M . X., Albert, S., Costa, R., Luchsinger, J., Manly, J., Stern, Y., and Mayeux, R. (2003). Stroke and the risk of Alzheimer disease. Arch Neurol 60, 1707-1712. Hsiao, K., Chapman, P., Nilsen, S., Eckman, C , Harigaya, Y., Younkin, S., Yang, F., and Cole, G. (1996). Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 274, 99-102. Huang, W., Qiu, C , Winblad, B., and Fratiglioni, L. (2002). Alcohol consumption and incidence of dementia in a community sample aged 75 years and older. J Clin Epidemiol 55, 959-964. Huse, J. T., Byant, D., Yang, Y., Pijak, D. S., D'Souza, I., Lah, J. J., Lee, V. M . , Doms, R. W., and Cook, D. G. (2003). Endoproteolysis of beta-secretase (beta-site amyloid 73 precursor protein-cleaving enzyme) within its catalytic domain. A potential mechanism for regulation. J Biol Chem 278, 17141-17149. Huse, J. T., and Doms, R. W. (2000). Closing in on the amyloid cascade: recent insights into the cell biology of Alzheimer's disease. Mol Neurobiol 22, 81-98. Husemann, J., Loike, J. D., Kodama, T., and Silverstein, S. C. (2001). Scavenger receptor class B type I (SR-BI) mediates adhesion of neonatal murine microglia to fibrillar beta-amyloid. J Neuroimmunol 114, 142-150. Hussain, I., Powell, D., Howlett, D. R., Tew, D. G., Meek, T. D., Chapman, C , Gloger, I. S., Murphy, K. E. , Southan, C. D., Ryan, D. M. , et al. (1999). Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol Cell Neurosci 14, 419-427. Hyman, B. T. (1997). The neuropathological diagnosis of Alzheimer's disease: clinical-pathological studies. Neurobiol Aging 18, S27-32. Diara, Y., Nukina, N., Miura, R., and Ogawara, M . (1986). Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer's disease. J Biochem (Tokyo) 99, 1807-1810. in't Veld, B. A., Ruitenberg, A., Hofman, A., Strieker, B. H., and Breteler, M . M . (2001). Antihypertensive drugs and incidence of dementia: the Rotterdam Study. Neurobiol Aging 22, 407-412. in f Veld, B. A., Ruitenberg, A., Hofman, A., Launer, L. J., van Duijn, C. M . , Stijnen, T., Breteler, M . M . , and Strieker, B. H. (2001). Nonsteroidal antiinflammatory drugs and the risk of Alzheimer's disease. N Engl J Med 345, 1515-1521. Iwata, N., Takaki, Y., Fukami, S., Tsubuki, S., and Saido, T. C. (2002). Region-specific reduction of A beta-degrading endopeptidase, neprilysin, in mouse hippocampus upon aging. J Neurosci Res 70,493-500. Iwata, N., Tsubuki, S., Takaki, Y., Shirotani, K., Lu, B., Gerard, N. P., Gerard, C , Hama, E. , Lee, H. J., and Saido, T. C. (2001). Metabolic regulation of brain Abeta by neprilysin. Science 292, 1550-1552. Jacobs, P. A., Baikie, A. G., Court Brown, W. M . , and Strong, J. A. (1959). The somatic chromosomes in mongolism. Lancet 1, 710. 74 Jellinger, K. A., and Attems, J. (2003). Incidence of cerebrovascular lesions in Alzheimer's disease: a postmortem study. Acta Neuropathol (Berl) 105,14-17. Jellinger, K. A., and Attems, J. (2005). Prevalence and pathogenic role of cerebrovascular lesions in Alzheimer disease. J Neurol Sci 229-230, 37-41. Jick, H. , Zornberg, G. L. , Jick, S. S., Seshadri, S., and Drachman, D. A. (2000). Statins and the risk of dementia. Lancet 356,1627-1631. Jimenez-Jimenez, F. J., de Bustos, F., Molina, J. A., Benito-Leon, J., Tallon-Barranco, A., Gasalla, T., Orti-Pareja, M . , Guillamon, F., Rubio, J. C., Arenas, J., and Enriquez-de-Salamanca, R. (1997). Cerebrospinal fluid levels of alpha-tocopherol (vitamin E) in Alzheimer's disease. J Neural Transm 104, 703-710. Jo, D. G., Kim, M . J., Choi, Y. H. , Kim, I. K., Song, Y. H. , Woo, H. N., Chung, C. W., and Jung, Y. K. (2001). Pro-apoptotic function of calsenilin/DREAM/KChIP3. Faseb J 75,589-591. Jo, D. G., Lee, J. Y., Hong, Y. M . , Song, S., Mook-Jung, I., Koh, J. Y., and Jung, Y. K. (2004). Induction of pro-apoptotic calsenilin/DREAM/KChIP3 in Alzheimer's disease and cultured neurons after amyloid-beta exposure. J Neurochem 88, 604-611. Johnson, G. V., and Stoothoff, W. H. (2004). Tau phosphorylation in neuronal cell function and dysfunction. J Cell Sci 777, 5721-5729. Juan, D., Zhou, D. H. , L i , J., Wang, J. Y., Gao, C , and Chen, M . (2004). A 2-year follow-up study of cigarette smoking and risk of dementia. Eur J Neurol 77, 277-282. Kaether, C , Capell, A., Edbauer, D., Winkler, E . , Novak, B., Steiner, H. , and Haass, C. (2004). The presenilin C-terminus is required for ER-retention, nicastrin-binding and gamma-secretase activity. Embo J 23,4738-4748. Kalmijn, S., Foley, D., White, L. , Burchfiel, C. M . , Curb, J. D., Petrovitch, H. , Ross, G. W., Havlik, R. J., and Launer, L. J. (2000). Metabolic cardiovascular syndrome and risk of dementia in Japanese-American elderly men. The Honolulu-Asia aging study. Arterioscler Thromb Vase Biol 20, 2255-2260. Kamboh, M . I. (2004). Molecular genetics of late-onset Alzheimer's disease. Ann Hum Genet 68, 381-404. 75 Kang, D. E. , Saitoh, T., Chen, X., Xia, Y., Masliah, E . , Hansen, L. A., Thomas, R. G., Thai, L. J., and Katzman, R. (1997). Genetic association of the low-density lipoprotein receptor-related protein gene (LRP), an apolipoprotein E receptor, with late-onset Alzheimer's disease. Neurology 49, 56-61. Kang, D. E. , Soriano, S., Frosch, M . P., Collins, T., Naruse, S., Sisodia, S. S., Leibowitz, G. , Levine, F., and Koo, E. H. (1999). Presenilin 1 facilitates the constitutive turnover of beta-catenin: differential activity of Alzheimer's disease-linked PS1 mutants in the beta-catenin-signaling pathway. J Neurosci 19, 4229-4237. Kang, D. E. , Soriano, S., Xia, X. , Eberhart, C. G., De Strooper, B., Zheng, H. , and Koo, E. H. (2002). Presenilin couples the paired phosphorylation of beta-catenin independent of axin: implications for beta-catenin activation in tumorigenesis. Cell 110, 751-762. Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M . , Masters, C. L. , Grzeschik, K. H. , Multhaup, G., Beyreuther, K., and Muller-Hill, B. (1987). The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature 325, 733-736. Karlinsky, H. , Vaula, G., Haines, J. L. , Ridgley, J., Bergeron, C , Mortilla, M . , Tupler, R. G., Percy, M . E. , Robitaille, Y., Noldy, N. E. , and et al. (1992). Molecular and prospective phenotypic characterization of a pedigree with familial Alzheimer's disease and a missense mutation in codon 717 of the beta-amyloid precursor protein gene. Neurology 42, 1445-1453. Kawas, C , Gray, S., Brookmeyer, R., Fozard, J., and Zonderman, A. (2000). Age-specific incidence rates of Alzheimer's disease: the Baltimore Longitudinal Study of Aging. Neurology 54, 2072-2077. Kayed, R., Head, E. , Thompson, J. L . , Mclntire, T. M . , Milton, S. C , Cotman, C. W., and Glabe, C. G. (2003). Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486-489. Kidd, M . (1963). Paired helical filaments in electron microscopy of Alzheimer's disease. Nature 197, 192-193. Kim, S. H., Leem, J. Y., Lah, J. J., Slunt, H. H. , Levey, A. I., Thinakaran, G., and Sisodia, S.S. (2001). Multiple effects of aspartate mutant presenilin 1 on the processing and trafficking of amyloid precursor protein. J Biol Chem 276, 43343-43350. 76 Kim, S. H. , and Sisodia, S. S. (2005). Evidence that the "NF" motif in transmembrane domain 4 of presenilin 1 is critical for binding with PEN-2. J Biol Chem 280, 41953-41966. Kimberly, W. T., Zheng, J. B., Guenette, S. Y., and Selkoe, D. J. (2001). The intracellular domain of the beta-amyloid precursor protein is stabilized by Fe65 and translocates to the nucleus in a notch-like manner. J Biol Chem 276, 40288-40292. King, G. D., and Scott Turner, R. (2004). Adaptor protein interactions: modulators of amyloid precursor protein metabolism and Alzheimer's disease risk? Exp Neurol 185, 208-219. Kitagawa, M . , Hatakeyama, S., Shirane, M . , Matsumoto, M . , Ishida, N., Hattori, K., Nakamichi, I., Kikuchi, A., Nakayama, K., and Nakayama, K. (1999). An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin. Embo J 18, 2401-2410. Kitazume, S., Nakagawa, K., Oka, R., Tachida, Y., Ogawa, K., Luo, Y., Citron, M . , Shitara, H., Taya, C , Yonekawa, H., et al. (2005). In vivo cleavage of alpha2,6-sialyltransferase by Alzheimer beta-secretase. J Biol Chem 280, 8589-8595. Kitazume, S., Tachida, Y., Oka, R., Kotani, N., Ogawa, K., Suzuki, M . , Dohmae, N., Takio, K., Saido, T. C , and Hashimoto, Y. (2003). Characterization of alpha 2,6-sialyltransferase cleavage by Alzheimer's beta -secretase (BACE1). J Biol Chem 278, 14865-14871. Kivipelto, M . , Helkala, E. L . , Hanninen, T., Laakso, M . P., Hallikainen, M . , Alhainen, K., Soininen, H., Tuomilehto, J., and Nissinen, A. (2001a). Midlife vascular risk factors and late-life mild cognitive impairment: A population-based study. Neurology 56, 1683-1689. Kivipelto, M . , Helkala, E. L . , Laakso, M . P., Hanninen, T., Hallikainen, M . , Alhainen, K., Soininen, H., Tuomilehto, J., and Nissinen, A. (2001b). Midlife vascular risk factors and Alzheimer's disease in later life: longitudinal, population based study. Bmj 322, 1447-1451. Koike, H. , Tomioka, S., Sorimachi, H., Saido, T. C , Maruyama, K., Okuyama, A., Fujisawa-Sehara, A., Ohno, S., Suzuki, K., and Ishiura, S. (1999). Membrane-anchored metalloprotease MDC9 has an alpha-secretase activity responsible for processing the amyloid precursor protein. Biochem J 343 Pt 2, 371-375. 77 Koo, E. H., and Kopan, R. (2004). Potential role of presenilin-regulated signaling pathways in sporadic neurodegeneration. Nat Med 10 Suppl, S26-33. Koo, E. H. , and Squazzo, S. L. (1994). Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J Biol Chem 269,17386-17389. Kosik, K. S., Joachim, C. L. , and Selkoe, D. J. (1986). Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease. Proc Natl Acad Sci U S A 83, 4044-4048. Kraepelin, E. (1910). Psychiatrie: Ein Lehrbuch fur Studierende und Arzte. Leipzig: . Barth, 593-632. Kumar-Singh, S., De Jonghe, C , Cruts, M . , Kleinert, R., Wang, R., Mercken, M . , De Strooper, B., Vanderstichele, H., Lofgren, A., Vanderhoeven, I., et al. (2000). Nonflbrillar diffuse amyloid deposition due to a gamma(42)-secretase site mutation points to an essential role for N-truncated A beta(42) in Alzheimer's disease. Hum Mol Genet 9, 2589-2598. Lalonde, R., Dumont, M . , Staufenbiel, M . , and Strazielle, C. (2005). Neurobehavioral characterization of APP23 transgenic mice with the SHIRPA primary screen. Behav Brain Res 757,91-98. Lamb, B. T., Sisodia, S. S., Lawler, A. M . , Slunt, H. H. , Kitt, C. A., Kearns, W. G., Pearson, P. L. , Price, D. L. , and Gearhart, J. D. (1993). Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice [corrected]. Nat Genet 5, 22-30. Lambert, J. C , Wavrant-De Vrieze, F., Amouyel, P., and Chartier-Harlin, M . C. (1998a). Association at LRP gene locus with sporadic late-onset Alzheimer's disease. Lancet 357, 1787-1788. Lambert, M . P., Barlow, A. K., Chromy, B. A., Edwards, C , Freed, R., Liosatos, M . , Morgan, T. E. , Rozovsky, I., Trommer, B., Viola, K. L., etai. (1998b). Diffusible, nonflbrillar ligands derived from Abetal-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A 95, 6448-6453. Lammich, S., Kojro, E. , Postina, R., Gilbert, S., Pfeiffer, R., Jasionowski, M . , Haass, C , and Fahrenholz, F. (1999). Constitutive and regulated alpha-secretase cleavage of 78 Alzheimer's amyloid precursor protein by a disintegrin metalloprotease. Proc Natl Acad Sci U S A 96, 3922-3927. Launer, L. J., Ross, G. W., Petrovitch, H. , Masaki, K., Foley, D., White, L. R., and Havlik, R. J. (2000). Midlife blood pressure and dementia: the Honolulu-Asia aging study. Neurobiol Aging 21, 49-55. LaVoie, M . J., Fraering, P. C., Ostaszewski, B. L. , Ye, W., Kimberly, W. T., Wolfe, M . S., and Selkoe, D. J. (2003). Assembly of the gamma-secretase complex involves early formation of an intermediate subcomplex of Aph-1 and nicastrin. J Biol Chem 278, 37213-37222. Lee, E. B., Zhang, B., Liu, K., Greenbaum, E. A., Doms, R. W., Trojanowski, J. Q., and Lee, V. M . (2005). B A C E overexpression alters the subcellular processing of APP and inhibits Abeta deposition in vivo. J Cell Biol 168, 291-302. Lee, R. K., Wurtman, R. J., Cox, A. J., and Nitsch, R. M . (1995). Amyloid precursor protein processing is stimulated by metabotropic glutamate receptors. Proc Nad Acad Sci U S A 92, 8083-8087. Leissring, M . A., Farris, W., Chang, A. Y. , Walsh, D. M . , Wu, X., Sun, X., Frosch, M . P., and Selkoe, D. J. (2003). Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death. Neuron 40, 1087-1093. Lejeune, J., Turpin, R., and Gautier, M . (1959). [Mongolism; a chromosomal disease (trisomy).]. Bull Acad Natl Med 143, 256-265. Lesser, G., Kandiah, K., Libow, L. S., Likourezos, A., Breuer, B., Marin, D., Mohs, R., Haroutunian, V., and Neufeld, R. (2001). Elevated serum total and L D L cholesterol in very old patients with Alzheimer's disease. Dement Geriatr Cogn Disord 12, 138-145. Levy-Lahad, E. , Wasco, W., Poorkaj, P., Romano, D. M . , Oshima, J., Pettingell, W. H. , Yu, C. E. , Jondro, P. D., Schmidt, S. D., Wang, K., and et al. (1995a). Candidate gene for the chromosome 1 familial Alzheimer's disease locus. Science 2f59, 973-977. Levy-Lahad, E. , Wijsman, E. M . , Nemens, E . , Anderson, L. , Goddard, K. A., Weber, J. L. , Bird, T. D., and Schellenberg, G. D. (1995b). A familial Alzheimer's disease locus on chromosome 1. Science 269, 970-973. 79 Levy, E. , Carman, M . D., Fernandez-Madrid, I. J., Power, M . D., Lieberburg, I., van Duinen, S. G., Bots, G. T., Luyendijk, W., and Frangione, B. (1990). Mutation of the Alzheimer's disease amyloid gene in hereditary cerebral hemorrhage, Dutch type. Science 248, 1124-1126. Lewin (2004). Saving Lives, Saving Money: Dividends for Americans Invest-ing in Alzheimer Research. (Washington, D.C.: A report from the Lewin Group, commissioned by the Alzheimer's Association.). Lewis, J., Dickson, D. W., Lin, W. L. , Chisholm, L. , Corral, A., Jones, G., Yen, S. H. , Sahara, N., Skipper, L. , Yager, D., et al. (2001). Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP. Science 293, 1487-1491. Lewis, J., McGowan, E. , Rockwood, J. , Melrose, H. , Nacharaju, P., Van Slegtenhorst, M . , Gwinn-Hardy, K., Paul Murphy, M . , Baker, M . , Yu, X., et al. (2000). Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein. Nat Genet 25, 402-405. Li , G., Higdon, R., Kukull, W. A., Peskind, E. , Van Valen Moore, K., Tsuang, D., van Belle, G., McCormick, W., Bowen, J. D., Teri, L., et al. (2004). Statin therapy and risk of dementia in the elderly: a community-based prospective cohort study. Neurology 63, 1624-1628. Li , J. J., Dickson, D., Hof, P. R., and Vlassara, H. (1998). Receptors for advanced glycosylation endproducts in human brain: role in brain homeostasis. Mol Med 4, 46-60. Li , Q., and Sudhof, T. C. (2004). Cleavage of amyloid-beta precursor protein and amyloid-beta precursor-like protein by B A C E 1. J Biol Chem 279, 10542-10550. Li , X., and Greenwald, I. (1996). Membrane topology of the C. elegans SEL-12 presenilin. Neuron 17, 1015-1021. Li , X. , and Greenwald, I. (1998). Additional evidence for an eight-transmembrane-domain topology for Caenorhabditis elegans and human presenilins. Proc Natl Acad Sci U S A 95, 7109-7114. Li , Y. , Zhou, W., Tong, Y. , He, G. , and Song, W. (2006). Control of APP processing and Abeta generation level by BACE1 enzymatic activity and transcription. Faseb J 20, 285-292. 80 Li , Y. M . , Lai, M . T., Xu, M . , Huang, Q., DiMuzio-Mower, J., Sardana, M . K., Shi, X. P., Yin, K. C , Shafer, J. A., and Gardell, S. J. (2000a). Presenilin 1 is linked with gamma-secretase activity in the detergent solubilized state. Proc Nad Acad Sci U S A 97, 6138-6143. Li , Y. ML, Xu, M . , Lai, M . T., Huang, Q., Castro, J. L. , DiMuzio-Mower, J., Harrison, T., Lellis, C , Nadin, A., Neduvelil, J. G., et al. (2000b). Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1. Nature 405, 689-694. Lichtenthaler, S. F., Dominguez, D. I., Westmeyer, G. G., Reiss, K., Haass, C , Saftig, P., De Strooper, B., and Seed, B. (2003). The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1. J Biol Chem 278, 48713-48719. Lilliehook, C , Bozdagi, O., Yao, J., Gomez-Ramirez, M . , Zaidi, N. F., Wasco, W., Gandy, S., Santucci, A. C , Haroutunian, V., Hundey, G. W., and Buxbaum, J. D. (2003). Altered Abeta formation and long-term potentiation in a calsenilin knock-out. J Neurosci 23,9097-9106. Lilliehook, C , Chan, S., Choi, E. K., Zaidi, N. F., Wasco, W., Mattson, M . P., and Buxbaum, J. D. (2002). Calsenilin enhances apoptosis by altering endoplasmic reticulum calcium signaling. Mol Cell Neurosci 19, 552-559. Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A., and Tang, J. (2000). Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci U S A 97,1456-1460. Lipton, S. A. (2005). The molecular basis of memantine action in Alzheimer's disease and other neurologic disorders: low-affinity, uncompetitive antagonism. Curr Alzheimer Res 2,155-165. Lipton, S. A. (2006). Paradigm shift in neuroprotection by N M D A receptor blockade: Memantine and beyond. Nat Rev Drug Discov, 1-11. Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G. W., and Gong, C. X. (2004). O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proc Natl Acad Sci U S A 101,10804-10809. Lott, I. T., and Head, E. (2001). Down syndrome and Alzheimer's disease: a link between development and aging. Ment Retard Dev Disabil Res Rev 7, 172-178. 81 Lucas, J. J., Hernandez, F., Gomez-Ramos, P., Moran, M . A., Hen, R., and Avila, J. (2001). Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice. Embo J 20, 27-39. Luchsinger, J. A., Tang, M . X. , Siddiqui, M . , Shea, S., and Mayeux, R. (2004). Alcohol intake and risk of dementia. J Am Geriatr Soc 52, 540-546. Lue, L. F., Walker, D. G., Brachova, L. , Beach, T. G., Rogers, J., Schmidt, A. M . , Stern, D. M . , and Yan, S. D. (2001). Involvement of microglial receptor for advanced glycation endproducts (RAGE) in Alzheimer's disease: identification of a cellular activation mechanism. Exp Neurol 171, 29-45. Luo, Y., Bolon, B., Damore, M . A., Fitzpatrick, D., Liu, H. , Zhang, J., Yan, Q., Vassar, R., and Citron, M . (2003). BACE1 (beta-secretase) knockout mice do not acquire compensatory gene expression changes or develop neural lesions over time. Neurobiol Dis 14, 81-88. Luo, Y., Bolon, B., Kahn, S., Bennett, B. D., Babu-Khan, S., Denis, P., Fan, W., Kha, H. , Zhang, J., Gong, Y., et al. (2001). Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci 4, 231-232. Lustig, B., and Behrens, J. (2003). The Wnt signaling pathway and its role in tumor development. J Cancer Res Clin Oncol 129, 199-221. Maccioni, R. B., and Cambiazo, V. (1995). Role of microtubule-associated proteins in the control of microtubule assembly. Physiol Rev 75, 835-864. Mann, D. M . , Yates, P. O., Marcyniuk, B., and Ravindra, C. R. (1986). The topography of plaques and tangles in Down's syndrome patients of different ages. Neuropathol Appl Neurobiol 72, 447-457. Marcinkiewicz, M . , and Seidah, N. G. (2000). Coordinated expression of beta-amyloid precursor protein and the putative beta-secretase B A C E and alpha-secretase A D A M 10 in mouse and human brain. J Neurochem 75, 2133-2143. Marcus, D. L. , Thomas, C , Rodriguez, C , Simberkoff, K., Tsai, J. S., Strafaci, J. A., and Freedman, M . L. (1998). Increased peroxidation and reduced antioxidant enzyme activity in Alzheimer's disease. Exp Neurol 150,40-44. 82 Markesbery, W. R., and Carney, J. M . (1999). Oxidative alterations in Alzheimer's disease. Brain Pathol 9,133-146. Masters, C. L. , Simms, G., Weinman, N. A., Multhaup, G., McDonald, B. L. , and Beyreuther, K. (1985). Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A 82, 4245-4249. Mattson, M . P., Guo, Z. FL, and Geiger, J. D. (1999). Secreted form of amyloid precursor protein enhances basal glucose and glutamate transport and protects against oxidative impairment of glucose and glutamate transport in synaptosomes by a cyclic GMP-mediated mechanism. J Neurochem 73, 532-537. McGeer, P. L. , and McGeer, E. G. (2006). NSAIDs and Alzheimer disease: Epidemiological, animal model and clinical studies. Neurobiol Aging. McGeer, P. L. , Schulzer, M . , and McGeer, E. G. (1996). Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer's disease: a review of 17 epidemiologic studies. Neurology 47,425-432. McKhann, G., Drachman, D., Folstein, M . , Katzman, R., Price, D., and Stadlan, E. M . (1984). Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 34, 939-944. McLean, C. A., Cherny, R. A., Fraser, F. W., Fuller, S. J., Smith, M . J., Beyreuther, K., Bush, A. I., and Masters, C. L. (1999). Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease. Ann Neurol 46, 860-866. Mecocci, P. (2004). Oxidative stress in mild cognitive impairment and Alzheimer disease: a continuum. J Alzheimers Dis 6, 159-163. Merchant, C , Tang, M . X., Albert, S., Manly, J., Stern, Y., and Mayeux, R. (1999). The influence of smoking on the risk of Alzheimer's disease. Neurology 52, 1408-1412. Meziane, H. , Dodart, J. C , Mathis, C , Little, S., Clemens, J., Paul, S. M . , and Ungerer, A. (1998). Memory-enhancing effects of secreted forms of the beta-amyloid precursor protein in normal and amnestic mice. Proc Natl Acad Sci U S A 95, 12683-12688. 83 Mhatre, M . , Floyd, R. A., and Hensley, K. (2004). Oxidative stress and neuroinflammation in Alzheimer's disease and amyotrophic lateral sclerosis: common links and potential therapeutic targets. J Alzheimers Dis 6, 147-157. Miech, R. A., Breitner, J. C , Zandi, P. P., Khachaturian, A. S., Anthony, J. C , and Mayer, L. (2002). Incidence of A D may decline in the early 90s for men, later for women: The Cache County study. Neurology 58, 209-218. Mielke, M . M . , Zandi, P. P., Sjogren, M . , Gustafson, D., Ostling, S., Steen, B., and Skoog, I. (2005). High total cholesterol levels in late life associated with a reduced risk of dementia. Neurology 64,1689-1695. Miller, B. C , Eckman, E. A., Sambamurti, K., Dobbs, N., Chow, K. M . , Eckman, C. B., Hersh, L. B., and Thiele, D. L. (2003). Amyloid-beta peptide levels in brain are inversely correlated with insulysin activity levels in vivo. Proc Natl Acad Sci U S A 100, 6221-6226. Moechars, D., Dewachter, I., Lorent, K., Reverse, D., Baekelandt, V. , Naidu, A., Tesseur, I., Spittaels, K., Haute, C. V., Checler, F., et al. (1999). Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain. J Biol Chem 274, 6483-6492. Monsonego, A., and Weiner, H. L. (2003). Immunotherapeutic approaches to Alzheimer's disease. Science 302, 834-838. Monsonego, A., Zota, V. , Karni, A., Krieger, J. I., Bar-Or, A., Bitan, G., Budson, A. E. , Sperling, R., Selkoe, D. J., and Weiner, H. L. (2003). Increased T cell reactivity to amyloid beta protein in older humans and patients with Alzheimer disease. J Clin Invest 772,415-422. Morris, M . S. (2003). Homocysteine and Alzheimer's disease. Lancet Neurol 2,425-428. Mortimer, J. A. , van Duijn, C. M . , Chandra, V. , Fratiglioni, L. , Graves, A. B., Heyman, A., Jorm, A. F., Kokmen, E. , Kondo, K., Rocca, W. A., and et al. (1991). Head trauma as a risk factor for Alzheimer's disease: a collaborative re-analysis of case-control studies. E U R O D E M Risk Factors Research Group. Int J Epidemiol 20 Suppl 2, S28-35. Moss, M . L. , Jin, S. L. , Milla, M . E. , Bickett, D. M . , Burkhart, W., Carter, H. L. , Chen, W. J., Clay, W. C , Didsbury, J. R., Hassler, D., et al. (1997). Cloning of a disintegrin 84 metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 385, 733-736. Mucke, L. , Masliah, E. , Yu, G. Q., Mallory, M . , Rockenstein, E. M . , Tatsuno, G., Hu, K., Kholodenko, D., Johnson-Wood, K., and McConlogue, L. (2000). High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation. J Neurosci 20,4050-4058. Mullan, M . , Crawford, F., Axelman, K., Houlden, H., Lilius, L. , Winblad, B., and Lannfelt, L. (1992). A pathogenic mutation for probable Alzheimer's disease in the APP gene at the N-terminus of beta-amyloid. Nat Genet 1, 345-347. Mulnard, R. A., Cotman, C. W., Kawas, C , van Dyck, C. H. , Sano, M . , Doody, R., Koss, E. , Pfeiffer, E . , Jin, S., Gamst, A., et al. (2000). Estrogen replacement therapy for treatment of mild to moderate Alzheimer disease: a randomized controlled trial. Alzheimer's Disease Cooperative Study. Jama 283, 1007-1015. Murphy, T., Yip, A., Brayne, C , Easton, D., Evans, J. G., Xuereb, J., Cairns, N., Esiri, M . M . , and Rubinsztein, D. C. (2001). The B A C E gene: genomic structure and candidate gene study in late-onset Alzheimer's disease. Neuroreport 12, 631-634. Nakai, T., Yamasaki, A., Sakaguchi, M . , Kosaka, K., Mihara, K., Amaya, Y., and Miura, S. (1999). Membrane topology of Alzheimer's disease-related presenilin 1. Evidence for the existence of a molecular species with a seven membrane-spanning and one membrane-embedded structure. J Biol Chem 274, 23647-23658. Nelson, W. J., and Nusse, R. (2004). Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303,1483-1487. Newcomer, J. W., Craft, S., Askins, K., Hershey, T., Bardgett, M . E. , Csernansky, J. G., Gagliardi, A. E. , and Vogler, G. (1998). Glucocorticoid interactions with memory function in schizophrenia. Psychoneuroendocrinology 23, 65-72. Newcomer, J. W., Selke, G., Melson, A. K., Hershey, T., Craft, S., Richards, K., and Alderson, A. L. (1999). Decreased memory performance in healthy humans induced by stress-level Cortisol treatment. Arch Gen Psychiatry 56, 527-533. Nicoll, J. A., Wilkinson, D., Holmes, C , Steart, P., Markham, H. , and Weller, R. O. (2003). Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med 9, 448-452. 85 Niimura, M . , Isoo, N., Takasugi, N., Tsuruoka, M . , Ui-Tei, K., Saigo, K., Morohashi, Y., Tomita, T., and Iwatsubo, T. (2005). Aph-1 contributes to the stabilization and trafficking of the gamma-secretase complex through mechanisms involving intermolecular and intramolecular interactions. J Biol Chem 280, 12967-12975. Nitsch, R. M . , Slack, B. E. , Wurtman, R. J., and Growdon, J. H. (1992). Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors. Science 258, 304-307. Nukina, N., and Ihara, Y. (1986). One of the antigenic determinants of paired helical filaments is related to tau protein. J Biochem (Tokyo) 99, 1541-1544. Nussbaum, R. L. , and Ellis, C. E. (2003). Alzheimer's disease and Parkinson's disease. N Engl J Med 348, 1356-1364. Ohno, M . , Sametsky, E. A., Younkin, L. H. , Oakley, H. , Younkin, S. G., Citron, M . , Vassar, R., and Disterhoft, J. F. (2004). BACE1 Deficiency Rescues Memory Deficits and Cholinergic Dysfunction in a Mouse Model of Alzheimer's Disease. Neuron 41, 21-33. Olin, J. S., L; Novit, A; Luczak, S (2004). Hydergine for dementia (The Cochrane Database of Systematic Reviews). Orgogozo, J. M . , Gilman, S., Dartigues, J. F., Laurent, B., Puel, M . , Kirby, L. C , Jouanny, P., Dubois, B., Eisner, L. , Flitman, S., etal. (2003). Subacute meningoencephalitis in a subset of patients with A D after Abeta42 immunization. Neurology 61, 46-54. Ostbye, T., and Crosse, E. (1994). Net economic costs of dementia in Canada. Cmaj 757, 1457-1464. Ostbye, T., Steenhuis, R., Wolfson, C , Walton, R., and Hill, G. (1999). Predictors of five-year mortality in older Canadians: the Canadian Study of Health and Aging. J Am Geriatr Soc 47, 1249-1254. Ott, A., Breteler, M . M . , van Harskamp, F., Claus, J. J., van der Cammen, T. J., Grobbee, D. E . , and Hofman, A. (1995). Prevalence of Alzheimer's disease and vascular dementia: association with education. The Rotterdam study. Bmj 310, 970-973. 86 Ott, A., Slooter, A. J., Hofman, A., van Harskamp, F., Witteman, J. C , Van Broeckhoven, C , van Duijn, C. M . , and Breteler, M . M . (1998). Smoking and risk of dementia and Alzheimer's disease in a population-based cohort study: the Rotterdam Study. Lancet 351, 1840-1843. Ownby, R. L. , Crocco, E . , Acevedo, A., John, V., and Loewenstein, D. (2006). Depression and risk for Alzheimer disease: systematic review, meta-analysis, and metaregression analysis. Arch Gen Psychiatry 63, 530-538. Pastorino, L. , Ikin, A. F., Lamprianou, S., Vacaresse, N., Revelli, J. P., Piatt, K., Paganetti, P., Mathews, P. M . , Harroch, S., and Buxbaum, J. D. (2004). B A C E (beta-secretase) modulates the processing of APLP2 in vivo. Mol Cell Neurosci 25, 642-649. Pedersen, N. L. , Gatz, M . , Berg, S., and Johansson, B. (2004). How heritable is Alzheimer's disease late in life? Findings from Swedish twins. Ann Neurol 55, 180-185. Perez, R. G., Soriano, S., Hayes, J. D., Ostaszewski, B., Xia, W., Selkoe, D. J., Chen, X., Stokin, G. B., and Koo, E. H. (1999). Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42. J Biol Chem 274, 18851-18856. Pericak-Vance, M . A., Bebout, J. L . , Gaskell, P. C , Jr., Yamaoka, L. H. , Hung, W. Y., Alberts, M . J., Walker, A. P., Bartlett, R. J., Haynes, C. A., Welsh, K. A., and et al. (1991). Linkage studies in familial Alzheimer disease: evidence for chromosome 19 linkage. Am J Hum Genet 48, 1034-1050. Perkins, P., Annegers, J. F., Doody, R. S., Cooke, N., Aday, L. , and Vernon, S. W. (1997). Incidence and prevalence of dementia in a multiethnic cohort of municipal retirees. Neurology 49, 44-50. Perusini, G. (1910). Uber klinisch und histologisch eigenartige psychische Erkrankungen des spateren Lebensalters. Histologische und Histopathologische Arbeiten 7/7,297-351. Petrovitch, H. , White, L. R., Izmirilian, G., Ross, G. W., Havlik, R. J., Markesbery, W., Nelson, J., Davis, D. G., Hardman, J., Foley, D. J., and Launer, L. J. (2000). Midlife blood pressure and neuritic plaques, neurofibrillary tangles, and brain weight at death: the HAAS. Honolulu-Asia aging Study. Neurobiol Aging 27, 57-62. 87 Phiel, C. J., Wilson, C. A., Lee, V. M . , and Klein, P. S. (2003). GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides. Nature 423, 435-439. Pomara, N., Greenberg, W. M . , Branford, M . D., and Doraiswamy, P. M . (2003). Therapeutic implications of HPA axis abnormalities in Alzheimer's disease: review and update. Psychopharmacol Bull 37, 120-134. Postina, R., Schroeder, A., Dewachter, I., Bohl, J., Schmitt, U., Kojro, E . , Prinzen, C., Endres, K., Hiemke, C., Blessing, M. , et al. (2004). A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Invest 113, 1456-1464. Price, J. L. (1997). Diagnostic criteria for Alzheimer's disease. Neurobiol Aging 18, S67-70. Prokop, S., Shirotani, K., Edbauer, D., Haass, C , and Steiner, H. (2004). Requirement of PEN-2 for stabilization of the presenilin N-/C-terminal fragment heterodimer within the gamma-secretase complex. J Biol Chem 279, 23255-23261. Qing, H. , Zhou, W., Christensen, M . A., Sun, X., Tong, Y., and Song, W. (2004). Degradation of B A C E by the ubiquitin-proteasome pathway. Faseb J 18,1571-1573. Rademakers, R., Dermaut, B., Peeters, K., Cruts, M . , Heutink, P., Goate, A., and Van Broeckhoven, C. (2003). Tau (MAPT) mutation Arg406Trp presenting clinically with Alzheimer disease does not share a common founder in Western Europe. Hum Mutat 22, 409-411. Raichle, M . E . (2001). Cognitive neuroscience. Bold insights. Nature 412,128-130. Rail, S. C , Jr., Weisgraber, K. H. , Innerarity, T. L. , and Mahley, R. W. (1982). Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects. Proc Natl Acad Sci U S A 79, 4696-4700. Raux, G., Guyant-Marechal, L. , Martin, C , Bou, J., Penet, C , Brice, A., Hannequin, D., Frebourg, T., and Campion, D. (2005). Molecular diagnosis of autosomal dominant early onset Alzheimer's disease: an update. J Med Genet 42, 793-795. 88 Ray, W. J., Yao, M . , Mumm, J., Schroeter, E. H., Saftig, P., Wolfe, M . , Selkoe, D. J., Kopan, R., and Goate, A. M . (1999). Cell surface presenilin-1 participates in the garnma-secretase-like proteolysis of Notch. J Biol Chem 274, 36801-36807. Reagan, L. P., and McEwen, B. S. (1997). Controversies surrounding glucocorticoid-mediated cell death in the hippocampus. J Chem Neuroanat 13, 149-167. Reines, S. A., Block, G. A., Morris, J. C , Liu, G., Nessly, M . L. , Lines, C. R., Norman, B. A., and Baranak, C. C. (2004). Rofecoxib: no effect on Alzheimer's disease in a 1-year, randomized, blinded, controlled study. Neurology 62, 66-71. Reisberg, B., Doody, R., Stoffler, A., Schmitt, F., Ferris, S., and Mobius, H. J. (2003). Memantine in moderate-to-severe Alzheimer's disease. N Engl J Med 348, 1333-1341. Reisberg, B., Doody, R., Stoffler, A., Schmitt, E , Ferris, S., and Mobius, H. J. (2006). A 24-week open-label extension study of memantine in moderate to severe Alzheimer disease. Arch Neurol 63, 49-54. Reitz, C , Tang, M . X., Luchsinger, J., and Mayeux, R. (2004). Relation of plasma lipids to Alzheimer disease and vascular dementia. Arch Neurol 61, 705-714. Richards, J. G., Higgins, G. A., Ouagazzal, A. M . , Ozmen, L. , Kew, J. N., Bohrmann, B., Malherbe, P., Brockhaus, M . , Loetscher, H., Czech, G , et al. (2003). PS2APP transgenic mice, coexpressing hPS2mut and hAPPswe, show age-related cognitive deficits associated with discrete brain amyloid deposition and inflammation. J Neurosci 23, 8989-9003. Robakis, N. K., Ramakrishna, N., Wolfe, G., and Wisniewski, H. M . (1987). Molecular cloning and characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid peptides. Proc Natl Acad Sci U S A 84, 4190-4194. Roberds, S. L. , Anderson, J., Basi, G., Bienkowski, M . J., Branstetter, D. G., Chen, K. S., Freedman, S. B., Frigon, N. L. , Games, D., Hu, K., et al. (2001). B A C E knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet 10, 1317-1324. Roberts, G. W., Gentleman, S. M . , Lynch, A., Murray, L. , Landon, M . , and Graham, D. I. (1994). Beta amyloid protein deposition in the brain after severe head injury: implications for the pathogenesis of Alzheimer's disease. J Neurol Neurosurg Psychiatry 57, 419-425. 89 Roher, A. E. , Esh, C , Kokjohn, T. A., Kalback, W., Luehrs, D. C , Seward, J. D., Sue, L. I., and Beach, T. G. (2003). Circle of willis atherosclerosis is a risk factor for sporadic Alzheimer's disease. Arterioscler Thromb Vase Biol 23, 2055-2062. Romas, S. N., Tang, M . X., Berglund, L. , and Mayeux, R. (1999). APOE genotype, plasma lipids, lipoproteins, and AD in community elderly. Neurology 53, 517-521. Rondeau, V. , Commenges, D., Jacqmin-Gadda, H. , and Dartigues, J. F. (2000). Relation between aluminum concentrations in drinking water and Alzheimer's disease: an 8-year follow-up study. Am J Epidemiol 152, 59-66. Rossouw, J. E . , Anderson, G. L. , Prentice, R. L. , LaCroix, A. Z., Kooperberg, C , Stefanick, M . L. , Jackson, R. D., Beresford, S. A., Howard, B. V., Johnson, K. C , et al. (2002). Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial. Jama 288, 321-333. Rovelet-Lecrux, A., Hannequin, D., Raux, G., Le Meur, N., Laquerriere, A., Vital, A., Dumanchin, C , Feuillette, S., Brice, A., Vercelletto, M. , et al. (2006). APP locus duplication causes autosomal dominant early-onset Alzheimer disease with cerebral amyloid angiopathy. Nat Genet 38, 24-26. Ruitenberg, A., Skoog, I., Ott, A., Aevarsson, O., Witteman, J. C , Lernfelt, B., van Harskamp, F., Hofman, A., and Breteler, M . M . (2001). Blood pressure and risk of dementia: results from the Rotterdam study and the Gothenburg H-70 Study. Dement Geriatr Cogn Disord 12, 33-39. Sadock, B. J. S., Virginia A., ed. (2004). Kaplan & Sadock's Comprehensive Textbook of Psychiatry (Lippincott Williams & Wilkins). Saito, T., Iwata, N., Tsubuki, S., Takaki, Y., Takano, J., Huang, S. M . , Suemoto, T., Higuchi, M . , and Saido, T. C. (2005). Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation. Nat Med 11, 434-439. Sano, M . , Ernesto, C , Thomas, R. G., Klauber, M . R., Schafer, K., Grundman, M . , Woodbury, P., Growdon, J., Cotman, C. W., Pfeiffer, E., et al. (1997). A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer's disease. The Alzheimer's Disease Cooperative Study. N Engl J Med 336, 1216-1222. 90 Santacruz, K., Lewis, J., Spires, T., Paulson, J., Kotilinek, L. , Ingelsson, M . , Guimaraes, A., DeTure, M . , Ramsden, M . , McGowan, E., et al. (2005). Tau suppression in a neurodegenerative mouse model improves memory function. Science 309,476-481. Saunders, A. M . , Strittmatter, W. J., Schrnechel, D., George-Hyslop, P. H. , Pericak-Vance, M . A., Joo, S. H., Rosi, B. L. , Gusella, J. F., Crapper-MacLachlan, D. R., Alberts, M . J., and et al. (1993). Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer's disease. Neurology 43, 1467-1472. Saunders, P. A., Copeland, J. R., Dewey, M . E. , Davidson, I. A., McWilliam, C., Sharma, V., and Sullivan, C. (1991). Heavy drinking as a risk factor for depression and dementia in elderly men. Findings from the Liverpool longitudinal community study. Br J Psychiatry 759, 213-216. Schellenberg, G. D., Bird, T. D., Wijsman, E. M . , Orr, H. T., Anderson, L. , Nemens, E. , White, J. A., Bonnycastle, L. , Weber, J. L. , Alonso, M . E. , and et al. (1992). Genetic linkage evidence for a familial Alzheimer's disease locus on chromosome 14. Science 258,668-671. Scheuner, D., Eckman, C., Jensen, M . , Song, X., Citron, M . , Suzuki, N., Bird, T. D., Hardy, J., Hutton, M . , Kukull, W., et al. (1996). Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease. Nat Med 2, 864-870. Schrnechel, A., Strauss, M . , Schlicksupp, A., Pipkorn, R., Haass, C , Bayer, T. A., and Multhaup, G. (2004). B A C E forms dimers and colocalizes with APP. J Biol Chem. Schneider, J. A., Wilson, R. S., Cochran, E. J., Bienias, J. L. , Arnold, S. E . , Evans, D. A., and Bennett, D. A. (2003). Relation of cerebral infarctions to dementia and cognitive function in older persons. Neurology 60, 1082-1088. Scholz, W. (1938). Studien zur Pathologie der Hirngefasse, II: die drusige Entartung der Hirnarterien und Kapillaren. Z Gesamte Nurol Psychiatr (Berlin) 162, 694-715. Selhub, J., Jacques, P. F., Bostom, A. G., D'Agostino, R. B., Wilson, P. W., Belanger, A. J., O'Leary, D. H. , Wolf, P. A., Schaefer, E. J., and Rosenberg, I. H. (1995). Association between plasma homocysteine concentrations and extracranial carotid-artery stenosis. N Engl J Med 332, 286-291. 91 Selkoe, D. J. (2001a). Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 81, 741-766. Selkoe, D. J. (2001b). Clearing the brain's amyloid cobwebs. Neuron 32, 177-180. Senior, K. (2002). Dosing in phase II trial of Alzheimer's vaccine suspended. Lancet Neurol 1,3. Serban, G., Kouchi, Z., Baki, L . , Georgakopoulos, A., Litterst, C. M . , Shioi, J., and Robakis, N. K. (2005). Cadherins mediate both the association between PS1 and beta-catenin and the effects of PS1 on beta-catenin stability. J Biol Chem 280, 36007-36012. Sergeant, N., Delacourte, A., and Buee, L. (2005). Tau protein as a differential biomarker of tauopathies. Biochim Biophys Acta 1739, 179-197. Seshadri, S., Beiser, A., Selhub, J., Jacques, P. F., Rosenberg, I. H. , D'Agostino, R. B., Wilson, P. W., and Wolf, P. A. (2002). Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med 346, 476-483. Seshadri, S., Wolf, P. A., Beiser, A., Au, R., McNulty, K., White, R., and D'Agostino, R. B. (1997). Lifetime risk of dementia and Alzheimer's disease. The impact of mortality on risk estimates in the Framingham Study. Neurology 49, 1498-1504. Shaywitz, B. A., and Shaywitz, S. E . (2000). Estrogen and Alzheimer disease: plausible theory, negative clinical trial. Jama 283, 1055-1056. Shepherd, J., Blauw, G. J., Murphy, M . B., Bollen, E. L . , Buckley, B. M . , Cobbe, S. M . , Ford, I., Gaw, A., Hyland, M . , Jukema, J. W., et al. (2002). Pravastatin in elderly individuals at risk of vascular disease (PROSPER): a randomised controlled trial. Lancet 360,1623-1630. Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G., Dceda, M . , Chi, H. , Lin, C , Li , G., Holman, K., and et al. (1995). Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature 375, 754-760. Shibata, M . , Yamada, S., Kumar, S. R., Calero, M . , Bading, J., Frangione, B., Holtzman, D. M . , Miller, C. A., Strickland, D. K., Ghiso, J., and Zlokovic, B. V. (2000). Clearance of Alzheimer's amyloid-ss(l-40) peptide from brain by L D L receptor-related protein-1 at the blood-brain barrier. J Clin Invest 106, 1489-1499. 92 Shumaker, S. A., Legault, C , Kuller, L. , Rapp, S. R., Thai, L. , Lane, D. S., Fillit, H. , Stefanick, M . L. , Hendrix, S. L. , Lewis, C. E., et al. (2004). Conjugated equine estrogens and incidence of probable dementia and mild cognitive impairment in postmenopausal women: Women's Health Initiative Memory Study. Jama 291, 2947-2958. Silverberg, G. D., Mayo, M . , Saul, T., Rubenstein, E. , and McGuire, D. (2003). Alzheimer's disease, normal-pressure hydrocephalus, and senescent changes in CSF circulatory physiology: a hypothesis. Lancet Neurol 2, 506-511. Siman, R., Reaume, A. G., Savage, M . J., Trusko, S., Lin, Y. G., Scott, R. W., and Flood, D. G. (2000). Presenilin-1 P264L knock-in mutation: differential effects on abeta production, amyloid deposition, and neuronal vulnerability. J Neurosci 20, 8717-8726. Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., Doan, M . , Dovey, H. F., Frigon, N., Hong, J., et al. (1999). Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402, 537-540. Sisodia, S. S. (1992). Beta-amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci U S A 89, 6075-6079. Skoog, I., and Gustafson, D. (2002). Hypertension and related factors in the etiology of Alzheimer's disease. Ann N Y Acad Sci 977, 29-36. Skoog, I., and Gustafson, D. (2003). Hypertension, hypertension-clustering factors and Alzheimer's disease. Neurol Res 25, 675-680. Skoog, I., Lernfelt, B., Landahl, S., Palmertz, B., Andreasson, L. A., Nilsson, L. , Persson, G., Oden, A., and Svanborg, A. (1996). 15-year longitudinal study of blood pressure and dementia. Lancet 347, 1141-1145. Slooter, A. J., Cruts, M . , Kalmijn, S., Hofman, A., Breteler, M . M . , Van Broeckhoven, C , and van Duijn, C. M . (1998). Risk estimates of dementia by apolipoprotein E genotypes from a population-based incidence study: the Rotterdam Study. Arch Neurol 55, 964-968. Snowdon, D. A., Greiner, L. H. , Mortimer, J. A., Riley, K. P., Greiner, P. A., and Markesbery, W. R. (1997). Brain infarction and the clinical expression of Alzheimer disease. The Nun Study. Jama 277, 813-817. 93 Solfrizzi, V. , Panza, F., and Capurso, A. (2003). The role of diet in cognitive decline. J Neural Transm 110, 95-110. Song, W., Nadeau, P., Yuan, M . , Yang, X., Shen, J., and Yankner, B. A. (1999). Proteolytic release and nuclear translocation of Notch-1 are induced by presenilin-1 and impaired by pathogenic presenilin-1 mutations. Proc Natl Acad Sci U S A 96, 6959-6963. Soriano, S., Lu, D. C , Chandra, S., Pietrzik, C. U. , and Koo, E. H. (2001). The amyloidogenic pathway of amyloid precursor protein (APP) is independent of its cleavage by caspases. J Biol Chem 276, 29045-29050. Sparks, D. L. , Hunsaker, J. C , 3rd, Scheff, S. W., Kryscio, R. J., Henson, J. L. , and Markesbery, W. R. (1990). Cortical senile plaques in coronary artery disease, aging and Alzheimer's disease. Neurobiol Aging 11, 601-607. Sparks, D. L. , Scheff, S. W., Liu, FL, Landers, T. M . , Coyne, C. M . , and Hunsaker, J. C , 3rd (1995). Increased incidence of neurofibrillary tangles (NFT) in non-demented individuals with hypertension. J Neurol Sci 131, 162-169. Steiner, H., Duff, K., Capell, A., Romig, H. , Grim, M . G., Lincoln, S., Hardy, J., Yu, X., Picciano, M . , Fechteler, K., et al. (1999). A loss of function mutation of presenilin-2 interferes with amyloid beta-peptide production and notch signaling. J Biol Chem 274, 28669-28673. Steiner, H. , Kostka, M . , Romig, H. , Basset, G., Pesold, B., Hardy, J., Capell, A., Meyn, L. , Grim, M . L. , Baumeister, R., et al. (2000). Glycine 384 is required for presenilin-1 function and is conserved in bacterial polytopic aspartyl proteases. Nat Cell Biol 2, 848-851. Stern, Y., Gurland, B., Tatemichi, T. K., Tang, M . X., Wilder, D., and Mayeux, R. (1994). Influence of education and occupation on the incidence of Alzheimer's disease. Jama 271, 1004-1010. Strittmatter, W. J., Saunders, A. M . , Schrnechel, D., Pericak-Vance, M . , Enghild, J., Salvesen, G. S., and Roses, A. D. (1993a). Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci U S A 90, 1977-1981. Strittmatter, W. J., Weisgraber, K. H., Huang, D. Y., Dong, L. M . , Salvesen, G. S., Pericak-Vance, M . , Schrnechel, D., Saunders, A, M . , Goldgaber, D., and Roses, A. D. 94 (1993b). Binding of human apolipoprotein E to synthetic amyloid beta peptide: isoform-specific effects and implications for late-onset Alzheimer disease. Proc Natl Acad Sci U S A 90, 8098-8102. Struhl, G., and Greenwald, I. (1999). Presenilin is required for activity and nuclear access of Notch in Drosophila. Nature 398, 522-525. Sturchler-Pierrat, C., Abramowski, D., Duke, M . , Wiederhold, K. H. , Misd, C., Rothacher, S., Ledermann, B., Burki, K., Frey, P., Paganetti, P. A., et al. (1997). Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. Proc Natl Acad Sci U S A 94, 13287-13292. Sung, S., Yao, Y., Uryu, K., Yang, H. , Lee, V. M . , Trojanowski, J. Q., and Pratico, D. (2004). Early vitamin E supplementation in young but not aged mice reduces Abeta levels and amyloid deposition in a transgenic model of Alzheimer's disease. Faseb J 18, 323-325. Suzuki, N., Cheung, T. T., Cai, X. D., Odaka, A., Otvos, L. , Jr., Eckman, C , Golde, T. E. , and Younkin, S. G. (1994). An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants. Science 264, 1336-1340. Tabet, N. B., J; Grimley Evans, J; Orrel, M ; Spector, A (2005). Vitamin E for Alzheimer's disease (The Cochrane Database of Systematic Reviews). Takashima, A., Murayama, M . , Murayama, O., Kohno, T., Honda, T., Yasutake, K., Nihonmatsu, N., Mercken, M . , Yamaguchi, H., Sugihara, S., and Wolozin, B. (1998). Presenilin 1 associates with glycogen synthase kinase-3beta and its substrate tau. Proc Natl Acad Sci U S A 95, 9637-9641. Takasugi, N., Tomita, T., Hayashi, I., Tsuruoka, M . , Niimura, M . , Takahashi, Y., Thinakaran, G., and Iwatsubo, T. (2003). The role of presenilin cofactors in the gamma-secretase complex. Nature 422, 438-441. Tan, Z. S., Seshadri, S., Beiser, A., Wilson, P. W., Kiel, D. P., Tocco, M . , D'Agostino, R. B., and Wolf, P. A. (2003). Plasma total cholesterol level as a risk factor for Alzheimer disease: the Framingham Study. Arch Intern Med 163, 1053-1057. 95 Tanahashi, H. , and Tabira, T. (2001). Three novel alternatively spliced isoforms of the human beta-site amyloid precursor protein cleaving enzyme (B ACE) and their effect on amyloid beta-peptide production. Neurosci Lett 307, 9-12. Tanzi, R. E. , and Bertram, L. (2005). Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective. Cell 120, 545-555. Tanzi, R. E. , Gusella, J. E , Watkins, P. C , Bruns, G. A., St George-Hyslop, P., Van Keuren, M . L. , Patterson, D., Pagan, S., Kurnit, D. M . , and Neve, R. L. (1987). Amyloid beta protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus. Science 235, 880-884. Taubes, G. (2003). Neuroscience. Insulin insults may spur Alzheimer's disease. Science 301,40-41. Terry, R. (1963). The fine structure of neurofibrillary tangles in Alzheimer's disease. J Neuropathol Exp Neurol 22, 629-641. Thai, L. J., Ferris, S. H. , Kirby, L. , Block, G. A., Lines, C. R., Yuen, E. , Assaid, C , Nessly, M . L. , Norman, B. A., Baranak, C. C , and Reines, S. A. (2005). A randomized, double-blind, study of rofecoxib in patients with mild cognitive impairment. Neuropsychopharmacology 30, 1204-1215. Thinakaran, G., Borchelt, D. R., Lee, M . K., Slunt, H. H. , Spitzer, L. , Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C , Seeger, M. , et al. (1996). Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo. Neuron 17, 181-190. Tierney, L. M . M . , Stephen J.; Papadakis, Maxine A., ed. (2006). Current Medical Diagnosis & Treatment, 45th Edition edn (McGraw-Hill). Tong, Y., Zhou, W., Fung, V., Christensen, M . A., Qing, H. , Sun, X., and Song, W. (2005). Oxidative stress potentiates BACE1 gene expression and Abeta generation. J Neural Transm 772, 455-469. Turner, P. R., O'Connor, K., Tate, W. P., and Abraham, W. C. (2003). Roles of amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory. Prog Neurobiol 70, 1-32. 96 Van Broeckhoven, C , Haan, J., Bakker, E. , Hardy, J. A., Van Hul, W., Wehnert, A., Vegter-Van der Vlis, M . , and Roos, R. A. (1990). Amyloid beta protein precursor gene and hereditary cerebral hemorrhage with amyloidosis (Dutch). Science 248,1120-1122. Van Dam, D., D'Hooge, R., Staufenbiel, M . , Van Ginneken, C., Van Meir, E , and De Deyn, P. P. (2003). Age-dependent cognitive decline in the APP23 model precedes amyloid deposition. Eur J Neurosci 77, 388-396. van Dijk, P. T., Dippel, D. W., and Habbema, J. D. (1991). Survival of patients with dementia. J Am Geriatr Soc 39, 603-610. van Duijn, C. M . , and Hofman, A. (1991). Relation between nicotine intake and Alzheimer's disease. Bmj 302, 1491-1494. van Duijn, C. M . , Tanja, T. A., Haaxma, R., Schulte, W., Saan, R. J., Lameris, A. J., Antonides-Hendriks, G., and Hofman, A. (1992). Head trauma and the risk of Alzheimer's disease. Am J Epidemiol 735, 775-782. van Duinen, S. G., Castano, E. M . , Prelli, E , Bots, G. T., Luyendijk, W., and Frangione, B. (1987). Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer disease. Proc Natl Acad Sci U S A 84, 5991-5994. Van Nostrand, W. E. , Melchor, J. P., Cho, H. S., Greenberg, S. M . , and Rebeck, G. W. (2001) . Pathogenic effects of D23N Iowa mutant amyloid beta -protein. J Biol Chem 276, 32860-32866. Van Nostrand, W. E. , Wagner, S. L. , Suzuki, M . , Choi, B. H., Farrow, J. S., Geddes, J. W., Cotman, C. W., and Cunningham, D. D. (1989). Protease nexin-II, a potent antichymotrypsin, shows identity to amyloid beta-protein precursor. Nature 341, 546-549. Van Uden, E. , Mallory, M . , Veinbergs, I., Alford, M . , Rockenstein, E. , and Masliah, E . (2002) . Increased extracellular amyloid deposition and neurodegeneration in human amyloid precursor protein transgenic mice deficient in receptor-associated protein. J Neurosci 22, 9298-9304. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A. , Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., et al. (1999). Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease B A C E . Science 286, 735-741. 97 Vehmas, A. K., Borchelt, D. R., Price, D. L. , McCarthy, D., Wills-Karp, M . , Peper, M . J., Rudow, G., Luyinbazi, J., Siew, L. T., and Troncoso, J. C. (2001). beta-Amyloid peptide vaccination results in marked changes in serum and brain Abeta levels in APPswe/PSlDeltaE9 mice, as detected by SELDI-TOF-based ProteinChip technology. DNA Cell Biol 20, 713-721. Vekrellis, K., Ye, Z., Qiu, W. Q., Walsh, D., Hartley, D., Chesneau, V., Rosner, M . R., and Selkoe, D. J. (2000). Neurons regulate extracellular levels of amyloid beta-protein via proteolysis by insulin-degrading enzyme. J Neurosci 20, 1657-1665. Verchere, C. B., D'Alessio, D. A., Palmiter, R. D., Weir, G. C , Bonner-Weir, S., Baskin, D. G., and Kahn, S. E . (1996). Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide. Proc Nati Acad Sci U S A 93, 3492-3496. Vermeer, S. E. , Prins, N. D., den Heijer, T., Hofman, A., Koudstaal, P. J., and Breteler, M . M . (2003). Silent brain infarcts and the risk of dementia and cognitive decline. N Engl J Med 348, 1215-1222. von Koch, C. S., Zheng, H. , Chen, H. , Trumbauer, M . , Thinakaran, G., van der Ploeg, L. H. , Price, D. L. , and Sisodia, S. S. (1997). Generation of APLP2 KO mice and early postnatal lethality in APLP2/APP double KO mice. Neurobiol Aging 18, 661-669. Walter, J., Fluhrer, R., Hartung, B., Willem, M . , Kaether, C , Capell, A., Lammich, S., Multhaup, G., and Haass, C. (2001). Phosphorylation regulates intracellular trafficking of beta-secretase. J Biol Chem 276,14634-14641. Wang, H. X., Fratiglioni, L. , Frisoni, G. B., Viitanen, M . , and Winblad, B. (1999). Smoking and the occurrence of Alzheimer's disease: cross-sectional and longitudinal data in a population-based study. Am J Epidemiol 149, 640-644. Wang, P., Yang, G., Mosier, D. R., Chang, P., Zaidi, T., Gong, Y. D., Zhao, N. M . , Dominguez, B., Lee, K. F., Gan, W. B., and Zheng, H. (2005). Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2. J Neurosci 25, 1219-1225. Watanabe, N., Tomita, T., Sato, C , Kitamura, T., Morohashi, Y., and Iwatsubo, T. (2005). Pen-2 is incorporated into the gamma-secretase complex through binding to transmembrane domain 4 of presenilin 1. J Biol Chem 280, 41967-41975. 98 Wavrant-DeVrieze, F., Lambert, J. C , Stas, L. , Crook, R., Cottel, D., Pasquier, F., Frigard, B., Lambrechts, M . , Thiry, E. , Amouyel, P., et al. (1999). Association between coding variability in the LRP gene and the risk of late-onset Alzheimer's disease. Hum Genet 104,432-434. Weggen, S., Eriksen, J. L. , Das, P., Sagi, S. A., Wang, R., Pietrzik, C. U., Findlay, K. A., Smith, T. E. , Murphy, M . P., Bulter, T., et al. (2001). A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature 414, 212-216. Weidemann, A., Eggert, S., Reinhard, F. B., Vogel, M . , Paliga, K., Baier, G., Masters, C. L. , Beyreuther, K., and Evin, G. (2002). A novel epsilon-cleavage within the transmembrane domain of the Alzheimer amyloid precursor protein demonstrates homology with Notch processing. Biochemistry 41, 2825-2835. Weiner, H. L. , Lemere, C. A., Maron, R., Spooner, E. T., Grenfell, T. J., Mori, C , Issazadeh, S., Hancock, W. W., and Selkoe, D. J. (2000). Nasal administration of amyloid-beta peptide decreases cerebral amyloid burden in a mouse model of Alzheimer's disease. Ann Neurol 48, 567-579. Weisgraber, K. H. , Rail, S. C , Jr., and Mahley, R. W. (1981). Human E apoprotein heterogeneity. Cysteine-arginine interchanges in the amino acid sequence of the apo-E isoforms. J Biol Chem 256, 9077-9083. Westmeyer, G. G., Willem, M . , Lichtenthaler, S. F., Lurman, G., Multhaup, G., Assfalg-Machleidt, I., Reiss, K., Saftig, P., and Haass, C. (2004). Dimerization of beta-site beta-amyloid precursor protein-cleaving enzyme. J Biol Chem 279, 53205-53212. White, L. , Petrovitch, H. , Hardman, J., Nelson, J., Davis, D. G., Ross, G. W., Masaki, K., Launer, L. , and Markesbery, W. R. (2002). Cerebrovascular pathology and dementia in autopsied Honolulu-Asia Aging Study participants. Ann N Y Acad Sci 977, 9-23. Whitehouse, P. J., Price, D. L. , Struble, R. G., Clark, A. W., Coyle, J. T., and Delon, M . R. (1982). Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. Science 215, 1237-1239. Wilcock, G. K. (2003). Memantine for the treatment of dementia. Lancet Neurol 2, SOS-SOS. Willem, M . , Dewachter, I., Smyth, N., Van Dooren, T., Borghgraef, P., Haass, C , and Van Leuven, F. (2004). beta-site amyloid precursor protein cleaving enzyme 1 increases 99 amyloid deposition in brain parenchyma but reduces cerebrovascular amyloid angiopathy in aging B A C E x APP[V717I] double-transgenic mice. Am J Pathol 165, 1621-1631. Wisniewski, K. E . , Wisniewski, H. M . , and Wen, G. Y. (1985). Occurrence of neuropathological changes and dementia of Alzheimer's disease in Down's syndrome. Ann Neurol 77,278-282. Wolfe, M . S., Xia, W., Ostaszewski, B. L. , Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999). Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398, 513-517. Wolfson, C., Wolfson, D. B., Asgharian, M . , M'Lan, C. E . , Ostbye, T., Rockwood, K., and Hogan, D. B. (2001). A reevaluation of the duration of survival after the onset of dementia. N Engl J Med 344, 1111-1116. Wolozin, B., Kellman, W., Ruosseau, P., Celesia, G. G., and Siegel, G. (2000). Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors. Arch Neurol 57, 1439-1443. Wyss-Coray, T., and Mucke, L. (2002). Inflammation in neurodegenerative disease—a double-edged sword. Neuron 35, 419-432. Yaffe, K., Barrett-Connor, E. , Lin, F., and Grady, D. (2002). Serum lipoprotein levels, statin use, and cognitive function in older women. Arch Neurol 59, 378-384. Yan, R., Bienkowski, M . J., Shuck, M . E . , Miao, H., Tory, M . C , Pauley, A. M . , Brashier, J. R., Stratman, N. C , Mathews, W. R., Buhl, A. E., et al. (1999). Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature 402, 533-537. Yan, S. D., Chen, X., Fu, J., Chen, M . , Zhu, H., Roher, A., Slattery, T., Zhao, L. , Nagashima, M . , Morser, J., et al. (1996). R A G E and amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature 382, 685-691. Ye, Y., Lukinova, N., and Fortini, M . E . (1999). Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants. Nature 398, 525-529. Yu, G., Chen, F., Levesque, G., Nishimura, M . , Zhang, D. M . , Levesque, L. , Rogaeva, E . , Xu, D., Liang, Y. , Duthie, M. , et al. (1998). The presenilin 1 protein is a component of a 100 high molecular weight intracellular complex that contains beta-catenin. J Biol Chem 273, 16470-16475. Yu, G., Nishimura, M . , Arawaka, S., Levitan, D., Zhang, L. , Tandon, A., Song, Y. Q., Rogaeva, E. , Chen, E , Kawarai, T., et al. (2000). Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing. Nature 407, 48-54. Zandi, P. P., Sparks, D. L. , Khachaturian, A. S., Tschanz, J., Norton, M . , Steinberg, M . , Welsh-Bohmer, K. A., and Breitner, J. C. (2005). Do statins reduce risk of incident dementia and Alzheimer disease? The Cache County Study. Arch Gen Psychiatry 62, 217-224. Zhang, Z., Nadeau, P., Song, W., Donoviel, D., Yuan, M . , Bernstein, A., and Yankner, B. A. (2000). Presenilins are required for gamma-secretase cleavage of beta-APP and transmembrane cleavage of Notch-1. Nat Cell Biol 2,463-465. Zhou, J., Liyanage, U., Medina, M . , Ho, C , Simmons, A. D., Lovett, M . , and Kosik, K. S. (1997). Presenilin 1 interaction in the brain with a novel member of the Armadillo family. Neuroreport 8, 2085-2090. Zhou, W., and Song, W. (2006). Leaky Scanning and Reinitiation Regulate BACE1 Gene Expression. Mol Cell Biol 26, 3353-3364. Zlokovic, B. V. (2004). Clearing amyloid through the blood-brain barrier. J Neurochem 89, 807-811. Zohar, O., Cavallaro, S., D'Agata, V., and Alkon, D. L. (2003). Quantification and distribution of beta-secretase alternative splice variants in the rat and human brain. Brain Res Mol Brain Res 115, 63-68. 101 CHAPTER 2: The human BACE2 gene has distinct transcriptional regulation from BACE1 gene 1 A version of this chapter was previously published: Sun X, Wang Y, Qing H, Christensen M A , Liu Y, Zhou W, Tong Y, Xiao C, Huang Y, Zhang S, Liu X , and Song W. (2005) Distinct Transcriptional Regulation and Function of The Human BACE2 and BACE1 Genes. The FASEB Journal 19 (7):739-749. 102 2.1. Introduction 2.1.1. Overview of eukaryotic gene promoters The faithful implementation of biological processes such as development, proliferation, differentiation, apoptosis and aging requires an orchestrated set of genes being precisely expressed in specific time and space. Many diseases are associated with the dysfunction and/or dysregulation of genes. The expression of structural genes can be regulated at several steps, including transcription, translation, and posttranslational modifications, with most of regulation occurring at transcription initiation. In eukaryotes, the transcription of protein coding gene is performed by RNA polymerase II (Pol II) and regulated by two distinct cis-regulatory elements. One is the promoter, which contains the core promoter and proximal regulatory elements. The other is the distal regulatory element including enhancers, silencers, insulators and locus control regions. The transcription initiation starts with the binding of gene-specific regulatory factors near the site of transcription initiation, by directly interacting with components of transcription machinery or indirectly by recruiting factors that modify chromatin structure. The eukaryotic transcriptional machinery contains general transcription factors (GTFs), activator and coactivators. GTFs assemble onto the core promoter to form a preinitiation complex, which directs Pol II to the transcription start site (Figure 2.1). Recognition of the core promoter by transcription machinery is essential for the assembly of the preinitiation complex and subsequent transcription initiation. Eukaryotic core promoters are composed of T A T A box, Initiator element (Inr), Downstream Promoter Element (DPE), Downstream Core Element (DCE), TFIIB recognition element (BRE), and Motif Ten Element (MTE) (Figure 2.2). 103 Figure 2.1. The eukaryotic transcriptional machinery. Factors involved in eukaryotic transcription by RNA polymerase II can be classified into three groups: general transcription factors (GTFs), activators, and coactivators. GTFs, which include RNA polymerase II itself and TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, assemble on the core promoter in an ordered fashion to form a preinitiation complex (PIC), which directs RNA polymerase II to the transcription start site (TSS). (Adapted fromMaston et al., 2006) | TATA TSS | Core promoter Figure 2.2. Core promoter elements. Metazoan core promoters are composed of a number of elements that may include a T A T A box, an Initiator element (Inr), a Downstream Promoter Element (DPE), a Downstream Core Element (DCE), a TFIIB-Recognition Element (BRE), and a Motif Ten Element (MTE). The human consensus sequence of these elements, their relative 104 positions, and the transcription factors that bind them are shown. The DCE is shown on a separate core promoter for illustration purposes only. Although the DCE can be present in promoters containing a T A T A box and/or Inr, it presumably does not occur with a DPE or MTE. (Adapted from Maston et al , 2006). B R E T A T A Inr M T E D P E Consensus Binding factors -37 to -32 -31 to -26 C C A c g c c TATAyAA^ TFIIB TBP -2 to +4 TT , . . T T T C C A N A C C T A F 1 / 2 + 18 to+27 +28 to+32 C C A G C C C A A C G C G G T T A TAF6/9 Consensus Binding factors n D C E +10 to+40 N 5 . 7 [CTTC]N 7 . 8 [CTGT]N 7 . 1 , [AGCJN, . 2 TAF1 2.1.2. Identification of BACE2 gene and its tissue-specific expression By computer-based search for BACE1 homologous gene, BACE2 was cloned as a homolog of BACE1 shortly after BACE1 was identified as the major (3-secretase (Aleister J. Saunders, 1999; Farzan et al., 2000). BACE2 was also independently identified as Down Region Aspartyl protease (DRAP) from DS critical region by genomic cloning (Acquati et al., 2000). BACE2 is also called membrane associated aspartyl protease 1 (ASP1 or memapsin-1) as it was identified together with Asp2 (BACE1) while searching for C. elegans aspartyl proteases orthologues in vertebrates (Yan et al., 1999). BACE2 is mapped to Down Syndrome Critical Region on Chromosome 21 q22.3, a region proposed to be important in DS pathogenesis (Shapiro, 1999). BACE2 gene spans about 109kb and encodes 518 amino acids with 9 exons. 105 BACE2 has several transcripts by alternative splicing, the full length of 518 amino acids, and 396 and 468 amino acids with exon 8 and 7 skipped respectively (Solans et al., 2000). Northern blot and RNA dot blot show that BACE2 has a broad distribution in adult and fetal tissues, with highest levels in heart and pancreas, and clear expression in the whole brain and some brain subregions including hippocampus, medulla oblongata, substantia nigra and spinal cord (Solans et al , 2000). Another report from Bennett (2000) shows that BACE2 mRNA is expressed at low levels in most adult tissues but appears to be higher in colon, kidney, pancreas, placenta, prostate, stomach, and trachea (Bennett et al., 2000a). In contrast to the high expression of BACE1 in neural tissues (Marcinkiewicz and Seidah, 2000; Vassar et al., 1999; Yan et al , 1999), adult whole brain and most brain subregions express very low or undetectable levels of BACE2 mRNA, with the exception of the medulla and spinal cord, which express slightly higher levels(Bennett et al., 2000a). The expression pattern of BACE2 is distinct from the predictions for P-secretase. Our lab previously characterized the BACE1 gene promoter and demonstrated that BACE1 gene transcription is tightly regulated at the transcriptional level. SP1 can upregulate BACE1 gene expression and subsequently regulate APP processing and A|3 production (Qing et al., 2004). There are few studies about the mechanism of BACE2 gene transcription. Understanding the mechanism of gene transcription can provide additional information of BACE2's function in cells under physiological and pathological conditions. In this study, we cloned and functionally characterized the BACE2 gene 106 promoter. BACE1 and BACE2 are homologous proteins. Gene expression is coordinately regulated to achieve an efficient process(Garcia-Martinez et al., 2004; Kosak and Groudine, 2004; Przyborski et al., 2003; Sullivan and Thummel, 2003; Yang et al., 2004). We compared the transcriptional regulation of BACE1 and BACE2 genes. 2.2. Materials and methods All reagents are from Sigma unless otherwise stated. 2.2.1. Cell culture Human neuroblastoma SH-SY5Y cells (ATCC number CRL-2266), human embryonic kidney HEK293 cells (ATCC number CRL-1573), murine neuroblastoma N2A cells (ATCC number CCL-131) and murine glial cell line C6 (ATCC number C C L -107) were grown in Dulbecco's modified Eagle's medium (DMEM, from Invitrogen) containing 10% FBS, ImM of sodium pyruvate, 2mM of L-glutamine and 50 unit of Penicillin and 50 pg of Streptomycin. Media were changed every 2-3 days. All cells were maintained at 37°C in an incubator containing 5% CO2. PC 12 cell line was derived from rat pheochromocytoma and can be induced by NGF for a neuronal phenotype (ATCC number CRL-1721). PC 12 cells were cultured in RPMI media plus 10% FBS, 5% horse serum, 2mM of L-glutamine (GIBCO, 25030-081) and 50 unit of Penicillin and 50 \ig of Streptomycin (GIBCO, 15070-063). The plate was coated with 50ug/ml collagen I (20ul in 35mm plate and dry for overnight). All cells were maintained at 37°C in an incubator containing 5% CO2. 107 SP1 wild type (SP1-WT) and SP1 knockout (SP1-KO) embryonic cells were fibroblast cells derived from SP1-WT and SP1-KO mice. SP1-WT and SP1-KO cells were cultured in D M E M media (containing 415ml D M E M , 75ml FBS, 5ml of sodium pyruvate (ImM), 5ml of L-glutamine (2mM final), 5ml Penicillin and Streptomycin, 25-50ul ESGRO, 4ul RME, 5ml M E M non-Essential Amino Acids Solution). Cells were maintained at 37°C in an incubator containing 5% CO2. 2.2.2. Molecular cloning of BACE2 promoter and construction of chimeric luciferase reporter plasmids General procedures of molecular cloning. 2-5ug of vector plasmid and 5-10ug insert plasmid or PCR product were digested with restriction enzymes (New England Biolabs) in 50ul system for overnight. The vector plasmid can be dephosphorylated with Alkaline phosphatase (CIAP) to prevent self-ligation. Agarose gel electrophoresis was used to separate the desired DNA. 1-2% of agarose gels with 0.5ug/ml Ethidium Bromide were run in lxTBE buffer (10 x TBE: 108 g Trizma, 55 g Boric acid, 40 ml E D T A (0.5 mM/pH 8.0), make up to 1000ml with dH 20) at 110V for 30-40 minutes. The DNA was visualized with U V light and photographed with Kodak Imaging system. Desired DNA was cut out form the agarose gel and isolated with phenol-chlorophorm method. 1/10th volume of 3M NaAC and 2-3 volumes of ethanol were added to the supernatant collected and incubated at -80 degree for 15minutes. The DNA were pelleted with top speed (13,000rpm) for 15 minutes at 4 degree and washed once with 75% or 95% ethanol. DNA was dried and dissolved in 20ul H 2 0 and OD280 was measured to estimate DNA 108 concentration. Optionally another agarose gel can be run to determine the concentration of DNA before ligation. The ligation was usually done with 50-100ng of vector and 100-200ng of insert depending on the size (molar ratio of vectoninsert = 1:3). lu T4 DNA ligase from Invitrogen or NE Biolabs was used in lOul system for cohesive ends ligation at room temperature for lh to overnight. Blunted ends ligation was done with 2-5u ligase at 16 degree for overnight. The ligated product was transformed into 50-100ul DH5a competent cells (from Invitrogen) by reacting on ice for 30 minutes, 41 degree for 1 minute and ice for 5 minutes. The transformation was mixed with 200ul LB and shake for 2 hours at 300rpm and 37 degree and plated onto ampicillin (or kanamycin) supplemented LB agar plates (Add 15 g agar to 25 g LB mix and make up to 1000 ml with dH 2 0. Autoclave and allow to cool to about 60 °C, then add antibiotic to a concentration of 60 ng/ml before making the plates). 6-10 colonies were picked up next day and inoculated into l-2ml LB supplemented with ampicillin or kanamycin (60ng/ml). Minipreparation was done with DNA minipreparation kit from Promega following manufacturer's protocol. The minipreparation was checked with enzyme digestion and confirmed by sequencing. The confirmed plasmid is quickly transformed with lul plasmid into lOul DH5a by on ice 1 minute, 37 degrees 1 minute and ice 1 minute and plates onto the ampicillin (or kanamycin) plate. Next day one colony is picked up and inoculated into 5ml LB supplemented with antibiotics and cultured for 8 hours before diluted into 300-500ml LB (25g LB in 1000ml dH20 and autoclaved for 25 minutes. Antibiotics was added right before use with a concentration of 60ng/ml). The culture was continued for 16 hours at 37 degree and 300rpm before extracting plasmid DNA with Maxipreparation kit from Qiagen following the manufacturer's instructions. 109 Cloning of BACE2 promoter and construction of chimeric luciferase reporter plasmids. Primers were designed to include restriction enzymes sites so that the resulting PCR-amplified fragments could be easily cloned into the multiple cloning site of vector pGL3-basic (Promega). Eight fragments of BACE2 from -1583 upstream to +442 bp downstream of the transcription start site at +1 (adenine) were amplified by PCR and inserted in front of the luciferase reporter gene (Luc) in the pGL3-basic expression vector. Primers used to generate different promoter deletion plasmids include: forward, -446Nhe I (5V-A T A G C T A G C G C A G C C A G A C C C G G C G A C T G - 3 ), -371Nhe I ( 5 -A T A G C T A G C T A G T T C A G G C C C T C G C T G C - 3 ),-200NheI (5 s-A T A G C T A G C G T A T C A G A T G A G C C T C G T C - 3 v ) , -54Nhe I ( 5 -A T A G C T A G C G A G G A A A T T C G G G A C T C G - 3 ), and reverse, +278Hind III ( 5 - A G A A G C T T C G C G C C C A G C C T A G C C G G - 3 ) and +442 Hind HI ( 5 -A T A A G C T T C G G G G T G G G C G C A A C T A C - 3 ). The primers -1582Kpn I ( 5 s-A T G G A T C C C T G C A T C G G T C A C C A T G G T - 3 ) and +442 Mlu I (5-A T A C G C G T C G G G G T G G G C G C A A C T AC-3 S) were used to amplify the -1582 to +442 region to get the longest promoter fragment which was ligated into pGL3-basic at the site Kpn I and Mlu I sites. The BACE2 promoter region and the inserts of the promoter-luciferase plasmids were sequenced by an automatic fluorescence-based DNA sequencer (ASI PRISM DNA analyzer; Applied Biosystems). 110 2.2.3. Primer extension A primer extension assay was performed to determine the transcription initiation site. Total RNA was extracted from HEK293 cells with TRI reagent following the manufacturer's protocol (Sigma). A reverse primer, corresponding to bp +20 to +39 of the 5' untranslated region (UTR), 5^ -G C A A G T T C T T C T C C G C T G C C - 3 ' , was synthesized and radioactively labeled by using [y-32P] ATP (6,000 Ci/mmol, Amersham Biosciences) and T4 polynucleotide kinase (Promega, Inc.). Eighty micrograms of RNA and 20 ul dilution of 32P-labeled primer (10 pmol) were precipitated and hybridized in 30 pi of hybridization buffer (Promega, Inc.) at 30°C for overnight. The hybridized RNA primer samples were precipitated and incubated in 20 pi of 2x reverse transcriptase buffer (10 pi of avian myeloblastosis virus [AMV] primer extension buffer, 1.4 pi of 40 mM sodium pyrophosphate, 6.6 pi of nuclease-free water, 1.0 pi of 1-U/ pi A M V reverse transcriptase, 1.0 pi of RNase I) at 42°C for 40 min. The same radiolabeled primer was also used for DNA sequencing with DNA Sequencing Kit (USB cooperation). The primer extension assay samples were analyzed on 8 % denaturing polyacrylamide gels, and the DNA sequencing sample with the same primer was loaded in the same gel and used as the sequence marker. I l l 2.2.4. Transfection SH-SY5Y cells, HEK293 cells, N2A cells and SP1 wild type and knockout cells can be transfected with Lipofectamine 2000 (Invitrogen). Cells were split one day before transfection and grown to approximately 70% confluence at the time of transfection. Cells were transfected with Lipofectamine 2000 following the manufacturer's instructions. For example, on one 35 mm plate, 4ul LF2000 was added into lOOul OptiMEM (serum-free media) and let sit for 5 minutes. In another tube, 2ug of DNA was added into lOOul OptiMEM media and the LF2000 mixture was then added into DNA mixture. The mixture was allowed to sit at room temperature for 15 minutes before adding into the culture media. The media can be changed with fresh growth media 6 hours after transfection optionally and if the faster growth of cells is required. The cells were usually harvested 48-72 hours after transfection. In case of luciferase assay, 24 hours harvest is satisfactory. To obtain the maximal expression of transfected gene, the media can be changed or the cells can be split to ensure enough nutrients. 2.2.5. Promoter assay The Renilla (sea pansy) luciferase vector pCMVRluc was cotransfected with the promoter constructs to normalize the transfection efficiency (ratio of Firefly .-Renilla luciferase construct=1:200). 24-48 hours after transfection, cells were washed with cold lxPBS once and lysed in 250 pi of lx passive lysis buffer (Promega) for 20 minutes in shaking for 35mm plates. 2ul of lysates were mixed with lOul of firefly luciferase assay reagent II and the luminescent signal was measured by the T D 20/20 luminometer (Turner designs). Then lOul of Stop & 112 Glo Reagent was added to the same tube and the luminescent signal from the Renilla luciferase was measured by the same luminometer. Each sample was measured three times. The firefly luciferase activity was normalized according to Renilla luciferase activity and expressed as relative luciferase units (RLU) to reflect the promoter activity. 2.3. Results 2.3.1. Cloning the human B A C E 2 gene promoter and computer analysis for putative transcription factors The 5' upstream region of BACE2 gene was amplified from a human genomic library. We cloned and sequenced a 2025-bp 5' flanking region of BACE2 gene and the N-terminus of the coding region (Figure 2.3A). The sequence was deposited to TM GenBank under accession number AY769996. Computer-based transcription factor binding site search revealed that this 2.0 kb 5' flanking region contains several putative regulatory elements, such as NF1, SP1, API, AP2, G A T A , OCT1, and USF (Fig 2.3A). The human BACE2 gene promoter lacks typical T A T A and C A A T boxes and has a very high GC content in its proximal region. The G C content of the 600 bp region surrounding the transcription start site is 78%. 2.3.2. Primer extension to identify the transcription start sites The primer extension assay was performed to map the transcription start site of the human BACE2 gene. An antisense primer (5 ' -CAAGTTCTTCTCCGCTGCC) located 262 bp upstream of the translation start codon A T G was used to hybridize with 113 RNA isolated from HEK293 cells. The primer extension assay yielded a ~ 38-bp major cDNA product. DNA sequencing gel analysis indicates that the major transcription start site of the human BACE2 gene is located at 301 bp upstream of the translation start site. This transcription start site begins with adenine and was designated as +1 (Figure 2.3B). Figure 2.3. The human BACE2 gene promoter sequence. (A). The nucleotide sequence of the human BACE2 gene promoter. A 1583 bp fragment of the 5' flanking region and the first exon of the human BACE2 gene was isolated from a human genomic library and sequenced by the primer walking strategy. The adenine +1 represents the major transcription start site. The positions of some of the unique and common restriction enzymes are indicated in italics. The putative transcription factor binding sites are underlined in bold face. The codon of the first exon is also indicated. TM Genbank accession number is AY769996. (B). Primer extension assay. HEK293 RNA 32 was extracted by TRI-Reagent and yeast tRNA was used as a control. A P-labeled reverse primer complementary to +20 to +39 was used for both primer extension and sequencing reaction. Plasmid pB2Luc-B was used as sequencing template. The samples were analyzed by 6% denaturing P A G E gel electrophoresis. * indicates the major transcription start site. 114 A -1583 acc ectcaagact ggacaccaag gag-.ataaat -1550 aatcatttta aaaaaaatca cattccctcc acaaccttga dtc-gaaacj "snx-i -!S0C tgaatatatg aatagaaggg ctctggaata actatgcsga tttatggags -1450 aut iaac t t M g t u t a t c Mgggatcca gctatgcctt tcttcatgja All II USF -UOC qtxqggqatt. tqttaaagtt gagatatag'. ugggggaag tgtgatttat HSF -13SC ggttaaagte atctgaaaat Ctttacstn q&tgatctrt gacataaagg Bgl II -1300 tcccctgcag agctagacqt gatrciaaaa ttgogaaeae a^ gaatadaa SK -1250 at^aaatctt gagtagaagt agetgaaaat tgragtgatt cggggaagxrt Hind III -1200 tggcttctaa ctccccactg rttgaagatg ggcttgtttg ttttrtaaaa HKF-3 -I15C -ajccaacat ia'.teagetjr gaggaggtac aaagaatttt ctattctttg HP-l Pro II -HOC tttctgtaga aatcgatgga ctttagcttg tctaattgtc ccccctgcct -105C ttagtatsta aaataaaata a-cctcgttg cttgcattac tcaa-gca+t -1O0C tctgcgtctt ggcgtctatq gctaaacgag tatiMttag acagtccgca ST-1 T F T -950 gagagctggc tgggga.aga aggggaggtg gqqgaqaagg gcagggatca -90C cagcagcgtg gactcgtggc cctgatttgg gatectgaca gcaactuct -85C aggtggcctg aqggcT.gggt gccaggggag gcagcgggtt ccagtagcat AP-2alpha -800 ctgaectcrca tcaqggacag ggqcgeggcg gaqgqqgcga aggflsgcggq EE SSI -750 9gtgggcsga aggtgqctig ggtgaaq-c: -qr-• r ig c t a g c i ; i — «he I -TOO gcaacagagg gagtaagggg gggcaatgag gctggggcc3 ggcgccagca -65C gcagccacgc cccccacctc ccccgattti tagggaaaat totccMAQC NF-kappaB -€00 tcccgcatcc tcctctgcct ccttccaccc tccaccctcc cagcctccac -55C tgagacctet t^ aaaaceac crcaggggccg ccgggggatg aggecgggga -SOC acgqgctgga g=o:gg gqgctcgqgg gcagcqgacg ggaaacgccu T P I --450 cgaaaucagc cagacccggc gactgaaaLg agacgqagga gcttggcqa} -40C aggaggcgca ggctcggaaa ggcqcgcqag gctccaggct -cttcccgat -350 ccaccgctct cctcgccgac ctccgagtca cccccggaag ctcccgcca: -300 tgcugggcga atagaccccc gcggaccccc aagcgrgcgg ggccggggcc -250 ccaqttcagg ccctcqctgc ccctttaagg gttctcgaaa ctttcccccc -200 ggtatcagat gagcrtcgtc acatccgttq gc-gtggceg -tggggcgct KF7" -15C ttccgaggaa a'tcqggact cgafltttccc gqggaaqagg cgcggcctga Xho I -100 gcccggcgag ggtggggagg gcaggcgcag gtggaccccg gcgccccccg -50 agecccgctg tgaccctggc cgcgggggag gggctgggcc gctgc +1 Agtcgcgjee gecagagggg qcagcqgasa agaacttgcc caact Set I •51 gcgrgggrtg gggrgg-rct gcgrcfccgr gccrgcctc.7 eegge *101 ccttctcccc tcccgcgagc etcctscect cccgcgagcc tcctc +151 ggccctcctc ccgtcctccc cgccgccgcc ggtcccggtg cgege +201 ectgcccgca gccccgcgcg ccggccgagt cgctgagccg egget +251 aegggaeggg aceggctagg ctgggcgcgc cccccgggcc ccgcc •>30l CAT6GGC8C* CWecCCOGG CGCTSCTGCT 8CCTCTGCT8 OCCCfc M G A L A R A I L L F L L A Q + 351 TCCTGCSCGC CSCCCCSGAG CTGSCCCCCG CGCCCTICAC GCTGC' L R A A P E L A P A P F T L +401 CGGGTGGCCG CGGCCACGAA CCGCGTAGTT SCGCCCACCC CG S V A A A T M S V V A P T P 2.3.3. Identification of BACE2 promoter and its transcriptional activity To determine if the 5' UTR fragment of the BACE2 gene contained the promoter of the BACE2gene, we subcloned the 5' flanking fragments of the BACE2 gene into the promoterless vector pGL3-Basic. The pGL3- Basic vector plasmid lacks a eukaryotic promoter or enhancer sequence upstream of the reporter firefly (Photinus pyralis) luciferase gene. Expression of luciferase indicated by luciferase activity therefore 115 depends on the promoter inserted upstream of the reporter gene. A series of deletion plasmids containing various fragments of the 5' upstream region of BACE2 gene was generated (Figure 2.4A). The plasmid constructs were checked by agarose gel electrophoresis after restriction enzyme digestion and confirmed by DNA sequencing (Figure 2.4B). The pB2Luc-A was generated to contain the 1858 bp 5'UTR from -1580 bp to +278 bp of the BACE2 gene upstream of the luciferase reporter gene. Compared with the empty pGL3-Basic control, the pB2Luc-A transfected cells had a significantly higher luciferase activity (52.52 ± 1.97 R L U vs. 5.06 ± 0.37, P <0.0001), indicating that the 1858 bp 5'UTR from -1580 bp to +278 bp contains a functional promoter of the human BACE2 gene (Figure 2.4C and D). To further investigate the transcriptional regulation of the BACE2 gene in different cell types, the deletion plasmids were transfected into a murine neuroblastoma cell line, N2A, and human embryonic kidney cell line, HEK293. Plasmids pB2Luc-A, pB2Luc-B, pB2Luc-C, pB2Luc-D, and pB2Luc-E contained the BACE2 promoter region from -1580, -446, -371, -200 and -54 to +278 bp, respectively. These plasmids were transfected into cells and luciferase activity was measured 48 hours after transfection. In HEK293 cells, pB2Luc-D had the highest promoter activity (79.06 ± 3.72 RLU) and pB2Luc-A possessed the lowest promoter activity (52.52 ± 1.97 RLU) (P<0.001). Further deletions from -446 to -54 bp region had no significant effect on BACE2 promoter activity in H E K cells. The promoter activity of plasmids pB2Luc-C, and pB2Luc-E were 77.37 ± 1.07 and 71.13 ± 0.65 RLU, respectively, similar to pB2Luc-B, 77.78 ± 6.11 R L U (P>0.05) (Figure 2.4C). However, BACE2 promoter activity in the neuroblastoma 116 N2A cells was much lower. pB2Luc-A had the lowest promoter activity of 5.86 ± 0.26 RLU, whereas pB2Luc- B, pB2Luc-C, pB2Luc-D, and pB2Luc-E had promoter activity of 9.32 ± 0.89, 10.74 ± 1.14,12.62 ± 0.33, and 19.04 ± 3.78 RLU, respectively. The fragment from -54 to +273 bp region had the highest promoter activity in neuronal cells (p < 0.001 relative to others) (Figure 2.4D). These data indicate that the -54 bp fragment is essential for BACE2 transcriptional activation in both neuronal and non-neuronal cells, and BACE2 gene is preferentially transcribed in non-neuronal cells. Figure 2.4. Functional analysis of the BACE2 gene promoter. (A) Schematic diagrams of the BACE2 promoter deletion constructs containing various fragments of the 5' flanking region of BACE2 gene in the promoter-less vector plasmid pGL3-Basic. The firefly (Photinus pyralis) luciferase gene (Luc) was used as a reporter gene. Arrows indicate the direction of transcription. The numbers represent the end points of the BACE2 insert. (B) The deletion plasmids were confirmed by sequencing and restriction enzyme digestion, and the digested.samples were analyzed on a 1 % agarose gel. Vector size is 4.7 kb, and the BACE2 gene 5' flanking fragment insert sizes range from 0.3 to 2.0 kb. The deletion plasmids were cotransfected with pCMV-Rluc into HEK293 cells (C) and N2A cells (D). Luciferase activity was measured at 48 hr by a luminometer. The Renilla luciferase activity was used to normalize transfection efficiency. The values represent means ± standard errors of the means (n = 4). *, P < 0.001 by A N O V A with the post hoc Newmann-Keuls test. 117 2.3.4. SP1 can regulate BACE2 gene expression Previously we demonstrated that SP1 regulates BACE1 gene transcription (Qing et al., 2004). Though we detected no sequence similarity in the promoters of BACE1 and BACE2, the transcription factor data mining program suggested that the human BACE2 promoter also had putative SP1 binding elements. To investigate if SP1 could also regulate BACE2 gene expression, we transfected the SP1 expression plasmid pCGN-SPl and the BACE2 promoter plasmid pB2Luc-A or pB2Luc-B into HEK293 cells. SP1 overexpression significantly upregulated the BACE2 promoter activity in pB2Luc-A transfected cells (149.83 ± 4 . 5 1 %, P < 0.001 relative to control), and had no significant 118 effect on the pB2Luc-B transfected cells (P>0.05) or control vector transfected cells (Figure 2.5A). These data indicate that the putative SP1 binding site at -755bp was functional to regulate BACE2 transcription. To further confirm these data, we transfected +/+ -/-pB2Luc-A plasmid into SP1 (SP1-WT) and SP1 (SP1-KO) embryonic cells (Qing et al., 2004). pB2Luc-A had significantly lower promoter activity in SP1 -KO cells (20.56 ±1.82 RLU) than in SP1-WT cells (55.92 ± 4.64 RLU) (P<0.005) (Figure 2.5B). This indicates that SP1 is required for adequate transcription of the BACE2 gene. Furthermore, when pB2Luc-A transfected HEK293 cells were treated with a SP1 binding inhibitor, Mithramycin A (Qing et al., 2004), the BACE2 promoter activity was significantly inhibited in a dose-dependent and time-dependent manner. Addition of 25 nM, 75 nM, 125 nM and 250 nM of Mithramycin A for 48 hours decreased the promoter activity from 55.06 ± 4.41 R L U in control to 27.77 ± 1.68, 9.11 dz 0.32, 7.10 ± 0.21 and 4.67 ± 0.10 RLU, respectively (P<0.001 by ANOVA) (Figure 2.5C). Treatment with 125 nM Mithramycin A for 24 and 48 hours inhibited the promoter activity from 55.06 ± 4.41 R L U in control to 34.71 ± 2.06 and 5.58 ± 0.22 RLU, respectively (P<0.001 by ANOVA) (Figure 2.5D). Taken together, these data clearly demonstrate that SP1 regulates the transcription of the human BACE2 gene. Figure 2.5. Potentiation of BACE2 Promoter Activity by SP1. (A). SP1 expression plasmid pCGN-SPl was cotransfected with pGL3-Basic, BACE2 promoter pB2Luc-A or pB2Luc-B BACE1 plasmids into HEK293 cells. Plasmid pCMV-Rluc was used to normalize the transfection efficiency. SP1 overexpression significantly increased the BACE2 promoter activity in pB2Luc-A transfected cells, but not in pB2Luc-B transfected cells. The values represent percentage of normalized luciferase activity (means ± standard errors of the means) (n=3 to 6). *, P < 0.001 by A N O V A with 119 the post hoc Newmann-Keuls test. (B). BACE2 transcriptional activation was markedly -/- +/+ reduced in SP1-KO cells (SP1 ). SP1-WT (SP1 ) and SP1-KO cells were transfected with the BACE2 promoter plasmid pB2Luc-A or pGL3-Basic and pCMV-Rluc. (C). and (D). SP1 binding inhibitor Mithramycin A reduced the BACE2 promoter activity. HEK293 cells were transfected with the BACE2 promoter plasmid pB2Luc-A and treated with vehicle solution control or Mithramycin A for 48 hrs at 25, 75, 125or 250 nM (C), or with Mithramycin A at 125 nM for 24 or 48 hrs (D). Cells were harvested at the same transfection endpoint and luciferase activity was measured and expressed as mean ± S.E.M. R L U of control promoter activity. * P < 0.001 relative to control by A N O V A with post-hoc Newmann-Keuls test. 25 75 125 250 " z * Dosage (r»M) T , m e <hr> 120 2.3.5. Comparative sequence analysis oiBACEl and BACE2 genes BACE2 is the homolog of BACE1. However, it was reported that the BACE2 gene might function differently from BACE1. To investigate if BACE2 and BACE1 gene expression were distinctly regulated, we first examined the sequence differences between the BACE2 and BACE1 genes. Sequence alignment analysis showed that the amino acid sequences of the human BACE1 and BACE2 gene coding regions are 45% identical and 75% homologous (Figure 2.6A). Both of the gene promoters contain many putative transcription factor binding sites (Table 2-1). Comparative sequence analysis has evolved as an essential technique to identify functional coding and noncoding elements conserved throughout evolution. To compare the transcriptional regulation of the BACE1 and BACE2 gene, we aligned the sequences of the 5' promoter regions and 3'UTR of the human BACE1 and BACE2 genes using zPicture alignment tool. The homology between the BACE1 and BACE2 genes was further analyzed using the rVista program. rVista is used to identify evolutionarily conserved regions (ECR) including transcription factor binding sites, and combines transcription factor binding site search with comparative sequence analysis to reduce false positive predictions by the normal database search (Loots and Ovcharenko, 2004; Loots et al , 2002; Ovcharenko et al , 2004). ECR has been used to discover novel genes, identify distant gene regulatory elements and predict transcription factor binding sites. As the homologous genes are likely due to a gene duplication, the promoter region of the two homologous genes might also have an evolutionary conservation. The alignment by zPicture identified three evolutionarily conserved regions (ECR) in the coding region, with an ECR length at least 100 bp and ECR similarity at least 70%. The analysis showed there is no similarity in the promoter 121 region or the 3'UTR of these two genes. The similarity is only localized in the coding region of the human BACE1 and BACE2 genes with a matching score of 63.20% (Figure 2.6B). We also analyzed the promoters of BACE2 and BACE1 by other alignment tools. No significant similarity was found. The results indicate that despite the homology between BACE2 and BACE1 protein sequences, the regulatory nucleic acid sequences that control these two genes are significantly different. 2.3.6. Distinct transcriptional activation ofBACEl and BACE2 genes in neuronal and non-neuronal cell lines Previous studies showed that BACE1 and BACE2 expression is different in the nervous system and peripheral tissues. BACE1 is highly expressed in neurons whereas BACE2 is highly expressed in non-neuronal tissues. To investigate the role of the promoter in regulating the cell-specific expression of these two genes, we transfected the BACE1 or BACE2 promoter plasmids into HEK293 or N2A cells. The result showed that the BACE2 promoter has higher activity in HEK293 cells (160.11 ± 2.55 %) than in N2A cells (p<0.05), whereas the BACE1 promoter activity is higher in the neuronal N2A cells than in HEK293 cells (44.05 ± 1.64 %) (p<0.05) (Figure 2.6C). Figure 2.6. Comparative sequence and promoter activity analysis of the human BACE1 and BACE2 gene. (A) The amino acid sequences of BACE1 and BACE2 were aligned by multiAlign tool. The dark shaded area indicates identical amino acid sequences and light gray represents similar sequences between two proteins. (B) The sequences of the BACE1 and BACE2 genes including the promoter region, the coding sequence and the 3'UTR are aligned 122 with the zPicture alignment tool and submitted to rVista for analysis of the conservative sequence and transcription factor binding sites. The promoter, coding and 3' UTR were indicated. The scores of similarity were plotted between 50 % and 100 %. The location of the black dots or lines represents the similarity score between BACE2 and BACE1 gene in the region. The black bars indicate the evolutionarily conserved region (ECR). (C). BACE1 and BACE2 promoter activity in the neuronal and non-neuronal cell. The pGL3-Basic and the BACE2 promoter construct pB21uc-A and the BACE1 promoter construct pBlP-G (21) were transfected into HEK293 and N2A cells. The pCMV-Rluc was cotransfected to normalize the transfection efficiency. Cells were harvested at 48 hrs post transfection and dual luciferase assay was performed to measure the luciferase activity. The values represent means ± S .E .M. (n = 4). * P < 0.001 by student t test. BACE1 U> -D A C E 2 <1 » HQJ&fU Consensus 11> 1 I L M £ 0 PA A Ft HVDXt C SO GYYtEM ICS Consensus u>i> p»o LNt I .VOTOX.WAVAA I»H n ir KSTVR V V YTQCS » G ua OI.VSIP c; K S NIA I ean m e w G I W I A «H;;;;; ff?!*ip^^ BAOE2 <497| 0fir.:r-F(arSVV»|E:'BvS ill • Consensus (Ml) B BACE2 BACE1 Ckb t.9kb 34kb 44kb Promoter • > -Cfldl ITT 0 1.S6 39 5ll) " >: iSli No Sniiaiity found Mattes = 63.20% No Sirrslarity found S UJ 5,1 C X P * JS B A C E 2 BACE1 123 Table 2-1. Putative Transcription Factor Binding Sites In The Human BACE1 And BACE2 Genes. The numbers represent the bp location of the putative core sequence of the transcription factor binding site with +1 as the major transcription start site. - indicates the location upstream of the major transcription start site and + indicates the location downstream of the major transcription start site. BACE1 Jiin^n^Sites^ Transcription BACE2 JBhid^n^Sites Transcription factors factors -1891 -1771. -1703 -1634 -1618 -1881 -1764 -1698 1424 -1119 -987 -911 -1628 -1613 -1418 -1111 -982 -906 HSF-1 HSF-1 PU-Box A P I PU-Box A P I AP-2 GATA-1 SP-1 -1551 -1431 1368 -1265 -1162 -1448 -992 -966 -847 -1446 -1426 -1360 -1255 -1558 -1444 -987 -962 -843 -794 GATA-1 USF M A F GR HNF-3 NF-1 NF-1 A P I AP-2 -877. -796 -738 -612 -309 -252 -208 -870 -789 -733 -605 -304 -247 -201 AP-2 -799 -758 -617 -408 -262 -166 +61 E R SP1 N F - K B NF-l W T - l AP-2 SP-1 -141 -97 +82 -135 -88 +87 A P I C R E B AP-3 C-myc ;•' PU-Box C7EBP -749 -609 -404 -255 -162 +66 c-fos-SRF. Lymphokine -•^•'AP-^^-V: 2.4. Discussion The regulation of gene transcription is closely related to the function of a particular gene. Some genes involved in common processes are often coordinately regulated to achieve efficiency. In order to define the molecular mechanism by which 124 BACE2 gene expression is regulated at the transcriptional level, we cloned the 2.0 kb 5' flanking region of the human BACE2 gene and identified the gene promoter. Sequence analysis showed that the BACE2 promoter, unlike most type II eukaryotic gene promoters, does not contain a typical T A T A and C A A T box and has a high GC content. This TATA-less and high GC feature of the BACE2 gene is similar to BACE1 gene, and is common in many housekeeping genes (Christensen et al., 2004; Ge et al., 2004). The 5' flanking region has various possible transcription factor binding elements such as NF1, SP1, API, AP2, G A T A , and OCT1. The region of -54 to +278 bp had similar promoter activity to -446 to +278 in H E K cells and the highest promoter activity in N2A cells, suggesting that this proximal region of -54 to the transcriptional start site was important in regulation of the BACE2 gene. Furthermore, our deletion assay showed that the region from -1580 to -446 contained negative regulatory element (s); removing this region significantly increased the BACE2 promoter activity in both neuronal and non-neuronal cells. Further deletions from -446 bp to -54 bp resulted in a gradual increase in the promoter activity in N2A cells but not in HEK293 cells. The deletion of 146 bp fragment from -200 to -54 bp region significantly increased the BACE2 promoter activity. These data indicate that there might be a neuronal specific repressive element in this region and BACE2gene expression is tightly regulated at the transcription level in neuronal and non-neuronal cells. Computer analysis for putative transcription factor binding sites showed several NF1 sites in this region. NF1 is believed to interfere with the binding of TBP to its recognition site, thus inhibiting transcription (Barath et al , 2004; Osada et al., 1997; Pham et al., 2004). NF1 is also important in tissue specific and developmentally specific gene expression. In the BACE2 promoter, NF1 might interact with other 125 regulators to achieve repression in neuronal cells. Future studies might be directed to identify the transcription factors in this region of the BACE2 promoter. To examine function of BACE2 and its relationship to BACE1, the transcriptional regulation of BACE2 and BACE1 genes was analyzed. We previously cloned and characterized the human BACE1 promoter (Christensen et al., 2004). Analysis of the BACE2 and BACE1 sequences showed that despite 75 % homology in the coding sequence, there is no similarity in the promoter regions. There are different putative transcription factor binding sites in these two promoters. BACE1 and BACE2 have distinct expression patterns. BACE1 mRNA is highly expressed in neurons of most brain regions. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA (Bennett et al., 2000a). Our study shows that the BACE2 promoter has higher activity in HEK293 cells, whereas the BACE1 promoter has higher activity in neuronal cells. Our study provides a mechanism underlying the distinct distribution of BACE1 and BACE2 in nervous and peripheral tissues. Previously we reported that transcription factor SP1 plays an important role in BACE1 gene expression and APP processing (Christensen et al., 2004). SP1 and TAFII 130 transcriptional activity are disrupted in early Huntington's disease (Dunah et al., 2002; Freiman and Tjian, 2002; Li et al., 2002; Ryu et al , 2003). In this study we also 126 discovered that SP1 regulates human BACE2 gene transcription. BACE2 gene transcriptional activation was markedly reduced in the SP1-KO cells relative to the SP1-WT cells. Treatment with a SP1 binding inhibitor, Mithramycin A, resulted in inhibition of the BACE2 promoter activity in a time- and dose-dependent manner. Although sequence analysis revealed that the BACE2 gene promoter contained two SP1 binding sites at -755 and +63 bp, SP1 overexpression only significandy upregulated the BACE2 promoter activity in pB2Luc-A transfected cells and had no significant effect on the pB2Luc-B transfected cells. These data indicate that the putative SP1 binding site at -755bp is functional in regulating the human BACE2 transcription. Our study demonstrates that SP1 regulates both BACE2 and BACE1 genes. SP1 has been shown to play an important role in the regulation of the expression of many genes. Its C-terminal domain interacts with other transcription factors in a synergistic manner, which controls gene expression in time and space (Li et al., 1991). Our data suggest that SP1 might work with other transcription factors to regulate the different tissue expression patterns of the BACE2 and BACE1 genes. 127 2.5. References Acquati, F., Accarino, M . , Nucci, C , Fumagalli, P., Jovine, L. , Ottolenghi, S., and Taramelli, R. (2000). The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the down critical region. FEBS Lett 468, 59-64. Aleister J. Saunders, T.-W. K., Rudolph E. Tanzi (1999). B A C E Maps to Chromosome 11 and a B A C E Homolog, BACE2, Reside in the Obligate Down Syndrome Region of Chromosome 21. Science, Vol 286, Issue 5443, 1255, 1255a. Barath, P., Poliakova, D., Luciakova, K., and Nelson, B. D. (2004). Identification of NF1 as a silencer protein of the human adenine nucleotide translocase-2 gene. Eur J Biochem 271, 1781-1788. Bennett, B. D., Babu-Khan, S., Loeloff, R., Louis, J. C , Curran, E. , Citron, M . , and Vassar, R. (2000). Expression analysis of BACE2 in brain and peripheral tissues. J Biol Chem 275, 20647-20651. Christensen, M . A., Zhou, W., Qing, H. , Lehman, A., Philipsen, S., and Song, W. (2004). Transcriptional regulation of BACE1, the beta-amyloid precursor protein beta-secretase, by Spl. Mol Cell Biol 24, 865-874. Dunah, A. W., Jeong, H. , Griffin, A., Kim, Y. M . , Standaert, D. G., Hersch, S. M . , Mouradian, M . M . , Young, A. B., Tanese, N., and Krainc, D. (2002). Spl and TAFII130 transcriptional activity disrupted in early Huntington's disease. Science 296, 2238-2243. Farzan, M . , Schnitzler, C. E. , Vasilieva, N., Leung, D., and Choe, H. (2000). BACE2, a beta -secretase homolog, cleaves at the beta site and within the amyloid-beta region of the amyloid-beta precursor protein. Proc Nad Acad Sci U S A 97, 9712-9717. Freiman, R. N., and Tjian, R. (2002). Neurodegeneration. A glutamine-rich trail leads to transcription factors. Science 296, 2149-2150. Garcia-Martinez, J., Aranda, A., and Perez-Ortin, J. E . (2004). Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms. Mol Cell 15, 303-313. 128 Ge, Y. W., Maloney, B., Sambamurti, K., and Lahiri, D. K. (2004). Functional characterization of the 5' flanking region of the B A C E gene: identification of a 91 bp fragment involved in basal level of B A C E promoter expression. Faseb J 18, 1037-1039. Kosak, S. T., and Groudine, M . (2004). Form follows function: The genomic organization of cellular differentiation. Genes Dev 18, 1371-1384. Li , R., Knight, J. D., Jackson, S. P., Tjian, R., and Botchan, M . R. (1991). Direct interaction between Spl and the BPV enhancer E2 protein mediates synergistic activation of transcription. Cell 65, 493-505. Li , S. FL, Cheng, A. L. , Zhou, FL, Lam, S., Rao, M . , Li , FL, and Li , X. J. (2002). Interaction of Huntington disease protein with transcriptional activator Spl. Mol Cell Biol 22, 1277-1287. Loots, G. G., and Ovcharenko, I. (2004). rVISTA 2.0: evolutionary analysis of transcription factor binding sites. Nucleic Acids Res 32, W217-221. Loots, G. G., Ovcharenko, I., Pachter, L. , Dubchak, I., and Rubin, E. M . (2002). rVista for comparative sequence-based discovery of functional transcription factor binding sites. Genome Res 12, 832-839. Marcinkiewicz, M . , and Seidah, N. G. (2000). Coordinated expression of beta-amyloid precursor protein and the putative beta-secretase B A C E and alpha-secretase ADAM10 in mouse and human brain. J Neurochem 75, 2133-2143. Maston, G. A., Evans, S. K., and Green, M . R. (2006). Transcriptional Regulatory Elements in the Human Genome. Annu Rev Genomics Hum Genet. Osada, S., Daimon, S., Jkeda, T., Nishihara, T., Yano, K., Yamasaki, M . , and Imagawa, M . (1997). Nuclear factor 1 family proteins bind to the silencer element in the rat glutathione transferase P gene. J Biochem (Tokyo) 121, 355-363. Ovcharenko, I., Loots, G. G., Hardison, R. C , Miller, W., and Stubbs, L. (2004). zPicture: dynamic alignment and visualization tool for analyzing conservation profiles. Genome Res 14,412-411. 129 Pham, N. L. , Franzen, A., and Levin, E. G. (2004). NF1 regulatory element functions as a repressor of tissue plasminogen activator expression. Arterioscler Thromb Vase Biol 24, 982-987. Przyborski, S. A., Smith, S., and Wood, A. (2003). Transcriptional profiling of neuronal differentiation by human embryonal carcinoma stem cells in vitro. Stem Cells 21, 459-471. Qing, H. , Zhou, W., Christensen, M . A., Sun, X., Tong, Y., and Song, W. (2004). Degradation of B A C E by the ubiquitin-proteasome pathway. Faseb J 18,1571-1573. Ryu, FL, Lee, J., Zaman, K., Kubilis, J., Ferrante, R. J., Ross, B. D., Neve, R., and Ratan, R. R. (2003). Spl and Sp3 are oxidative stress-inducible, antideath transcription factors in cortical neurons. J Neurosci 23, 3597-3606. Shapiro, B. L. (1999). The Down syndrome critical region. J Neural Transm Suppl 57, 41-60. Solans, A., Estivill, X., and de La Luna, S. (2000). A new aspartyl protease on 21q22.3, BACE2, is highly similar to Alzheimer's amyloid precursor protein beta-secretase. Cytogenet Cell Genet 89, 177-184. Sullivan, A. A., and Thummel, C. S. (2003). Temporal profiles of nuclear receptor gene expression reveal coordinate transcriptional responses during Drosophila development. Mol Endocrinol 17, 2125-2137. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., et al. (1999). Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease B A C E . Science 286, 735-741. Yan, R., Bienkowski, M . J., Shuck, M . E. , Miao, H., Tory, M . C , Pauley, A. M . , Brashier, J. R., Stratman, N. C , Mathews, W. R., Buhl, A. E., et al. (1999). Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature 402, 533-537. Yang, G. X., Jan, A., Shen, S. Ff., Yazaki, J., Ishikawa, M . , Shimatani, Z., Kishimoto, N., Kikuchi, S., Matsumoto, H. , and Komatsu, S. (2004). Microarray analysis of brassinosteroids- and gibberellin-regulated gene expression in rice seedlings. Mol Genet Genomics 271,468-478. 130 C H A P T E R 3: B A C E 2 , as a novel APP 0-secretase, is not responsible for the pathogenesis of Alzheimer's Disease in Down Syndrome 2 A version of this part was previously published: Sun X, He G and Song W. (2006) BACE2, as a novel APP 9-secretase, is not responsible for the pathogenesis of Alzheimer's Disease in Down Syndrome. The F A S E B Journal 20 (9): 1369-76. 131 3.1. Introduction BACE2 is a homolog of BACE1 and is mapped to DS critical region on Chromosome 21q22.3(Acquati et al , 2000; Bennett et al., 2000a; Lin et al., 2000; Solans et al., 2000; Yan et al., 1999). The coding sequence of BACE2 and BACE1 is 45% identical and 75% homologous. Similar to BACE1, BACE2 also has two D T/S G sites and a transmembrane domain (Solans et al., 2000). BACE1 and BACE2 are more homologous with each other than with other aspartyl proteases. Both BACE1 and BACE2 have a transmembrane domain that distinguishes them from other family members. BACE2 also features a 20 amino acid signal peptide at N-terminal and two O-glycosylation sites at Asnl70 (NTSF) and Asn366 (NSSR)(Acquati et al., 2000; Solans et al., 2000). A Prosite domain search also revealed that BACE2 has two leucine-zipper domain, several PKC and CK2 phosphorylation sites, N-myristoylation sites and tyrosine sulfuration sites (http://ca.expasy.org/prosite/). BACE2 is predominantly localized to post-Golgi membranes and on the cell surface (Acquati et al., 2000; Ehehalt et al., 2002). In contrast to BACE1, pro-BACE2 is not active and prodomain processing is required for activation (Motonaga et al., 2002). Prodomain processing of BACE2 is autocatalytic (Hussain et al., 2001). The structure of BACE2 follows the general fold of A l aspartic proteases (Ostermann et al., 2006). The crystal structure of BACE2 reveals differences in the S3, S2, SI' and S2' active site substrate pockets compared to BACE1 (Ostermann et al., 2006). There is no similarity in the 5'UTR or 3'UTR of BACE1 and BACE2 and their transcription is distincdy regulated (Sun et al., 2005), which can explain why BACE1 and 132 BACE2 have distinct expression patterns. BACE1 mRNA is highly expressed in neurons of most brain regions. Northern analysis reveals that BACE2 mRNA is expressed at low levels in most human peripheral tissues and at higher levels in the colon, kidney, pancreas, placenta, prostate, stomach, and trachea. Human adult and fetal whole brain and most adult brain subregions express very low or undetectable levels of BACE2 mRNA (Bennett et al., 2000a). Furthermore, the BACE2 promoter has higher activity in HEK293 cells, whereas the BACE1 promoter has higher activity in neuronal cells (Sun et al., 2005). Although there are extensive studies about the function of BACE1, the function of BACE2 remains unknown. Several studies show that BACE2 can cleave APP at the P-secretase cleavage site in vitro (Farzan et al., 2000; Hussain et al., 2000). BACE2, but not BACE1, was reported to be responsible for the production of Ap in the Flemish mutant APP transfected cells (Farzan et al., 2000). However, other studies show that BACE2 +19 +20 cleaves APP at the Phe and Phe sites that are adjacent to the a-secretase cleavage site and BACE2 functions as an alternative a-secretase and as an antagonist of BACE1 (Basi et al., 2003; Yan et al., 2001). Furthermore, there is no compensatory upregulation of BACE2 in the BACE1 knockout mice which have a deficiency in generating AP (Luo et al., 2003) and knockout of BACE2 gene by deletion of exon 6 in mice did not show any phenotypic alterations (Dominguez et al., 2005). The BACE1 gene is localized on chromosome 1 lq23.3. There have been no mutations in the BACE1 gene coding sequence genetically associated with A D (Murphy et al., 2001). However, the BACE2 gene is located on chromosome 21q22.3, a critical region of DS. Nearly all DS patients 133 will develop A D neuropathology including neuritic plaques and neurofibrillary tangles in their brains after middle age (Glenner and Wong, 1984a; Glenner and Wong, 1984b; Hardy and Selkoe, 2002a). Ap, the major component of neuritic plaques (Glenner and Wong, 1984b), is derived from APP by the sequential cleavages by P- and y-secretase at the P- and y-site respectively. BACE1 is the major p-secretase in vivo (Cai et al., 2001; Hussain et al , 1999; Luo et al., 2001; Roberds et al., 2001b; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999). The levels of APP C-terminal fragment C99, the major P-secretase product, and Ap are increased in DS (Busciglio et al., 2002; Sun et al., 2006), suggesting increased P-secretase activity in these patients. Although a genotyping study has shown no association between A D and the intronic polymorphism in BACE2 (Nowotny et al., 2001), immunoreactivity for BACE2 was detected in neurofibrillary tangle-bearing neurons from elderly DS brains with Alzheimer-type neuropathology, but not in DS brains without Alzheimer-type neuropathology or in control brains of any age (Motonaga et al., 2002). Beta-site APP cleaving enzyme 2 (BACE2) is a homolog of BACE1 (Acquati et al , 2000; Bennett et al., 2000a; Lin et al., 2000; Solans et al., 2000; Yan et al., 1999), and both the APP and BACE2 genes are located on chromosome 21. The extra copy of APP and BACE2 may play a role in the abnormal processing of APP in DS; however, the mechanism by which A D neuropathology develops in DS remains elusive. Therefore, it is very important to elucidate the function of BACE2 and its involvement in the pathogenesis of A D in DS. 134 In this study, we clarified the function of BACE2 in APP processing. We identified BACE2 as a novel 9-secretase that cleaves APP at a novel 9-site downstream of the a site to generate APP C80 fragments. Cleavage of APP at the 0-site by BACE2 abolished AR production. Despite the fact that BACE2 mRNA levels were increased by approximately 1.5 fold in DS patients, immunoblotting analysis showed that BACE2 protein expression is unchanged in DS. Overexpression of BACE2 by lentivirus markedly reduced AR production in primary neurons derived from Swedish mutant APP transgenic mice. Our data exclude the involvement of BACE2 in AD pathogenesis in DS, and support the therapeutic potential of overexpressing BACE2 for A D and DS therapy. 3.2. Materials and methods 3.2.1. Plasmids construction BACE2 was cut out from pcDNABACE2 by Nhel and Kpnl. The fragment was cloned into pcDNA3.1 (-)/mycHis (A) at Nhel and Kpnl sites. MycHis epitope tag is fused with BACE2 at its C-terminus. BACE2 was cut from pcDNA3.1BACE2mychis with Pmel and cloned into pcDNA4mycHis (A) at EcoRl and Pmel sites blunted by Klenow. PcDNA4BACE2-mychis also has myc tag fused at its C-terminal. L26Syn-BACE2 was cloned by inserting blunt BACE2 digested from pcDNA3.1BACE2-mychis with Pmel into L26FSGW cut with BamHl and Xbal and blunted with Klenow. Xbal site in the new plasmid was converted to a stop codon. L26Syn-GFP BACE2 was made by inserting EGFP-BACE2 cut from pEGFP-BACE2 with Nhel and Xbal and blunted with Klenow into the L26FSGW cut with BamHl and Xbal and blunted with Klenow. 135 APP C99 and C83 cDNA were amplified by PCR and then cloned into pcDNA3 vector (Invitrogen) to generate mammalian expression plasmid pAPP-C99 and pAPP-C83. pAPP-C99Flag was generated by PCR with C-terminus of C99 fragment tagged with Flag epitope. 3.2.2. Transient and stable transfection Transfection was performed with LipofectAMJNE2000 (Invitrogen) according to the manufacturer's instructions and as previously described (see chapter 2.2.4). BACE1 and BACE2 antisenses were transfected with Oligofectamine from Invitrogen following the manufacturer's instructions. HEK293FT cells were transfected with calcium-phosphate method to generate lentivirus. HEK293FT Cells were splitted one day before transfection to achieve 80% confluency at the day of transfection. The media was changed one hour before transfection. 10-20ug DNA for 100mm plate was mixted in 500ul 0.25M CaCI 2 and droped into 500ul bubbled 2xHBS (0.28M NaCl, 0.05M HEPES, 1.5mM Na2HP04, PH 7.0). The mixture reacted at room temperature for 30 minutes before adding to culture media dropwise. The cells can be incubated in 3% CO2 for 7-12 hours optionally to increase the transfection efficiency. The transfection media were changed the next day and the lentivirus was collected 48 hours later. 20E2 cell line is a Swedish mutant APP stable HEK293 cell line under the selection of geneticin. pBACEl-mycHis was stably transfected into 20E2 to generate 2EB2 (Christensen et al., 2004). BACE2 was stably transfected into HEK293 cells to get 4B25 cells by the selection of zeocine. 4EB2 cell line stably expressing BACE2 and 136 Swedish APP695 was established by transfecting plasmid pZ-BACE2mycHis into 20E2 cells under selection of Zeocin (Sun et al., 2005). The stable cell lines were maintained in complete Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), ImM sodium pyruvate, 2 mM L-glutamine, 50 unit/ml penicillin G sodium, 50 pg/ml streptomycin sulfate, and supplemented with 100 p.g/ml Zeocin and/or Geneticin (Invitrogen). 3.2.3. Immunoblotting and immunbprecipitation Buffer recipes RIPA Lysis buffer: 30ml 5M NaCl, 50ml IM Tris-HCl pH7.2, 10ml Triton X -100, 10ml 10% SDS, lOg Sodium Deoxycholate to a total volume of 1L with distilled water. (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.05M Tris-HCl, pH 7.2). Supplement with protease inhibitor cocktail Complete (Roche) right before use. SDS sample buffer (2x): 2.5ml 4xTris-HCI/SDS, pH6.8 (0.1M); 2ml glycerol; 0.4g SDS; 0.31g DTT; O.lmg bromphenol Blue; Add H 2 0 to 10ml and mix. Aliquot to 1ml and store at -80 degree. SDS sample buffer (6x): 7ml 4xTris-HCI7SDS, pH6.8 (0.28M); 3ml glycerol, lg SDS, 0.93g DTT, 1.2mg brophenol blue, add H 2 0 to 10ml. Aliquot to 1ml and store at -80 degree. 4x Tris -HCl , pH 6.8: Dissolve 6.05g Trisbase in 40ml H 2 0 . Adjust pH to 6.8 with IN HCI. Add H 2 0 to 500ml total volume. Filter the solution through a 0.45um filter and store at 4 degree up to 1 month. 137 ' 4x Tris-HCI/SDS, pH 8.8: Dissolve 91g Tris Base (1.5M) in 300ml H 2 0 . Adjust pH to 8.8 with IN HCI. Add H 2 0 to 500ml total volume. Filter the solution through a 0.45um filter, add 2g SDS and store at 4 degree up to 1 month. Tris-HCI/SDS, pH 8.45: Dissolve 182g Tris base (3.0M) in 300ml H20. Add H 2 0 to 500ml total volume. Filter the solution through a 0.45um filter, add 1.5g SDS and store at 4 degree up to 1 month. 10 x Tris-glycine running buffer: 29 g Trizma, 144 g Glycine, 10 g SDS, Make up to 1000ml with dH 2 0. lOx Tris-tricine running buffer: 121g Tris, 179g Tricine, lOg SDS in 1000ml dH 2 0. 10 x blotting buffer: 30.3 g Trizma, 144 g Glycine, make up to 1000ml with dH 2 0. 1 x blotting buffer: Dilute 100 ml lOx blotting buffer in 200 ml methanol + 700 ml dH 2 0. 10 x Phosphate buffered saline (PBS), 80 g NaCl, 2 g KC1, 14.4 g Na 2 HP0 4 , 2.4 g K H 2 P 0 4 . Add 800 ml dH 2 0 and adjust pH to 7.4, then make up to 1000ml. Ix PBS-T buffer: Dilute lOx PBS to lx and add 1ml Tween-20. Blocking buffer: 5% milk and 3% BSA in lxPBS-T. Store in -20 degree. ECL chemiluminescent reagents: Solution I, 8.9ml H 2 0 , lOOul 250mM luminol (0.44g in 10ml DMSO), 44ul 90mM p-coumaric acid (0.15g in 10ml), 1ml IM Tris pH 8.5. Solution II, 9ml H 2 0 , 6ul 30% H 2 0 2 , 1ml I M Tris pH 8.5. Mix solution I and solution II just before detection and incubate the blot in the mixture for 1 minute. Tris-glycine (SDS) separating gel: x ml 40 % acrylamide/bis (37.5:1), 2.5 ml 1.5 M Tris (pH 8.8), y ml dH 2 0, 100 pi 10 % SDS, 5 pi T E M E D , 50 pi 10 % ammonium persulphate (freshly made) for total volume of 10ml. That is enough for two 1mm thick 138 gels, x + y = 7.4. e.g. 10 % gel: x = 2.5, y = 4.9; 12% gel, x=3ml, y=4.4ml; 15% gel, x=3.75ml, y=3.65ml. Tris-glycine (SDS) stacking gel: 0.5 ml 40 % acrylamide/bis, 1.26 ml 0.5 M Tris (pH 6.8), 3.18 ml dH 2 0, 50 pi 10 % SDS, 5 pi T E M E D , 25 pi 10 % APS. 16% Tris-tricine separating gel: 0.28ml H 2 0 , 5.36ml 30% Acrylamide/Bis, 1ml glycerol, 3.35ml Tris-HCl (pH8.45), lOul T E M E D , 50ul 10% APS. Tris-tricine stacking gel: 3.89ml H20, 0.81ml 30% Acrylamide/Bis, 1.55ml Tris-HCl (pH8.45), lOul TEMED, 50ul 10% APS. Sample preparation. Cells were washed twice with cold lxPBS and harvested into cold RTPA lysis buffer supplemented with protease inhibitors cocktail Complete (Roche). Cell pellets in the lysis buffer were sonicated with Sonicator (Fisher Scientific). The frozen brain tissues were homogenized in RIPA lysis buffer (containing 1% SDS instead) supplemented with protease inhibitors and sonicated. Cell and tissue lysates were centrifuged at top speed for 15 minutes to pellet cell debris. The protein concentration was determined by B C A method from BioRad according to manufacturer's control. Gradient concentration of 0-50ug/ml of BSA (Sigma) was used as protein standard. The supernatant can be stored at -20 or -80 degree. SDS-PAGE electrophoresis and western blot. The gels were poured with gel assembling cassette from BioRad and let stand at RT for at least 30 minutes or 4 degree for overnight. The cell or tissue lysates were mixed with glycine SDS loading buffer and boiled for 5 minutes at 95 degree to denature the proteins (except detection of presenilin). 139 BACE1 and BACE2 were separated with 12% or 15% tris-glycine SDS-PAGE gel. Gel was run in lx SDS glycine running buffer at 60voltage for 30 minutes then 120voltage for 90-120 minutes. APP C-terminal C99 and C83 were separated with 16%tris-tricine SDS-PAGE gel. The samples were prepared with 2x tricine sample buffer (from Invitrogen). Tris-tricine gel was run in lx Tris-Tricine running buffer at 30 miliampere/gel constantly for 2-3 hours. Proteins separated on the gel were transferred to PVDF membrane at 105voltage for 2 hours in lx blotting buffer. The PVDF membrane was blocked with blocking buffer for 1 hour at room temperature with shaking and blotted with primary antibody at 4 degree with shaking for overnight. The membrane was washed three times with lxPBS- Tween (0.1%) for 15 minutes (monoclonal antibody) or 30 minutes (polyclonal antibody) each time. The biotinylated secondary anti-mouse or anti-rabbit (1:2000) was added and incubated for 1 hour at room temperature. Then the membrane was washed three times with lxPBS- Tween (0.1%) for 15 minutes each time. The western blot was developed with E C L method. Immunoprecipitation. Harvest cells of 10 cm plate into 1ml RJPA (0.1% SDS) and sonicate. lOOul CL-4B was added into the cell lysate and shaked for one hour at 4 degree. The cell lysates were pelleted at top speed for 10 minutes at 4 degree. 8ul of the capture antibody M2 and 20ul protein A / G were added into the supernatant in a new 1.5ml Eppendorf tube. The reaction was incubated for overnight at 4 degree with shaking. The precipitates were centrifuged and washed three times with RIP A buffer. lOul 2x tris-tricine sample buffer was added into 140 the precipitate and boiled for 5 minutes before loading onto the 16% tris-tricine gel to resolve the proteins. Antibodies. The antibodies applied to the PVDF membranes were diluted in lx PBS-T or blocking buffer. BACE1 and BACE2 cloned in pcDNA3.1mychis or pcDNA4mychis have myc tag fused with BACE1 and BACE2. The myc tag fused with the protein was recognized with 9E10 as primary antibody and anti-mouse as secondary antibody. A peptide corresponding to C-terminus of BACE1 (RCLRCLRQQHDDFADD) was injected into rabbit and a polyclonal antibody 208 was developed to target the BACE1. A polyclonal antibody 210 against BACE2 C-terminal (PRDPEVVNDESSLVRH) was raised in rabbit. The secondary antibody for 208 and 210 is anti-rabbit. APP and its C-terminal C99 and C83 were resolved by C20 antibody targeting the 20 amino acids of APP C-terminus. 22C11 antibody, a mouse monoclonal antibody against amino acids 66-81 of the APP N-terminus (Chemicon), was used to analyze the secreted APP in the conditioned media. Internal control p-actin expression was analyzed using monoclonal anti-3-actin antibody AC-15 (1:3000, Sigma) and antimouse secondary antibody. 3.2.4. N-terminal sequencing pZBACE2mychis was stably transfected into HEK293 cells to establish the stable cell line 4B25. 4B25 cells were transfected with pAPP-C99FIag and harvested 72 hours 141 after transfection. Transfection was performed using Lipofectamine 2000 as previously described. The cell lysates were immunoprecipitated with monoclonal M2 anti-Flag antibody (Sigma). The immunoprecipitates were resolved by 16% Tris-Tricine gel and stained with Gelcode blue stain reagent (Pierce). The C80 band, confirmed by C20 antibody detection, was subjected to N-terminal sequencing using Applied Biosystems Precise Sequencer (UBC NAPS). The N-terminal sequence of the sequenced peptide was subject to blast search and mapped to APP sequence to identify the BACE2 cleavage site. 3.2.5. APP23 mice and genotyping APP23 transgenic mice were shipped from Novartis in Switzerland. APP23 mice have human APP751 Swedish mutant gene driven by murine thy 1.2 promoter which has a neuronal specific expression pattern (Sturchler-Pierrat et al , 1997). APP23 mice were crossed with C57BL/6 mice to generate APP new born mice. Breeding of APP23 mice can start as early as 2-3 months old to 6-8 months old. Wild type C57BL/6 mice were preferred as mothers because the APP23 mothers have a tendency to eat their pups. The litter size is usually about 6-10. Ear punch is used to identify the mice. Weaning and genotyping are done 3 weeks after birth. Genotyping was done with PCR from the DNA isolated from mouse tails or the tissues from ear punch. The DNA can be isolated with Qiagen genomic DNA isolation kit following the manufacturer's instructions (Cat # 69504). Alternatively, the tissue from ear punch or tail tips can be digested in 250-500ul of Tris buffer containing lOmM Tris-HCl pH 8.0, lOmM E D T A pH 8.0, 150mM NaCl and 0.5%SDS and 250ug/ml proteinase K (from Sigma). The mixture was digested at 56 degrees overnight and directly used as the template for PCR genotyping. 142 Mice were genotyped with ThylE2 primer 5' -C A C C A C A G A A T C C A A G T C G G -3' and APP1082r 5 ' -CTTGACGTTCTGCCTCTTCC-3' by PCR method. I designed the thylE2 primer that was annealed to exon2 of thyl gene in the transgene construct (pUC18Thyl). The lkb band from PCR indicates the existence of the APP transgene. As the homozygous APP23 cannot survive after birth, the appearance of the transgene band indicates the hemizygous APP23. And the negative of the PCR band indicates the wild type, p-actin was amplified by primers Actin-IF 5 ' - G A C A G G A T G C A G A A G G A G A T -3' and Actin-IR 5 ' -TTGCTGATCC A C ATCTGCTG-3' to serve as the internal control. 3.2.6. Primary neuronal culture Dayl APP23 new born mice were killed by cervical dislocation and the dissected hippocampal and neocortical tissues were gently digested with trypsin (0.025%EDTA; Gibco). The cells were suspended in neurobasal media supplemented with 2.25% B27 (Gibco), 1% L-glutamine, 1% penicillin/streptomycin. The cells were plated at a density of 2xl0 6 cells per 35mm plates (for 96 wells, 15-20xl03; 24 wells, 200xl03; 60mm plate 2.5-3xl06; 100mm plate 6-8xl06) coated by poly-D-lysine (0.01 mg/ml; Sigma). Tails from the new-born mice were used to isolate the genomic DNA and genotyped by PCR method. The cultures were maintained at 37 °C in a humidified incubator containing 5% C02 and the media were changed every 2-3 days. The culture was used for experiments 5 days later and up to 10 days. 143 3.2.7. Lentivirus generation and infection The BACE2 lentivirus construct L26Syn-BACE2 was cotransfected with helper plasmids V S V G and 58.87 into HEK293FT cells by calcium-phosphate method. 48hours after transfection, the supernantants were harvested, filtered at a 0.45-u.m pore size, and ultracentrifuged with MLS50 at 45,000 for 2hours. The pellet was resuspended in lOOul of PBS. Primary neuronal culture in 35mm plates was infected with lOul virus and the media was changed 6 hours after infection. 36 hours after infection, the conditioned media was harvested for AR ELISA. 3.2.8. AR40/42 sandwich ELISA assay Conditioned media was collected from cells and protein inhibitors and AEBSF (lmg/ml, Sigma) were added to prevent degradation of Ap. The concentration of AP40/42 was detected by P-amyloid 1-40 or 1-42 Colorimetric ELISA kit (Biosource International, Inc) according to the manufacturer's instructions. Briefly, 50ul AP40/42 standard of 0, 15.63, 31.25, 62.5, 125, 250, 500, lOOOpg/ml and 50ul of samples were added to the wells. 50ul detection antibody was added into wells immediately and shaken at room temperature for 3 hours. The wells were washed with 400ul lx washing buffer supplied four times with 20 seconds each. lOOul secondary antibody was added and further incubated for 30 minutes. Do another four times washing and add lOOul stabilized chymogen and incubate in dark for 30 minutes at room temperature. lOOul stop solution was added and the OD at 450nm wavelength was read with photometer and Multiscan software. The standard curve was made using excel with an order of 4 in polynomial type. 144 3.3. Results 3.3.1. BACE2 and BACE1 distinctly regulate APP processing and AR generation A3 is generated from APP by B-secretase and y-secretase. Previous studies showed that BACE1 is the major (3- secretase in vivo. However, the function of BACE2 protein was not fully defined. To address this issue, we generated stably-transfected cell lines 2EB2 and 4EB2. pBACEl-mycHis plasmid containing mycHis-tagged BACE1 cDNA was stably transfected into the Swedish mutant APP695 cells 20E2 to generate an APP-BACE1 double stable cell line 2EB2. The 4EB2 cell line was established by stably transfecting pZ-B ACE2mycHis into 20E2 cells so the cells stably express both Swedish mutant APP695 and human BACE2 genes. To detect BACE2 protein, a synthetic peptide B2CT with sequence PRDPEVVNDESSLVRH corresponding to the C-terminus of human BACE2 protein was used to immunize a rabbit and a polyclonal antibody 210 was raised against BACE2 protein. To characterize the BACE2 antibody, HEK293 cells were transfected with empty vector, pBACEl-mycHis, or pZ-BACE2mycHis plasmids. Figure 3.1 A shows that our antibody 210 detected BACE2 protein, but not BACE1 protein in the transfected cells. Overexpressed BACE2 protein could not be detected with the pre-immunization sera Pre210. Furthermore, preincubation of 210 with excess B2CT peptides resulted in clearance of BACE2-specific antibody and the precleared antibody could not detect BACE2 protein. These data clearly demonstrated that our 210 antibody is specific to detect BACE2 protein. Western blot analysis showed that 2EB2 cells had robust expression of BACE1 detected by anti-myc antibody 9E10 and anti-C-terminal BACE1 145 antibody 208, and 4EB2 cells had robust expression of BACE2 detected by 9E10 and 210 (Figure 3.IB). To examine the role of BACE2 and BACE1 in APP processing, cell lysates from the stable cells 4EB2 and 2EB2 were subject to Western immunoblot analysis with C20 antibody to detect APP C-terminal fragments (CTFs) (Figure 3.IB). In 20E2 cells the major APP CTF was C83, whereas in 2EB2 cells, the majority of CTFs were APP C99 fragments, indicating that the overexpression of the BACE1 gene significantly increased APP processing at the p-secretase site, resulting in markedly increased generation of APP C99 fragments. In contrast, the majority of the CTFs in 4EB2 cells were C80 fragments, and the level of APP C99 in 4EB2 cells was slightly decreased relative to that in 20E2 cells, suggesting that overexpression of the human BACE2 gene has no effect on the P-secretase cleavage of APP. To further define the role of BACE2 and BACE1 in regulating APP processing and Ap generation, the levels of Ap production in 20E2, 2EB2 and 4EB2 cells were determined. The conditioned media from the cells were collected and AP colorimetric sandwich ELISA assay was performed to measure Ap levels. The levels of AP40 and Ap42 were significantly increased in 2EB2 cells by 734 ± 33 % and 3791 ± 746 % (p < 0.001), respectively, but drastically decreased in 4EB2 cells to 6.57 ± 1.55 % and 39.06 ± 5.58 % (p < 0.001), respectively, relative to control cells 20E2 (Figure 3.1C). 22C11, an APP N-terminal antibody, was used to further check the secreted forms of APP (sAPP) in these cells. sAPP species include a-secretase-generated sAPPa, P-secretase-generated 146 sAPPp and BACE2-generated sAPP. The conditioned media from the cells were subject to 12% Tris-Glycine PAGE analysis. Overexpression of BACE1 significantly increased the secretion of sAPPp into the conditioned media and overexpression of B ACE2 markedly increased the production of BACE2-generated sAPP, resulting in an overall elevation of total sAPP levels in the conditioned media, relative to control 20E2 cells (Fig. 3.ID). The significant increase in sAPP together with a reduction in AP generation indicates that B ACE2 functions not as a P-secretase but instead cleaves APP within the Ap domain to preclude AP generation. To further confirm the distinct role of BACE2 in APP processing, BACE1 stable cells 2EB2 were transfected with BACE2 cDNA. Due to the significant increase in the S-secretase activity in 2EB2 cells, the majority of the APP CTFs were the major P-secretase cleavage product C99 and the minor product C83 in 2EB2 cell lysates. However, overexpression of BACE2 significantly changed the APP processing pattern in BACE1 stable cells, resulting in a decrease in C99 and C83 generation, but a significant increase in C80 production (Figure 3.IE). This suggests that BACE2 cleaves APP at a site downstream of the P-secretase sites. Consistent with this observation, AP production was inhibited by overexpression of BACE2 in the BACE1 stable cells 2EB2. The levels of Ap40 and 42 were markedly reduced to 1.99 ± 0.45 % and 2.40 ± 0.87 % in the BACE2 transfected cells relative to controls (p < 0.001) (Figure 3. IE). Furthermore, transfection ofBACE2 antisense oligos in BACE2 stable cell 4EB2 cells resulted in significant reduction in the level of BACE2 expression (Figure 3.IF). Such knockdown 147 of BACE2 expression markedly increased the levels of C83 fragment in 4EB2 cells but had no significant effect on C99 generation in BACE1 stable cell 2EB2 (Figure 3. IF). These data clearly demonstrate that BACE2 processes APP not at the p-secretase sites as BACE1 does, but rather, cleaves APP at a site downstream of a-secretase site. This cleavage of APP within the Ap domain significantly reduced Ap generation. Figure 3.1. Regulation of APP processing and Ap production by BACE1 and BACE2. (A). Characterization of BACE2 antibody 210. pBACE 1 -mycHis, pZ-BACE2mycHis or pcDNA4mycHis were transfected into H E K 293 cells. The cell lysates were separated by 12% SDS-Glycine gel and immunoblotted with specific BACE2 C-terminal antibody 210, rabbit serum drawn before immunization with BACE2 peptide B2CT, or 210 antibody preincubated with B2CT peptides. (B). The lysate samples from 20E2, 2EB2 and 4EB2 cells were subject to Western blot analysis with anti-myc antibody 9E10, anti-BACE1 antibody 208, anti-BACE2 antibody 210 or APP C-terminal antibody C20. (C). Ap40 and Ap42 levels in the conditioned media from 20E2, 2EB2 and 4EB2 cells were measured by colorimetric ELISA assay, n = 4. * P <0.001 by A N O V A . Details described in Materials and Methods section. (D). sAPP in the conditioned media was analyzed by 12% Tris-Glycine gel with an N-terminal APP antibody 22C11. P-actin was detected by AC-15 antibody (Sigma). (E). The BACE1 stable cells 2EB2 were transfected with empty vector as control or pZ-BACE2mycHis. APP CTFs were analyzed on 16 % Tris-Tricine gel with C20 antibody. The conditioned media were used for Ap measurement by ELISA. n = 3. * P <0.001 by student t test. (F). 2EB2 and 4EB2 cells were transfected with BACE2 antisense oligos. BACE2 level in 4EB2 was detected by antibody 210. APP CTFs in 2EB2 cells (middle gel) and in 4EB2 cells (bottom gel) were separated by 16% Tris-Tricine gel and blotted with C20. 148 3.3.2. N-terminal sequencing analysis shows that B A C E 2 cleaves APP at a novel 9 site To determine the identity of the new fragment and the APP cleavage site of the BACE2 protease, pAPP-C99Flag expression plasmid was transfected into 4B25 cells which stably express BACE2. APP CTFs in the transfected cells were immunoprecipitated with anti-Flag antibody M2, resolved on SDS-PAGE gels and stained by Gelcode blue (Figure3.2A). The APP CTFs were confirmed by C20 antibody immunoblotting. The lower molecular weight band generated by B ACE2 was subjected to N-terminal sequencing for identification of the first eight amino acids (Figure 3.2B), which revealed that the cleavage product sequence is F A E D V G S N (Figure 3.2B and C). The sequencing data clearly demonstrates that BACE2 cleaved APP between Phe + 1 9 and Phe + 2 0 of the A(3 domain, and that this site is distinct from both the (3- or a-secretase cleavage sites (Figure 3.2E). To further confirm BACE2's role as a novel site secretase in APP processing, a C99-flag expression plasmid was transfected into the BACE2 stable 4B.25 cells. Overexpression of BACE2 in these cells markedly increased its secretase activity, resulting in decreased C99-flag production and the generation of a new C80-flag fragment (Figure 3.2D), indicating that BACE2 can process APP at a site downstream of the P-secretase site and can also cleave the P-secretase product in cells. We designate this novel site as the APP 0-secretase cleavage site. BACE2, as a 9-secretase, cleaves APP at the 0-site within AP domain, precluding AP production and generating the APP C80 fragment (Figure 3.IB, E). These data suggest that potentiation of BACE2 0-secretase 150 activity may be a potential pharmaceutical strategy for preventing overgeneration of A(3 leading to Ap deposition in AD. Figure 3.2. N-terminal sequencing of the APP C-terminal cleaved by B A C E 2 showed that BACE2 cleaves APP at a novel 0 site. (A) For identification of the APP cleavage site by BACE2, BACE2 stably-transfected 4B25 cells were transfected with pAPP-C99Flag and harvested 72 hours after transfection. Anti-Flag M2 antibody was used to immunoprecipitate APP CTFs and the samples were resolved by 16% Tris-Tricine gel. The C80 band was subjected to N-terminal sequencing (Applied Biosystems Precise Sequencer, UBC NAPS). (B) The N-terminal sequencing result of the first six residues of the C80 fragment. Each panel represents an amino acid residue, denoted by the letter above the peak signal of the amino acid. (C) The sequence of the first eight amino acids of the C80 N-terminus sequencing. (D) Plasmid pAPP-C99Flag was transfected into HEK293 control and BACE2 stably-transfected cells 4B25. Flag-tagged C99 protein and its derivatives were detected with anti-Flag M2 antibody. Overexpression of BACE2 induces a cleavage within the C99 fragment, resulting in a decrease in C99 levels and the generation of C80 proteins. (E) The schematic diagram shows that B ACE2 has a distinct APP cleavage site compared with BACE1 and a-secretase. BACE2 cleaves APP between Phe + 1 9 and Phe + 2 0 of Ap at the 0 site. BACE1 processes APP at the major P-secretase site at Asp + 1 and the minor P-secretase site at G l u + n of the AP domain. The number represents the position of amino acids in Ap. The arrows point to the protease cleavage sites. 151 3.3.3. Overexpression of BACE2 markedly reduced Af$ generation in primary neurons from A D transgenic mice Lentivirus is a type of retrovirus that can infect both dividing and non-dividing cells. When the Synapsin I promoter is located in the lentivirus vector it can direct expression of the target gene such that it is specifically expressed in neurons (Dittgen et al , 2004; Lois et al., 2002). APP23 mice carry the human Swedish mutant APP751 transgene driven by the Thy 1.2 promoter which can drive the transgene to be specifically expressed in neurons, making APP23 a valued A D mice model. Neuritic plaques can be detected in the brains of APP23 mice at 6 months of age and memory deficits can be detected as early as 3 months (Sturchler-Pierrat et al , 1997; Van Dam et al., 2003). We dissected primary neurons from day 1 newborn APP23 mice, and infected them with empty vector or EGFP-BACE2 lentivirus. Infection with BACE2-containing lentivirus resulted in a significant 75±0.5% decrease in ApUO and 53±11% decrease in Af342 production in neurons derived from APP23 transgenic mice, relative to controls (p<0.001) (Figure 3.3). Our results clearly demonstrate that BACE2 overexpression can 152 reduce AR production in primary APP23 neuronal cultures. These results are consistent with our previous report using BACE2 and Swedish APP double stable cell line showing that BACE2 overexpression drastically inhibited AR generation. Figure 3.3. Overexpression of BACE2 by lentivirus decreased AR production in primary transgenic neurons. Primary APP23 neurons were infected with EGFP-BACE2 or empty vector lentivirus. AR40 and 42 ELISA was performed using media 36 hours after infection. EGFP-BACE2 lentivirus infection significantly decreased AR40 (A) and 42 (B) production by approximately 75% and 53% (p<0.01). Values are means ± S.E.M. (n = 3). * p < 0.01 by student t test. 3.3.4. BACE2 Transcription is elevated in DS patients. The BACE2 gene is located in Down Syndrome Critical Region on chromosome 21, thus we examined whether the transcription of BACE2 was increased in DS due to its triplication. Fetal cortical tissues from Trisomy-21 and gestation age-matched control brains were homogenized for RNA extraction, and total RNA was isolated from frozen 153 brain tissue using TRI-Reagent. Quantitative RT-PCR was used to measure BACE2 mRNA levels in both DS brain tissues and normal controls (Figure 3.4A). BACE2 mRNA levels were increased by 147.7 ± 11.46 % in DS brain tissues relative to controls (P < 0.05) (Figure 3.4B). Such increases may be due to an extra copy of the BACE2 gene on chromosome 21 in DS. Figure 3.4. Increased BACE2 mRNA levels in DS patients. (A) A representative blot of the Quantitative RT-PCR products. RNA was isolated from the cerebral cortical tissues of 16 to 20 week gestational fetal abortuses in seven DS and seven age-matched controls. Quantitative RT-PCR was performed to measure the endogenous levels of BACE2 mRNA. Specific BACE2 and (3-actin coding sequence primers were used to amplify the cDNA. Different cycles and amounts of PCR products were analyzed and the DNA gel represents 25 cycles of RT-PCR products separated on 1 % agarose gel. (B) The ratio of BACE2 to (3-Actin mRNA in DS and age-matched controls were quantitated by Kodak Image Analysis. Endogenous BACE2 mRNA levels were increased by approximately 1.5 fold in DS brain tissues relative to controls. *p < 0.05 relative to controls by student t test. Shown are the mean ± S.E.M (N=7). 154 C C C DS DS DS - B A C E 2 —p-actin 13 J 5 0 "o5 " 5 1 2 5 > *— 0) c 1 0 0 — i o < O 7 5 ^ o 5 0 E ^ 2 5 0 Control 3.3.5. BACE2 protein level remains unchanged in DS patients Trisomy-21 and gestation age-matched control brains were homogenized for protein extraction. BACE2 levels in DS brain tissue lysates were assayed by Western blot with BACE2 C-terminus antibody 210 (Figure 3.5A) (Sun et al., 2005). B A C E 2 protein levels were not significandy increased in DS brains relative to controls (103.10 ± 1.24 %, p > 0.05) (Figure 3.5B). These data indicate that despite an extra copy of the BACE2 gene in DS and the increase in its transcription, BACE2 has little effect on APP processing in DS brains and cannot account for increased C99 production. These data are also consistent with our results demonstrating that BACE2 is a 9-secretase that cleaves APP within the 155 Aft domain. Increased BACE2 protein level would have elevated 9-secretase activity and prevented AR overproduction and neuritic plaque formation in DS. Figure 3.5. Protein level of B A C E 2 remains unchanged in D S . (A) A representative blot of BACE2 detection by 210 antibody. Cerebral cortical tissues of 16 to 20 week gestational fetal abortuses in seven DS and seven age-matched controls were lysed in RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.05M Tris-HCl, pH 7.2) supplemented with Complete (Roche) protease inhibitors cocktail. 150 p.g of the brain tissue lysates was separated on a 12% Tris-Glycine gel. P-actin was detected by AC-15 was used as the loading control. Brain tissues from DS and controls were lysed in RIPA (1% Triton X100, 1% sodium deoxycholate, 4% SDS, 0.15M NaCl, 0.05M Tris-HCl, pH 7.2) buffer supplemented with protease inhibitors. (B) Quantitative analysis of BACE2 proteins by Kodak Image Analysis. Values are means ± S.E.M. (n = 7). Protein levels are expressed as a percentage of control levels. There was no significant difference in BACE2 protein levels between DS and Control (p > 0.05 by student t test). c c C C D S D S D S D S -BACE2 -p-actin Control 156 3.4. Discussion Neuritic plaques and neurofibrillary tangles are the two pathological hallmarks of AD. The neuritic plaques consist of the fibrillary form of Ap. The production of Ap, especially AP42, is elevated in familial AD bearing dominant mutations in the APP or presenilin genes. The levels of AP and APP C99 are also elevated in the brains of DS patients which have three copies of chromosome 21 (Busciglio et al., 2002; Lott and Head, 2001). Since the APP and BACE2 genes are located on chromosome 21 and there are extra copies of these two genes in DS, it was speculated that the extra gene copies may play a role in abnormal processing of APP in DS. The elevation of APP C99 and AP in DS is partially due to an extra copy of the APP gene in chromosome 21. While the additional copy of the APP gene is seen in 99% of DS patients, the overexpression of APP cannot fully explain the AD pathogenesis in DS patients (D'Hooge et al., 1996; Koistinaho et al., 2001; Podlisny et al., 1987). Thus several other genes on chromosome 21, including BACE2, a homolog of BACE1, have also been implicated in the AD pathogenesis. BACE1 is the major P-secretase in vivo. siRNA suppression of BACE1 reduced CTFP and AP production in neurons derived from both wild-type and the Swedish APP mutant transgenic mice (Kao et al., 2004b). Disruption of the presenilin genes inhibits y-secretase activity and abolishes Ap production. Presenilin deficiency also inhibits Notch signaling and embryonic development (De Strooper et al., 1999; Shen et al., 1997; Song et al., 1999; Wong et al , 1997; Zhang et al , 2000). In contrast, BACE1-KO mice have 157 abolished Ap generation, but exhibit a normal phenotype without any observed developmental deficits (Cai et al., 2001; Luo et a l , 2001; Roberds et al., 2001b). Furthermore, disruption of the BACE1 gene rescues memory deficits and cholinergic dysfunction in Tg2576 Swedish APP mutant transgenic mice (Ohno et al., 2004). These results suggest that inhibition of BACE1 is a valid therapeutic target for AD. BACE2 and BACE1 share many similarities including 63% matching in amino acids, and two aspartic protease active site motifs, six conserved lumenal cysteine residues, a C-terminal transmembrane domain, N-linked glycosylation sites, and other structural features. Like BACE1, BACE2 undergoes a complex set of post-translational modifications including a 62 63 prodomain processing between Leu and Ala . Prodomain processing of B ACE2 is autocatalytic (Charlwood et al., 2001). The high degree of similarity between BACE2 and BACE1 protein sequences suggested that B ACE2 might also function as a P-secretase. It was reported that B ACE2, but not BACE1, was responsible for the production of Ap in the Flemish mutant APP transfected cells (Farzan et al., 2000). BACE2 cleaves at the P site and more efficiently at a different site within Ap. The familial AD-associated Flemish mutant APP is adjacent to this latter site. BACE1 and BACE2 respond identically to conservative P-site mutations, and alteration of a common active site Arg inhibits p-site cleavage but not cleavage within AP by both enzymes (Farzan et al., 2000). However, other studies showed that 62 63 BACE2 mainly cleaved between Leu and Ala in the middle of the AP sequence, after th th the 19 and 20 residues that are adjacent to the a-secretase cleavage site (Andrau et al., 2003; Fluhrer et al., 2002), leading to accumulation of the N-terminal truncated product 158 C79 in BACE2-expressing cells (Andrau et al., 2003). BACE2 functions as an alternative a-secretase and as an antagonist of BACE1 (Basi et al., 2003; Yan et al., 2001). Furthermore, while in situ hybridization showed the coexpression of APP, BACE1, and ADAM10, expression of BAGE2 and ADAM17 only partially overlapped with that of APP, suggesting that ADAM10 and BACE1 are authentic a- and P-secretases (Motonaga et al., 2002). In this report, our data show that BACE2 gene transcription is indeed increased in DS, but BACE2 protein levels are not significantly changed. These studies suggest that BACE2 might have distinct functions from BACE1. Using the cell line stably expressing both human BACE2 and Swedish mutant APP genes our experiments show that the cleavage product of BACE2 is C80, not C83 as is generated by a-secretase, and N-terminal sequencing clearly demonstrates that the BACE2 cleavage site is located between AP-Phe+19 and Ap-Phe+20. Our data suggest that BACE2 is a new class of APP cleaving enzyme, neither a P-secretase nor an alternative a-secretase, and that BACE2 functions as a novel 9-secretase to cleave APP within the AP domain, thus precluding AP generation. This finding indicates that BACE2, despite its extra gene copy in DS, cannot account for the increased AP production in the AD pathogenesis of DS; instead, BACE2, as an APP 0-secretase inhibiting AP generation, may be a potential drug target, and upregulation of BACE2 activity may be a useful pharmaceutical strategy for AD therapy. Consistent with our studies, BACE1 knockout mice do not acquire compensatory gene expression changes or develop neural lesions over time. In particular, BACE2 expression is not upregulated (Luo et al., 2003). Selective inactivation of BACE2 by RNAi results in increased P-cleaved secreted APP 159 and AP secretion from cells (Basi et al., 2003). The crystal structures of BACE2 and BACE1 reveal some key differences between these two enzymes which may allow for the design of selective BACE1 or BACE2 inhibitors (Hong et al., 2000; Hong et al , 2002; Ostermann et al., 2006). As BACE1 is the major P-secretase and an important drug target, inhibitors which can specifically inhibit BACE1 without affecting BACE2 should be more selectively potent in preventing AP production. Previous linkage studies suggest that chromosome 21q may harbor a susceptibility gene for late-onset AD (Goldin and Gershon, 1993; Heston et al., 1991; Kehoe et al., 1999; Myers et al., 2002; Myllykangas et al., 2005; Pericak-Vance et al., 1991a). Recently, in a high resolution genome screen, a maximum linkage peak was found with marker D21S1440 which is mapped between the APP and BACE2 genes, 5 Mb from BACE2 and 12 Mb from APP (Blacker et al., 2003). BACE2 haplotype analyses showed that a haplotype H5 was associated with AD, and a second haplotype H7 was associated with protection from AD, providing further evidence for an AD susceptibility locus on chromosome 21q within or close to BACE2 (Myllykangas et al., 2005). These linkage studies are consistent with our results, which show that the expression of BACE2 by lentivirus in primary APP23 neurons can significantly reduce Ap production. Further studies are needed to determine if overexpression of BACE2 in vivo can decrease plaque formation and ameliorate the memory deficits seen in APP23 mice. Our study suggests that potentiation of BACE2 in the elderly may protect against AD pathogenesis. 160 3.5. Reference Acquati, F., Accarino, M. , Nucci, C , Fumagalli, P., Jovine, L., Ottolenghi, S., and Taramelli, R. (2000). The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the down critical region. FEBS Lett 468, 59-64. Andrau, D., Dumanchin-Njock, C , Ayral, E., Vizzavona, J., Farzan, M. , Boisbrun, M. , Fulcrand, P., Hernandez, J. F., Martinez, J., Lefranc-Jullien, S., and Checler, F. (2003). BACE1- and BACE2-expressing human cells: characterization of beta-amyloid precursor protein-derived catabolites, design of a novel fluorimetric assay, and identification of new in vitro inhibitors. J Biol Chem 278, 25859-25866. Basi, G., Frigon, N. , Barbour, R., Doan, T., Gordon, G., McConlogue, L., Sinha, S., and Zeller, M . (2003). Antagonistic effects of beta-site amyloid precursor protein-cleaving enzymes 1 and 2 on beta-amyloid peptide production in cells. J Biol Chem 278, 31512-31520. Bennett, B. D., Babu-Khan, S., Loeloff, R., Louis, J. C , Curran, E., Citron, M. , and Vassar, R. (2000). Expression analysis of BACE2 in brain and peripheral tissues. J Biol Chem 275, 20647-20651. Blacker, D., Bertram, L., Saunders, A. J., Moscarillo, T. J., Albert, M . S., Wiener, H., Perry, R. T., Collins, J. S., Harrell, L. E., Go, R. C , et al. (2003). Results of a high-resolution genome screen of 437 Alzheimer's disease families. Hum Mol Genet 12, 23-32. Busciglio, J., Pelsman, A., Wong, C , Pigino, G., Yuan, M. , Mori, H., and Yankner, B. A. (2002). Altered metabolism of the amyloid beta precursor protein is associated with mitochondrial dysfunction in Down's syndrome. Neuron 33, 677-688. Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D. R., Price, D. L., and Wong, P. C. (2001). BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci 4, 233-234. Charlwood, J., Dingwall, C , Matico, R., Hussain, L, Johanson, K., Moore, S., Powell, D. J., Skehel, J. M. , Ratcliffe, S., Clarke, B., etal. (2001). Characterization of the glycosylation profiles of Alzheimer's beta -secretase protein Asp-2 expressed in a variety of cell lines. J Biol Chem 276, 16739-16748. 161 Christensen, M . A., Zhou, W., Qing, H., Lehman, A., Philipsen, S., and Song, W. (2004). Transcriptional Regulation of BACE 1, the beta-Amyloid Precursor Protein beta-Secretase, by Spl. Mol Cell Biol 24, 865-874. D'Hooge, R., Nagels, G., Westland, C. E., Mucke, L., and De Deyn, P. P. (1996). Spatial learning deficit in mice expressing human 751-amino acid beta-amyloid precursor protein. Neuroreport 7, 2807-2811. De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M . S., Ray, W. J., et al. (1999). A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 598,518-522. Dittgen, T., Nimmerjahn, A., Komai, S., Licznerski, P., Waters, J., Margrie, T. W., Helmchen, F., Denk, W., Brecht, M. , and Osten, P. (2004). Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc Natl Acad Sci U S A 101, 18206-18211. Dominguez, D., Tournoy, J., Hartmann, D., Huth, T., Cryns, K., Deforce, S., Serneels, L., Camacho, I. E., Marjaux, E., Craessaerts, K., et al. (2005). Phenotypic and biochemical analyses of BACE1- and BACE2-deficient mice. J Biol Chem 280, 30797-30806. Ehehalt, R., Michel, B., De Pietri Tonelli, D., Zacchetti, D., Simons, K., and Keller, P. (2002). Splice variants of the beta-site APP-cleaving enzyme BACE1 in human brain and pancreas. Biochem Biophys Res Commun 293, 30-37. Farzan, M. , Schnitzler, C. E., Vasilieva, N. , Leung, D., and Choe, H. (2000). BACE2, a beta -secretase homolog, cleaves at the beta site and within the amyloid-beta region of the amyloid-beta precursor protein. Proc Natl Acad Sci U S A 97, 9712-9717. Fluhrer, R., Capell, A., Westmeyer, G., Willem, M. , Hartung, B., Condron, M . M. , Teplow, D. B., Haass, C , and Walter, J. (2002). A non-amyloidogenic function of BACE-2 in the secretory pathway. J Neurochem 81, 1011-1020. Glenner, G. G., and Wong, C. W. (1984a). Alzheimer's disease and Down's syndrome: sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res Commun 122, 1131-1135. 162 Glenner, G. G., and Wong, C. W. (1984b). Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120, 885-890. Goldin, L. R., and Gershon, E. S. (1993). Linkage of Alzheimer's disease to chromosome 21 and chromosome 19 markers: effect of age of onset assumptions. Genet Epidemiol 10, 449-454. Hardy, J., and Selkoe, D. J. (2002). The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road to Therapeutics. Science 297, 353-356. Heston, L. L., Orr, H. T., Rich, S. S., and White, J. A. (1991). Linkage of an Alzheimer disease susceptibility locus to markers on human chromosome 21. Am J Med Genet 40, 449-453. Hong, L., Koelsch, G., Lin, X. , Wu, S., Terzyan, S., Ghosh, A. K., Zhang, X. C , and Tang, J. (2000). Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor. Science 290,150-153. Hong, L., Turner, R. T., 3rd, Koelsch, G., Shin, D., Ghosh, A. K., and Tang, J. (2002). Crystal structure of memapsin 2 (beta-secretase) in complex with an inhibitor OM00-3. Biochemistry 41, 10963-10967. Hussain, I., Christie, G., Schneider, K., Moore, S., and Dingwall, C. (2001). Prodomain processing of Aspl (BACE2) is autocatalytic. J Biol Chem 276, 23322-23328. Hussain, I., Powell, D., Howlett, D. R., Tew, D. G., Meek, T. D., Chapman, C , Gloger, I. S., Murphy, K. E., Southan, C. D., Ryan, D. M., et al. (1999). Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol Cell Neurosci 14, 419-427. Hussain, I., Powell, D. J., Howlett, D. R., Chapman, G. A., Gilmour, L., Murdock, P. R., Tew, D. G., Meek, T. D., Chapman, C , Schneider, K., et al. (2000). ASP1 (BACE2) cleaves the amyloid precursor protein at the beta-secretase site. Mol Cell Neurosci 16, 609-619. Kao, S. C , Krichevsky, A. M. , Kosik, K. S., and Tsai, L. H. (2004). BACE1 suppression by RNA interference in primary cortical neurons. J Biol Chem 279, 1942-1949. 163 Kehoe, P., Wavrant-De Vrieze, F., Crook, R., Wu, W. S., Holmans, P., Fenton, I., Spurlock, G., Norton, N. , Williams, H., Williams, N., et al. (1999). A full genome scan for late onset Alzheimer's disease. Hum Mol Genet 8, 237-245. Koistinaho, M. , Ort, M. , Cimadevilla, J. M. , Vondrous, R., Cordell, B., Koistinaho, J., Bures, J., and Higgins, L. S. (2001). Specific spatial learning deficits become severe with age in beta -amyloid precursor protein transgenic mice that harbor diffuse beta -amyloid deposits but do not form plaques. Proc Natl Acad Sci U S A 98, 14675-14680. Lin, X. , Koelsch, G., Wu, S., Downs, D., Dashti, A., and Tang, J. (2000). Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci U S A 97, 1456-1460. Lois, C , Hong, E. J., Pease, S., Brown, E. J., and Baltimore, D. (2002). Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868-872. Lott, I. T., and Head, E. (2001). Down syndrome and Alzheimer's disease: a link between development and aging. Ment Retard Dev Disabil Res Rev 7, 172-178. Luo, Y., Bolon, B., Damore, M . A., Fitzpatrick, D., Liu, H., Zhang, J., Yan, Q., Vassar, R., and Citron, M . (2003). BACE1 (beta-secretase) knockout mice do not acquire compensatory gene expression changes or develop neural lesions over time. Neurobiol Dis 14, 81-88. Luo, Y., Bolon, B., Kahn, S., Bennett, B. D., Babu-Khan, S., Denis, P., Fan, W., Kha, H., Zhang, J., Gong, Y., et al. (2001). Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci 4, 231-232. Motonaga, K., Itoh, M. , Becker, L. E., Goto, Y., and Takashima, S. (2002). Elevated expression of beta-site amyloid precursor protein cleaving enzyme 2 in brains of patients with Down syndrome. Neurosci Lett 326, 64-66. Murphy, T., Yip, A., Brayne, C , Easton, D., Evans, J. G., Xuereb, J., Cairns, N. , Esiri, M . M. , and Rubinsztein, D. C. (2001). The BACE gene: genomic structure and candidate gene study in late-onset Alzheimer's disease. Neuroreport 12, 631-634. 164 Myers, A., Wavrant De-Vrieze, F., Holmans, P., Hamshere, M. , Crook, R., Compton, D., Marshall, H., Meyer, D., Shears, S., Booth, J., et al. (2002). Full genome screen for Alzheimer disease: stage II analysis. Am J Med Genet 114, 235-244. Myllykangas, L., Wavrant-De Vrieze, F., Polvikoski, T., Notkola, I. L., Sulkava, R., Niinisto, L., Edland, S. D., Arepalli, S., Adighibe, O., Compton, D., etal. (2005). Chromosome 21 BACE2 haplotype associates with Alzheimer's disease: a two-stage study. J Neurol Sci 236, 17-24. Nowotny, P., Kwon, J. M. , Chakraverty, S., Nowotny, V., Morris, J. C , and Goate, A. M . (2001). Association studies using novel polymorphisms in BACE1 and BACE2. Neuroreport 12,1799-1802. Ohno, M. , Sametsky, E. A., Younkin, L. H., Oakley, H., Younkin, S. G., Citron, M. , Vassar, R., and Disterhoft, J. F. (2004). BACE1 Deficiency Rescues Memory Deficits and Cholinergic Dysfunction in a Mouse Model of Alzheimer's Disease. Neuron 41, 27-33. Ostermann, N. , Eder, J., Eidhoff, U., Zink, F., Hassiepen, U., Worpenberg, S., Maibaum, J., Simic, O., Hommel, U., and Gerhartz, B. (2006). Crystal structure of human BACE2 in complex with a hydroxyethylamine transition-state inhibitor. J Mol Biol 355, 249-261. Pericak-Vance, M . A., Bebout, J. L., Gaskell, P. C , Jr., Yamaoka, L. H., Hung, W. Y., Alberts, M . J., Walker, A. P., Bartlett, R. J., Haynes, C. A., Welsh, K. A., and al., e. (1991). Linkage studies in familial Alzheimer disease: evidence for chromosome 19 linkage. Am J Hum Genet 48, 1034-1050. Podlisny, M . B., Lee, G., and Selkoe, D. J. (1987). Gene dosage of the amyloid beta precursor protein in Alzheimer's disease. Science 238, 669-671. Roberds, S. L., Anderson, J., Basi, G., Bienkowski, M. J., Branstetter, D. G., Chen, K. S., Freedman, S. B., Frigon, N. L., Games, D., Hu, K., et al. (2001). BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet 10,1317-1324. Shen, J., Bronson, R. T., Chen, D. F., Xia, W., Selkoe, D. J., and Tonegawa, S. (1997). Skeletal and CNS defects in Presenilin-1-deficient mice. Cell 89, 629-639. 165 Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., Doan, M. , Dovey, H. F., Frigon, N. , Hong, J., et al. (1999). Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402, 537-540. Solans, A., Estivill, X. , and de La Luna, S. (2000). A new aspartyl protease on 21q22.3, B ACE2, is highly similar to Alzheimer's amyloid precursor protein beta-secretase. Cytogenet Cell Genet 89, 177-184. Song, W., Nadeau, P., Yuan, M. , Yang, X., Shen, J., and Yankner, B. A. (1999). Proteolytic release and nuclear translocation of Notch-1 are induced by presenilin-1 and impaired by pathogenic presenilin-1 mutations. Proc Natl Acad Sci U S A 96, 6959-6963. Sturchler-Pierrat, C , Abramowski, D., Duke, M. , Wiederhold, K. H., Mistl, C , Rothacher, S., Ledermann, B., Burki, K., Frey, P., Paganetti, P. A., et al. (1997). Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. Proc Natl Acad Sci U S A 94, 13287-13292. Sun, X., Tong, Y., Qing, H., Chen, C , and Song, W. (2006). Increased BACE1 maturation contributes to Alzheimer's Disease pathogenesis in Down Syndrome. Faseb J In Press. Sun, X., Wang, Y., Qing, H., Christensen, M . A., Liu, Y., Zhou, W., Tong, Y., Xiao, C , Huang, Y., Zhang, S., et al. (2005). Distinct transcriptional regulation and function of the human BACE2 and BACE1 genes. FASEB J 19, 739-749. Van Dam, D., D'Hooge, R., Staufenbiel, M. , Van Ginneken, C , Van Meir, F., and De Deyn, P. P. (2003). Age-dependent cognitive decline in the APP23 model precedes amyloid deposition. Eur J Neurosci 17, 388-396. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., et al. (1999). Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286, 735-741. Wong, P. C , Zheng, H., Chen, H., Becher, M . W., Sirinathsinghji, D. J., Trumbauer, M . E., Chen, H. Y., Price, D. L., Van der Ploeg, L. H., and Sisodia, S. S. (1997). Presenilin 1 is required for Notchl and D i l l expression in the paraxial mesoderm. Nature 387, 288-292. 166 Yan, R., Bienkowski, M . J., Shuck, M . E., Miao, H., Tory, M . C , Pauley, A. M. , Brashier, J. R., Stratman, N . C , Mathews, W. R., Buhl, A. E., et al. (1999). Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature 402, 533-537. Yan, R., Munzner, J. B., Shuck, M . E., and Bienkowski, M . J. (2001). BACE2 functions as an alternative alpha-secretase in cells. J Biol Chem 276, 34019-34027. Zhang, Z., Nadeau, P., Song, W., Donoviel, D., Yuan, M. , Bernstein, A., and Yankner, B. A. (2000). Presenilins are required for gamma-secretase cleavage of beta-APP and transmembrane cleavage of Notch-1. Nat Cell Biol 2,463-465. 167 CHAPTER 4: Increased B A C E 1 Maturation contributes to Alzheimer's Disease pathogenesis in Down Syndrome 3 A version of this part was previously published: Sun X, Tong Y, Qing H, Chen A, and Song W (2006) Increased BACE1 Maturation contributes to Alzheimer's Disease pathogenesis in Down Syndrome. The FASEB Journal 20 (9):1361-8. 168 4.1. Introduction Down Syndrome (DS) is the most common genetic cause of mental retardation, affecting about 1 in 700 to 1000 live births (Down, 1866; Jacobs et al., 1959; Lejeune J, 1969). The majority of DS cases are caused by an extra copy of chromosome 21, thereby also known as Trisomy 21. AD is the most common neurodegenerative disease leading to dementia. Deposition of Ap in the brain is the hallmark of the AD pathology (Mattson, 2004). Ap, the major component of neuritic plaques, is derived from APP following sequential cleavage by P-secretase and y-secretase (Selkoe, 2001). BACE1 has been identified as the major P-secretase in vivo (Cai et al., 2001; Hussain et al., 1999; Luo et al., 2001; Roberds et al., 2001b; Sinha et al., 1999; Vassar et al., 1999; Yan et al , 1999). BACE1 cleaves APP at the major Asp+1 site and a minor Glu+11 of Ap. BACE1 is the p-secretase to process APP (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001a; Sun et al., 2005). BACE2 is a homolog of BACE1 and located on critical region of chromosome 21 (Bennett et al., 2000a; Lin et al., 2000; Yan et al., 1999). However, we recently reported that despite being homologous in amino acid sequence, BACE2 and BACE1 have distinct functions and transcriptional regulation, and BACE2 is not a P-secretase (Sun et al., 2005). In the previous chapter, we showed that BACE2 processes APP at a novel 9-secretase site within the AP domain, which precludes Ap production. BACE1 undergoes a complex set of post-translational modifications during its maturation. Pro-BACEl is cleaved by furin and other members of the furin family of convertases to remove the 24-amino acid N-terminal region of the pro-peptide within the trans-Golgi network (TGN) (Benjannet et al., 2001; Bennett et al., 2000b; Capell et al., 169 2000; Creemers et al., 2001). The 24-amino acid prodomain is required for the efficient exit of pro-BACEl from the endoplasmic reticulum (Benjannet et al., 2001). The majority of BACE1 is located in Golgi and endosomal compartments. Mature BACE1 has four TV-glycosylation sites at Asnl53, 172, 223, and 354, and the p-secretase activity is dependent on the extent of N-glycosylation (Capell et al., 2000; Charlwood et al., 2001; Haniu et al., 2000; Huse et al., 2000). The cytoplasmic domain of B ACE1 and its phosphorylation are required for efficient maturation and its intracellular trafficking through the TGN and endosomal system (Capell et al., 2000; Huse et al., 2000; Walter et al., 2001). BACE1 is also processed between Leu 2 2 8 and Ala 2 2 9 to generate stable N- and C-terminal fragments that remain covalently associated via a disulfide bond (Huse et al., 2003). BACE1 forms a dimer prior to its full maturation and pro-peptide cleavage and dimerization of BACE may help APP binding and cleavage (Schmechel et al., 2004; Westmeyer et al., 2004). BACE1 also interacts with reticulon family member proteins, and reticulon proteins block access of BACE 1 to APP and reduce the APP cleavage (He et al., 2004). The degradation of BACE 1 is mediated by the ubiquitin proteasome pathway and the proteasomal degradation of BACE 1 regulates APP processing and Ap generation (Qing et al., 2004). BACE1 gene is tightly regulated at the transcription level (Christensen et al., 2004; Sun et al., 2005). Although genetic analysis has failed to uncover any BACE1 coding sequence mutation in the patients with familial AD (Cruts et al., 2001; Nicolaou et al., 2001), increased P-secretase activity was reported in some FAD brains (Russo et al., 2000) and greater expression level of B ACE1 in the cortex of sporadic AD patients versus age-matched controls (Fukumoto et al., 2002; Fukumoto et al., 2004; Holsinger et al., 2002; Yang et al., 2003). BACE1-KO mice, without 170 developmental deficits, have abolished Ap generation (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001a). Disruption of BACE 1 gene rescues memory deficits and cholinergic dysfunction in the Swedish APP mutant mice (Ohno et al., 2004). Suppression of BACE1 siRNA also reduced Aft production in APP mutant transgenic neurons (Kao et al., 2004). In addition to APP, BACE1 substrates also include the low density lipoprotein receptor-related protein (LRP) (von Arnim et al., 2005), APLP1 (Li and Sudhof, 2004), APLP2 (Pastorino et al., 2004), a Golgi-resident sialyltransferase ST6Gal I (Kitazume et al., 2001), the cell adhesion protein P-selectin glycoprotein ligand-1 (PSGL-1), (Lichtenthaler et al., 2003). Brains of DS patients are characteristically small, rounded, and exhibit an extreme narrowing of the superior temporal gyri. After middle age, people with DS inevitably develop characteristic AD neuropathology including neuritic plaques, neurofibrillary tangles and cerebral angiopathy (Glenner and Wong, 1984; Hardy and Selkoe, 2002). The levels of APP C-terminal fragment C99, the major p-secretase product, and Ap are increased in DS (Busciglio et al., 2002). While the additional copy of the APP gene is seen in 99% of DS, the onset age of AD in DS varies significantly (Mutton et al., 1996), and the gene dosage effect cannot fully account for the occurrence of AD in DS (Podlisny et al., 1987). The mechanism underlying the pathogenesis of AD in DS remains unknown. To investigate the molecular mechanism by which AD neuropathology develops in DS patients, we examined the role of BACE 1 gene in APP processing and Ap generation in DS using cerebral cortical tissues. In this report, the level of total BACE1 171 proteins, and particularly the mature form of BACE1, is significantly increased in DS. Time-lapse live image analysis revealed that BACE1 proteins were predominantly immobile and accumulated in Golgi in DS cells, while trafficking in controls was normal. Our study demonstrates that the abnormal BACE1 protein trafficking and accumulation contribute to the increased P-secretase activity, and subsequently AP generation in DS. Our results provide a novel molecular mechanism by which AD develops in DS. 4.2. Materials and methods 4.2.1. Cell culture, plasmids and transfection Human embryonic kidney HEK293 cells (ATCC number CRL-1573), were grown in Dulbecco's modified Eagle's medium (DMEM, from Invitrogen) containing 10% FBS, ImM of sodium pyruvate, 2mM of L-glutamine and 50 unit of Penicillin and 50 pg of Streptomycin. Media were changed every 2-3 days. 2EB2 cells were HEK293 cells stably overexpressing APP Swedish mutant and BACE1 under selection of Geneticin and Zeocine (from Invitrogen) respectively. Al l cells were maintained at 37°C in an incubator containing 5% C O 2 . For transient transfection, cells were grown to approximately 70% confluency and transfected with plasmids using LipofectAMINE2000 (Invitrogen) according to the manufacturer's instructions. The cells were harvested 48-72 hrs following transfection. Human Trisomy-21 (47, X X , +21) and control (46, XX) fibroblast cells were derived from 18 weeks of gestation fetal abortuses. The cells were cultured in same media with HEK293 cells. The identities of the cell lines were further 172 confirmed by karyoptyping. Trisomy 21 cells and control cell line were transfected with LipofectaminePlus reagent (from Invitrogen) following the manufacture's protocol. APP C99 and C83 cDNA were amplified by PCR and then cloned into pcDNA3 vector (Invitrogen) to generate mammalian expression plasmid pAPP-C99 and pAPP-C83. pAPP-C99 and pAPP-C83 were transfected into cells and the expressed fragments were used as the APP CTF protein markers. BACE1 was PCRed and cloned into pEGFP-Nl to get plasmid pBACEl-EGFP with EGFP fused with BACE1 at its C-terminus. 4.2.2. Western blot The frozen brain tissues were homogenized in RIPA lysis buffer (1% Triton X100,1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.05M Tris-HCl, pH 7.2) supplemented with protease inhibitors cocktail Complete (Roche) and sonicated. Tissue lysates were resolved by 12% Tris-Glycine or 16% Tris-Tricine gels. Immunoblotting was performed as previously described (Qing et al., 2004). BACE1 was detected by 208 antibody against C-terminus of BACE1 (RCLRCLRQQHDDFADD) (Qing et a l , 2004). APP was detected by the C20 antibody against the last 20 amino acids of its C-terminus. Internal control (3-actin expression was analyzed using monoclonal anti-beta-actin antibody AC-15 (Sigma). 173 4.2.3. Immunoprecipitation Cells of 10 cm plate were harvested into 1ml RIPA (0.1% SDS) and sonicated. lOOul CL-4B was added into the cell lysate and shaken for one hour at 4 degrees. The cell lysates were pelleted at top speed for 10 minutes at 4 degree. 20ul of the capture antibody 208 and 25ul protein A/G were added into the supernatant in a new Eppendorf tube. The reaction was incubated for overnight at 4 degrees with shaking. The precipitates were centrifuged and washed three times with RIPA buffer. lOul 2x tris-tricine sample buffer was added into the precipitate and boiled for 5 minutes before loading onto the 15% tris-glycine gel to resolve the protein. 4.2.4. Ap40/42 sandwich ELISA assay ELISA assay was performed as previously described (Qing et al., 2004). In brief, protein inhibitors and AEBSF (Sigma) were added to tissue lysates to prevent the degradation of Ap. The concentration of AP40/42 was measured using the P-amyloid 1-40 or 1-42 Colorimetric ELISA kit (Biosource International, Inc.) according to the manufacturer's protocol. 4.2.5. Quantitative R T - P C R and real time P C R Total RNA was isolated from frozen brain tissue using TRI-Reagent (Sigma). PowerScript™ reverse transcriptase (Invitrogen) was used to synthesize the first strand cDNA from equal amount of the RNA sample following the manufacturer's instruction. The newly synthesized cDNA templates were further amplified by Platinum Tag DNA polymerase (Invitrogen) in a 50ul reaction. 25 to 35 cycles of PCR reaction were used to 174 cover the linear range of the PCR amplification. The BACE1 gene-specific primers 5'-cggaattcgccaccatgaccgacgaagagcccgag-3' and 5'-cgggatcccacaatgctcttgtcatag-3' were used to amplify a 725 bp fragment of the BACE1 coding region. The APP gene-specific primers 5'-cggaattcccttggtgttctttgcagaag-3' and 5'-cggaattccgttctgcatctgctcaaag-3' were used to amplify a 248bp fragment of the APP coding region. (3-Actin was used as an internal control. A pair of gene-specific primers 5'-ggacttcgagcaagagatgg-3' and 5'-gaagcatttgcggtggag-3' was used to amplify a 462 bp fragment of beta-Actin. The samples were further analyzed on 1 % agarose gel. Kodak Image Station 1000 software (Perkin Elmer) was used to analyze the data. Real-time PCR was performed with the TaqMan® Universal PCR Master Mix (Applied Biosystems) using the Smart Cycler II (Cephoid) according to the manufacturer's instructions. The sequence of BACE1 primers are 5'-gcccaagaaagtgtttgaagct-3' and 5' -gccagaaaccatcagggaact -3'. The Taqman probe for BACE1 is FAM™ -5'-aatccatcaaggcagcctcctccac-3' -TAMRA™ . The sequence for actin primers are 5'-aggccaaccgcgagaag-3' and 5'-acagcctggatagcaacgtacat-3'. The probe for actin is TET™-5' tgacccagatcatgtttgagacctt-3' TAMRA™. 4.2.6. Karyotyping of DS cell lines 5ul of lmg/ml colchicines solution (5ug/ml final) was added to each culture (1.5ml) and incubate for another 3 hours. The cells was harvested by centrifugation and cells were swelled in 0.075% KCI for 30 minutes and then fixed in 3ml fresh made fixative (methanol: glacial acetic acid, 3: 1) for 30 minutes. Fixing was repeated for three 175 times before 300-500ul fixative was added to suspend the nuclei. Cell suspension was dropped onto the pre-cooled slides (washed with sulfate and ddHiO and store at -20 degree) in a height of about 30cm. The slides were aged at 75 degree for at least 3 hours. The slides were trypsinized with 0.025% trypsin (from Signma) for 1 minute to be determined. The slides were rinsed with 0.9% NaCl solution twice and stained with Giesma solution (from Sigma) for 10 min. The karyotype of the cells was analyzed under a microscope. 4.2.7. Time-lapse live cell image analysis Trisomy-21 and control cells were plated onto 35mm glass-bottom micro well dish (MatTek) at a density of 100,000 per plate. Plasmid pBACEl-EGFP with EGFP fused with BACE1 at its C-terminus was transfected into Trisomy-21 and control cells using Lipofectamine Plus (from Invitrogen). Live cell image analysis was performed 48 hours after transfection. Time-lapse images were recorded with the Axiovert 200M microscope using an AxioCam HRm Rev.2 camera. The objective lens of the microscope was Plan Apochromat 63x/1.40 Oil (DIC III). The images were acquired every 500 milliseconds. 4.2.8. Cell fractionation The brain tissues were homogenized in FfB Buffer (0.25M sucrose, 5mM Hepes PH7.4, ImM EDTA, 3mM imidazole with protease/phosphotase inhibitors) on ice by passing a 23G1 needle 5 times. A discontinuous 4 step sucrose gradient was made by loading 1ml 2M, 1.5ml 1.3M, 1.5ml 1.0M and 1ml 0.6M sucrose into an ultracentrifuge tube. The homogenate was loaded onto the sucrose cushion and centrifuged at 45,000rpm 176 for 2 hours using a SW50.1 Bechman rotor. Golgi fraction located at the 1.0M/0.6M sucrose interface was collected and lysed with the 6x SDS loading buffer. The fractions were separated on a 15% SDS-glycine gel and blotted with 208 antibody to detect BACE1 in the Golgi fractions. 4.2.9. Pulse-chase assay Cells were transfected with BACEl-mychis cDNA in a 10-cm plate with LipofectaminePlus. 48 hours after transfection, the cells were starved in Methionine-free DMEM for 30 minutes and then radiolabeled in a medium containing 125pCi/ml of 3 5 S-methionine and 35S-cysteine for 2 hours. Subsequently the cells were chased for 0, 4 and 9 hours with non-radioactive media with 10 X excess of methionine and cysteine. The cell lysates were irnmunoprecipitated with the anti-BACEl 208 antibody. The radiolabeled proteins were separated on a 12% SDS-Glycine gel and quantitated by INSTANT-IMAGER (Packard Bioscience Company). 4.2.10. Tunicamycin treatment 2EB2 cells were treated with 10 p,g/ml tunicamycin (Sigma) for 0, 3 and 6 hours. The cells were harvested in RIPA lysis buffer and sonicated. The lysates were separated using a 16% Tris-Tricine gel. APP C99 and C89 fragments were detected by the C20 antibody, p-actin detected by anti-P-actin (AC-15) was used as the control. 177 4.3. Results 4.3.1. Markedly elevated P-secretase activity in DS brains Abnormal APP processing has been implicated in DS. To investigate the role of R-secretase in APP processing and A p generation in DS, we first measured the APP, C99 and A P levels in DS and control samples. The fetal cortical tissues from Trisomy-21 and gestation age-matched control brains were homogenized for protein extraction. The Ap level in the DS and control tissues were measured by a colorimetric ELISA method. ELISA results showed that the AP40 and AP42 levels were significantly elevated by 172.50 ± 8.90 % and 152.70 ± 10.10 %, respectively in the DS patients compared to the normal controls (P < 0.0001) (Figure 4.1 A and B). To assay the levels of APP and APP C-terminal fragments (CTFs), the tissue lysates were separated in SDS-PAGE gels and immunoblotted with an anti-APP C-terminus C20 antibody. The level of APP protein was slightly elevated in DS by 121.2 ± 18.47 %, relative to control (P > 0.05) (Figure 4.1C and E). Compared to control samples, the level of C99 in DS was markedly increased by 490.0 ± 96.29 % (P < 0.0001) (Figure 4.1D and F). These results are consistent with previous report that C99 and A P are increased in DS (Busciglio et al., 2002). The increased APP levels in DS can be attributed to the extra copy of the APP gene in Trisomy-21. However, the drastic increase in the level of C99, the major P-secretase product, suggests a significant increase in p-secretase activity. The increased APP substrate level only partially contributes to the overgeneration of Ap in DS. 178 Figure 4.1. Marked elevation of Ap and C 9 9 , the major P-secretase product in the DS brains. The cerebral cortical tissues from 16 to 20 weeks of gestation fetal abortuses of seven DS and seven age-matched controls were lysed in RIPA buffer (1% Triton XI00, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.05M Tris-HCl, pH 7.2) supplemented with protease inhibitors cocktail Complete (Roche) and AEBSF (Sigma). The concentrations of AP40 (A) and AP42 (B) were measured by P-amyloid 1-40 or 1-42 Colorimetric ELISA kit (Biosource International, Inc.). The values represent mean ± S.E.M., n = 7. (C). 150 p.g of total protein from control and DS brain tissue lysates was separated by 12% SDS-PAGE with C20 antibody to detect APP proteins, p-actin was detected by the monoclonal anti P-actin antibody AC-15 (Sigma). C represents control . samples. (D) The brain samples were separated in 16% Tris-Tricine gel and immunoblotted with C20 to detect the APP C-terminal fragments. Plasmid pAPP-C99 and pAPP-C83 were transfected into HEK293 cells and the cell lysates were used as the protein size markers for C99 and C83 fragments. Quantitative analysis of full length APP (E) and APP C99 (F). Western blots were quantified using Kodak Image Analysis. 179 > | 1 5 t H 0 O100-w 0 B C o n t r o l D S 0 9 15 - 1 , 9 iooi CM ~ S. O 50] < ^ ^ 0 C o n t r o l D S C C C D S D S D S • • • I - A P P f i - a c t i n D C C C C D S D S D S - C 9 9 - C 8 3 4.3.2. Increased B A C E 1 total and glycosylated protein levels in the DS brains APP C99 is produced by P-secretase cleavage of APP at the P site. Since the marked increase in C99 in DS indicated increased P-secretase activity, we then examined whether BACE1 is upregulated and responsible for the abnormal APP processing in DS. 180 The brain tissue lysates from DS and control were analyzed by SDS-PAGE gel and immunoblotted with BACE1 antibody 208 (Figure 4.2A). The results showed that the BACE1 protein level was markedly elevated in DS (215.31 ± 39.40 %) compared to control (P < 0.01) (Figure 4.2B). More importantly, there was a change in the ratio of the mature and immature forms of BACE1 protein in DS. The mature form of BACE1 was predominant in DS, while the immature form of BACE 1 was predominant in the control tissues. The ratio of mature to immature forms of BACE 1 was significantly higher in DS (389.30 ± 27.56 %) than in control (Figure 4.2C) (P < 0.0001). The results clearly demonstrate that abnormal BACE1 protein level causes upregulated p-secretase activity, resulting in more AP generation leading to the AD neuropathology in DS. Figure 4.2. Significant increase in total and mature B A C E 1 protein levels in the DS brains (A) The brain tissue lysates were separated on a 15% Tris-Glycine gel and immunoblotted with the 208 antibody to detect BACEl.(B). Quantitative analysis of total BACE1 protein levels by Kodak Image Analysis. (C) Quantitative analysis of the ratio of mature BACE1 vs. immature BACE1 levels. Values are means ± S.E.M. and n-l. The total protein levels are expressed as a percentage of the levels in control. * p < 0.05 by the student t test. 181 c c c DS DS DS BACE1 -mature --immature actin B ^ 2 5 0 1 * 2 0 0 m 150 too aj 50 o 0 Q_ Control DS Control 4.3.3. BACE1 transcription was unchanged in DS To investigate if the abnormal BACE1 protein increase resulted from upregulation of BACE1 gene transcription in DS, quantitative Reverse Transcription-PCR was used to measure its mRNA levels in DS and normal controls (Figure 4.3A). The APP level was increased in DS brain tissues by 142.2 ± 17.61 % relative to controls (P < 0.05) (Figure 4.3B). Such increases might be due to the extra copies of the APP gene on chromosome 21 in DS. The BACE1 mRNA levels were unchanged (92.32 ±2.154 %) (P> 0.05) (Figure 4.3B). Similar result was obtained by real-time PCR. In Figure 4.2 we show that the protein level of BACE1 was significantly increased, resulting in upregulated R-secretase activity. Since the BACE1 mRNA levels were not changed in DS, this suggests that the elevation of BACE1 in DS is not due to abnormal BACE1 transcription. 182 Figure 4.3. BACE1 transcription was unchanged in DS patients whereas APP mRNA was increased by about 1.5 times. (A) RNA was isolated from DS and control brain tissues. Quantitative RT-PCR was performed to measure the endogenous levels of the APP, BACE1 mRNA. Specific APP, BACE1 and P-actin coding sequence primers were used to amplify the cDNA. Different cycles and amounts of PCR products were analyzed and the DNA gel represents 25 cycles of RT-PCR products separated on 1 % agarose gel. (B). The ratio of APP, BACE1 to P-Actin mRNA in DS and age-matched controls were quantitated by Kodak Image Analysis. Endogenous APP mRNA level was increased in DS relative to controls. *p < 0.05 relative to controls by student t test. Endogenous BACE1 mRNA level is similar between DS and controls (p > 0.05 by the student t test). Shown are the mean ± S.E.M. C DS DS DS mmJtgtjgm^ jga-jjfagjBigeg. .^^^ajtajj^. TMlfn tf tl |TI fiJf ill ^ B P B W W flP|HWP!BB»Bg. iwHBInBRIW^ •ISISU-"--"1* -—p-actin -APP BACE1 p-actin • Con t ro l • D S APP BACE1 183 4.3.4. Time-lapse analysis of BACE1 trafficking in DS cell lines To define the mechanism by which the mature BACE1 protein is abnormally increased in DS, we examined the trafficking and post-translational modifications of BACE1. To examine the trafficking of BACE1 protein, the Trisomy-21 and control fibroblast cells derived from 18 weeks of gestation fetal abortuses were transfected with EGFP-tagged BACE1 cDNA and time-lapse images were recorded on the live cells to monitor the BACE1-EGFP protein trafficking (Figure 4.4). In the control cells, we observed normal patterns of BACE 1 protein trafficking; BACE1 fluorescent particles moved smoothly and rapidly through subcellular compartments, mostly inside ER and Golgi complex (Figure 4.4, top panel). However, in the Trisomy-21 cells, BACE1 fluorescent proteins were predominantly immobile in Golgi-like complexes (Figure 4.4, lower panel), suggesting abnormal protein trafficking. Transient fluorescent signals were observed in the ER and Golgi complexes of control cells, while abnormally prolonged signals were observed in Trisomy-21 cells (Figure 4.4). Figure 4.4. Time-lapse live imaging analysis of BACE1 trafficking in DS cell lines. Live image analysis of the cells transfected with pBACEl-EGFP. Time-lapse images were recorded with Axiovert 200M microscope using the Plan Apochromat 63x/1.40 Oil (DIC III) objective lens. The images were acquired every 500 milliseconds. Arrows indicate the trafficking of BACE1-EGFP particles. Top panel: Representative images of the live control cells at the 1st, 10th, 20 th and 30 th frames (C-l to C30) illustrating quick BACE1 movement; Lower panel: the images of the live Trisomy-21 cells at the 1st, 10th, 20 th and 30 th frames (DS-1 to DS-30) illustrating immobile EGFP-BACE1 particles. 184 C-t 0 0 0 • 0 * • '•«P-"'i-;::-0 0 H H H H H H H H i NHMSMNNHM DS-1 0 0 DS-K5 0 > DS-20 0 .:.r',,-.; 0 f 4.3.5. Abnormal BACE1 protein trafficking and accumulation in Golgi of DS cells To examine the abnormal accumulation of BACE1 in DS, the Golgi samples were extracted from brain tissues. Subcellular fractionation experiment revealed that while there were similar levels in mature and immature BACE1 protein in the Golgi fraction of controls, mature form of BACE1 proteins were markedly increased in the Golgi fraction of DS samples (Figure 4.5A). Furthermore, pulse-chase experiments showed that both mature and immature BACE1 proteins were detected and the half-life of the newly-synthesized proteins were about 9 hours in the control fibroblast cells. However, the predominant BACE1 proteins in the DS cell samples were mature form and there was slightly longer half-life of the protein (Figure 4.5B). These data indicated that BACE1 proteins underwent abnormal trafficking and accumulated in the DS cells. BACE1 undergoes a complex set of posttranslational modifications including phosphorylation, glycosylation and ubiquitination during its maturation and degradation (Capell et al., 2000; Huse et al., 2000; Walter et al., 2001). BACE1 has 4 N-glycosylation sites at Asn-153, -172, -223 and -354 (Capell et al., 2000; Charlwood et al., 2001; Huse et al., 2000). 185 Our data indicate that there are more glycosylated mature BACE1 proteins in DS than controls. To determine if glycosylation affects the BACE1 protease activity, BACE1 stable cells were treated with Tunicamycin, an inhibitor of N-acetylglucosamine transferases. Tunicamycin treatment significantly reduced the generation of the (3-secretase cleavage product, C99 and C89 fragments in 2EB2 cells (Figure 4.5C). This result suggests that inhibition of the BACE1 glycosylation reduces APP processing at the P-secretase site. This is consistent with previous reports that the BACE1 activity depends on the extent of N-glycosylation (Capell et al., 2000; Charlwood et al., 2001). Figure 4.5. Abnormal trafficking and accumulation of mature BACE1 protein in DS. (A). Fractionation of brain tissue lysates from Trisomy-21 and control. Golgi fractions were collected and separated by 15% SDS-Glycine gel. 208 antibody was used to detect BACE1. Monoclonal anti p7actin antibody AC 15 was used to detect P-actin. Mature BACE1 is significantly increased in the Golgi compartment of DS samples. (B) . BACE1 Pulse-Chase experiment. Trisomy-21 and control cells transfected with pBACEl-mychis were incubated with 35S-methione and 35S-cystine labeling media for 2 hrs, and then replaced with non-radioactive chasing media. The cell lysates were immunoprecipitated with the 208 antibody and separated with 15% SDS-glycine gel. While control cells show the newly-synthesized mature and immature 3 5 S-BACE proteins, Trisomy-21 cells predominantly show mature form. (C). Tunicamycin inhibits P-secretase activity. BACE1 stable 2EB2 cells were treated with Tunicamycin at 10 pg/ml for 0, 3 and 6 hours. The cell lysates were analyzed by 16% Tris-Tricine gel with C20 antibody to detect APP C99 and C89. Tunicamycin treatment markedly reduced P-secretase activity, resulting in lower levels of C99 and C89 generation, without affecting P-actin. 186 B A C E 1 M a t u r e I m m a t u r e [ 3 - a c t i n B C o n t r o l 0 4 T r i s o m y 2 1 0 4 9 C h a s i n g (hr) — M a t u r e I m m a t u r e T u n i c a m y c i n 0 3 6 ( h r ) — 1 — l l i l l l l l l l l l M l l l i l II11II l O l l l i l g B M M T K - C 9 9 - C 8 9 -p-actin 4.4. Discussion AD is the most common neurodegenerative disease leading to dementia. Almost all Down Syndrome (DS) patients inevitably develop characteristic AD neuropathology. The mechanism by which AD neuropathogenesis develops in DS is previously unknown. It was reported that altered APP metabolism was associated with mitochondrial dysfunction in DS (Busciglio et al., 2002). Since the APP and BACE2 genes are on chromosome 21 and there are extra copies of these two genes in DS, it was speculated that the extra copy of the genes might therefore play a role in the abnormal processing of APP in DS. While the additional copy of the APP gene is seen in 99% of DS, the gene 187 dosage effect cannot fully account for the occurrence of AD in DS (Podlisny et al., 1987). In this report, our data show that the transcription of the APP was indeed increased in DS; however, the APP protein level was not significantly changed in DS. The slightly increased APP protein level cannot fully explain the AD pathogenesis in DS patients. Our data show that P-secretase activity is upregulated in DS. Increased BACE1 protein levels, and particularly the higher levels of N-glycosylated mature BACE1 proteins in DS, result in upregulated P-secretase activity, leading to higher C99 production and Ap generation. The cause of the BACE1 elevation and increased glycosylation in DS is unknown. The BACE1 elevation is not due to increased transcription, and may therefore be explained by its post-translational modifications, and particularly the reduction of BACE 1 degradation. We previously showed that BACE1 is degraded by the ubiquitin-proteasome pathway (Qing et al., 2004). Further study on the proteasomal degradation of BACE1 in DS is needed. The abnormal trafficking and drastic elevation of BACE1 and subsequent increase in Ap deposition in DS would explain the development of AD pathogenesis in DS. Our study in previous chapters excluded the involvement of BACE2 in AD pathogenesis in DS, by showing that BACE2 cleaves within AP as a novel 0-secretase and decreases Ap production. Thus, our results provide a novel mechanism by which AD develops in DS and suggest the therapeutic potential of inhibiting BACE1 or potentiating BACE2 in AD and DS. 188 4.5. Reference Benjannet, S., Elagoz, A., Wickham, L., Mamarbachi, M. , Munzer, J. S., Basak, A., Lazure, C , Cromlish, J. A., Sisodia, S., Checler, R, etal. (2001). Post-translational processing of beta-secretase (beta-amyloid-converting enzyme) and its ectodomain shedding. The pro- and transmembrane/cytosolic domains affect its cellular activity and amyloid-beta production. J Biol Chem 276, 10879-10887. Bennett, B. D., Babu-Khan, S., Loeloff, R., Louis, J. C , Curran, E., Citron, M. , and Vassar, R. (2000a). Expression analysis of BACE2 in brain and peripheral tissues. J Biol Chem 275, 20647-20651. Bennett, B. D., Denis, P., Haniu, M. , Teplow, D. B., Kahn, S., Louis, J. C , Citron, M. , and Vassar, R. (2000b). A furin-like convertase mediates propeptide cleavage of BACE, the Alzheimer's beta -secretase. J Biol Chem 275, 37712-37717. Busciglio, J., Pelsman, A., Wong, C , Pigino, G., Yuan, M. , Mori, H., and Yankner, B. A. (2002). Altered metabolism of the amyloid beta precursor protein is associated with mitochondrial dysfunction in Down's syndrome. Neuron 33, 677-688. Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D. R., Price, D. L., and Wong, P. C. (2001). BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci 4, 233-234. Capell, A., Steiner, H., Willem, M. , Kaiser, H., Meyer, C , Walter, J., Lammich, S., Multhaup, G., and Haass, C. (2000). Maturation and pro-peptide cleavage of beta-secretase. J Biol Chem 275, 30849-30854. Charlwood, J., Dingwall, C , Matico, R., Hussain, I , Johanson, K., Moore, S., Powell, D. J., Skehel, J. M. , Ratcliffe, S., Clarke, B., et al. (2001). Characterization of the glycosylation profiles of Alzheimer's beta -secretase protein Asp-2 expressed in a variety of cell lines. J Biol Chem 276, 16739-16748. Christensen, M . A., Zhou, W., Qing, H., Lehman, A., Philipsen, S., and Song, W. (2004). Transcriptional regulation of BACE 1, the beta-amyloid precursor protein beta-secretase, by Sp 1. Mol Cell Biol 24, 865-874. Creemers, J. W., Ines Dominguez, D., Plets, E., Serneels, L., Taylor, N. A., Multhaup, G., Craessaerts, K., Annaert, W., and De Strooper, B. (2001). Processing of beta-secretase by furin and other members of the proprotein convertase family. J Biol Chem 276,4211-4217. 189 Cruts, M. , Dermaut, B., Rademakers, R., Roks, G., Van den Broeck, M. , Munteanu, G., van Duijn, C. M. , and Van Broeckhoven, C. (2001). Amyloid beta secretase gene (BACE) is neither mutated in nor associated with early-onset Alzheimer's disease. Neurosci Lett 313,105-107. Down, J. L. H. (1866). Observations on an ethnic classification of idiots. London Hospital Reports 3, 259-262. Fukumoto, H., Cheung, B. S., Hyman, B. T., and Irizarry, M . C. (2002). Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease. Arch Neurol 59, 1381-1389. Fukumoto, H., Rosene, D. L., Moss, M . B., Raju, S., Hyman, B. T., and Irizarry, M . C. (2004). Beta-secretase activity increases with aging in human, monkey, and mouse brain. Am J Pathol 164, 719-725. Glenner, G. G., and Wong, C. W. (1984). Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120, 885-890. Haniu, M. , Denis, P., Young, Y., Mendiaz, E. A., Fuller, J., Hui, J. O., Bennett, B. D., Kahn, S., Ross, S., Burgess, T., et al. (2000). Characterization of Alzheimer's beta -secretase protein BACE. A pepsin family member with unusual properties. J Biol Chem 275,21099-21106. Hardy, J., and Selkoe, D. J. (2002). The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road to Therapeutics. Science 297, 353-356. He, W., Lu, Y., Qahwash, I., Hu, X . Y., Chang, A., and Yan, R. (2004). Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation. Nat Med. Holsinger, R. M. , McLean, C. A., Beyreuther, K., Masters, C. L., and Evin, G. (2002). Increased expression of the amyloid precursor beta-secretase in Alzheimer's disease. Ann Neurol 57,783-786. Huse, J. T., Byant, D., Yang, Y., Pijak, D. S., D'Souza, I., Lah, J. J., Lee, V. M. , Doms, R. W., and Cook, D. G. (2003). Endoproteolysis of beta-secretase (beta-site amyloid precursor protein-cleaving enzyme) within its catalytic domain. A potential mechanism for regulation. J Biol Chem 278, 17141-17149. 190 Huse, J. T., Pijak, D. S., Leslie, G. J., Lee, V. M. , and Doms, R. W. (2000). Maturation and endosomal targeting of beta-site amyloid precursor protein-cleaving enzyme. The Alzheimer's disease beta-secretase. J Biol Chem 275, 33729-33737. Hussain, I., Powell, D., Howlett, D. R., Tew, D. G., Meek, T. D., Chapman, C , Gloger, I. S., Murphy, K. E., Southan, C. D., Ryan, D. M., et al. (1999). Identification of a novel aspartic protease (Asp 2) as beta-secretase. MOl Cell Neurosci 14,419-427. Jacobs, P. A., Baikie, A. G., Court Brown, W. M. , and Strong, J. A. (1959). The somatic chromosomes in mongolism. Lancet 1, 710. Kao, S.-C, Krichevsky, A. M. , Kosik, K. S., and Tsai, L.-H. (2004). BACE1 Suppression by RNA Interference in Primary Cortical Neurons: J Biol Chem 279, 1942-1949. Kitazume, S., Tachida, Y., Oka, R., Shirotani, K., Saido, T. C , and Hashimoto, Y. (2001). Alzheimer's beta-secretase, beta-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransferase. Proc Nad Acad Sci U S A 98,13554-13559. Lejeune J, T. R., Gautier M . (1969). Mongolism: a chromosomal disease (trisomy). Bull Acad Natl Med 143, 256-265. Li , Q., and Sudhof, T. C. (2004). Cleavage of amyloid-beta precursor protein and amyloid-beta precursor-like protein by BACE 1. J Biol Chem 279, 10542-10550. Lichtenthaler, S. F., Dominguez, D. I., Westmeyer, G. G., Reiss, K., Haass, C , Saftig, P., De Strooper, B., and Seed, B. (2003). The cell adhesion protein P-selectin glycoprotein ligand-1 is a substrate for the aspartyl protease BACE1. J Biol Chem 278,48713-48719. Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A., and Tang, J. (2000). Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Nad Acad Sci U S A 97, 1456-1460. Luo, Y., Bolon, B., Kahn, S., Bennett, B. D., Babu-Khan, S., Denis, P., Fan, W., Kha, H., Zhang, J., Gong, Y., et al. (2001). Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci 4, 231-232. 191 Mattson, M . P. (2004). Pathways towards and away from Alzheimer's disease. Nature 430,631-639. Mutton, D., Alberman, E., and Hook, E. B. (1996). Cytogenetic and epidemiological findings in Down syndrome, England and Wales 1989 to 1993. National Down Syndrome Cytogenetic Register and the Association of Clinical Cytogeneticists. J Med Genet 33, 387-394. Nicolaou, M. , Song, Y. Q., Sato, C. A., Orlacchio, A., Kawarai, T., Medeiros, H., Liang, Y., Sorbi, S., Richard, E., Rogaev, E. I., et al. (2001). Mutations in the open reading frame of the beta-site APP cleaving enzyme (BACE) locus are not a common cause of Alzheimer's disease. Neurogenetics 3, 203-206. Ohno, M. , Sametsky, E. A., Younkin, L. H., Oakley, H., Younkin, S. G., Citron, M. , Vassar, R., and Disterhoft, J. F. (2004). BACE1 Deficiency Rescues Memory Deficits and Cholinergic Dysfunction in a Mouse Model of Alzheimer's Disease. Neuron 41, 27-33. Pastorino, L., Ikin, A. R, Lamprianou, S., Vacaresse, N. , Revelli, J. P., Piatt, K., Paganetti, P., Mathews, P. M. , Harroch, S., and Buxbaum, J. D. (2004). BACE (beta-secretase) modulates the processing of APLP2 in vivo. Mol Cell Neurosci 25, 642-649. Podlisny, M . B., Lee, G., and Selkoe, D. J. (1987). Gene dosage of the amyloid beta precursor protein in Alzheimer's disease. Science 238, 669-671. Qing, H., Zhou, W., Christensen, M . A., Sun, X. , Tong, Y., and Song, W. (2004). Degradation of BACE by the ubiquitin-proteasome pathway. Faseb J 18,1571-1573. Roberds, S. L., Anderson, J., Basi, G., Bienkowski, M . J., Branstetter, D. G., Chen, K. S., Freedman, S., Frigon, N. L., Games, D., Hu, K., et al. (2001a). BACE knockout mice are healthy despite lacking the primary {beta}-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet 10, 1317-1324. Roberds, S. L., Anderson, J., Basi, G., Bienkowski, M . J., Branstetter, D. G., Chen, K. S., Freedman, S. B., Frigon, N . L., Games, D., Hu, K., et al. (2001b). BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet 10, 1317-1324. 192 Russo, C , Schettini, G., Saido, T. C , Hulette, C., Lippa, C , Lannfelt, L., Ghetti, B., Gambetti, P., Tabaton, M. , and Teller, J. K. (2000). Presenilin-1 mutations in Alzheimer's disease. Nature 405, 531-532. Schmechel, A., Strauss, M. , Schlicksupp, A., Pipkorn, R., Haass, C., Bayer, T. A., and Multhaup, G. (2004). BACE forms dimers and colocalizes with APP. J Biol Chem. Selkoe, D. J. (2001). Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 81, 741-766. Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., Doan, M. , Dovey, H. E , Frigon, N. , Hong, J., et al. (1999). Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402, 537-540. Sun, X. , Wang, Y., Qing, H., Christensen, M . A., Liu, Y., Zhou, W., Tong, Y., Xiao, C , Huang, Y., Zhang, S., et al. (2005). Distinct transcriptional regulation and function of the human BACE2 and BACE1 genes. FASEB J 19, 739-749. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., et al. (1999). Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286,735-741. von Arnim, C. A. F., Kinoshita, A., Peltan, I. D., Tangredi, M . M. , Herl, L., Lee, B. M. , Spoelgen, R., Hshieh, T. T., Ranganathan, S., Battey, F. D., et al. (2005). The Low Density Lipoprotein Receptor-related Protein (LRP) Is a Novel {beta}-Secretase (BACE1) Substrate. J Biol Chem 280, 17777-17785. Walter, J., Fluhrer, R., Hartung, B., Willem, M. , Kaether, C , Capell, A., Lammich, S., Multhaup, G., and Haass, C. (2001). Phosphorylation regulates intracellular trafficking of beta-secretase. J Biol Chem 276, 14634-14641. Westmeyer, G. G., Willem, M. , Lichtenthaler, S. F., Lurman, G., Multhaup, G., Assfalg-Machleidt, I., Reiss, K., Saftig, P., and Haass, C. (2004). Dimerization of beta-site beta-amyloid precursor protein-cleaving enzyme. J Biol Chem 279, 53205-53212. Yan, R., Bienkowski, M . J., Shuck, M . E., Miao, H., Tory, M . C , Pauley, A. M. , Brashier, J. R., Stratman, N . C , Mathews, W. R., Buhl, A. E., et al. (1999). Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature 402, 533-537. 193 Yang, L. B., Lindholm, K., Yan, R., Citron, M. , Xia, W., Yang, X. L., Beach, T., Sue, L., Wong, P., Price, D., et al. (2003). Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease. Nat Med 9, 3-4. 194 C H A P T E R 5: General discussion 195 AD is the most common neurodegenerative disease leading to dementia. Also literally depicted in numerous novels and movies, in his best-selling book, "The Notebook", Nicholas Sparks calls AD "It is a barren disease, as empty and lifeless as a desert. It is a thief of hearts and souls and memories." Not only AD patients suffer from the memory deficit, it is also a great burden for their families and caregivers when they gradually lose their relationships with the patients. There has been no cure for this devastating disease. As the population ages, AD becomes a great burden to the medicare system. Our studies in AD pathogenesis may contribute to the discovery of new treatments for AD. FAD caused by mutations in APP, presenilin 1 and 2 only accounts for about 5% of AD population. How A(3 deposits in brains of sporadic AD patients remains unknown. Nearly all DS patients develop AD neuropathological abnormalities after their 30s (Lott and Head, 2001). The molecular mechanism of AD pathogenesis in DS is poorly defined. DS represents a special AD population, in which the development of AD may give clues about how A(3 is accumulated in sporadic AD cases. DS is caused by an extra copy of chromosome 21. Both of APP and BACE2 are located on chromosome 21; however, APP duplication alone can not account for AD pathogenesis in DS (Arai et al., 1997). Our studies find that BACE1 is the molecule that is attributable for A(3 overgeneration in DS patients. Moreover, our data show that BACE2 is the novel 8-secretase cleaving APP within Ap and contradicts with the function of BACE1 in Ap generation. 196 Described by Dr. Alzheimer in 1906, the neuritic plaques and neurofibrillary tangles are the two hallmarks of AD neuropathology (Alzheimer, 1906). Extensive studies have focused on these two neuropathological changes, about how these changes develop in the brain and how they lead to memory deficit in AD patients. The genetic studies have led to the p amyloid cascade hypothesis, which can explain most though not all of the phenomena in AD (Hardy and Selkoe, 2002b). Ap, the major component of neuritic plaques, is generated from APP by sequential cleavage by (3- and y-secretases (Selkoe, 2001a). As the identity of y-secretase remains a puzzle and its substrates include some vital signaling factors such as Notch, inhibition of y-secretase may have many undesirable side effects. BACE1 is the major p-secretase in vivo (Hussain et al., 1999; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999), knockout of which in mice abolishes Ap production without obvious phenotype (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001b). Most people believe that P-secretase is the prime drug target for AD. Furthermore, our studies on BACE2, as the novel 0-secretase inhibiting Ap production, implicates that BACE2 is a new drug target for AD therapy. 5.1. Molecular rationale of B A C E 1 as the prime drug target BACE1 is a prime drug target for AD therapy development. There are several approaches that can decrease BACE1 enzymatic activity in AD patients. The first strategy is to develop specific BACE1 inhibitors that can decrease abnormal BACE1 enzymatic activity in AD. Another approach is to target the molecules whose dysfunctions lead to abnormal BACE1 expression in AD patients. Increased BACE1 expression and enzymatic activity in AD cases can come from transcriptional regulation, 197 translational regulation and posttranslational modifications, or combination of the above. Our study of AD pathogenesis in DS patients suggests that abnormal posttranslational modifications of BACE1 may be a common pathophysiological pathway in AD pathogenesis. Inhibiting P-secretase activity with selective BACE1 inhibitors is the direct way to reduce A(3 production and deposition. Broad-spectrum protease inhibitors such as pepstatin, known aspartic protease inhibitors from renin, and HIV protease inhibitors, as well as cocktails thereof, have little inhibitory effect on BACE1 activity. Most of BACE1 inhibitors developed are hydroxyethyl transition state analogues based on the sequence of APP around p-secretase cleavage site. OM99-2 (EVNL*AAEF) and OM00-3 (ELDL*AVEF), with a Ki of InM and 0.3nM respectively, are the two most potent inhibitors(Hong et al., 2000; Hong et al., 2002). The inhibitor bound BACE1 structure has been crystallized and determined. The bilobal structure of BACE1 has the conserved general folding of aspartic proteases. Active-site Asp and Asp and the surrounding hydrogen bond network are conserved and located in the center of the cleft(Hong et al., 2000). Many of the BACE1 inhibitors are still at the stage of preclinical trials. Further studies are needed to reduce the molecular weight and increase the cell and blood brain barrier permeability(John et al., 2003; Roggo, 2002; Vassar, 2002). Nonpeptidic inhibitors identified from a screening approach were also reported. The tetralin derivatives and latifolin have an IC50 of 0.35-3uM and 180uM respectively(Roggo, 2002). As functions of BACE1 and BACE2 are contradictory and there is subtle 198 difference in their crystal structures, it is important to develop inhibitors that can selectively inhibit BACE1 without affecting BACE2. A slight increase in the BACE1 protein level is able to drastically increase BACE1 enzymatic activity (Li et al., 2006), suggesting that some slight increase in BACE1 protein by some environmental risk factors with aging may remarkably increase P-secretase activity and AP deposition, and subsequently lead to AD. Specific transcription factors are potential drug targets in that it can inhibit BACE1 gene expression and decrease AP production. BACE1 gene is a tightly regulated gene (Christensen et al., 2004; Sun et al., 2005; Tong et al., 2004). The mechanism underlying the neuronal expression of BACE1 is still elusive. Future studies will investigate the cis-regulatory elements and transcription factors responsible for the neuronal expression of BACE1. BACE1 gene is located on chromosome llq23.3. Several studies have shown that BACE1 expression and enzymatic activity are elevated in brains of sporadic AD patients, which are also correlated with Ap production (Olin, 2004; Yang et al., 2003); however, no familial AD is found to be linked to BACE1 gene locus (Cruts et al., 2001; Nicolaou et al., 2001; Olin, 2004). Most single nucleotide polymorphism studies did not identify any significant association between BACE1 and AD occurrence (Gold et al., 2003; Liu et al., 2003), whereas other studies showed thatBACEl gene polymorphism C786G and G1239C are associated with late onset sporadic AD in a Chinese population (Kan et al., 2005; Shi et al., 2004). Further genetic screening is needed to resolve this discrepancy. 199 The other physiological functions of BACE1 except APP cleavage remain to be elucidated. BACE1 interacts with reticulon family member proteins, and reticulon proteins can block access of BACE1 to APP and reduce the APP cleavage (He et al., 2004). A brain-specific type II membrane protein BRI3 and a phospholipid scramblase 1 (PLSCR1) were identified to interact with BACE1 by a yeast two-hybrid system (Kametaka et al., 2003; Wickham et al., 2005). Identification of more BACE1 interacting proteins may disclose other functions of BACE1, studies of which will contribute to understanding the side effects of inhibiting BACE1 as a drug target. 5.2. Molecular rationale of BACE2 as a novel drug target BACE2 is closely related to BACE1 although the expression of BACE2 is low in the brain. Our data show that BACE2 physiologically counteracts the function of BACE1 in Ap production (Figure 5.1). Overexpression of BACE 1 markedly increases Ap production, while overexpression of BACE2 can significantly decrease Ap generation both in cell lines and in primary neurons. BACE2 may be the potential weapon the body reserved for AD self-limitation. Figure 5.1. BACE2, as a novel APP 9-secretase, is not responsible for AD pathogenesis in DS. BACE2 cleaves APP at LVF'FAED between 19th and 20** amino acid of Ap. Cleavage of APP by BACE2 decreases Ap production, which is opposite to the function of BACE1. APP cleavage by BACE1 generates Ap, leading to plaque formation and AD. 200 1 10 20 s A P P I S E V K M D A E F R H D S G Y E V H H Q K L V F s A P P p t P(BACE1) t Y 30 40 KGAIIGLMVGGWIATVIVITLVMLKK 7 7 40 '42 CTFy s A P P O AP Alzheimer 's d isease NoAp + Both the crystal structures of BACE1 and BACE2 have been defined (Hong et al., 2002; Ostermann et al., 2006). The sequence alignment of BACE1 and BACE2 shows 45% identical and 75% homologous, and the structural alignment shows a root-mean-square deviation (RMSD) of 0.82 over 352 C a atoms (Ostermann et al , 2006). The structure of BACE1 and BACE2 is very similar; however, the crystal structure also reveals conformational differences between BACE1 and BACE2 in three loops close to the non-prime binding sites comprising amino acid residues Gly58-Tyr67, Phel24-Trpl31 and Ile319-Tyr332 as well as in the two loops of the C-terminal extension (Cys371-Ala378 and Phe386-Cys395) (Ostermann et al., 2006). It will be interesting to study why these two homologous proteins have contradictory functions in APP processing. Chromosome 21q has consistently been linked to late-onset AD by several genetic studies (Goldin and Gershon, 1993; Heston et al., 1991; Kehoe et al., 1999; Myers et al., 201 2002; Myllykangas et al , 2005; Pericak-Vance et al., 1991a). A maximum linkage peak is found with marker D21S1440 which is mapped between APP and BACE2 genes, 5 Mb from BACE2 and 12 Mb from APP (Blacker et a l , 2003). BACE2 haplotype analysis shows that a haplotype H5 is associated with AD, and a second haplotype H7 is associated with protection from AD, providing further evidence for an AD susceptibility locus on chromosome 21q within or close to BACE2 (Myllykangas et al., 2005). Further genetic screening is needed to elucidate if B ACE2 gene mutation or polymorphism is associated with AD. 5.3. Future directions Our study shows that abnormal BACE1 trafficking leads to BACE1 accumulation in the Golgi and subsequendy increased A(3 production in DS (Figure 5.2). BACE1 protein undergoes a complex set of post-translational modifications such as prodomain casting, palmotylation, phosphorylation, cystein bridge formation, N-glycosylation and ubiquitination. It is known that N-glycosylation and ubiquitination can significantly affect BACE1 enzymatic activity and subsequent APP processing. The p-secretase activity greatly depends on the extent of N-glycosylation (Capell et al., 2000b; Charlwood et al., 2001; Haniu et al., 2000; Huse and Doms, 2000). The ubiquitination of BACE1 targets it to proteasome degradation pathway, inhibition of which greatly elevates P-secretase activity and Ap production (Qing et al., 2004). Further studies are needed to investigate how the N-glycosylation and ubiquitination are regulated to affect BACE1 function. 202 Figure 5.2. Increased B A C E 1 maturation contributes to AD pathogenesis in DS. Overproduction of A|3 in DS is caused by abnormal BACE1 protein trafficking and maturation. Increased BACE1 protein levels, and particularly the higher levels of N -glycosylated mature BACE1 proteins in DS, result in the up-regulated P-secretase activity, leading to higher AP generation and neurodegeneration. ( Trisomy 2 1 ' . Normal cef lsN XXX XX / Normal B A C E 1 Abnormal B A C E 1 * * * * * * / ' 7 m S T g B A C E 1 More mature B A C E 1  / 1 10 20 30 40 KM OA, EFR HDSG YEVH H QKLVF££Q2VGSAfKG Al IGLMVG GWIATV l t f 4 ft P ( B A C E 1 ) p o: V Increased p - | s e c r e t a s e activity Alzheimer's disease Our data have clearly showed that overexpression of BACE2 can reduce AP generation in vitro. Further studies will be designed to test if BACE2 overexpression can also decrease AP generation in vivo. BACE2 transgenic mice will be generated and crossed with APP23 mice. Ap immunostaning and Morris water-maze tests will be 203 performed to examine if BACE2 overexpression will delay plaque formation and improve memory impairment in APP23 mice. 5.4. Conclusions The research described here addresses the hypothesis that abnormal R-secretase is involved in AD pathogenesis in DS. Transcriptional regulation of BACE2 gene is distinct from the transcription of BACE1 gene. Though they are homologous in amino acid sequence and in structure, the functions of BACE 1 and BACE2 contradict each other in AR generation. Further studies provide evidence that BACE2 cleaves APP at 9-site within AR domain and decreases AR production when overexpressed. The elevation of BACE1, particularly its mature form, is attributable to the drastic increase of C99 and AR in brains of DS patients. Further study of BACE1 trafficking shows that BACE1 is abnormally accumulated in the Golgi of DS cells. The body of work presented here suggests that BACE1 is the molecule that is responsible for the early AR accumulation and subsequent plaque deposition in DS patients. Though the transcription of BACE2 is elevated in DS, the protein level of BACE2 remains unchanged. The identification of BACE2 as a novel G-secretase further excludes the association of BACE2 in the pathogenesis of AD in DS. Results herein suggest that therapeutic interventions that inhibit BACE1 or potentiate BACE2 may prevent AD pathogenesis and benefit AD patients. 204 5.5. References Alzheimer, A. (1906). fiber einen eigenartigen schweren ErkrankungsprozeB der Hirnrinde. Neurologisches Centralblatt 23, 1129-1136. Arai, Y., Suzuki, A., Mizuguchi, M. , and Takashima, S. (1997). Developmental and aging changes in the expression of amyloid precursor protein in Down syndrome brains. Brain Dev 19, 290-294. Blacker, D., Bertram, L., Saunders, A. J., Moscarillo, T. J., Albert, M . S., Wiener, H., Perry, R. T., Collins, J. S., Harrell, L. E., Go, R. C , et al. (2003). Results of a high-resolution genome screen of 437 Alzheimer's disease families. Hum Mol Genet 12, 23-32. Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D. R., Price, D. L., and Wong, P. C. (2001). BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci 4, 233-234. Capell, A., Steiner, H., Willem, M. , Kaiser, H., Meyer, C , Walter, J., Lammich, S., Multhaup, G., and Haass, C. (2000). Maturation and pro-peptide cleavage of beta-secretase. J Biol Chem 275, 30849-30854. Charlwood, J., Dingwall, C , Matico, R., Hussain, I., Johanson, K., Moore, S., Powell, D. J., Skehel, J. M. , Ratcliffe, S., Clarke, B., et al. (2001). Characterization of the glycosylation profiles of Alzheimer's beta -secretase protein Asp-2 expressed in a variety of cell lines. J Biol Chem 27f5, 16739-16748. Christensen, M . A., Zhou, W., Qing, H., Lehman, A., Philipsen, S., and Song, W. (2004). Transcriptional regulation of BACE 1, the beta-amyloid precursor protein beta-secretase, by Spl. Mol Cell Biol 24, 865-874. Cruts, M. , Dermaut, B., Rademakers, R., Roks, G., Van den Broeck, M. , Munteanu, G., van Duijn, C. M. , and Van Broeckhoven, C. (2001). Amyloid beta secretase gene (BACE) is neither mutated in nor associated with early-onset Alzheimer's disease. Neurosci Lett 313, 105-107. Gold, G., Blouin, J. L., Herrmann, F. R., Michon, A., Mulligan, R., Duriaux Sail, G., Bouras, C , Giannakopoulos, P., and Antonarakis, S. E. (2003). Specific BACE1 genotypes provide additional risk for late-onset Alzheimer disease in APOE epsilon 4 carriers. Am J Med Genet 119B, 44-41. 205 Goldin, L. R., and Gershon, E. S. (1993). Linkage of Alzheimer's disease to chromosome 21 and chromosome 19 markers: effect of age of onset assumptions. Genet Epidemiol 10, 449-454. Haniu, M. , Denis, P., Young, Y., Mendiaz, E. A., Fuller, J., Hui, J. O., Bennett, B. D., Kahn, S., Ross, S.,.Burgess, T., et al. (2000). Characterization of Alzheimer's beta -secretase protein BACE. A pepsin family member with unusual properties. J Biol Chem 275,21099-21106. Hardy, J., and Selkoe, D. J. (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353-356. He, W., Lu, Y., Qahwash, I., Hu, X . Y., Chang, A., and Yan, R. (2004). Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation. Nat Med. Heston, L. L., Orr, H. T., Rich, S. S., and White, J. A. (1991). Linkage of an Alzheimer disease susceptibility locus to markers on human chromosome 21. Am J Med Genet 40, 449-453. Hong, L., Koelsch, G., Lin, X. , Wu, S., Terzyan, S., Ghosh, A. K., Zhang, X. C , and Tang, J. (2000). Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor. Science 290, 150-153. Hong, L., Turner, R. T., 3rd, Koelsch, G., Shin, D., Ghosh, A. K., and Tang, J. (2002). Crystal structure of memapsin 2 (beta-secretase) in complex with an inhibitor OM00-3. Biochemistry 41, 10963-10967. Huse, J. T., and Doms, R. W. (2000). Closing in on the amyloid cascade: recent insights into the cell biology of Alzheimer's disease. Mol Neurobiol 22, 81-98. Hussain, I., Powell, D., Howlett, D. R., Tew, D. G., Meek, T. D., Chapman, C , Gloger, I. S., Murphy, K. E., Southan, C. D., Ryan, D. M., et al. (1999). Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol Cell Neurosci 14, 419-427. John, V., Beck, J. P., Bienkowski, M . J., Sinha, S., and Heinrikson, R. L. (2003). Human beta-secretase (BACE) and BACE inhibitors. J Med Chem 46, 4625-4630. Kametaka, S., Shibata, M. , Moroe, K., Kanamori, S., Ohsawa, Y., Waguri, S., Sims, P. J., Emoto, K., Umeda, M. , and Uchiyama, Y. (2003). Identification of phospholipid 206 scramblase 1 as a novel interacting molecule with beta -secretase (beta -site amyloid precursor protein (APP) cleaving enzyme (BACE)). J Biol Chem 278, 15239-15245. Kan, R., Wang, B., Zhang, C , Jin, R, Yang, Z., Ji, S., Lu, Z., Zheng, C , and Wang, L. (2005). Genetic Association of BACE1 Gene Polymorphism C786G With Late-Onset Alzheimer's Disease in Chinese. J Mol Neurosci 25, 127-132. Kehoe, P., Wavrant-De Vrieze, E , Crook, R., Wu, W. S., Holmans, P., Fenton, I., Spurlock, G., Norton, N. , Williams, H., Williams, N., et al. (1999). A full genome scan for late onset Alzheimer's disease. Hum Mol Genet 8, 237-245. Li , Y., Zhou, W., Tong, Y., He, G., and Song, W. (2006). Control of APP processing and Abeta generation level by BACE1 enzymatic activity and transcription. Faseb J 20, 285-292. Liu, H. C , Leu, S. J., Chang, J. G., Sung, S. M. , Hsu, W. C , Lee, L. S., and Hu, C. J. (2003). The association of beta-site APP cleaving enzyme (BACE) C786G polymorphism with Alzheimer's disease. Brain Res 961, 88-91. Lott, I. T., and Head, E. (2001). Down syndrome and Alzheimer's disease: a link between development and aging. Ment Retard Dev Disabil Res Rev 7, 172-178. Luo, Y., Bolon, B., Kahn, S., Bennett, B. D., Babu-Khan, S., Denis, P., Fan, W., Kha, H., Zhang, J., Gong, Y., et al. (2001). Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci 4,231-232. Myers, A., Wavrant De-Vrieze, F., Holmans, P., Hamshere, M. , Crook, R., Compton, D., Marshall, H., Meyer, D., Shears, S., Booth, J., et al. (2002). Full genome screen for . Alzheimer disease: stage II analysis. Am J Med Genet 114, 235-244. Myllykangas, L., Wavrant-De Vrieze, F., Polvikoski, T., Notkola, I. L., Sulkava, R., Niinisto, L., Edland, S. D., Arepalli, S., Adighibe, O., Compton, D., et al. (2005). Chromosome 21 BACE2 haplotype associates with Alzheimer's disease: a two-stage study. J Neurol Sci 236,17-24. Nicolaou, M. , Song, Y. Q., Sato, C. A., Orlacchio, A., Kawarai, T., Medeiros, H., Liang, Y., Sorbi, S., Richard, E., Rogaev, E. I., et al. (2001). Mutations in the open reading frame of the beta-site APP cleaving enzyme (BACE) locus are not a common cause of Alzheimer's disease. Neurogenetics 3, 203-206. 207 Olin, J. S., L; Novit, A; Luczak, S (2004). Hydergine for dementia (The Cochrane Database of Systematic Reviews). Ostermann, N. , Eder, J., Eidhoff, U., Zink, R, Hassiepen, U., Worpenberg, S., Maibaum, J., Simic, O., Hommel, U., and Gerhartz, B. (2006). Crystal structure of human BACE2 in complex with a hydroxyethylamine transition-state inhibitor. J Mol Biol 355, 249-261. Pericak-Vance, M . A., Bebout, J. L., Gaskell, P. C , Jr., Yamaoka, L. H., Hung, W. Y., Alberts, M . J., Walker, A. P., Bartlett, R. J., Haynes, C. A., Welsh, K. A., and al., e. (1991). Linkage studies in familial Alzheimer disease: evidence for chromosome 19 linkage. Am J Hum Genet 48, 1034-1050. Qing, H., Zhou, W., Christensen, M . A., Sun, X. , Tong, Y., and Song, W. (2004). Degradation of BACE by the ubiquitin-proteasome pathway. Faseb J 18,1571-1573. Roberds, S. L., Anderson, J., Basi, G., Bienkowski, M . J., Branstetter, D. G., Chen, K. S., Freedman, S. B., Frigon, N . L., Games, D., Hu, K., et al. (2001). BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet 10, 1317-1324. Roggo, S. (2002). Inhibition of BACE, a promising approach to Alzheimer's disease therapy. Curr Top Med Chem 2, 359-370. Selkoe, D. J. (2001). Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 81, 741-766. Shi, J., Zhang, S., Tang, M. , Liu, X., L i , T., Wang, Y., Han, H., Guo, Y., Hao, Y., Zheng, K., et al. (2004). The 1239G/C polymorphism in exon 5 of BACE1 gene may be associated with sporadic Alzheimer's disease in Chinese Hans. Am J Med Genet 124B, 54-57. Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., Doan, M. , Dovey, H. F., Frigon, N. , Hong, J., et al. (1999). Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402, 537-540. Sun, X. , Wang, Y., Qing, H., Christensen, M . A., Liu, Y., Zhou, W., Tong, Y., Xiao, C , Huang, Y., Zhang, S., et al. (2005). Distinct transcriptional regulation and function of the human BACE2 and BACE1 genes. FASEB J 19, 739-749. 208 Tong, Y., Zhou, W., Fung, V., Christensen, M . A., Qing, FL, Sun, X. , and Song, W. (2004). Oxidative stress potentiates BACE1 gene expression and Abeta generation. J Neural Transm. Vassar, R. (2002). Beta-secretase (BACE) as a drug target for Alzheimer's disease. Adv Drug Deliv Rev 54, 1589-1602. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., et al. (1999). Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science 285,735-741. Wickham, L., Benjannet, S., Marcinkiewicz, E., Chretien, M. , and Seidah, N. G. (2005). Beta-amyloid protein converting enzyme 1 and brain-specific type II membrane protein BRI3: binding partners processed by furin. J Neurochem 92, 93-102. Yan, R., Bienkowski, M . J., Shuck, M . E., Miao, H., Tory, M . C , Pauley, A. M. , Brashier, J. R., Stratman, N . C , Mathews, W. R., Buhl, A. E., et al. (1999). Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity. Nature 402, 533-537. -Yang, L. B., Lindholm, K., Yan, R., Citron, M. , Xia, W., Yang, X . L., Beach, T., Sue, L., Wong, P., Price, D., et al. (2003). Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease. Nat Med 9, 3-4. 209 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0100546/manifest

Comment

Related Items