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Regulation of APP processing and Aβ production by BACE1 Deng, Yu 2011

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Regulation of APP processing and Aβ production by BACE1  by Yu Deng BSc. (Biotechnology) Beijing Institute of Technology, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)  April 2011  © Yu Deng, 2011  Abstract Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease among elderly people. The main symptoms of AD are memory loss and cognitive deficits. One of the hallmarks of AD is neuritic plaques in the brain. Amyloid β protein (Aβ), a peptide of 39–42 amino acids, forms the predominant component of plaques. Aβ is generated from amyloid-β precursor protein (APP) by sequential cleavages mediated by β-secretase and γ-secretase.  Previous studies have shown that BACE1 can cleave APP at the ASP +1 site or at the Glu+11 site of Aβ domain. Cleavage at ASP+1 is required for generating full length Aβ and increases in cleavage at ASP+1 site has been considered as one of the major pathological pathways in AD cases. Swedish mutant APP, carrying double mutations (Lys595-Met596 to Asn595-Leu596) close to the ASP+1 site in APP gene, causes early onset of familial AD. The Swedish mutation increases BACE1 cleavage at ASP+1 site, resulting in significant increase of Aβ production. However, how BACE1 regulates APP processing at ASP+1 and Glu+11 remains elusive. Our preliminary studies indicated that majority of wild type APP is cleaved by BACE1 at Glu+11 site, in contrast, Swedish mutation shifts the major cleavage site from Glu+11 to ASP+1 , resulting in significant increase of Aβ production under pathological condition. This work provides new insights in the pathological pathway of AD and suggests a major potential for the pharmaceutical development.  ii  Preface Animal experiment protocols were approved by The University of British Columbia Animal Care and Use committee. The protocol number is A090274.  Atomic Force Microscope result shown in figure 3.2.1.B is based on the work conducted by Peng Zhang in Dr. Hongbin Li’s laboratory at department of chemistry of UBC.  Figure 3.2E (d) Western Blot’s result was performed by Xiaozhu Zhang in Dr.Weihong Song’s laboratory at department of psychiatry of UBC.  iii  Table of Contents Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iii Table of Contents .................................................................................................................... iv List of Tables .......................................................................................................................... vii List of Figures........................................................................................................................ viii List of Abbreviations ............................................................................................................... ix Acknowledgements ................................................................................................................. xii Chapter 1.  Introduction .......................................................................................................1  1.1 An Overview of Alzheimer’s Disease .................................................................................1 1.1.1  Familial Form of AD ...............................................................................................1  1.1.2  Sporadic Form of AD ..............................................................................................4  1.1.3  Classic Features of AD ............................................................................................7  1.2 Amyloid Cascade Hypothesis of AD ..................................................................................8 1.2.1  Amyloid Precursor Protein (APP) ..........................................................................8  1.2.2  Enzymes in APP Processing ................................................................................. 10  1.2.3  APP Processing .....................................................................................................14  1.2.4  Amyloid βProtein (Aβ) ........................................................................................ 17  1.3. Hypotheses and Specific Aims ......................................................................................... 19 1.3.1 Hypotheses ...................................................................................................................... 19 1.3.2 Specific Aims .................................................................................................................. 19 Chapter 2.  Materials and Methods.................................................................................... 21  2.1 Cell Culture ...................................................................................................................... 21 iv  2.1.1  Culture Media Preparation .................................................................................. 21  2.1.2  Trypsinization .......................................................................................................21  2.1.3  Cell Transfection ................................................................................................... 21  2.2 Western Blotting............................................................................................................... 22 2.3 Primary Rat Cortical Neuronal Culture .........................................................................23 2.4 Preparation of Aβ Fibrils .................................................................................................24 2.5 MTT(Thiazolyl Blue Tetrazolium Bromide) Assay ........................................................ 24 2.6 Aβ ELISA ......................................................................................................................... 25 2.7 Data Collection and Statistics .......................................................................................... 25 Chapter 3.  Results .............................................................................................................. 27  3.1 Examination of APP Processing and Aβ Production by BACE1 in vitro ....................... 27 3.1.1  Generation of Stable Cell Lines ............................................................................27  3.1.2  Examination of APP Processing and Aβ Production by BACE1 in Swedish and  Wildtype APP Cells ................................................................................................................. 30 3.1.3 3.2  Aβ Production was Affected by APP Mutation and BACE1 ............................... 35 In vitro Study of Aggregation and Neurotoxicity of Different Aβ Species Generated by  Differential BACE1 Cleavages ............................................................................................... 37 3.2.1  Aβ Aggregation .....................................................................................................37  3.2.2  Neurotoxicity of Aβ Species .................................................................................. 40  3.3 Determination of the Role of BACE1 Cleavage Sites in AD Pathogenesis in vivo .........41 v  3.4 Structural Study of APP ..................................................................................................42 Chapter 4. General Discussion ............................................................................................... 45 4.1 Examination of APP Processing and Aβ Production by BACE1 in vitro. ....................... 45 4.2 In vitro Study of Function and Aggregation of Aβ Species .............................................46 4.3 In vivo Studies ................................................................................................................... 47 4.4 Conclusion ........................................................................................................................ 48 4.5 Future Direction ............................................................................................................... 49 References................................................................................................................................ 50  vi  List of Tables  Table 3.1  Sequence of Synthesized Aβ Peptides ............................................................... 37  Table 3.2 A  AWildtype APP 695 Amino Acids Sequence ................................................ 43  Table 3.2 B  Swedish Mutant APP 695 Amino Acids Sequence .......................................43  Table 3.3 C  Primer Design for APP Fragments Cloning. ................................................ 44  vii  List of Figures Figure 1.1 APP Processing………………………….………………..…..………..…..16 Figure 3.1 A Generation of HAW Cells. ................................................................................ 28 Figure 3.1 B Generation of BAW Cells ................................................................................. 28 Figure 3.1 C Generation of 20E2 Cells. ................................................................................. 29 Figure 3.1 D Generation 2EB2 Cells. .................................................................................... 29 Figure 3.2 A C83 is the Main APP Processing Product in Cells Expressing Wildtype APP.... 30 Figure 3.2 B CTF in Cells Expressing Wildtpe APP and BACE1. .........................................31 Figure 3.2 C CTF in Cells Expressing Swedish APP.. ........................................................... 31 Figure 3.2 D CTF in Cells Expressing Swedish APP and BACE1. ........................................32 Figure 3.2 E APP is Cleaved by BACE1 Differentially ......................................................... 33 Figure 3.2 F CTF Generations. .............................................................................................. 34 Figure 3.2 G CTF Generation in BAW and 2EB2 Cells. …………………………………....35 Figure 3. 3 Aβ Level Have were by ELISA ...........................................................................36 Figure 3.4 A Aggregation of Aβ Species. .............................................................................. 39 Figure 3.4 B Observation of Aβ Species Aggregation under Atomic Force Microscope.. .......40 Figure 3.5 MTT Assay………………………………………………………………………41 Figure 3.6 Aβ Level of Human Brain Tissue were Measured by ELISA……….……..….42  viii  List of Abbreviations o  C………………………………………Degrees centigrade  μg………………………………………Micro-gram μL………………………………………Micro-liter HEK293 cells  …………………….....Human embryonic kidney 293 cells  20E2 cells……………………………….HEK293 cells stably expresses Swedish mutant APP695 Aβ………………………………………Amyloid-β AD………………………………………Alzheimer’s Disease ADAM…………………………………..A disintegrin and metalloproteinase domain Aph-1……………………………………Anterior pharynx-defective-1 apoE……………………………………..Apolipoprotein E APP……………………………………...Amyloid Precursor Protein Asp………………………………………Aspartic acid BACE ………………………………….β-site APP cleaving enzyme C83………………………………………83-residue CTF of APP C99………………………………………99-residue CTF of APP CNS……………………………………...Central Nervous System C-terminus……………………………….Carboxyl-terminus CSF………………………………………Cerebrospinal fluid CTF………………………………………Carboxyl-terminal fragment Cys……………………………………….Cysteine ix  DS………………………………………...Down syndrome ECL……………………………………….Enhanced chemiluminescence EDTA …………………………………….Ethylenediaminetetraacetic acid ELISA …………………………………...Enzyme Linked-Immuno-Sorbent Assay HBSS………………………………………Hanks Balanced Salt Solution HEPES……………………………………..4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HRP………………………………………..Horseradish peroxidase kDa…………………………………………Kilo Dalton LDL………………………………………...Low-density lipoprotein Leu……………………………………….....Leucine LPS ………………………………………...Lipopolysaccharide LRP………………………………………...LDL-receptor-related protein Lys……………………………………….....Lysine Mg…………………………………………..Magnesium MgCl2……………………………………….Magnesium chloride M……………………………………….........Molarity Met…………………………………………..Methionine nm…………………………………………....Nanometer NaCl………………………………………....Sodium chloride Na2HPO4…………………………………….Di-sodium phosphate NaOH ……………………………………...Sodium hydroxide x  Nct…………………………………………...Nicastrin NFT……………………………………….....Neurofibrillary tangles N-terminus…………………………………..Amino acids of N-terminus NTF……………………………………….....Amino-terminal fragment O2-……………………………………….......Superoxide anion OH. ………………………………………….Hydroxyl radical PBS-T……………………………………….Phosphate buffered saline-Tween 20 Pen-2………………………………………...Presenilin enhancer-2 Phe………………………………………......Phenylalanine Pro…………………………………………...Proline PS………………………………………........Presenilin PTEN………………………………………...Phosphatase and tensin homolog PVDF………………………………………...Polyvinylidene fluoride rpm……………………………………….......Revolutions per minute sAPP………………………………………....Soluble APP SEM………………………………………….Standard errors of the means SH-SY5Y…………………………………….Human neuroblastoma  xi  Acknowledgements I would like to give my sincerest thanks to my supervisor Dr. Weihong Song, for providing me with this great opportunity to accomplish my graduate studies in his laboratory. I truly appreciate his encouragements, suggestion and support all through my projects and graduate studies. I would also like to thank the members of my supervisory committee: Dr. Honglin Luo, Dr. William Jia, and Dr. Peter Reiner for all their helpful suggestions and comments.  I appreciate all the members of Townsend Family Laboratories for their friendship and help. Especially, I would like to sincerely thank Xiaozhu Zhang, Ruitao Wang, Philip Ly, Kelley Bromley-Brits, Fang Cai, Yili Wu, Haiyan Zhou and Kristy Song for their support and assistant, as well as their patience along the way. I would also like to thank Dr. Hongbin Li sincerely for opening his laboratory to me for the experiment. I would also like to extend my sincerest thanks to Mr. Qing Peng and Mr. Peng Zheng in Dr. Li’s lab for their cheerful technical assistance.  Finally, I would like to give my sincerest thanks to my parents and friends for their unconditional support, encouragements and love for me.  xii  Chapter 1. Introduction An Overview of Alzheimer’s Disease  1.1  Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases among seniors. Due to substantial changes in life expectancy and demographic parameters in recent years, it is anticipated that by 2050, over 25% of the population over 65 will be affected by AD (Sisodia, 1999). AD was first described by Alois Alzheimer in 1906 as a disease exhibiting characteristics of senile plaques, neurofibrillary tangles and neuronal loss (Alzheimer, 1906, 1907). Since then, substantial efforts were made to understand the pathogenesis of AD and to develop more effective therapeutics. Currently, cholinergic and glutamatergic neurotransmission modulators are the main drug for AD (Birks, 2006; Mangialasche et al., 2010; Nordberg, 2006; Wang et al., 2009; Winblad, 2009). However, their neuroprotective effects are controversial and there is no effective means to prevent and treat AD.  1.1.1  Familial Form of AD  Two forms of AD, familial (FAD) and sporadic (SAD), are recognized (Evans et al., 1989; Goedert and Spillantini, 2006; Pereira et al., 2005; Selkoe and Podlisny, 2002). Clinically, less than 10% of the cases are FAD and the rest are SAD. FAD exhibits an autosomal dominant phenotype and has an early disease onset (Goate et al., 1991; Schutte, 2006; Selkoe, 2001). Currently, four genetic factors (APP, PS1, PS2 and ApoE4) predisposing to AD have been confirmed. 1  1.1.1.1  Mutations on Amyloid-β Precursor Protein (APP) The first specific identified genetic cause of AD was the occurrence of missense  mutations in APP (Goate et al., 1991). Today, nine missense mutations in APP are known to increase Amyloid Beta (Aβ) production via various mechanisms. The location of the mutations dictates the effect of the mutation. A double mutation at codons at 670 and 671 was identified to cause early onset AD in two large Swedish families (Mullan et al., 1992). This Swedish mutation (KM to NL) was located right before the β-secretase cleavage site, and it significantly increases β-secretase cleavage and neurotoxic Aβ42 production (Cai et al., 1993; Citron et al., 1992). In contrast, a mutation after the α-secretase cleavage site or downstream of the γ-secretase cleavage site (Meredith, 2005; Selkoe and Podlisny, 2002) can increase hydrophobicity of the transmembrane region or modify the translational regulation of the APP mRNA(Goate et al., 1991; Tanzi and Hyman, 1991). Individuals exhibiting these mutations are predisposed to an increased production of amyloid β proteins (Aβ) and an onset of AD before the age of 65(Meredith, 2005; Selkoe and Podlisny, 2002). In addition, Down syndrome (DS) patients have extra copy of chromosome 21 where APP gene locates, and thus, over-expression of normal APP can also predispose individuals to develop AD (Kang et al., 1987; Olson and Shaw, 1969; Robakis et al., 1987; Tanzi et al., 1987). Clinical studies suggest that the majority of DS patients display an increased generation of Aβ from birth, which leads to the occurrence of classic AD neuropathology in their middle age (Goate, 2006). Our lab found that Aβ production is significantly increased in DS brain tissues and increased maturation of BACE1 contributes to neuritic plaque formation in the AD 2  pathogenesis in DS (Sun et al., 2006c). Furthermore, our lab showed that over expressed RCAN1 in DS resulted in neuronal apoptosis (Sun et al., 2011).  1.1.1.2  Presenilin Mutations In addition to APP gene, Presenilin 1(PS1) on chromosome 14q and Presenilin 2 (PS2)  on chromosome 1 also contribute to an early-onset of AD (Levy-Lahad et al., 1995; Schellenberg et al., 1992; Sherrington et al., 1995). Humans with PS1 and PS2 missense mutations exhibit a selective two fold increase in Aβ42 in plasma and skin fibroblast as measured in direct assays for Aβ40 and Aβ42 (Scheuner et al., 1996). Further investigations have identified over 100 PS1 missense mutations and three PS2 mutations worldwide that causes early-onset AD (Hardy, 1997). Moreover, studies indicate FAD typically correlate with a mutation on the PS1 gene. Often, this also causes the earliest AD onset (at age 50) with the most aggressive form (death at age 60) (Selkoe, 1997). Studies have also shown that PS1 mutation in transgenic mice crossed with human APP can substantially accelerate AD-like phenotypes in the offspring, where Aβ42 plaques are detected in the mice as early as 3–4 month after birth (Borchelt et al., 1996; Citron et al., 1997; Duff et al., 1996; Holcomb et al., 1998; Scheuner et al., 1996).  1.1.1.3  ApoE Polymorphism The ε4 allele of Apolipoprotein E gene (ApoE4) on chromosome 19q is another major  risk factor for late-onset of AD (Strittmatter et al., 1993). Immunohistochemistry studies of AD brain tissues from patients showed high percentages of ApoE protein in Aβ42 rich areas (Namba 3  et al., 1991). Genetic analyses also showed AD patients have a higher ε4 expression profiles as compared to the normal population (Namba et al., 1991). Individuals with the ε4 alleles have a significantly higher Aβ plaque burden and probability to develop AD in their 60s or 70s as compared to individuals with the ε2 and/or ε3 alleles (Saunders et al., 1993; Scheuner et al., 1996). Other studies have shown that offspring’s of APP transgenic mice and ApoE-deficient mice displayed a substantial reduction in cerebral Aβ level (Bales et al., 1997). Presently, however, the mechanism as to how ApoE4 increases Aβ deposition remains to be determined.  1.1.2  Sporadic Form of AD Efforts have been made during the past decades to indentify other risk factors in sporadic  AD cases while genetic risk factors contributing to the majority of familial AD cases have been clarified. 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).  1.1.2.1  Apolipoprotein Human apolipoprotein (apo) E is a major lipoprotein responsible for the metabolism, and  redistribution of lipids and the gene coding for it located on chromosome 19, generating three major protein isoforms, apoE2, apoE3 and apoE4 (Mahley, 1988; Rall et al., 1982). In particular, ApoE4 is associated with both late-onset FAD and SAD. ApoE4 has been particularly indicated to be associated with both late-onset familial and sporadic cases of AD, and the frequency of the 4  apoE ε4 allele is augmented in early-onset sporadic, late-onset familial, and common late-onset sporadic AD (Farrer et al., 1997; Poirier et al., 1995; Strittmatter et al., 1993; Wisniewski et al., 1994). ApoE is produced primarily by astrocytes and binds to lipid particles. It directs their catabolism through association with the low-density lipoprotein (LDL) receptor, the very low-density lipoprotein (VLDL) receptor, and the LDL-receptor-related protein (LRP) (Boyles et al., 1985; Pitas et al., 1987; 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 which play a role in the integrity and remodeling of the CNS (Mahley and Huang, 1999; Mahley and Rall, 2000). ApoE co-localizes with the extracellular amyloid deposits, affecting the polymerization and accumulation of Aβ (Fassbender et al., 2001; Ma et al., 1994; Namba et al., 1991; Saunders et al., 1993). Moreover, apoE4 can increase the number and density of amyloid fibers in brain (Schmechel et al., 1993). Additionally, it has been suggested that secreted Aβ is internalized through an apoE-dependent mechanism since the binding of full-length APP, sAPP, and Aβ to apoE is through the LRP-mediated pathway (Scharnagl et al., 1999). Further studies also indicate that apoE4 could modulate APP processing and Aβ generation through both the LRP-mediated pathway and domain interaction (Ye et al., 2005). These evidence supports the idea that apoE4 is a major risk factor for the development of AD (Corder et al., 1993).  5  1.1.2.2  Angiotensin I-Converting Enzyme 1 Since apoE can only contribute to 45-60% of the genetic risk for developing SAD and  7-9% for developing FAD, 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) was proposed as a linkage between AD and vascular risk (Elkins et al., 2004; Farrer et al., 1989; Korten et al., 1993). ACE1 is located on chromosome 17 within an insert/deletion (I/D) polymorphism in its 16th intron (Chapman et al., 1998; Farrer et al., 2000). Early studies demonstrated that 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, however, its levels are reduced in the cerebrospinal fluid (CSF) of AD patients (Kehoe, 2003). In vitro studies showed that ACE plays a role in degrading synthetic forms of Aβ and precluding the production of Aβ aggregates (Hu et al., 2001).  1.1.2.3  Non-genetic AD Risk Factors Since the risk of AD doubles every 5 years after the age of 65, aging is a major and  primary non-genetic risk factor for AD. Mutations on mRNA and oxidative damage associated with aging are proposed contributing factors to AD (Ganguli et al., 2000; Smith et al., 1995; van Leeuwen et al., 1998). Other non-genetic risk factors such as diabetes mellitus, atherosclerosis, high-calorie and high fat diets are also considered to contribute to the development of AD  6  (Mattson, 2003; Mayeux, 2003; Ott et al., 1999; Skoog, 1994; Stern et al., 1994). However, the relationship between some of these non-genetic factors and AD pathogenesis has not yet been fully established.  1.1.3  Classic Features of AD Several clinical and pathological features have been characterized in AD, including  neuronal and synapses loss, brain atrophy, extracellular Aβ deposition and intracellular neurofibrillary tangles. People with this disease will eventually develop progressive cognitive and functional impairments. The loss of neuron and synapses that mainly occurs in the cerebral cortex and certain sub-cortical regions results in the atrophy of those affected brain regions. MRI and PET studies in AD patients have shown the size reduction in their brains comparing to normal population. Neurofibrillary tangles (NFT) are abnormal fibers, which are the aggregation of hyperphosphorylated microtubule-associated tau protein (Grundke-Iqbal et al., 1986; Kosik et al., 1986; Wood et al., 1986). The aggregation of tau protein is attributed to the later stages of AD (Baker et al., 2000).  In addition to the tau protein hypothesis, the amyloid hypothesis is at the center of the research on pathogenesis of AD (Baker et al., 2000).  7  1.2  1.2.1  Amyloid Cascade Hypothesis of AD  Amyloid Precursor Protein (APP) Aβ is generated through a sequential cleavage of a protein called APP by β-secretase and  γ-secretase (Kang et al., 1987). APP is a type I integral membrane glycoprotein within a single transmembrane domain, a large exocytoplasmic domain and a short cytoplasmic tail. In the human genome, the APP gene is located on chromosome 21q21.2-3 and contains18 exons (Lamb et al., 1993). Among these amino acids, the Aβ sequence spans portions of the exocytoplasmic and transmembrane domain (Checler, 1995; Selkoe, 1991). APP is synthesized on membrane bound polysomes and is transported into the endoplasmic reticulum (ER). There, APP will undergo post-translation modification such as glycosylation, phosphorylation and tyrosin-sulfatation. Ultimately, the majority of the APP is transported to the Golgi with a small portion transported to the plasma membrane (De Strooper et al., 1993; Kuentzel et al., 1993; Sambamurti et al., 2002; Schubert et al., 1989b; Weidemann et al., 1989). Eight variants of APP are generated by alternative splicing of exons 7, 8, and 15 (Sandbrink et al., 1996). An APP that lacks exon 15 is a chondroitin sulfate proteoglycan. Exon 7 is a protease inhibitor while the function of exon 8 is not known. (Pangalos et al., 1995; Shioi et al., 1992; Tanzi et al., 1988). APP695, APP751 and APP770 are the dominant isoforms in cells and are cell specific, with disparaging ratios between tissue types (Goldgaber et al., 1987; Matsui et al., 2007; Selkoe et al., 1988; Tanaka et al., 1989; Yoshikai et al., 1990). APP695 exhibits a  8  heightened expression profile in neuronal cells as compared to APP751 and APP770 expression profiles in general. (Haass et al., 1991). The homologous proteins of APP have been indentified in human and other species. APP like protein 1 (APLP1) and APLP2 have been found in human, appl1 in Drosophila and apl-1 in C.elegans. All of these APP proteins share a similar structure, including a large extracellular domain and a short cytoplasmic region, however, only APP contains the coding sequences for Aβ (Thinakaran and Koo, 2008; Zheng and Koo, 2006). Early studies demonstrated that APP processing and secretion are highly regulated. 30% of APP is cleaved to sAPPα or sAPPβ and C-terminal fragments (CTFs). sAPPα and sAPPβ are excreted whereas CTFs are retained in the cells. The remainder uncleaved APP is degraded in lysosomes or other cellular organelles (De Strooper et al., 1999; Esch et al., 1990; Farzan et al., 2000; Weidemann et al., 1989). Previous studies showed that APP contains heparin-binding sites and integrin-binding sites, suggesting that it might have a function in cell adhesion (Clarris et al., 1997; Ghiso et al., 1992; Yamazaki et al., 1997). APP might also be a receptor that signals via trimetric Go involving in AD pathogenesis (Yamatsuji et al., 1996). Transgenic studies in mouse showed that knockout of the APP gene only produces mild effects on neuronal function. In contrast, triple knockouts of the APP gene and its two homologues resulted in abnormal layering of the cerebral cortex (Herms et al., 2004; Kerr and Small, 2005; Zheng et al., 1995). These results suggest that the APP family of proteins plays a role in the development of the nervous system (Kerr and Small, 2005). Presently, the precise function of APP is not known. 9  APP695 isoform lacks the Kunitz-type protease inhibitor (KPI) domain and the OX-2 homology domain (Donnelly et al., 1989). APP contains a larger ectodomain and a short cytoplasmic tail with the N-terminus toward the lumen (Kang et al., 1987). The transmembrane domain is suggested to function either as a receptor or a mediator of extracellular interaction (Breen et al., 1991; Schubert et al., 1989a). The ectodomain of APP has orphic functions. The heparin-binding domain near the N-terminus has been implicated to stimulate the neurite outgrowth-promoting effects of APP (Small et al., 2001).  1.2.2  1.2.2.1  Enzymes in APP Processing α-Secretase Under normal conditions, the majority of APP is cleaved by α-secretase, which is  recognized as the principal pathway to process APP (Sisodia, 1992; Walter et al., 2001). This principal pathway is non-amyloidogenical. APP is cleaved by α-secretase at the α site within the critical Aβ peptide region generating sAPPα and C83. Thus, through this pathway, the formation of Aβ is prevented (Busciglio et al., 1993; Hartmann, 1999). α-secretase activity is mediated by one or more enzymes from the family of disintegrin and metalloproteinase domain proteins (ADAM). These enzymes are involved in many biological processes including fertilization and neurogenesis (Asai et al., 2006; Evans et al., 1998; Weskamp et al., 2002). Studies have shown that ADAM9, 10, 17 and 19 exert α-secretase activities (Asai et al., 2003; Fahrenholz et al., 2000; Tanabe et al., 2007). In vitro evidence has 10  shown that ADAM10 can cleave APP within Aβ between APP residues Lys 16 and Leu17 and the Flemish mutation of APP (Ala21 to Gly21) can reduce this cleavage event (Lammich et al., 1999). Further investigations in an AD mouse model demonstrated that over-expression of ADAM 10 reduces Aβ level and cognitive deficits (Postina et al., 2004).  1.2.2.2  β-Secretase β-site amyloid precursor protein cleaving enzyme (BACE1) is a 501 amino acid type 1  transmembrane aspartic protease with the highest activity in neuronal cells in the brain (Haniu et al., 2000; Walter et al., 2001; Yan et al., 1999). BACE1 is predominantly localized in acidic intracellular compartments with its active site in the lumen of the vesicles (Vassar et al., 2009). The majority of BACE1 protein is detected in the Glogi and in endosomal compartments where APP is located (Huse et al., 2000). In AD brain, BACE1 protein level and its activity are higher than normal humans. Soon after the discovery of BACE1, its homolog protein BACE2 is identified. This promoted speculation that BACE2 is also a β-secretase. Later research on BACE1 knocked out (BACE1-/-) mice, however, excluded this possibility and concluded that BACE1 was the only β-secretase in vivo. When BACE1-/- mice were bred with APP transgenic mice, Aβ production and cognitive deficits were disappeared. Subsequent studies characterized BACE2 as a novel α-secretase without involving in Aβ production (Sun et al., 2006b). Presently, BACE1 is considered as the only β-secretase in vivo.  11  The 30kb BACE1 gene of nine exons is a strong candidate responsible for sporadic AD. BACE1 promoter contains multiple transcription factor binding sites such as interferon, perxisome proliferate activated receptor and hypoxia inducible factor (Cole and Vassar, 2008). These sites regulate transcription activity of BACE1. Furthermore, stress, TBI and cellular consequences of vascular disease like hypoxia and oxidative stress can elevate BACE1 mRNA levels to affect APP processing and Aβ levels (Cole and Vassar, 2007; Sun et al., 2006a; Tong et al., 2005). Therefore, BACE1 is an important therapeutic target for AD treatment (Cole and Vassar, 2008; Sambamurti et al., 2004). BACE1 cleaves APP at the N-terminus of the Aβ domain releasing a soluble APPβ fragment (Turner et al., 2003). Two BACE1 cleavage site are present on APP: a Asp+1 site and a Glu+11 site. BACE1 cleavage at the Asp+1 site generates a 4kDa Aβ1-40/42 peptide via the amyloidogenic pathway (Cai et al., 2001; Farzan et al., 2000; Vassar et al., 1999). BACE1 cleavage at the Glu+11site generates a 3kDa Aβ11-40/42 peptide. This peptide is likely processed by the Golgi apparatus in a manner that is species specific (Fluhrer et al., 2002; Vassar et al., 1999). Matured BACE1 is a 70KDa protein. It is synthesized in the endoplasmic reticulum and transported to Golgi to be processed by a furin-like protein convertase and a series of glycosylation to produce the mature BACE1(Haniu et al., 2000; Vassar, 2004). Glycosylation can affect BACE1’s protease activity while palmitoylation at C-terminal affects BACE1’s localization. BACE1, exhibits enhanced catalytic activities in its homodimeric form and functions with other proteins such as the reticulum/Nogo proteins and SorLA/LR11 to catalyze  12  APP. Ubiquitin proteasome has been suggested to be involved in the degradation of BACE1 (Cole and Vassar, 2007).  1.2.2.3  γ-Secretase γ-secretase is a membrane-bound aspartyl proteases that can cleave APP at its  intra-membrane region on the γ site to release Aβ1-40/42. Besides APP, more than 50 γ-secretase substrates have been discovered (Beel and Sanders, 2008), including Notch, the neuregulin binding partner ErbB4, N-cadherin, p75NTR, and others (Beel and Sanders, 2008). γ-secretase is a complex of at least four proteins: PS1 or 2, Nct, Aph-1 and Pen 2. γ-secretase matures in discrete steps. Nct and Aph-1 form a complex responsible for substrates reorganization. The heterodimer then binds sequentially to PS and Pen2 (Li et al., 2009; Winkler et al., 2009). Studies in early-onset AD indicated that PS mutations elevate the amyloidogenic Aβ42 deposition, suggesting that PS may play a role in γ-secretase cleavage (Cruts et al., 1995; Hardy and Selkoe, 2002; Hutton et al., 1996; Sherrington et al., 1995). PS1 and PS2 are highly homologous. Both proteins are important in maintaining cortical function in the brain and might be involved in calcium signaling (Feng et al., 2004; Newman et al., 2007; Tu et al., 2006). PS1 and PS2 are essential to γ-secretase’s proteolytic activities. Inhibitors of PS1 or PS2 destroy γ-secretase’s activities (Esler et al., 2000; Kimberly et al., 2000; Li et al., 2000; Steiner et al., 1999). Furthermore, PS1 and PS2 knockouts reduceAβ1-40/42 levels, at the same time elevate  13  γ-secretase substrate levels (De Strooper et al., 1998; Herreman et al., 2000; Zhang et al., 2000b).  1.2.3  APP Processing  APP is processed by at least two distinct proteolytic pathways (Nunan and Small, 2000). In one pathway, APP is cleaved by the enzyme called α-secretase at α site that is within the Aβ region (Esch et al., 1990). The cleavage of APP by α-secretase releases 83 amino acids (C83), which remains membrane associated, and secretes N-terminal fragment sAPPα. Recent research has indicated that ADAM10, which could process both constitutive and a regulated secretase activity, is the most likely to be α-secretase (Lammich et al., 1999). Additionally, increasing the activity of ADAM10 in double-transgenic mouse prevents the formation of Aβ. sAPPα will be secreted to the extracellular space while C83 will be cleaved by γ-secretase. Through this pathway, the formation of Aβ is prevented and thus, it is called non-amyloidogenic pathway. APP could also be cleaved by another enzyme called β-secretase. BACE1 has been shown to be β-secretase. There are two cleavage sites for BACE1. One site is at the N-terminal side of Asp+1 of Aβ sequence, by which C99 and sAPPβ will be released. sAPPβ is secreted to the extracellular while C99 is cleaved by γ-secretase generating Aβ within 40-42 amino acids that is Aβ40 and Aβ42. The aggregation of Aβ42 will form Aβ plaque in hippocampus and cortical region of brain resulting in neuronal death, dysfunction of synapses and memory loss. Thereby, this amyloidogenic pathway has been considered contributive to pathogenesis pathway of AD.  14  Another cleavage site by BACE1 is at Glu+11 within the Aβ region on APP. This cleavage will generate C89 and sAPPβ’. γ-secretase will cleave C89 releasing short Aβ peptide within 30-32 amino acids which are Aβ30 and Aβ32. The functions of these two shorter peptides are still unknown. Undergoing this pathway, no Aβ 42 has been generated, and thus, BACE1 cleaved APP at Glu+11 is also non-amyloidogenic.  15  Figure 1.1 APP processing Figure 1.1 APP processing. APP is sequentially processed by three enzymes through two different pathways. One is non-amyloidogenic in which APP is cleaved by α- secretase generating C83 which is subsequently cleaved by γ-secretase producing p3. In this pathway, the generation of Aβ is prevented. In amyloidogenic pathway, APP is first cleaved by β-secretase at Asp+1 site releasing C99 which is later cleaved by gamma-secretase producing Aβ40 and Aβ42. Another cleavage site for BACE1 has been characterized as Glu +11 on APP. BACE1 cleaved APP on Glu+11 will generate C89 which is then cleaved by γ- secretase releasing shorter Aβ peptide 11-40 or 11-42. Cleavage at Glu+11 site is non-amyloidogenic since no full length of Aβ has been generated. 16  1.2.4  Amyloid βProtein (Aβ)  Aβ is a 39-42 amino acid peptides generated through APP processing. It is a normal soluble product of cellular protein degradation. However, the extracellular deposition of aggregated Aβ has been considered to be pathological in AD. Aβ was first considered as an abnormal and toxic peptide generated only in the brains of aged people. APP transgenic mice also developed an AD phenotype (Haass et al., 1992; Hardy and Selkoe, 2002; Kang et al., 1987; Sandbrink et al., 1996; Selkoe, 2004). However, later researches suggest that Aβ is soluble and also exists in the body fluids of various species, and is secreted in the cell medium (Haass et al., 1992; Seubert et al., 1992). Aβ is generated through sequential cleavage of APP by β-secretase and γ-secretases. Its length can range from 39 to 42 amino acids. Aβ40 is the major species whereas only approximately 10% is Aβ42. In PS mutation, this ratio has been increased due to the mutations (Borchelt et al., 1996; Seubert et al., 1992). Evidence indicated that most Aβ is generated in subcellular localizations and subsequently secreted through exocytosis (Chow et al., 2010). Previous study indicated that the optimal BACE1 activity requires an acidic environment. Additionally, APP is decorated in Golgi which has been shown as the major site of APP residence in neurons at steady state with an ideal acidic environment and also as the major site for Aβ generation (Hartmann et al., 1997; Xu et al., 1997). Thus, this evidence suggests that the generation of Aβ requires a mature APP form and an acidic environment. After being generated in trans-Golgi network, Aβ could be subjected to the secretory pathway and released into the extracellular fluid (Caporaso et al., 1994; Nordstedt et al., 1993). The accumulation of Aβ42 in the hippocampus causing neuron loss and dysfunction of 17  synapses are prone to the development of early AD pathology in patients with mild cognitive impairments (Gouras et al., 2000). In familial AD cases and animal models of AD, Aβ level is increased .The deposition of Aβ can cause neuronal death and dysfunction of synapses. The sequence of Aβ was first determined in AD and trisomy 21 patients meningeal blood vessels (Glenner and Wong, 1984). It has been reported that the sequence of Aβ is amphipathic with a hydrophilic N-terminal and a hydrophobic C-terminal. Residues 1-28 locate in a soluble extracellular APP domain, while residues 20-42 locate within the transmembrane domain of APP (Selkoe, 2004). Several factors such as oxidative stress, inflammation and apoptosis have been reported affecting the formation and accumulation of Aβ (Hardy and Selkoe, 2002; Nakagawa et al., 2000; Roberson and Mucke, 2006; Soto, 2003). Previous studies indicated that Aβ undergoes conformational change from monomers to plaques. Different species within varies conformation have been isolated from postmortem tissue and body fluid such as cerebrospinal fluid (Cleary et al., 2005; Lesne et al., 2006; Lue et al., 1999; McLean et al., 1999). The monomers of Aβ are soluble amphipathic molecule generated from APP, in which random β-sheet and coil form have been observed (Barrow and Zagorski, 1991; Garzon-Rodriguez et al., 1997; Lazo et al., 2005; Selkoe, 2004; Xu et al., 2005; Zhang et al., 2000a). Aβ dimmers are localized intracellularly in vivo and in vitro with a hydrophobic core structure (Garzon-Rodriguez et al., 1997; Hilbich et al., 1992; Nakabayashi et al., 1998; Podlisny et al., 1995; Roher et al., 1996; Walsh et al., 2000). Evidence shows that soluble Aβ oligomers inhibit NMDA-mediated synaptic transmission causing spine and synapse loss. The extract from AD patients brain showed that Aβ dimmers are the most potent forms of Aβ oligomers (Selkoe, 18  1991). Comparing to Aβ40, Aβ42 is more hydrophobic and prone to fibril formation(Burdick et al., 1992). However, the function of other Aβ species and their aggregation still remain elusive.  1.3. Hypotheses and Specific Aims BACE1 can cleave APP at both β-secretase sites, Asp+1 and Glu+11, and generate different species of Aβ. However, the underlying mechanism of the cleavage site selection of BACE1 on APP processing is not well defined and its role in AD pathogenesis remains elusive. This thesis seeks to address these issues. Following are my working hypotheses and specific aims:  1.3.1 Hypotheses A. Aβ Glu+11 site is the major cleavage site of BACE1 on wild type APP. B. Shifting BACE1 cleavage site from Glu+11 to Asp+1 site plays an important role in AD pathogenesis.  1.3.2 Specific Aims Specific Aim 1 To examine APP processing and Aβ production by BACE1 in vitro.  Specific Aim 2 To study the aggregation and neurotoxicity of Aβ species generated through different BACE1 cleavage site in vitro  19  Specific Aim 3 To determine the role of BACE1 cleavage sites in AD pathogenesis in vivo  20  Chapter 2. Materials and Methods 2.1  Cell Culture  2.1.1  Culture Media Preparation 20E2 stable cell line which is embryonic kidney (HEK293) cells stably expressing  Swedish mutant APP695) (Qing et al., 2004) and HAW stable cell line which is HEK293 cells stably expressing Wild type APP695 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 1 mM of sodium pyruvate, 2 mM of L-glutamine and 50 units of Penicillin and 50 g of Streptomycin and 75μg/mL Geneticin (all from GIBCO BRL, Burlington, Ontario). BAW stable cell line which is HEK293 cells stably expressing Wild type APP695 and BACE1, and 2EB2 cell line that is (HEK293) cells stably expressing Swedish mutant APP695 and BACE1 were cultured in the same cell medium described above with additional 75μg/mL Zeocin.  2.1.2  Trypsinization Once cells reached a confluency of 80-90%, culture medium was removed and cells were  washed by room-temperature Dulbecco's Modified Eagle Medium (Gibco) and treated with Trypsin-EDTA (Gibco). Cells were suspended in fresh culture medium, counted and seeded for transfection. All cell lines were maintained in 37oC incubator containing 5% CO2.  2.1.3  Cell Transfection  2.1.3.1  Calcium Phosphate Transfection  Cells were maintained at 37 C in an incubator containing 5% CO2. Cells were seeded onto 35cm plate one day prior to transfection and grown to approximately 70% confluence by 21  the day of transfection. Cells were transfected with 0.5 μg of plasmid DNA per well using 1 μl Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). For HEK293 cells: 24 hours before transfection, HEK293 cells were seeded in 60mm plates at the density of 1.0 ×10 6 cells in 4 mL culture media. Mixed 10μg DNA with 125μL 0.5M CaCl2 to make up to 250μL DNA-CaCl2 with autoclaved distilled water. DNA-CaCl2 was added to “bubbled” 2X HEBS (1.636g NaCl,1.19g HEPES ,0.0213g Na2HPO4, anhydrous in 100mL distilled water and pH was adjusted to 7.00 with NaOH solution) solution drop by drop. DNA-CaCl2-HEBS mixture was placed at room temperature for 25 minutes and then added to each plate. Culture media were changed after 24 h and cells were harvested 48 h after transfection.  2.2  Western Blotting Cells were harvested and lysed in RIPA-DOC lysis buffer containing 0.15M NaCl,  0.05M Tris-HCl pH 7.2, 0.1% SDS, 1% sodium deoxycholate, and 1% triton x-100. Lysates were sonicated and cellular debris was pelleted by centrifugation at 14,000xg for 10 minutes. SDS-PAGE gel electrophoresis was performed using 16% tris-tricine and 10% glycine gels. For Western blotting, proteins were transferred from the gel onto a PVDF membrane (Immobilon-FL, Millipore, MA, USA). Membranes were probed with polyclonal C20 (1:1000) antibody against the last 20 C-terminal amino acids of APP antibodies or anti-9E10 (1:10) in milk containing PBS with 0.1% Tween-20 and 0.01% sodium azide, overnight at 4ºC. The membranes were then washed and incubated with goat anti-mouse 800CW or goat anti-rabbit 680CW (LI-COR  22  Biosciences).  Membranes were then scanned at 700 and 800nm wavelengths using an Odyssey  Infrared Imaging System (LI-COR Biosciences).  2.3  Primary Rat Cortical Neuronal Culture The day before plating of the isolated neurons, 96-well plates were coated with a  10ug/mL solution of poly-D-lysine (PDL) (Sigma) and incubated in the dark overnight at room temperature. On the day of plating, PDL was removed from the plates, wells were washed with sterile ddH2O to remove traces of PDL solution, after which the ddH2O was removed and plates were left to dry. For the culture of primary neurons, E18-E19 embryos were isolated from timed pregnant Sprague-Dawley rats. The study complied with all institutional policies and was approved by the ethics committee of the Animal Care Centre of The University of British Columbia. The heads of the embryos were separated from the bodies and were placed in a separate sterile dish where the skulls were opened and the brains removed. The freshly isolated brains were placed immediately in ice-cold dissection buffer containing 1X Hank’s Balanced Salt Solution without Ca+2 and Mg+2 (GIBCO), 20mg/mL glucose, 5mg/mL sucrose and 3.56mg/mL Hepes (Sigma, St. Louis, MO). The cortex of each hemisphere was removed and placed in fresh ice-cold dissection buffer.  The dissection buffer was removed and the cortices  were incubated with a 0.25% solution of trypsin at 37ºC for 30 minutes.  The cortices were then  washed with DMEM containing 10% FBS to inhibit and remove traces of trypsin.  The tissue  was then suspended in plating media containing 0.5mM Glutamax-I, 2% B-27 (GIBCO), and 25μM glutamic acid (Sigma) in Neurobasal Media (GIBCO). The suspension was centrifuged at  23  2,000 rpm for 1 minute. Plating media was removed and the tissue was resuspended in fresh plating media. The tissue was passed through a 10mL pipette several times to facilitate dissociation of the tissue after which it was further triturated by placing a 100μL pipette tip onto a 10mL pipette.  Once dissociation was observed to be complete, cell viability was assessed by  trypan blue staining. Cells were seeded onto 96-well plates at a density of 5 x 104 cells per well and cultured in a 37o C incubator containing 5% CO2and 21% O2. This was counted as day in vitro 1 (DIV1). The next day, and every 3rd day thereafter, half of the culture media was replaced with feeding media containing 0.5mM Glutamax-I and 2% B-27 in Neurobasal Media.  2.4  Preparation of Aβ Fibrils Sterile ddH2O was added to lyophilized A to have a final concentration of 1mM. Pippet  up and down 40-50 times to ensure that A completely dissolves in ddH2O. Incubate A mixture at 37 oC for 1 hour. Add same volume of sterile 1 X PBS (1:1 dilution) to the mixture and pippet up and down to have it mixed well. The stock concentration of A is 0.5 mM. Age A at 37 oC for 4 days to allow the stable growth of A fibrils in water/PBS. To check A fibrils: dilute 2 l of 0.5 mM aged A in 200 l ddH2O and check under microscope at 100 X objective lens. 2.5  MTT(Thiazolyl Blue Tetrazolium Bromide) Assay Plate 500-10,000 cells in 200ul media per well in a 96 well plate. Leave 8 wells empty for  blank controls. Incubate (37C, 5% CO2) overnight to allow the cells to attach to the wells. Add aggregated Aβ species at different concentration to each well. Incubate (37C, 5% CO2) for 3 days. Make 2ml or more of MTT solution per 96 well plate at 5mg/ml in PBS. Add 20ul MTT solution to 24  each well. Place on a shaking table, 150rpm for 5 minutes, to thoroughly mix the MTT into the media. Incubate (37C, 5% CO2) for 1-5 hours to allow the MTT to be metabolized. Dump off the media and then resuspend formazan (MTT metabolic product) in 200ul DMSO (Dimethyl sulfoxide). Place on a shaking table, 150rpm for 5 minutes, to thoroughly mix the formazan into the solvent. Read optical density at 560nm and subtract background at 670nm. Optical density should be directly correlated with cell quantity.  2.6  Aβ ELISA Count cells for each of the cell lines and seed them equally to 10cm plate. Once cells  reached a confluency of 80%, change the cell media to DMEM only and culture for another 3 days. Collect cells medium within 15ml tube and centrifugate at 2000rpm for 5 mins in order to get rid of dead cells in the medium. Then collect the medium to new 15ml tubes. The cortical region of human brain has been cut and homogenized within 2% SDS RIPA DOC buffer (1% Triton X-100, 2% SDS, 1% sodium deoxycholate, 0.15M NaCl, 0.05 M Tris-HCl pH 7.2 and Roche protease inhibitor tablet). BetaMark™ Beta-Amyloid x-40 Chemiluminescent ELISA Kit (SIG-38950) and BetaMark™ Beta-Amyloid x-42 Chemiluminescent ELISA Kit (SIG-38952) from Covance were used to detect Aβ 40 and Aβ 42 level. 2.7  Data Collection and Statistics Data were collected in the following ways: using the Fluoroskan Ascent software for  analysis of MTT assays (Thermo Fisher Scientific, Waltham, MA), Data were analyzed,  25  organized into graphical form, and statistics generated by using GraphPad Prism 4 software (GraphPad Software, La Jolla, CA).  26  Chapter 3. Results 3.1 3.1.1  Examination of APP Processing and Aβ Production by BACE1 in vitro Generation of Stable Cell Lines It has been known that APP processing undergoes at least two distinct pathways. In one  pathway, APP is cleaved by α-secretase whereas in the other pathway, APP is processed by β-secretase. In order to examine APP processing and Aβ production in vitro, we have first established four HEK293-based stable cell lines. HAW stably expresses wild type (wt) APP695 (8.7 fold increase of wtAPP695 expression relative to non stable cells, p<0.001) (Figure 3.1A). BAW stably expresses wtAPP695 and BACE1 (8.3 fold increase of wtAPP695 expression and 21 fold increase of BACE1 expression in BAW cell line relative to non-transfected cell line, p<0.001) (Figure 3.1 B). 20E2 stably expresses Swedish mutant (swe) APP695 (7.6 fold increase of sweAPP695 expression relative to non-stable cells,p<0.01) (Figure 3.1 C), and 2EB2 stably expresses sweAPP695 and BACE1 (comparing to non-transfected cell lines, 6.4 fold increase of sweAPP695 expression in 2EB2 cell line, p<0.01, for the expression of BACE1, 9.1 fold increase in 2EB2 cell line, p<0.001) (Figure 3.1 D).  27  Figure 3.1. A Generation of HAW cells. HEK293 cell were stably transfected with APPwt plasmid generating HAW cell. The cells have robust expression of APPwt. APP protein was detected by C20 antibody. ***p<0.001, by student’s t-test, n=3  Figure 3.1. BGeneration of BAW cells. HEK293 cell were transfected with APPwt and BACE1 plasmid generating BAW cell. The cells stably expresses APPwt and BACE1 . APP was detected by C20 antibody and BACE1 was detected by 9E10..***p<0.001, by student’s t-test,n=3  28  Figure 3.1. C Generation of 20E2 cells. HEK293 cell was stably transfected with APPswe plasmid and the cells overexpress APPswe. APP protein was detected by C20 antibody.**p<0.01, by student’s t-test, n=3  Figure 3.1. D Generation of 2EB2 cells. HEK293 cells were stably transfected with APPswe and BACE1. The 2EB2 cells overexpress APPswe and BACE1. APP and BACE1 protein were detected by C20 and 9E10 antibody, respectively. **p<0.01, by student’s t-test, n=3  29  3.1.2  Examination of APP Processing and Aβ Production by BACE1 in Swedish and Wildtype APP Cells To study the processing of wild-type and Swedish mutant APP, the production of APP CTFs  generated by different stable cell lines were examined by Western blot. Our results showed that major CTF products in HAW cells, the wtAPP over expressing HEK293 cells, were C83, the α-secretase product (Figure 3.2A). In BAW cells, over expressing wtAPP 695and BACE1, the majority of CTFs are C89 while minors of C99 has also been detected and very few C83 was generated (Figure 3.2 B). In 20E2 cell, over expressing sweAPP695, the main CTF is C83 (Figure 3.2 C). In 2EB2 cell, over expressing swe APP695 and BACE1, the major CTFs are C99 whereas minors are C89 (Figure 3.2 D).  Figure 3.2. A C83 is the main APP processing product in cells expressing wild type APP. Cell lysates from HAW cells were analyzed by Western blot. C83 and C89 protein were used as markers for CTF. CTFs were detected by C20 antibody and β-actin was used as control.  30  Figure 3.2. B The majority of CTFs are C89 and minors of C99 in cells expressing wild type APP and BACE1. Cell lysates from BAW cells were analyzed by Western blot. C83 and C89 protein were used as markers for CTF. CTFs were detected by C20 antibody and beta-actin was used as control.  Figure 3.2.C C83 is the main APP processing product in cells expressing Swedish APP. Cell lysates from 20E2 cells were analyzed by Western blot. C99 protein was used as markers for CTF. CTFs were detected by C20 antibody and beta-actin was used as control.  31  Figure 3.2. D The majority of CTFs are C99 and minors of C89 in cells expressing Swedish APP and BACE1. Cell lysates from 2EB2 cells were analyzed by Western blot. C83 and C89 protein were used as markers for CTF. CTFs were detected by C20 antibody and beta-actin was used as control  Comparing CTFs generated by these four stable cell lines, APP is cleaved by BACE1 differentially. The main CTFs generated by HAW and 20E2 cells are C83. BAW cells mainly generate C89 (1.7 fold increase of C89 relative to C99 in BAW cells, p<0.01) (Figure 3.2 E-b). 2EB2 cells produce the vast majority of C99 (2.2 fold increase of C99 relative to C89, p<0.01) ((Figure 3.2 E-c). To confirm our CTFs result, N-terminal fragments which are secreted form of APP (sAPP) are also detected by Western blot. Cell medium from each of the cell lines have been collected after 3 days culturing with FBS-free DMEM cell medium. HAW and 20E2 cells generate the longest secreted APP form which is sAPPα (Figure 3.2 E-d). BAW cell produce sAPPβ’, 2EB2 cell generate the shortest secreted APP form which is sAPPβ.  32  Figure 3.2.E APP is cleaved by BACE1 differentially. (a) The majority of CTFs in BAW cells are C89 (b), while the main CTFs in 2EB2 cells are C99 (c). (d) For the secreted form of APP, HAW generates sAPPα, BAW generates sAPPβ’, sAPPα is longer than sAPPβ’; 20E2 generates sAPPα. 2EB2 generates sAPPβ. sAPPα is longer than sAPPβ.**P<0.01, by student’s t-test, n=3. Further comparing CTFs results between each of the cell lines, we found that without BACE1 over-expression, the major CTFs are C83. In wt APP695 and BACE1 over-expression cell lines, β’-Glu+11 site is preferred by BACE1 (Figure 3.2F-a). In sweAPP695 and BACE1 over-expression cell lines, β-Asp+1 site is the major cleavage site for BACE1 (Figure 3.2 F-b). To confirm this result, we compared the CTFs of BAW and 2EB2 shown in Figure 3.2G-a. C89 level significantly increase in BAW cells comparing to 2EB2 cells (2.4 fold increase of C89 level relative in BAW cells relative to 2EB2 cells, p<0.01) (Figure 3.2G-b). The level of C99 33  dramatically increase in 2EB2 comparing to BAW cells (1.7 fold increase of C99 level in 2EB2 cells compare to BAW cells, p<0.01) (Figure 3.2-c)  Figure 3.2. F CTF generations. Majority of wt APP695 is cleaved by BACE1 at β’-Glu+11 site in BAW cells, while the majority of swe APP695 is cleaved by BACE1 at β-Asp+1 site in 2EB2 cells. C83 is major products in HAW and 20E2 cells, CTF is detected by C20 antibody.  34  Figure 3.2. G CTF generation in BAW and 2EB2 cells. The cell lysates were analyzed by Western Blot and the C89 level was significantly increased in BAW cells (b). C99 was dramatically increased in 2EB2 cells compared to BAW cells(c). **p<0.01, by student’s t-test, n=3  3.1.3  Aβ Production was Affected by APP Mutation and BACE1 To examine the effect of APP Swedish mutation and BACE1 overexpression on Aβ  generation, conditioned media were collected from the stable cells. Aβ level was measured by ELISA analysis. In HAW and BAW cells, both Aβ 40 and Aβ42 significantly increased in BAW cells shown in Figure 3.3A (for Aβ40, 1816+ 157 and 5876 + 426 pg/ml of HAW and BAW, respectively, p<0.05, for the level of Aβ42, 1454+ 581 and 5361+ 319 pg/ml of HAW and BAW respectively, p<0.05). In 20E2 and 2EB2 cells, Aβ 40 and Aβ42 increase in 2EB2 cells, however,  35  there is no significantly difference in between these two cell lines (5541+ 88 and 12964+ 3390 pg/ml of 20E2 and 2EB2 for Aβ40 level, p=0.24 For Aβ42, 1283+ 266 and 5143 + 1231 pg/ml of 20E2 and 2EB2, p=0.3) (Figure 3.3 B).  AA  B  *  *  *  *  *  D  C  **  ** **  Figure 3.3  Figure 3.3 Aβ level was measured by ELISA. Aβ production analysis. Both Aβ 40 and Aβ42 level were significantly increased in BAW cells compared to HAW cells (A). Compared to 20E2 both Aβ 40 and Aβ42 level have dramatically increased in 2EB2 cells (B). Aβ 40 and Aβ42 levels have proportionally increased in 20E2 cells comparing to HAW cells (C). Additionally, Aβ 40 and Aβ42 were also significantly increased in 2EB2 cells compared to BAW cells (D). *P<0.05, **P<0.01, by student’s t-test, n=3  36  3.2  In vitro Study of Aggregation and Neurotoxicity of Different Aβ Species Generated by Differential BACE1 Cleavages  3.2.1 Aβ Aggregation Since our studies showed that BACE1 cleave mutant and wild type APP differentially, resulting in the generation of different Aβ species are also generated. In order to study the aggregation and neurotoxicity of the different Aβ species, five Aβ peptides based on the different cleavage sites were designed and synthesized. Cleavage of APP at Asp+1 site by BACE1 generates Aβ40 and Aβ42 containing 1 to 40 or 42 of Aβ, whereas cleavage of APP at Glu+11 generates Aβ30 and Aβ32 containing 1 to 30 or 32 of Aβ. Aβ42G and Aβ32G are Aβ42 and Aβ32 labelled with FITC at its N-terminus, respectively; and Aβ40R and Aβ40R are Aβ40 and Aβ30 labelled with rhodamine at its N-terminus, respectively shown in table 3.1 Peptides Aβ42G Aβ40R Aβ32G Aβ30R  Sequences FITC-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA Rhodamine-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV FITC-EVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA Rhodamine-EVHHQKLVFFAEDVGSNKGAIIGLMVGGVV  Table 3.1 Sequence of synthesized Aβ peptides  Aβ is generated from APP by sequential cleavage mediated by β-secretase and γ-secretase. Most Aβ form secreted from cells under normal condition is soluble and non-toxic. The fibrilar form of Aβ resulted from its aggregation causes neuronal degeneration during AD 37  pathogenesis (Yankner et al., 1990). Increased Aβ level may trigger its aggregation. However the underlying mechanism and its dynamic are still unclear. To examine the dynamic of aggregation of the Aβ species generated from two β-secretase cleavage sites by BACE1, we incubate Aβ peptides at 37°C for 4 days at the concentration of 0.5mM. The morphology change was observed by 100x objective lens during 4-days incubation period. The results are shown in Figure 3.4 A. On day 1and day 2, no aggregated peptides have been observed. On day 3, Aβ42 and Aβ40 have been found partially aggregated to form fibrillized formation. On day 4, large amount of Aβ42 and Aβ40 have been found aggregated as fibrillar forms. However, no aggregated forms of Aβ32 and Aβ30 have been observed. To further confirm our findings, after 4-days incubation at 37°C, Atomic Force Microscope has been used to detect the aggregation form of Aβ species (Figure 3.4 B). These results suggest that the majority for amyloid form Aβ32 or Aβ30, the cleavage products of wildtype APP by BACE1 under normal condition, is soluble form, while the full length amyloid form Aβ42 or Aβ40, the main cleavage products of Swedish APP by BACE1 is prone to aggregation.  38  Figure 3.4 A Aggregation of Aβ species. The morphology change has been observed by 100x objective lens during 4-days incubation. On day 1and day 2, no aggregated peptides were observed. On day 3, Aβ42 and Aβ40 have been found partially aggregated to form fibrillized formation. On day 4, large amount of Aβ42 and Aβ40 have been found aggregated as fibrillized form. However, no aggregated forms of Aβ32 and Aβ30 observed under 100x objective lens. 39  Figure 3.4 B Observation of Aβ species aggregation under Atomic Force Microscope. a)and b) No fibirlized form of Aβ32 were observed. c) and d) Aβ42 is highly aggregated and fibrilized.  3.2.2  Neurotoxicity of Aβ Species To study the neurotoxicity of Aβ species, MTT assay was performed. Aggregated Aβ was  diluted to different concentration: 10nM, 50nM, 250nM and 500nM to treat rat primary neuron at day 10. Figure 3.5 shows that 500 nM Aβ42 and Aβ40 treatment cause dramatically neuronal death in primary neurons.100nM non-fibrilized Aβ peptides were used as control. 40  Figure 3.5 MTT assay. Primary neuron cells were treated with different Aβ species at concentration of 50nM, 100nM,2 50nM, 500nM. 100 nM non-fibrilized Aβ peptides were used as control.** p<0.01, n=3,by student’s t-test.  3.3 Determination of the Role of BACE1 Cleavage Sites in AD Pathogenesis in vivo To further confirm our in vitro data which suggest that BACE1 cleaved wt APP695 at Glu+11 while Asp+1 is the major site for BACE1 to process sweAPP695, human brain tissues have been used to evaluate CTFs in AD patients and control. Control group are the samples from normal subjects (n=16), AD group are the samples from sporadic AD patients (n=16). However, in these two groups, CTFs signal are too weak to have any meaningful results.  To examine the Aβ production in the brain, I performed ELISA assay. The brain tissues from cortical region of human brain were homogenized within 2% SDS RIPA DOC. The results show  41  a robust increase of Aβ42 level in AD patients samples (313+35 and 16804+972 pg/ml in control and AD, respectively, p<0.0001, n=16) (Figure 3.6)  Figure 3.6 The Aβ Elisa results show that Aβ42 level dramatically increased in AD patients samples.***p<0.0001,by student’s t-test, n=16.  3.4 Structural Study of APP To determine if Swedish mutation in APP changes the protein structure, resulting in BACE1 cleavage site shifting, my co-worker Xiaozhu Zhang cloned different APP fragments in wild type or carrying Swedish mutation and purified the proteins for structural analysis. Wt APP695 (sequence shown in table 3.2 A) and sweAPP 695 (sequence shown in table 3.2 B) will be used as template for each for the cloning. pET28 and pET22 vectors will be used as cloning vector. In collaboration with Structural Genomic Center Toronto the crystal structure of the proteins and its relation to BACE1 cleavage will be examined. 42  Wild type APP 695 Amino Acids Sequence 1 61 121 181 241 301 361 421 481  MLPGLALLLL TCIDTKEGIL EFVSDALLVP GVEFVCCPLA EADDDEDDED DKYLETPGDE QEKVESLEQE KYVRAEQKDR EEIQDEVDEL  AAWTARALEV QYCQEVYPEL DKCKFLHQER EESDNVDSAD GDEVEEEAEE NEHAHFQKAK AANERQQLVE QHTLKHFEHV LQKEQNYSDD  PTDGNAGLLA QITNVVEANQ MDVCETHLHW AEEDDSDVWW PYEEATERTT ERLEAKHRER THMARVEAML RMVDPKKAAQ VLANMISEPR  EPQIAMFCGR PVTIQNWCKR HTVAKETCSE GGADTDYADG SIATTTTTTT MSQVMREWEE NDRRRLALEN IRSQVMTHLR ISYGNDALMP  LNMHMNVQNG GRKQCKTHPH KSTNLHDYGM SEDKVVEVAE ESVEEVVRVP AERQAKNLPK YITALQAVPP VIYERMNQSL SLTETKTTVE  KWDSDPSGTK FVIPYRCLVG LLPCGIDKFR EEEVAEVEEE TTAASTPDAV ADKKAVIQHF RPRHVFNMLK SLLYNVPAVA LLPVNGEFSL  541 DDLQPWHSFG ADSVPANTEN EVEPVDARPA ADRGLTTRPG SGLTNIKTEE ISEV KMDAEF 601 RHDSGYEVHH QKLVFFAEDV GSNKGAIIGL MVGGVVIATV IVITLVMLKK KQYTSIHHGV 661 VEVDAAVTPE ERHLSKMQQN GYENPTYKFF EQMQN Table 3.2 A Wild type APP 695 Amino Acids Sequence  Swedish mutant APP 695 Amino Acids Sequence 1 61 121 181 241 301 361 421 481  MLPGLALLLL TCIDTKEGIL EFVSDALLVP GVEFVCCPLA EADDDEDDED DKYLETPGDE QEKVESLEQE KYVRAEQKDR EEIQDEVDEL  AAWTARALEV QYCQEVYPEL DKCKFLHQER EESDNVDSAD GDEVEEEAEE NEHAHFQKAK AANERQQLVE QHTLKHFEHV LQKEQNYSDD  PTDGNAGLLA QITNVVEANQ MDVCETHLHW AEEDDSDVWW PYEEATERTT ERLEAKHRER THMARVEAML RMVDPKKAAQ VLANMISEPR  EPQIAMFCGR PVTIQNWCKR HTVAKETCSE GGADTDYADG SIATTTTTTT MSQVMREWEE NDRRRLALEN IRSQVMTHLR ISYGNDALMP  LNMHMNVQNG GRKQCKTHPH KSTNLHDYGM SEDKVVEVAE ESVEEVVRVP AERQAKNLPK YITALQAVPP VIYERMNQSL SLTETKTTVE  KWDSDPSGTK FVIPYRCLVG LLPCGIDKFR EEEVAEVEEE TTAASTPDAV ADKKAVIQHF RPRHVFNMLK SLLYNVPAVA LLPVNGEFSL  541 DDLQPWHSFG ADSVPANTEN EVEPVDARPA ADRGLTTRPG SGLTNIKTEE ISEV NLDAEF 601 RHDSGYEVHH QKLVFFAEDV GSNKGAIIGL MVGGVVIATV IVITLVMLKK KQYTSIHHGV 661 VEVDAAVTPE ERHLSKMQQN GYENPTYKFF EQMQN Table 3.2 B Swedish mutant APP 695 Amino Acids Sequence  43  Construct ID  Vector  Primer name  APP:V289-K624  pET-28b  NH289~624F NH289~624R  APP:V289-K612  pET-28b  NH289-612F NH289-612R  APP:V289-K624:CH  pET-28b  CH289~624F CH289~624R  APP:V289-K612:CH  pET-28b  CH289-612F CH289-612R  APP:E19-K624:PM  pET-22b(+)  CH19~624F CH19~624R  APP: E19-K612  pET-22b(+)  CH19-612F CH19-612R  Primer sequence aaccacc cat atg GTT CCT ACA ACA GCA GCC AG ccgg aag ctt TTA TTT GTT TGA ACC CAC ATC aaccacc cat atg GTT CCT ACA ACA GCA GCC AG ccgg aag ctt TTA TTT TTG ATG ATG AAC TTC AT aca cca tgg aa GTT CCT ACA ACA GCA GCC AG ccgg aag ctt TTT GTT TGA ACC CAC ATC aca cca tgg aa GTT CCT ACA ACA GCA GCC AG ccgg aag ctt TTT TTG ATG ATG AAC TTC AT aca cca tgg ca GAG GTA CCC ACT GAT GGT AAT ccgg aag ctt TTT GTT TGA ACC CAC ATC aca cca tgg ca GAG GTA CCC ACT GAT GGT AAT ccgg aag ctt TTT TTG ATG ATG AAC TTC AT  Table 3.2 C Primer design for APP fragments cloning.  44  Chapter 4. General Discussion 4.1 Examination of APP Processing and Aβ Production by BACE1 in vitro. Previous studies indicated that APP is processed by at least two distinct proteolytic pathways (Nunan and Small, 2000). In one pathway, APP is cleaved by the enzyme called α-secretase at α site within the Aβ region (Esch et al., 1990). The cleavage of APP by α-secretase releases 83 amino acids (C83), which remains membrane associated , and secreted N-terminal fragment sAPPα. sAPPα is secreted to the extracellular space while C83 is cleaved by γ-secretase. APP could also be cleaved by BACE1 at the N-terminus of the Aβ domain releasing a soluble APPβ fragment (Turner et al., 2003). There are two BACE1 cleavage sites on APP, which are Asp+1 and Glu+11. APP cleaved by BACE1 at Asp+1 followed by γ-secretase could generate a 4kDa Aβ1-40/42, therefore, this is amyloidogenic pathway (Cai et al., 2001; Farzan et al., 2000; Vassar et al., 1999). In order to examine how APP is processed by BACE1 in vitro, we first establish four stable cell lines. Our CTFs Western blot results indicated that APP is cleaved by BACE1 differentially. Without overexpression of BACE1, both wt APP 695 over-expressing cell line and sweAPP695 cell line, shown as HAW and 20E2 cells respectively, the major CTF product is C83. This result indicates that APP is mainly cleaved by α-secretase. In BAW cells that overexpress both wt APP695 and BACE1, the major CTF product is also C89. These results support that the dominant BACE1 site in wt APP695 is the Glu+11 site. In contrast, 2EB2 cells, which over-express sweAPP695 and BACE1, the major CTF product is C99. This result suggests that the dominant BACE1 site in sweAPP695 cells is the ASP+1 site. Due to over-expression of BACE1, majority of  45  APP is cleaved by BACE1 instead of α-secretase. Therefore, no C83 has been detected in BAW cells and 2EB2 cells. Moreover, these results also suggest that the Swedish mutation of APP shifts the BACE1 cleavage site from the Glu+11 site to the ASP+1 site. Although it has been shown that BACE1 have two distinct cleavage sites on APP, known as Asp+1 and Glu+11 site, previous studies suggested that Asp+1 is the major cleavage site for BACE1 leading to AD(Farzan et al., 2000; Kerr and Small, 2005; Vassar, 2004). However, our findings suggest that Asp+1 is the major site for BACE1 only in cleaving Swedish APP695, whereas for BACE1 cleavage of wtAPP695 Glu+11 is the major cleavage site. Aβ ELISA results show that shifting of BACE1 cleavage site from Glu +11 to Asp+1 increase the production of Aβ40/42. Both Aβ40 and Aβ 42 levels dramatically increase in sweAPP695 over-expressing cell lines without affecting the ratio of Aβ 42 to Aβ40. This finding indicates that Swedish mutation of APP causes the proportionally increasing of Aβ level, resulting in formation of Aβ fibrillar in AD pathogenesis.  4.2 In vitro Study of Function and Aggregation of Aβ Species Aβ is generated through sequential cleavages of APP by β-secretase and γ-secretase. Cleavage at ASP+1 site by BACE1 will produce C99, which is subsequently cleaved by γ-secretase releasing Aβ 42 and Aβ40. Aβ32 and Aβ30 are released by the cleavage of C89 by γ-secretase, which is generated by BACE1 cleavage at Glu+11 site. It has been known that Aβ 42 and Aβ40 are able to aggregate to form fibril and become neurotoxic. Previous studies indicated  46  that Aβ undergoes conformational change from monomers to oligomers and forms plaques (Cleary et al., 2005; Lesne et al., 2006; Lue et al., 1999; McLean et al., 1999). Further, soluble Aβ oligomers inhibit NMDA-mediated synaptic transmission and causes spine and synapse loss. Aβ dimmers extracted from brain of AD patients are the most potent form of Aβ oligomers (Selkoe, 1991). Aβ42 is considered to be pathological in AD and compared to Aβ40, it is more hydrophobic and prone to fibril formation. Presently, however, the functions of the different Aβ species and the effects of their aggregation are still not known. To study the aggregation and neurotoxicity of the four Aβ species (Aβ42, Aβ40 Aβ32 Aβ30) in vitro, we designed and fluorescently labelled the four Aβ peptides. After a four day incubation period, we observed, under a 100X fluorescence microscope, that the Aβ42 is more prone to fibril formation than Aβ40. The shorter peptides, Aβ32 and Aβ30, are less prone to fibril formation. To study the function of aggregated Aβ species in cells, rat primary neuron was treated with Aβ species at different doses. MTT assay in rat primary neuron showed that 100 nM, 250 nM and 500 nM of Aβ42 and Aβ40 treatment result in neuronal death as compared to Aβ32 and Aβ30. This data indicates that high concentration of Aβ42 and Aβ40 are neuro-toxic, while Aβ32 and Aβ30 are less of neurotoxicity.  4.3 In vivo Studies To investigate the regulation of APP processing and Aβ production by BACE1, efforts have been made to detect CTFs by using Western blot in human brain tissues from the cortical region. However, our results showed that in both control and AD group, no CTFs were detected.  47  This is expected because under normal conditions, neither wtAPP 695 nor BACE1 is overexpressed. Therefore, detection of CTFs should be minimal. Our Aβ ELISA result showed that both Aβ40 and Aβ42 dramatically increased in AD group as compared to the controls. These increases were probably a result of heighted γ-secretase activities.  4.4 Conclusion In conclusion, our data clearly demonstrated that APP undergoes differential processing in the BACE1 pathway. With sweAPP695, the major cleavage site by BACE1 is ASP+1 site. Wt APP695 is cleaved by BACE1 at Glu+11 site. Therefore, Swedish mutant shifts the cleavage site of BACE1 from Glu+11 site to ASP+1 site. As a result, both Aβ40 and Aβ42 level have proportionally increased in Swedish mutant cell lines. However, the ratio of Aβ42 to Aβ40 is not affected. Comparing to Aβ32 and Aβ30, Aβ42 and Aβ40 are prone to fibril formation. High dosage of Aβ42 and Aβ40 are neuronal toxic to primary neuron. In vivo data indicate that the major CTFs are C99, which might be due to several factors such as mutations, polymorphisms, as well as non-genetic factors. Increasing of Aβ42 deposition might involve factors, such as increasing of γ-secretase activity. Our results provide a new insight on the function of BACE1 and the pathological pathway of AD and a major potential for the pharmaceutical development.  48  4.5 Future Direction While this thesis provides a novel insight: under normal conditions, the major BACE1 cleavage site is the Glu+11 site. More importantly, the results of this thesis provoke several interesting questions. First, according to our in vitro data, Swedish mutation shifts the cleavage site of BACE1. It will be interesting to study how does this mutation causes the shift of the cleavage site. We have cloned the APP fragments and suggest further crystal structural analysis will provide structural explanations for this shifting. Secondly, our in vitro data show that the vast majority of wt APP695 is cleaved by BACE1 through the non-amyloidogenic pathway, however, the in vivo data suggest that most of wt APP695 is cleaved by BACE1 via amyloidogenic pathway causing an increase of C99 levels. Further experiment should be done to investigate the underlying cause of this difference. ApoE4 is potentially an important factor, however, other factors might also contribute to this difference. Finally, we have collected the cell media from our four stable cell lines for mass spectrophotometer analysis. We want to characterize Aβ species from different APP processing pathways. This analysis will support our previous studies and aid in our understanding of how BACE1 differentially processes APP.  Aβ generation is initiated by BACE1, and thus, further investigation of the role of BACE1 in APP processing will provide valid drug target for lowering Aβ levels in prevention and treatment of AD. Furthermore, understanding the molecular mechanisms of APP processing will enhance the development of novel AD therapies and provide insights of etiology of AD.  49  References Alzheimer, A. (1906). Über einen eigenartigen schweren Erkrankungsprozeßder Hirnrinde. Neurologisches Centralblatt 23, 1129-1136. Alzheimer, A. (1907). Über eine eigenartige Erkrankung der Hirnrinde. Allgemeine Zeitschrift für Psychiatrie und Psychisch-Gerichtliche Medizin 64, 146-148 Asai, M., Hattori, C., Iwata, N., Saido, T.C., Sasagawa, N., Szabo, B., Hashimoto, Y., Maruyama, K., Tanuma, S., Kiso, Y., et al. (2006). 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