Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Regulation of human BACE1 gene expression by NF-kappa B signaling Chen, Chia-Hsiung Alan 2007

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

Item Metadata


831-ubc_2007-0054a.pdf [ 10.53MB ]
JSON: 831-1.0100990.json
JSON-LD: 831-1.0100990-ld.json
RDF/XML (Pretty): 831-1.0100990-rdf.xml
RDF/JSON: 831-1.0100990-rdf.json
Turtle: 831-1.0100990-turtle.txt
N-Triples: 831-1.0100990-rdf-ntriples.txt
Original Record: 831-1.0100990-source.json
Full Text

Full Text

REGULATION OF HUMAN  BACE1 G E N E E X P R E S S I O N B Y N F - K A P P A B SIGNALING  by CHIA-HSIUNG, A L A N C H E N B.Sc, McGill University, 2004  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR T H E D E G R E E OF  M A S T E R OF SCIENCE  in  THE F A C U L T Y OF G R A D U A T E STUDIES  (Neuroscience)  THE UNIVERSITY OF BRITISH C O L U M B I A April 2007  © Chia-Hsiung, Alan Chen, 2007  ABSTRACT Nuclear Factor-Kappa B  (NF-KB)  signaling plays an important role in gene regulation  and is implicated in cell apoptosis, inflammation and oxidative stress. Binding of Inhibitor of Kappa B (IKB) to  NF-KB  dimers, mostly RelA (p65)/p50, causes the dimers to be sequestered  in cytoplasm and remain inactive. When NF-KB-activating stimuli activate the Inhibitor of Kappa B Kinase (IKK) complex, I K K phosphorylates translocates to the nucleus, where it interacts with NF-KB  IKB,  NF-KB  and  NF-KB  is released and  binding elements in the promoter of  target genes to modulate gene expression.  One of the hallmark neuropathological features of Alzheimer's disease (AD) is the senile plaques formed by the deposition of amyloid-P peptide (AP) in the brain. Ap generation is achieved by sequential proteolytic cleavage of P-amyloid precursor protein (APP) by P-site APP cleaving enzyme (BACE1) and y-secretase. Regulation of B A C E 1 activity is efficient to modify AP production and B A C E 1 inhibition has become a potential target in A D treatment. BACE1 is reported to be tightly regulated at both the transcriptional and translational level. Several putative  NF-KB  binding elements in the human BACE1 promoter region have been  identified, but the precise role of N F - K B in BACE1 gene expression remains to be defined.  To investigate whether functional  NF-KB  NF-KB  modulates BACE1 gene expression, we first identified  binding elements in the BACE1 promoter region. Co-expression of  NF-KB  p65 and BACE1 reporter plasmid in HEK293 cells resulted in increased BACE1 promoter activity and dual-luciferase reporter assays and RT-PCR analysis revealed that N F - K B p65 regulates BACE1 gene at the transcriptional level. Neurons are speculated to be the major source of AP generation, contributing to the elevated Ap burden in A D pathogenesis, and NF-  KB p65 was also demonstrated to elevate BACE1 promoter activity in neuronal-like cells. In addition, NF-KB p65 overexpression leads to elevation of C99 protein levels and increased production of Ap . Therefore, our results show that by modulating NF-KB signalling, BACE1 4u  gene transcription is regulated and amyloidogenic APP processing is altered.  iii  T A B L E OF CONTENTS ABSTRACT  ii  T A B L E OF C O N T E N T S  iv  LIST OF F I G U R E S  viii  LIST OF A B B R E V I A T I O N S  x  ACKNOWLEDGEMENTS CHAPTER I  xvi 1  Introduction  1.1. A n Overview of Alzheimer's Disease  1  1.1.1. Familial Form of A D  1 Amyloid Precursor Protein Mutations Presenilin Mutations A p o E Polymorphism 1.1.2. Sporadic Form of A D  1  \  3 3 4 Apolipoprotein  4 Angiotensin I-Converting Enzyme 1  6 Nongenetic A D Risk Factors  7  1.1.3. Classic Lesions of A D 1.2. Amyloid Cascade Hypothesis of A D  8 9  1.2.1. Amyloid Precursor Protein  9  1.2.2. Amyloid Precursor Protein Processing.  9  1.3. A P P Cleavage Secretases  13  1.3.1. a-secretase  14  1.3.2. y-secretase  15 Presenilins  16  iv Nicastrin  17 Anterior Pharynx defective-land Presenilin enhancer-2  19  1.3.3. P-secretase  20 P-Site APP Cleaving Enzyme 1  20 BACE2  24  1.4. Transcriptional and Translational Regulation of BACE1 Expression  26  1.4.1. Transcriptional Modulation of BACE1  26  1.4.2. Translational Modulation of BACE1  27  1.5. Nuclear Factor Kappa B  29  1.5.1. The Identity and Function of N F - K B Components 1.5.2. Regulation of N F - K B Activity by I K B 1.5.3. N F - K B Activation  29 ,  30 33 Canonical and Non-Canonical Pathways  33 Other Factors in the Regulation of N F - K B Activity  35 IKK Complex  36  1.5.4. Biological Functions of N F - K B  37 N F - K B and Apoptosis  37 N F - K B and Abnormal Cell Growth  38 N F - K B and Immunity and Learning  38  1.6. Oxidative Stress  40  1.6.1. Free Radical Generation and Elimination in the Brain  40  1.6.2. Neuronal Cell Death by AP Neurotoxicity  43  1.6.3. Membrane Lipoperoxidation  45  1.7. Neuroinflammation  46  V  1.7.1. Microglial Activation  47  1.7.2. Astrocyte Recruitment  49  1.7.3. Cytokine and Chemokine Production  50 Interleukin-1  51 Interleukin-6  52 Tumor Necrosis Factor-a  54 Transforming Growth Factor-P  55  CHAPTER II  Materials and Methods  2.1. Generation of Human B A C E 1 Gene Promoter Constructs 2.1.1. Generation of B A C E - N F - K B Plasmid  57 57 57  2.2. Site-Directed Mutagenesis  58  2.3. Cell Culture  59  2.3.1. Culture Media Preparation  59  2.3.2. Trypnization  60  2.4. Cell Transfection  60  2.4.1. Calcium Phosphate Transfection  60  2.4.2. Lipofectamine 2000 Transfection  61  2.5. Dual-Luciferase Reporter Assay  62  2.6. Reverse-Transcription-Polymerase Chain Reaction  62  2.6.1. Cell Transfection  62  2.6.2. R N A Extraction  62  2.6.3. c D N A Synthesis  63  2.6.4. P C R Amplification  63  2.7. Electrophoretic Mobility Shift Assay  64  VI  2.8. Western Blotting  66  2.9. Enzyme Linked-Immuno-Sorbent Assay  67  CHAPTER I I I  Results  68  3.1. Identification a Functional Interaction Between N F - K B p65 and N F - K B Binding Elements in the Human BACE1 Promoter Region  68  3.2. N F - K B p65 Regulates Human BACEl  74  Promoter Activity  3.3. BACEl Promoter Activity is Regulated Through Two Distinct N F - K B Binding Elements  79  3.4. Human BACEl Gene Transcription is Modulated by N F - K B Signaling  83  3.5. The Role of N F - K B in Regulation of BACEl Transcriptional Activity in Neurons  84  3.6. N F - K B p65 Elevates |3-secretase Processing of APP and AP Production  88  3.7. Summary  90  CHAPTER I V  General Discussion  4.1. Regulation of BACEl  Gene Transcription by N F - K B  94 94  4.2. The Role of N F - K B Activity in A D  96  4.3. Concluding Remarks  98  REFERENCES  99  LIST OF FIGURES Figure 1-1. Amyloid Cascade Hypothesis of AD and A|3 Sequence  11  Figure 1-2. Sequence Alignment of BACE1 and BACE2  25  Figure 1-3. A Schematic Model of N F - K B Activation  34  Figure 1-4. Oxidative Stress in AD  41  Figure 1-5. The Role of Inflammation Cytokines Produced by Glial Cells in AD Pathogenesis  48  Figure 3-1. Identification of N F - K B Binding Sequence in the Human BACE1 Promoter Region  69  Figure 3-2. Physical Interaction between N F - K B p65 and N F - K B Binding Elements in the Human BACE1 Promoter  70  Figure 3-3. Specific Interaction between the First and Fourth N F - K B Binding Elements and  NF-KB  p65  71  Figure 3-4. N F - K B p65 Binding Elements on the Human BACE1 Promoter are Functional  73  Figure 3-5. Illustrations of BACE1 Reporter Plasmids  75  Figure 3-6. BACEl Promoter Activity is Elevated by N F - K B p65  78  Figure 3-7. Illustrations of BACE1 Reporter Plasmids Containing a Mutated N F - K B Binding Element  80  Figure 3-8. BACE1 Promoter Activity is Modulated by N F - K B Binding Elements  82  Figure 3-9. N F - K B p65 Upregulates Human BACE1 at the Transcriptional Level  84  Figure 3-10. Human BACE1 Promoter Activity in Neurons is Upregulated by N F KB  87  Figure 3-11. N F - K B p65 potentiates C99 Production  89  Figure 3-12. N F - K B p65 Increase A R  4 0  Generation  90  IX  LIST O F ABBREVIATIONS  °C  Degrees centigrade  pg  micro-gram  (tL  micro-liter  X  Mole fraction  20E2 cells  HEK293 cells stably expressing Swedish mutant APP695  AP  Amyloid-P  ACE1  Angiotensin I-converting enzyme 1  AD  Alzheimer's Disease  ADAM  a disintegrin and metalloproteinase domain  AEBSF  4-(2-aminoethyl)-benzenesulfonyl fluoride  Ala  alanine  Aph-1  Anterior pharynx-defective-1  apoE  Apolipoprotein E  APP  Amyloid Precursor Protein  Arg  Arginine  Asn  Asparagine  Asp  aspartic acid  ATP  Adenosine triphosphate  bcl  B-cell lymphoma  BACE  P-site APP cleaving enzyme  bp  base pair  BSA  Bovine serum albumin  ci  curie  x  C83  83-residue CTF of APP  C99  99-residue CTF of APP  C. elegans  Caenorhabditis elegans  CaCl  Calcium chloride  2  CO2  Carbon dioxide  cDNA  Complementary deoxyribonucleic acid  CNS  Central Nervous System  C-terminus  carboxyl-terminus  COX  Cyclooxygenase  CSF  cerebrospinal fluid  CTF  Carboxyl-terminal fragment  Cys  Cysteine  DEPC  Diethylpyrocarbonate  DNA  -  Deoxyribonucleic acid  D-PBS  Dulbecco's phosphate-buffered saline  DS  Down syndrome  ECL  Enhanced chemiluminescence  EDTA  Ethylenediaminetetraacetic acid  ELISA  Enzyme Linked-Immuno-Sorbent Assay  EMSA  Electrophoretic Mobility Shift Assay  g  gram  gp80/130  glycoprotein 80/130  Gly  glycine  GSH  Glutathione  xi  h  hour  hGAPDH  Human Glyceraldehyde 3-phosphate dehydrogenase  H 0  Water  H2O2  Hydrogen peroxide  HBSS  Hanks Balanced Salt Solution  HEBS  HEPES buffered saline  HEK293 cells  Human embryonic kidney 293 cells  HEPES  4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid  HRP  Horseradish peroxidase  IKB  Inhibitor of Kappa B  I-CliPs  Intramembrane-cleaving proteases  IKK  IKB kinase  IL-1  Interleukin-1  IL-6  Interleukin-6  He  isoleucine  Kb  Kilo basepair  kDa  kilo Dalton  LDL  Low-density lipoprotein  Leu  leucine  LPS  Lipopolysaccharide  LRP  LDL-receptor-related protein  Lys  lysine  mA  milli ampere  mei  milli curie  2  xii  mL  milli-liter  mm  milli-mole  mM  milli-molar  mRNA  messenger R N A  Mg  Magnesium  MgCE  Magnesium chloride  M  Molarity  MAIL  Molecule possessing ankyrin-repeats induced by lipopolysaccharide  MDC  metalloprotease, disintegrin, cysteine-rich proteins  MEM  Minimum essential medium  Met  Methionine  MHCII  Major histocompatibility complex type II  nm  nanometer  N2a  Mouse neuroblastoma  NaCl  Sodium chloride  Na2HP04  Di-sodium phosphate  NaOH  Sodium hydroxide  Net  Nicastrin  NES  Nuclear export sequences  NF-KB  Nuclear factor kappa B  NFT  Neurofibrillary tangles  N-terminus  amino-terminus  NICD  Notch intracellular domain  xiii  NIH-3T3 cells  Mouse Fibroblast cells  NLS  Nuclear localization sequences  NO  Nitric oxide  NSAIDs  Nonsteroidal inflammatory drugs  NTF  Amino-terminal fragment  O2"  Superoxide anion  OH'  Hydroxyl radical  pmole  pico mole  PBS-T  Phosphate buffered saline-Tween 20  PCR  Polymerase chain reaction  Pen-2  Presenilin enhancer-2  Phe  phenylalanine  RNA  Ribonucleic acid  Pro  Proline  PS  Presenilin  PTEN  Phosphatase and tensin homolog (mutated in multiple advanced cancers 1)  PVDF  Polyvinylidene fluoride  RelA-KO  RelA(p65) Knockout  RHD  Rel homology domain  ROS  Reactive Oxygen Species  rpm  Revolutions per minute  1 RT-PCR sAPP  Reverse transcription-polymerase chain reaction soluble APP  xiv  SDS-PAGE  Sodium dodecyl sulfate polyacrylamide gel electrophoresis  SEM  Standard errors of the means  SH-SY5Y  Human neuroblastoma  Smad  Mothers against decapentaplegic  SOD  Superoxide dismutase  SPP  Signal peptide peptidase  TACE  TNF-a converting enzyme  TBE  Tris-Borate-EDTA  TGF-P  Transforming growth factor-|3  TNF-a  Tumor necrosis factor-a  Tris  2-amino-2-hydroxymethyl-1,3-prc>panediol  U  unit  UTR  Untranslated region  V  Volt  Val  valine  VLDL  Very low-density lipoprotein  YY1  Yin Yang 1  XV  ACKNOWLEDGEMENTS First I would like to thank Dr. Weihong Song for his support and guidance during the course of my study. Dr. Song provided me with well-equipped research environment to investigate science and introduced me with creative ideas. He has been the best model for me as a successful scientist.  I would also like to thank Dr. Weihui Zhou, who has significant contribution on my thesis project and instructed me how to design flawless experiments. I appreciate the work with all the past and present members in Dr. Song lab, especially Dr. Xiulian Sun, Dr. Guiqiong He, Dr. Hong Qing and Dr. Fang Cai for advice and help on my experiments, as well as Ke Wong, Dr. Bin Chen, Dr. Shengcai Wei, Dr. Yu Li, and Dr. Yigang Tong, all of whom are great colleagues and friends to work with. I am thankful for the time and effort Kelly Bromley, Odysseus Zis, and Andrea Human put on proofreading my thesis.  I am grateful for the suggestions for my thesis from my supervisory committee members, Dr. William G. Honer, Dr. Katerina Dorovini-Zis and Dr. Robert A. Holt, as well as program professors Dr. Neil Cashman and Dr. William Jia. Moreover, I would like to thank Dr. W.C. Greene of UCSF for IKBCI expression plasmid, Dr. Y. Tone of University of Oxford for N F - K B p65 expression plasmid and Dr. T.D. Gilmore of Boston University for RelA-KO cells.  Lastly, but not least, I deeply appreciate my family members for their moral support and encouragement throughout my study in Canada.  Chapter I. Introduction 1.1. An Overview of Alzheimer's Disease As the life expectancy of humans has risen during the 20 century, neurodegenerative th  disorders have become a common issue and Alzheimer's disease (AD) is the most prevalent form of neurodegenerative disease leading to dementia in the aged population (Sisodia, 1999). AD wasfirstdescribed by Alois Alzheimer in 1906 and the typical neuropathological features include senile plaques, neurofibrillary tangles, and neuronal loss (Alzheimer, 1906; Alzheimer, 1907). Based on the cholinergic hypothesis of AD, cholinergic neurons are predominantly affected in this neurodegenerative disease; hence, several pharmacotherapeutic strategies have been developed to stabilize cholinergic neurotransmission in the brain (Bartus et al., 1982; Bowen et al., 1992; Michaelis, 2003; Perry, 1986).  1.1.1. Familial Form of AD Amyloid Precursor Protein Mutations Two forms of AD have been recognized: familial and sporadic. Less than 10% of all AD cases are the familial form, and while the remaining AD cases are predominantly sporadic and have a late-onset, they are neuropathologically and clinically indistinguishable from familial forms of AD (Evans et al., 1989a; Goedert and Spillantini, 2006; Pereira et al., 2005; Selkoe and Podlisny, 2002). Familial AD can be inherited in an autosomal dominant fashion within families and is normally observed in the early-onset cases of AD (Goate et al., 1991; Schutte, 2006; Selkoe, 2001).  Polymorphic alleles can be the genetic determinants that predispose an individual to AD in some late-onset cases (Chapman et al., 1998; McGeer and McGeer, 2001; Rocchi et al.,  1  2003). The first specific genetic cause of AD was identified as missense mutations in Amyloid Precursor Protein (APP) (Goate et al., 1991; Hardy, 1992; Hendriks et al., 1992; Mullan et al., 1992). The mutations are located either before the P-secretase site, after the a-secretase site, or downstream of the y-secretase site. Individuals inherited with these mutations are predisposed to an increased production of amyloid P-proteins (AP) and an onset of AD before the age of 65 (Selkoe and Podlisny, 2002). Several missense mutations in the APP gene have been identified in familial AD, and some of them located outside the Ap region of the APP gene lead to enhanced production of neurotoxic AP42 by augmented P-secretase proteolytic processing of APP (Meredith, 2005; Selkoe and Podlisny, 2002). However, the Artie APP mutation (Glu  693  to Gly ) occurring within the AP region has been reported to cause decreased AP levels in 693  plasma (Nilsberth et al., 2001).  Overexpression of normal APP in Down syndrome can also predispose individuals to develop AD (Olson and Shaw, 1969). Down syndrome patients harbor an extra copy of chromosome 21, the chromosome in which the APP gene is located (Kang et al., 1987; Robakis et al., 1987; Tanzi et al., 1987). The majority of Down syndrome patients display an increased generation of AP from birth, which leads to the occurrence of classic AD neuropathology in their middle age (Goate, 2006). A clinicopathological study was conducted on a patient with Down syndrome with partial trisomy 21 in which the obligate Down's region in the distal portion of chromosome 21 was duplicated but the break point was telomeric to the APP gene (Prasher et al., 1998). Autopsy and behavioral data did not indicate any Alzheimertype neuropathological changes; thus, the development of early-onset AD owing to elevated AP deposition can also be caused by overexpression of the APP gene (Goate, 2006). Presenilin Mutations Since only a few familial AD are associated with mutations in APP, studies have been focused on identifying other genes responsible for inherited forms of AD. Both linkage analysis and genetic surveys have revealed that mutations of Presenilin 1 (PSI) on chromosome 14q and Presenilin 2 (PS2) on chromosome 1 commonly lead to early-onest of this disorder (Levy-Lahad et al., 1995; Schellenberg et al., 1992; Sherrington et al., 1995). PS mutations are linked to an elevated cleavage of the APP-C99 fragment by y-secretase, generating more A p  4 2  than A p  4 0  (Lemere et al., 1996; Mann et al., 1996). The majority of  mutations associated with familial AD are located within the PSI gene (Larner and Doran, 2006). More than 100 missense mutations in the PSI gene are associated with early-onset AD and the ratio of A p / A p 4 2  4 0  is increased due to AD-associated PSI mutations (De Strooper,  2007; Menendez, 2004; Wolfe, 2007). ApoE Polymorphism While missense mutations in APP, PSI or PS2 result in early-onset AD, the apoE4 (e4 allele of Apolipoprotein E) gene on chromosome 19q is one of the major risk factors in some late-onset AD families (Strittmatter et al., 1993a). Autopsy studies report that the frequency of the apoE e4 allele varied from 0.33 to 0.4 in AD patients and from 0.05 to 0.14 in healthy individuals (Nalbantoglu et al., 1994). This suggests that the s4 allele of apoE increases the likelihood of developing familial and sporadic late-onset AD (Saunders et al., 1993).  When compared to individuals who inherited the E2 and/or E3 alleles, those who inherited E4 alleles had a significantly higher A P plaque burden and a higher likelihood to develop AD in their 60s or 70s (Saunders et al., 1993; Scheuner et al., 1996). Although the  3  mechanisms by which the apoE4 protein elevates A0 deposition have not been fully defined, offspring resulting from APP transgenic mice and apoE-deficient mice showed a substantial decrease in cerebral A|3 deposition (Bales et al., 1997). Though some individuals with a single or pair of c4 alleles have an increased risk of developing AD, other individuals homozygous for E4 alleles don't show AD symptoms after the age of 70; thus, harboring e4 alleles of the apoE gene is only a risk factor for developing common late-onset AD (Selkoe and Podlisny, 2002).  Although it appears that alterations of APP, PS, or apoE4 genes may cause a familial form of AD, numerous families have been reported to develop AD without mutations in any of these genes, indicating that other genetic risk factors may be responsible for these cases of familial AD.  1.1.2. Sporadic Form of AD While genetic risk factors contributing to the majority of familial AD cases have been clarified, attempts to identify environmental risk factors associated with AD have not been conclusive. Several factors, such as mutations, polymorphisms, as well as non-genetic factors, can lead to the development of familial and sporadic late-onset AD (Behl, 1999). Apolipoprotein Human apolipoprotein (apo) E is one of the major lipoproteins responsible for the metabolism and redistribution of lipids (Mahley, 1988). Aside from the liver, apoE is synthesized in the brain and play a role in the integrity and remodeling of the central nervous system (CNS) (Mahley and Huang, 1999; Mahley and Rail, 2000). ApoE is produced  primarily from astrocytes within the CNS (Boyles et al., 1985; Pitas et al., 1987). Following synthesis, apoE binds to lipid particles and directs their catabolism through association with the low-density lipoprotein (LDL) receptor, very low-density lipoprotein (VLDL) receptor, and the LDL-receptor-related protein (LRP) (Takahashi et al., 1992; Wyne et al., 1996). Astrocytes express the LDL receptors, while neurons express LRP (Bu et al., 1994; Poirier et al., 1993). ApoE and its complex are produced by glial cells and internalized by neurons following interactions with surface receptors.  ApoE is an essential cholesterol carrier in both the vascular system and neurons, and is responsible for transporting membrane lipids during neuronal plasticity processes (Mahley, 1988). Located on chromosome 19, human apoE has three major protein isoforms, apoE2, apoE3 and apoE4, which differ in arg-cys substitution at positions 112 and 158 (Rail et al., 1982). Particularly, apoE4 has been described to associate with both late-onset familial and sporadic cases of AD and the frequency of the apoE e4 allele is augmented in early-onset sporadic, late-onset familial, and common late-onset sporadic AD (Fairer et al., 1997; Poirier et al., 1995; Strittmatter et al., 1993a; Wisniewski et al., 1994). ApoE has been found to colocalize with extracellular amyloid deposits and affect Ap accumulation in an isoformspecific manner (Fassbender et al., 2001; Namba et al., 1991; Strittmatter et al., 1993b). Moreover, apoE has a strong stimulatory role in the polymerization of AP into amyloid filaments (Ma et al., 1994). AD patients who were homozygous for the E4 allele of the apoE gene had a greater brain amyloid burden than AD patients homozygous for the e3 allele (Rebeck et al., 1993). Furthermore, apoE4 was examined to increase the number and density of cerebral and cerebrovascular AP deposits (Schmechel et al., 1993). Full-length APP, sAPP, and Ap bind to apoE through the LRP-mediated pathway. Thus, the secreted Ap was  5  suggested to be internalized by an apoE-dependent mechanism (Scharnagl et al., 1999). Since Ap can inhibit cellular production and secretion of apoE, disruption of cholesterol homeostasis by AP may be responsible for the development of AD pathology.  ApoE is a major cholesterol transport protein and cholesterol increases AP generation by modifying the activity of APP secretase (Fassbender et al., 2001; Kojro et al., 2001; Simons et al., 1998). Although the mechanism by which apoE influences plaque formation remains elusive, it is known that apoE facilitates Ap plaque build-up by binding to soluble Ap and enhancing P-pleated sheet formation and amyloid fibril stability (Berr et al., 1994). In addition, apoE4 has been considered to modulate APP processing and Ap generation through both the LRP-mediated pathway and domain interaction (Ye et al., 2005). These findings and others support the idea that apoE4 is a major risk factor for the development of AD (Corder et al., 1993). Angiotensin I-Conyerting E n z y m e 1  It is estimated that apoE only contributes to only 45-60% of the genetic risk for developing sporadic AD and 7-9% for developing familial AD; therefore, additional risk factors are involved in the development of late-onset AD (Daw et al., 2000; Rubinsztein and Easton, 1999). Angiotensin I-converting enzyme 1 (ACE1) has been targeted based on genetic candidate linkage studies between AD and vascular risk (Elkins et al., 2004; Farrer et al., 1989; Korten et al., 1993). ACE1 is essential for blood pressure regulation and electrolyte balance. ACE activity is increased in tissue from brain regions involved in acetylcholine production in AD cases and ACE levels are reduced in the cerebrospinal fluid (CSF) of AD patients (Kehoe, 2003).  6  ACE1 is located on chromosome 17 and contains an insert/deletion (I/D) polymorphism in its 16 intron (Chapman et al., 1998; Fairer et al., 2000). Individuals bearing th  one of two copies of the ACE1*D allele have higher circulating and tissue ACE levels than those with two copies of the ACE1*I allele (Tiret et al., 1992). This polymorphism has been associated with an increased risk to develop sporadic AD and numerous studies have supported the finding that inheritance of the ACE1*I allele is a risk factor for late-onset AD (Cheng et al., 2002; Kehoe et al., 1999; Kehoe et al., 2003; Narain et al., 2000). In vitro studies have showed that ACE can degrade synthetic forms of Ap and preclude the production of Ap aggregates (Hu et al., 2001). As possessing of the ACE1*I allele results in lower ACE enzyme activity and ACE may play a role in removing Ap plaques, the ACE 1*1 polymorphism may lead to a higher chance of developing AD (Kehoe et al., 2004; Kehoe et al., 2003). Nongenetic AD Risk Factors Aging is the primary nongenetic risk factor of AD. The prevalence of AD can be doubled every 5 years after the age of 65, and oxidative damage and messenger RNA mutations associating with aging may contribute to the development of AD (Ganguli et al., 2000; Smith et al., 1995b; van Leeuwen et al., 1998). Other nongenetic risk factors, such as diabetes mellitus and atherosclerosis can also predispose individuals to AD development (Ott et al., 1999; Skoog, 1994). Several epidemiological studies have revealed that poor education, high-calorie and high-fat diets, and other environmental factors may affect the risk of AD (Mattson, 2003; Mayeux, 2003; Stern et al., 1994). Nevertheless, the relationship between some of these nongenetic factors and AD pathogenesis has not yet been fully established.  7  1.1.3. Classic Lesions of AD Extracellular amyloid plaques and intracellular neurofibrillary tangles are the pathological characteristics of AD (Blessed et al., 1968). Ap generation is more likely to be involved in the early stage of AD pathology, whereas the aggregation of tau proteins is attributed to the later stages of AD (Baker et al., 2000). One of the hallmarks of AD is neuritic plaques, or filamentous extracellular amyloid deposits. Amyloid fibrils are formed from the aggregation of Ap (Glenner, Wong 1984).  Dystrophic neurites have been found within amyloid deposits and activated glial cells are also associated with the neuritic plaques (Dickson, 1997). Microglia reside within or adjacent to the amyloid core of plaques, whereas astrocytes are often found in the surrounding vicinity (Itagaki et al., 1989). Fibrillar Ap peptides are either 40 or 42 amino acids in length with Ap being more hydrophobic and prone to aggregation and A p being produced more 42  40  abundantly by neurons (Glenner and Wong, 1984; Masters et al., 1985).  In addition to extracellular neuritic plaques, neurons located in AD-affected brain regions contain bundles of abnormal fibers, recognized as neurofibrillary tangles (NFT) composed of microtubule-associated protein, tau (Grundke-Iqbal et al., 1986; Kosik et al., 1986; Wood et al., 1986). Tau protein is hyperphosphorylated and aggregates as insoluble paired helical filaments within NFTs. Many kinases have been shown to phosphorylate tau at different sites and yet the kinases responsible for tau hyperphosphorylation have not been identified (Avila, 2006). According to the amyloid cascade hypothesis of AD, the development of Ap aggregates precedes and participates in the formation of NFTs as a result of invoked cellular changes (Hardy and Higgins, 1992).  8  1.2. Amyloid Cascade Hypothesis of AD 1.2.1. Amyloid  Precursor Protein  The process of Ap generation involves sequential proteolysis of APP by P-secretase and y-secretase (Kang et al., 1987). The APP gene is located on chromosome 21q21.2-3 and the 19-exon-containing mRNA of APP can be spliced into multiple isoforms, the dominant transcript isoforms being APP , APP | and APP 695  75  77u  with different isoform ratios seen in  specific tissue types (Goldgaber et al., 1987; Selkoe et al., 1988; Tanaka et al., 1989; Yoshikai et al., 1990). The A P P 7 5 1 and APP770 isoforms are expressed in non-neuronal and neuronal cells, whereas a higher level of the APP695 isoform is found in neurons (Haass et al., 1991). Compared to APP  75]  and APP , the APP9.<s isoform lacks an exon that encodes for a 56770  6  amino acid motif homologous to the Kunitz-type of serine protease inhibitors (Tanaka et al., 1988). Studies of AD brains have revealed that detection of an increased production of APP695 mRNA in nucleus basalis and locus ceruleus neurons may be implicated in the deposition of cerebral Ap in AD (Palmert et al, 1988).  1.2.2.  Amyloid Precursor Protein Processing With a large extracellular N-terminal domain and a small intracellular cytoplasmic C-  terminal domain, APP is a single transmembrane domain protein and is expressed ubiquitously as a type I membrane glycoprotein (Haass et al., 1994; Kang et al., 1987; Kowalska, 2004). APP protein includes the AP region from parts of exons 16 and 17 (Johnstone et al., 1996; Yoshikai et al., 1990). At its N-terminus, a signal peptide leads APP to undergo posttranslational modification through the secretory pathway via the endoplasmic reticulum and Golgi apparatus (Turner et al., 2003; Weidemann et al., 1989). As APP undergoes trafficking through the secretory pathway, it is subject to various proteolytic cleavages and  9  releases its metabolic derivatives into vesicles (Hartmann, 1999; Turner et al., 2003) (Figure 1la). At 12 amino acids N-terminal to the transmembrane domain, APP is first cleaved by asecretase to generate soluble sAPPa and a 83-residue C-terminal fragment (CTF) (Esch et al., 1990; Sisodia, 1992; Weidemann et al., 1989). While sAPPa is released into the extracellular space, the 83-residue CTF (C83) remains bound to the membrane (Selkoe, 1989; Turner et al., 2003). Alternatively, APP can be processed by p secretase, which cleaves APP at 16 residues N-terminal to the a-cleavage site (Figure 1-lb), to produce soluble sAPPp and membrane bound, 99-residue CTF (C99) (De Strooper and Annaert, 2000; Seubert et al., 1993; Turner et al., 2003).  Following cleavage of APP by either a- or p-secretase, y-secretase can cleave C83 and C99 in the middle of the transmembrane domain (Maruyama et al., 1994; Mattson, 2004). Since a-secretase cuts APP within the region of Ap (lys - leu ), a non-pathogenic peptide, P3, 16  17  is produced following further cleavage of C83 by y-secretase (Haass et al., 1993b; Hartmann, 1999; Lammich et al., 1999). Similarly, y-secretase processes C99 at the C- terminus of the AP region to generate AP (Haass et al., 1993b; Haass et al., 1994; Steiner and Haass, 2000).  In familial AD, APP mutations near the secretase cleavage sites influence the efficiency of secretase cleavage (Haass et al., 1994; Haass and Selkoe, 1993; Kowalska, 2004; Selkoe, 1998). For example, the Swedish APP mutations near the p-cleavage site (Lys -Met 595  596  to  Asn -Leu ) (Figure 1-lb) lead to abnormal metabolism of APP which shifts APP processing 595  396  in favor of the P-secretase cleavage pathway, resulting in increased Ap generation (Cai et al., 1993; Citron et al., 1992; Haass et al., 1994; Mullan et al., 1992).  10  a membrane  •  a-secretase  p-secretase (BACE1)  CTFp (C99)  CTFa (C83)  CTFy  •  ^^^^^^^  y-secretase  j  y-secretase P3  sAPPa  sAPPp  Ap BACE1  I I  y-secretase  a-secretase  I /1\  4042  ...ISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKK...  NL Swedish  5 Flemish  • N Q Iowa Dutch G Arctic  *  Membrane  Figure 1-1. Amyloid Cascade Hypothesis of AD and Ap Sequence (a) Senile plaque was characterized in A D pathogenesis and was formed by the accumulation of Ap. A P P would undergo serial proteolytic processes to generate Ap peptides in the brain. A P P could go through a non-amyloidogenic pathway to be cleaved by a-secretase to generate a soluble N-terminal fragment, sAPPa and C-terminal fragment, C83. Since a-secretase cleaves A P P within AP region, subsequent y-secretase cleavage will preclude Ap formation and generate the p3 fragment. Alternatively, through amyloidogenic pathway, P-secretase (BACE1) cleaves A P P to produce a secreted fragment. sAPPp and a membrane-bound fragment. C99. C99 fragment will be subsequently cleaved by y-secretase to produce A p or A p . (b) Partial sequence of A P P and A P P cleavage sites of a-, p- and y-secretase are provided. The sequence representing AP domain in A P P is shown in bold and the sequence for transmembrane domain is underlined. Some familial A P P mutations were also indicated. Following BACE1 cleavage of A P P , y-secretase cleaves at amino acid 40 to generate A p and cleaves at amino acid 42 to generate A p . On the contrary, a-secretase cleaves A P P in the middle of the Ap domain to prevent AP production. 4 0  42  4fl  42  I 1  In addition, familial AD cases with APP mutations near the y-secretase cleavage sites can shift APP processing to generate the more neurotoxic Ap , and cases with APP mutations 42  near the a-secretase cleavage site can allow more APP to be processed by P-secretase, as opposed to a-secretase (Hutton et al., 1998; Jarrett and Lansbury, 1993; Selkoe, 1998; Suzuki et al., 1994).  Even though Ap generation is assumed to be a pathogenic event, AP can be constitutively released from cells under normal conditions and is detected in both cerebrospinal fluid and plasma in healthy subjects throughout life (Haass et al., 1992b; Seubert et al., 1992; Shoji et al., 1992). With regard to where APP undergoes sequential cleavages, the localization of membrane anchored aspartyl proteases has been determined. Immunocytochemical analysis has showed that APP cleavage at the a-site occurs within the endoplasmic reticulum and transGolgi apparatus, and a-secretase activity could also be detected at the cell surface where the membrane-spanning receptors were processed (Nunan and Small, 2000; Parvathy et al., 1998; Parvathy et al., 1999). The ADAM (A Disintegrin And Metalloproteinase domain) family are membrane-anchored cell surface proteins and are proposed as a-secretase candidates (Asai et al., 2003; Buxbaum et al., 1998; Walter et al., 2001). Since removal of the prodomain of ADAM family proteins occurs in the late Golgi, the cleavage of APP by a-secretase is likely to occur in the later stages of the APP's translocation to the cell membrane (Lammich et al., 1999).  Similar to a-secretase, P-secretase activity is also localized at the endoplasmic reticulum, Golgi, trans-Golgi and plasma membrane (Huse et al., 2000). A high level of ysecretase activity is localized to endosomes and the plasma membrane, while there are a  12  limited amount of presenilins in these areas, in fact, the majority of presenilins reside in the endoplasmic reticulum and early Golgi (Kovacs et al., 1996; Weidemann et al., 1997; Xia et al., 1997). However, limited amount of y-secretase cleavage of APP occurs at the endoplasmic reticulum and this finding may be explained by the uncompleted assembly of the y-secretase complex (Turner et al., 2003). Other essential components of the y-secretase complex may originate from a post-endoplasmic reticulum compartment to form a functional y-secretase complex at the end of the APP secretory pathway (Cupers et al., 2001). The identities of the essential y-secretase complex components are still under investigation and this clarification may be crucial to the characterization of y-secretase processing of APP through the amyloidogenic cascade. Recent evidence indicates that A|3 is not destined for secretion, however, the levels of Ap in extracellular fluids is much higher than in plasma (Motter et al., 1995; Scheuneret al., 1996).  1.3. A P P Cleavage Secretases  Both P- and y-secretases are aspartyl proteases (Steiner and Haass, 2000; Vassar et al., 1999; Yan et al., 1999). Aspartyl proteases are defined by the presence of two conserved aspartate residues that are essential to catalyze the hydrolysis of a peptide bond. Aspartyl proteases contain the classic D(T/S)G(T/S) signature motif, and two active site motifs are present in BACEl: DTGS (residues 93-96) and DSGT (residue 289-292) (Rentmeister et al., 2006; Walter et al., 2001). The identification of two aspartate residues and a highly conserved GxGD active site motif in PS has led to the speculation that PS may be identical to y-secretase (Haass and Steiner, 2002; Steiner et al., 2000; Yamasaki et al., 2006). If either one of the two aspartate residues is mutated, proteolytic activity of p and y-secretases is inhibited (Hussain et al., 1999; Wolfe et al., 1999c).  13  1.3.1.  a-secretase In most cells, a-secretase cleavage of APP is recognized as the principal pathway to  process APP (Sisodia, 1992; Walter et al., 2001). a-secretase cleaves APP within the critical AP peptide region and by increasing their a-secretase enzymatic activity, neuronal cells can preclude the generation of Ap peptides and prevent the formation of neuritic plaques (Busciglio et al., 1993; Hartmann, 1999). As such, estrogen and testosterone, which upregulate cellular a-secretase activity, have been included in the therapeutic treatments of AD patients (Xu et al., 1998). Based on the characterized functions of a-secretase, zinc-dependent metalloproteases, ADAMs, also called MDCs (Metalloprotease/Disintegrin/Cysteine-rich proteins), have been suggested as potential candidates (Kojro and Fahrenholz, 2005). The ADAM family of membrane-anchored glycoproteins have been implicated in many biological processes, such as, fertilization and neurogenesis (Asai et al., 2006; Evans et al., 1998; Weskamp et al., 2002). ADAM9 (MDC9), ADAM 10, and ADAM 17, also termed TACE (TNF-a converting enzyme) can affect the concentration of cellular sAPPa and may also cleave APP (Allinson et al., 2003; Hotoda et al., 2002; Koike et al., 1999; Lammich et al., 1999). In vitro evidence has shown that ADAM 10 can indeed cleave APP within Ap between residues Lys and Leu of APP, and the Flemish mutation of APP (Ala to Gly ) can reduce 16  17  21  21  such a cleavage event (Lammich et al., 1999). Another potential candidate for a-secretase protein is BACE2, which is located distal of APP in the obligate Down syndrome region of chromosome 21 (21q22.3) (Acquati et al., 2000). BACE2 cleaves APP within the Ap domain to abrogate amyloid peptide formation, and polymorphisms of BACE2 may be linked to familial AD cases that are associated with chromosome 21 and normal APP (Farzan et al., 2000; Solans et al., 2000; Sun et al., 2006b).  1.3.2. y-secretase As an unusual membrane-bound aspartyl protease, y-secretase is capable of cleaving within the hydrophobic environment of the lipid bilayer (Wolfe et al., 1999a). This cleavage releases the intracellular domain of the protein to trigger specific biological responses (Brown et al., 2000). Genetic linkage analysis of familial, early-onset AD suggests that PS mutations may shift y-secretase cleavage to enhance production of highly amyloidogenic Ap , suggesting 42  that PS may be involved in the y-secretase cleavage event, or may be y-secretase itself (Cruts et al., 1995; Hardy and Selkoe, 2002; Hutton et al., 1996; Sherrington et al., 1995). In humans, PSI resides on chromosome 14 and PS2 resides on chromosome 1 (Levy-Lahad et al., 1995; Rogaev et al., 1995; Sherrington et al., 1995). PSI and PS2 are highly homologous and share 63% amino acid identity, and both are important in maintaining cortical function in the brain and have been implicated in calcium signaling; however, the complete functions of PSI and PS2 have not yet been fully determined (Feng et al., 2004; Newman et al, 2007; Tu et al., 2006).  When both PS 1 and PS2 were knocked out, higher levels of y-secretase substrates C83 and C99 were detected, but the generation of both A P 4 0 and Ap were decreased, suggesting 42  that PS was essential for the proteolytic function of the y-secretase complex (De Strooper et al., 1998; Herreman et al., 2000; Zhang et al., 2000). The presence of two conserved aspartate residues within transmembrane domains 6 and 7 of PS provides the catalytic core of the aspartyl protease activity (Leimer et al., 1999; Wolfe et al., 1999c). When these conserved transmembrane aspartyl residues were mutated in PSI or PS2, or bound to aspartyl proteases inhibitors, y-secretase activity was lost (Esler et al., 2000; Kimberly et al., 2000; Li et al., 2000b; Steiner et al., 1999b). The active site motif of PS is also present in signal peptide  15  peptidase (SPP), SPP-like proteases, and bacterial type 4 prepilin proteases (Ponting et al., 2002; Steiner et al., 2000; Weihofen et al., 2002). PS and SPP represent intramembranecleaving proteases (I-CliPs), a polytopic aspartyl protease family characterized by two active sites located within two adjacent transmembrane domains (Weihofen et al., 2002).  While APP is a type I transmembrane protein, PS 1 and PS2 have eight membranespanning segments, with an intracellular N and C-terminus and a cytoplasmic loop between segments 6 and 7 (Steiner and Haass, 2000). Recently, another intramembrane cleavage site, the e-site, located close to the C-terminus of the APP transmembrane domain is also suggested to be cleaved by the y-secretase complex (Chen et al., 2002; Gu et al., 2001; Sastre et al., 2001; Yu et al., 2001). Presenilins Presenilin 1 is synthesized as a holoprotein (~50-kDa) and undergoes constitutive endoproteolysis within a hydrophobic portion of the cytoplasmic loop between transmembrane domains 6 and 7 in endoplasmic reticulum vesicles (Doan et al., 1996; Li and Green wald, 1998; Steiner et al., 1999c). Such proteolytic cleavage produces a presenilin heterodimer composed of the N-terminal fragment (NTF) and C-terminal fragment (CTF) (Capell et al., 1998; Podlisny et al., 1997; Thinakaran et al., 1996; Wolfe et al., 1999c). Currently, since this heterodimeric form of presenilin is detected in the active y-secretase complex, the presenilin heterodimer is suggested to function as the mature and active aspartyl protease (Cervantes et al., 2001; Levitan et al., 2001; Schroeter et al., 2003). Peptidomimetic compounds used to inhibit y-secretase activity are not only able to decrease the production of A p , but also able to 42  inhibit the intramembranous cleavage of Notch receptors at the e-site (Schroeter et al., 2003; Wolfe et al., 1999b).  Presenilin is also involved in Notch signaling, whereby cell surface signals are transmitted to the nucleus to alter expression of various downstream genes (Schroeter et al., 1998; Turner et al., 2003). Upon ligand binding, Notch is initially processed by ADAMdependant cleavage events, which are followed by PS-mediated intramembrane cleavage (Brou et al, 2000; De Strooper et al., 1999; Hartmann et al., 2002; Song et al., 1999; Struhl and Adachi, 1998). Notch then releases Notch intracellular domain (NICD), which translocates to the nucleus and initiates gene transcription essential for cell-fate determination during development (Greenwald, 1998; Schroeter et al., 1998). When the evolutionarily conserved aspartate residues within the transmembrane of PS are mutated, the ability of y-secretase to process C83 and C99 is inhibited, and NICD release from Notch receptors is blocked (Ray et al., 1999a; Ray et al., 1999b; Steiner et al., 1999b; Wolfe et al., 1999c). For certain familial AD cases caused by PS mutations, Notch cleavage by PS is reduced while  AP42  production is  enhanced (Moehlmann et al., 2002; Song et al., 1999; Walter et al., 2001). Nicastrin Despite its importance, PS alone is not sufficient for y-secretase activity, but rather requires other cofactors to assemble a functional y-secretase complex (De Strooper, 2003). The purified y-secretase protein mass is estimated at 250-2000kDa, a size far larger than the 50kDa PS alone (Periz and Fortini, 2004; Steiner et al., 2002; Yu et al., 1998; Yu et al., 2000). After intensive biochemical and genetic studies of the y-secretase complex, three additional conserved components, Nicastrin, as well as Aph-1 (Anterior Pharynx defective-1) and Pen-2  17  (Presenilin enhancer-2), have been proposed to regulate y-secretase activity (Francis et al., 2002; Takasugi et al., 2003; Yu et al., 2000). This proposition is supported by the observation of a physical interaction between PS and Nicastrin in C. elegans (Yu et al., 2000).  Nicastrin (Net) is a ~80kDa type I transmembrane glycoprotein which undergoes glycosylation and sialylation to create a ~150kDa mature form (Edbauer et al., 2002; Periz and Fortini, 2004; Tomita et al., 2002). Mature Net is present from the Golgi apparatus to the cell membrane and is associated with y-secretase activity by an interaction with PS (Kaether et al., 2002; Leem et al., 2002; Yang et al., 2002). When PSI is ablated, Net does not undergo further trafficking through the secretory pathway and the availability of mature Net is reduced (Leem et al., 2002). Proteasome and lysosome have been described to be involved in Net degradation and low Net availability may affect the activation of y-secretase complex (He et al., 2007). When Net activity is downregulated by RNA interference, a similar loss of ysecretase activity is observed and the formation of the PSI /y-secretase complex is significantly reduced (Edbauer et al., 2002; Francis et al., 2002). It is then suggested that Net may be essential for the stability of PS fragments and prevent PS from rapid degradation (Hu et al., 2002). Net also associates directly with type I transmembrane proteins, such as APP and Notch, to affect the efficiency of intramembranous proteolysis (Shah et al., 2005; Yu et al., 2000). Hence, PS and Net are essential components of the y-secretase complex and their interaction is necessary in order to exert y-secretase activity and process APP (Capell et al., 2003; Chung and Struhl, 2001; Edbauer et al., 2002).  18 Anterior Pharynx defective-land Presenilin enhancer-2 Genetic screens of mutations in C. elegans that affect Notch and PS functions have revealed that Aph-1 and Pen-2 are associated with the PS-Nicastrin complex and regulate ysecretase activity (De Strooper, 2003; Francis et al., 2002; Goutte et al., 2002). Aph-1 is predicted to have a multipass membrane-spanning structure while the smaller-sized Pen-2 contains two predicted membrane-spanning segments (Francis et al., 2002; Goutte et al., 2002; Periz and Fortini, 2004). Both Aph-1 and Pen-2 have been determined to be degraded by ubiquitin-proteasome pathway (He et al., 2006). When all four components are expressed in yeast Saccharomyces cerevisiae, an organism which lacks y-secretase activity, fully functional y-secretase activity is reconstituted (Edbauer et al., 2003). When Net, Aph-1 or Pen-2 is eliminated in vitro or in vivo, mature PS-cleaved NTF and CTF fail to aggregate and functional y-secretase activity is not detected (De Strooper, 2003; Fortini, 2002).  Overexpression of PS does not enhance y-secretase activity, and the overexpressed holoproteins are degraded; thus, it is speculated that there may be other limiting factors which stabilize PS holoprotein and promote its maturation (Takasugi et al., 2003; Thinakaran et al., 1996; Thinakaran et al., 1997). The expression of all four known components of the ysecretase complex leads to an increased amount of mature PS NTF and CTF (Edbauer et al., 2003; Kimberly et al., 2003; Takasugi et al., 2003). It was also examined that an increased level of mature y-secretase complex is accompanied by a significantly higher degree of Notch and APP proteolysis (Hu and Fortini, 2003; Kim et al., 2003; Luo et al., 2003). Although the composition of the y-secretase complex has been elucidated, the role of each individual component still remains unknown.  19  1.3.3. P-secretase The Ap sequence in APP can start from lie" , Vaf , Asp and Glu ", but P-secretase is 6  3  +1  +  only responsible for producing Ap peptides starting at Asp (major) and Glu " (minor) +I  +  (Busciglio et al., 1993; Haass et al., 1993a; Haass et al., 1992b; Shoji et al., 1992). Following Y-secretase cleavage at the C-terminus of the Ap domain of APP, Ap is released and aggregated to form neuritic plaques (Mattson, 2004). P-secretase cleaves the APP 5 isoform at 69  Met , the N-terminus of Ap, and the Swedish APP mutations make APP a better substrate for 596  P-secretase cleavage (Citron et al., 1992). The Swedish APP mutations shift proteolytic processing of APP towards the P-secretase pathway, and this may explain the accelerated Ap deposition in some familial AD individuals inherited with APP missense mutations (Cai et al., 1993; Citron et al., 1992; Haass et al., 1995). p-Site APP Cleaving Enzyme 1 The pepsin family is a family of endopeptidases which require aspartic residues for their catalytic activity, including cathepsin D and cathepsin E, which are related by extensive sequence homology (Tang and Wong, 1987). A new member of this family, p-site APP Cleaving Enzyme 1 (BACE1), has been described to have a unique transmembrane spanning domain and cleave APP to produce membrane bound C99 (Hussain et al., 1999; Morgan et al., 1992; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999). BACE1 is a N-glycosylated integral membrane protein of 501 amino acids, belongs to the eukaryotic aspartic protease family of proteins, and has two active site motifs at its luminal end (Haniu et al., 2000; Walter et al., 2001; Yan et al., 1999). BACE1 has a single transmembrane domain near the Cterminus, contains many N-linked glycosylation sites, and six luminal cysteine residues (Cys -Cys , Cys -Cys 216  420  278  443  and Cys -Cys ), which form three intramolecular disulfide 330  380  20  bonds (Haniu et al., 2000). When comparing its disulfide structure to aspartic proteases in the pepsin family, BACEl does not show significant homology to other pepsin family members; thus, structural differences may contribute to the substrate specificity of the proteases (Haniu et al., 2000).  Pro-BACEl, produced in the endoplasmic reticulum, weighs ~60kDa and is short-lived. After addition of a carbohydrate complex and removal of the propeptide domain by cleavage between Arg and Glu inside the Golgi apparatus, it becomes longer-lived and weighs 45  46  ~70kDa (Bennett et al., 2000b; Capell et al., 2000; Haniu et al., 2000). Four N-glycosylation 1 53  sites (Asn ~ , Asn  172  , Asn  223  354  and Asn" ) on BACEl are essential for its protease activity, but  the function of BACEl glycosylation has not been well characterized (Charlwood et al., 2001). Glycosylation may contribute to proper protein folding to stabilize BACEl. BACEl is sulfated at mature N-glycosylated moieties and palmitoylated at three cysteine residues within its transmembrane/cytosolic tail (Benjannet et al., 2001). Palmitoylation of BACEl is important not only to control BACEl intracellular trafficking, but also to prevent BACEl from ectodomain shedding. The presence of the shed or soluble form of BACEl enhances A|3 generation by favoring amyloidogenic processing of APP (Benjannet et al., 2001). In addition, BACEl has been determined to be degraded by ubiquitin-proteasome pathway and consequently APP proteolytic cleavage by BACEl in A[3 generation may be affected (Qing et al., 2004).  The majority of aspartic proteases can cleave their propeptide domain autocatalytically in response to changes in pH or ionic strength; for example, BACEl homologue BACE2 can undergo propeptide cleavage autocatalytically in an acidic environment of pH4.5 (Hussain et  21  al., 2001). However, BACE1 requires another protease to remove the propeptide domain (Capell et al., 2000; Creemers et al., 2001). The propeptide domain of immature BACE1 contains a proprotein convertase cleavage recognition sequence (Arg-Leu-Pro-Arg) and the mutation of the arginine consensus residues can inhibit BACE1 maturation by preventing propeptide removal (Bennett et al., 2000b; Creemers et al., 2001). Though other proprotein convertases have been reported to be able to cleave immature BACE1, furin, a ubiquitous proprotein convertase inside the Golgi, is the major mediator of BACE1 propeptide removal (Bennett et al., 2000b).  Normally, a prodomain on an immature protein can suppress the protein's functions; however, immature proBACEl still exhibits P-secretase activity despite presence of the prodomain (Creemers et al., 2001). Therefore, APP can be cleaved by proBACEl and Ap can be generated in the endoplasmic reticulum (Shi et al., 2001). Compared to healthy individuals, Down syndrome patients have been shown to have higher levels of mature BACE1 proteins in Golgi, which leads to increased P-secretase activity and AP generation (Sun et al., 2006c).  BACE1 mRNA levels are highest in pancreas and brain and P-secretase activity is highly localized within the neuronal cells (Hussain et al., 1999; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999). The majority of BACE1 protein is detected in the Golgi and in endosomal compartments where APP is located (Huse et al., 2000). These acidic secretory compartments allow access to the ideal subcellular sites to process APP (Haass et al., 1992a; Lin et al., 2000). P-secretase activity can be detected in many different cell and tissue types. The maximal P-secretase activity can only be measured in the brain and BACE1 is the major proteolytic enzyme that cleaves APP at the P-site (Cai et al., 2001; Seubert et al., 1993).  22  BACEl cleaves APP at the N-terminus of the Ap domain and releases a soluble APPp fragment that is 16 amino acids shorter than the soluble APPa fragment (Turner et al., 2003). BACEl cleavage at Asp or Glu 1  3kDa A P i  1-40/42  11  within the Ap sequence of APP generates a 4kDa A Pi.40/42 or  peptide, respectively (Cai et al., 2001; Farzan et al., 2000; Vassar et al., 1999).  Cleavage of APP at Glu" by BACEl is species specific and is favored to be processed within the Golgi apparatus (Fluhrer et al., 2002; Vassar et al., 1999). In some AD cases, together with the presence of mutated APP which is more efficiently cleaved by BACEl, an elevated level and activity of p-secretase may lead to excessive Ap generation and plaque formation (Steiner et al., 1999a).  Though the brain displays a high level of BACEl mRNA, little or no BACEl mRNA expression was detected in glial cells from in-situ hybridization experiments (HartlageRubsamen et al., 2003; Rossner et al., 2005; Vassar et al., 1999; Yan et al., 1999). A high expression of BACEl protein is found in neurons but not in glial cells (Hussain et al., 1999). Although neurons are believed to be the major source of BACEl enzymatic activity in the brain, p-secretase activity is also detected in astrocytes (Hartlage-Rubsamen et al., 2003; Rossner et al., 2001; Rossner et al., 2005). BACEl mRNA is expressed in the pancreas and peripheral tissues, but little or no BACEl enzymatic activity is detectable in these tissues (Ehehalt et al., 2002; Yan et al., 1999). A novel splice variant of BACEl mRNA which is missing two-thirds of exon 3, was identified in the pancreas (Bodendorf et al., 2001). This novel variant of BACEl is expressed as both mRNA and protein, but it lacks the functional ability to process APP. Due to the partial exon deletion, this particular BACEl protein may be misfolded and remain in the endoplasmic reticulum (Bodendorf et al., 2001; Ehehalt et al., 2002). As a result, the presence of BACEl protein formed alternative splicing of BACEl  23  mRNA may explain the lack of C-terminal amyloidogenic intermediate C99 in the pancreas, despite high BACE1 mRNA levels. B A C E 2  Following the identification of BACE1 as P-secretase, BACE2, located on chromosome 21q22.3, was also suggested to cleave APP at the p-site and be implicated in the Alzheimertype neuropathology of Down syndrome (Farzan et al., 2000; Solans et al., 2000). However, despite the evidence of elevated Ap generation, BACE2 cleaves APP within the Ap domain and may not be responsible for the AD pathogenesis in Down syndrome patients (Basi et al., 2003; Sun et al., 2006b; Sun et al., 2005). Similar to other mammalian aspartic proteases, BACE2 and its homologue BACE1 have a conserved catalytic domain and DTG and DSG active site motifs (Walter et al., 2001) (Figure 1-2). Both BACE2 and BACE1 show a typical transmembrane region and disulfide bond structure at the C-terminus (Acquati et al., 2000; Lin et al., 2000). BACE2 also undergoes similar post-translational modification as BACE1, including N-glycosylation and prodomain removal (Hussain et al., 2001; Solans et al., 2000; Sun et al., 2005). While the prodomain of BACE1 is cleaved by furin or furin-like proteases, the prodomain of BACE2 is removed autocatalytically (Capell et al., 2000; Creemers et al., 2001; Yan et al., 2001). BACE2 mRNA has a low expression in the central nervous system and has a higher expression in peripheral tissues, such as the pancreas and stomach (Bennett et al., 2000a; Solans et al., 2000). Unlike BACE1 brain mRNA distribution, BACE2 mRNA is selectively expressed in the neurons in a limited number of brain nuclei, including the ventromedial hypothalamus nucleus and the mammilary body (Bennett et al., 2000a).  PRO-PEPTIDE DOMAIN BACEl BACE2  MAQALPW LLLWMGAGVLPAHGTQHGIRLPLRSGL-GGAPLGLRLPRE MGALARALLLPLLAQWLLRAAPELAPAPFTLPLRVAAATNRVVAPTPGP GTPAERHA  •  * N-TERMINAL ACTIVE SITE  BACEl BACE 2  TD—EEPEEPGR RGSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFA DGIJULiALEPAIASPAGAANFLAMVCbTLQ  BACEl BACE2  VGAAPH PFLHRYYQRQL S S TYRD LRKGVYVPYTQGKWE GE LGTDLVSIPHGPNVTVRANI VAGTPHSYIDTYFDTERSSTYRSKGFDVTVKYTQGSWTGFVGEDLVTIPKGFNTSFLVNI * ** * ***** * * **** * * * *** ** * * **  BACEl BACE 2  AAITE SDKFFINGSNWEGILGLAYAEIARPDDSLE PFFDSLVKQTHVPNLFSLQLCGAGF ATIFESENFFLPGIKWNGILGIJ^YATLAKPSSSI^TFFDSLWQANIPNvTSMCiM  BACEl BACE2  PLNQSEVIASVGGSMIIGGIDHSLYTGSLWYTPIRREWYYEVTIYRVEINGQDLKMDCKE P-VAGSGTN—GGSLVLGGIEPSLYKGDIV^TPIKEEWYYQIEILKLEIGGQSLNLDCRE * *** *** *** * ***** **** * ***** ** *  BACEl BACE 2  •  * * **  *  **  *  ***** * ** *** **  * ********  * *  *** ****** *  C-TERMINAL ACTIVE SITE YNYDKSIVDSGTTNIJ^PKKVFEAAVKSIKAA^ YNADKAIVDSGTTLLRLPQKVTDAVVEA^  ** ** ******* **** *** * *  * *** * ***********  **  * * * * * ****  * **** * ** **  ***  BACEl BACE 2  FPVT SL YLMGEVTNQS FRITILPQQYLRPVE DVAT S QDD CYKFAIS QS S T GTVMGAVIME FPKISIYLRDENSSRSFRITILPQLYIQPMMGAGLNY-ECYRFGISPSTNALVIGATVME ** ** ** * ********* * * ****** * ** **  BACEl BACE 2  GFYVVPDRARKRIGFAVSACHVHDEFRTAAVEGPFVTLDMEDCGYNIPQTDESTLMTIAY GFYVIFDRAQKRVGFAASPCAEIAGAAVSEISGPFSTEDVASNCVPAQSLSE PILWIVSY **** **** ** *** * * *** * * * * *  BACEl BACE 2  TM DOMAIN VMAAIC^FMLPLCI>IVCQWCCLRCLRQQHDDFADDISLLK ALMSVCGAILLVLIVLLLLPFRCQRRPRDPE WNDE SSLVRHRWK * * * * **  Figure 1-2. Sequence Alignment of B A C E l and B A C E 2 Protein sequence of B A C E l (ENSP00000318585/1-501) and B A C E 2 (ENSP00O00332979/1-518). Hyphens denote gaps in the polypeptide sequence and * donates a single, fully conserved residue. The sequences of aspartic protease active site motifs, pro-peptide domain and transmembrane domain are indicated below the lines. B A C E l shows -64% similar in amino acid sequence with B A C E 2 , but B A C E l or B A C E 2 shares only -40% amino acid similarity to other pepsin family members. B A C E l has two predicted N-linked glycosylation sites and six luminal cysteine residues that are closely at position with B A C E 2 . In addition, a single transmembrane domain at the C-terminal extension was characterized in both B A C E l and B A C E 2 , but not in closely related pepsin family members. So B A C E l and B A C E 2 are defined as novel transmembrane aspartic proteases.  25  APP cleavage by BACE2 can occur in the Golgi and in later secretory compartments (Fluhrer et al., 2002; Hussain et al., 2000). In addition to cleaving at the P-secretase site, BACE2 is also determined to have a similar function as a-secretase and cleave more efficiently near the a-secretase cleavage site within the Ap region of APP (Phe -Phe and Phe -Ala ) 19  20  20  21  (Fluhrer et al., 2002; Yan et al., 2001). However, there are a few familial AD cases where a number of missense mutations near the P-site can affect APP processing by BACE1 or BACE2. For example, the Flemish APP mutation (Ala to Gly in the AP region of APP) is a 21  21  missense mutation adjacent to the BACE2 cleavage site within the Ap domain and APP, in this case, is preferentially processed by BACE2 at the P-secretase site to result in a markedly increased Ap production (Farzan et al., 2000; Lammich et al., 1999). BACE2 is also proposed to perturb AP generation in the brain by participating in the cleavage of wild-type APP at a novel 0-site, downstream of the a-site (Sun et al., 2006b). While the catalytic role of BACE1 is well characterized, the role of BACE2 as a proteolytic secretase in APP processing remains to be examined.  1.4. Transcriptional and Translational Regulation of BACE1 Expression 1.4.1. Transcriptional Modulation of BACE1 Based on the characterization of several cis-acting transcription factor binding elements in TATA-less BACE1 promoter region, BACE1 is considered to be tightly regulated at the transcriptional level (Christensen et al., 2004; Ge et al., 2004; Sambamurti et al., 2004). Though BACE1 and BACE2 are highly homologous, limited sequence similarity is found between the promoters of BACE1 and BACE 2 (Solans et al., 2000; Sun et al., 2005). In addition to its role in regulating of BACE2 gene transcription, Spl has been demonstrated to associate with human BACE1 promoter and enhance BACE1 gene transcription (Christensen  26  et al., 2004; Sun et al., 2005). By increasing BACE1 promoter activity, Spl elevates BACE1 protein levels and enzymatic activity, leading to augmented A(3 generation.  Limited interspecies similarity is determined between human and rat BACE1 sequence; however, several transcription factors binding elements, such as Spl, found on human BACE1 promoter are also identified on rat BACE1 promoter (Christensen et al., 2004; Sambamurti et al, 2004). Rat BACE1 promoter is determined to be regulated by Peroxisome proliferatoractivated receptor-y and Yin Yang 1 (YY1) at the transcriptional level (Lange-Dohna et al., 2003). As vascular abnormalities have been considered as a risk factor in AD development, several studies have focused on defining the link between AD pathology and vascular disorders (Kalaria et al., 1998; Kehoe, 2003; Panza et al., 2004; Sun et al., 2006a). For example, hypoxia caused consequently by reduced cerebral perfusion is implicated in neurodegeneration (Halterman et al., 1999; Pugh and Ratcliffe, 2003). Hypoxia initiates gene transcription regulation by Hypoxia inducible factor-1, which has been demonstrated to associate with hypoxia-responsive element on human BACE1 promoter to elevate BACE1 gene transcription and AP production (Sun et al., 2006a; Zhang et al., 2007). HaCVinduced oxidative stress has also been determined to upregulate BACE1 gene transcription and alter APP processing (Tong et al., 2005).  1.4.2. Translational Modulation of B A C E 1 Recent reports have shown that increased BACE1 protein levels in AD brain are accompanied by unchanged BACE1 mRNA levels; hence, post-transcriptional modulations of BACE1, such as translation control and protein degradation, have been suggested to regulate BACE1 expression (De Pietri Tonelli et al., 2004; Holsinger et al., 2002; Preece et al., 2003;  27  Puglielli et al., 2003; Rogers et al., 2004). Due to the unique sequence features of 5' UTR of BACE1 mRNA, BACE1 translation is significantly reduced (Lammich et al., 2004; Zhou and Song, 2006). Certain physiological or pathological stimuli may alleviate the repressed BACE1 translation; thus elevated BACE1 expression contributes to increased AP generation in AD pathogenesis (Gingras et al., 2001; Li et al., 2006).  In normal conditions, BACE1 mRNA levels have been demonstrated to be lower than APP mRNA levels and only small amount of APP is processed through p-secretase cleavage pathway (Li et al., 2006). It has been suggested that leaky scanning mechanism by ribosome and reinitiation of BACE1 translation may result in low BACE1 expression (Zhou and Song, 2006). However, a slight increase in BACE1 levels has been determined to lead to a dramatic elevation in Ap production (Li et al., 2006). BACE1 is tightly regulated at both the transcriptional and translational levels, and since higher BACE1 expression has been detected in sporadic AD brain and elevated P-secretase activity has been found in some familial AD brains, BACE1 inhibition has been targeted for the development of AD therapeutic treatment (Fukumoto et al., 2002; Fukumoto et al., 2004; Holsinger et al., 2002; Li et al., 2006; Marcinkeviciene et al., 2001; Russo et al., 2000; Vassar, 2002; Yang et al., 2003b). Despite some minor developmental and behavioral deficits, AP generation is abolished in BACE1knockout mice; therefore, defining the mechanisms by which BACE1 is regulated in the brain provides insights into the strategies to reduce Ap burden in AD pathogenesis (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001).  Based on the sequence analysis of the human BACE1 promoter region (GenBank AY 162468) (Christensen et al., 2004), we have identified four putative N F - K B binding  28  elements. N F - K B transcription factor regulates gene expression in response to several pathological conditions, such as inflammation and oxidative stress (Baeuerle and Henkel, 1994; Schreck et al., 1992), and is implicated in neurological disorders, such as A D (O'Neill and Kaltschmidt, 1997). An increased N F - K B activity has been detected in the nuclei of neurons in the vicinity of neurotoxic A p plaques (Behl et al., 1994b; Kaltschmidt et al., 1997; Terai et al., 1996), which cause oxidative stress-induced neuronal injury and neuroinflammation as described in AD (Coyle and Puttfarcken, 1993; Huang et al., 1999; McGeer and McGeer, 2003). Therefore, N F - K B may be involved in gene regulation during the development of AD pathogenesis.  1.5. Nuclear Factor Kappa B 1.5.1.  The Identity and Function of NF-KB  NF-KB  Components  is a transcription factor composed of two subunits, including N F - K B 1 (p50),  N F - K B 2 (p52), RelA (p65), RelB and C-Rel (Baldwin, 1996; Karin and Ben-Neriah, 2000; Siebenlist et al., 1994). N F - K B was first reported as a B-cell nuclear factor that binds to a site in the immunoglobin K enhancer (Sen and Baltimore, 1986). Due to the presence of a highly conserved Rel homology domain (RHD), N F - K B subunits are able to form dimers and associate with DNA and I K B (Ghosh et al., 1998; Karin and Ben-Neriah, 2000). N F - K B dimers can exist as homodimers or heterodimers, but the dominant form of N F - K B is composed of p65 and p50 (Baeuerle and Baltimore, 1989; Kawakami et al., 1988; O'Neill and Kaltschmidt, 1997).  NF-KB  is a sequence-specific transcription factor and different forms of N F - K B dimers  express distinct DNA binding preferences (Kunsch et al., 1992). In the promoter region of NF-  29  KB-targeted genes, p65/p50 dimers bind to the sequence 5'-GGGRNNYYCC-3' whereas p65/C-Rel dimers recognize the sequence 5'-HGGARNYYCC-3' (Baldwin, 1996; Miyamoto and Verma, 1995). Moreover, the subunits of N F - K B have a preferential interaction with different I K B isoforms. For example, p65-containing dimers prefer to associate with IKBCC and I K B P , but p50-containing dimers have a preference for IicBy and IKB£ (Moorthy and Ghosh, 2003; Siebenlist et al, 1994; Yamazaki et al., 2001). N F - K B are constitutively expressed in all cell types with the exception of RelB, the expression of which is only found in certain cell types, such as interdigitating dendritic cells in lymphoid tissues (Carrasco et al., 1993; Ryseck et al., 1996).  Though homodimers of p52, p50 or p65 are considered to repress gene transcription activity, the general role of N F - K B in transcriptional regulation is as an activator (Ghosh and Karin, 2002). Homodimers of p50 or p65 associate with histone deacetylase-1, bind to the gene promoter, and suppress NF-KB-dependent gene expression (Zhong et al., 2002). In terms of transcriptional regulation, N F - K B proteins containing the RelB subunit can either activate or repress gene transcription (Ryseck et al., 1992). For example, the RelB/p50 or RelB/p52 dimer activates gene transcription, whereas the p65/RelB heterodimer acts as a suppressor (Marienfeld et al., 2003). Hence, the distinct functions of individual N F - K B subunits provide NF-KB  complexes with the capacity to modulate NF-KB-dependent gene expression based on  physiological necessities (Siebenlist et al., 1994).  1.5.2.  Regulation of N F - K B Activity by IKB NF-KB  activity is modulated by proteins of the I K B family, which consist of IKBCC,  I K B P , IKBY, I K B E , I K B ^ ,  Bcl-3, pl05, plOO and molecule possessing ankyrin-repeats induced  30  by lipopolysaccharide (MAIL) (Baeuerle and Baltimore, 1996; Kitamura et al., 2000; Yamazaki et al., 2001). The pl05 and plOO proteins are precursors of p50 and p52, respectively, and both of their C-termini contain the structural characteristics of the I K B family (Karin and Ben-Neriah, 2000). After pl05 and plOO undergo proteasome-mediated proteolysis at their C-termini, the intact N-terminals become p50 and p52 (Betts and Nabel, 1996; Lin and Ghosh, 1996; Moorthy and Ghosh, 2003).  Because of the presence of six to eight copies of the ankyrin repeat domain, all members of the I K B family are able to interact with N F - K B dimers and mask their nuclear translocation sequence (Huxford et al., 1998). Furthermore, the unique structure of certain I K B members contributes to the differences in binding preference, function and mode of activation of N F - K B dimer. IKBOI, I K B P and I K B C have a N-terminal regulatory region which is essential for stimulation-induced I K B degradation (Siebenlist et al., 1994). In particular, both IKBOI and Ixfip show a preference to interact with N F - K B dimers containing the transactivating p65 subunit, whereas IKBY and I K B ^ prefer to associate with p50-containing N F - K B complexes (Baldwin, 1996; Moorthy and Ghosh, 2003; Yamazaki et al., 2001). Other I K B family members, such as I K B E and plOO, are also reported to interact exclusively with N F - K B complexes composed of particular subunits (Baeuerle and Baichwal, 1997).  The regulatory mechanism for IKB protein expression is different for each family member. hcBa, I K B P and I K B E are constitutively expressed in all cell types, but IKBY is only present in certain cell types, such as pre-B-cells (Inoue et al., 1992; Karin and Ben-Neriah, 2000). Being the only inducible nuclear proteins in the I K B family, I K B ^ and MAIL are induced by lipopolysaccharide (LPS) and pro-inflammatory cytokines, and their transcriptional  31  activities are regulated by N F - K B (Eto et al., 2003; Ito et al., 2004). Contrary to the inhibitory functions of other I K B proteins, MAIL is recognized as a transcriptional enhancer and mediates LPS-induced IL-6 expression (Kitamura et al., 2000).  The response of I K B proteins is dependent on the identity of N F - K B activators. For example, I K B P can only undergo degradation in response to LPS or IL-1, but not to TNF-a or phorbol myristate acetate. In contrast, IKBCI is degraded by all of the aforementioned N F - K B activating stimuli (Thanos and Maniatis, 1995). So, it has been proposed that I K B P can form a iKBp/NF-KB/Ras complex, which interferes with I K B P phosphorylation and degradation (Chen et al., 2004). In terms of biological functions, IKBOI is involved in negative feedback mechanisms which turn off the rapid N F - K B response. Conversely, I K B P and I K B E function to keep the N F - K B response stabilized during long-term stimulation (Hoffmann et al., 2002). As such, each I K B protein member works through its unique functions to regulate N F - K B activity.  As inhibitors of N F - K B activity, I K B proteins sequester N F - K B in the cytoplasm, facilitate the dissociation of N F - K B dimers from their DNA binding sites, and export N F - K B from the nucleus (Ghosh and Karin, 2002). N F - K B activation can increase the transcription of MBa, I K B ^ and MAIL genes (Eto et al., 2003; Ito et al., 2004; Karin and Ben-Neriah, 2000). Inducible I K B ^ associates with N F - K B dimers in the nucleus to inhibit NF-KB-responsive gene transcription (Yamazaki et al., 2001). Due to its nuclear export sequences (NES), IicBa exports N F - K B dimers from the nucleus after dissociating them from DNA binding sites (Arenzana-  Seisdedos et al., 1997). By masking the nuclear localization sequences (NLS) on p65 and exposing NLS on p50, the I K B O / N F - K B complex is able to shuttle between the nucleus and the cytoplasm and the dominant NES effect of IKBO. causes the complex to remain in the  32  cytoplasm (Baeuerle, 1998; Cramer and Muller, 1999; Ghosh and Karin, 2002; Jacobs and Harrison, 1998).  1.5.3. N F - K B NF-KB  Activation can be activated by various stimuli, such as oxidant stress, mitogens, apoptotic  mediators, and bacterial products (Ghosh et al., 1998; Siebenlist et al., 1994). There are multiple transduction pathways leading to N F - K B activation and some pathways are associated with particular stimuli; therefore, a great amount of signaling mediators are involved in the N F K B activation pathways (Mercurio and Manning, 1999; Rothwarf and Karin, 1999).  Canonical and Non-Canonical Pathways Although the upstream signaling pathways of N F - K B activation are complicated, these  pathways can be converged into canonical and non-canonical pathways at some nodal points. While the majority of N F - K B activators work through canonical pathways, there are N F - K B stimulators that can work through both (Pomerantz and Baltimore, 2002).  I K B kinase, IKK, is the converging point of the canonical pathways and numerous N F K B activators initiate signaling pathways that result in activation of the IKK complex (Rothwarf and Karin, 1999; Zandi and Karin, 1999). Activated IKK complex causes a rapid phosphorylation on serine residues of I K B proteins and subsequently, phosphorylated I K B proteins undergo polyubiquitination by Skpl-Cullinl-Rocl-F-box ubiquitin ligase complex (Hayden and Ghosh, 2004; Karin and Ben-Neriah, 2000). After I K B proteins undergo the serial process of phosphorylation, ubiquitination, and 26S proteasomal degradation, released  33  NF-KB  dimers are translocated into the nucleus and regulate  NF-KB  responsive gene  transcription (Alkalay et al., 1995) (Figure 1-3).  F i g u r e 1-3. A Schematic M o d e l of N F - K B A c t i v a t i o n Due to their binding to IKB, N F - K B dimers (mostly RelA/p65:p50) are sequestered in cytoplasm and remain inactive. After NF-KB-activating stimuli activate the I K K complex, I K K phosphorylates IKB at specific serine residues. Phosphorylated IKB is ubiquitinated and degraded by proteasome pathway. Released N F - K B dimers are translocated into nucleus and regulate N F KB responsive genes by associating to the cis-acting binding elements in the promoter regions.  Despite the involvement of I K K activation, IKB serine phosphorylation and IKB degradation in the signaling pathways leading to N F - K B translocation, N F - K B can also be activated through non-canonical pathways, which are divergent signaling pathways without a defined converging point (Pomerantz and Baltimore, 2 0 0 2 ) . Hypoxia-reoxygenation, for example, can evoke N F - K B activation by tyrosine-phosphorylation of I K B without complex involvement, and  H2O2  IKK  activates N F - K B proteins without eliciting IKB(X degradation 34  (Canty et al., 1999; Fan et al., 2003). Hepatitis C virus nonstructural protein 5A can also induce tyrosine phosphorylation of IKBCI protein, but in this case, IKBOI degradation is mediated by the protease calpain, instead of the proteasome (Gong et al., 2001; Waris et al., 2003). In the case of N F - K B activation induced by mitochondrial stress, the function of I K B P can be turned off by calcineurin-mediated dephosphorylation; hence, the translocation of N F - K B dimers into the nucleus can results from the activation of many physiological pathways, and the role of N F - K B in gene regulation is implicated in numerous pathological conditions (Biswas et al., 2003; O'Neill and Kaltschmidt, 1997). Other Factors in the Regulation of N F - K B Activity  Besides iKB-mediated nuclear translocation, N F - K B activity can also be regulated by other mechanisms involving nuclear import and export of N F - K B . A decrease in intracellular zinc concentration reduces the nuclear import of N F - K B dimers, whereas an elevation in intracellular calcium concentration promotes N F - K B responsive transcription by accelerating N F - K B dimer translocation (Komarova et al., 2003; Mackenzie et al., 2002). In addition, N F K B activity can be controlled by regulating the recruitment of the N F - K B dimers to their target  genes to modulate NF-KB-mediated transcription.  Following posttranslational modifications of N F - K B protein subunits, such as phosphorylation and acetylation, the interaction between N F - K B and chromatin is enhanced (Chen and Greene, 2003; Ghosh and Karin, 2002; Hou et al., 2003). N F - K B subunit, p65, has been examined to be phosphorylated by protein kinase A, Serine/Threonine protein kinase, GSK-3P,  and NF-KB-activating kinase in order to drive gene transcription (Ghosh and Karin,  2002). Numerous kinases can phosphorylate p65 directly or through IKK activation, and each  kinase works on different phospho-acceptor sites; therefore, the transcriptional regulation of NF-KB  on gene expression has been suggested to be modulated by.phosphorylating N F - K B p65  subunit in a cis-acting element and promoter-specific manner (Anrather et al., 2005; Buss et al., 2004; Chantome et al., 2004; Hayden and Ghosh, 2004). In addition to phosphorylation modification, N F - K B undergoes reversible acetylation, S-nitrosylation and glutathionylation processes which regulate N F - K B activity (Chen and Greene, 2003; delaTorre et al., 1999; Furia et al., 2002; Pineda-Molina et al., 2001).  Various transcriptional co-activators or co-repressors can directly or indirectly interact with N F - K B . This cross-talk can be either synergistic or antagonistic and can be either reciprocal or nonreciprocal; thus, this type of protein interaction may be responsible for maintaining balancing N F - K B responsive gene transcription (Gao et al., 2005; Rothwarf and Karin, 1999). In addition, positive and negative feedback loops are reported to regulate N F - K B activity. In the case of positive feedback mechanisms, p65 induces hcBa degradation allowing more N F - K B is able to translocate into the nucleus; on the other hand, N F - K B activation upregulates the production of I K B proteins, such as hcBa and I K B ^ , such that released N F - K B is sequestered in the cytoplasm (Eto et al., 2003; Karin and Ben-Neriah, 2000; Nelson et al., 2004; Yang et al, 2003a). I K K Complex  As the converging point of canonical pathways leading to N F - K B activation, IKK is composed of three subunits: IKKa, IKKP and IKKy (Regnier et al., 1997; Rothwarf et al., 1998). Since both IKKa and IKKp have leucine zipper motifs, they can form a dimer and subsequently, associate with IKKy to complete IKK complex assembly (Zandi et al., 1997).  36  Although IKKa and IKKP function as catalytic subunits in IKK activation, the absence of regulatory subunit IKKy results in the disconnection of the IKK complex from the upstream signaling pathways (Rothwarf et al., 1998). While they are responsible for IKK complex formation and IKK catalytic function, both the IKKa and IKKP subunits have distinct functions in events implicated in N F - K B activation. In response to inflammation, IKKP is involved in the stimulation of innate immunity and induces the activation of N F - K B dimers by mediating I K B phosphorylation and degradation, while IKKa mediates the termination of inflammatory responses and promotes N F - K B responsive gene transcription by phosphorylating p65 and posttranslationally modifying histone H3 (Anest et al., 2003; Bonizzi and Karin, 2004; Yamamoto et al., 2003).  Phosphorylation of serine residues on the activation loop of the IKKa or IKKp subunit is essential for IKK activation and can be achieved by either an upstream kinase or IKK itself (Karin and Ben-Neriah, 2000). Various kinases, such as NF-KB-inducing kinase, N F - K B activating kinase and MEK kinase 1, are able to activate IKK, but a specific kinase for IKK phosphorylation has not yet been identified (Hayden and Ghosh, 2004).  1.5.4. B i o l o g i c a l F u n c t i o n s of N F - K B N F - K B a n d A b n o r m a l C e l l G r o w t h N F - K B proteins mediate the expression of many genes involved in tumor cell growth  and survival, and N F - K B activation leads to tumor cell proliferation and angiogenesis; however, N F - K B activation can also suppress tumor repressor genes PTEN and p53 which can exert anti-tumor effects by inhibiting cellular N F - K B activity (Culmsee et al., 2003; Gustin et al., 2001; Shishodia and Aggarwal, 2004). Though N F - K B can function either as a tumor  37  suppressor or a tumor promoter, constitutive N F - K B activity has been detected in tumor cell lines and several reports have supported N F - K B to be oncogenic (Shishodia and Aggarwal, 2004). N F - K B NF-KB  and Apoptosis is involved in numerous cellular functions, and in particular, N F - K B regulates  inflammatory responses and apoptotic processes (Baeuerle and Henkel, 1994; Baldwin, 1996; Ghosh et al., 1998). Generally, activated N F - K B proteins modulate the expression of antiapoptotic or cell survival genes and thus N F - K B is able to mediate cell survival pathways (Mistry et al., 2004; Shishodia and Aggarwal, 2004). By inhibiting N F - K B activity, apoptotic events can be promoted in cells (Watabe et al., 2004). Studies of N F - K B subunit knockout mice showed massive apoptosis with mice dying at embryonic day 12.5-14.5 (Li and Verma, 2002; Tanaka et al., 1999). Although recent reports have revealed that N F - K B is able to induce expression of proapoptotic genes such as Fas ligand and c-Myc, timing and cellular environment changes should also be included to define the role of N F - K B in mediating apoptotic events (Shishodia and Aggarwal, 2004). N F - K B  in Immunity and Learning  Several studies have been conducted to study the importance of N F - K B proteins in immune and inflammatory responses (Baldwin, 1996). In response to inflammation in the nervous system, increased N F - K B activity is detected in acute and chronic neurodegenerative disorders (Barger and Mattson, 1996; Cheng et al., 1994; Hunot et al., 1997; Lukiw and Bazan, 1998; Nonaka et al., 1999; O'Neill and Kaltschmidt, 1997; Yu et al., 1999). N F - K B activity in microglia can promote neurodegenerative processes, whereas activated N F - K B can also protect  38  neurons from further insults (Mattson and Camandola, 2001; O'Neill and Kaltschmidt, 1997). NF-KB  activity is necessary for normal development of neurons and is also associated with  long-term potentiation of synaptic transmission for learning and memory; thus, N F - K B activation is essential for the biological functions of neurons (Albensi and Mattson, 2000; Meberg et al., 1996; Worley et al., 1993).  NF-KB  deficient mice exhibit deficits not only in selective learning, but also in adaptive  immunity, as they have impaired B cell and T cell functions (Baldwin, 1996; Li and Verma, 2002). N F - K B modulates adaptive immunity by regulating immune cell production, which includes hematopoiesis and differentiation and maturation of myeloid and lymphoid immune cells (Denk et al., 2000; Siebenlist et al., 2005). Moreover, N F - K B activity elicits antiapoptotic effects by mediating survival pathways in T lymphocytes, B lymphocytes, and dendritic cells (Denk et al., 2000; Siebenlist et al., 2005).  NF-KB  activity can also be responsible for the regulation of innate immunity and  cellular defense against bacterial invasion (Ghosh et al., 1998). Macrophages and neutrophils, for example, are the front line of innate immunity and their development and maturation require N F - K B activity (Denk et al., 2000). When N F - K B activity is absent, the host can become more susceptible to bacterial infections and exhibit defective bacterial clearance mechanisms (Lavon et al., 2000; Sha et al., 1995). In addition, evidence has highlighted the involvement of N F - K B in stimulating inflammatory responses, and N F - K B is responsible for the expression of pro-inflammatory genes such as cytokines and chemokines (Baeuerle and Baichwal, 1997; Pahl, 1999). Even though N F - K B inhibition appears to be a potential strategy to resolve issues of inflammation, N F - K B proteins composed of different subunits may be  39  involved in different phases of the inflammatory responses; therefore, inhibiting one subunit of the N F - K B proteins may not be able to prevent the inflammatory development in these pathological conditions (Baeuerle and Baichwal, 1997).  1.6. Oxidative Stress 1.6.1. Free Radical Generation and Elimination in the Brain Free radicals are defined as molecules with unpaired electrons which readily accept either an electron or a hydrogen atom in order to achieve stability. There are numerous types of free radicals that can be formed within the human body; in particular, reactive oxygen species (ROS), including the superoxide anion (CV), the hydroxyl radical (OH '), and hydrogen peroxide (H2O2), are thought to be involved in redox cell signaling pathways leading to stress or cell death (Chong et al., 2005; Gutierrez et al., 2006; Harman et al., 1976; Taylor and Crack, 2004).  When a ground-state oxygen molecule is excited, one unpaired electron changes its spin, resulting in the formation of 0 \ which can produce other free radicals to mediate 2  oxidative chain reactions (Chong et al., 2005). For example, O2" can react with nitric oxide (NO) to form peroxynitrite. One major source of 0 " comes from mitochondria, since 1% to 2  5% of electrons in the electron transport chain are lost to ROS production (Reddy, 2006). Once mitochondria are exposed to ROS, the function of mitochondrial enzymes is altered and the mitochondrial adenosine triphosphate (ATP) generation system is impaired (Agar and Durham, 2003; Blass et al., 2000; Reddy and Beal, 2005; Yamamoto et al., 2002) (Figure 1-4).  40  Normal Pathway  mitochondrion  P r o p o s e d A P pathway  (decreased TCA/ETC enzyme activities)  Neuronal death Figure 1-4. Oxidative Stress in A D Schematic view of proposed mechanism implicated in the cytoplasmic oxidative stress in Alzheimer's disease. When the activities of Tricarboxylic Acid cycle components in mitochondria matrix are normal, 0 ~ can be eliminated by converting to H 0 by superoxide dismutase (SOD) and catalase. It was proposed that in A D , this pathway can not completely remove 0 ". Due to the deficiency in key mitochondrial enzymes and increased SOD activity, the concentration of H 0 was increased in mitochondria and the surrounding cytoplasm. Furthermore, catalase activity is decreased and only a certain amount of H 0 is removed. H 0 undergoes Fenton reaction to converted into highly reactive OH', which is a strong oxidant that can react with nucleic acid, lipids and proteins to cause oxidative damage in the cytoplasm. 2  2  2  2  2  2  2  2  2  During the respiratory process that occurs in mitochondria, three ROS, 0 \ OH ', and 2  H 0 , are formed as the byproducts of oxidative phosphorylation reactions that break down 2  2  glucose to synthesize ATP (Behl, 1999; Christen, 2000):  (+4e~/+2H ) +  0 -> 0 ~ -> H 0 -> OH ' -^H 0 2  2  2  2  2  41  Glucose is the major nutrient for brain function and a high glucose metabolism can be detected in neurons; therefore, neurons are almost dependent on mitochondrial oxidative phosphorylation reactions for the production of ATP. Moreover, due to a high concentration of iron in the brain, H2O2 can react with iron to produce OH ' via the Fenton reaction (Behl, 1999; Taylor and Crack, 2004):  Fe  2+  + H 0 -> F e 2  2  3+  + HO ~ + OH '  Neurons appears to be particularly vulnerable to attack by free radicals because of a higher proportion of polyunsaturated fatty acids in neuronal membranes, the high oxygen demand of brain, and a low content of natural antioxidant glutathione (GSH) (Hazel and Williams, 1990; Raps et al., 1989; Smith et al., 1995a). Therefore, the free radical hypothesis of aging has been proposed that accumulation of ROS may result in damage to neurons and may be involved in A D pathogenesis (Christen, 2000; Harman et al., 1976).  Although oxidative phosphorylation reactions in mitochondria are considered to be the primary source of ROS production, other enzymatic or non-enzymatic cellular reactions can also generate ROS in the brain (Behl, 1999; Halliwell et al., 1992). To protect the brain from free radical damage, enzymatic and non-enzymatic antioxidants are present to maintain a fineturned balance between the physiological production of ROS and their detoxification (Behl, 1999). For example, O2" leaked from mitochondria can be converted to H2O2 by superoxide dismutase (SOD), and then H 2 0 2 c a n be eliminated by intracellular antioxidant GSH and glutathione peroxidase. Non-enzymatic or chain-breaking antioxidants, such as the lipophilic free radical scavenger a-tocopherol (Vitamin E) and the hydrophilic ascorbate (Vitamin C), can remove ROS through direct interactions (Halliwell et al., 2001). 42  However, when there is an increased production of free radicals resulting from either over-production of endogenous ROS or exogenous oxidative insults, the intracellular free radical defense mechanisms will be impaired. The imbalance between ROS generation and free radical elimination has been speculated to be involved in the development of neurodegenerative diseases, such as A D (Evans et al., 1989b; Olanow and Arendash, 1994; Volicer and Crino, 1990). Since ROS accumulate in an age-dependent manner, neurons from aged individual may experience mitochondrial D N A and protein damage caused by these free radicals (Christen, 2000).  1.6.2. Neuronal Cell Death by A p Neurotoxicity Since the findings of neurotoxic A p in patients with A D , numerous reports have highlighted that A p is associated with the necrosis and apoptosis of neurons (Behl et al., 1994a; Cotman and Anderson, 1995; Yankner et al., 1989; Yankner et al., 1990). From in vivo experiments, antioxidants were able to inhibit amyloidogenic APP processing (Harman et al., 1976). In addition, neuronal cell death induced by the cytotoxic A p peptides can be prevented in vitro by treatment of antioxidants, such as free radical scavengers Vitamin E and propyl gallate (Behl et al., 1992; Behl et al., 1994b). A P toxicity is also able to cause lipid peroxidation of vascular endothelial cells by the generation of free O2" (Suo et al., 1997; Thomas et al., 1996). Based on these findings, aggregated A p peptides may result in neuronal cell death via free radical mediated pathways (Behl, 1999). Free radical scavengers, such as EGb 761, have been demonstrated to protect hippocampal cells from p-amyloid toxicity (Bastianetto and Quirion, 2002). H 02-mediated oxidative stress has also been examined to 2  modulate B A C E 1 gene transcription and increase A p generation (Li et al., 2004; Tong et al.,  43  2005). So oxidative stress induced by  neurotoxicity may increase BACEl activity and  enhance A(3 production.  Indeed, A P aggregates can induce oxidative stress and attract inflammatory mediators to generate NO and increase the free radical accumulation within Ap-insulted neurons (Beckman et al., 1994; Harris et al., 1995; Hensley et al., 1994). Ap aggregates have also been proposed to enhance the accumulation of H2O2 in neurons by the induction of cellular O2" generating enzyme systems which are sensitive to flavin-containing oxidase inhibitors; therefore, A P peptides may directly or indirectly induce oxidative stress and the use of antioxidants in preventing neuronal cell death has been included in the treatment for AD (Behl et al., 1994b; Dykens et al., 2005; Goodman et al., 1994; Markesbery, 1997; Rodriguez-Franco et al., 2006; Schubert et al., 1995).  As one of the hallmarks of AD, Ap plaques have been hypothesized to cause neuronal damage by the generation of free radicals, mitochondrial oxidative damage, synaptic failure, and inflammation (Mattson, 2004; Tanzi and Bertram, 2005). Both in vivo and ex vivo experiments revealed that Ap can increase free radical production and induce oxidative stress (Mattson et al., 1997; McLellan et al., 2003; Pappolla et al., 1998). Furthermore, because free radicals can promote protein cross-linking, they may mediate A P deposition and, in turn, increased Ap plaque formation lead to more free radical generation (Mattson, 1995). From in vitro investigations, the level of enzymes involved in mitochondrial respiration can be reduced by Ap peptides and further diminished by the introduction of free radicals (Casley et al., 2002). Mutated APP and soluble Ap peptides have also been indicated to enter mitochondria and cause mitochondrial dysfunction (Anandatheerthavarada et al., 2003; Caspersen et al., 2005;  44  Lustbader et al., 2004). As such, AP accumulation inside mitochondria may cause energy production deficits in neurons by impairing the critical enzymatic activity of respiratory chain complexes III and IV (Caspersen et al., 2005).  1.6.3. M e m b r a n e L i p o p e r o x i d a t i o n  Neuronal cell death by Ap neurotoxocity is reported to be caused by the peroxidation of neuronal membrane lipids (Qi et al., 2005). In addition to the increased peroxidation of membrane lipids found in AD brain, alternation of phospholipid metabolism and plasma membrane abnormalities were also observed (Miatto et al., 1986; Nitsch et al., 1991; Nitsch et al., 1992; Qi et al., 2005; Subbarao et al., 1990). Because of these membrane defects, an abnormal calcium distribution in cells, free radical insults, and cell death pathways are considered to be initiated (Praprotnik et al., 1996).  Moreover, apoE4 was shown to be more susceptible to attack by free radicals and thus antioxidants, such as EGb 761 were developed to protect this specific isoform of apoE (Christen, 2000). The expression of apoE is associated with the degree of lipoperoxidation in AD brain and the level of peroxidation is inversely proportional to the concentration of apoE in the brain (Ramassamy et al., 1999). By protecting apoE with antioxidants, the level of lipoperoxidation is reduced (Christen, 2000). Since apoE reduces the production of Ap~ induced free radicals and prevents neuronal death through antioxidant activity, these findings support the hypothesis that the presence of apoE has beneficial effects against oxidative stress in the brain (Bastianetto et al., 2000; Miyata and Smith, 1996).  45  The aggregation of AP peptides not only increases free radical generation, but also triggers AD-related inflammation processes and microglial activation. Particularly, neurotoxic AP aggregates can stimulate microglial cells to release free radicals, such as 0 ~ (McDonald et 2  al., 1997; Meda et al., 1995).  1.7. Neuroinflammation The majority of neurons residing in the hippocampus and frontal cortex of A D patients are undergoing cell death, and because these brain regions are important for memory, A D patients experience a progressive inability to form new memories and access existing ones (Turner et al., 2003). In the brain, microglia play an essential role in CNS immune defense and it has been reported that activated microglial cells surround neurotoxic Ap plaques (Sheng et al., 1998). Once they are activated, microglial cells synthesize and release cytokines, such as Interleukin-1 (IL-1), Interleukin-6 (IL-6) and Tumor Necrosis Factor-a (TNF-a), as well as chemokines, to initiate inflammatory pathways (Akiyama et al., 2000; Cacquevel et al., 2004) (Figure 1-6). A high level of cytokines has been detected in the brain tissue and cerebrospinal fluid of A D patients and both IL-1 and TNF-a can elevate the expression of APP and AP (Benzing et al., 1999; Blasko et al., 1999; Rogers et al., 1999; Tuppo and Arias, 2005).  Numerous reports have revealed the involvement of inflammation during the development of A D pathogenesis, and anti-inflammatory drugs, such as COX-2 inhibitors, have the potential to treat A D patients (Aisen, 2002; Sugaya et al., 2000). Despite some successful studies of anti-inflammatory drugs in treating A D patients, the application of nonsteroidal inflammatory drugs (NSAIDs), such as a selective COX-2 inhibitors, have failed to show improvements in cognitive function during clinical trials (Aisen et al., 2003; McGeer  46  and McGeer, 2006). In addition, patients taking NSAIDs have an increased incidence of cardiovascular side effects (Couzin, 2004; Konstam and Weir, 2002). Nevertheless, the implication of increased immune responses in A D pathogenesis is well substantiated and it will be necessary to further define the association between inflammation and A D in the future.  1.7.1.  Microglial Activation Microglia are composed mostly of mesodermally derived macrophages and act as  immunocompetent defense cells that are responsible for regulating the endogenous CNS immune responses to protect neurons (Streit and Kincaid-Colton, 1995). Under pathological conditions, such as neurodegenerative disease, these glial cells become activated and surround damaged neuronal cells (Fetler and Amigorena, 2005). In addition to phagocytic and scavenger properties, microglia are able to express major histocompatibility complex type II (MHCII) and release pro-inflammatory cytokines, ROS, and complement proteins (Griffin et al., 1998; Liu and Hong, 2003; Moore and O'Banion, 2002). Activated microglia also upregulate a variety of surface receptors, including MHCII and complement receptors (Liu and Hong, 2003). Dramatic morphological changes were observed in microglia as they evolve from resting ramified state to motile activated amoeboid state (Kreutzberg, 1996).  Since activated microglia were claimed to be found within pathological regions of A D brain, microglia have been speculated to participate in the neurotoxic inflammatory responses of A D (Luber-Narod and Rogers, 1988; Rogers et al., 1988). Ap peptides can attract microglia to the sites of Ap accumulations in the brain. Once microglia are activated upon exposure to AP, an increase in MHCII expression on the cell surface is detected (Rogers and Lue, 2001). Microglia can also enhance secretion of pro-inflammatory cytokines, chemokines, and TNF-a  47  in response to neurotoxic A p  42  introduction (Fiala et al., 1998; Lue et al., 2001; Walker et al.,  2001) (Figure 1-5). Such an increase in the production of pro-inflammatory mediators can contribute to severe neuronal dysfunction and cell death (Akiyama et al., 2000).  A p generation  A p plaques  Astrocyte recruitment  (Astrocytes)  - —  (  Microglia activation  N  e  u  r  o  n  - ( IL-6  s  )  y  (Microglia)  - —  -  F i g u r e 1-5. T h e Role of I n f l a m m a t i o n Cytokines P r o d u c e d by G l i a l Cells i n A D Pathogenesis Ap aggregation in the brain has been proposed to stimulate brain cells, such as microglia, astrocytes and oligodendrocytes, to produce cytokines. Particularly, pro-inflammatory cytokines, like I L - 1 , IL-6 and T N F - a , have been reported to relate to neuroinflammation implicated in A D pathogenesis. T h e over-production o f those cytokines leads to increased Ap generation. Hence, a positive feedback loop is created to accelerate Ap accumulation.  By being able to reduce AP plaque deposition through phagocytosis, clearance and degradation, activated microglia are important to the survival of neurons (Bard et al., 2000; Gelinas et al., 2004; Weiner and Selkoe, 2002). Due to the phagocytotic capability of microglia, insoluble Ap aggregates have been detected within microglia in both cell and animal studies (Frackowiak et al., 1992; Frautschy et al., 1992). Microglial phagocytosis was only  48  observed in models receiving fibrillar AP (Weldon et al., 1998). According to in vitro studies, cultured microglia migrate to Ap deposit sites and within 2 to 4 weeks, Ap is removed and localized in phagosome-like intracellular vesicles (Ard et al., 1996). In addition, microglia and other neural cells are able to release a metalloprotease, insulin-degrading enzyme, which degrades secreted Ap o and A P 4 2 peptides (Qiu et al., 1998; Qiu et al., 1997). 4  When human or rodent microglia are cultured on unfixed sections of AD cortex containing Ap aggregates, they are found to phagocytose Ap deposits once the tissue sections are treated with anti-Ap antibodies; hence, antibodies against Ap peptides may be able to cross the blood-brain barrier and trigger Ap clearance by microglia (Bard et al., 2000; Weiner and Selkoe, 2002). The development of anti-Ap antibodies for human AP immunization has been approached to induce local activated microglia to uptake AP-antibody complex via Fc receptormediated phagocytosis (Bard et al., 2003; Lee et al., 2005). In other protein aggregation disorders, such as prion disorders, astrocytes encircle scrapie plaques and protease resistant proteins are found within lysosomes of both microglia and astrocytes; therefore, a better understanding of the responses of microglia and astrocytes to abnormal protein depositions is essential in order to investigate the role of glial cells in many neurodegenerative diseases (Jeffrey et al., 1994).  1.7.2. Astrocyte Recruitment Aside from neurons, astrocytes are the most common cells in the brain and are involved in the connective tissue and skeletal functions of the brain, the maintenance of neuronalsynapse integrity, and neuronal activity (Tuppo and Arias, 2005). Astrocytes are also implicated in the inflammatory process associated with AD as they form a protective barrier  49  between Ap deposits and neurons (Rossner et al., 2005; Wyss-Coray et al., 2003). Ap plaques are capable of astrocyte activation and reactive astrocytes are observed to cluster at Ap deposit sites (Dickson, 1997). Similar to activated microglia, reactive astrocytes are able to secrete pro-inflammatory molecules, such as interleukins, prostaglandins, leukotrienes, thromboxanes, coagulation factors and complement factors; however, under certain circumstances, reactive astrocytes can be involved in chronic stress and may not be beneficial to neurons (Heneka et al., 2001; McGeer and McGeer, 1995). When astrocytes are recruited to A P plaque sites, they may cause prolonged neuroinflammation which could result in NO-mediated neurotoxicity (Heneka et al., 2001). Moreover, astrocytes can be another source of Ap peptide production in the CNS because their B A C E l expression is elevated in response to chronic stress (Rossner et al., 2005).  Analysis of A D brain tissue by electron microscopy revealed that Ap peptides can be found within astrocytes, which may accumulate Ap via phagocytosis of local degenerated dendrites and synapses (Kurt et al., 1999; Nagele et al., 2003). Furthermore, reports of Ap degradation by mouse astrocytes suggest that reactive astrocytes around Ap deposits actively phagocytose Ap, and that a deficiency in the clearance of Ap peptides by astrocytes can result in the development of A D pathogenesis (Wyss-Coray et al., 2003). Thus, while microglia are the key mediators contributing to elevated Ap plaque formation, astrocytes are the major brain cells involved in Ap plaque degradation (Wegiel et al., 2000).  1.7.3. C y t o k i n e a n d C h e m o k i n e P r o d u c t i o n  Cytokines are a family of proteins which include the interleukins, TNF-a, and T G F - P (Transforming Growth Factor-P) and are synthesized by both microglia and astrocytes in the  50  CNS (Hopkins and Rothwell, 1995). In response to an inflammatory state, cytokines production is increased to regulate immune responses (Lucas et al., 2006). Chemokines are a family of small pro-inflammatory cytokine proteins that are involved in the recruitment of inflammatory cells (Akiyama et al., 2000). Following injury, cells release chemokines to attract leucocytes to the site of inflammation (Glabinski and Ransohoff, 1999). Cytokines are able to transcriptionally upregulate levels of BACE1 mRNA, protein, and enzymatic activity (Sastre et al., 2003). In addition to presenilin-1, BACE1 is the critical enzyme in AB generation in neurons and its absence can lead to considerably reduced Ap synthesis (Walter et al., 2001). An increase in BACE1 expression and activity is observed in neuronal cells after exposure to oxidative stress and also in reactive astrocytes in chronic models of gliosis (Hartlage-Rubsamen et al., 2003; Tamagno et al., 2002).  Four major cytokine family members have been extensively studied in relation to AD onset or progression, including the pro-inflammatory cytokines IL-1, IL-6, TNF-a and antiinflammatory cytokine TGF-P (Cacquevel et al., 2004). Also, an association between AD pathogenesis and gene polymorphisms has been highlighted in IL-1, IL-6 and TNF-a genes (McCusker et al., 2001; Nicoll et al., 2000; Papassotiropoulos et al., 1999). These polymorphisms increase the risk of developing AD and may be useful to screen populations which are more susceptible to AD (Licastro and Chiappelli, 2003). Interleukin-1 Cytokine IL-1 has been described as a mediator in several forms of neurodegeneration with pleiotropic actions (Rothwell et al., 1997; Touzani et al, 1999). In AD, an increase of IL1 production has been detected in microglia by immunohistochemistry (Griffin et al., 1989). In  51  response to exposure to AP plaques, microglia and astrocytes display an increased IL-1 expression; and, IL-1 overexpression can be detected during the early stages of Ap plaque formation (Griffin and Mrak, 2002; Griffin et al., 1995; Johnstone et al., 1999).  IL-1 has been implicated in the production and proteolytic processing of APP and in glial cell activation (Mrak and Griffin, 2001). IL-1 overexpression is also coupled with an increased number of IL-1 immunoreactive microglia associated with amyloid plaques; thus, the secreted and soluble forms of APP are proposed to activate microglia and induce IL-1 production (Barger and Harmon, 1997; Griffin et al., 1998; Mrak and Griffin, 2005). IL-1, in turn, activates astrocytes to induce production of S100B (Mrak and Griffin, 2001; Mrak and Griffinbc, 2001). S100B is a neurite growth-promoting cytokine and its expression is elevated in AD brain (Li et al., 2000a; Marshak et al., 1992). The cross-sectional area of dystrophic neurites in Ap plaques correlates closely to the number of SlOOB-associated reactive astrocytes (Mrak et al., 1996). This suggests that dystrophic neurite growth in the vicinity of Ap deposits can be caused by the enhanced expression of S100B; therefore, when microglia are activated by secreted APP to induce IL-1 expression, reactive astrocytes promoted S100B secretion to cause dystrophic neurite outgrowth (Griffin et al., 1998). In addition, S100B is an inducer of neuronal APP expression and its overexpression may account for an increased AP generation (Li et al., 1998; Mrak and Griffinbc, 2001). This demonstrates a close relationship among glial cell signaling pathways through the action of cytokines. Interleukin-6 Through its interaction with a specific membrane-bound receptor, IL-6 mediates immune responses and inflammatory reactions (Graeve et al., 1993; Hirano et al., 1997). Both  52  gp80 and gpl30 membrane glycoproteins have been described to promote the association between the IL-6 receptor and IL-6 (Hibi et al., 1990; Yasukawa et al., 1990). By interacting with the IL-6 receptor, gpl30 has the implication in the transduction of IL-6 signals and in the activation of Janus Kinase/Signal Transducer and Activator of Transcription signaling pathways (Hibi et al., 1996; Ip et al., 1992).  IL-6 is a pro-inflammatory cytokine which can induce the production of acute phase proteins and increase vascular permeability, lymphocyte activation, and antibody synthesis (Baumann and Gauldie, 1994). In vivo experiments have shown that an elevated concentration of IL-6 can lead to CNS damage and behavioral deficits; however, the absence of IL-6 expression in mice can cause sensory impairments and delay axonal regeneration (Campbell et al., 1993; Heyser et al., 1997; Zhong et al., 1999). This indicates that an adequate level of IL-6 is essential for the regulation of neuronal function and survival. IL-6 is also expressed in the nervous system during development but is barely detected in adult brain (Akiyama et al., 2000). IL-6 expression can only be induced under certain pathological events (Vallieres and Rivest, 1997). In the CNS, both IL-6 and gpl30 expression are detected in neuronal and glial cells (Marz et al., 1998; Van Wagoner et al., 1999; Watanabe et al., 1996). In the CSF of AD patients, the levels of soluble gp80 and gpl30 are lower than controls, but IL-6 protein levels are not altered (Hampel et al., 1997; Hampel et al., 1998). Nevertheless, mRNA levels of IL-6 are increased in the entorhinal cortex and the superior temporal gyrus of AD patients (Luterman et al., 2000). Ap peptides can increase production of IL-6 from astrocytes directly or indirectly through IL-l-directed mechanisms; thus, IL-6 may act as a secondary mediator to amplify the inflammatory responses induced by IL-1 (Aloisi et al., 1992; Cacquevel et al., 2004; Eriksson et al., 1998; Holmlund et al., 2002; Toro et al., 2001).  53 T u m o r Necrosis F a c t o r - a  TNF-a is known to associate with two distinct receptors, p55 and p75 TNF receptors, to elicit TNF-a mediated responses (Goate, 1994; Louis et al., 1993). Both receptors share low homology in the extracellular and intracellular regions and particularly, the intracellular region of p55 TNF receptor contains a death domain (Sipe et al., 1996; Tartaglia et al., 1993). Once TNF-a binds to the p55 TNF receptor, the activated death domain can initiate cell death pathways (Leist et al., 1995; Monsma et al., 1993). Conversely, from studies involving knockout of the p75 TNF receptor, p75 TNF receptor is involved in the protection of neurons (McKee et al., 1998). TNF-a levels are increased in AD brains, as evident by the increased TNF-a expression in AD brain parenchyma (Tarkowski et al., 1999; Tarkowski et al., 2000). Microglia and astrocytes are activated by Ap peptides and their activation causes an elevated TNF-a production (Akama and Van Eldik, 2000; Tan et al., 2000; Yates et al., 2000). Even though Ap exposure can lead to an elevated expression of TNF-a in AD serum, CSF, cortex and glial cell cultures, the role of TNF-a in AD brain is still not well defined (Fillit et al., 1991; Lue et al., 2001).  TNF-a may promote the induction of inflammation signals, which are involved in CNS disorders, such as multiple sclerosis and Parkinson's disease (Antel et al., 1996; Hsu et al., 1996). TNF-a generation has also been demonstrated to induce the expression of NO and complement factors (Combs et al., 2001; Veerhuis et al., 1999). Despite the findings that TNFa overexpression can damage human cortical neurons, TNF-a displays neuroprotective properties against toxic agents (Akiyama et al., 2000; Barger et al., 1995; Mogi et al., 1994). TNF-a, in addition, can induce survival factors, such as manganese superoxide dismutase,  through N F - K B stimulation (Bruce-Keller et al., 1999; Keller et al., 1998; Sullivan et al., 1999). Transforming Growth Factor-P By associating with a heteromeric complex of transmembrane serine/threonine Kinase receptors (type I and type II), T G F - P is able to orchestrate many physiological and pathological events (de Caestecker et al., 2000; Massague and Chen, 2000). The constitutively active TGFP  type II receptor binds to T G F - P by its own, recruits and phosphorylates type I receptor in the  GS box (Wrana et al., 1994). Activated type I receptor subsequently leads to Smad2 and Smad3 phosphorylation, which allows Smad4 to form heterodimeric complexes with phosphorylated Smad2 and Smad3 (Massague, 1998). Smad belongs to the members of the Mother Against Decapentaplegic family and Smad4 is closely linked to T G F - P signaling pathways (Heldin et al., 1997; Lagna et al., 1996; Zhang et al., 1996). Heterodimeric complexes of Smad are translocated into the nucleus and bind to the CAGA box in the promoter region of TGF-P-targeted DNA; hence, T G F - P is able to regulate the expression of genes essential for cell cycle regulation and extracellular matrix formation (Derynck et al., 1998).  TGF-P  overexpression is observed in plasma, CSF, intrathecal compartments, and brain  parenchyma of AD patients (De Servi et al., 2002; Luterman et al., 2000; Tarkowski et al., 2002). T G F - P is overexpressed in astrocytes in the vicinity of A  P  deposits, and is considered  to increase APP metabolism (Frautschy et al., 1996; Wyss-Coray et al., 1997). Several in vitro studies have shown that TGF-P upregulates APP expression in human or rodent astrocytes through two proposed mechanisms (Burton et al., 2002; Gray and Patel, 1993). By associating  55  with a region located in the 5' UTR of the APP promoter, TGF-P can promote a dramatic expression of APP in astrocytes (Lesne et al., 2003). TGF-P can also markedly elevate APP mRNA levels in astrocytes and increases the half-life of the APP message; therefore, TGF-P overexpression can increase AP burden during the development of AD pathogenesis (Amara et al., 1999; Frautschy et al., 1996; Harris-White et al., 1998). However, research from in vitro models of neurons has indicated neuroprotective properties of TGF-P against AP-induced neuronal death (Prehn et al., 1996; Ren and Flanders, 1996). TGF-P upregulates the antiapoptotic genes bcl-xL and bcl-2 to protect neurons from Ap-induced neurotoxicity (Kim et al., 1998; Ren et al., 1997). Moreover, TGF-P activates microglia to promote the clearance of Ap peptides (Wyss-Coray et al., 2001). These findings support a beneficial role of TGF-P in AD pathogenesis.  An increased ROS load has been described in AD (Smith et al., 1992) and H 0 has 2  2  been reported to increase AP generation by increasing human BACE1 transcriptional activity (Tong et al., 2005). Since the implication of N F - K B activation in H 0 -induced oxidative 2  2  stress and the detection of increased N F - K B activity in AD brain (Kaltschmidt et al., 1997; Schreck et al., 1991), we suggest that by interacting with the N F - K B binding elements on human BACE1 promoter, N F - K B may be involved in the regulation of BACE1 gene expression and the modulation of AP production in AD. In non-neuronal and neuronal-like cells, we determined the changes in BACE1 promoter activity and transcription in response to  NF-KB  p65 overexpression. We also measured C99 protein levels and AP production to detect the alternation in amyloidogenic APP processing. Because  NF-KB  has not been reported in human  BACE1 gene regulation, this study allows us to investigate the role of N F - K B activation in AP generation in AD pathogenesis.  56  Chapter II. Materials and Methods 2.1. Generation of Human B A C E l Gene Promoter Constructs We have previously cloned the 5' upstream region of human BACEl gene by a 5' genome walking strategy using the BACEl gene from the human fetus brain genomic library (Clonthch) and a specific primer corresponding to the BACEl coding sequence (Christensen et al., 2004). In order to analyze BACEl promoter activity, we designed the specific BACEl primers with restriction enzyme sites and inserted the PCR-resulted DNA fragments into the vector pGL3-basic (Promega). Previously, we have constructed three 5' upstream fragments of the BACEl gene amplified by PCR in front of the firefly luciferase gene of pGL3-basic vector (pBlP-G, B1P-H and pBlP-J) (Christensen et al., 2004). The pBlP-N4 reporter plasmid was constructed by cloning the BACEl promoter fragment -145-+292 generated by PCR, using primers: -145XhoI (5'-ccgctcgagcctagatgtccctccaa-3') and +292rHindIII (5'cacaagcttccaccataatccagctcg-3') and pBlP-H as template, into pGL3-basic vector at Xho I and Hind III.  2.1.1. Generation of B A C E - N F - K B Plasmid  To remove multiple Spl enhancer sequence within SV40 promoter of the vector pGL3promoter (Promega), PCR reaction was carried out with primers: pGLP136fBgl (5'gaagatctccatcgctgactaatttttt-3') and pGLP270r (5'-accaacagtaccggaatgccaag-3') and pGL3promoter vector as template. The PCR-resulted DNA fragment was then cloned into pGL3promoter vector at Bgl II and Hind III (pGL-pL). The resulting plasmid pGL-pL contains the SV40 basic promoter sequence without any Spl enhancers. Double-stranded oligonucleotide, 4NF-KB, was generated by the oligonucleotide primers 4NF-KBf (5'ctagcgtgaaaccccggtgaaatcccacgaagattcccttgtggaacccc-3') and 4NF-KBr (5'-  57  tcgaggggttccacaagggaatcttcgtgggatttcaccggggtttcacg-3'). This oligonucleotide consists of four putative N F - K B p65 binding elements in the human BACE1 promoter region from -1466 to +292 and was inserted in front of the firefly luciferase gene of the pGL-pL plasmid at Nhe I and Xho I (pBACE-NF-KB).  2.2. Site-Directed Mutagenesis Forward primers: BACE1U-1466 (5'-gctagctagctttccaacatatataac-3'), NFlmf (5'acacgTtCaaaGTTcgtctctactaaaaat-3') and NF4mf, (5'-attgtTTCTaaGGTTactgcggcaggaatcac-3') and reverse primers: BACE1U292 (5'-cacaagcttccaccataatccagctcg-3'), NFlmr (5'gAACtttGaAcgtgttagccaagatggt-3') and NF4mr (5'-gtAACCttAGAAacaatacgatgtggca-3'), were used to generate the human BACE1 promoter fragments with a mutated N F - K B binding element.  To construct the BACE1 promoter fragment containing the mutation of the first N F - K B binding element, PCR reactions were carried out with template pBlP-G and two sets of primers BACElU-1466/NFlmr and NFlmf/BACElU292. These PCR-resulted DNA fragments were annealed and served as the template in a PCR reaction with BACE1U-1466/ BACE1U292 primers to generate a mutation in the first N F - K B binding element of the BACE1 promoter fragment. The DNA fragment was confirmed with gel electrophoresis and was cloned in front of the firefly luciferase gene of pGL3-basic vector at Nhe I and Hind III (pBlPNlm). The mutation of the first N F - K B binding element of the BACE1 promoter fragment was confirmed by sequencing. The BACE1 promoter fragment containing the mutation of the fourth N F - K B binding site was generated by a similar method and inserted into pGL3-basic at Nhe I and Hind III (pBlP-N4m).  58  2.3. Cell Culture 2.3.1. Culture Media Preparation Culture medium for HEK293 (Human Embryonic Kidney), N2a (Mouse neuroblastoma), SHSY5Y (Human neuroblastoma) and NIH-3T3 (Mouse fibroblast) cell lines: 1 bottle 1%  Dulbecco's Modified Eagle Medium (500mL) (Gibco 11960-069) Sodium Pyruvate (5mL) (Gibco 11360-070)  1%  Penicillin-Streptomycin (5mL) (Gibco 15070-063)  1%  L-glutamine (5mL) (Gibco 25030-081)  10%  Fetal Bovine Serum (50mL) (Gibco 26140-079)  A Swedish mutant APP cell line (Sun et al, 2006a), SH-SY5Y cells overexpressing Swedish mutant APP, was selected by lOOOpg/mL zeocin (Invitrogen).  Culture medium for RelA knockout (RelA-KO) cell line (Gapuzan et al., 2005): 415mL  Dulbecco's Modified Eagle Medium (Gibco 11960-069)  75mL  Fetal Bovine Serum (Gibco 26140-079)  5mL  Sodium Pyruvate (Gibco 11360-070)  5mL  Penicillin-Streptomycin (Gibco 15070-063)  5mL  L-glutamine (Gibco 25030-081)  4pL  P-mercaptoethanol (Fisher 034461-100)  25pL  ESGRO® (LIF) (Chemicon ESG1106)  5mL  MEM Non-Essential Amino Acids Solution (Gibco 11140-050)  59  Culture medium for 20E2 stable cell line (HEK293 stably expressing Swedish mutant APP695) (Qing et al., 2004): 1 bottle  Dulbecco's Modified Eagle Medium (500mL) (Gibco 11960-069)  1%  Sodium Pyruvate (5mL) (Gibco 11360-070)  1%  L-glutamine (5mL) (Gibco 25030-081)  10%  Fetal Bovine Serum (50mL) (Gibco 26140-079)  75pg/mL  Geneticin (Gibco 11811-031)  2.3.2. Trypnization Once cells reached a confluency of 80-90%, culture medium was removed and cells were washed by room-temperature Hanks Balanced Salt Solution (HBSS) (Gibco 14170-112) and treated with Trypsin-EDTA (Gibco 25200-072). Cells were suspended in fresh culture medium, counted and seeded for transfection. A l l cell lines were maintained in 37°C incubator containing 5% CO2.  2.4. Cell Transfection 2.4.1. Calcium Phosphate Transfection For HEK293 cells: Reagents: 1) 0.5M C a C l  2  3.675g C a C l - 2 H 0 in 50mL distilled water. 2  2  60  2) 2X HEBS 1.636g NaCl 1.19g HEPES 0.0213g Na HP0 , anhydrous 2  4  In lOOmL distilled water and pH was adjusted to 7.00 with NaOH solution.  1. 24 h before transfection, HEK293 cells were seeded in 60mm plates at the density of 1.0 xlO cells in 4 mL culture media. 6  2. Mixed lOpg DNA with 125pL 0.5M CaCL to make up to 250pL DNA-CaCL with autoclaved distilled water. 3. DNA-CaCl was added to "bubbled" 2X HEBS solution drop by drop. 2  4. DNA-CaC^-HEBS mixture was placed at room temperature for 25 minutes and then added to each plate.  5. Culture media were changed after 24 h and cells were harvested 48 h after transfection.  2.4.2. Lipofectamine 2000 Transfection 1. 2-4pg plasmid DNA was mixed with 200-250uL Opti-MEM I (Gibco 31985-070). Same amount of Opti-MEM I was mixed gently with 4-8pL Lipofectamine 2000 (Invitrogen 11668-019) (DNA:Lipofectamine 2000 ratio = 1:2) and left at room temperature for 5 minutes. 2. Mix Opti-MEM I-Lipofectamine with Opti-MEM I-DNA solution and incubate the mixture at room temperature for 20 minutes. 3. Before adding the mixture to each plate, culture medium was changed. Cells would be harvested 24-48 h after transfection. 61  2.5. Dual-Luciferase Reporter Assay 48 h after transfection, cells were washed twice with Dulbecco's Phosphate-Buffered Saline (D-PBS) (Gibco 14190-136) and suspended in D-PBS. Following a 2 minute centrifuge, the supernatant was removed and IX Passive Lysis Buffer was used to lyse the cells. The cell lysis reaction was preceded at room temperature for 20-30 minutes. In order to detect the firefly luciferase activity, 2 pL of the cell lysate was mixed with 10 pL of luciferase assay reagent II (Promega E1910) and luminescent signal was detected by Luminometer (Turner Designs, TD20/20). The pRL-CMV plasmid expressing the Renilla Luciferase was also included in cell transfection to serve as an internal control and used to normalize the transfection efficiency. To measure Renilla luciferase activity, the addition of 10 pL of Stop & Glo reagent was followed immediately after the reading of firefly luciferase activity. Firefly luciferase measurement was normalized by Renilla luciferase measurement.  2.6. Reverse Transcription-Polymerase Chain Reaction 2.6.1. Cell Transfection Cells (1.0X10 ) were seeded in 60mm plates and Calcium Phosphate transfection was 6  used to transfect cells with desired DNA plasmids. Cells were harvested with 0.5mL TriReagent (Sigma, T9424) 48 h after transfection and frozen in -80°C.  2.6.2. RNA Extraction Cell samples were treated with 0.1 mL chloroform and left at room temperature for 10 minutes. The mixture was then centrifuged at 4°C at 13000rmp for 15 minutes. The top colorless aqueous phase was collected and mixed with 0.25mL isopropanol. The samples were centrifuged at 13000rpm for 10 minutes at 4°C after 7-minute room temperature incubation.  62  The supernatant was removed and the pellet was washed with 0.5mL 75% Ethanol (prepared with DEPC-treated water). After centrifuging at 13000rpm for 5 minute, RNA pellet was airdried, dissolved in 30pL DEPC-treated water and incubated in 55°C water bath for 10 minutes.  2.6.3. cDNA Synthesis ThermoScript RT-PCR System kit (Invitrogen, 11146-016) was used to synthesize cDNA from the extracted RNA. RNA-primer denaturation reaction included 50pM 01igo(dT) o 2  primer, RNA, lOmM dNTP Mix and DEPC-treated water. In a 0.2mL PCR tube, cDNA synthesis was initiated by incubating 0.9pg denatured RNA and master reaction mix at 50°C for 50 minutes in the PCR machine (Eppendorf, Mastercycler gradient 5331) and terminated by incubating at 85°C for 5 minutes. The cDNA synthesis master reaction mix contained 5X cDNA Synthesis Buffer, 0.1M Dithiothreitol, RNaseOUT™ (40U/uL), DEPC-treated water and ThermoScript™RT (15U/pL). The remaining RNA templates in the reaction were removed by incubating at 37°C for 20 minutes after adding 1 uL RNase H.  2.6.4. PCR Amplification PCR reaction consisting of Taq DNA Polymerase, Native (Invitrogen, 18038-042) and a set of primers specific for the 576 bp fragment of human BACEl gene: BACEl-288fEco (5cggaattcgccaccatgctggtggatacaggcagc-3') and BACEl-864rBam (5cgggatcccacaatgctcttgtcatag-3') was carried out. In addition, the 324 bp fragment of Human Glyceraldehyde-3 phosphate dehydrogenase (hGAPDH) gene was amplified as an internal control with the primers: hGAPDH -305Bam (5'-tctggatcctcaccaccatggagaaggc-3') and hGAPDH -629rXho (5'-atactcgaggcagggatgatgttctg-3'). PCR Master Mix included 10X PCR Buffer Minus Mg, 50mM MgCl , lOmM dNTP Mix, sense/anti-sense primers and Taq DNA 2  63  polymerase. In a 0.2mL PCR tube, 4.45uL of PCR Master Mix and 0.75uL cDNA were added and the volume was brought up to 25pL with DEPC-treated water. PCR amplification was achieved by running 26-28 PCR cycles at the annealing temperature 60°C. PCR-resulted DNA fragments were loaded onto a 1.5% agarose gel and the result was analyzed by Kodak Image Station 1000 software (Perkin-Elmer).  2.7. Electrophoretic M o b i l i t y Shift Assay 4NF-KB  probe was labelled with [y- P]-ATP by phosphorylation reaction (Promega, 32  E3050) at 37°C for 10 minutes:  3.6pmole  4NF-KB  luL  T4 Polynucleotide Kinase 10X buffer  6.6pL  Nuclease-free water  lpL  T4 Polynucleotide Kinase (lOunit/uL)  1 pL  probe  [y-P ] - ATP (3000Ci/mmole at 1 OmCi/mL) 32  After the reaction was terminated by adding lpL 0.5M EDTA, the labelled  4NF-KB  probe concentration was adjusted to 0.035pmole/uL with TE buffer. HEK293 cells were transfected with lOpg N F - K B p65 expression DNA plasmid by Lipofectamine 2000. 48 h after transfection, HEK293 cells were harvested and treated with Buffer C and Buffer A. A brief sonication was followed to obtain the nuclear extract from N F - K B transfected cells.  64  After glass plates were cleaned with Acrylease™ Nonstick Plate Coating (Stratagene, 300132), 6% nondenaturing acrylamide gel was prepared as following:  lmL  TBE 10X buffer  2.4mL  50% Long Range gel solutions (Cambrex, 50611)  1.2mL  80% Glycerol  15.4mL  Distilled water  20pL  Tetramethylethylenediamine (Bio-Rad, 161-0801)  150uL  10% Ammonium Persulfate  The binding reaction was induced in sterile microcentrifuge tubes containing Gel Shift binding 5X buffer, HEK293 nuclear extract, and labelled oligonucleotide probe. The labelled probe was incubated with NF-KB-transfected HEK293 nuclear extract in Gel Shift Binding 5X Buffer at room temperature for 20 minutes. Before loading, the samples were mixed with gel loading 10X buffer. The gel was run at room temperature in 0.5X TBE buffer at 60V for 110 minutes and further analyzed by autoradiography.  In the competition assays, N F - K B consensus oligonucleotide (5'agttgaggggactttcccaggc-3') (Promega, E3292) and unlabelled 4 N F - K B probe served as competitors of the labeled 4 N F - K B probe. Moreover, four short double-stranded oligonucleotide probes (S-KB1, S-KB2, S-KB3 and S-KB4) corresponding to individual putative N F - K B binding elements in the human BACEl promoter region were synthesized by annealing  the primers: s-KBlf (5'-acacggtgaaaccccgtctc-3'), S - K B I T (5'-gagacggggtttcaccgtgt-3'), s-KB2f (5'-aactggtgaaatcccatctc-3'), s-KB2r (5'-gagatgggatttcaccagtt -3'), s-KB3f (5'aaaacgaagattccctttca-3'), s-KB3r (5'-tgaaagggaatcttcgtttt-3'), s-KB4f (5'-attgtgtggaaccccactgc-3'), 65  and s-KB4r (5'-gcagtggggttccacacaat-3'). These four short competitor probes were used to determine which N F - K B binding element in the human B A C E 1 promoter region interacts with NF-KB  p65. Before the addition of [y- P]-labelled 4 N F - K B probes, the nuclear extract of NF32  KB-transfected HEK293 cells was incubated with 40pm (100X excess) unlabelled competitor probe for 10 minutes. In the super shift assays, the nuclear extract was treated with anti-NF-KB p65 antibody (Sigma, N8523) prior to the addition of labelled 4 N F - K B probes.  2.8. Western Blotting After 48 h Calcium Phosphate transfection, cells were washed with D-PBS and harvested in RIPA-DOC cell lysis buffer containing protease inhibitor. After sonicating and centrifuging, cell lysates were mixed with loading buffer and heated at 95°C for 5 minutes. The samples were then loaded in 12% Tris-Glycine or 16% Tris-Tricine gels and run at constant current 55mA in Mini-PROTEAN 3 cell (Bio-Rad). P V D F (Polyvinylidene fluoride) membrane (Millipore, IPVH00010) was briefly incubated with Methanol and proteins were transferred from SDS-PAGE (Polyacrylamide gel electrophoresis) gel to the membrane by wet transfer system (Bio-Rad, Mini Trans-Blot cell). After 2-hour transfer at 110V, the membrane was incubated in blocking buffer (5% milk and 3% B S A in PBS-T) for 30 minutes.  After blocking, the membrane was incubated with the primary antibody at 4°C overnight. Monoclonal anti-luciferase antibody (Novus Biologicals, Inc, NB 600-307) against the first 258 amino acids of firefly luciferase protein was used to measure firefly luciferase protein expression (1:2000 working dilution in blocking buffer). Polyclonal C20 antibody against the last 20 amino acids of APP C-terminus was used to detect the full-length APP and  66  C99 fragment. Internal control p-actin protein was detected by monoclonal anti-^-actin antibody, AC 15 (Sigma).  The membrane was washed with PBS-T (Phosphate Buffered Saline-Tween 20) and then incubated with the secondary antibody (1:5000 dilution for anti-mouse or 1:10000 dilution for anti-rabbit in PBS-T) at room temperature for one hour. Enhanced Chemiluminescent detection method was applied for western blotting analysis. The membrane was treated with Enhanced Chemiluminescence system (ECL) (18mL H 0, 2mL 1M Tris pH8.5, lOOpL 2  250mM luminal, 44uL 90mM p-coumaric acid and 6pL 3% H 0 ). Emitting luminescence 2  2  was captured by photographic films and the images were analyzed by densitometry.  2.9. Enzyme Linked-Immuno-Sorbent Assay After cells were transfected by Lipofectamine 2000 for 48 h, culture medium was collected and treated with ImM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) (Sigma) to prevent protein degradation. The concentration of A p was measured using Colorimetric 40  ELISA kit for A P i _  4  o  (Biosource Internationa], Inc., Camarillo, CA, USA). 50pL sample was  incubated with 50pL detection antibody, Rabbit anti-Human Ap o antibody, in the antibody4  coated well (mouse antibody to N-terminus of A P ) at 4°C overnight.  The next day, before incubating with lOOuL Anti-rabbit Ig's-Peroxidase (HRP) for 30 minutes, the wells were washed with working washing buffer. Before the absorbance of each well at 450nm was measured, lOOpL Stabilized Chromogen was added in dark and lOOuL stop solution was added to each well after 30-minute incubation. The concentration of the sample in pg/mL was calculated according to the standard absorbance-concentration curve.  Chapter III. Results 3.1. Identification of a Functional Interaction Between N F - K B p65 and N F - K B Binding Elements in the Human BACE1 Promoter Region. Human BACE1 gene is tightly regulated at both transcriptional and translational levels and its promoter region contains several transcription factor binding elements, such as Spl and YY1 (Christensen et al., 2004; Nowak et al., 2006; Rossner et al., 2006; Sambamurti et al., 2004). To further examine transcriptional regulation of BACE1 gene expression, we analyzed the sequence of the human BACE1 promoter (GenBank A Y 162468) and identified four putative N F - K B binding elements. Though N F - K B has been described to modify the expression of many genes, there is still no report on the involvement of N F - K B in human BACE1 regulation (Liu and Malik, 2006; Shishodia and Aggarwal, 2004). Our results show the association between N F - K B p65 and BACE1 promoter region and clarify the role of N F - K B p65 in BACE1 transcriptional regulation and APP processing; hence, we hypothesize that N F - K B signaling regulates APP processing and Ap generation during the development of AD pathogenesis by modifying BACE1 gene activity.  (  Based on the consensus binding sequence of N F - K B 5'-GGGRNNYYCC-3', sequence analysis of human BACE1 reveals four putative N F - K B binding elements in the promoter region (Figure 3-1). To explore whether these N F - K B binding elements physically interact with transcription factor N F - K B , Electrophoretic Mobility Shift Assay (EMSA) was performed. A double-stranded oligonucleotide probe ( 4 N F - K B ) containing all four putative N F - K B binding  elements of the BACE1 promoter was synthesized and labelled with [y- P]. The labelled P32  4NF-KB  32  probe was incubated with nuclear extract obtained from N F - K B p65 transfected cells  to induce a DNA-protein complex binding reaction. Unlabelled competitor N F - K B consensus  68  oligonucleotide (5 '-agttgaggggactttcccaggc-3') was also included in the experiment to determine a direct association between N F - K B p65 and putative N F - K B binding elements of human BACE1. -1941 -1881 -1821 -1761 -1701 -1641 -1581 -1521 -14 6 1 -1401 -1341 -1281  gtgggctctc ctgattctac ttaccttcta ttttcctgta ggaatgaaat agtttttgaa attcccatag ttattgatta ccaacatata ccgtgtagga ccagcacttt gctaacacgg  ccagttacta agaagtttta gggtttgtta gttttatttt tggatttggt taatgcgtcc atacatttct gggctttcta taacactttt taagagtaga gggaggccga txpinnryTry-yj  -1221 -1161 -1101 -1041 -981 -921 -861  aggcgcctgt ggcagagctt acgcctcctc tgcctttaat tgtttaaaaa aggccgacgt aaataccatc  agtcccagct gcagtgagct aaaaaaaaaa gggagtaacg gtttcctggc gggcggatca tctactaaaa  N F - K B  N F - K B  attgaaccag aatatttaag gggaggtctt aatgttaaat tttttttcct attttcctct ctattttgat tccatatcat tccattttct gataatgatc ggcgggagga tctctactaa  aattagaaca tcatcaaatc cttcaatata tctttaatct tccaaatggg aatacgaatg ttcgattttt ataatctgca tgtcttttga ggccgggcgc tcacgaggtc aaatacaaaa  ctggtggtat tggaatactt catccagttt ttgggtttgt agatcatgaa a a t a t t c t a t atctggagtt tattttcaga ctgccagacg tcccaacaac ccatctctat catatctaca ttaacttgtg tctaatcatc actaatgatt cctttgcttt gtcaatcaga ccggtctgaa ggtggctcac gcctgtaatc aggagatcga g a c c a t c t t g aattagcggg gcgtggttgc  attcgggagg gagatcccac aaaaaaagag tagcctgtta tgggtacagt tttgaggcca atacaaaaat  ctgaggcagg cactgcactc tagagataat ttactacatt ggtccatgcc ggagttcgag tagctgggta  agaatagcct gaacccggga cagcctggcg acaaagcgag gatctttcct ttccaaagcc tgttgttgtt gtcgttatct tataatcccagcattttggg accagcctgg ccaactqgtg tggtggcgca t g c t t g t a a t  (1)  (2)  -801 -741 -681  c c c a g c t a c t cagaggctga ggcatgagaa ctgcatgaac acgggaggcg a c t t g a g a c c a t g c c a c t g c a c t c c a g c c t gggtgacaga g c a a g t c t c c a a a a a c g a a g attooctttc a t t t t t t a a a aaaattaaat cagagtaact  -621 -561 -501 -441 -381 -321 -261 -201 -141  atcaatggct tttaagctat tagagatact tggctgatgg aacaggttca gagaaaataa gaaaaactct catttttaaa gatgtccctc  ctccacattt tagaactctt tttcggagtg tgcagaaaat gatgggagaa gacaactgca tcctcccctt cgctcaaatt caaatcccta  gataaacaat acgcttttgg ttaacacttt cagaggagcc gacccactgg ctttcccctt tcatcctgct aaataccacc ctccttaatg  atattgttaa ttcaaggctt aaagctatca atgaggattc ctcttacctg ttggaccctg ccctcaatct ttctcccact ccacatcgta  aaccataccg taagctctta acagccattt aagtagagct cccgcagatc ctccgctggt ctgctcgtga gagtcttccc ttgtgtayyda,  -81 -21 +41 +101 +161 +221 +281 +341 +401 +461 +521 +581 +641 +701  gcaggaatca gcctggtagt gacgggaggt actccacctc ccatgggaag cagccgccgc tggattatgg ccgcgccgcc tcccagcccc gcgggctccg tctccacagc ccccaagctc ggacggacgt agccctgccc  cttaactgtc gtgcctggca gtgcccctct ggcagagggc actacacctc cacaagtctt tggcctgagc gcccgccggg gccgggagcc gatcccagcc ccggacccgg cctctcatga gggccagtgc tggctcctgc  ccgtgtagaa tacagtgggt ccatccgtct atcccagacc ccagcgatcc tccgcctccc agccaacgca gggaccaggg cgcgcccgct tctcccctgc gggctggccc gaagccacca gagcccagag tgtggatg  tcaccgttta gctccttcca agcccttccc cctctccagc cagggaaaag cagcccgccc gccgcaggag aagccgccac gcccgggctg tcccgtgctc agggccctgc gcaccaccca ggcccgaagg  tttctctctg tgctgaaaga gccagggcct cccggaagcc cgaaaacctt gggagctgcg cccggagccc tggcccgcca gccgccgccg tgcggatctc aggccctggc gacttgggag ccggggccca  N F - K B  gaggttgcag ttttcaaaag gctgaatttt  (3)  N F - K B  gcttttctcc ccagttaata ctcctcagtt caagggcaca gcaggcgcag ttgcagcctg aaatcctacc agatccccta ococactgcg (4)  tatctccagc aagactgaca tgcagggcgg ggattgcctg ttggctttga agccgcgagc ttgcccctgc tgcccacccc tgccggtgta ccctgaccgc gtcctgatgc caggcgccag ccatggccca  Figure 3-1: Identification of N F - K B Binding Sequence in the Human BACE1 Promoter Region. N u c l e o t i d e s e q u e n c e o f t h e 2 6 6 8 b p h u m a n BACE1  p r o m o t e r region was o b t a i n e d f r o m the h u m a n g e n o m i c l i b r a r y ( G e n B a n k  A Y 1 6 2 4 6 8 ) . T h e +1 n u c l e o t i d e r e p r e s e n t s t h e t r a n s c r i p t i o n start s i t e a n d t h e n u c l e o t i d e s e q u e n c e o f f o u r p u t a t i v e N F - K B b i n d i n g e l e m e n t s , N F - K B ( 1 ) , N F - K B ( 2 ) , N F - K B (3) a n d N F - K B (4), a r e b o l d e d a n d u n d e r l i n e d .  69  32  A shifted protein-DNA complex band was detected after incubating the labelled" P 4 N F - K B probe with nuclear extract (Figure 3-2, Lane 2). The shifted band was completely abolished by addition of N F - K B consensus oligonucleotide (Figure 3-2, Lane 3). Super gel shift assay was also performed to further confirm the  binding element in the  NF-KB  4NF-KB  probe. A slower-migrating super shifted band was detected after anti-NF-KB p65 antibody was incubated with the P-labeled 4 N F - K B probe and nuclear extract (Figure 3-2, Lane 4). The 32  detection of a complex shifted band indicates that putative  NF-KB  NF-KB  p65 directly associates with the  binding elements in the human BACEl promoter region. Anti-NF-KB 65 antibody  -  -  N F - K B c o n s e n s u s o l i g o (100X)  -  +  Nuclear Extract  -  P - 4 N F - K B probe  +  + + + + + +  3 2  Lane  1  2  3  +  -  Anti-NF-KB p65  -  NF-KB p65  -  Non-specific  —  Free Probe  4  Figure 3-2: Physical Interaction between N F - K B p65 and N F - K B Binding Elements in the Human BACEl Promoter. The association between N F - K B p65 and the human BACEl promoter region was confirmed by E M S A . A [y-- P]-labeled double-stranded oligonucleotide probe, 4 N F - K B , consisting of all four putative N F - K B binding elements of the BACEl gene, was incubated with nuclear extract obtained from N F - K B p65-transfected H E K 2 9 3 cells. Two slow migrating complexes were detected after the labeled probe was incubated with nuclear extract (Lane 2). If 40pm (100X excess) N F - K B consensus oligonucleotide was also included in the incubation, one slow migrating complex was abolished (Lane 3). This indicates that N F - K B interacts with the putative N F - K B binding elements in the BACEl promoter region. Furthermore, the association of N F - K B p65 with 4NF-KB probe was examined by a super shift assay using anti-NF-KB p65 antibody (Lane 4). 12  70  To investigate which putative N F - K B binding elements of BACEl KB  p65, we synthesized four short unlabelled probes  (S-KB1,  interacted with NF-  2, 3, 4) as competitors of the  4 N F - K B probe in our EMSAs. Each short probe covered the sequence of an individual putative NF-KB  binding element identified in the  between nuclear extract and labelled  BACEl  4NF-KB  promoter region. Complex formation  probe was abolished when either  S-KB  1  or  S-KB4  was applied (Figure 3-3; Lane 4 and 7); therefore, the data suggests that the first and the fourth NF-KB  binding elements are able to interact with  NF-KB  p65 in BACEl  gene regulation.  Competitor (short NF-KB probe) (100X)  & ****# / «a? «T - ,•*»  /**•  <c  <b  <b <o  + +  + +  + + + +  Competitor (4NF-KB probe) (100X)  -  +  Nuclear Extract  +  +  +  +  32 . P  4 N F  - K B probe  +  - N F - K B p65 — Non-specific  Free Probe Lane  1  Figure 3-3: Specific Interaction between the First and Fourth N F - K B Binding Elements and N F - K B  p65.  Specific N F - K B p65 binding elements in the human BACEl promoter region were determined by E M S A . Nuclear extract was collected from HEK293 cells transfected with N F - K B 65 expression plasmid. After incubating nuclear extract with labeled P - 4 N F - K B probe, two complex formations were detected (Lane 2). In the competition assays, the addition of 40pm (100X excess) unlabeled 4 N F - K B probe abolished the complex formations (Lane 3). Four short oligonucleotides representing four putative N F - K B binding elements of the human BACEl gene were synthesized. When 40pm (100X excess) S-KBI or S-KB4 probe was applied in the competition assays, the complex formation, recognized as the specific interaction between putative N F - K B binding elements and N F - K B p65, was abolished (Lane 4 & 7). The results reveal that the first and the fourth putative N F - K B binding elements of the human BACEl promoter directly associate with N F - K B p65. ,2  71  To investigate whether the identified N F - K B binding elements of BACEl were functional, we inserted 4 N F - K B double-stranded oligonucleotide in front of the firefly luciferase gene of pGL-pL (pBACE-NF-KB) and measured promoter activity by a dualluciferase reporter assay. The plasmid, pGL-pL, was modified to eliminate multiple Spl enhancer sequence within the SV40 promoter. Compared to the control pGL-pL, a 10.0 fold increase in luciferase activity (0.97 ± 0.012-fold to 9.69 ± 0.26-fold; P<0.001) was detected in RelA-KO cells co-expressing pBACE-NF-KB and N F - K B p65 (Figure 3-4a) and a 13.1 fold increase in luciferase activity (4.00 ± 0.044-fold to 52.54 ± 0.41-fold; P<0.001) was measured in wild-type 3T3 cells co-expressing pBACE-NF-KB and N F - K B p65 (Figure 3-4b). In empty vector cotransfections, no significant difference in luciferase activity was determined in both cell lines transfected with either the control pGL-pL or pBACE-NF-KB plasmid. These results suggest that the human BACEl promoter region contains functional N F - K B p65 binding elements.  72  NF-KB p65  AST  Empty vector  ^°  Figure 3-4: N F - K B p65 Binding Elements on the Human BACE1 Promoter are Functional. The functional activity of putative N F - K B binding elements was analyzed by dual-luciferase reporter assays in (a) R e l A - K O and (b) wild-type 3T3 cells. By inserting 4 N F - K B oligonucleotide in front of firefly luciferase reporter gene of p G L - p L ( p B A C E - N F - K B ) , the level of luciferase expression represents the promoter activity of the oligonucleotide. Luciferase measurements generated from control plasmid pGL-pL were also included. Along with p B A C E - N F - K B reporter plasmid, N F KB p65 expression plasmid or empty vector p M T F was co-transfected into the cells. Firefly luciferase measurement was normalized by internal Renilla luciferase expression. The values are expressed as means ± S E M of Renilla luciferase activity in relative fold (n=3). *. P < 0.001 relative to control by Student's I test.  73  3.2. N F - K B p65 Regulates Human BACE1 Promoter Activity. In order to determine the effect of N F - K B p65 on BACE1 gene expression, we generated four BACE1 reporter plasmids, pBlP-Nl, -N2, -N3, and -N4 (Figure 3-5a). pBlPNl reporter plasmid was developed by inserting BACE1 promoter fragment (-1466-+292) covering all four putative  NF-KB  binding elements into the pGL3-basic plasmid, which has no  defined eukaryotic promoter or enhancer sequence in front of the firefly luciferase reporter gene. BACE1 promoter deletion constructs with sequential eliminations of one upstream putative  NF-KB  binding element were also cloned into promoter-less pGL3-basic plasmid  (Figure 3-5a, b). The inserted promoter fragment drives the expression of luciferase protein, which corresponds to its promoter activity.  74  ATG 1  N F - K B (1)  N F - K B (2)  N F - K B (3)  •  N F - K B (4)  pB1P-N1  (+292)  1  Luciferase W  (-1466) Luciferase  pB1P-N2 (-932)  w W  Luciferase  pB1 P-N3 (-757)  w W  pB1 P-N4 (-145)  Luciferase  Luciferase  w  —•  b  Figure 3-5: Illustrations of BACEl Reporter Plasmids. (a) Schematic diagram of BACEl promoter fragments, covering from bp-1466 to +292, bp -932 to +292, bp -757 to +292 or bp -145 to +292, cloned in front of the firefly luciferase gene of the promoter-less pGL3-basic plasmid. (b) The size of constructed plasmids were confirmed by restriction enzyme digestion ( p B l P - N l by Nhe MHind III digestion and p B l P - N 2 ~ N 4 and pGL3-basic plasmids by Xho MHind III digestion) and analyzed on agarose gel. The size of pGL3-basic vector is 4.82kb and the size of the inserted BACEl gene 5' flanking fragments range from 0.44 to 1.76kb.  75  The BACE1 reporter plasmid was cotransfected into HEK293 cells with either  NF-KB  p65 expression plasmid or empty vector pMTF. Luciferase expression was normalized by internal P-actin protein levels. Western blot analysis revealed a significant increase in luciferase expression in cell lysates from cells cotransfected with BACE1 reporter plasmid and NF-KB  p65 expression plasmid (Figure 3-6a, b). When N F - K B p65 was co-expressed,  luciferase expression of cells transfected with pBlP-Nl increased from 44.79 ± 1.11% to 94.29 ± 3.46% (P<0.001). A similar elevation in luciferase expression was detected in cells transfected with pBlP-N2 (53.05 ± 1.64% to 106.8 ± 3.23%; P<0.001), with pBlP-N3 (43.85 ± 0.81% to 79.15 ± 3.29%; P<0.001) and with pBlP-N4 (47.54 ± 1.70% to 86.52 ± 3.01%; P<0.001) reporter plasmid transfections in the presence of N F - K B p65 overexpression; however, no significant difference in luciferase activity was shown in pGL3-basic transfected cells with or without N F - K B p65 expression.  To confirm our Western blot results, we used a dual-luciferase reporter assay to examine luciferase expression of the BACE1 reporter plasmids in HEK293 cells co-transfected with N F - K B p65 expression plasmid or empty vector pMTF. Firefly luciferase activity was normalized by internal Renilla luciferase expression. Consistent with our Western blot results, a significant elevation of promoter activity was detected in cells co-expressing BACE1 reporter plasmid and  NF-KB  p65 (Figure 3-6c). When N F - K B p65 expression plasmid was  cotransfected, cells transfected with pBlP-Nl resulted in a 1.61 fold increase in luciferase reporter activity (13.09 ± 0.036-fold to 21.02 ± 0.072-fold; P<0.001), cells transfected with pBlP-N2 resulted in a 2.38 fold increase in luciferase reporter activity (15.88 ± 0.20-fold to 37.75 ± 0.35-fold; P<0.001), cells transfected with pBlP-N3 resulted in a 2.43 fold increase in luciferase reporter activity (9.81 ± 0.15-fold to 23.87 ± 0.02-fold; P<0.001) and cells  76  transfected with pBlP-N4 resulted in a 2.94 fold increase in luciferase reporter activity (9.148 ± 0.28-fold to 26.90 ± 0.99-fold; P<0.001). Compared to luciferase activity of cells cotransfected with empty vector, no significant difference in luciferase activity was detected in cells co-expressing pGL3-basic and  NF-KB  p65 plasmids. Taken together, these results  demonstrate that N F - K B p65 upregulates human BACE1 promoter activity.  77  Figure 3-6: BACEl  P r o m o t e r A c t i v i t y is Elevated by N F - K B p65.  (a) HEK293 cells were cotransfected with BACEl reporter plasmid ( p B l P - N l , N2. N3 or N4), and NF-KB p65 expression plasmid or empty vector pMTF. Cell lysates were collected 48 h after transfection and electrophoresed by 12% Tris-glycine S D S - P A G E . Western blot analysis used monoclonal anti-luciferase antibody to detect the level of luciferase protein and monoclonal anti-P-actin antibody (AC 15) was included to measure internal P-actin protein levels, (b) Image J was used to quantitatively analyze luciferase expression. The values are means ± S E M relative to P-actin expression (n=3). Protein level is expressed as a percentage of internal P-actin protein levels. *, P<0.001 relative to control by Student's / test, (c) After BACEl reporter plasmids were cotransfected into HEK293 cells with N F - K B p65 expression plasmid or empty vector, cells were treated with passive lysis buffer and luciferase activity was measured. Renilla luciferase activity was used to normalize transfection efficiency. The values are expressed as means ± S E M of Renilla luciferase activity in relative fold (n=3). *. P<0.001 relative to control by Student's I test.  7S  3.3. BACE1  P r o m o t e r A c t i v i t y is Regulated T h r o u g h T w o Distinct N F - K B B i n d i n g  Elements.  EMSA results indicated that the first and fourth putative N F - K B binding elements of the human BACE1 promoter are able to bind to N F - K B p65 (Figure 3-3). To investigate if these two cis-acting binding elements regulate BACE1 gene expression, we generated BACE1 reporter plasmids containing a mutated N F - K B binding element by site-directed mutagenesis. Five nucleotides of the first N F - K B binding element sequence were altered from (5'gtgaaaccccg-3') to (5'-TtCaaaGTTcg-3') to generate pBlP-Nlm, and seven nucleotides of the fourth N F - K B binding element sequence were changed from (5'-gtggaacccc-3') to (5'TtCTaaGGTT-3') to generate pBlP-N4m (Figure 3-7a). These BACE1 reporter plasmids have the same DNA fragment size as the control pBlP-Nl reporter plasmid and their luciferase expression corresponds to the transcriptional activity of the inserted BACE1 promoter fragment.  79  Human BACE1 promoter NF-KB (1) GTGAAACCCCG  i TTCAAAGTTCG  ATG  NF-KB (4) GTGGAACCCC  i TTCTAAGGTT  luc pB1P-N1m  NF-KB (4) luc NF-KB (1)  pB1P-N4m  Figure 3-7: Illustrations of BACE1 Reporter Plasmids Containing a Mutated Binding Element.  NF-KB  (a) Position and sequence of thefirstand fourth putative NF-KB binding sites of the human BACE1 promoter region, with nucleotides mutated by site-directed mutagenesis in boldface, (b) Luciferase reporter plasmid constructs with a mutated NFKB binding element (pBlP-Nlm and pBlP-N4m), where "X" indicates the mutated NF-KB binding element.  Western blot analysis revealed that mutations of the N F - K B binding element in the BACE1 promoter region reduce promoter activity (Figure 3-8a, b). Luciferase measurements from cells transfected with BACE1 reporter plasmid and empty vector pMTF are all normalized to approximate 100% protein level. When N F - K B p65 expression plasmid was cotransfected with pBlP-Nl into HEK293 cells, luciferase protein level increased by 1.85 fold (97.72 ± 1.34% to 180.8 ± 0.33%; P<0.05). A mutation in the first N F - K B binding element (pBlPNlm) caused the luciferase protein level to increase by only 1.56 fold (95.90 ± 1.66% to 149.6 ± 1.29%; P<0.05), and a mutation in the fourth N F - K B binding element (pBlP-N4m) led  80  luciferase expression to go up 1.58 fold (103.0 ± 0.66% to 163.5 ± 0.25%; P<0.05). These results indicate that mutating the functional N F - K B p65 binding elements in the BACEJ promoter region causes a significant reduction in the ability of N F - K B p65 to increase luciferase expression.  We performed a dual-luciferase reporter assay to test whether mutating the N F - K B binding elements affected the activating effect of N F - K B p65 on human BACE1 promoter activity. Luciferase measurements from cells transfected with BACE1 reporter plasmid and empty vector pMTF were normalized to near 100 fold relative luciferase activity (Figure 3-8c). Luciferase expression detected in cells transfected with pBlP-Nl and N F - K B p65 was increased by 6.63 fold (107.9 ± 2.68-fold to 715.5 ± 11.54-fold; P<0.05). Cotransfection of pBlP-Nlm and N F - K B p65 expression plasmid resulted in a smaller 3.76 fold increase (95.73 ± 1.25-fold to 360.3 ± 5.54-fold; P<0.05) in luciferase activity, and cotransfection of pBlPN4m and N F - K B p65 expression plasmid resulted in a similar 3.70 fold increase (113.8 ± 1.15fold to 420.5 ± 5.02-fold; P<0.05) in luciferase expression. Therefore, these two N F - K B binding elements of the human BACE1 promoter are important for the regulation of BACE1 promoter activity by N F - K B p65.  81  b  200  <r  <r  <r +  -  +  -  > NF-KB p65  +  g  —Luciferase  = «ar  150 100  o D_  50  — N F - K B p65  <?'^  —P-Actin  N  <r  J* x9  J> <T  ^  <?~ <y  ^  ^  x  <?>  Empty vector  NF-KB (1)  SS  AT  NF-KB (4)  <5 &  X  #  NF-KB p65  pB1P-N1  luc  ^^Xh^H  NF-KB (4)  |—L  P  B 1 P -N1m  luc NF-KB (1)  IP-N4m  T 0  100  200  300  400  500  r 600  700  800  900  Luciferase activity (Fold)  Figure 3-8: BACEl Promoter Activity is Modulated by N F - K B Binding Elements. (a) Western blot analysis was used to determine the level of luciferase protein generated in HEK293 cells after cotransfection of BACEl reporter plasmids with or without N F - K B p65 expression plasmid. Cell lysates were collected 48 h after transfection and electrophoresed by 12% Tris-glycine SDS-PAGE. Comparing to the control plasmid p B l P - N l , mutation of the NF-KB binding elements leads to a relatively smaller increase in BACEl promoter activity when NF-KB p65 is present. Monoclonal anti-luciferase antibody was used to detect the level of luciferase protein. Monoclonal anti-NF-KB p65 antibody was also used to detect the level of NF-KB p65 in cells transfected with empty vector pMTF and in cells transfected with NF-KB p65 expression plasmid. A robust protein level of N F - K B p65 was detected in cells expressing NF-KB p65. Monoclonal anti-Pactin antibody (AC 15) was included to measure internal P-actin protein levels, (b) Quantitative analysis of luciferase expression was achieved by using Image J software. Luciferase levels from cells transfected with reporter plasmid and empty vector were normalized to near 100% protein level. The luciferase measurements from cells transfected with p B ! P - N l and NF-KB p65 expression plasmid served as the positive control. Values are means ± S E M relative to P-actin expression (n=3). The protein level is expressed as a percentage of internal P-actin protein expression. *, P<0.05 relative to control by Student's t test, (c) Illustration of BACEl reporter plasmids where mutated NF-KB binding elements are marked with an " X " . BACEl reporter plasmids containing mutated NF-KB binding sites were cotransfected into HEK293 cells with NF-KB p65 expression plasmid or empty vector pMTF. Luciferase activity was measured after 48 h. Renilla luciferase activity was used to normalize transfection efficiency. Luciferase levels from cells transfected with reporter plasmid and empty vector were normalized to near 100 fold relative luciferase activity. Luciferase measurements from cells transfected with p B l P - N l and N F - K B p65 expression plasmid served as the positive control. The values are expressed as means ± S E M of Renilla luciferase activity in relative fold (n=3). *, P<0.05 relative to control by Student's / test. 82  3.4. H u m a n BACEl Gene T r a n s c r i p t i o n is M o d u l a t e d by N F - K B Signaling.  To further investigate if N F - K B regulates human BACEl gene at the transcriptional level, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to measure endogenous BACEl mRNA levels in HEK293 cells transfected with empty vector or N F - K B p65 expression plasmid (Figure 3-9a). Human GAPDH mRNA was used as the control: When cells were transfected with N F - K B p65 expression plasmid, the mRNA level of BACEl was elevated to 476.6 ± 21.68%, which was significantly higher than the control mRNA level, 100 ± 4.734% (P<0.001). Herein, we demonstrate that human BACEl is regulated by N F - K B p65 at the transcriptional level and by elevating BACEl mRNA expression,  NF-KB  can result in an increased BACEl protein level.  83  Empty vector  NF-KB p65  Figure 3-9: N F - K B p65 Upregulates Human BACE1 at the Transcriptional Level. (a) HEK293 cells were first transfected with empty vector p M T F or N F - K B p65 expression plasmid and R N A was extracted 48 h after transfection. Semi-quantitative RT-PCR was carried out to measure endogenous human BACE1 m R N A level with specific primers recognizing coding sequences of the BACE1 gene and human G A P D H m R N A level as an internal control. Different cycles and amounts of PCR products were tested, and 26-cycle PCR products were analyzed on 1% agarose gel. (b) The ratio of human B A C E 1 to human G A P D H m R N A in transfected HEK293 cells was analyzed by Image J software. Endogenous BACE1 mRNA was significantly increased in cells transfected with N F - K B p65 expression plasmid. The values are shown as means ± S E M (n=3). *, /><0.001 relative to control by the Student's / test.  3.5. The Role of N F - K B in Regulation of BACE1 Transcriptional Activity in Neurons. Our data show that N F - K B increases human BACE1 gene transcription in HEK293 cells. In order to determine the implication of N F - K B in A D pathogenesis, BACE1 reporter plasmids p B l P - N l ~ N 4 with or without N F - K B p65 expression plasmid were cotransfected into  84  neuronal-like cells. We used mouse neuroblastoma (N2a) and human neuroblastoma (SHSY5Y) cell lines to examine BACEl transcriptional regulation by N F - K B in neurons.  To characterize the effect of N F - K B p65 overexpression on BACEl promoter activity in neurons, BACEl reporter plasmid was cotransfected into N2a cells with either empty vector pMTF or N F - K B p65 expression plasmid. Luciferase activity was measured 48 h after transfection. Compared to the luciferase expression from cells cotransfected with empty vector, N F - K B p65 cotransfected cells showed a significant elevation in BACEl promoter activity (Figure 3-10a). With N F - K B p65 overexpression, cells cotransfected with the BACEl reporter plasmid pBlP-Nl, containing all four putative N F - K B binding elements, had a 3.40 fold increase in luciferase expression (3.631 ± 0.014-fold to 12.34 ± 0.113-fold; P<0.001). Similarly, when N F - K B p65 was present, a 3.74 fold increase in luciferase activity (4.382 ± 0.068-fold to 16.40 ± 0.223-fold; P<0.001) was detected in pBlP-N2 cotransfected cells and a 4.09 fold increase in luciferase activity (3.531 ± 0.013-fold to 14.45 ± 0.168-fold P<0.001) in pBlP-N3 cotransfected cells, as well as a 3.67 fold increase in luciferase activity (5.198 ± 0.066-fold to 19.06 ± 0.352-fold P<0.001) in pBlP-N4 cotransfected cells. No significant difference in luciferase activity was detected in pGL3-basic cotransfected cells with N F - K B p65 plasmid or empty vector plasmid co-expression. Together, these results revealed that similar to the HEK293 cells, N F - K B p65 can upregulate human BACEl promoter activity in neuronal-like cells.  Also, BACEl reporter plasmid pBlP-Nlm or pBlP-N4m, which contains a mutated NF-KB  binding element, was cotransfected into SH-SY5Y cells with or without N F - K B p65  expression plasmid (Figure 3-10b). Luciferase measurements from cells transfected with  85  BACE1 reporter plasmid and empty vector pMTF were normalized to near 100 fold relative luciferase activity. In the presence of N F - K B p65, luciferase activity of cells transfected with control pBlP-Nl was increased by approximately 1.5 fold (131.2 ± 14.93-fold to 191.3 ± 14.11-fold; P<0.05); however, when N F - K B p65 expression plasmid was cotransfected, luciferase activity of cells transfected with pBlP-Nlm was only elevated by approximately 1.14 fold (103.6 ± 1.704-fold to 118.0 ± 7.287-fold; P>0.05), whereas luciferase expression of cells transfected with pBlP-N4m increased by 1.3 fold (111.4 ± 7.026-fold to 142.9 ± 7.615fold; P<0.05). No significant change in BACEl promoter activity by N F - K B p65 expression was observed when the first N F - K B binding element was mutated while the mutation of the fourth N F - K B binding element resulted in a small increase in BACEl promoter activity in the presence of N F - K B p65. Thus, by altering thefirstor fourth binding element sequence of N F KB  p65 in the human BACEl promoter region, the activating effect of N F - K B p65 on BACEl  transcriptional activity can be diminished.  86  r* . 0  5  .  .  10  I  . 15  20  25  Luciferase activity (Fold) b  0  50  100  150  200  250  Luciferase activity (Fold)  Figure 3-10: Human  BACEl  Promoter Activity in Neurons is Upregulated by  NF-KB.  (a) BACEl reporter plasmids p B l P - N I ~ N 4 were cotransfected into N2a cells with N F - K B p65 expression plasmid or empty vector pMTF. Luciferase activity was measured 48 h after transfection and Renilla luciferase activity was used to normalize transfection efficiency. The values are expressed as means ± S E M of Renilla luciferase activity in relative fold (n=3). *, P<0.001 relative to control by Student's I test, (b) BACEl reporter plasmid, p B l P - N l m or p B l P - N 4 m , which contains a mutated N F - K B binding element, was cotransfected into S H - S Y 5 Y cells with or without N F - K B p65 expression plasmid. Luciferase measurements of p B l P - N l transfected cells with or without N F - K B p65 expression were included as the control. Luciferase measurements from cells transfected with BACEl reporter plasmid and empty vector were normalized to near 100 fold relative luciferase activity. After the first or the fourth NF-KB binding element was mutated, BACEl gene transcription was reduced significantly. The values are expressed as means ± S E M of Renilla luciferase activity in relative fold (n=3). *, P< 0.05 relative to control by Student's / test. 87  3.6.  NF-KB  p65 Elevates p-secretase Processing of APP and Ap Production.  We speculate that N F - K B p65 increases P-secretase activity by upregulating BACEl gene transcription, and more APP is processed through the p-secretase cleavage pathway according to the Amyloid Cascade Hypothesis of AD (Hardy and Higgins, 1992). To test whether APP processing is modulated by N F - K B , we measured protein levels of C99, the major cleavage product of APP by BACEl in 20E2 cells, after transfection of empty vector pMTF or NF-KB  p65 expression plasmid (Figure 3-1 la). The 20E2 cell line was generated by stably  expressing Swedish mutant APP s in HEK293 cells (Qing et al., 2004). C99 protein 69  expression in transfected cells was analyzed by Western blotting with APP antibody C20, which specifically recognizes the last 20 amino acids of the APP C-terminus, and P-actin protein was used as the internal control. The results revealed an increased amount of C99 in 20E2 cells transfected with N F - K B p65 expression plasmid (Figure 3-1 lb). C99 protein levels were increased by approximately 1.5 fold in the presence of N F - K B p65, from an endogenous level of 100.3 ± 4.192%, or from an empty vector transfection level of 99.72 ± 1.772% to 152.3 ± 6.032% (P<0.005). Moreover, though the amount of APP protein was slightly increased tol 13.4 ± 2.881% in  NF-KB  p65 transfected cells, no significant difference in APP  level was found in empty vector transfected cells, 105.8 ± 3.011% (P>0.05); therefore,  NF-KB  p65 may shift APP processing toward the P-secretase cleavage pathway by elevating BACEl gene expression to result in increased C99 production.  88  b 1 Control  C99  APP  Figure 3-11: N F - K B p65 Potentiates C 9 9 P r o d u c t i o n . After transfection of 20E2 cells with empty vector p M T F or N F - K B p65 expression plasmid, cell lysates were treated with protease inhibitor cocktail Complete (Roche) and electrophoresed by 16% Tris-tricine S D S - P A G E . C20 antibody recognizing the C-terminus of A P P was applied to detect protein levels of full-length APP, C83 and C99. Cell lysates from HEK293 cells transfected with pC99 or pC83 expression plasmid were used as positive controls for the C99 and C83 fragments. Moreover, (3-actin was detected by monoclonal anti-P-actin antibody AC-15 as an internal control, (b) Quantitative analysis of C99 and full-length APP protein levels was done by Image J software. The values are expressed as means ± S E M (n=3) and the protein level is calculated as percentages of internal p-actin protein level. *, / <0.005 relative to the control by the Student's ; test. >  To investigate the effect of N F - K B p65 on AP generation, we measured Ap o protein 4  levels by colorimetric Enzyme Linked-Immuno-Sorbent Assay (ELISA). SH-SY5Y cells which overexpress Swedish mutant APP (Sun et al., 2006a) were transfected with empty  vector, MBa expression plasmid or  NF-KB  p65 expression plasmid and 48 h after transfection,  the cultured media were collected and treated with ImM A0 inhibitor (AEBSF). The results showed that N F - K B p65 expression significantly increased A(3o protein concentration from 4  100.0 ± 2.586% in empty vector-transfected cells to 132.1 ± 4.489% (P<0.001) (Figure 3-12). Meanwhile, MBa transfection reduced A p  40  protein levels to 92.24 ± 2.680% (P<0.05).  Hence, Ap o production was altered by N F - K B p65 in neuronal cell line. 4  Empty  NF-KB  vector  p65  ,  p  Figure 3-12:  N F - K B p65 Increases A p 4 0 Generation. S H - S Y 5 Y cells that overexpress Swedish mutant A P P were transfected with empty vector, N F - K B p65 expression plasmid or IKB<X expression plasmid and the culture supernatant was collected and treated with A E B S F . The concentration of A p was determined by colorimetric E L I S A kit specific for Af} . The values are expressed as means ± S E M (n=12). *, /><0.001 relative to the control by the Student's t test. 4()  40  3.7. Summary According to consensus N F - K B binding sequence 5 ' - G G G R N N Y Y C C - 3 ' , we identified four putative N F - K B binding elements in the human BACEl promoter region. An oligonucleotide probe containing the sequence of all four putative N F - K B binding elements was synthesized to conduct EMSAs. The results show that these N F - K B binding elements are able to interact with transcription factor N F - K B p65. Furthermore, to investigate which  NF-KB  binding element could associate with N F - K B p65, we synthesized four short oligonucleotide probes corresponding to the sequences of the four individual N F - K B binding elements. By  90  applying EMSA competition assays, we demonstrate that the first and fourth  NF-KB  binding  elements can interact with N F - K B p65. Dual-luciferase reporter assay was carried out to determine whether these N F - K B binding elements were functional. We cloned the doublestranded oligonucleotide that contained the sequence of all four N F - K B binding sites in front of the firefly luciferase reporter gene of the modified pGL3-promoter vector pGL-pL (pBACENF-KB).  Luciferase activity correlates with the promoter activity of the inserted  oligonucleotide and the analysis revealed that N F - K B p65 significantly increases luciferase activity in RelA-KO cells; therefore,  NF-KB  signaling can regulate human BACEl  transcriptional activity by associating with the first or fourth N F - K B binding element.  To clarify the role of N F - K B in regulating BACEl promoter activity, we constructed BACEl reporter plasmids pBlP-Nl~N4 by cloning various BACEl promoter fragments in front of the firefly luciferase gene of promoter-less pGL3-basic plasmid. BACEl reporter plasmids were cotransfected with or without N F - K B p65 expression plasmid into HEK293 cells to detect luciferase expression, which corresponds to the activity of the inserted BACEl promoter fragment. Both Western blotting and dual-luciferase reporter assay analyses show that luciferase expression is significantly elevated when N F - K B p65 is co-expressed, and a similar effect of N F - K B p65 on BACEl promoter activity was observed in every reporter construct transfection. This suggests that N F - K B p65 upregulates human BACEl gene transcription.  Site-directed mutagenesis was used to construct BACEl reporter plasmids containing a mutated N F - K B binding element (pBlP-Nlm and pBlP-N4m) in order to determine whether the first or fourth N F - K B binding site was important in modulating BACEl promoter activity.  91  After cotransfection of BACEl reporter plasmid with N F - K B p65 expression plasmid or empty vector pMTF into HEK293 cells, luciferase activity was analyzed by Western blotting and dual-luciferase reporter assay. Contrary to the dramatic increase in luciferase activity when cells were transfected with positive control pBlP-Nl and  NF-KB  p65 expression plasmids, a  smaller increase of luciferase expression was measured in cells transfected with mutant plasmid (pBlP-Nlm or pBlP-N4m) in the presence of N F - K B p65. These studies clearly support that N F - K B p65 plays an important role in modulating BACEl transcriptional activity.  Semi-quantitative RT-PCR was used to confirm that BACEl was regulated by p65 at the transcriptional level. HEK293 cells were transfected with empty vector or  NF-KB NF-KB  p65 expression plasmid and RNA was extracted for RT-PCR quantification. A robust increase of endogenous BACEl mRNA was detected in cells expressing N F - K B p65 compared to cells transfected with empty vector, thus N F - K B p65 modifies BACEl gene expression at the transcriptional level.  The role of N F - K B p65 in regulating the BACEl promoter was examined in neuronal cells and we cotransfected BACEl reporter plasmid with or without N F - K B p65 expression plasmid into N2a cells. Luciferase reporter assay analysis revealed that N F - K B p65 increases BACEl promoter activity in neuronal cells.  According to the amyloid cascade hypothesis of AD pathogenesis, the C99 fragment is generated following APP cleavage by P-secretase (Hardy and Higgins, 1992). To demonstrate whether N F - K B p65 increased C99 protein production, we transfected 20E2 cells, which are HEK293 cells stably expressing Swedish mutant APP695, with empty vector pMTF or  NF-KB  p65 expression plasmid. Cell lysates were analyzed by Western blotting to detect C99 protein levels by the C20 antibody, which recognizes the last 20 amino acids of the APP C-terminus. Despite a similar APP expression level, a significant increase in the C99 fragment was detected in N F - K B p65 expressing cells relative to empty vector controls.  APP undergoes sequential proteolytic processes to generate Ap n or Ap , and as more 4  42  C99 fragments are produced, there should be more substrates for y-secretase to generate AP (Li et al., 2006). We transfected empty vector, N F - K B expression plasmid, or i K B a expression plasmid into SH-SY5Y cells that overexpress Swedish mutant APP. The concentration of Ap was measured by colorimetric ELISA and the analysis showed that Ap was significantly 40  increased in cells transfected with N F - K B p65 expression plasmid compared to cells transfected with empty vector; therefore, N F - K B p65 may alter amyloidogenic APP processing by regulating BACEl gene transcription to cause elevated AP generation.  93  C h a p t e r I V . G e n e r a l Discussion 4.1. Regulation of BACEl  Gene T r a n s c r i p t i o n b y N F - K B  AD, the most common neurodegenerative disease leading to dementia, is pathologically characterized by Ap deposition in the brain. Ap is generated by sequential cleavages of APP by P- and y-secretases. Though homologous BACEl and BACE2 are transmembrane aspartic proteases, BACEl exerts P-secretase activity by cleaving APP at the N-terminus of the AP domain and BACE2 process APP by cleaving within the AP domain (Hussain et al., 1999; Sinha et al., 1999; Sun et al., 2006b; Yanet al., 1999; Yan et al., 2001). Despite the detection of APP protein in both neuronal and non-neuronal cells, higher APP mRNA levels and more efficient APP metabolism have been examined in neurons (LeBlanc et al., 1991; Zhao et al., 1996). Comparing to non-neuronal cells, BACEl promoter activity and BACEl enzymatic activity are higher in human neurons and contribute to enhanced P-secretase cleavage of APP and AP generation (Cai et al., 2001; LeBlanc et al, 1997; Yan et al., 1999). Since elevated BACEl activity has been described in AD pathogenesis, the regulation of BACEl gene expression is critical to AP generation (Li et al., 2006).  BACEl gene expression has been reported to be regulated at the transcriptional level by several transcription factors, and at the translational level by post-transcriptional mechanisms (Christensen et al., 2004; Zhou and Song, 2006). In our previous studies, we have characterized the human BACEl promoter region and determined that Spl is essential for BACEl promoter activity (Christensen et al., 2004). The study also showed that by elevating BACEl transcription, Spl increases BACEl activity in APP processing and enhances Ap production. In addition to Spl binding element, other transcription factor binding elements have also been described in the promoter region of human BACEl  (Ge et al., 2004).  94  In this study, we show, for the first time, that N F - K B physically interacts with the human  promoter region and regulates BACEl  BACEl  gene expression at the transcriptional  level. According to the consensus binding sequence of N F - K B , we have identified four putative NF-KB  NF-KB  p65.  binding elements on the human BACEl  NF-KB  promoter, two of which interact with  p65 overexpression elicits a functional interaction with these  NF-KB  binding elements and results in elevated promoter activity in RelA-KO cells. The presence of NF-KB  p65 significantly increases BACEl  promoter activity and after mutating these  binding elements, diminishes the increase in BACEl elements on the BACEl  NF-KB  promoter activity; hence, N F - K B binding  promoter are able to regulate  BACEl  gene transcription.  Dimeric N F - K B complex is composed of five distinct subunits and all subunits share the RHD at their N-termini (Siebenlist et al., 1994). Since RelA (p65), RelB and c-Rel all have a transactivating domain at their C-termini, p65/p65, RelB/RelB and c-Rel/c-Rel homodimers are capable of gene transcription activation (Malek et al., 1998; Ruben et al., 1992). Although the predominantly active form of N F - K B is mostly p65/p50 heterodimeric, p5G7p50, p52/p52 and p65/p65 homodimers are also involved in gene regulation (Ballard et al., 1992; Ghosh and Karin, 2002; Urban et al., 1991). Homodimeric p50 complexes have been observed to inhibit IL-2 promoter activity in T cells, whereas p65/p65 homodimers bind to the CD28 response element to activate IL-2 promoter expression in T cells (Kang et al., 1992; Lai et al., 1995). In addition to the results from promoter assays, semi-quantitative RT-PCR analysis reveals that NF-KB  BACEl  p65 upregulates human BACEl mRNA levels; thus, N F - K B p65 activates human gene expression.  95  Our results have shown that overexpression of N F - K B p65 can increase BACEl gene transcription and elevate AP production. Since we identified the dimeric N F - K B in our study was composed of N F - K B p65 by Super-shift assay, we should investigate whether N F - K B p65 homodimers or N F - K B p65-containing heterodimers are responsible for BACEl gene regulation. We could use antibodies of other N F - K B subunits in Super-shift assays to define the identity of dimeric N F - K B composition. Different combinations of N F - K B subunits have been described to express specific transcriptional activation of NF-KB-dependent genes (Lin et al., 1995; Perkins et al., 1992); therefore, by clarifying the composition of dimeric N F - K B , we could define the precise role of N F - K B activation in amyloidogenic APP processing in AD.  Since H2O2 is able to induce N F - K B activation and has been reported to increase BACEl gene transcription (Schreck et al., 1991; Takuma et al., 1999; Tong et al., 2005), we could include H2O2 and the N F - K B inhibitor pyrrolidinedithiocarbamate to modulate N F - K B activation and determine whether a similar activating effect of N F - K B on human BACEl promoter activity can be observed. In addition, we should investigate the identity of N F - K B dimers activated by H 0 ; thus, these findings could provide insights into the strategies to 2  2  suppress N F - K B activity induced by oxidative stress in AD.  4.2. T h e R o l e of N F - K B A c t i v i t y i n A D NF-KB  activity can be induced by various stimuli, such as T N F , IL-1 and glutamate  and, particularly,  Ap and secreted forms of APP were reported to activate N F - K B in neuronal  and glial cells in AD (Barger et al., 1995; Behl et al., 1994b; Guo et al., 1998). Increased N F K B activity has been detected in neurons and astrocytes in the vicinity of AP plaques in AD brains, and compared to the particulate fractions of temporal cortex from control brain, those  96  from AD brains showed augmented N F - K B p65 protein levels (Kitamura et al., 1997; Terai et al., 1996). AD is a chronic neurodegenerative disease and a low level of oxidative stress induced by AP may depend on transcription factors to exert neurotoxic effects. Neurotoxic Ap has been found to induce H2O2 generation and the regulation of N F - K B dependent gene transcription may mediate the neurodegenerative effect attributed by Ap toxicity in AD (Behl et al., 1994b; Yankner et al., 1990). By inducing N F - K B activity, reactive oxygen intermediates can activate gene expression of IL-1, IL-6 and inducible NO synthase to provoke neurodegenerative damage (Campbell et al., 1993; Minc-Golomb et al., 1994). Anti-apoptotic Bcl-2 overexpression is suggested to prevent AP-induced cell death in neurons, and this protective effect is correlated with the inhibition of AP-stimulated N F - K B activation (Saille et al., 1999; Song et al., 2004; Vinet et al., 2002). Some NSAIDs have also been demonstrated to have therapeutic potentials in AD treatment by altering amyloidogenic APP processing and reducing AP generation (Blasko et al., 2001; Sastre et al., 2006; Weggen et al., 2001; Yan et al., 2003). We have previously demonstrated that H2O2 is able to upregulate BACEl gene transcription and increase Ap generation (Tong et al., 2005).  NF-KB  p65 overexpression in  our current study significantly increases BACEl mRNA and facilitates C99 and Ap production. Due to the vital role of BACEl in AP generation in the pathological progression of AD (Li et al., 2006), inhibition of N F - K B signaling could be a valid therapeutic AD treatment.  As  NF-KB  activity induced by neurotoxic Ap can increase AP generation, a vicious  positive-feedback loop may be implicated in the enhanced AP burden observed in AD pathogenesis. In addition to the involvement of N F - K B signaling in regulating BACEl gene transcription, N F - K B p50 has also been reported to increase APP gene expression and this NF-  97  K B binding activity can be enhanced by IL-lp and glutamate (Grilli et al., 1996; Grilli et al., 1995). Although Ap and sAPP are likely candidates for stimulating N F - K B activity in neurons, we should investigate the signals which are responsible for increased N F - K B activity in AD. So the molecular mechanism by which N F - K B activation elevates Ap generation could be elucidated.  4.3. C o n c l u d i n g R e m a r k s  In this study, we have identified functional N F - K B binding elements on the human BACEl promoter. Our results reveal that N F - K B p65 significantly upregulates BACEl gene transcription through physical interaction with N F - K B binding elements on the BACEl promoter, and when the binding elements are mutated, the ability of N F - K B p65 to enhance BACEl promoter activity is diminished.  NF-KB  p65 not only elevates BACEl mRNA levels,  but also increases C99 and Ap o generation; hence, 4  NF-KB  may have a regulatory role in  BACEl gene transcription and Ap production during the development of AD pathogenesis.  98  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. Agar, J., and Durham, H. (2003). Relevance of oxidative injury in the pathogenesis of motor neuron diseases. Amyotroph Lateral Scler Other Motor Neuron Disord 4, 232-242. Aisen, P. S. (2002). Evaluation of selective COX-2 inhibitors for the treatment of Alzheimer's disease. J Pain Symptom Manage 23, S35-40. 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. Akama, K. T., and Van Eldik, L. J. (2000). Beta-amyloid stimulation of inducible nitric-oxide synthase in astrocytes is interleukin-lbeta- and tumor necrosis factor-alpha (TNFalpha)dependent, and involves a TNFalpha receptor-associated factor- and NFkappaB-inducing kinase-dependent signaling mechanism. J Biol Chem 275, 7918-7924. Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G. M., Cooper, N. R., Eikelenboom, P., Emmerling, M., Fiebich, B. L., et al. (2000). Inflammation and Alzheimer's disease. Neurobiol Aging 21, 383-421. Albensi, B. C , and Mattson, M. P. (2000). Evidence for the involvement of TNF and NFkappaB in hippocampal synaptic plasticity. Synapse 35, 151-159. Alkalay, I., Yaron, A., Hatzubai, A., Orian, A., Ciechanover, A., and Ben-Neriah, Y. (1995). Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A 92, 1059910603. Allinson, T. M., Parkin, E. T., Turner, A. J., and Hooper, N. M. (2003). ADAMs family members as amyloid precursor protein alpha-secretases. J Neurosci Res 74, 342-352. Aloisi, F., Care, A., Borsellino, G., Gallo, P., Rosa, S., Bassani, A., Cabibbo, A., Testa, U., Levi, G., and Peschle, C. (1992). Production of hemolymphopoietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-1 beta and tumor necrosis factor-alpha. J Immunol 149, 2358-2366. Alzheimer, A. (1906). Uber einen eigenartigen schweren ErkrankungsprozeB der Hirnrinde. Neurologisches Centralblatt 23, 1129-1136. Alzheimer, A. (1907). fiber eine eigenartige Erkrankung der Hirnrinde. Allgemeine Zeitschrift fiir Psychiatrie und Psychisch-Gerichtliche Medizin 64, 146-148.  99  Amara, F. M., Junaid, A., Clough, R. R., and Liang, B. (1999). TGF-beta(l), regulation of alzheimer amyloid precursor protein mRNA expression in a normal human astrocyte cell line: mRNA stabilization. Brain Res Mol Brain Res 71, 42-49. Anandatheerthavarada, H. K., Biswas, G., Robin, M. A., and Avadhani, N. G. (2003). Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells. J Cell Biol 161, 41-54. Anest, V., Hanson, J. L., Cogswell, P. C , Steinbrecher, K. A., Strahl, B. D., and Baldwin, A. S. (2003). A nucleosomal function for IkappaB kinase-alpha in NF-kappaB-dependent gene expression. Nature 423, 659-663. Anrather, J., Racchumi, G., and Iadecola, C. (2005). cis-acting, element-specific transcriptional activity of differentially phosphorylated nuclear factor-kappa B. J Biol Chem 280, 244-252. Antel, J. P., Becher, B., and Owens, T. (1996). Immunotherapy for multiple sclerosis: from theory to practice. Nat Med 2, 1074-1075. Ard, M. D., Cole, G. M., Wei, J., Mehrle, A. P., and Fratkin, J. D. (1996). Scavenging of Alzheimer's amyloid beta-protein by microglia in culture. J Neurosci Res 43, 190-202. Arenzana-Seisdedos, F., Turpin, P., Rodriguez, M., Thomas, D., Hay, R. T., Virelizier, J. L., and Dargemont, C. (1997). Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B from the nucleus to the cytoplasm. J Cell Sci 110 (Pt 3), 369-378. Asai, M., Hattori, C , Iwata, N., Saido, T. C , Sasagawa, N., Szabo, B., Hashimoto, Y., Maruyama, K., Tanuma, S., Kiso, Y., and Ishiura, S. (2006). The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice. J Neurochem 96, 533-540. Asai, M., Hattori, C , Szabo, B., Sasagawa, N., Maruyama, K., Tanuma, S., and Ishiura, S. (2003). Putative function of ADAM9, ADAM 10, and ADAM 17 as APP alpha-secretase. Biochem Biophys Res Commun 301, 231-235. Avila, J. (2006). Tau phosphorylation and aggregation in Alzheimer's disease pathology. FEBS Lett 580, 2922-2927. Baeuerle, P. A. (1998). IkappaB-NF-kappaB structures: at the interface of inflammation control. Cell 95, 729-731. Baeuerle, P. A., and Baichwal, V. R. (1997). NF-kappa B as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv Immunol 65, 111-137. Baeuerle, P. A., and Baltimore, D. (1989). A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB. Genes Dev 3, 1689-1698. Baeuerle, P. A., and Baltimore, D. (1996). NF-kappa B: ten years after. Cell 87, 13-20.  100  Baeuerle, P. A., and Henkel, T. (1994). Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 12, 141-179. Baker, M , Graff-Radford, D., Wavrant DeVrieze, F., Graff-Radford, N., Petersen, R. C., Kokmen, E., Boeve, B., Myllykangas, L., Polvikoski, T., Sulkava, R., et al. (2000). No association between TAU haplotype and Alzheimer's disease in population or clinic based series or in familial disease. Neurosci Lett 285, 147-149. Baldwin, A. S., Jr. (1996). The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol 14, 649-683. Bales, K. R., Verina, T., Dodel, R. C., Du, Y., Altstiel, L., Bender, M., Hyslop, P., Johnstone, E. M., Little, S. P., Cummins, D. J., et al. (1997). Lack of apolipoprotein E dramatically reduces amyloid beta-peptide deposition. Nat Genet 17, 263-264. Ballard, D. W., Dixon, E. P., Peffer, N. J., Bogerd, H., Doerre, S., Stein, B., and Greene, W. C. (1992). The 65-kDa subunit of human NF-kappa B functions as a potent transcriptional activator and a target for v-Rel-mediated repression. Proc Natl Acad Sci U S A 89, 1875-1879. Bard, F., Barbour, R., Cannon, C , Carretto, R., Fox, M., Games, D., Guido, T., Hoenow, K., Hu, K., Johnson-Wood, K., et al. (2003). Epitope and isotype specificities of antibodies to beta -amyloid peptide for protection against Alzheimer's disease-like neuropathology. Proc Natl Acad Sci U S A 100, 2023-2028. Bard, F., Cannon, C , Barbour, R., Burke, R. L., Games, D., Grajeda, H., 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. Barger, S. W., and Harmon, A. D. (1997). Microglial activation by Alzheimer amyloid precursor protein and modulation by apolipoprotein E. Nature 388, 878-881. Barger, S. W., Horster, D., Furukawa, K., Goodman, Y., Krieglstein, J., and Mattson, M. P. (1995). Tumor necrosis factors alpha and beta protect neurons against amyloid beta-peptide toxicity: evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation. Proc Natl Acad Sci U S A 92, 9328-9332. Barger, S. W., and Mattson, M. P. (1996). Induction of neuroprotective kappa B-dependent transcription by secreted forms of the Alzheimer's beta-amyloid precursor. Brain Res Mol Brain Res 40, 116-126. Bartus, R. T., Dean, R. L., 3rd, Beer, B., and Lippa, A. S. (1982). The cholinergic hypothesis of geriatric memory dysfunction. Science 217, 408-414. 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.  101  Bastianetto, S., and Quirion, R. (2002). EGb 761 is a neuroprotective agent against betaamyloid toxicity. Cell Mol Biol (Noisy-le-grand) 48, 693-697. Bastianetto, S., Ramassamy, C , Dore, S., Christen, Y., Poirier, J., and Quirion, R. (2000). The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by beta-amyloid. Eur J Neurosci 12, 1882-1890. Baumann, H., and Gauldie, J. (1994). The acute phase response. Immunol Today 15, 74-80. Beckman, J. S., Chen, J., Crow, J. P., and Ye, Y. Z. (1994). Reactions of nitric oxide, superoxide and peroxynitrite with superoxide dismutase in neurodegeneration. Prog Brain Res 103, 371-380. Behl, C. (1999). Alzheimer's disease and oxidative stress: implications for novel therapeutic approaches. Prog Neurobiol 57, 301-323. Behl, C , Davis, J., Cole, G. M., and Schubert, D. (1992). Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochem Biophys Res Commun 186, 944-950. Behl, C , Davis, J. B., Klier, F. G., and Schubert, D. (1994a). Amyloid beta peptide induces necrosis rather than apoptosis. Brain Res 645, 253-264. Behl, C , Davis, J. B., Lesley, R., and Schubert, D. (1994b). Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77, 817-827. 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 betasecretase (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. Benzing, W. C , Wujek, J. R., Ward, E. K., Shaffer, D., Ashe, K. H., Younkin, S. G., and Brunden, K. R. (1999). Evidence for glial-mediated inflammation in aged APP(SW) transgenic mice. Neurobiol Aging 20, 581-589. Berezovska, O., Ramdya, P., Skoch, J., Wolfe, M. S., Bacskai, B. J., and Hyman, B. T. (2003). Amyloid precursor protein associates with a nicastrin-dependent docking site on the presenilin 1-gamma-secretase complex in cells demonstrated by fluorescence lifetime imaging. J Neurosci 23, 4560-4566.  102  Berr, C , Hauw, J. J., Delaere, P., Duyckaerts, C , and Amouyel, P. (1994). Apolipoprotein E allele epsilon 4 is linked to increased deposition of the amyloid beta-peptide (A-beta) in cases with or without Alzheimer's disease. Neurosci Lett 178, 221-224. Betts, J. C , and Nabel, G. J. (1996). Differential regulation of NF-kappaB2(pl00) processing and control by amino-terminal sequences. Mol Cell Biol 16, 6363-6371. Biswas, G., Anandatheerthavarada, H. K., Zaidi, M., and Avadhani, N. G. (2003). Mitochondria to nucleus stress signaling: a distinctive mechanism of NFkappaB/Rel activation through calcineurin-mediated inactivation of IkappaBbeta. J Cell Biol 161, 507-519. Blasko, I., Apochal, A., Boeck, G., Hartmann, T., Grubeck-Loebenstein, B., and Ransmayr, G. (2001). Ibuprofen decreases cytokine-induced amyloid beta production in neuronal cells. Neurobiol Dis 8, 1094-1101. Blasko, I., Marx, F., Steiner, E., Hartmann, T., and Grubeck-Loebenstein, B. (1999). TNFalpha plus fPNgamma induce the production of Alzheimer beta-amyloid peptides and decrease the secretion of APPs. Faseb J 13, 63-68. Blass, J. P., Sheu, R. K., and Gibson, G. E. (2000). Inherent abnormalities in energy metabolism in Alzheimer disease. Interaction with cerebrovascular compromise. Ann N Y Acad Sci 903, 204-221. Blessed, G., Tomlinson, B. E., and Roth, M. (1968). The association between quantitative measures of dementia and of senile change in the cerebral grey matter of elderly subjects. Br J Psychiatry 114, 797-811. 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. Bonizzi, G., and Karin, M. (2004). The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol 25, 280-288. Bowen, D. M., Francis, P. T., Pangalos, M. N., Stephens, P. H., and Procter, A. W. (1992). Treatment strategies for Alzheimer's disease. Lancet 339, 132-133. Boyles, J. K., Pitas, R. E., Wilson, E., Mahley, R. W., and Taylor, J. M. (1985). Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J Clin Invest 76, 1501-1513. 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 TACE. Mol Cell J, 207-216. Brown, M. S., Ye, J., Rawson, R. B., and Goldstein, J. L. (2000). Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell 100, 391-398.  103  Bruce-Keller, A. J., Geddes, J. W., Knapp, P. E., McFall, R. W., Keller, J. N., Holtsberg, F. W., Parthasarathy, S., Steiner, S. M., and Mattson, M. P. (1999). Anti-death properties of TNF against metabolic poisoning: mitochondrial stabilization by MnSOD. J Neuroimmunol 93, 5371. Bu, G., Maksymovitch, E. A., Nerbonne, J. M., and Schwartz, A. L. (1994). Expression and function of the low density lipoprotein receptor-related protein (LRP) in mammalian central neurons. J Biol Chem 269, 18521-18528. Burton, T., Liang, B., Dibrov, A., and Amara, F. (2002). Transcriptional activation and increase in expression of Alzheimer's beta-amyloid precursor protein gene is mediated by TGF-beta in normal human astrocytes. Biochem Biophys Res Commun 295, 702-712. Busciglio, J., Gabuzda, D. FL, Matsudaira, P., and Yankner, B. A. (1993). Generation of betaamyloid in the secretory pathway in neuronal and nonneuronal cells. Proc Natl Acad Sci U S A 90, 2092-2096. Buss, H., Dorrie, A., Schmitz, M. L., Hoffmann, E., Resch, K., and Kracht, M. (2004). Constitutive and interleukin-1-inducible phosphorylation of p65 NF-{kappa}B at serine 536 is mediated by multiple protein kinases including I{kappa}B kinase (IKK)-{alpha}, IKK{beta}, IKK{epsilon}, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor 1131-mediated interleukin-8 transcription. J Biol Chem 279, 55633-55643. 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. (1998). 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'. Cacquevel, M., Lebeurrier, N., Cheenne, S., and Vivien, D. (2004). Cytokines in neuroinflammation and Alzheimer's disease. Curr Drug Targets 5, 529-534. Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D. R., Price, D. L., and Wong, P. C. (2001). BACEl is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci 4, 233-234. Cai, X. D., Golde, T. E., and Younkin, S. G. (1993). Release of excess amyloid beta protein from a mutant amyloid beta protein precursor. Science 259, 514-516. Campbell, I. L., Abraham, C. R., Masliah, E., Kemper, P., Inglis, J. D., Oldstone, M. B., and Mucke, L. (1993). Neurologic disease induced in transgenic mice by cerebral overexpression of interleukin 6. Proc Natl Acad Sci U S A 90, 10061-10065. Canty, T. G., Jr., Boyle, E. M., Jr., Fair, A., Morgan, E. N., Verrier, E. D., and Pohlman, T. H. (1999). Oxidative stress induces NF-kappaB nuclear translocation without degradation of IkappaBalpha. Circulation 100,11361-364.  104  Capell, A., Grunberg, J., Pesold, B., Diehlmann, A., Citron, M., Nixon, R., Beyreuther, K., Selkoe, D. J., and Haass, C. (1998). The proteolytic fragments of the Alzheimer's diseaseassociated presenilin-1 form heterodimers and occur as a 100-150-kDa molecular mass complex. J Biol Chem 273, 3205-3211. Capell, A., Kaether, C , Edbauer, D., Shirotani, K., Merkl, S., Steiner, H., and Haass, C. (2003). Nicastrin interacts with gamma-secretase complex components via the N-terminal part of its transmembrane domain. J Biol Chem 278, 52519-52523. 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. Carrasco, D., Ryseck, R. P., and Bravo, R. (1993). Expression of relB transcripts during lymphoid organ development: specific expression in dendritic antigen-presenting cells. Development 118, 1221-1231. Casley, C. S., Canevari, L., Land, J. M., Clark, J. B., and Sharpe, M. A. (2002). Beta-amyloid inhibits integrated mitochondrial respiration and key enzyme activities. J Neurochem 80, 91100. Caspersen, C , Wang, N., Yao, J., Sosunov, A., Chen, X., Lustbader, J. W., Xu, H. W., Stern, D., McKhann, G., and Yan, S. D. (2005). Mitochondrial Abeta: a potential focal point for neuronal metabolic dysfunction in Alzheimer's disease. Faseb J 19, 2040-2041. Cervantes, S., Gonzalez-Duarte, R., and Marfany, G. (2001). Homodimerization of presenilin N-terminal fragments is affected by mutations linked to Alzheimer's disease. FEBS Lett 505, 81-86. Chantome, A., Pance, A., Gauthier, N., Vandroux, D., Chenu, J., Solary, E., Jeannin, J. F., and Reveneau, S. (2004). Casein kinase II-mediated phosphorylation of NF-kappaB p65 subunit enhances inducible nitric-oxide synthase gene transcription in vivo. J Biol Chem 279, 2395323960. Chapman, J., Wang, N., Treves, T. A., Korczyn, A. D., and Bornstein, N. M. (1998). ACE, MTHFR, factor V Leiden, and APOE polymorphisms in patients with vascular and Alzheimer's dementia. Stroke 29, 1401-1404. 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. Chen, F., Gu, Y., Hasegawa, H., Ruan, X., Arawaka, S., Fraser, P., Westaway, D., Mount, H., and St George-Hyslop, P. (2002). Presenilin 1 mutations activate gamma 42-secretase but reciprocally inhibit epsilon-secretase cleavage of amyloid precursor protein (APP) and S3cleavage of notch. J Biol Chem 277, 36521-36526.  105  Chen, L. F., and Greene, W. C. (2003). Regulation of distinct biological activities of the NFkappaB transcription factor complex by acetylation. J Mol Med 81, 549-557. Chen, Y., Vallee, S., Wu, J., Vu, D., Sondek, J., and Ghosh, G. (2004). Inhibition of NFkappaB activity by IkappaBbeta in association with kappaB-Ras. Mol Cell Biol 24, 3048-3056. Cheng, B., Christakos, S., and Mattson, M. P. (1994). Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis. Neuron 72, 139-153. Cheng, C. Y., Hong, C. J., Liu, H. C , Liu, T. Y., and Tsai, S. J. (2002). Study of the association between Alzheimer's disease and angiotensin-converting enzyme gene polymorphism using DNA from lymphocytes. Eur Neurol 47, 26-29. Chong, Z. Z., Li, F., and Maiese, K. (2005). Stress in the brain: novel cellular mechanisms of injury linked to Alzheimer's disease. Brain Res Brain Res Rev 49, 1-21. Christen, Y. (2000). Oxidative stress and Alzheimer disease. Am J Clin Nutr 71, 621S-629S. Christensen, M. A., Zhou, W., Qing, H., Lehman, A., Philipsen, S., and Song, W. (2004). Transcriptional regulation of BACEl, the beta-amyloid precursor protein beta-secretase, by Spl. Mol Cell Biol 24, 865-874. Chung, H. M., and Struhl, G. (2001). Nicastrin is required for Presenilin-mediated transmembrane cleavage in Drosophila. Nat Cell Biol 3, 1129-1132. 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, 672-674. Combs, C. K., Karlo, J. C , Kao, S. C , and Landreth, G. E. (2001). beta-Amyloid stimulation of microglia and monocytes results in TNFalpha-dependent expression of inducible nitric oxide synthase and neuronal apoptosis. J Neurosci 21, 1179-1188. Corder, E. H., 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. Cotman, C. W., and Anderson, A. J. (1995). A potential role for apoptosis in neurodegeneration and Alzheimer's disease. Mol Neurobiol 10, 19-45. Couzin, J. (2004). Clinical trials. Nail-biting time for trials of COX-2 drugs. Science 306, 1673-1675. Coyle, J. T., and Puttfarcken, P. (1993). Oxidative stress, glutamate, and neurodegenerative disorders. Science 262, 689-695.  106  Cramer, P., and Muller, C. W. (1999). A firm hand on NFkappaB: structures of the IkappaBalpha-NFkappaB complex. Structure 7, Rl-6. 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. Cruts, M., Backhovens, FL, Wang, S. Y., Van Gassen, G., Theuns, J., De Jonghe, C. D., Wehnert, A., De Voecht, J., De Winter, G., Cras, P., and et al. (1995). Molecular genetic analysis of familial early-onset Alzheimer's disease linked to chromosome 14q24.3. Hum Mol Genet 4, 2363-2371. Culmsee, C , Siewe, J., Junker, V., Retiounskaia, M., Schwarz, S., Camandola, S., El-Metainy, S., Behnke, H., Mattson, M. P., and Krieglstein, J. (2003). Reciprocal inhibition of p53 and nuclear factor-kappaB transcriptional activities determines cell survival or death in neurons. J Neurosci 23, 8586-8595. Cupers, P., Bentahir, M., Craessaerts, K., Orlans, I., Vanderstichele, H., Saftig, P., De Strooper, B., and Annaert, W. (2001). The discrepancy between presenilin subcellular localization and gamma-secretase processing of amyloid precursor protein. J Cell Biol 154, 731-7'40. Daw, E. W., Payami, H., Nemens, E. J., Nochlin, D., Bird, T. D., Schellenberg, G. D., and Wijsman, E. M. (2000). The number of trait loci in late-onset Alzheimer disease. Am J Hum Genet 66, 196-204. de Caestecker, M. P., Piek, E., and Roberts, A. B. (2000). Role of transforming growth factorbeta signaling in cancer. J Natl Cancer Inst 92, 1388-1402. De Pietri Tonelli, D., Mihailovich, M., Di Cesare, A., Codazzi, F., Grohovaz, F., and Zacchetti, D. (2004). Translational regulation of BACE-1 expression in neuronal and non-neuronal cells. Nucleic Acids Res 32, 1808-1817. De Servi, B., La Porta, C. A., Bontempelli, M., and Comolli, R. (2002). Decrease of TGFbetal plasma levels and increase of nitric oxide synthase activity in leukocytes as potential biomarkers of Alzheimer's disease. Exp Gerontol 37, 813-821. De Strooper, B. (2003). Aph-1, Pen-2, and Nicastrin with Presenilin generate an active gammaSecretase complex. Neuron 38, 9-12. De Strooper, B. (2007). Loss-of-function presenilin mutations in Alzheimer disease. Talking Point on the role of presenilin mutations in Alzheimer disease. EMBO Rep 8, 141-146. De Strooper, B., and Annaert, W. (2000). Proteolytic processing and cell biological functions of the amyloid precursor protein. J Cell Sci 113 (Pt 11), 1857-1870.  107  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 gammasecretase-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. delaTorre, A., Schroeder, R. A., Punzalan, C , and Kuo, P. C. (1999). Endotoxin-mediated Snitrosylation of p50 alters NF-kappa B-dependent gene transcription in ANA-1 murine macrophages. J Immunol 162, 4101-4108. Denk, A., Wirth, T., and Baumann, B. (2000). NF-kappaB transcription factors: critical regulators of hematopoiesis and neuronal survival. Cytokine Growth Factor Rev 11, 303-320. Derynck, R., Zhang, Y., and Feng, X. H. (1998). Smads: transcriptional activators of TGF-beta responses. Cell 95, 737-740. Dickson, D. W. (1997). The pathogenesis of senile plaques. J Neuropathol Exp Neurol 56, 321339. 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. Dykens, J. A., Moos, W. H., and Howell, N. (2005). Development of 17alpha-estradiol as a neuroprotective therapeutic agent: rationale and results from a phase I clinical study. Ann N Y Acad Sci 1052, 116-135. Edbauer, D., Winkler, E., Haass, C , and Steiner, H. (2002). 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 BACEl in human brain and pancreas. Biochem Biophys Res Commun 293, 30-37. Elkins, J. S., Douglas, V. C , and Johnston, S. C. (2004). Alzheimer disease risk and genetic variation in ACE: a meta-analysis. Neurology 62, 363-368. Eriksson, G., Zetterstrom, M., Cortes Toro, V., Bartfai, T., and Iverfeldt, K. (1998). Hypersensitive cytokine response to beta-amyloid 25-35 in astroglial cells from IL-1 receptor type I-deficient mice. Int J Mol Med 1, 201-206.  108  Esch, F. S., Keim, P. S., Beattie, E. C , Blacher, R. W., Culwell, A. R., Oltersdorf, T., McClure, D., and Ward, P. J. (1990). Cleavage of amyloid beta peptide during constitutive processing of its precursor. Science 248, 1122-1124. Esler, W. P., Kimberly, W. T., Ostaszewski, B. L., Diehl, T. S., Moore, C. L., Tsai, J. Y., Rahmati, T., Xia, W., Selkoe, D. J., and Wolfe, M. S. (2000). Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1. Nat Cell Biol 2, 428-434. Eto, A., Muta, T., Yamazaki, S., and Takeshige, K. (2003). Essential roles for NF-kappa B and a Toll/IL-1 receptor domain-specific signal(s) in the induction of I kappa B-zeta. Biochem Biophys Res Commun 301, 495-501. Evans, D. A., Funkenstein, H. Ff., Albert, M. S., Scherr, P. A., Cook, N. R., Chown, M. J., Hebert, L. E., Hennekens, C. H., and Taylor, J. O. (1989a). Prevalence of Alzheimer's disease in a community population of older persons. Higher than previously reported. Jama 262, 25512556. Evans, J. P., Schultz, R. M., and Kopf, G. S. (1998). Roles of the disintegrin domains of mouse fertilins alpha and beta in fertilization. Biol Reprod 59, 145-152. Evans, P. H., Klinowski, J., Yano, E., and Urano, N. (1989b). Alzheimer's disease: a pathogenic role for aluminosilicate-induced phagocytic free radicals. Free Radic Res Commun 6, 317-321. Fan, C , Li, Q., Ross, D., and Engelhardt, J. F. (2003). Tyrosine phosphorylation of I kappa B alpha activates NF kappa B through a redox-regulated and c-Src-dependent mechanism following hypoxia/reoxygenation. J Biol Chem 278, 2072-2080. 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 275, 13491356. Farrer, L. A., O'Sullivan, D. M., Cupples, L. A., Growdon, J. H., and Myers, R. H. (1989). Assessment of genetic risk for Alzheimer's disease amongfirst-degreerelatives. Ann Neurol 25,485-493. Farrer, L. A., Sherbatich, T., Keryanov, S. A., Korovaitseva, G. I., Rogaeva, E. A., Petruk, S., Premkumar, S., Moliaka, Y., Song, Y. Q., Pei, Y., et al. (2000). Association between angiotensin-converting enzyme and Alzheimer disease. Arch Neurol 57, 210-214. 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 amyloidbeta precursor protein. Proc Natl Acad Sci U S A 97, 9712-9717.  109  Fassbender, K., Simons, M., Bergmann, C , Stroick, M., Lutjohann, D., Keller, P., Runz, FL, Kuhl, S., Bertsch, T., von Bergmann, K., et al. (2001). Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo. Proc Natl Acad Sci U S A 98, 5856-5861. Feng, R., Wang, H., Wang, J., Shrom, D., Zeng, X., and Tsien, J. Z. (2004). Forebrain degeneration and ventricle enlargement caused by double knockout of Alzheimer's presenilin-1 and presenilin-2. Proc Natl Acad Sci U S A 101, 8162-8167. Fetler, L., and Amigorena, S. (2005). Neuroscience. Brain under surveillance: the microglia patrol. Science 309, 392-393. Fiala, M., Zhang, L., Gan, X., Sherry, B., Taub, D., Graves, M. C , Hama, S., Way, D., Weinand, M., Witte, M., et al. (1998). Amyloid-beta induces chemokine secretion and monocyte migration across a human blood—brain barrier model. Mol Med 4, 480-489. Fillit, H., Ding, W. H., Buee, L., Kalman, J., Altstiel, L., Lawlor, B., and Wolf-Klein, G. (1991). Elevated circulating tumor necrosis factor levels in Alzheimer's disease. Neurosci Lett 729,318-320. 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. Fortini, M. E. (2002). Gamma-secretase-mediated proteolysis in cell-surface-receptor signalling. Nat Rev Mol Cell Biol 3, 673-684. Frackowiak, J., Wisniewski, H. M., Wegiel, J., Merz, G. S., Iqbal, K., and Wang, K. C. (1992). Ultrastructure of the microglia that phagocytose amyloid and the microglia that produce betaamyloid fibrils. Acta Neuropathol (Berl) 84, 225-233. 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. Frautschy, S. A., Cole, G. M., and Baird, A. (1992). Phagocytosis and deposition of vascular beta-amyloid in rat brains injected with Alzheimer beta-amyloid. Am J Pathol 140, 1389-1399. Frautschy, S. A., Yang, F., Calderon, L., and Cole, G. M. (1996). Rodent models of Alzheimer's disease: rat A beta infusion approaches to amyloid deposits. Neurobiol Aging 17, 311-321. 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.  110  Fukumoto, FL, 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 764,719-725. Furia, B., Deng, L., Wu, K., Baylor, S., Kehn, K., Li, H., Donnelly, R., Coleman, T., and Kashanchi, F. (2002). Enhancement of nuclear factor-kappa B acetylation by coactivator p300 and HIV-1 Tat proteins. J Biol Chem 277, 4973-4980. Ganguli, M., Dodge, H. FL, Chen, P., Belle, S., and DeKosky, S. T. (2000). Ten-year incidence of dementia in a rural elderly US community population: the MoVIES Project. Neurology 54, 1109-1116. Gao, Z., Chiao, P., Zhang, X., Zhang, X., Lazar, M. A., Seto, E., Young, H. A., and Ye, J. (2005). Coactivators and corepressors of NF-kappaB in IkappaB alpha gene promoter. J Biol Chem 250,21091-21098. Gapuzan, M. E., Schmah, O., Pollock, A. D., Hoffmann, A., and Gilmore, T. D. (2005). Immortalized fibroblasts from NF-kappaB RelA knockout mice show phenotypic heterogeneity and maintain increased sensitivity to tumor necrosis factor alpha after transformation by v-Ras. Oncogene 24, 6574-6583. Ge, Y. W., Maloney, B., Sambamurti, K., and Lahiri, D. K. (2004). Functional characterization of the 5' flanking region of the BACE gene: identification of a 91 bp fragment involved in basal level of BACE promoter expression. Faseb J 75, 1037-1039. Gelinas, D. S., DaSilva, K., Fenili, D., St George-Hyslop, P., and McLaurin, J. (2004). Immunotherapy for Alzheimer's disease. Proc Natl Acad Sci U S A 101 Suppl 2, 14657-14662. Ghosh, S., and Karin, M. (2002). Missing pieces in the NF-kappaB puzzle. Cell 109 Suppl, S81-96. Ghosh, S., May, M. J., and Kopp, E. B. (1998). NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16, 225-260. Gingras, A. C , Raught, B., and Sonenberg, N. (2001). Regulation of translation initiation by FRAP/mTOR. Genes Dev 15, 807-826. Glabinski, A. R., and Ransohoff, R. M. (1999). Chemokines and chemokine receptors in CNS pathology. J Neurovirol 5, 3-12. 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. Goate, A. (1994). The future of Alzheimer's disease research: a molecular genetic perspective. Neurobiol Aging 15 Suppl 2, S161-164.  ill  Goate, A. (2006). Segregation of a missense mutation in the amyloid beta-protein precursor gene with familial Alzheimer's disease. J Alzheimers Dis 9, 341-347. 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. Goedert, M., and Spillantini, M. G. (2006). A century of Alzheimer's disease. Science 314, 777-781. Goldgaber, D., Lerman, M. I., McBride, W. O., Saffiotti, U., and Gajdusek, D. C. (1987). Isolation, characterization, and chromosomal localization of human brain cDNA clones coding for the precursor of the amyloid of brain in Alzheimer's disease, Down's syndrome and aging. J Neural Transm Suppl 24, 23-28. Gong, G., Waris, G., Tanveer, R., and Siddiqui, A. (2001). Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-kappa B. Proc Natl Acad Sci U S A 98, 9599-9604. Goodman, Y., Steiner, M. R., Steiner, S. M., and Mattson, M. P. (1994). Nordihydroguaiaretic acid protects hippocampal neurons against amyloid beta-peptide toxicity, and attenuates free radical and calcium accumulation. Brain Res 654, 171-176. 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. Graeve, L., Baumann, M., and Heinrich, P. C. (1993). Interleukin-6 in autoimmune disease. Role of IL-6 in physiology and pathology of the immune defense. Clin Investig 71, 664-671. Gray, C. W., and Patel, A. J. (1993). Induction of beta-amyloid precursor protein isoform mRNAs by bFGF in astrocytes. Neuroreport 4, 811-814. Greenwald, I. (1998). LIN-12/Notch signaling: lessons from worms and flies. Genes Dev 12, 1751-1762. Griffin, W. S., and Mrak, R. E. (2002). Interleukin-1 in the genesis and progression of and risk for development of neuronal degeneration in Alzheimer's disease. J Leukoc Biol 72, 233-238. Griffin, W. S., Sheng, J. G., Roberts, G. W., and Mrak, R. E. (1995). Interleukin-1 expression in different plaque types in Alzheimer's disease: significance in plaque evolution. J Neuropathol Exp Neurol 54, 276-281. Griffin, W. S., Sheng, J. G., Royston, M. C , Gentleman, S. M., McKenzie, J. E., Graham, D. I., Roberts, G. W., and Mrak, R. E. (1998). Glial-neuronal interactions in Alzheimer's disease: the potential role of a 'cytokine cycle' in disease progression. Brain Pathol 8, 65-72.  112  Griffin, W. S., Stanley, L. C., Ling, C., White, L., MacLeod, V., Perrot, L. J., White, C. L., 3rd, and Araoz, C. (1989). Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc Natl Acad Sci U S A 86, 7611-7615. Grilli, M., Goffi, F., Memo, M., and Spano, P. (1996). Interleukin-1 beta and glutamate activate the NF-kappaB/Rel binding site from the regulatory region of the amyloid precursor protein gene in primary neuronal cultures. J Biol Chem 271, 15002-15007. Grilli, M., Ribola, M., Alberici, A., Valerio, A., Memo, M., and Spano, P. (1995). Identification and characterization of a kappa B/Rel binding site in the regulatory region of the amyloid precursor protein gene. J Biol Chem 270, 26774-26777. Grundke-Iqbal, I., Iqbal, K., Tung, Y. C , Quinlan, M., Wisniewski, H. M., and Binder, L. I. (1986). Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci U S A 83, 4913-4917. Gu, Y., Misonou, H., Sato, T., Dohmae, N., Takio, K., and lhara, Y. (2001). Distinct intramembrane cleavage of the beta-amyloid precursor protein family resembling gammasecretase-like cleavage of Notch. J Biol Chem 276, 35235-35238. Guo, Q., Robinson, N., and Mattson, M. P. (1998). Secreted beta-amyloid precursor protein counteracts the proapoptotic action of mutant presenilin-1 by activation of NF-kappaB and stabilization of calcium homeostasis. J Biol Chem 273, 12341-12351. Gustin, J. A., Maehama, T., Dixon, J. E., and Donner, D. B. (2001). The PTEN tumor suppressor protein inhibits tumor necrosis factor-induced nuclear factor kappa B activity. J Biol Chem 276, 21140-21144. Gutierrez, J., Ballinger, S. W., Darley-Usmar, V. M., and Landar, A. (2006). Free radicals, mitochondria, and oxidized lipids: the emerging role in signal transduction in vascular cells. Circ Res 99, 924-932. Haass, C , Hung, A. Y., Schlossmacher, M. G., Oltersdorf, T., Teplow, D. B., and Selkoe, D. J. (1993a). Normal cellular processing of the beta-amyloid precursor protein results in the secretion of the amyloid beta peptide and related molecules. Ann N Y Acad Sci 695, 109-116. Haass, C , Hung, A. Y., Schlossmacher, M. G., Teplow, D. B., and Selkoe, D. J. (1993b). betaAmyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms. J Biol Chem 268, 3021-3024. Haass, C , Hung, A. Y., and Selkoe, D. J. (1991). Processing of beta-amyloid precursor protein in microglia and astrocytes favors an internal localization over constitutive secretion. J Neurosci 11, 3783-3793. Haass, C , Hung, A. Y., Selkoe, D. J., and Teplow, D. B. (1994). Mutations associated with a locus for familial Alzheimer's disease result in alternative processing of amyloid beta-protein precursor. J Biol Chem 269, 17741-17748.  113  Haass, C , Koo, E. H., Mellon, A., Hung, A. Y., and Selkoe, D. J. (1992a). Targeting of cellsurface beta-amyloid precursor protein to lysosomes: alternative processing into amyloidbearing fragments. Nature 357, 500-503. Haass, C , Lemere, C. A., Capell, A., Citron, M., Seubert, P., Schenk, D., Lannfelt, L., and Selkoe, D. J. (1995). The Swedish mutation causes early-onset Alzheimer's disease by betasecretase cleavage within the secretory pathway. Nat Med 1, 1291-1296. Haass, C , Schlossmacher, M. G., Hung, A. Y., Vigo-Pelfrey, C , Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E. H., Schenk, D., Teplow, D. B., and et al. (1992b). Amyloid betapeptide is produced by cultured cells during normal metabolism. Nature 359, 322-325. Haass, C , and Selkoe, D. J. (1993). Cellular processing of beta-amyloid precursor protein and the genesis of amyloid beta-peptide. Cell 75, 1039-1042. Haass, C , and Steiner, H. (2002). Alzheimer disease gamma-secretase: a complex story of GxGD-type presenilin proteases. Trends Cell Biol 12, 556-562. Halliwell, B., Gutteridge, J. M., and Cross, C. E. (1992). Free radicals, antioxidants, and human disease: where are we now? J Lab Clin Med 119, 598-620. Halliwell, B., Zhao, K , and Whiteman, M. (2001). The Gastrointestinal Tract: A Major Site of Antioxidant Action? Free Radic Res 33, 819-830. Halterman, M. W., Miller, C. C , and Federoff, H. J. (1999). Hypoxia-inducible factor-lalpha mediates hypoxia-induced delayed neuronal death that involves p53. J Neurosci 19, 68186824. Hampel, H., Schoen, D., Schwarz, M. J., Kotter, H. U., Schneider, C , Sunderland, T., Dukoff, R., Levy, J., Padberg, F., Stubner, S., et al. (1997). Interleukin-6 is not altered in cerebrospinal fluid of first-degree relatives and patients with Alzheimer's disease. Neurosci Lett 228, 143146. Hampel, H., Sunderland, T., Kotter, H. U., Schneider, C , Teipel, S. J., Padberg, F., Dukoff, R., Levy, J., and Moller, H. J. (1998). Decreased soluble interleukin-6 receptor in cerebrospinal fluid of patients with Alzheimer's disease. Brain Res 780, 356-359. 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. (1992). Framing beta-amyloid. Nat Genet 1, 233-234. 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. Hardy, J. A., and Higgins, G. A. (1992). Alzheimer's disease: the amyloid cascade hypothesis. Science 256, 184-185. 114  Harman, D., Eddy, D. E., and Noffsinger, J. (1976). Free radical theory of aging: inhibition of amyloidosis in mice by antioxidants; possible mechanism. J Am Geriatr Soc 24, 203-210. Harris-White, M. E., Chu, T., Balverde, Z., Sigel, J. J., Flanders, K. C , and Frautschy, S. A. (1998). Effects of transforming growth factor-beta (isoforms 1-3) on amyloid-beta deposition, inflammation, and cell targeting in organotypic hippocampal slice cultures. J Neurosci 18, 10366-10374. Harris, M. E., Hensley, K., Butterfield, D. A., Leedle, R. A., and Carney, J. M. (1995). Direct evidence of oxidative injury produced by the Alzheimer's beta-amyloid peptide (1-40) in cultured hippocampal neurons. Exp Neurol 131, 193-202. Hartlage-Rubsamen, M., Zeitschel, U., Apelt, J., Gartner, U., Franke, H., Stahl, T., Gunther, A., Schliebs, R., Penkowa, M., Bigl, V., and Rossner, S. (2003). Astrocytic expression of the Alzheimer's disease beta-secretase (BACEl) is stimulus-dependent. Glia 41, 169-179. Hartmann, D., de Strooper, B., Serneels, L., Craessaerts, K., Herreman, A., Annaert, W., Umans, L., Lubke, T., Lena Illert, A., von Figura, K., and Saftig, P. (2002). The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alphasecretase activity in fibroblasts. Hum Mol Genet 11, 2615-2624. Hartmann, T. (1999). Intracellular biology of Alzheimer's disease amyloid beta peptide. Eur Arch Psychiatry Clin Neurosci 249, 291-298. Hayden, M. S., and Ghosh, S. (2004). Signaling to NF-kappaB. Genes Dev 18, 2195-2224. Hazel, J. R., and Williams, E. E. (1990). The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment. Prog Lipid Res 29, 167-227. He, G., Qing, H., Cai, F., Kwok, C , Xu, H., Yu, G., Bernstein, A., and Song, W. (2006). Ubiquitin-proteasome pathway mediates degradation of APH-1. J Neurochem 99, 1403-1412. He, G., Qing, H., Tong, Y., Cai, F., Ishiura, S., and Song, W. (2007). Degradation of nicastrin involves both proteasome and lysosome. J Neurochem. Heldin, C. H., Miyazono, K., and ten Dijke, P. (1997). TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465-471. 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. Heneka, M. T., Wiesinger, H., Dumitrescu-Ozimek, L., Riederer, P., Feinstein, D. L., and Klockgether, T. (2001). Neuronal and glial coexpression of argininosuccinate synthetase and inducible nitric oxide synthase in Alzheimer disease. J Neuropathol Exp Neurol 60, 906-916.  115  Hensley, K., Carney, J. M., Mattson, M. P., Aksenova, M., Harris, M., Wu, J. F., Floyd, R. A., and Butterfield, D. A. (1994). A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease. Proc Natl Acad Sci U S A 91, 3270-3274. Herreman, A., Serneels, L., Annaert, W., Collen, D., Schoonjans, L., and De Strooper, B. (2000). Total inactivation of gamma-secretase activity in presenilin-deficient embryonic stem cells. Nat Cell Biol 2, 461-462. Heyser, C. J., Masliah, E., Samimi, A., Campbell, I. L., and Gold, L. H. (1997). Progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin 6 in the brain. Proc Natl Acad Sci U S A 94, 1500-1505. Hibi, M., Murakami, M., Saito, M., Hirano, T., Taga, T., and Kishimoto, T. (1990). Molecular cloning and expression of an IL-6 signal transducer, gpl30. Cell 63, 1149-1157. Hibi, M., Nakajima, K., and Hirano, T. (1996). IL-6 cytokine family and signal transduction: a model of the cytokine system. J Mol Med 74, 1-12. Hirano, T., Nakajima, K., and Hibi, M. (1997). Signaling mechanisms through gpl30: a model of the cytokine system. Cytokine Growth Factor Rev 8, 241-252. Hoffmann, A., Levchenko, A., Scott, M. L., and Baltimore, D. (2002). The IkappaB-NFkappaB signaling module: temporal control and selective gene activation. Science 298, 12411245. Holmlund, L., Cortes Toro, V., and Iverfeldt, K. (2002). Additive effects of amyloid beta fragment and interleukin-lbeta on interleukin-6 secretion in rat primary glial cultures. Int J Mol Med 10, 245-250. 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 51, 783-786. Hopkins, S. J., and Rothwell, N. J. (1995). Cytokines and the nervous system. I: Expression and recognition. Trends Neurosci 18, 83-88. Hotoda, N., Koike, H., Sasagawa, N., and Ishiura, S. (2002). A secreted form of human ADAM9 has an alpha-secretase activity for APP. Biochem Biophys Res Commun 293, 800805. Hou, S., Guan, H., and Ricciardi, R. P. (2003). Phosphorylation of serine 337 of NF-kappaB p50 is critical for DNA binding. J Biol Chem 278, 45994-45998. Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. (1996). TRADD-TRAF2 and TRADDFADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84, 299-308.  116  Hu, J., Igarashi, A., Kamata, M., and Nakagawa, H. (2001). Angiotensin-converting enzyme degrades Alzheimer amyloid beta-peptide (A beta); retards A beta aggregation, deposition, fibril formation; and inhibits cytotoxicity. J Biol Chem 276, 47863-47868. Hu, Y., and Fortini, M. E. (2003). Different cofactor activities in gamma-secretase assembly: evidence for a nicastrin-Aph-1 subcomplex. J Cell Biol 767, 685-690. Hu, Y., Ye, Y., and Fortini, M. E. (2002). Nicastrin is required for gamma-secretase cleavage of the Drosophila Notch receptor. Dev Cell 2, 69-78. Huang, X., Atwood, C. S., Hartshorn, M. A., Multhaup, G., Goldstein, L. E., Scarpa, R. C , Cuajungco, M. P., Gray, D. N., Lim, J., Moir, R. D., et al. (1999). The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry 38, 7609-7616. Hunot, S., Brugg, B., Ricard, D., Michel, P. P., Muriel, M. P., Ruberg, M., Faucheux, B. A., Agid, Y., and Hirsch, E. C. (1997). Nuclear translocation of NF-kappaB is increased in dopaminergic neurons of patients with parkinson disease. Proc Natl Acad Sci U S A 94, 75317536. 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., 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. Hutton, M., Busfield, F., Wragg, M., Crook, R., Perez-Tur, J., Clark, R. F., Prihar, G., Talbot, C , Phillips, H., Wright, K., et al. (1996). Complete analysis of the presenilin 1 gene in early onset Alzheimer's disease. Neuroreport 7, 801-805. Hutton, M., Perez-Tur, J., and Hardy, J. (1998). Genetics of Alzheimer's disease. Essays Biochem35, 117-131. Huxford, T., Huang, D. B., Malek, S., and Ghosh, G. (1998). The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation. Cell 95, 759-770.  117  Inoue, J., Kerr, L. D., Kakizuka, A., and Verma, I. M. (1992). I kappa B gamma, a 70 kd protein identical to the C-terminal half of pi 10 NF-kappa B: a new member of the I kappa B family. Cell 68, 1109-1120. Ip, N. Y., Nye, S. FL, Boulton, T. G., Davis, S., Taga, T., Li, Y., Birren, S. J., Yasukawa, K., Kishimoto, T., Anderson, D. J., and et al. (1992). CNTF and LIF act on neuronal cells via shared signaling pathways that involve the fL-6 signal transducing receptor component gpl30. Cell 69, 1121-1132. Itagaki, S., McGeer, P. L., Akiyama, H., Zhu, S., and Selkoe, D. (1989). Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease. J Neuroimmunol 24, 173182. Ito, T., Morimatsu, M., Oonuma, T., Shiina, T., Kitamura, H., and Syuto, B. (2004). Transcriptional regulation of the MAIL gene in LPS-stimulated RAW264 mouse macrophages. Gene 342, 137-143. Jacobs, M. D., and Harrison, S. C. (1998). Structure of an IkappaBalpha/NF-kappaB complex. Cell 95, 749-758. Jarrett, J. T., and Lansbury, P. T., Jr. (1993). Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell 73, 1055-1058. Jeffrey, M., Goodsir, C. M., Bruce, M. E., McBride, P. A., and Farquhar, C. (1994). Morphogenesis of amyloid plaques in 87V murine scrapie. Neuropathol Appl Neurobiol 20, 535-542. Johnstone, E. M., Babbey, L. E., Stephenson, D., Paul, D. C , Santerre, R. F., Clemens, J. A., Williams, D. C , and Little, S. P. (1996). Nuclear and cytoplasmic localization of the betaamyloid peptide (1-43) in transfected 293 cells. Biochem Biophys Res Commun 220, 710-718. Johnstone, M., Gearing, A. J., and Miller, K. M. (1999). A central role for astrocytes in the inflammatory response to beta-amyloid; chemokines, cytokines and reactive oxygen species are produced. J Neuroimmunol 93, 182-193. Kaether, C , Lammich, S., Edbauer, D., Ertl, M., Rietdorf, J., Capell, A., Steiner, H., and Haass, C. (2002). Presenilin-1 affects trafficking and processing of betaAPP and is targeted in a complex with nicastrin to the plasma membrane. J Cell Biol 158, 551-561. Kalaria, R. N., Cohen, D. L., Premkumar, D. R., Nag, S., LaManna, J. C , and Lust, W. D. (1998). Vascular endothelial growth factor in Alzheimer's disease and experimental cerebral ischemia. Brain Res Mol Brain Res 62, 101-105. Kaltschmidt, B., Uherek, M., Volk, B., Baeuerle, P. A., and Kaltschmidt, C. (1997). Transcription factor NF-kappaB is activated in primary neurons by amyloid beta peptides and in neurons surrounding early plaques from patients with Alzheimer disease. Proc Natl Acad Sci U S A 94, 2642-2647.  118  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. Kang, S. M., Tran, A. C., Grilli, M., and Lenardo, M. J. (1992). NF-kappa B subunit regulation in nontransformed CD4+ T lymphocytes. Science 256, 1452-1456. Karin, M., and Ben-Neriah, Y. (2000). Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol 18, 621-663. Kawakami, K., Scheidereit, C , and Roeder, R. G. (1988). Identification and purification of a human immunoglobulin-enhancer-binding protein (NF-kappa B) that activates transcription from a human immunodeficiency virus type 1 promoter in vitro. Proc Natl Acad Sci U S A 85, 4700-4704. 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. Kehoe, P. G. (2003). The renin-angiotensin-aldosterone system and Alzheimer s disease? J Renin Angiotensin Aldosterone Syst 4, 80-93. Kehoe, P. G., Katzov, H., Andreasen, N., Gatz, M., Wilcock, G. K., Cairns, N. J., Palmgren, J., de Faire, U., Brookes, A. J., Pedersen, N. L., et al. (2004). Common variants of ACE contribute to variable age-at-onset of Alzheimer's disease. Hum Genet 114, 478-483. Kehoe, P. G., Katzov, H., Feuk, L., Bennet, A. M., Johansson, B., Wiman, B., de Faire, U., Cairns, N. J., Wilcock, G. K., Brookes, A. J., et al. (2003). Haplotypes extending across ACE are associated with Alzheimer's disease. Hum Mol Genet 12, 859-867. Keller, J. N., Kindy, M. S., Holtsberg, F. W., St Clair, D. K., Yen, H. C , Germeyer, A., Steiner, S. M., Bruce-Keller, A. J., Hutchins, J. B., and Mattson, M. P. (1998). Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J Neurosci 18, 687-697. Kim, E. S., Kim, R. S., Ren, R. F., Hawver, D. B., and Flanders, K. C. (1998). Transforming growth factor-beta inhibits apoptosis induced by beta-amyloid peptide fragment 25-35 in cultured neuronal cells. Brain Res Mol Brain Res 62, 122-130. Kim, S. H., Ikeuchi, T., Yu, C , and Sisodia, S. S. (2003). Regulated hyperaccumulation of presenilin-1 and the "gamma-secretase" complex. Evidence for differential intramembranous processing of transmembrane subatrates. J Biol Chem 278, 33992-34002. Kimberly, W. T., LaVoie, M. J., Ostaszewski, B. L., Ye, W., Wolfe, M. S., and Selkoe, D. J. (2003). Gamma-secretase is a membrane protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2. Proc Natl Acad Sci U S A 100, 6382-6387.  119  Kimberly, W. T., Xia, W., Rahmati, T., Wolfe, M. S., and Selkoe, D. J. (2000). The transmembrane aspartates in presenilin 1 and 2 are obligatory for gamma-secretase activity and amyloid beta-protein generation. J Biol Chem 275, 3173-3178. Kitamura, H., Kanehira, K., Okita, K., Morimatsu, M., and Saito, M. (2000). MAIL, a novel nuclear I kappa B protein that potentiates LPS-induced IL-6 production. FEBS Lett 485, 53-56. Kitamura, Y., Shimohama, S., Ota, T., Matsuoka, Y., Nomura, Y., and Taniguchi, T. (1997). Alteration of transcription factors NF-kappaB and STAT1 in Alzheimer's disease brains. Neurosci Lett 237, 17-20. Koike, H., Tomioka, S., Sorimachi, H., Saido, T. C , Maruyama, K , Okuyama, A., FujisawaSehara, 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. Kojro, E., and Fahrenholz, F. (2005). The non-amyloidogenic pathway: structure and function of alpha-secretases. Subcell Biochem 38, 105-127. Kojro, E., Gimpl, G., Lammich, S., Marz, W., and Fahrenholz, F. (2001). Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the alpha -secretase ADAM 10. Proc Natl Acad Sci U S A 98, 5815-5820. Komarova, S. V., Pilkington, M. F., Weidema, A. F., Dixon, S. L, and Sims, S. M. (2003). RANK ligand-induced elevation of cytosolic Ca2+ accelerates nuclear translocation of nuclear factor kappa B in osteoclasts. J Biol Chem 278, 8286-8293. Konstam, M. A., and Weir, M. R. (2002). Current perspective on the cardiovascular effects of coxibs. Cleve Clin J Med 69 Suppl 1, SI47-52. Korten, A. E., Jorm, A. F., Henderson, A. S., Broe, G. A., Creasey, H., and McCusker, E. (1993). Assessing the risk of Alzheimer's disease in first-degree relatives of Alzheimer's disease cases. Psychol Med 23, 915-923. 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. Kovacs, D. M., Fausett, H. J., Page, K. J., Kim, T. W., Moir, R. D., Merriam, D. E., Hollister, R. D., Hallmark, O. G., Mancini, R., Felsenstein, K. M., et al. (1996). Alzheimer-associated presenilins 1 and 2: neuronal expression in brain and localization to intracellular membranes in mammalian cells. Nat Med 2, 224-229. Kowalska, A. (2004). Genetic basis of neurodegeneration in familial Alzheimer's disease. Pol J Pharmacol 56, 171-178. Kreutzberg, G. W. (1996). Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19, 312-318. 120  Kunsch, C , Ruben, S. M., and Rosen, C. A. (1992). Selection of optimal kappa B/Rel DNAbinding motifs: interaction of both subunits of NF-kappa B with DNA is required for transcriptional activation. Mol Cell Biol 72, 4412-4421. Kurt, M. A., Davies, D. C , and Kidd, M. (1999). beta-Amyloid immunoreactivity in astrocytes in Alzheimer's disease brain biopsies: an electron microscope study. Exp Neurol 158, 221-228. Lagna, G., Hata, A., Hemmati-Brivanlou, A., and Massague, J. (1996). Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature 383, 832-836. Lai, J. H., Horvath, G., Subleski, J., Bruder, J., Ghosh, P., and Tan, T. H. (1995). RelA is a potent transcriptional activator of the CD28 response element within the interleukin 2 promoter. Mol Cell Biol 75, 4260-4271. 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 Alzheimer's amyloid precursor protein by a disintegrin metalloprotease. Proc Natl Acad Sci U S A 96, 3922-3927. Lammich, S., Schobel, S., Zimmer, A. K., Lichtenthaler, S. F., and Haass, C. (2004). Expression of the Alzheimer protease BACEl is suppressed via its 5'-untranslated region. EMBO Rep 5, 620-625. Lange-Dohna, C , Zeitschel, U., Gaunitz, F., Perez-Polo, J. R., Bigl, V., and Rossner, S. (2003). Cloning and expression of the rat BACEl promoter. J Neurosci Res 73, 73-80. Larner, A. L, and Doran, M. (2006). Clinical phenotypic heterogeneity of Alzheimer's disease associated with mutations of the presenilin-1 gene. J Neurol 253, 139-158. Lavon, I., Goldberg, I., Amit, S., Landsman, L., Jung, S., Tsuberi, B. Z., Barshack, I., Kopolovic, J., Galun, E., Bujard, H., and Ben-Neriah, Y. (2000). High susceptibility to bacterial infection, but no liver dysfunction, in mice compromised for hepatocyte NF-kappaB activation. Nat Med 6, 573-577. LeBlanc, A. C , Chen, H. Y., Autilio-Gambetti, L., and Gambetti, P. (1991). Differential APP gene expression in rat cerebral cortex, meninges, and primary astroglial, microglial and neuronal cultures. FEBS Lett 292, 171-178. LeBlanc, A. C , Papadopoulos, M., Belair, C , Chu, W., Crosato, M., Powell, J., and Goodyer, C. G. (1997). Processing of amyloid precursor protein in human primary neuron and astrocyte cultures. J Neurochem 68, 1183-1190. Lee, M., Bard, F., Johnson-Wood, K., Lee, C , Hu, K., Griffith, S. G., Black, R. S., Schenk, D., and Seubert, P. (2005). Abeta42 immunization in Alzheimer's disease generates Abeta Nterminal antibodies. Ann Neurol 58, 430-435.  121  Leem, J. Y., Vijayan, S., Han, P., Cai, D., Machura, M., Lopes, K. 0., Veselits, M. L., Xu, H., and Thinakaran, G. (2002). Presenilin 1 is required for maturation and cell surface accumulation of nicastrin. J Biol Chem 277, 19236-19240. Leimer, U., Lun, K., Romig, H., Walter, J., Grunberg, J., Brand, M., and Haass, C. (1999). Zebrafish (Danio rerio) presenilin promotes aberrant amyloid beta-peptide production and requires a critical aspartate residue for its function in amyloidogenesis. Biochemistry 38, 13602-13609. Leist, M., Gantner, F., Jilg, S., and Wendel, A. (1995). Activation of the 55 kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release. J Immunol 154, 1307-1316. Lemere, C. A., Lopera, F., Kosik, K. S., Lendon, C. L., Ossa, J., Saido, T. C , Yamaguchi, H., Ruiz, A., Martinez, A., Madrigal, L., et al. (1996). The E280A presenilin 1 Alzheimer mutation produces increased A beta 42 deposition and severe cerebellar pathology. Nat Med 2, 11461150. Lesne, S., Docagne, F., Gabriel, C , Liot, G., Lahiri, D. K., Buee, L., Plawinski, L., Delacourte, A., MacKenzie, E. T., Buisson, A., and Vivien, D. (2003). Transforming growth factor-beta 1 potentiates amyloid-beta generation in astrocytes and in transgenic mice. J Biol Chem 278, 18408-18418. Levitan, D., Lee, J., Song, L., Manning, R., Wong, G., Parker, E., and Zhang, L. (2001). PSI N- and C-terminal fragments form a complex that functions in APP processing and Notch signaling. Proc Natl Acad Sci U S A 98, 12186-12190. 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. (1995). Candidate gene for the chromosome 1 familial Alzheimer's disease locus. Science 269, 973-977. Li, Q., and Verma, I. M. (2002). NF-kappaB regulation in the immune system. Nat Rev Immunol 2, 725-734. Li, R., Lindholm, K , Yang, L. B., Yue, X., Citron, M., Yan, R., Beach, T., Sue, L., Sabbagh, M., Cai, H., et al. (2004). Amyloid beta peptide load is correlated with increased beta-secretase activity in sporadic Alzheimer's disease patients. Proc Natl Acad Sci U S A 101, 3632-3637. 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., Barger, S. W., Liu, L., Mrak, R. E., and Griffin, W. S. (2000a). SlOObeta induction of the proinflammatory cytokine interleukin-6 in neurons. J Neurochem 74, 143-150. Li, Y., Wang, J., Sheng, J. G., Liu, L., Barger, S. W., Jones, R. A., Van Eldik, L. J., Mrak, R. E., and. Griffin, W. S. (1998). S100 beta increases levels of beta-amyloid precursor protein and its encoding mRNA in rat neuronal cultures. J Neurochem 71, 1421-1428. 122  Li, Y., Zhou, W., Tong, Y., He, G., and Song, W. (2006). Control of APP processing and Abeta generation level by BACEl enzymatic activity and transcription. Faseb J 20, 285-292. Li, Y. M., 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. Licastro, F., and Chiappelli, M. (2003). Brain immune responses cognitive decline and dementia: relationship with phenotype expression and genetic background. Mech Ageing Dev 124, 539-548. Lin, L., and Ghosh, S. (1996). A glycine-rich region in NF-kappaB pl05 functions as a processing signal for the generation of the p50 subunit. Mol Cell Biol 16, 2248-2254. Lin, R., Gewert, D., and Hiscott, J. (1995). Differential transcriptional activation in vitro by NF-kappa B/Rel proteins. J Biol Chem 270, 3123-3131. 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. Liu, B., and Hong, J. S. (2003). Role of microglia in inflammation-mediated neurodegenerative diseases: mechanisms and strategies for therapeutic intervention. J Pharmacol Exp Ther 304, 17. Liu, S. F., and Malik, A. B. (2006). NF-kappa B activation as a pathological mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol Physiol 290, L622-L645. Louis, J. C , Magal, E., Takayama, S., and Varon, S. (1993). CNTF protection of oligodendrocytes against natural and tumor necrosis factor-induced death. Science 259, 689692. Luber-Narod, J., and Rogers, J. (1988). Immune system associated antigens expressed by cells of the human central nervous system. Neurosci Lett 94, 17-22. Lucas, S. M., Rothwell, N. J., and Gibson, R. M. (2006). The role of inflammation in CNS injury and disease. Br J Pharmacol 147 Suppl 1, S232-240. Lue, L. F., Rydel, R., Brigham, E. F., Yang, L. B., Hampel, H., Murphy, G. M., Jr., Brachova, L., Yan, S. D., Walker, D. G., Shen, Y., and Rogers, J. (2001). Inflammatory repertoire of Alzheimer's disease and nondemented elderly microglia in vitro. Glia 35, 72-79. Lukiw, W. J., and Bazan, N. G. (1998). Strong nuclear factor-kappaB-DNA binding parallels cyclooxygenase-2 gene transcription in aging and in sporadic Alzheimer's disease superior temporal lobe neocortex. J Neurosci Res 53, 583-592.  123  Luo, W. J., Wang, H., Li, H., Kim, B. S., Shah, S., Lee, H. J., Thinakaran, G., Kim, T. W., Yu, G., and Xu, H. (2003). PEN-2 and APH-1 coordinately regulate proteolytic processing of presenilin 1. J Biol Chem 278, 7850-7854. 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 BACEl, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci 4, 231-232. Lustbader, J. W., Cirilli, M., Lin, C , Xu, H. W., Takuma, K., Wang, N., Caspersen, C , Chen, X., Pollak, S., Chaney, M., et al. (2004). ABAD directly links Abeta to mitochondrial toxicity in Alzheimer's disease. Science 304, 448-452. Luterman, J. D., Haroutunian, V., Yemul, S., Ho, L., Purohit, D., Aisen, P. S., Mohs, R., and Pasinetti, G. M. (2000). Cytokine gene expression as a function of the clinical progression of Alzheimer disease dementia. Arch Neurol 57, 1153-1160. Ma, J., Yee, A., Brewer, H. B., Jr., Das, S., and Potter, H. (1994). Amyloid-associated proteins alpha 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-protein intofilaments.Nature 372, 92-94. Mackenzie, G. G., Zago, M. P., Keen, C. L., and Oteiza, P. I. (2002). Low intracellular zinc impairs the translocation of activated NF-kappa B to the nuclei in human neuroblastoma IMR32 cells. J Biol Chem 277, 34610-34617. Mahley, R. W. (1988). Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 240, 622-630. Mahley, R. W., and Huang, Y. (1999). Apolipoprotein E: from atherosclerosis to Alzheimer's disease and beyond. Curr Opin Lipidol 10, 207-217. Mahley, R. W., and Rail, S. C , Jr. (2000). Apolipoprotein E: far more than a lipid transport protein. Annu Rev Genomics Hum Genet 1, 507-537. Malek, S., Huxford, T., and Ghosh, G. (1998). Ikappa Balpha functions through direct contacts with the nuclear localization signals and the DNA binding sequences of NF-kappaB. J Biol Chem 273, 25427-25435. Mann, D. M., Iwatsubo, T., Cairns, N. J., Lantos, P. L., Nochlin, D., Sumi, S. M., Bird, T. D., Poorkaj, P., Hardy, J., Hutton, M., et al. (1996). Amyloid beta protein (Abeta) deposition in chromosome 14-linked Alzheimer's disease: predominance of Abeta42(43). Ann Neurol 40, 149-156. Marcinkeviciene, J., Luo, Y., Graciani, N. R., Combs, A. P., and Copeland, R. A. (2001). Mechanism of Inhibition of beta-site amyloid precursor protein-cleaving enzyme (BACE) by a statine-based peptide. J Biol Chem 276, 23190-2319A.  124  Marienfeld, R., May, M. J., Berberich, I., Serfling, E., Ghosh, S., and Neumann, M. (2003). RelB forms transcriptionally inactive complexes with RelA/p65. J Biol Chem 278, 1985219860. Markesbery, W. R. (1997). Oxidative stress hypothesis in Alzheimer's disease. Free Radic Biol Med 23, 134-147. Marshak, D. R., Pesce, S. A., Stanley, L. C , and Griffin, W. S. (1992). Increased S100 beta neurotrophic activity in Alzheimer's disease temporal lobe. Neurobiol Aging 13, 1-7. Maruyama, K., Kawamura, Y., Asada, H., Ishiura, S., and Obata, K. (1994). Cleavage at the Nterminal site of Alzheimer amyloid beta/A4 protein is essential for its secretion. Biochem Biophys Res Commun 202, 1517-1523. Marz, P., Cheng, J. G., Gadient, R. A., Patterson, P. H., Stoyan, T., Otten, U., and Rose-John, S. (1998). Sympathetic neurons can produce and respond to interleukin 6. Proc Natl Acad Sci U S A 95, 3251-3256. Massague, J. (1998). TGF-beta signal transduction. Annu Rev Biochem 67, 753-791. Massague, J., and Chen, Y. G. (2000). Controlling TGF-beta signaling. Genes Dev 14, 627644. 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. (1995). Free radicals and disruption of neuronal ion homeostasis in AD: a role for amyloid beta-peptide? Neurobiol Aging 16, 679-682; discussion 683. Mattson, M. P. (2003). Gene-diet interactions in brain aging and neurodegenerative disorders. Ann Intern Med 139, 441-444. Mattson, M. P. (2004). Pathways towards and away from Alzheimer's disease. Nature 430, 631-639. Mattson, M. P., Begley, J. G., Mark, R. J., and Furukawa, K. (1997). Abeta25-35 induces rapid lysis of red blood cells: contrast with Abetal-42 and examination of underlying mechanisms. Brain Res 777, 147-153. Mattson, M. P., and Camandola, S. (2001). NF-kappaB in neuronal plasticity and neurodegenerative disorders. J Clin Invest 107, 247-254. Mayeux, R. (2003). Epidemiology of neurodegeneration. Annu Rev Neurosci 26, 81-104. McCusker, S. M., Curran, M. D., Dynan, K. B., McCullagh, C. D., Urquhart, D. D., Middleton, D., Patterson, C. C , Mcllroy, S. P., and Passmore, A. P. (2001). Association between polymorphism in regulatory region of gene encoding tumour necrosis factor alpha and risk of Alzheimer's disease and vascular dementia: a case-control study. Lancet 357, 436-439. 125  McDonald, D. R., Brunden, K. R., and Landreth, G. E. (1997). Amyloid fibrils activate tyrosine kinase-dependent signaling and superoxide production in microglia. J Neurosci 17, 2284-2294. McGeer, E. G., and McGeer, P. L. (2003). Inflammatory processes in Alzheimer's disease. Prog Neuropsychopharmacol Biol Psychiatry 27, 741-749. McGeer, P. L., and McGeer, E. G. (1995). The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res Brain Res Rev 21, 195-218. McGeer, P. L., and McGeer, E. G. (2001). Polymorphisms in inflammatory genes and the risk of Alzheimer disease. Arch Neurol 58, 1790-1792. McGeer, P. L., and McGeer, E. G. (2006). NSAIDs and Alzheimer disease: Epidemiological, animal model and clinical studies. Neurobiol Aging. McKee, A. C., Kowall, N. W., Schumacher, J. S., and Beal, M. F. (1998). The neurotoxicity of amyloid beta protein in aged primates. Amyloid 5, 1-9. McLellan, M. E., Kajdasz, S. T., Hyman, B. T., and Bacskai, B. J. (2003). In vivo imaging of reactive oxygen species specifically associated with thioflavine S-positive amyloid plaques by multiphoton microscopy. J Neurosci 23, 2212-2217'. Meberg, P. J., Kinney, W. R., Valcourt, E. G., and Routtenberg, A. (1996). Gene expression of the transcription factor NF-kappa B in hippocampus: regulation by synaptic activity. Brain Res Mol Brain Res 38, 179-190. Meda, L., Cassatella, M. A., Szendrei, G. I., Otvos, L., Jr., Baron, P., Villalba, M., Ferrari, D., and Rossi, F. (1995). Activation of microglial cells by beta-amyloid protein and interferongamma. Nature 374, 647-650. Menendez, M. (2004). Pathological and clinical heterogeneity of presenilin 1 gene mutations. J Alzheimers Dis 6, 475-482. Mercurio, F., and Manning, A. M. (1999). Multiple signals converging on NF-kappaB. Curr Opin Cell Biol 11, 226-232. Meredith, S. C (2005). Protein denaturation and aggregation: Cellular responses to denatured and aggregated proteins. Ann N Y Acad Sci 1066, 181-221. Miatto, O., Gonzalez, R. G., Buonanno, F., and Growdon, J. H. (1986). In vitro 31P NMR spectroscopy detects altered phospholipid metabolism in Alzheimer's disease. Can J Neurol Sci 13, 535-539. Michaelis, M. L. (2003). Drugs targeting Alzheimer's disease: some things old and some things new. J Pharmacol Exp Ther 304, 897-904. 126  Minc-Golomb, D., Tsarfaty, I., and Schwartz, J. P. (1994). Expression of inducible nitric oxide synthase by neurones following exposure to endotoxin and cytokine. Br J Pharmacol 112, 720722. Mistry, P., Deacon, K., Mistry, S., Blank, J., and Patel, R. (2004). NF-kappaB promotes survival during mitotic cell cycle arrest. J Biol Chem 279, 1482-1490. Miyamoto, S., and Verma, I. M. (1995). Rel/NF-kappa B/I kappa B story. Adv Cancer Res 66, 255-292. Miyata, M., and Smith, J. D. (1996). Apolipoprotein E allele-specific antioxidant activity and effects on cytotoxicity by oxidative insults and beta-amyloid peptides. Nat Genet 14, 55-61. Moehlmann, T., Winkler, E., Xia, X., Edbauer, D., Murrell, J., Capell, A., Kaether, C , Zheng, H., Ghetti, B., Haass, C , and Steiner, H. (2002). Presenilin-1 mutations of leucine 166 equally affect the generation of the Notch and APP intracellular domains independent of their effect on Abeta 42 production. Proc Natl Acad Sci U S A 99, 8025-8030. Mogi, M., Harada, M., Riederer, P., Narabayashi, H., Fujita, K., and Nagatsu, T. (1994). Tumor necrosis factor-alpha (TNF-alpha) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients. Neurosci Lett if55, 208-210. Monsma, F. J., Jr., Shen, Y., Ward, R. P., Hamblin, M. W., and Sibley, D. R. (1993). Cloning and expression of a novel serotonin receptor with high affinity for tricyclic psychotropic drugs. Mol Pharmacol 43, 320-327. Moore, A. H., and O'Banion, M. K. (2002). Neuroinflammation and anti-inflammatory therapy for Alzheimer's disease. Adv Drug Deliv Rev 54, 1627-1656. Moorthy, A. K., and Ghosh, G. (2003). pl05.Ikappa Bgamma and prototypical Ikappa Bs use a similar mechanism to bind but a different mechanism to regulate the subcellular localization of NF-kappa B. J Biol Chem 278, 556-566. Morgan, J. G., Dolganov, G. M., Robbins, S. E., Hinton, L. M., and Lovett, M. (1992). The selective isolation of novel cDNAs encoded by the regions surrounding the human interleukin 4 and 5 genes. Nucleic Acids Res 20, 5173-5179. Motter, R., Vigo-Pelfrey, C , Kholodenko, D., Barbour, R., Johnson-Wood, K., Galasko, D., Chang, L., Miller, B., Clark, C , Green, R., and et al. (1995). Reduction of beta-amyloid peptide42 in the cerebrospinal fluid of patients with Alzheimer's disease. Ann Neurol 38, 643648. Mrak, R. E., and Griffin, W. S. (2001). Interleukin-1, neuroinflammation, and Alzheimer's disease. Neurobiol Aging 22, 903-908. Mrak, R. E., and Griffin, W. S. (2005). Glia and their cytokines in progression of neurodegeneration. Neurobiol Aging 26, 349-354.  127  Mrak, R. E., and Griffinbc, W. S. (2001). The role of activated astrocytes and of the neurotrophic cytokine S100B in the pathogenesis of Alzheimer's disease. Neurobiol Aging 22, 915-922. Mrak, R. E., Sheng, J. G., and Griffin, W. S. (1996). Correlation of astrocytic S100 beta expression with dystrophic neurites in amyloid plaques of Alzheimer's disease. J Neuropathol Exp Neurol 55, 273-279. 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 Nterminus of beta-amyloid. Nat Genet 1, 345-347. Murphy, M. P., Das, P., Nyborg, A. C , Rochette, M. J., Dodson, M. W., Loosbrock, N. M., Souder, T. M., McLendon, C , Merit, S. L., Piper, S. C , et al. (2003). Overexpression of nicastrin increases Abeta production. Faseb J77, 1138-1140. Nagele, R. G., D'Andrea, M. R., Lee, H., Venkataraman, V., and Wang, H. Y. (2003). Astrocytes accumulate A beta 42 and give rise to astrocytic amyloid plaques in Alzheimer disease brains. Brain Res 971, 197-209. Nalbantoglu, J., Gilfix, B. M., Bertrand, P., Robitaille, Y., Gauthier, S., Rosenblatt, D. S., and Poirier, J. (1994). Predictive value of apolipoprotein E genotyping in Alzheimer's disease: results of an autopsy series and an analysis of several combined studies. Ann Neurol 36, 889895. Namba, Y., Tomonaga, M., Kawasaki, H., Otomo, E., and Ikeda, K. (1991). Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer's disease and kuru plaque amyloid in Creutzfeldt-Jakob disease. Brain Res 541, 163-166. Narain, Y., Yip, A., Murphy, T., Brayne, C , Easton, D., Evans, J. G., Xuereb, J., Cairns, N., Esiri, M. M., Furlong, R. A., and Rubinsztein, D. C. (2000). The ACE gene and Alzheimer's disease susceptibility. J Med Genet 37, 695-697. Nelson, D. E., Ihekwaba, A. E., Elliott, M., Johnson, J. R., Gibney, C. A., Foreman, B. E., Nelson, G., See, V., Horton, C. A., Spiller, D. G., et al. (2004). Oscillations in NF-kappaB signaling control the dynamics of gene expression. Science 306, 704-708. Newman, M., Musgrave, F. I., and Lardelli, M. (2007). Alzheimer disease: Amyloidogenesis, the presenilins and animal models. Biochim Biophys Acta 1772, 285-297. Nicoll, J. A., Mrak, R. E., Graham, D. I., Stewart, J., Wilcock, G., MacGowan, S., Esiri, M. M., Murray, L. S., Dewar, D., Love, S., et al. (2000). Association of interleukin-1 gene polymorphisms with Alzheimer's disease. Ann Neurol 47, 365-368. Nilsberth, C , Westlind-Danielsson, A., Eckman, C. B., Condron, M. M., Axelman, K., Forsell, C , Stenh, C , Luthman, J., Teplow, D. B., Younkin, S. G., et al. (2001). The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation. Nat Neurosci 4, 887-893. 128  Nitsch, R., Pittas, A., Blusztajn, J. K., Slack, B. E., Growdon, J. H., and Wurtman, R. J. (1991). Alterations of phospholipid metabolites in postmortem brain from patients with Alzheimer's disease. Ann N Y Acad Sci 640, 110-113. Nitsch, R. M., Blusztajn, J. K., Pittas, A. G., Slack, B. E., Growdon, J. H., and Wurtman, R. J. (1992). Evidence for a membrane defect in Alzheimer disease brain. Proc Natl Acad Sci U S A 89, 1671-1675. Nonaka, M., Chen, X. H., Pierce, J. E., Leoni, M. J., Mcintosh, T. K., Wolf, J. A., and Smith, D. H. (1999). Prolonged activation of NF-kappaB following traumatic brain injury in rats. J Neurotrauma 16, 1023-1034. Nowak, K., Lange-Dohna, C , Zeitschel, U., Gunther, A., Luscher, B., Robitzki, A., PerezPolo, R., and Rossner, S. (2006). The transcription factor Yin Yang 1 is an activator of BACEl expression. J Neurochem 96, 1696-1707. Nunan, J., and Small, D. H. (2000). Regulation of APP cleavage by alpha-, beta- and gammasecretases. FEBS Lett 483, 6-10. O'Neill, L. A., and Kaltschmidt, C. (1997). NF-kappa B: a crucial transcription factor for glial and neuronal cell function. Trends Neurosci 20, 252-258. Olanow, C. W., and Arendash, G. W. (1994). Metals and free radicals in neurodegeneration. Curr Opin Neurol 7, 548-558. Olson, M. I., and Shaw, C. M. (1969). Presenile dementia and Alzheimer's disease in mongolism. Brain 92, 147-156. Ott, A., Stolk, R. P., van Harskamp, F., Pols, H. A., Hofman, A., and Breteler, M. M. (1999). Diabetes mellitus and the risk of dementia: The Rotterdam Study. Neurology 53, 1937-1942. Pahl, H. L. (1999). Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18, 6853-6866. Palmert, M. R., Golde, T. E., Cohen, M. L., Kovacs, D. M., Tanzi, R. E., Gusella, J. F., Usiak, M. F., Younkin, L. H., and Younkin, S. G. (1988). Amyloid protein precursor messenger RNAs: differential expression in Alzheimer's disease. Science 241, 1080-1084. Panza, F., DTntrono, A., Colacicco, A. M., Basile, A. M., Capurso, C , Kehoe, P. G., Capurso, A., and Solfrizzi, V. (2004). Vascular risk and genetics of sporadic late-onset Alzheimer's disease. J Neural Transm 111, 69-89. Papassotiropoulos, A., Bagli, M., Jessen, F., Bayer, T. A., Maier, W., Rao, M. L., and Heun, R. (1999). A genetic variation of the inflammatory cytokine interleukin-6 delays the initial onset and reduces the risk for sporadic Alzheimer's disease. Ann Neurol 45, 666-668.  129  Pappolla, M. A., Chyan, Y. J., Omar, R. A., Hsiao, K., Perry, G., Smith, M. A., and Bozner, P. (1998). Evidence of oxidative stress and in vivo neurotoxicity of beta-amyloid in a transgenic mouse model of Alzheimer's disease: a chronic oxidative paradigm for testing antioxidant therapies in vivo. Am J Pathol 152, 871-877. Parvathy, S., Hussain, I., Karran, E. H., Turner, A. J., and Hooper, N. M. (1998). Alzheimer's amyloid precursor protein alpha-secretase is inhibited by hydroxamic acid-based zinc metalloprotease inhibitors: similarities to the angiotensin converting enzyme secretase. Biochemistry 37, 1680-1685. Parvathy, S., Hussain, I., Karran, E. H., Turner, A. J., and Hooper, N. M. (1999). Cleavage of Alzheimer's amyloid precursor protein by alpha-secretase occurs at the surface of neuronal cells. Biochemistry 38, 9728-9734. Pereira, C , Agostinho, P., Moreira, P. I., Cardoso, S. M., and Oliveira, C. R. (2005). Alzheimer's disease-associated neurotoxic mechanisms and neuroprotective strategies. Curr Drug Targets CNS Neurol Disord 4, 383-403. Periz, G., and Fortini, M. E. (2004). Functional reconstitution of gamma-secretase through coordinated expression of presenilin, nicastrin, Aph-1, and Pen-2. J Neurosci Res 77, 309-322. Perkins, N. D., Schmid, R. M., Duckett, C. S., Leung, K., Rice, N. R., and Nabel, G. J. (1992). Distinct combinations of NF-kappa B subunits determine the specificity of transcriptional activation. Proc Natl Acad Sci U S A 89, 1529-1533. Perry, E. K. (1986). The cholinergic hypothesis-ten years on. Br Med Bull 42, 63-69. Pineda-Molina, E., Klatt, P., Vazquez, J., Marina, A., Garcia de Lacoba, M., Perez-Sala, D., and Lamas, S. (2001). Glutathionylation of the p50 subunit of NF-kappaB: a mechanism for redox-induced inhibition of DNA binding. Biochemistry 40, 14134-14142. Pitas, R. E., Boyles, J. K., Lee, S. H., Foss, D., and Mahley, R. W. (1987). Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta 917, 148-161. Podlisny, M. B., Citron, M., Amarante, P., Sherrington, R., Xia, W., Zhang, J., Diehl, T., Levesque, G., Fraser, P., Haass, C , et al. (1997). Presenilin proteins undergo heterogeneous endoproteolysis between Thr291 and Ala299 and occur as stable N- and C-terminal fragments in normal and Alzheimer brain tissue. Neurobiol Dis 3, 325-337. Poirier, J., Baccichet, A., Dea, D., and Gauthier, S. (1993). Cholesterol synthesis and lipoprotein reuptake during synaptic remodelling in hippocampus in adult rats. Neuroscience 55,81-90. Poirier, J., Delisle, M. C , Quirion, R., Aubert, I., Farlow, M., Lahiri, D., Hui, S., Bertrand, P., Nalbantoglu, J., Gilfix, B. M., and Gauthier, S. (1995). Apolipoprotein E4 allele as a predictor of cholinergic deficits and treatment outcome in Alzheimer disease. Proc Natl Acad Sci U S A 92, 12260-12264. 130  Pomerantz, J. L., and Baltimore, D. (2002). Two pathways to NF-kappaB. Mol Cell 10, 693695. Ponting, C P., Hutton, M., Nyborg, A., Baker, M., Jansen, K., and Golde, T. E. (2002). Identification of a novel family of presenilin homologues. Hum Mol Genet 11, 1037-1044. Praprotnik, D., Smith, M. A., Richey, P. L., Vinters, H. V., and Perry, G. (1996). Plasma membrane fragility in dystrophic neurites in senile plaques of Alzheimer's disease: an index of oxidative stress. Acta Neuropathol (Bed) 91, 1-5. Prasher, V. P., Farrer, M. J., Kessling, A. M., Fisher, E. M., West, R. J., Barber, P. C., and Butler, A. C. (1998). Molecular mapping of Alzheimer-type dementia in Down's syndrome. Ann Neurol 43, 380-383. Preece, P., Virley, D. J., Costandi, M., Coombes, R., Moss, S. J., Mudge, A. W., Jazin, E., and Cairns, N. J. (2003). Beta-secretase (BACE) and GSK-3 mRNA levels in Alzheimer's disease. Brain Res Mol Brain Res 116, 155-158. Prehn, J. H., Bindokas, V. P., Jordan, J., Galindo, M. F., Ghadge, G. D., Roos, R. P., Boise, L. H., Thompson, C. B., Krajewski, S., Reed, J. C , and Miller, R. J. (1996). Protective effect of transforming growth factor-beta 1 on beta-amyloid neurotoxicity in rat hippocampal neurons. Mol Pharmacol 49, 319-328. Pugh, C. W., and Ratcliffe, P. J. (2003). Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med 9, 677-684. Puglielli, L., Ellis, B. C , Saunders, A. J., and Kovacs, D. M. (2003). Ceramide stabilizes betasite amyloid precursor protein-cleaving enzyme 1 and promotes amyloid beta-peptide biogenesis. J Biol Chem 278, 19777-19783. Qi, X. L., Xiu, J., Shan, K. R., Xiao, Y., Gu, R., Liu, R. Y., and Guan, Z. Z. (2005). Oxidative stress induced by beta-amyloid peptide(l-42) is involved in the altered composition of cellular membrane lipids and the decreased expression of nicotinic receptors in human SH-SY5Y neuroblastoma cells. Neurochem Int 46, 613-621. 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. Qiu, W. Q., Walsh, D. M., Ye, Z., Vekrellis, K., Zhang, J., Podlisny, M. B., Rosner, M. R., Safavi, A., Hersh, L. B., and Selkoe, D. J. (1998). Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J Biol Chem 273, 32730-32738. Qiu, W. Q., Ye, Z., Kholodenko, D., Seubert, P., and Selkoe, D. J. (1997). Degradation of amyloid beta-protein by a metalloprotease secreted by microglia and other neural and nonneural cells. J Biol Chem 272, 6641-6646. Rail, S. C , Jr., Weisgraber, K. H., and Mahley, R. W. (1982). Human apolipoprotein E. The complete amino acid sequence. J Biol Chem 257, 4171-4178. 131  Ramassamy, C , Averill, D., Beffert, U., Bastianetto, S., Theroux, L., Lussier-Cacan, S., Cohn, J. S., Christen, Y., Davignon, J., Quirion, R., and Poirier, J. (1999). Oxidative damage and protection by antioxidants in the frontal cortex of Alzheimer's disease is related to the apolipoprotein E genotype. Free Radic Biol Med 27, 544-553. Raps, S. P., Lai, J. C , Hertz, L., and Cooper, A. J. (1989). Glutathione is present in high concentrations in cultured astrocytes but not in cultured neurons. Brain Res 493, 398-401. Ray, W. J., Yao, M., Mumm, J., Schroeter, E. H., Saftig, P., Wolfe, M., Selkoe, D. J., Kopan, R., and Goate, A. M. (1999a). Cell surface presenilin-1 participates in the gamma-secretaselike proteolysis of Notch. J Biol Chem 274, 36801-36807. Ray, W. J., Yao, M., Nowotny, P., Mumm, J., Zhang, W., Wu, J. Y., Kopan, R., and Goate, A. M. (1999b). Evidence for a physical interaction between presenilin and Notch. Proc Natl Acad Sci U S A 96, 3263-3268. Rebeck, G. W., Reiter, J. S., Strickland, D. K , and Hyman, B. T. (1993). Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions. Neuron 11, 575-580. Reddy, P. H. (2006). Amyloid precursor protein-mediated free radicals and oxidative damage: implications for the development and progression of Alzheimer's disease. J Neurochem 96, 113. Reddy, P. H., and Beal, M. F. (2005). Are mitochondria critical in the pathogenesis of Alzheimer's disease? Brain Res Brain Res Rev 49, 618-632. Regnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. (1997). Identification and characterization of an IkappaB kinase. Cell 90, 373-383. Ren, R. F., and Flanders, K. C. (1996). Transforming growth factors-beta protect primary rat hippocampal neuronal cultures from degeneration induced by beta-amyloid peptide. Brain Res 732, 16-24. Ren, R. F., Hawver, D. B., Kim, R. S., and Flanders, K. C. (1997). Transforming growth factor-beta protects human hNT cells from degeneration induced by beta-amyloid peptide: involvement of the TGF-beta type II receptor. Brain Res Mol Brain Res 48, 315-322. Rentmeister, A., Bill, A., Wahle, T., Walter, J., and Famulok, M. (2006). RNA aptamers selectively modulate protein recruitment to the cytoplasmic domain of beta-secretase BACEl in vitro. Rna 12, 1650-1660. 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.  132  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. Rocchi, A., Pellegrini, S., Siciliano, G., and Murri, L. (2003). Causative and susceptibility genes for Alzheimer's disease: a review. Brain Res Bull 61, 1-24. Rodriguez-Franco, M. I., Fernandez-Bachiller, M. I., Perez, C , Hernandez-Ledesma, B., and Bartolome, B. (2006). Novel tacrine-melatonin hybrids as dual-acting drugs for Alzheimer disease, with improved acetylcholinesterase inhibitory and antioxidant properties. J Med Chem 49, 459-462. Rogaev, E. I., Sherrington, R., Rogaeva, E. A., Levesque, G., Ikeda, M., Liang, Y., Chi, H., Lin, C , Holman, K., Tsuda, T., and et al. (1995). Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene. Nature 376, 775-778. Rogers, G. W., Jr., Edelman, G. M., and Mauro, V. P. (2004). Differential utilization of upstream AUGs in the beta-secretase mRNA suggests that a shunting mechanism regulates translation. Proc Natl Acad Sci U S A 101, 2794-2799. Rogers, J., Luber-Narod, J., Styren, S. D., and Civin, W. H. (1988). Expression of immune system-associated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer's disease. Neurobiol Aging 9, 339-349. Rogers, J., and Lue, L. F. (2001). Microglial chemotaxis, activation, and phagocytosis of amyloid beta-peptide as linked phenomena in Alzheimer's disease. Neurochem Int 39, 333-340. Rogers, J. T., Leiter, L. M., McPhee, J., Cahill, C. M., Zhan, S. S., Potter, H., and Nilsson, L. N. (1999). Translation of the alzheimer amyloid precursor protein mRNA is up-regulated by interleukin-1 through 5'-untranslated region sequences. J Biol Chem 274, 6421-6431. Rossner, S., Apelt, J., Schliebs, R., Perez-Polo, J. R., and Bigl, V. (2001). Neuronal and glial beta-secretase (BACE) protein expression in transgenic Tg2576 mice with amyloid plaque pathology. J Neurosci Res 64, A31-AA6. Rossner, S., Lange-Dohna, C , Zeitschel, U., and Perez-Polo, J. R. (2005). Alzheimer's disease beta-secretase BACEl is not a neuron-specific enzyme. J Neurochem 92, 226-234. Rossner, S., Sastre, M., Bourne, K., and Lichtenthaler, S. F. (2006). Transcriptional and translational regulation of BACEl expression—implications for Alzheimer's disease. Prog Neurobiol 79, 95-111. Rothwarf, D. M., and Karin, M. (1999). The NF-kappa B activation pathway: a paradigm in information transfer from membrane to nucleus. Sci STKE 1999, RE1.  133  Rothwarf, D. M., Zandi, E., Natoli, G., and Karin, M. (1998). IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex. Nature 395, 297-300. Rothwell, N., Allan, S., and Toulmond, S. (1997). The role of interleukin 1 in acute neurodegeneration and stroke: pathophysiological and therapeutic implications. J Clin Invest 100, 2648-2652. Ruben, S. M., Narayanan, R., Klement, J. F., Chen, C. H., and Rosen, C. A. (1992). Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative. Mol Cell Biol 12, 444-454. Rubinsztein, D. C , and Easton, D. F. (1999). Apolipoprotein E genetic variation and Alzheimer's disease, a meta-analysis. Dement Geriatr Cogn Disord 10, 199-209. 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. Ryseck, R. P., Bull, P., Takamiya, M., Bours, V., Siebenlist, U., Dobrzanski, P., and Bravo, R. (1992). RelB, a new Rel family transcription activator that can interact with p50-NF-kappa B. Mol Cell Biol 12, 674-684. Ryseck, R. P., Weih, F., Carrasco, D., and Bravo, R. (1996). RelB, a member of the Rel/NFkappa B family of transcription factors. Braz J Med Biol Res 29, 895-903. Saille, C , Marin, P., Martinou, J. C , Nicole, A., London, J., and Ceballos-Picot, I. (1999). Transgenic murine cortical neurons expressing human Bcl-2 exhibit increased resistance to amyloid beta-peptide neurotoxicity. Neuroscience 92, 1455-1463. Sambamurti, K., Kinsey, R., Maloney, B., Ge, Y. W., and Lahiri, D. K. (2004). Gene structure and organization of the human beta-secretase (BACE) promoter. Faseb J 18, 1034-1036. Sastre, M., Dewachter, I., Landreth, G. E., Willson, T. M., Klockgether, T., van Leuven, F., and Heneka, M. T. (2003). Nonsteroidal anti-inflammatory drugs and peroxisome proliferatoractivated receptor-gamma agonists modulate immunostimulated processing of amyloid precursor protein through regulation of beta-secretase. J Neurosci 23, 9796-9804. Sastre, M., Dewachter, I., Rossner, S., Bogdanovic, N., Rosen, E., Borghgraef, P., Evert, B. O., Dumitrescu-Ozimek, L., Thai, D. R., Landreth, G., et al. (2006). Nonsteroidal antiinflammatory drugs repress beta-secretase gene promoter activity by the activation of PPARgamma. Proc Natl Acad Sci U S A 103, 443-448. Sastre, M., Steiner, H., Fuchs, K., Capell, A., Multhaup, G., Condron, M. M., Teplow, D. B., and Haass, C. (2001). Presenilin-dependent gamma-secretase processing of beta-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch. EMBO Rep 2, 835-841.  134  Saunders, A. M., Strittmatter, W. J., Schmechel, 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. Scharnagl, H., Tisljar, U., Winkler, K., Huttinger, M., Nauck, M. A., Gross, W., Wieland, H., Ohm, T. G., and Marz, W. (1999). The betaA4 amyloid peptide complexes to and enhances the uptake of beta-very low density lipoproteins by the low density lipoprotein receptor-related protein and heparan sulfate proteoglycans pathway. Lab Invest 79, 1271-1286. 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. Schmechel, D. E., Saunders, A. M., Strittmatter, W. J., Crain, B. J., Hulette, C. M., Joo, S. H., Pericak-Vance, M. A., Goldgaber, D., and Roses, A. D. (1993). Increased amyloid beta-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease. Proc Natl Acad Sci U S A 90, 9649-9653. Schreck, R., Albermann, K., and Baeuerle, P. A. (1992). Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Radic Res Commun 17, 221-237. Schreck, R., Rieber, P., and Baeuerle, P. A. (1991). Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. Embo J 10, 2247-2258. Schroeter, E. H., Hagan, M. X., Brunkan, A. L., Hecimovic, S., Li, Y. M., Xu, M., Lewis, H. D., Saxena, M. T., De Strooper, B., Coonrod, A., et al. (2003). A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis. Proc Natl Acad Sci U S A 100, 13075-13080. Schroeter, E. H., Kisslinger, J. A., and Kopan, R. (1998). Notch-1 signalling requires ligandinduced proteolytic release of intracellular domain. Nature 393, 382-386. Schubert, D., Behl, C , Lesley, R., Brack, A., Dargusch, R., Sagara, Y., and Kimura, H. (1995). Amyloid peptides are toxic via a common oxidative mechanism. Proc Natl Acad Sci U S A 92, 1989-1993. Schutte, D. L. (2006). Alzheimer disease and genetics: anticipating the questions. Am J Nurs 106, 40-47; quiz 47-48.  135  Selkoe, D. J. (1989). Molecular pathology of amyloidogenic proteins and the role of vascular amyloidosis in Alzheimer's disease. Neurobiol Aging 10, 387-395. Selkoe, D. J. (1998). The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer's disease. Trends Cell Biol 8, 447-453. Selkoe, D. J. (2001). Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 81, 741766. Selkoe, D. J., and Podlisny, M. B. (2002). Deciphering the genetic basis of Alzheimer's disease. Annu Rev Genomics Hum Genet 3, 67-99. Selkoe, D. J., Podlisny, M. B., Joachim, C. L., Vickers, E. A., Lee, G., Fritz, L. C , and Oltersdorf, T. (1988). Beta-amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues. Proc Natl Acad Sci U S A 85, 7341-7345. Sen, R., and Baltimore, D. (1986). Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46, 705-716. Seubert, P., Oltersdorf, T., Lee, M. G., Barbour, R., Blomquist, C , Davis, D. L., Bryant, K., Fritz, L. C , Galasko, D., Thai, L. J., and et al. (1993). Secretion of beta-amyloid precursor protein cleaved at the amino terminus of the beta-amyloid peptide. Nature 361, 260-263. Seubert, P., Vigo-Pelfrey, C , Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M., Whaley, J., Swindlehurst, C , and et al. (1992). Isolation and quantification of soluble Alzheimer's beta-peptide from biological fluids. Nature 359, 325-327. Sha, W. C , Liou, H. C , Tuomanen, E. I., and Baltimore, D. (1995). Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. Cell 80, 321-330. Shah, S., Lee, S. F., Tabuchi, K., Hao, Y. H., Yu, C , LaPlant, Q., Ball, H., Dann, C. E., 3rd, Sudhof, T., and Yu, G. (2005). Nicastrin functions as a gamma-secretase-substrate receptor. Cell 122, 435-447. Sheng, J. G., Griffin, W. S., Royston, M. C , and Mrak, R. E. (1998). Distribution of interleukin-1-immunoreactive microglia in cerebral cortical layers: implications for neuritic plaque formation in Alzheimer's disease. Neuropathol Appl Neurobiol 24, 278-283. Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G., Ikeda, 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. Shi, X. P., Chen, E., Yin, K. C , Na, S., Garsky, V. M., Lai, M. T., Li, Y. M., Platchek, M., Register, R. B., Sardana, M. K., et al. (2001). The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain. J Biol Chem 276, 10366-10373.  136  Shishodia, S., and Aggarwal, B. B. (2004). Nuclear factor-kappaB: a friend or a foe in cancer? Biochem Pharmacol 68, 1071-1080. Shoji, M., Golde, T. E., Ghiso, J., Cheung, T. T., Estus, S., Shaffer, L. M., Cai, X. D., McKay, D. M., Tintner, R., Frangione, B., and et al. (1992). Production of the Alzheimer amyloid beta protein by normal proteolytic processing. Science 258, 126-129. Siebenlist, U., Brown, K., and Claudio, E. (2005). Control of lymphocyte development by nuclear factor-kappaB. Nat Rev Immunol 5, 435-445. Siebenlist, U., Franzoso, G., and Brown, K. (1994). Structure, regulation and function of NFkappa B. Annu Rev Cell Biol 10, 405-455. Simons, M., Keller, P., De Strooper, B., Beyreuther, K., Dotti, C. G., and Simons, K. (1998). Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons. Proc Natl Acad Sci U S A 95, 6460-6464. 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. Sipe, K. J., Srisawasdi, D., Dantzer, R., Kelley, K. W., and Weyhenmeyer, J. A. (1996). An endogenous 55 kDa TNF receptor mediates cell death in a neural cell line. Brain Res Mol Brain Res 38, 222-232. Sisodia, S. S. (1992). Beta-amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci U S A 89, 6075-6079. Sisodia, S. S. (1999). Alzheimer's disease: perspectives for the new millennium. J Clin Invest 104, 1169-1170. Skoog, I. (1994). Risk factors for vascular dementia: a review. Dementia 5, 137-144. Smith, C. D., Carney, J. M., Tatsumo, T., Stadtman, E. R., Floyd, R. A., and Markesbery, W. R. (1992). Protein oxidation in aging brain. Ann N Y Acad Sci 663, 110-119. Smith, M. A., Rudnicka-Nawrot, M., Richey, P. L., Praprotnik, D., Mulvihill, P., Miller, C. A., Sayre, L. M., and Perry, G. (1995a). Carbonyl-related posttranslational modification of neurofilament protein in the neurofibrillary pathology of Alzheimer's disease. J Neurochem 64, 2660-2666. Smith, M. A., Sayre, L. M., Monnier, V. M., and Perry, G. (1995b). Radical AGEing in Alzheimer's disease. Trends Neurosci 18, 172-176. 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.  137  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. Song, Y. S., Park, H. J., Kim, S. Y., Lee, S. H., Yoo, H. S., Lee, H. S., Lee, M. K., Oh, K. W., Kang, S. K., Lee, S. E., and Hong, J. T. (2004). Protective role of Bcl-2 on beta-amyloidinduced cell death of differentiated PC12 cells: reduction of NF-kappaB and p38 MAP kinase activation. Neurosci Res 49, 69-80. Steiner, H., Capell, A., Leimer, U., and Haass, C. (1999a). Genes and mechanisms involved in beta-amyloid generation and Alzheimer's disease. Eur Arch Psychiatry Clin Neurosci 249, 266270. Steiner, H., Duff, K., Capell, A., Romig, H., Grim, M. G., Lincoln, S., Hardy, J., Yu, X., Picciano, M., Fechteler, K., et al. (1999b). A loss of function mutation of presenilin-2 interferes with amyloid beta-peptide production and notch signaling. J Biol Chem 274, 2866928673. Steiner, H., and Haass, C. (2000). Intramembrane proteolysis by presenilins. Nat Rev Mol Cell Biol 1, 217-224. 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. Steiner, H., Romig, H., Pesold, B., Philipp, U., Baader, M., Citron, M., Loetscher, H., Jacobsen, H., and Haass, C. (1999c). Amyloidogenic function of the Alzheimer's diseaseassociated presenilin 1 in the absence of endoproteolysis. Biochemistry 38, 14600-14605. Steiner, H., Winkler, E., Edbauer, D., Prokop, S., Basset, G., Yamasaki, A., Kostka, M., and Haass, C. (2002). PEN-2 is an integral component of the gamma-secretase complex required for coordinated expression of presenilin and nicastrin. J Biol Chem 277, 39062-39065. 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 277, 1004-1010. Streit, W. J., and Kincaid-Colton, C. A. (1995). The brain's immune system. Sci Am 273, 5455,58-61. Strittmatter, W. J., Saunders, A. M., Schmechel, 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.  138  Strittmatter, W. J., Weisgraber, K. H., Huang, D. Y., Dong, L. M., Salvesen, G. S., PericakVance, M., Schmechel, D., Saunders, A. M., Goldgaber, D., and Roses, A. D. (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 Adachi, A. (1998). Nuclear access and action of notch in vivo. Cell 93, 649660. Subbarao, K. V., Richardson, J. S., and Ang, L. C. (1990). Autopsy samples of Alzheimer's cortex show increased peroxidation in vitro. J Neurochem 55, 342-345. Sugaya, K., Uz, T., Kumar, V., and Manev, H. (2000). New anti-inflammatory treatment strategy in Alzheimer's disease. Jpn J Pharmacol 82, 85-94. Sullivan, P. G., Bruce-Keller, A. J., Rabchevsky, A. G., Christakos, S., Clair, D. K , Mattson, M. P., and Scheff, S. W. (1999). Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury. J Neurosci 19, 62486256. Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., Staufenbiel, M., Huang, L. E., and Song, W. (2006a). Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACEl gene expression. Proc Natl Acad Sci U S A 103, 18727-18732. Sun, X., He, G , and Song, W. (2006b). BACE2, as a novel APP theta-secretase, is not responsible for the pathogenesis of Alzheimer's disease in Down syndrome. Faseb J 20, 13691376. Sun, X., Tong, Y., Qing, H., Chen, C. H., and Song, W. (2006c). Increased BACEl maturation contributes to the pathogenesis of Alzheimer's disease in Down syndrome. Faseb J 20, 13611368. 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 BACEl genes. Faseb J 19, 739-749. Suo, Z., Fang, C , Crawford, F., and Mullan, M. (1997). Superoxide free radical and intracellular calcium mediate A beta(l-42) induced endothelial toxicity. Brain Res 762, 144152. 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. Takahashi, S., Kawarabayasi, Y., Nakai, T., Sakai, J., and Yamamoto, T. (1992). Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity. Proc Natl Acad Sci U S A 89, 9252-9256.  139  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. Takuma, K., Lee, E., Kidawara, M., Mori, K., Kimura, Y., Baba, A., and Matsuda, T. (1999). Apoptosis in Ca2 + reperfusion injury of cultured astrocytes: roles of reactive oxygen species and NF-kappaB activation. Eur J Neurosci 11, 4204-4212. Tamagno, E., Bardini, P., Obbili, A., Vitali, A., Borghi, R., Zaccheo, D., Pronzato, M. A., Danni, O., Smith, M. A., Perry, G., and Tabaton, M. (2002). Oxidative stress increases expression and activity of BACE in NT2 neurons. Neurobiol Dis 10, 279-288. Tan, J., Town, T., Mori, T., Wu, Y., Saxe, M., Crawford, F., and Mullan, M. (2000). CD45 opposes beta-amyloid peptide-induced microglial activation via inhibition of p44/42 mitogenactivated protein kinase. J Neurosci 20, 7587-7594. Tanaka, M., Fuentes, M. E., Yamaguchi, K., Durnin, M. H., Dalrymple, S. A., Hardy, K. L., and Goeddel, D. V. (1999). Embryonic lethality, liver degeneration, and impaired NF-kappa B activation in IKK-beta-deficient mice. Immunity 10, 421-429. Tanaka, S., Nakamura, S., Ueda, K., Kameyama, M., Shiojiri, S., Takahashi, Y., Kitaguchi, N., and Ito, H. (1988). Three types of amyloid protein precursor mRNA in human brain: their differential expression in Alzheimer's disease. Biochem Biophys Res Commun 157, 472-479. Tanaka, S., Shiojiri, S., Takahashi, Y., Kitaguchi, N., Ito, H., Kameyama, M., Kimura, J., Nakamura, S., and Ueda, K. (1989). Tissue-specific expression of three types of beta-protein precursor mRNA: enhancement of protease inhibitor-harboring types in Alzheimer's disease brain. Biochem Biophys Res Commun 165, 1406-1414. Tang, J., and Wong, R. N. (1987). Evolution in the structure and function of aspartic proteases. J Cell Biochem 33, 53-63. Tanzi, R. E., and Bertram, L. (2005). Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective. Cell 720, 545-555. Tanzi, R. E., Gusella, J. F., 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. Tarkowski, E., Blennow, K., Wallin, A., and Tarkowski, A. (1999). Intracerebral production of tumor necrosis factor-alpha, a local neuroprotective agent, in Alzheimer disease and vascular dementia. J Clin Immunol 19, 223-230. Tarkowski, E., Issa, R., Sjogren, M., Wallin, A., Blennow, K., Tarkowski, A., and Kumar, P. (2002). Increased intrathecal levels of the angiogenic factors VEGF and TGF-beta in Alzheimer's disease and vascular dementia. Neurobiol Aging 23, 237-243.  140  Tarkowski, E., Liljeroth, A. M., Nilsson, A., Ricksten, A., Davidsson, P., Minthon, L., and Blennow, K. (2000). TNF gene polymorphism and its relation to intracerebral production of TNFalpha and TNFbeta in AD. Neurology 54, 2077-2081. Tartaglia, L. A., Pennica, D., and Goeddel, D. V. (1993). Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor. J Biol Chem 268, 18542-18548. Taylor, J. M., and Crack, P. J. (2004). Impact of oxidative stress on neuronal survival. Clin Exp Pharmacol Physiol 31, 397-406. Terai, K., Matsuo, A., and McGeer, P. L. (1996). Enhancement of immunoreactivity for NFkappa B in the hippocampal formation and cerebral cortex of Alzheimer's disease. Brain Res 735, 159-168. Thanos, D., and Maniatis, T. (1995). NF-kappa B: a lesson in family values. Cell 80, 529-532. Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. FI., 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. Thinakaran, G., Harris, C. L., Ratovitski, T., Davenport, F., Slunt, H. H., Price, D. L., Borchelt, D. R., and Sisodia, S. S. (1997). Evidence that levels of presenilins (PSI and PS2) are coordinately regulated by competition for limiting cellular factors. J Biol Chem 272, 2841528422. Thomas, T., Thomas, G., McLendon, C , Sutton, T., and Mullan, M. (1996). beta-Amyloidmediated vasoactivity and vascular endothelial damage. Nature 380, 168-171. Tiret, L., Rigat, B., Visvikis, S., Breda, C , Corvol, P., Cambien, F., and Soubrier, F. (1992). Evidence, from combined segregation and linkage analysis, that a variant of the angiotensin Iconverting enzyme (ACE) gene controls plasma ACE levels. Am J Hum Genet 57, 197-205. Tomita, T., Katayama, R., Takikawa, R., and Iwatsubo, T. (2002). Complex N-glycosylated form of nicastrin is stabilized and selectively bound to presenilin fragments. FEBS Lett 520, 117-121. Tong, Y., Zhou, W., Fung, V., Christensen, M. A., Qing, H., Sun, X., and Song, W. (2005). Oxidative stress potentiates BACEl gene expression and Abeta generation. J Neural Transm 772, 455-469. Toro, V. C , Tehranian, R., Zetterstrom, M., Eriksson, G , Langel, U., Bartfai, T., and Iverfeld, K. (2001). Increased gene expression of interleukin-1 alpha and interleukin-6 in rat primary glial cells induced by beta-amyloid fragment. J Mol Neurosci 77, 341-350. Touzani, O., Boutin, H., Chuquet, J., and Rothwell, N. (1999). Potential mechanisms of interleukin-1 involvement in cerebral ischaemia. J Neuroimmunol 100, 203-215.  141  Tu, H., Nelson, O., Bezprozvanny, A., Wang, Z., Lee, S. F., Hao, Y. H., Serneels, L., De Strooper, B., Yu, G., and Bezprozvanny, I. (2006). Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations. Cell 126, 981-993. Tuppo, E. E., and Arias, H. R. (2005). The role of inflammation in Alzheimer's disease. Int J Biochem Cell Biol 37, 289-305. 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. Urban, M. B., Schreck, R., and Baeuerle, P. A. (1991). NF-kappa B contacts DNA by a heterodimer of the p50 and p65 subunit. Embo J 10, 1817-1825. Vallieres, L., and Rivest, S. (1997). Regulation of the genes encoding interleukin-6, its receptor, and gpl30 in the rat brain in response to the immune activator lipopolysaccharide and the proinflammatory cytokine interleukin-lbeta. J Neurochem 69, 1668-1683. van Leeuwen, F. W., de Kleijn, D. P., van den Hurk, H. H., Neubauer, A., Sonnemans, M. A., Sluijs, J. A., Koycu, S., Ramdjielal, R. D., Salehi, A., Martens, G. J., et al. (1998). Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients. Science 279, 242-247. Van Wagoner, N. J., Oh, J. W., Repovic, P., and Benveniste, E. N. (1999). Interleukin-6 (IL-6) production by astrocytes: autocrine regulation by IL-6 and the soluble IL-6 receptor. J Neurosci 19, 5236-5244. 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 286, 735741. Veerhuis, R., Janssen, I., De Groot, C. J., Van Muiswinkel, F. L., Hack, C. E., and Eikelenboom, P. (1999). Cytokines associated with amyloid plaques in Alzheimer's disease brain stimulate human glial and neuronal cell cultures to secrete early complement proteins, but not CI-inhibitor. Exp Neurol 160, 289-299. Vinet, J., Bernier, P. J., and Parent, A. (2002). Bcl-2 expression in thalamus, brainstem, cerebellum and visual cortex of adult primate. Neurosci Res 42, 269-277. Volicer, L., and Crino, P. B. (1990). Involvement of free radicals in dementia of the Alzheimer type: a hypothesis. Neurobiol Aging 11, 567-571. Walker, D. G., Lue, L. F., and Beach, T. G. (2001). Gene expression profiling of amyloid beta peptide-stimulated human post-mortem brain microglia. Neurobiol Aging 22, 957-966. 142  Walter, J., Kaether, C , Steiner, H., and Haass, C. (2001). The cell biology of Alzheimer's disease: uncovering the secrets of secretases. Curr Opin Neurobiol 11, 585-590. Waris, G., Livolsi, A., Imbert, V., Peyron, J. F., and Siddiqui, A. (2003). Hepatitis C virus NS5A and subgenomic replicon activate NF-kappaB via tyrosine phosphorylation of IkappaBalpha and its degradation by calpain protease. J Biol Chem 278, 40778-40787. Watabe, M., Hishikawa, K., Takayanagi, A., Shimizu, N., and Nakaki, T. (2004). Caffeic acid phenethyl ester induces apoptosis by inhibition of NFkappaB and activation of Fas in human breast cancer MCF-7 cells. J Biol Chem 279, 6017-6026. Watanabe, D., Yoshimura, R., Khalil, M., Yoshida, K., Kishimoto, T., Taga, T., and Kiyama, H. (1996). Characteristic localization of gpl30 (the signal-transducing receptor component used in common for IL-6/fL-l l/CNTF/LIF/OSM) in the rat brain. Eur J Neurosci 8, 16301640. 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. Wegiel, J., Wang, K. C , Tarnawski, M., and Lach, B. (2000). Microglia cells are the driving force in fibrillar plaque formation, whereas astrocytes are a leading factor in plague degradation. Acta Neuropathol (Berl) 100, 356-364. Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L., and Beyreuther, K. (1989). Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein. Cell 57, 115-126. Weidemann, A., Paliga, K., Durrwang, U., Czech, C , Evin, G., Masters, C. L., and Beyreuther, K. (1997). Formation of stable complexes between two Alzheimer's disease gene products: presenilin-2 and beta-amyloid precursor protein. Nat Med 3, 328-332. Weihofen, A., Binns, K., Lemberg, M. K., Ashman, K., and Martoglio, B. (2002). Identification of signal peptide peptidase, a presenilin-type aspartic protease. Science 296", 2215-2218. Weiner, H. L., and Selkoe, D. J. (2002). Inflammation and therapeutic vaccination in CNS diseases. Nature 420, 879-884. Weldon, D. T., Rogers, S. D., Ghilardi, J. R., Finke, M. P., Cleary, J. P., O'Hare, E., Esler, W. P., Maggio, J. E., and Mantyh, P. W. (1998). Fibrillar beta-amyloid induces microglial phagocytosis, expression of inducible nitric oxide synthase, and loss of a select population of neurons in the rat CNS in vivo. J Neurosci 18, 2161-2173. Weskamp, G., Cai, H., Brodie, T. A., Higashyama, S., Manova, K., Ludwig, T., and Blobel, C. P. (2002). Mice lacking the metalloprotease-disintegrin MDC9 (ADAM9) have no evident major abnormalities during development or adult life. Mol Cell Biol 22, 1537-1544.  143  Wisniewski, T., Castano, E. M . , Golabek, A., Vogel, T., and Frangione, B. (1994). Acceleration of Alzheimer's fibril formation by apolipoprotein E in vitro. A m J Pathol 145, 1030-1035. Wolfe, M . S. (2007). When loss is gain: reduced presenilin proteolytic function leads to increased Abeta42/Abeta40. Talking Point on the role of presenilin mutations in Alzheimer disease. E M B O Rep 8, 136-140. Wolfe, M . S., De Los Angeles, J., Miller, D. D., Xia, W., and Selkoe, D. J. (1999a). Are presenilins intramembrane-cleaving proteases? Implications for the molecular mechanism of Alzheimer's disease. Biochemistry 38, 11223-11230. Wolfe, M . S., Xia, W., Moore, C. L., Leatherwood, D. D., Ostaszewski, B., Rahmati, T., Donkor, I. O., and Selkoe, D. J. (1999b). Peptidomimetic probes and molecular modeling suggest that Alzheimer's gamma-secretase is an intramembrane-cleaving aspartyl protease. Biochemistry 38, 4720-4727. Wolfe, M . S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999c). Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398, 513-517. Wood, J. G., Mirra, S. S., Pollock, N . J., and Binder, L. I. (1986). Neurofibrillary tangles of Alzheimer disease share antigenic determinants with the axonal microtubule-associated protein tau (tau). Proc Natl Acad Sci U S A 83, 4040-4043. Worley, P. F., Bhat, R. V., Baraban, J. M . , Erickson, C. A., McNaughton, B . L., and Barnes, C. A. (1993). Thresholds for synaptic activation of transcription factors in hippocampus: correlation with long-term enhancement. J Neurosci 13, 4776-4786. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massague, J. (1994). Mechanism of activation of the TGF-beta receptor. Nature 370, 341-347. Wyne, K. L., Pathak, K., Seabra, M . C , and Hobbs, H. H . (1996). Expression of the V L D L receptor in endothelial cells. Arterioscler Thromb Vase Biol 16, 407-415. Wyss-Coray, T., Lin, C., Yan, F., Yu, G. Q., Rohde, M . , McConlogue, L., Masliah, E., and Mucke, L. (2001). TGF-betal promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice. Nat Med 7, 612-618. Wyss-Coray, T., Loike, J. D., Brionne, T. C , Lu, E., Anankov, R., Yan, F., Silverstein, S. C , and Husemann, J. (2003). Adult mouse astrocytes degrade amyloid-beta in vitro and in situ. Nat Med 9, 453-457. Wyss-Coray, T., Masliah, E., Mallory, M . , McConlogue, L., Johnson-Wood, K., Lin, C , and Mucke, L. (1997). Amyloidogenic role of cytokine TGF-betal in transgenic mice and in Alzheimer's disease. Nature 389, 603-606.  144  Xia, W., Zhang, J., Perez, R., Koo, E. H., and Selkoe, D. J. (1997). Interaction between amyloid precursor protein and presenilins in mammalian cells: implications for the pathogenesis of Alzheimer disease. Proc Natl Acad Sci U S A 94, 8208-8213. Xu, H., Gouras, G. K., Greenfield, J. P., Vincent, B., Naslund, J., Mazzarelli, L., Fried, G., Jovanovic, J. N., Seeger, M., Relkin, N. R., et al. (1998). Estrogen reduces neuronal generation of Alzheimer beta-amyloid peptides. Nat Med 4, 447-451. Yamamoto, T., Maruyama, W., Kato, Y., Yi, H., Shamoto-Nagai, M., Tanaka, M., Sato, Y., and Naoi, M. (2002). Selective nitration of mitochondrial complex I by peroxynitrite: involvement in mitochondria dysfunction and cell death of dopaminergic SH-SY5Y cells. J Neural Transm 109, 1-13. Yamamoto, Y., Verma, U. N., Prajapati, S., Kwak, Y. T., and Gaynor, R. B. (2003). Histone H3 phosphorylation by IKK-alpha is critical for cytokine-induced gene expression. Nature 423, 655-659. Yamasaki, A., Eimer, S., Okochi, M., Smialowska, A., Kaether, C., Baumeister, R., Haass, C , and Steiner, H. (2006). The GxGD motif of presenilin contributes to catalytic function and substrate identification of gamma-secretase. J Neurosci 26, 3821-3828. Yamazaki, S., Muta, T., and Takeshige, K. (2001). A novel IkappaB protein, HcappaB-zeta, induced by proinflammatory stimuli, negatively regulates nuclear factor-kappaB in the nuclei. J . Biol Chem 276, 27657-27662. Yan, Q., Zhang, J., Liu, H., Babu-Khan, S., Vassar, R., Biere, A. L., Citron, M., and Landreth, G. (2003). Anti-inflammatory drug therapy alters beta-amyloid processing and deposition in an animal model of Alzheimer's disease. J Neurosci 23, 7504-7509. 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. Yang, D. S., Tandon, A., Chen, F., Yu, G., Yu, H., Arawaka, S., Hasegawa, H., Duthie, M., Schmidt, S. D., Ramabhadran, T. V., et al. (2002). Mature glycosylation and trafficking of nicastrin modulate its binding to presenilins. J Biol Chem 277, 28135-28142. Yang, L., Ross, K., and Qwarnstrom, E. E. (2003a). RelA control of IkappaBalpha phosphorylation: a positive feedback loop for high affinity NF-kappaB complexes. J Biol Chem 278, 30881-30888. Yang, L. B., Lindholm, K., Yan, R., Citron, M., Xia, W., Yang, X. L., Beach, T., Sue, L., Wong, P., Price, D., et al. (2003b). Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease. Nat Med 9, 3-4.  145  Yankner, B. A., Dawes, L. R., Fisher, S., Villa-Komaroff, L., Oster-Granite, M. L., and Neve, R. L. (1989). Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer's disease. Science 245, 417-420. Yankner, B. A., Duffy, L. K., and Kirschner, D. A. (1990). Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science 250, 279-282. Yasukawa, K., Saito, T., Fukunaga, T., Sekimori, Y., Koishihara, Y., Fukui, H., Ohsugi, Y., Matsuda, T., Yawata, FL, Hirano, T., and et al. (1990). Purification and characterization of soluble human IL-6 receptor expressed in CHO cells. J Biochem (Tokyo) 108, 673-676. Yates, S. L., Burgess, L. FL, Kocsis-Angle, J., Antal, J. M., Dority, M. D., Embury, P. B., Piotrkowski, A. M., and Brunden, K. R. (2000). Amyloid beta and amylin fibrils induce increases in proinflammatory cytokine and chemokine production by THP-1 cells and murine microglia. J Neurochem 74, 1017-1025. Ye, S., Huang, Y., Mullendorff, K., Dong, L., Giedt, G., Meng, E. C., Cohen, F. E., Kuntz, I. D., Weisgraber, K. H., and Mahley, R. W. (2005). Apolipoprotein (apo) E4 enhances amyloid beta peptide production in cultured neuronal cells: apoE structure as a potential therapeutic target. Proc Natl Acad Sci U S A 102, 18700-18705. Yoshikai, S., Sasaki, H., Doh-ura, K., Furuya, H., and Sakaki, Y. (1990). Genomic organization of the human amyloid beta-protein precursor gene. Gene 87, 257-263. Yu, C , Kim, S. H., Dceuchi, T., Xu, H., Gasparini, L., Wang, R., and Sisodia, S. S. (2001). Characterization of a presenilin-mediated amyloid precursor protein carboxyl-terminal fragment gamma. Evidence for distinct mechanisms involved in gamma -secretase processing of the APP and Notch 1 transmembrane domains. J Biol Chem 276, 43756-43760. 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 high molecular weight intracellular complex that contains beta-catenin. J Biol Chem 273, 1647016475. Yu, G., Nishimura, M., Arawaka, S., Levitan, D., Zhang, L., Tandon, A., Song, Y. Q., Rogaeva, E., Chen, F., Kawarai, T., et al. (2000). Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing. Nature 407, 48-54. Yu, Z., Zhou, D., Bruce-Keller, A. J., Kindy, M. S., and Mattson, M. P. (1999). Lack of the p50 subunit of nuclear factor-kappaB increases the vulnerability of hippocampal neurons to excitotoxic injury. J Neurosci 19, 8856-8865. Zandi, E., and Karin, M. (1999). Bridging the gap: composition, regulation, and physiological function of the IkappaB kinase complex. Mol Cell Biol 19, 4547-4551. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997). The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Cell 91, 243-252. 146  Zhang, X., Zhou, K., Wang, R., Cui, J., Lipton, S. A., Liao, F. F., Xu, FL, and Zhang, Y. W. (2007). Hypoxia inducible factor 1 alpha (HIF-lalpha)-mediated hypoxia increases BACEl expression and beta -amyloid generation. J Biol Chem. Zhang, Y., Feng, X., We, R., and Derynck, R. (1996). Receptor-associated Mad homologues synergize as effectors of the TGF-beta response. Nature 383, 168-172. 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. Zhao, J., Paganini, L., Mucke, L., Gordon, M., Refolo, L., Carman, M., Sinha, S., Oltersdorf, T., Lieberburg, I., and McConlogue, L. (1996). Beta-secretase processing of the beta-amyloid precursor protein in transgenic mice is efficient in neurons but inefficient in astrocytes. J Biol Chem 277, 31407-31411. Zhong, H., May, M. J., Jimi, E., and Ghosh, S. (2002). The phosphorylation status of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1. Mol Cell 9, 625-636. Zhong, J., Dietzel, I. D., Wahle, P., Kopf, M., and Heumann, R. (1999). Sensory impairments and delayed regeneration of sensory axons in interleukin-6-deficient mice. J Neurosci 19, 4305-4313. Zhou, W., and Song, W. (2006). Leaky scanning and reinitiation regulate BACEl gene expression. Mol Cell Biol 26, 3353-3364.  147  


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



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"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items