Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The role of inflammation and amyloid beta in Alzheimer disease pathology Dickstein, Dara L. 2004

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

Item Metadata

Download

Media
831-ubc_2005-99466x.pdf [ 16.13MB ]
Metadata
JSON: 831-1.0092329.json
JSON-LD: 831-1.0092329-ld.json
RDF/XML (Pretty): 831-1.0092329-rdf.xml
RDF/JSON: 831-1.0092329-rdf.json
Turtle: 831-1.0092329-turtle.txt
N-Triples: 831-1.0092329-rdf-ntriples.txt
Original Record: 831-1.0092329-source.json
Full Text
831-1.0092329-fulltext.txt
Citation
831-1.0092329.ris

Full Text

THE R O L E OF INFLAMMATION AND A M Y L O I D BETA IN A L Z H E I M E R DISEASE P A T H O L O G Y by Dara L . Dickstein B . S c , Y o r k University, 1997 A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR T H E D E G R E E OF D O C T O R OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Genetics) W e accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A November 2004 © Dara L y n n Dickstein, 2004  Abstract Alzheimer disease ( A D ) is the most common form o f dementia.  Due to longer  life-spans the number o f affected individuals is expected to triple over the next few decades. A s a consequence, a great deal o f research is focused on determining the many processes by which the disease manifests as well as in discovering biomarkers and therapeutics to aid in diagnosis and disease prevention. The neuropathological hallmarks of A D include extracellular deposits o f amyloid into senile plaques, accumulation o f abnormal Tau filaments into neurofibrillary tangles, extensive neurodegeneration and inflammation. Although significant advances have been made in A D neurodegeneration, there still remain many unanswered and unforeseen aspects to the disease. It has been established that microglia, the immune cells o f the brain, become activated in response  to amyloid; however, the precise intracellular responses o f  microglia to amyloid and the relationship between microglia and amyloid deposition or clearance is unresolved.  There have been many genes identified whose expression is  upregulated in activated microglia and many o f them have been proposed to be used as markers for inflammation.  It has been demonstrated in humans that serum levels o f  melanotransferrin (p97), an iron binding molecule, is elevated in individuals affected with A D and that it is the activated, plaque-associated microglia that are responsible for this upregulation.  This thesis further investigated the association between microglial  activation and p97 gene expression and found that the levels o f p97, both m R N A and protein, are increased in activated microglia in culture. The change in gene expression occurred largely in response to amyloid treatment possibly by the regulation o f the AP-1 transcription factor downstream o f the p38 mitogen-activated protein kinase pathway.  n  Moreover, p97 expression was altered by the treatment o f activated microglia with antiinflammatory drugs indicating that p97 may be used as a marker specific for amyloidinduced inflammation. The production and degradation o f amyloid in the brain appears to be in a strict equilibrium. In A D , it is thought that the production o f amyloid occurs at a faster rate than its removal and degradation and it is this shift in equilibrium that leads to plaque development.  This study addressed the role o f microglia in amyloid plaque formation  using an A D transgenic model mouse that exhibits dysfunctional microglia. These mice accumulated amyloid deposits at the same rate as A D model mice however, limited numbers o f mice did not allow for definite conclusions.  Interestingly, these mice also  displayed a shift in amyloid distribution, as indicated by increased vascular deposits, whereas normal A D model mice did not. Microglial activation and subsequent removal o f amyloid deposits is one of the mechanisms suggested to explain the success o f the amyloid beta vaccination treatment protocols. Immunization with amyloid and anti-human amyloid antibodies has resulted in the decrease in amyloid plaque  burden, neurodegeneration,  pathology and cognitive and memory deficits.  gliosis, early Tau  One aspect o f A D not previously  investigated was the effect o f immunization on the integrity o f the blood-brain barrier (BBB).  The studies performed in this thesis show that there was a decrease in B B B  permeability  after  amyloid immunization.  These  data  further  support  amyloid  immunization as a treatment for A D as well as provide an explanation o f the mechanism by which immunization effectively reduces A D pathology.  111  Table of Contents Abstract Table of Contents List o f Figures List o f Tables List o f Abbreviations Acknowledgements and Dedication Chapter 1: Introduction 1.1 Alzheimer disease 1.2 Genetics o f Alzheimer disease 1.2.1 Early onset gene candidates 1.2.2 Late onset gene candidates 1.3 The amyloid precursor protein and amyloid beta 1.3.1 The amyloid precursor protein 1.3.2 A P P processing 1.3.3 A m y l o i d beta, structure and function 1.3.4 Putative amyloid beta receptors 1.3.5 A n i m a l models 1.4 Microglia, inflammation and Alzheimer disease 1.4.1 M i c r o g l i a as immune cells o f the brain 1.4.2 Morphological plasticity o f microglia 1.4.3 The function o f microglia in the central nervous system 1.4.4 The role o f activated microglia in Alzheimer disease 1.4.5 Signal transduction pathways and microglial activation 1.5 The blood-brain barrier and Alzheimer disease 1.5.1 The blood-brain barrier, structure and function 1.5.2 B B B integrity and Alzheimer disease 1.6 Therapeutic strategies 1.7 Project rationale and general approach Chapter 2: Materials and Methods 2.1 Mice 2.1.1 Tg2576 A D model mice 2.1.2 Colony stimulating factor-1 deficient mice (op/op) 2.1.3 Generation o f Tg/+;op/op mice 2.2 Preparation o f reagents 2.3 C e l l culture 2.4 C e l l stimulation 2.5 Creation o f stable B V - 2 transfectant cell lines 2.6 R N A Isolation 2.7 Reverse transcriptase and Polymerase Chain Reaction 2.8 Real-time Polymerase Chain Reaction 2.9 T N F - a E L I S A assay 2.10 Western blot analysis 2.11 Immunohistochemistry 2.12 A(3 and antibody injection 2.13 Vaccination protocol  ii iv vi viii ix xii 1 1 2 3 5 7 7 9 14 20 22 27 27 28 30 31 34 36 36 39 41 44 45 45 45 45 46 46 48 48 49 53 53 54 55 55 57 58 59  iv  2.14 Evans blue assay 2.15 Statistical analysis Chapter 3: P97 expression in activated microglia 3.1 Rationale 3.2 Results 3.2.1 Microglial activation 3.2.2 P97 expression in B V - 2 cells 3.2.3 P97 expression in Tg2576 A D model mice 3.2.4 M A P K pathways control the expression o f p97 3.2.5 P97 expression in B V - 2 cells after treatment with N S A I D s 3.3 Discussion Chapter 4: The role o f microglia in amyloid deposition 4.1 Rationale 4.2 Results 4.2.1 Characterization o f Tg/+;op/op mice 4.2.2 A m y l o i d burden in Tg/+;op/op mice 4.2.3 Microgliosis in Tg/+;op/op mice 4.3 Discussion Chapter 5: Ap immunization and the blood-brain barrier 5.1 Rationale 5.2 Results 5.2.1 Ap peptide and anti-Ap antibodies and their ability to cross the B B B . . . 5.2.2 Anti-Ap antibody titres in immunized animals 5.2.3 A m y l o i d plaque burden in immunized animals 5.2.4 Microgliosis in immunized animals 5.2.5 B B B permeability in immunized animals 5.3 Discussion Chapter 6: Concluding remarks and future directions Appendix I: Domain Structure o f A P P Appendix II: Regional diagram o f the brain References  60 61 62 62 67 67 69 72 74 76 80 85 85 88 88 90 95 98 104 104 107 107 113 115 119 122 126 132 138 139 140  v  List of Figures Figure 1.1. Mutations in A P P genetically linked to E O F A D  4  Figure 1.2. Schematic diagram of A P P and its metabolic derivatives  13  Figure 1.3. Morphological and functional plasticity of microglia  29  Figure 1.4. Schematic of the B B B  38  Figure 2.1. p E G F P - 1 vector and multiple cloning site  51  Figure 2.2. G e l o f digested p E G F P and p97 promoter construct  52  Figure 3.1. T N F - a production increased in B V - 2 cells treated with various known activators  68  Figure 3.2. P97 expression in treated cells  70  Figure 3.3. p97 expression is increased in affected brain regions in Tg2576 mice  73  Figure 3.4. The p97 promoter appears to be regulated by the p38 M A P K pathway  75  Figure 3.5. N S A I D treatment decreased T N F - a production in activated B V - 2 cells  77  Figure 3.6. p97 expression is decreased in B V - 2 cells treated with Ibuprofen  78  Figure 3.7. p97 expression is decreased in B V - 2 cells treated with Nimesulide  79  Figure 4.1. P C R genotyping of Tg2576 A D model mice  89  Figure 4.2. Tg/+;op/op mice are smaller than control littermates  90  Figure 4.3. A m y l o i d plaque burden in 9 month Tg/+;op/op mice compared to controls. 91 Figure 4.4. A m y l o i d accumulation in cerebral blood vessels of Tg/+;op/op mice  93  Figure 4.5. Reduced number and altered morphology of microglia in Tg/+;op/op mice. 96 Figure 5.1. A p peptides can cross the B B B in both transgenic and wild-type mice  109  Figure 5.2. anti-Ap antibodies cannot cross the B B B in both transgenic and wild-type mice  Ill  vi  Figure 5.3. Antibody titre in serum of transgenic and non-transgenic mice immunized with either A p or P B S  114  Figure 5.4. A m y l o i d Pathology in Tg2576 M i c e Immunized with A p or P B S  116  Figure 5.5. Cerebral amyloid levels are reduced in Tg2576 mice following A p immunization  118  Figure 5.6. Microgliosis in Immunized M i c e  120  Figure 5.7. B B B permeability as determined by Evans Blue in cortical regions of A p and P B S immunized mice  124  vii  List of Tables Table 1. Summary o f the primary APP-based transgenic mouse models o f A D  26  Table 2. List o f primer sequences and product  47  viii  List of Abbreviations A m y l o i d beta A|3l-40  40-residue C-terminal variant o f amyloid beta  AP..42  42-residue C-terminal variant o f amyloid beta  AD  Alzheimer disease  ADAMs  A disintegrin and metalloproteinase  ApoE  Apolipoprotein E  APP  A m y l o i d precursor protein  Aph-1  Anterior pharynx-defective-1  BACE  P-site A P P cleavage enzyme  BBB  Blood-brain barrier  bp  Base pair  BSA  Bovine serum albumin  CAA  Cerebral amyloid angiopathy  Caspases  Cysteine aspartyl proteases  CC  Cerebral cortex  C-terminal  Carboxy-terminal  CFA  Complete Freund's adjuvant  CNS  Central nervous system  COX  Cyclo-oxygenase  CREB  c A M P responsive element binding protein  CSF-1  Colony stimulating factor 1  Cu(II)  Copper II  DMEM  Dulbecco's modified Eagle's medium  DMSO  dimethylsulfoxide  cDNA  Complementary deoxyribonucleic acid  dNTP  Deoxyribonucleotide triphosphate  DTT  dithiothreitol  ELISA  Enzyme linked immuno-absorbant assay  EOF A D  Early onset familial Alzheimer disease  ER  ,  Endoplasmic reticulum  ERK  extracellular signal-regulated kinases  FBS  Fetal bovine serum  FcR  Fc receptor  Fe(III)  Iron III  FPRL1  formyl peptide receptor-like 1  GFP  Green fluorescent protein  hAPP  Human amyloid precursor protein  HI  hippocampus  HRP  Horse radish peroxidase  Ibu  Ibuprofen  ICFA  Incomplete Freund's adjuvant  IL  Interleukin  IFN-y  Interferon gamma  i.p.  intraperitoneal  i.v.  Intravenous  INK  c-jun N-terminal kinases  LOAD  Late-onset Alzheimer disease  LPS  Lipopolysaccharide  LRP-1  Low-density lipoprotein receptor related protein  MAPK  Mitogen-activated protein kinase  mRNA  Messenger ribonucleic acid  N.D.  Not determined  N-terminal  A m i n o terminal  Nim  Nimesulide  NFKB  Nuclear factor K B  NFTs  Neurofibrillary tangles  NSAIDs  Non-steroidal anti-inflammatory drugs  OD  Optical density  op/op  CSF-1 deficient mouse  PBS  Phosphate buffered saline  PCR  Polymerase chain reaction  PD  Parkinson's disease  Pen2  Presenilin enhancer 2  PFA  Paraformaldehyde  PPARy  Peroxisome proliferators-activated receptor gamma  PS  Permeability coefficient x surface area  PS1  Presenilin 1  PS2  Presenilin 2  RAGE  Receptor for advanced glycation end product  ROS  Reactive oxygen species  sAPPa  Secreted amyloid precursor protein alpha  sAPPp  Secreted amyloid precursor protein beta  SDS page  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis  SR  Scavenger receptor  Smac  Second-mitochondria derived activator o f caspase  TfR  Transferrin receptor  TGF-p  Transforming growth factor beta  Tg/+  Tg2576 A D model mouse  TNF-a  Tumor necrosis factor alpha  VEGF  Vascular endothelial growth factor  VLDL-R  Very low-density lipoprotein receptor  XIAP  X chromosome linked inhibitor o f apoptosis protein  Zn(II)  Zinc II  ZO  Zonula occudens proteins  +/+  Wild-type mouse  xi  Acknowledgements and Dedication This thesis could not have been completed without the help o f many people. First and foremost I would like to thank my supervisor, Dr. Wilfred Jefferies, for bringing me into his lab and giving me the opportunity to work on this exciting project.  His  encouragement and enthusiasm in this project, through the good times and the bad, have propelled me to excel and have taught me how to face things head on, to not give up and to think beyond the obvious. He has taught me how to be a successful scientist and for that I am eternally thankful.  I am grateful to my committee members, Dr. Keith  Humphries, Dr. Peter Reiner, Dr. Gerry Krystal and Dr. Elizabeth Simpson for their invaluable guidance and assistance, to the Genetics Graduate Program, the Biotechnology Laboratory and the Biomedical Research Centre.  I would like to thank all o f my lab  members, past and present. In particular, D r . M a y a Kotfuri, Dr. Cheryl Pfeifer, Dr. M a k i Ujiie, and Jason Grant for their technical advice, their help with proof reading my thesis and most importantly for their friendship.  A special thanks to A n d y Jeffries and Ray  Gopaul, for their assistance in experimental procedures, and their exceptional care o f my animals, Brian Chung and Janet Lee for their help with many experiments and Dr. Aruna Somasiri, Arthur Legg, and Kenny To o f Wax-it Histology for all their hard work and technical assistance.  I also acknowledge the Alzheimer Society o f Canada who granted  me a Doctoral research scholarship for 4 years o f my Ph.D. Finally I would like to recognize my family and friends for all their love and support and constant encouragement throughout the years. In particular I would like to express my gratitude to my parents, who have always inspired me to strive for what I believe in and to attain each goal I put before myself.  xn  This work is dedicated to the memory o f my grandmother, Ruth Arbuck, 1914-2003  xiii  Chapter 1: Introduction 1.1 Alzheimer  disease  Alzheimer disease ( A D ) was first described in 1907 by Alios Alzheimer as a neuropathological syndrome characterized by progressive dementia and deterioration o f cognitive function along with neurological lesions identified as dark staining plaques and fiber-like tangles \ elderly . 1  A D accounts for approximately 65% of all dementia cases in the  It is estimated that there are approximately 20 million people affected  worldwide with either A D or mild cognitive impairment. The prevalence o f A D , in the general population, increases with age as the rate is 3% in those between 65 and 74 years compared with 47% among those over 85 years o f age . 3  Progressive memory and  cognitive decline as well as difficulty in language, praxis and visual perception are the clinical manifestations which characterize the disease.  The decline in intellectual  function progresses at a slow but inexorable rate and leads to severe debilitation and death within 12 years after onset . 4  Pathologically, A D selectively damages brain regions and neural circuits critical for cognition and memory and is distinguished by the presence o f proteinaceous deposits in the brain, comprised of extracellular amyloid plaques and accumulated paired helical filaments of hyper-phosphorylated Tau in intracellular neurofibrillary tangles (NFTs), dystrophic neuritis and neuropil threads ' . 5  6  Inflammation also plays a key role in A D  and appears to be facilitated by activated microglia, the immune cells o f the central nervous system ( C N S ) . It took 75 more years to determine that the main constituent o f the plaques was a 40-42 amino acid peptide referred to as amyloid p (AP). . A P is a 7  1  metabolic product resulting from the proteolytic cleavage o f the amyloid precursor protein ( A P P ) and its aggregation into fibrils is thought to be the central event o f A D . The "amyloid cascade hypothesis" proposes that A P precipitation into fibrils initiates the formation o f amyloid plaques which in turn contribute to the formation o f neurofibrillary tangles, initiate complement cascades  and inflammatory processes  and ultimately  culminate in cell death . However, this hypothesis is not consistent with recent advances 8  that implicate inflammation, N F T s and oxidative stress as independent processes that may even be upstream o f A P aggregation.  1.2 Genetics of Alzheimer  disease  The inheritance o f predisposing genetic factors appears to play an important role in AD.  After age, family history is the second greatest risk factor for A D .  There are two  classes of A D : early-onset familial A D ( E O F A D ) , where the age o f onset is less than 60 years o f age; and late-onset A D ( L O A D ) , where age of onset is greater than 60 years. The genetics o f A D are complex since A D is a heterogeneous genetic disorder in which numerous  genetic factors, with both minor and major effects, play  independent,  simultaneous and interdependent roles. Further complexity arises since mutations and polymorphisms in multiple genes are acting together with many environmental factors. Moreover, A D represents a dichotomous situation where genes which cause E O F A D are rare in prevalence but 100% penetrant, while genes which confer increased risk for L O A D are highly prevalent with low penetrance  9  .  Present research is focused on  elucidating the genes responsible for the various aspects o f A D . This w i l l make it  2  possible to create and estimate a person's genetic susceptibility profile that w i l l aid in both early diagnosis and the development o f preventative treatment.  1.2.1 Early onset gene candidates The first candidate gene for A D was discovered in the early 1980's. analysis  and  subsequent  positional  cloning  multigenerational families who all had E O F A D .  techniques  were  Linkage  performed  on  The inheritance pattern in all these  families appeared to be autosomal dominant and highly penetrant.  The findings from  these families showed linkage to chromosome 21 and focused on a candidate gene encoding the A P P . Although association was later found to be a false positive, it did 1 0  lead investigators to a compelling candidate gene.  More persuasive evidence for the  involvement o f A P P in A D was the fact that D o w n ' s Syndrome patients, who have trisomy 21, had strikingly similar brain pathology to those suffering from A D " .  In  addition, D o w n ' s patients had increased A P P messenger ribonucleic acid ( m R N A ) expression along with elevated A P P and A P in the serum and brain ' . 12  13  Finally, mice  overexpressing mutant forms of A P P developed A D - l i k e pathology, further implicating A P P as a genetic determinant for A D . The first A P P mutation was found in 1990 in a Dutch family where individuals were affected with cerebral hemorrhage with significant amyloid deposits in accordance with an autosomal dominance inheritance pattern . Since then 20 mutations in the A P P 9  gene have been found, all of which are missense mutations located close to or within the coding region o f the A p fragment in the P and y-secretase cleavage sites (Figure 1.1)  1 0  .  Overall, mutations in the A P P gene result in an increase in the production o f Api_42 peptide, the more amyloidogenic and toxic species o f A p . Other mutations, such as the  3  London mutation (V7171), cause an increase in the ratio o f APi_42 peptide to peptide  l 4  APMO  , whereas the Swedish mutation ( K 6 7 0 N / M 6 7 1 L ) causes an increase in the  production o f both species o f A p M o and AP142 ' . Individuals with A P P mutations have 5  an average year o f onset o f 49 ± 8 years and disease duration o f approximately 12 years . Mutations in the A P P gene account for 5-7% o f all E O F A D cases which account for less than 2% o f all A D cases.  p-secretase  a-secretase  y-secretase G (Arctic) K (Italian) N (Iowa)  (Belgian) A (French) MV (Florida)  i  t It ...SEVKM DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVI gTVIVITLVMLKK...  Hi (Swedish) N L  / /|\  ii (Flemish) G Q (Dutch)  I  |S  IFG P  (Austrian) (London) (Australian)  Figure 1.1. Mutations i n A P P genetically linked to E O F A D The A P coding region within the A P P is expanded and shown by amino acid code. The arrows indicate residues with known missense mutations which have been identified in E O F A D and/or hereditary cerebral hemorrhage with amyloidosis. Mutations o f A P P near the positions 670, 693 and 715 have been found to increase the risk o f A D . A double mutation at K670 and M671 increases the production o f both A P M O and Ap42, while mutations near the y-secretase cleavage site favors the production o f Api_42- Three-digit numbers refer to the residue number o f A P P (Adapted from Selkoe ) . 1 6  A year after the discovery o f mutations in the A P P gene, another F A D locus was found to be linked to chromosome 14 and later identified as the presenilin 1 (PS1) gene . 9  Shortly after, another gene encoding a second member o f the presenilin family called presenilin 2 (PS2) located on chromosome 1 was also identified  . T o date there are  4  approximately 140 mutations in PS1 and 10 mutations in PS2, and together presenilin mutations account for the majority of EOFAD cases  10  " . 16  PS1 encodes a 7  transmembrane spanning protein that functions as an aspartyl protease and is required for y-secretase activity . At first it was thought that PS1 was the y-secretase. However, 17  many studies have demonstrated that PS1 is part of a protease complex consisting of at 18 19 20  least three other proteins including, nicastrin, APH-1, and Pen-2 presenilin genes result in the over production of  AP1-42,  ' ' . Mutations in  presumably by altering  y-  secretase activity . Individuals with PS1 mutations have an earlier age of disease onset 21  (43.5 ± 0.7 years) and have shorter disease, duration (4.5 ± 0.7 years), while PS2 S 22  mutations have a later age of onset and longer duration  . Interestingly, PS2 mutations  are not highly penetrant compared to PS1 mutations which are completely penetrant. Overall, mutations in PS genes account for greater than 55% of all EOFAD mutations. 1.2.2 Late onset gene candidates LOAD accounts for approximately 95% of AD cases and is classified as sporadic AD. The genetic contributions to LOAD are more difficult to address due to the lack of complete family histories and fewer living subjects from which to obtain blood samples. There are a few gene candidates for LOAD which have been identified by association or linkage disequilibrium studies.  Co-segregation of particular allele(s) and disease  phenotype was one of the strategies employed to identify an allele of the gene encoding apolipoprotein E (ApoE) as a risk factor for AD . There are three alleles of the ApoE 23  gene; s2, s3, and s4.  Individuals who have the e4 allele, either heterozygous or  homozygous, have a greater risk of developing AD; however, there are many individuals who are homozygous for the 84 allele and do not develop dementia. Therefore, the e4 5  allele is neither necessary nor sufficient for the development o f A D .  A p o E is a 299  amino acid glycoprotein that normally functions in cholesterol and lipid metabolism . It 2 4  is thought that the s4 allele acts as a modifying gene by decreasing the age o f onset in a dose dependent manner  1 0  .  The s4 isoform has also been reported to promote A p  aggregation by mechanisms not yet elucidated.  It is thought that e4 is less capable o f  clearing A P from the neuropil and that 84 binds to A p with a lower affinity than the other isoforms  2 5  .  Genetic linkage studies have also given rise to many more candidate genes including a -macroglobulin, low-density lipoprotein receptor related protein (LRP-1), 2  insulin degrading enzyme, urokinase plasminogen activator and the very low-density lipoprotein  receptor  (VLDL-R).  a2-Macroglobulin  and  LRP-1,  both  located on  chromosome 12, appear to play a role in A P clearance and degradation. LRP-1 can also serve as a receptor for A p o E ,  a2-macroglobulin and secreted forms o f A P P and  polymorphisms in exon 3 and 6 have been linked to A D ' . Insulin degrading enzyme 9  2 6  and urokinase plasminogen activator, located on chromosome 10, have also been suggested to play a role in A P degradation  2 1  . Finally, V L D L - R , located on the short arm  of chromosome 9, may be a receptor for lipoproteins such as A p o E . 9  There are also  many inflammatory factors that are deemed to have high risk alleles. Polymorphisms in interleukin  (IL)-la,  IL-1 P,  IL-6, tumor  necrosis  factor  antichymotrypsin have all been shown to influence A D risk  2 8  a  (TNF-a)  and  d\-  . There are approximately  10 polymorphisms that have been found in the general population and individuals carrying one or more of these alleles are hypersensitive to oddities resulting in inflammatory processes that can cause increased degeneration  28  .  Since the genes  6  identified to date are believed to only account for approximately 30% of the genetic variance in A D , the search for genetic factors associated with A D is an ongoing research effort.  The amyloid  1.3  precursor  protein  and amyloid  beta  1.3.1 The amyloid precursor protein With the identification o f A P P being a genetic determinant for E O F A D , research began into clarifying the biological role of A P P and its proteolytic cleavage products. APP  is a type 1 transmembrane glycoprotein that is localized to chromosome 21  (21q21.2-3), contains  18 exons and spans 170 kilobases  2 9  .  A P P is expressed  ubiquitously throughout the body and can exist in at least three isoforms: APP770, APP751, and APP695, arising from alternative splicing with different isoforms expressed in specific tissues  3 0  . The full length protein contains zinc and copper binding sites, a cysteine rich  subdomain and an acidic region at the amino terminus. There is also a kunitz protease inhibitor domain, although the functional significance o f this domain is unclear . In the 4  brain the APP695 isoform is the predominant form and is expressed in glial, endothelial and  neuronal cells.  A P P has been observed to be localized to many membranous  structures including the endoplasmic reticulum ( E R ) , Golgi compartments, plasma membrane, postsynaptic densities, axons, and dendrites.  In addition, A P P has been  found in cholesterol and G M 1 ganglioside rich membrane microdomains  .  The exact function of A P P is not clear and many studies have focused on the role of A P P and its fragments. It had been demonstrated that A P P and its fragments, such as  7  secreted APPa (sAPPa), secreted APPP (sAPPP) and Ap, are powerful regulators of neuronal functions including synaptic plasticity and transmission, neuritic outgrowth, cell excitability, cholesterol metabolism, cell adhesion, cell death and gene transcription '. One possible function of full length APP is as a cell surface G-protein-coupled receptor. Binding of G-proteins to APP causes the G-protein to dissociate into its dimeric form resulting in the activation of multiple signal transduction molecules such as phospholipase C, voltage dependant calcium channels, adenylyn cyclase and many enzymes involved in apoptotic signaling cascades. However, the role of APP mediated G-protein activation is not fully understood.  APP has also been implicated in cell  adhesion. Immunohistochemical studies have demonstrated that cell surface APP colocalizes with adhesion proteins such as P-l integrin  31  and telencephalin . Moreover, 32  APP contains several domains that facilitate the binding of heparin, collagen and laminin Other possible functions include synaptic transmission and plasticity and more recently memory and cognition. Nerve process outgrowth is thought to be regulated, in part, by APP. Studies focusing oh embryonic development and APP expression have shown that APP expression is at its highest between embryonic day 6-9, when neuritic outgrowth is at its maximum . Moreover, in vitro experiments with synthetic APP 4  demonstrate that APP is able to stimulate neuritic outgrowth in PC 12 neuronal cells . 4  Research on APP function is conflicting since most AD animal models are transgenic for the human form of the protein that may interfere in the evaluation of the endogenous mouse protein function.  8  1.3.2 A P P processing The processing o f A P P into its metabolites involves three different enzymes: P-, a-, and y-secretases and two cleavage pathways (Figure 1.2). In A P P , there are two main cleavage sites on the extracellular side o f the membrane and one site within the transmembrane domain. P- and a-secretase cleavage appears to be mutually exclusive events,  each  generating  soluble carboxyl-terminal (C-terminal) fragments.  The  remaining amino-terminal (N-terminal) fragment o f A P P is then cleaved by y-secretase . 4  In the non-amyloidogenic pathway, A P P is cleaved by a-secretase at a cleavage site located within the A P peptide. This cleavage precludes the production o f A p producing a 612 amino acid soluble protein, s A P P a , which has been shown to have neuroprotective and memory enhancing effects  3 3  .  Cleavage at this site is dependent on secondary  structure and on the location of the cleavage site from the membrane.  In addition, a-  secretase activity can be regulated by protein kinase C activity as well as by other molecules such as estrogen, testosterone, various neurotransmitters and growth factors  3 4  .  Although the exact identity o f a-secretase is still unknown, three proteins have been described that appear to have a-secretase-like activities. Studies with protease inhibitors have shown that a-secretase is a zinc metalloproteinase and belongs to the family o f a disintegrin and metalloproteinase ( A D A M s ) . The three candidate proteins are A D A M I 7 (also known as T N F - a convertase), A D A M 10 and A D A M 9 (also known as M D C 9 )  3 3  .  A D A M s are type 1 integral membrane proteins with a multi-domain structure and function in cell adhesion, cell fusion, intracellular signaling and proteolysis  3 4  .  The  expression and localization o f each these proteins in A D are altered and gives rise to the potential role of each in A P P processing.  A D A M I 7 has been found to localize to  9  neurons as well as to senile plaques and tangles, although there is no change in its expression in A D . In contrast, A D A M 10 protein levels are significantly reduced in A D , as are levels o f s A P P a and a-secretase a c t i v i t y . The emerging hypothesis, as suggested 33  by studies involving knock out mice and enzymatic inhibition, is that all three A D A M enzymes are involved to a similar extent in A P P processing acting together at the asecretase cleavage site to varying degrees depending on cell type.  It is possible that  A D A M 10 is involved in both constitutive and regulative pathways o f a-secretase, whereas A D M A 1 7 may be involved solely in the regulatory pathway and A D A M 9 in the actual cleavage e v e n t . 33  In the amyloidogenic pathway o f A P P processing, A P P is first cleaved by Psecretase followed by cleavage with y-secretase. These events generate two metabolites, s A P P p and the AP peptide.  T w o enzymes capable o f cleavage at the P-secretase  cleavage site are the P-site A P P cleavage enzymes ( B A C E 1 ) and B A C E 2 . B A C E 1 , also 6  referred to as Asp2 or memapsin2, is a transmembrane aspartyl protease and exists in three isoforms. B A C E 1 is expressed at high levels in the pancreas, moderate levels in the brain and at low levels in most peripheral tissue. However, B A C E 1 activity is highest in neuronal tissue and is increased in A D . Intracellularly, B A C E 1 can be found primarily 3:>  in the trans-Golgi network, and the endosomal compartments, although it can also be found in the endoplasmic reticulum and on the cell surface  3 4  . Cleavage by B A C E 1 is the  rate limiting step in the generation o f A p . B A C E 1 cleaves at two positions depending on intracellular localization, either at the A s p l (P-site) or at G l u l 1 (p'-site) o f AP, generating an  APi_4o/42 fragment  or  an  APi 1.40/42 fragment,  respectively.  p-site  predominantly occurs in the E R while P'-site cleavage occurs in the Golgi  3 4  cleavage .  Further  10  evidence to support the role o f B A C E 1 as p-secretase comes from studies on various A D animal models. In A P P transgenic mice expressing the Swedish mutation, there is a 10 fold increase in B A C E cleavage activity. B A C E have a significant increase in  Moreover, mice which overexpress human  A01.40/42  levels and knock out o f this gene perturbs  A P P processing and prevents A P generation . Finally, in B A C E l ' / A P P mice there is a 3 6  lack o f A P compared to other A P P transgenics  3 6  .  B A C E 2 , another aspartyl protease, is 55% identical to B A C E 1 and shows similar substrate specificity to B A C E 1 . B A G E 2 is widely expressed in peripheral tissues but is not highly expressed in the brain. It cleaves A P at position A s p l and at position P h e l 9 or Phe-20 of the A p peptide  3 4  . The precise role o f B A C E 2 in A P P processing is unclear.  There is no direct evidence for the role o f B A C E 2 in A D however, cleavage at positions 19 and 20 located within A p is affected by the F A D Flemish mutation  3 4  and B A C E 2  maps to chromosome region 21q22.2-22.3 . 3 7  The final event in A P P processing is the cleavage o f the C83 or C99 A P P fragments  by y-secretase.  Cleavage occurs at a site  located within  the A P P  transmembrane domain. It was first thought that PS1 and PS2 were y-secretases since mutations in PS1 and PS2 cause an increase in the production of A P i ^  3S 2  - In addition,  PS1 and PS2 contain aspartic acid residues in transmembrane domain 6 and 7, sites necessary for aspartyl protease activity and hence y-secretase activity  17  .  PS1 is  expressed in its full length form as an integral membrane protein with 8 membrane spanning regions.  In this form, PS1 is thought to be inactive.  To form an active  presenilin complex, the presenilin protein is cleaved between membrane domains 6 and 7. This  yields C - and N-terminal  fragments,  which  remain coupled and  form  a  11  heterodimeric active complex \  If one or both o f the aspartic residues in presenilin are  mutated or knocked out, the production o f A(3 and the P3 peptide is greatly reduced along with an associated increase in the amount o f C-terminal fragments, s A P P a and sAPP(3 . 17  This theory was questioned and an alternative hypothesis proposed that states that ysecretase is multifaceted and is made up of several proteins. The role o f presenilins in this complex would be in A P P trafficking to sites where cleavage by the complete ysecretase complex can take place. This viewpoint was corroborated by biochemical and genetic studies which have identified four membrane proteins as components o f ysecretase: heterodimeric presenilin, (composed o f its N - and C-terminal fragments); anterior pharynx-defective-1 (Aph-1); nicastrin, (glycosylated); and presenilin enhancer 2 (Pen-2)  2 0  .  12  .SEVKM DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVI  ft-secretase  TVIVITLVML..  a-secretase  sAPPB  sAPPa  y-secretase  Y-secretase  Figure 1.2. Schematic d i a g r a m of APP and its metabolic derivatives A P P is a type 1 membrane protein and exists in three major isoforms, A P 7 7 0 , A P 7 5 1 , and AP695. Enlargement o f the A p peptide region shows the amino acid sequence and primary sites of cleavage by the various secretases. Cleavage o f A P P by either P- or asecretase results in soluble N-terminal fragments, s A P P p and s A P P a and two membranebound C-terminal fragments, C99 and C 8 3 , respectively. Subsequent cleavage by ysecretase gives rise to a non-pathogenic A p peptide and P3 (Adapted from Small, ) . 4  Nicastrin is a type 1 transmembrane glycoprotein that is 709 amino acids long and contains 4 conserved cysteine residues at the N-terminus. It is encoded on chromosome lq23, a region previously identified as a susceptibility locus for L O A D  3 4  . Nicastrin has  been shown to interact with PS1 and PS2 as well as with the C-terminal A P P fragments, C83 and C99. Mutations in either PS 1/2 directly effect nicastrin trafficking and posttranslational modifications thereby preventing its maturation and function. In this state, the y-secretase complex cannot form and A P production is abolished . Aph-1 and Pen-2 3 4  were two proteins discovered through a genetic screen o f Caenorhabditis elegans. These  13  genes both encode multi-pass transmembrane proteins that have the capability o f binding to P S 1 , PS2, and nicastrin with high affinity. The specific role o f Aph-1 and Pen-2 and the protein-protein interactions responsible for assembling Aph-1 and Pen-2 into the multimeric protease complex remains unknown. Recently, it was revealed that Aph-1 binds to nicastrin in its immature, non-glycosylated form and remains bound after glycosylation and maturation  3 9  . In regards to how this complex forms, it is thought that  Aph-1 and nicastrin initially form a subcomplex. Once nicastrin is modified and matured the Aph-1/nicastrin complex binds to and stabilizes presenilin. Pen-2 then binds, confers the y-secretase activity and facilitates endoproteolysis o f presenilin which serves as the catalytically active core o f the y-secretase complex . There are many details about the 3 9  active y-secretase complex that remain to be uncovered These include determining which y-secretase co-factors physically interact with one another and identifying the proteinprotein binding domains that govern these interactions.  1.3.3 A m y l o i d beta, structure and function A P is a 4 kiloDalton peptide and is a normal cellular product and exists in two predominant forms with different C-termini, A p i ^ o and Api42-  A P contains seven  positively and seven negatively charged residues at the N-terminal region and a 21  hydrophobic domain at residues 17-21 and 29-40/42 (Figure 1.2)  .  The A P protein  exists as monomers, dimers, and oligomers and can undergo further aggregation to yield full  fibrils.  The conformation o f A P appears to be in a dynamic flux and is dependant  upon environmental conditions, metal binding and interactions with various other proteins.  In its monomeric state A P exists as a random coil containing two a-helices  between residues 15-23 and 31-3 5 . Transition o f these a-helical coils into P-pleated 4 0  14  sheets facilitates fertilization where the p-pleated sheets are anti-parallel to one another. It is these full fibrils that are found to make up the bulk o f the senile plaques and are thought to have neurotoxic properties  4 1  . However, pools o f soluble A p have been found  in the brains of A D patients indicating that monomeric, dimeric and oligomeric forms of A p may facilitate the pathology o f A D  4 1  . The proportion o f soluble A P M 2 in these pools  is significantly higher than soluble A P M O levels. Soluble A P i ^ 2 dimers have also been shown to have a higher magnitude of neurotoxicity than A p i ^ o  4 1  •  The deposition o f amyloid is not found in all regions o f the brain. A P plaques exhibit a spatio-temporal distribution pattern where regions such as the cortex and hippocampus contain many plaques and regions such as the cerebellum exhibit little to no Ap.  One possible explanation for the distribution pattern o f A p has been proposed  stating that the presence o f A p in the neuropil o f neurons sets in motion a cascade o f reactions  including inflammation, neuritic sprouting and the upregulation of A P P  expression . 4  This model is based on a positive feedback loop which relies on neuritic  sprouting and A P P expression. It is known that as the brain ages, certain areas become myelinated However,  and  mechanisms  regions  o f the  which  brain  facilitate  responsible  neuritic for  outgrowth  memory  have  are  suppressed.  ongoing  synaptic  remodeling, continuation o f neuritic outgrowth and are thus the most vulnerable.  These  include the cortex, hippocampus and olfactory system all o f which are affected in A D . 4  In addition, regions o f the brain that are the last to undergo myelination, such as the temporal cortex, are the first regions to be affected.  Therefore, myelin proteins may  prevent neuritic sprouting and subsequent A P P expression thereby protecting a given brain region from degeneration  4 2  .  15  The aggregation o f A|3 into fibrils and toxicity can occur in response to a variety of actions, including a shortage in the supply o f energy in the cell, oxidative stresses, calcium dysregulation and apoptosis.  Studies have shown that a disruption in  mitochondrial energy metabolism and dysregulation o f calcium homeostasis can up regulate the expression o f A P P as well as promote the pro-amyloidigenic processing o f A P P to A p and subsequent plaque formation  4 3  ' . 4 4  Oxidative stresses like hydrogen  peroxide, ultraviolet light, and superoxide radicals have been shown to increase the production o f neuronal A p . Moreover, high concentrations o f extracellular A P had been shown to induce oxidative stress and in turn render cells vulnerable to exocitoxicity and apoptosis through the dysregulation o f calcium homeostasis. Metal ions have also been shown to cause A P aggregation and promote the formation o f diffuse plaques. In the A D brain, there are significant amounts o f transition metals, such as copper II (Cu(II)), zinc II (Zn(II)) and iron III (Fe(III)), in both A p plaques and neuropil . 4 5  Histidine residues  located in the A p peptide bind to Cu(II), Zn(Il) and Fe(III) as sequester it in the brain during times of oxidative stress and inflammation, since the interaction of these metals with hydrogen peroxide, which is generated in both processes, would cause production or reactive oxygen species  4 5  .  the  Treatment of post-mortem A D brains with  metal-chelators confirmed this finding since the chelators were able to enhance the solubilization o f A P .  The generation o f reactive oxygen species has many harmful  46  consequences including disruption o f calcium homeostasis by impairing ATPase activity, increase lipid peroxidation and altering the activity o f the anti-oxidant enzyme, superoxide dismutase  4 1  .  16  It was originally demonstrated in non-neuronal cells, that APP metabolism can elevate intracellular calcium levels which enhances A P production. This creates a feed back loop since an increase in A p results in the increase of intracellular calcium . Soon 47  after studies in neuronal cells, other studies reported that exposure to fibrillar A P causes a disruption in calcium homeostasis generally leading to an increase in cytosolic calcium 47,48,49  jj  i e  m  fl  u x  0  f calcium is thought to be mediated by L-type voltage-gated calcium 48  channels and is regulated by the mitogen-activated protein kinase (MAPK) pathways  .  Under normal physiological conditions, calcium influx in neuronal cells has been implicated in promoting synaptic plasticity and thus beneficial. However, if too much calcium enters the cell or if the entry of calcium occurs at a dramatic rate, it could cause down-regulation of neuronal plasticity mechanisms due to the self-inhibitory mechanisms of calcium '.  Pierrot et al. have recently established that high cytosolic calcium  concentrations favour the amyloidogenic pathway of APP by preventing the cleavage of APP by a-secretase possibly through modifications of APP by phosphorylation . In 49  addition, high calcium levels resulted in the increase in AP142, with no change in A p ^ o , suggesting that calcium may also regulate y-secretase activity. Nonetheless, alterations in calcium homeostasis are detrimental to neuronal survival due to their high polarized nature. Recent advances in AD research suggest that in addition to extracellular A p , intracellular neuronal A P plays a significant role in neurodegeneration.  Increasing  evidence indicates that A P is more toxic to cells in its protofibrillar state rather than in its P-pleated conformation . Initial studies by Wertkin et al. demonstrated that NT2N 16  neuronal cells produced A P and either stored it in the cell or secreted it into the medium  17  .  Examination o f brain sections from young and old animal models illustrated an  anterior to posterior gradient o f intra-neuronal A p immunoreactivity in the hippocampus which increased with age in distal processes and synaptic compartments . What effect 5 1  intraneuronal A p exerts on neurons is unclear.  Current studies in h A P P and PS  transgenic rats propose that physiological levels of A p play a role in synaptic plasticity via activation o f c A M P Responsive Element Binding Protein ( C R E B ) directed gene expression  5 2  . A pathological increase in intraneuronal A p would cause abnormal  phosphorylation patterns that end up dysregulating these pathways.  This is noteworthy  since C R E B - d r i v e n gene expression has been found to be important for learning and memory.  It was also found that A p caused an upregulation o f extracellular signal-  regulated kinases ( E R K ) , and E R K then phosphorylates a number o f proteins, including Tau, inferring a further link between A p and Tau pathways  5 2  . In addition, both aged  animal models and humans exhibit high levels o f A P immunoreactivity in the cholinergic neuron o f the basal forebrain. The accumulation o f intracellular A p in neuronal processes and synapses is suggested to be associated with the manifestation o f cognitive decline seen in A D , since accumulation o f A p is concordant architecture and synaptic dysfunction causes o f cognitive decline.  5 3  .  with abnormal  cytoskeletal  Synaptic dysfunction is one o f the proposed  Moreover, oligomer A p neuronal accumulation  and  cognitive decline occur before plaque deposition. These findings proposed the "Synaptic A p Hypothesis" that proposes A P accumulates in neuronal synapses in the brain. Once the A p load in the neuron becomes too cumbersome, the cell dies and releases the amyloid into the extracellular space. The released amyloid can then act as a seed and attract soluble A P MO/42 thereby forming a plaque  5 3  .  18  Apoptotic processes are viewed to play a major role in A D pathology. Evidence suggests that selective neuronal loss seen in A D involves the activation of cysteine aspartyl proteases (caspases), which initiate and execute apoptosis  5 4  . Both extracellular  A p oligomers and intracellular A p may activate caspases. Extracellular A P initiates caspase activity via activation o f cell surface death receptors. This pathway involves signaling through cell surface death receptors, such as the T N F receptor, which are regulated by decoy receptors and Fas-associated death domain proteins, however, direct binding of A p or A p oligomers to death receptors remains to be shown . It is speculated 5 5  that the binding of A p to these receptors causes downstream activation of various caspases such as caspases 2 and 8, leading to the activation o f caspases 3, 6 and 7, all o f which are important initiators of the apoptotic signaling cascade  5 4  .  Alternatively,  intracellular A P may activate caspases through a process that involves E R stress or mitochondrial stress. This leads to the downstream activation o f caspase 9 and caspases 3, 6 and 7  5 6  .  Caspase activation is thought to facilitate the cleavage o f Tau thereby  favouring conformational changes of the protein into paired helical filaments. accumulation o f the altered Tau proteins causes  cytoskeletal disruption and  The the  consequent failure o f axoplasmic and dendritic transport that culminates in neuronal death  5 4  . A P has also been shown to elicit its toxic effects by the initiation of host immune  responses.  The action o f A P on glial cells results in an inflammatory reaction and  progressive amyloid deposition promotes the chemotaxis and subsequent activation o f microglia . Studies by M c D o n a l d et al. have established that exposure of fibrillar A P to 2 6  microglia or monocytes activates M A P K pathways involving p38 and the E R K , as well  19  as tyrosine kinase-dependent signaling pathways, involving L y n , Syk and F A K  ' . The  activation o f these pathways leads to changes in the expression o f various cytokine and pro-inflammatory genes and generates reactive oxygen intermediates,  respectively,  leading to further neurotoxicity and degeneration. The extensive effect o f A p on the inflammatory response elicited by glial cells w i l l be discussed in greater detail later.  1.3.4 Putative amyloid beta receptors The binding of amyloid to plasma membranes has been implicated in many o f the pathological features o f A D . Binding of A P to various receptors elicits neurotoxicity in neurons and cerebral vascular endothelia and activation o f inflammation in microglia. Due  to its structure, A p can bind to a variety o f molecules including proteins,  proteoglycans, and lipids . O n microglia, scavenger receptor A ( S R - A ) and B I (SR-BI), 5 9  C D 3 6 , heparin sulfate proteoglycan, formyl peptide receptor-like 1 ( F P R L 1 ) and a complex o f C D 3 6 , a 6 p 1-integrin, and C D 4 7 have been shown to bind to A p . Neuronal receptors include the N-methyl-D-aspartate receptor, the a7-nicotinic acetylcholine receptor, the p75 neurotrophin receptor and the C L A C - P / c o l l a g e n type X X V receptor  5 9  .  Receptors common to glial cells, neurons and cerebral endothelia include the receptor for advanced glycation end product ( R A G E ) , L R P , the insulin receptor, integrins and the serpine-enzyme complex receptor  5 9  .  These receptors vary in their ability to bind  monomeric or fibrillar A p . A few o f these receptors w i l l be discussed below. S R - A and S R - B I receptors are expressed on microglia, bind fibrillar A p and mediate the clearance o f amyloid aggregates  6 0  .  These receptors have also been  implicated in inflammatory responses and the production of reactive oxygen species;  20  however they are not needed for microglial activation.  Expression o f S R - A and S R - B I  are developmental^ regulated and are not expressed normally in adult mouse brain  6 1  ;  however, expression o f S R - A and S R - B I m R N A and protein are both upregulated invitro and in-vivo upon exposure to lipopolysaccharide (LPS), interferon gamma (IFN-y) and IL-1 a  6 2  . Another microglial receptor for A p is C D 3 6 . C D 3 6 is a class B scavenger  receptor that has been shown to bind to fibrillar A p and contribute to microglial activation.  A p binding to C D 3 6 elicits many signaling cascades involving Src family  kinases L y n and Fyn, and the mitogen-activated protein kinases ( M A P K ) as well as the production of reactive oxygen species.  C D 3 6 is also involved in a multi-receptor  complex with a6pl-integrin and C D 4 7 , and this complex mediates the binding o f microglia to fibrillar A p and the subsequent activation o f pro-inflammatory pathways, respiratory bursts, adhesion and cell migration  . Both S R - A and C D 3 6 are elevated in  microglia in brains o f A D patients compared to controls  6 4  .  F P R L 1 is a seven  transmembrane, G-protein coupled protein and is thought to initiate pro-inflammatory effects in response to both soluble and fibrillar A P . 65  Activation o f F P R L 1 can lead to  the production o f reactive oxygen species and pro-inflammatory cytokines. In addition, F P R L 1 is involved in mediating the chemotactic activity o f A p on microglia.  Other  studies have shown that F P R L 1 can form a complex with A p and can be internalized into cytoplasmic compartments resulting in the accumulation  o f intracellular amyloid  aggregates . Under normal condition F P R L 1 is expressed at low levels. In A D , F P R L 1 6 6  expression is increased and is seen at high levels in plaque infiltrating m i c r o g l i a . 65  The R A G E receptor is a multi-ligand receptor in the immunoglobulin superfamily found in neurons, microglia and cerebral endothelial cells. It binds many ligands such as  21  A P , the S1 OO/calgranulin family of pro-inflammatory cytokine-like mediators and the high mobility group 1 D N A binding protein amphoterin ' . The interaction of Ap with 67  67  R A G E initiates signaling cascades that result in oxidative stress via the production of reactive oxygen species and lipid peroxides as well as the enhanced expression of macrophage colony stimulating factor, which stimulates microglial proliferation and receptor expression. It has also been implicated in Ap internalization on microglia . Its 68  expression is ligand dependent and is up-regulated in A D , particularly in the vasculature. R A G E expression is also increased in neurons and microglia, but not as much as in cerebral endothelia. Many in vitro and in vivo studies.have demonstrated that R A G E mediates AP transport, therefore, R A G E is thought to be the major influx receptor for peripheral A p at the B B B  69  ' ' . 70  67  LRP is a member of the L D L receptor family and is a multifunctional scavenger receptor and signaling receptor. Its ligands include biomolecules such as ApoE and 0C2macroglobulin, tissue plasminogen activator, APP and lactoferrin  71  .  LRP has been  genetically linked to L O A D , but the exact mechanism by which LRP affects disease onset in not known . Expression of LRP in A D is decreased and many animal studies 9  involving LRP deficient mice and A D mice results in increased cerebral amyloid load and increase in parenchymal amyloid plaques  72  ' . 73  As a result, LRP is thought to 70  regulate A P clearance by controlling its efflux from brain to blood .  1.3.5 Animal models With the multiple genetic mutations associated with the A P P gene, many transgenic mouse models have been created to mimic the diverse pathological features of A D . To date, there are approximately 20 APP transgenic mice made, some of which are 22  described in detail below (Table 1). The first generation o f A D model mice involved expression o f the wild-type human A P P (hAPP) complementary deoxyribonucleic acid (cDNA)  7 4  .  These mice, with the exception o f the N S E A P P mouse, did not develop  amyloid deposits. In the N S E A P P mouse the h A P P i isoform is expressed under control 7 5  of the neuron-specific enolase promoter.  These mice develop amyloid pathology;  however, the deposits in the brain were diffuse and did not resemble the compact plaques seen in the brains o f A D patients  7 5  . The next generation o f mice focused on  overexpressing the mutated h A P P protein at levels well above the endogenous gene level. This required vectors that provide transgene expression specific to the C N S . Examples o f these promoters include the Thy-1 promoter, which exhibits neuron specific gene expression  7 6  , and the hamster prion promoter, which is largely C N S specific with  expression in peripheral organs such as the heart  77  . More recently, transgenic mice have 7X  been generated expressing the complete h A P P by using a yeast artificial chromosome Second generation A D mice include P D A P P , Tg2576 and A P P 2 3 .  .  The P D A P P  transgenic mouse expresses all isoforms o f the h A P P gene and contains the Indiana mutation, V717F, under control o f the platelet-derived growth factor P-chain minipromoter  7 9  .  These mice exhibit many of the pathological features o f A D including  neuritic plaques in the hippocampus and neocortical regions, dystrophic neurites, astroand  microgliosis  and  synaptic  degeneration.  However,  NFTs  and  clear  79 80  neurodegeneration have not been identified in these animals  * .  Radial arm maze  testing and Morris water maze testing showed that these mice also exhibit memory impairment as early as 3 months of age, in the pre-plaque stage, and that these  23  impairments were correlated with increased age and plaque load  81 82  ' . The pathological  features observed in these animals are evident as early as 8 months of age  8 3  .  A second transgenic model, which is one o f the most widely used A D model, is the Tg2576 mouse. The Tg2576 mouse expresses the hAPP695 transgene containing the Swedish double mutation K 6 7 0 N / M 6 7 1 L under control o f the prion promoter at levels approximately 5 fold higher than the endogenous gene ' . These mice develop amyloid 8 4  8 5  plaques at approximately 9 months o f age, with the first deposition in the entorhinal and piriform cortices ; exhibit increased gliosis, as well as a decrease in synaptic activity in 8 6  the hippocampal C A 1 region, dentate gyrus; and exhibit a reduction in long term potentiation  8 7  (see Appendix II for diagram of brain regions) . Cognitive deficits, as  determined by Y-maze alternation and water maze learning, occur at 9-10 months o f age 8 5  . The one shortfall o f these animals is that there is a lack o f neurodegeneration  8 3  . This  may be due to genetic background since genetics influences susceptibility to excitotoxic cell death  8 8  .  Another mouse model which expresses the Swedish mutation A P P 2 3 mouse.  APP751  gene is the  This mouse differs from the Tg2576 by expression o f the gene under  control o f the Thy-1 promoter, resulting in a 7 fold increase in gene expression. A s early as 6 months o f age, these mice develop plaques and have astro- and microgliosis, memory deficits as well as hyperphosphorylated Tau > ' . 89  90  91  in contrast to other mouse  models, A P P 2 3 mice exhibit a 14% decrease in the number o f C A 1 neurons compared to control mice and have deposits o f cerebral A p , preferentially in capillaries and arterioles, and develop cerebral amyloid angiopathy ( C A A )  9 2  ' . 9 3  24  In addition to transgenic mice expressing mutated h A P P , mice expressing PS1 mutations or PS-1 knock out mice have been generated.  Expression of the presenilin  gene did not result in the formation o f plaques, but did result in an increase in the production o f A P 1 4 2 • These mice were then crossed with the aforementioned mouse 38  models to develop mouse strains that would better represent human pathology.  In  PS 1/hAPP mice there is an elevation in the levels o f A p and most interestingly, acceleration in the rate o f amyloid plaque depositions with plaque formation occurring at 6 months o f age  9 4  .  In addition, other transgenic mice, for example A p o E knock out,  A p o E 4 , B A C E overexpressing, B A C E knockout mice and Tau mice have been crossed to A P P mice in the hope to provide models which accurately mimic A D and help aid in future diagnosis, treatment and prevention.  To date no single mouse model faithfully  demonstrates the classic triad o f human A D pathology; A p containing plaques, N F T s and wide spread neuronal loss in the hippocampus and cortical areas, and as such none can be considered a complete model o f the disease.  25  Tg line (background)  Transgene promoter  Mutation  Tg2576 (C57BL/6 x SJL) PDAPP (C57BL/6 x DBA/2 x Swiss Webster) TgAPP23 (C57BL/6 x DBA/2)  Hamster PrP  (HUAPP69S) APP n  l  1  PDGF-p  Murine Thy-1  K670M/N671L  APP  APP r  Y  1  L  V7171  ;  K670M/N671L  Age at onset of AP deposits 9-12 months (Congo red Positive) 6 months (diffuse and compact)  6 months (congophilic and diffuse)  Plaque location  Micro- and Astrogliosis  Neuronal loss  Cognitive deficits  NFTs  Ref  HI, CC, amygdala  yes  Not detectable  yes  AT8 positive  84,  HI, corp call, CC  yes  Not detectable  Yes  AT8 positive  79, 80,  HI, neocortex  yes  Yes, -25% in CA1 at  N.D.  AT8 positive  89,92  N.D.  AT8 positive around Congo red plaques (no NFTs) AT8 positive  89  85,  95,83  82  M I So 1 H-1  TgAPP22 (C57BL/6)  Human Thy-1  APP  Tg2576/PSl (PSAPP) (C57BL/6 x SJL) TgAPP/Ld/2 (FVB/N)  Hamster PrP  APP  TgAPP/Sw/1 (FVB/N)  Murine Thy-1  K670M/N671L  18 months  HI  yes  months N.D.  6 months (Congo red Positive)  HI, CC, amygdala  yes  N.D.  Yes  13-18 months (diffuse, mostly APi.  HI, CC  yes  No overt loss  yes  AT8 positive  96,97  HI, CC  yes  No over loss  N.D.  AT8 positive  98  andv7ni  K670M/N671L  and PS1 p MI46L  Murine Thy-1  APP  V717I  38,94  42)  APP  ^ "  K.670M/N671L  18 months (diffuse, mostly Ap,_ 40)  Table 1. Summary of the primary APP-based transgenic mouse models of AD  Genetic parameters, onset of Ap plaque formation and summary of inflammation and cognitive deficits are described above for some of the APP transgenic mice generated. Abbreviations: HI - hippocampus, CC - cerebral cortex, NFTs - Neurofibrillary tangles, N.D. - not determined (Adapted from Janus et al. °, Gotz et al. ). 10  m  26  1.4 Microglia, inflammation  and Alzheimer  disease  The involvement o f inflammatory processes in A D pathology has been established by multiple lines o f evidence.  The upregulation o f many inflammatory markers co-  localize to regions o f the brain affected by A D , in close proximity to A p deposits and 26  NFTs  .  There is also direct evidence of inflammatory mediated neurotoxicity as  illustrated by the extensive amount o f neuronal death in areas with dense microglia. Finally, many epidemiological studies have suggested that the use o f non-steroidal antiinflammatory drugs ( N S A I D s ) reduces the risk and delay the onset of A D inflammatory processes seen in A D appear to be mediated by microglia  1.4.1  1 0 4  1 0 2  '  1 0 3  .  The  .  Microglia as immune cells of the brain Microglia were first functionally described by del Rio-Hortega in 1932 as  mesodermal pial cells that invaded the brain during embryonic development.  They have  migratory and phagocytic properties and exist in different resting and activated states  1 0 5  .  It is estimated that microglia make up 5-15% o f the brain and are considered to be permanent residents that do not move like macrophages to the ontogenic origin o f microglia.  1 0 6  . There is still much debate as  Initially, microglia were thought to be o f  mesodermal origin. Further studies confirmed this and found that microglial precursor cells are found in the yolk sac and in the mesenchymal tissue associated with the neuroepithelium.  These cells enter the brain, become amoeboid microglia and spread  throughout the brain  1 0 7  . Alternatively, carbon particle tracing and immunohistochemical  techniques showed that ramified microglia originated from circulating blood monocytes that entered the developing brain during development and assumed the form o f amoeboid microglia that ultimately evolve to become the ramified microglia  1 0 7  . Lastly, others state  27  that microglia are not derived from monocytes, rather they are derived during fetal development from neuroectodermal cell precursors; glioblasts or cells of the germinal matrix  1 0 5  .  Regardless o f their origin, it is agreed that microglial cells constitute the  immune system of the brain. They carry out the innate immune response observed in the central nervous system during injury and infection.  1.4.2 Morphological plasticity of microglia It has been established that microglia can assume  various morphological  appearances that can correlate to a distinct functional state (Figure 1.3).  Studies  performed in the facial nerve system first demonstrated this phenomenon by showing that motor neuron regeneration occurred after injury as a result of a microglial response. Microglia appeared at the site o f injury within minutes in response to neuronal signals, became  activated  and  assumed  a highly ramified morphology, proliferated  and  surrounded the injured neuron. During the degenerative phase o f the response, microglia became rounded and phagocytosed the dying neuron  1 0 8  .  In the normal adult brain,  microglia exist in a resting state and also in a highly ramified state. Amoeboid microglia are also seen but these are present predominantly in the developing brain and can transform into ramified cells with age  1 0 7  . Resting microglia are small with flattened or  angular nuclei and have been shown to be non-dividing. Once activated, microglia take on a different shape that is dependent on environment and brain architecture  1 0 5  .  28  Hyper-ramified microglia (itiicrmediate stage)  Figure 1.3. Morphological and functional plasticity of microglia  Microglia can assume different types of morphological shapes in response to environmental stimuli. Signals from damaged or dying neurons cause resting microglia to become active. The amount of signal dictates what conformational state the microglia adopt. In a resting state, microglia have small cell bodies and thin ramified processes with few branches. Intermediate signals from mildly injured neurons cause a hyperramified state but in'most cases microglia become reactive in response to stimuli. This activation causes the cell to adopt a larger cell body and thicker more highly branched processes. In some instances microglia can become amoeboid in shape and take on a phagocytic role, removing dying cells from the brain (Adapted from Streit et al. ). 104  When activated, microglia adopt a reactive conformation with ramified thick processes and abundant cytoplasm. These microglia are usually found in the gray matter of the brain. The hyper-ramified state represents an intermediate stage between the resting and reactive form and signifies the beginning of microglial activation  104  ' . 105  Microglia have also been seen as rod-shaped with elongated bodies and thin ramified processes. These can be found in the brain white matter as well as in the aged brains of normal individual. In this instance, aged microglia lose contact inhibition and fuse with one another to create rod cells  1 0 4105  . Finally, upon neuronal cell death, microglia morph  into brain macrophages, with even more cytoplasm than the reactive state and short thick  29  processes. It is this conformation that microglia are able to phagocytose and degrade cell debris  1 0 5  .  1.4.3 The function of microglia in the central nervous system Microglia are a highly responsive population o f cells with the potential to engage in various functions. These include response  to invading pathogens  and  injury,  elimination of dying cells and debris, initiation in inflammatory processes as well as regulation o f homeostatic mechanisms and neurodevelopment. When microglia respond to neuronal stress signals ( A T P , neuropeptides and neurotransmitters) they become activated, release several secretory proteins such as complement (classical and alternative pathway proteins), proteinases, cytokines, chemokines, excitotoxins, reactive nitrogen and oxygen intermediates, and have altered gene expression  2 6  -  1 0 9  .  Microglia can also  function as antigen presenting cells, albeit not as well as peripheral macrophage. Microglia residing in the C N S rarely encounter T cells and as a result, may not need to present antigen efficiently  1 0 9  . Microglial activation in response to acute C N S injury is  short lived and is a beneficial process. Within 24 hours of insult, microglia arrive to the area and upregulate surface protein expression. Proliferation occurs within 2-3 days and immunophenotypic changes are evident by day 7  m  .  Microglia maintains a very close  physical association with injured neurons in order to facilitate targeted delivery o f growth factors and to displace afferent synapses thereby aiding in neuronal regeneration. In nonreversible injury, such as ischemic damage, reactive microglia secrete neurotoxic factors that aid in killing the damaged neuron preparing it for removal and degradation by phagocytic microglia  1 0 4  .  30  In circumstances that engage chronic activation, microglia may exert detrimental effects by transforming into autoaggressive effector cells that attack healthy cells, also known as bystander damage. Chronic C N S activation, as in A D , is primarily caused by bystander damage and is characterized by slow, progressive neurodegeneration that takes decades to develop.  Exposure o f cells to the various proteases and toxic molecules,  produced by activated microglia for a long period o f time causes extensive neuronal damage and death. Moreover, many o f the pro-inflammatory cytokines and chemokines that are secreted contribute to a positive feedback mechanism, causing further attraction and activation o f more microglia.  These same factors have also been implicated in  initiating signal transduction cascades on both microglia and neuronal cells that results in the transcription o f I L - l p , transforming growth factor-P ( T G F - P ) and more cytotoxic agents via the nuclear factor K B ( N F K B ) transcription factor  1 0 5  . Finally, many o f the  inflammatory products secreted by microglia can alter the processing o f A P P in neurons to favour the production o f  AP1-42,  activation and neuronal death  thereby resulting in more plaque formation, microglial  .  1.4.4 The role of activated microglia in Alzheimer disease Many reports imply that microglia may exacerbate  the pathological states in  neurodegenerative diseases. Persistent activation o f microglia is an accepted hallmark of A D and a substantial amount o f the neuronal damage observed in A D may be due to the inflammatory response mediated by microglia. A p , N F T s and degenerating neurons are most likely the stimulants for inflammation in A D . A s in the periphery, there is not just one system of inflammation that is involved in a response.  In the A D brain there is  substantial evidence for the multiple interactions o f complement pathways, cytokine and  31  chemokine pathways, cyclo-oxygenase ( C O X ) and acute phase proteins.  In vitro  experiments with A p aggregates, in vivo experiments in A D mice and A p infused animals, and in situ studies on post-mortem A D brains have all demonstrated that A p can induce microglia to upregulate the expression o f cytokines ( I L - i p , T N F - a ,  IL-6),  I  chemokines chemoattractant  (IL-8,  macrophage  peptide-1)  inflammatory  chemokine receptors  protein-la  ( C C R 3 and C C R 5 ) ,  proteins ( C l q and C 3 , C4) and major histocompatibility complex II such as melanotransferrin (p97)  1 1 0 1 1 1  and  heme oxygenase 1  1 1 2  macrophage complement  and other genes  and urokinase plasminogen-  activating receptor " . Activated microglia also release the excitotoxins quinolinic acid 3  1 1 4  and glutamate  1 1 5  . Since endotoxins only cause limited damage to synapses and  dendrites, it is thought that they may contribute to the neuronal dysfunction rather than complete neuronal death  2 6  . Together, all of these factors contribute to the inflammatory  mechanisms observed in the disease. It has been shown in-situ that microglia cluster around the sites of amyloid deposition both in humans and in A P P transgenic mice  116  - > > li7  90  118  The clustering o f microglia  around the senile plaques can be explained due to the chemotactic signaling o f a number of molecules including A p , signals from damaged/dying neurons and also the many proinflammatory mediators which are found in the area o f the plaque. Still under debate is the relationship between microglia and the development o f A P plaques. One possibility is that microglia may directly contribute to amyloidosis by participating in the synthesis and processing of A P P to A p fibrils. It has been demonstrated that cultured microglia are able to secrete A p ; however, in vivo, microglia do not appear to express A P P m R N A  2 6  Therefore the likelihood that microglia secrete A P and contribute to A P plaque formation  32  .  is debatable and requires more research.  Another possible mechanism relates to the  potential of microglia to convert soluble AP into a more fibrillar and aggregative form. Many studies have stated that most activated microglia appear to associate around neuritic plaques rather than diffuse plaques in both mice and humans ' m  1 1 8  ' ; however, 9 0  there are some microglia associated with diffuse plaques - . These data suggest that 120  121  microglia play a role in transforming nonfibrillar amyloid into the more insoluble fibrillar form and aid in the transformation from diffuse plaques to neuritic plaques. Finally, microglia may contribute to plaque formation by their ability to phagocytose and degrade Ap.  It is proposed that there is a dynamic balance between amyloid  deposition and removal in the brain. It has been established in culture, that microglia migrate to and internalize microaggregates of A p ^ , via a type A scavenger receptor, and deliver it to late endosomes and lysosomes, where it is degraded  122  ' . This uptake 60  123  appears to occur rapidly; however, the degradation process occurs at a slow rate Furthermore, it has been reported that microglia found in the cortex of AD patients contain fragments of A P , most notably C-terminal fragments, inferring that the A p was phagocytosed  124,125  . This rapid internalization of A p microaggregates coupled with the  slow degradation can lead to the accumulation of A p inside the cell. Therefore, in the case of AD, the overproduction and thus the persistence of A P may become too overwhelming for the microglia and thereby disrupting the dynamic balance between A p deposition and removal. This was demonstrated in a study by Rogers et al. where microglia cultured with A p were able to migrate to and phagocytose the A P ; however, degradation of the fibrils took between 2-4 weeks  126  . Another study found that although  some degradation of A p by microglia was observed over 3 days, no further degradation 33  was observed over the next 9 days. Instead, there was a slow release o f intact A P Understanding the mechanism o f A p clearance by microglia is important in determining the steps o f A p plaque formation. It is possible that there is a fine balance between A p processing and deposition and degradation.  A n y disruption o f the A p equilibrium can  thereby lead to the accumulation o f A p and the formation o f amyloid plaques. A n alternative theory to the role o f microglia in A D was offered by Streit  1 0 9  and  states that microglia become senescent and/or dysfunctional with normal aging, therefore their ability to support neurons would be diminished resulting in neuronal degradation. The rate o f deterioration can be affected by genetic and environmental risk factors thereby causing some individuals to develop A D .  Some indication o f dysfunction may be  in the structural abnormalities seen in microglia as they age and in A D .  Microglia which  cluster around plaques have bulbous swellings in their cytoplasmic processes, called spheroids, as well as the formation o f long stringy processes  1 0 9  . However, there is still  little experimental evidence o f microglial dysfunction in A D .  1.4.5 Signal transduction pathways and microglial activation Many in vitro studies have demonstrated that the exposure o f microglia to fibrillar Ap  results in the activation o f complex kinase and phosphatase signal transduction  pathways that lead to the activation o f various transcription factors  involved in  inflammation. These include: the S T A T family members, peroxisome proliferatoractivated receptor y (PPAR-y), c-fos, and c-jun; N F k B ; and members of the family o f transcription factors > - > 58  128  129  130  C/EBP  Subsequent immunohistochemistry studies in  A D brains o f both human and animal models have revealed the upregulation o f many  34  signal transduction proteins in microglia and neurons that are in close proximity to the plaque  1 3 1  .  One of the most important kinase families involved in the inflammatory response and consequent neuronal damage/death is the M A P K family.  The complex molecular  interactions between M A P K s and proteins associated with the evolution o f A D form a cornerstone in the knowledge o f a still burgeoning field. The M A P K . family comprises four subfamilies: 1) E R K 1 / 2 (p44/42 M A P K ) ; 2) c-jun N-terminal kinases ( I N K ) ; 3) p38 M A P K ; and 4) E R K 5 / b i g M A P kinase 1  1 3 0  . Activation and subsequent phosphorylation  of E R K 1 / 2 , I N K and p38 have been detected in A D . In the brain, the E R K 1 / 2 kinase pathway has been implicated in eliciting stress responses, including oxidative stress, and in the regulation of intracellular calcium levels ° . J N K pathways have also been shown l 3  to  respond  to  cell  stresses and  in addition, evidence  has  confirmed that  phosphorylation o f J N K leads to the activation o f death domain receptors by T N F - a  the 1 3 2  .  The p38 M A P K family is activated by inflammatory molecules and is involved in cell cycle, cell growth and cell differentiation  I 3 0  . In A D mice, phosphorylated p38 levels are  increased approximately 3 fold in plaque-associated microglia. Many cell culture experiments have demonstrated that exposure o f microglia and co-cultures o f microglia and neurons to both oligomer and fibrillar A p leads to the activation o f p38, E R K and J N K in microglia  5 7 1 3 3  '  1 2 8  .  Moreover, M A P K s have been found to be integral to the  induction o f I L - i p , T N F - a and reactive nitrogen intermediates from L P S and A p treated cells  5 7 , 1 3 3  .  The molecular mechanisms by which M A P K signaling contribute to glial  mediated responses in A D are not fully elucidated. It is thought that the different M A P K pathways are activated in response to different mediators, leading to a differential  35  response by the microglia.  Therefore, delineation o f the downstream targets o f these  signaling cascades is also critical in order to further unravel their role in A D as well as in creating potential therapeutics.  1.5  The blood-brain  barrier  and Alzheimer  disease  1.5.1 The blood-brain barrier, structure and function The blood-brain barrier ( B B B ) , found in all vertebrates,  prevents the  free  diffusion o f circulating molecules and cells into the brain interstitial space. The barrier is formed by the presence o f epithelial-like, high resistance tight junctions that fuse brain capillary endothelia together into a continuous cellular layer separating blood and brain interstitial space. There are fine structural differences from the endothelium in the brain compared to that o f other organs.  These include tight junctions between  endothelial cells, paucity o f pinocytotic vesicles and lack o f fenestrations endothelial cells o f the B B B have high mitochondrial content.  adjacent 1 3 4  .  The  It is thought that the  energy generated in the mitochondria provide energy to the cell in order to create and sustain high-resistant tight junctions  1 3 4  . The junctions that comprise the B B B are made  up o f adherens junctions and tight junctions.  Adherens junctions are made up o f  cadherins which form adhesive contacts between cells by binding to cadherins on the surface o f neighboring cells. Inside the cell, cadherins bind to the actin cytoskeleton via proteins such as catenins  1 3 4  . Tight junctions consist o f three integral membrane proteins,  claudin, occludin and junction adhesion molecules.  There are also many cytoplasmic  accessory proteins that link tight junctions to the cytoskeleton o f the cell.  Claudins are  36  the major proteins found in tight junctions and bind to other claudins on adjacent cells to create the primary junction.  The carboxy terminus of claudins also associates with  cytoplasmic proteins, such as zonula occudens proteins (ZO) which, in turn, bind to actin. Occludins are also found with claudins in the cell membrane. These proteins also bind to Z O proteins and are implicated in the regulation o f cell permeability  1 3 5  . The B B B is also  comprised of a basement membrane, astrocyte foot processes that surround the vessel, and pericytes embedded within the basement membrane (Figure 1.4)  l 3 4  . The basement  membrane is a heterogeneous mixture o f proteins that are secreted from the endothelial cells and glial cells and include laminin, fibronectin, collagen type I V and proteoglycans. The presence of the basement membrane in the B B B is to provide elasticity to the vessels as well as to aid in the selective transport o f highly charged molecules  l 3 6  .  The  contribution o f astrocytes to the B B B appears to be that o f influencing the morphogenesis and organization o f the endothelial cells that make up the vessel wall. Studies involving the transplantation o f cultured astrocytes into areas o f the brain with leaky vessels have demonstrated that astrocytes induced the tightening o f the junctions between endothelial cells  1 3 7  the  . This is thought to be mediated by astrocyte derived soluble factors,  such as TGF-P and IL-6, and may require direct contact between the astrocytes and the endothelium  138  > . in contrast, it has been shown that upon glial cell activation, many 134  cytokines and chemokines, such as T N F - a and IL-8, are produced resulting in an increase in vascular permeability and lymphocyte entry into the brain  1 3 6  . Pericytes wrap around  endothelial cells and appear to play a role in structural integrity, formation o f tight junctions and the differentiation o f the intact vessel as well as in angiogenesis. studies have shown that pericyte loss can result in microaneurysm formation  Some 1 3 9 1 4 0  37  However, little is known how these processes are mediated since few studies on pericyte function have been conducted  1 3 4  .  F i g u r e 1.4. Schematic of the BBB. The B B B is created by the tight apposition o f endothelial cells lining blood vessels in the brain, forming a barrier between the circulation and the brain parenchyma. The B B B is permeable to small and lipophilic molecules. Larger and hydrophilic molecules can only be transported across via an active transport system. Blood-borne immune cells (lymphocytes, monocytes and neutrophils) cannot penetrate this barrier. A thin basement membrane, comprised o f laminin, fibronectin and other proteins, surrounds the endothelial cells, associated astrocytes and pericytes, providing both mechanical support and a barrier function  The fluid o f the central nervous system differs in composition from the non-neural extracellular fluid due to the selective permeability o f the B B B  1 4 1  . The functional events  that define the B B B occur at the capillary level. The B B B , under normal physiological conditions, regulates the passage o f peptides, proteins and other molecules between the  38  periphery and the brain. The physiochemical properties o f the penetrating substance or solute, determine whether it can be transported across the B B B . Hydrophobic solutes can penetrate across the B B B while hydrophilic substances are preferentially transported across the B B B by specific membrane carrier proteins for which they have a high affinity 1 4 2  .  In contrast, most foreign substances cannot penetrate the B B B since they are not  recognized by the carrier systems.  The barrier works in both directions. In addition to  keeping unwanted substances out, the B B B retains brain synthesized compounds, such as neurotransmitters, in the brain. The cerebral endothelia forms tighter junctions than other endothelia and contains specific proteins, which are specifically expressed in the brain. These include alkaline phosphatase, y-glutamyl transpeptidase, the glucose transporter (Glut-1)  1 4 3  , and the molecules involved in metal transport such as the transferrin receptor  (TfR) and p97  1 4 4  .  1.5.2 BBB integrity and Alzheimer disease The integrity o f the B B B is an area o f great contention in A D research. comparing the vasculature o f A D patients to controls have conflicting results.  Studies Upon  analysis of vasculature from post-mortem A D brains there was a decreased mitochondrial content and increased pinocytotic vesicles as compared to values obtained previously in endothelium from multiple sclerosis patients and in A D animal models. Other findings such as accumulation of collagen in vascular basement membranes and focal necrotic changes in endothelial cells were present in A D patients indicating B B B disruption In contrast,  1 4 5  .  Caserta et al. found that there was no difference in B B B integrity between  A D cases and controls as indicated by blood-to-brain transport (KI) and tissue to blood  39  efflux (k2) o f meglumine iothalamate, a radiopaque medium that binds to plasma proteins and is commonly used for angiography and venography  1 4 6  .  In animals models the role o f the B B B integrity in A D is also debated.  Increased  B B B permeability for some proteins, such as insulin, is evident and suggests some structural alterations at the B B B , however, these alterations do not support the concept o f extensive B B B damage  l 4 7  . What's more, it appears that leukocytes are able to cross an  intact B B B without causing concomitant B B B breakdown  . Alternatively, it has  recently been established that the B B B integrity is compromised in h A P P overexpressing A D mice compared to controls and that the breach in B B B integrity precedes plaque formation  1 4 9  . Many studies have indicated that A p peptides promote pro-apoptotic and  pro-angiogenic  signals  in  endothelial  cells  150  > > l51  152 ?  a  n  (  i that  systemic-derived  inflammation, either triggered by A P or neuronal signals, causes B B B tight junction disruption and increased paracellular permeability  153  > > 154  155  In addition to the neurodegeneration observed in A D , there are also considerable cerebrovascular abnormalities. It is becoming evident that the relationship between A D and C A A is growing more complex and it is harder to distinguish one disease being distinct from the other. It is estimated that the prevalence o f C A A in A D patients varies from 70 to 100% primarily A p j  3 9  156  > . > . ,57  and A P  158  M 0  C A A is characterized by the deposition o f amyloid,  159  , in the cerebral vessel wall  1 6 0  .  Since A D and vascular  disease share common risk factors and since a history o f strokes may be a risk factor for AD  1 6 1  ,  it  is  important  to  understand  the  relationship  between  dementia,  neurodegeneration and cerebrovascular abnormalities. It is possible that this relationship is attributed to alterations in the B B B .  Furthermore, as evidenced by the first phase o f  40  human clinical trials o f the amyloid vaccine where cerebral complications ensued  1 6 2  '  1 6 3  '  there is still much to learn about the relationship between neurodegeneration, dementia and cerebral abnormalities induced by A p .  1.6 Therapeutic strategies The treatment o f A D up until now has focused on the cholinergic system and symptoms thereof.  Cholinergic therapeutics, such as Aricept, an acetylcholinesterase  inhibitor, and psychotrophic drugs have had relative success in stabilizing the cognitive decline and behavioral deficits attributed to A D . Nevertheless, these therapies only treat the symptoms and do not delay or prevent disease progression.  Based on the widely  accepted "amyloid cascade hypothesis", research is now centered on the generation o f anti-amyloid therapies.  Therapeutic strategies directed at lowering A p levels and  decreasing levels o f toxic A p aggregates through: (1) inhibition of A P P processing to A P , (2) inhibition, reversal or clearance o f A P aggregation, (3) cholesterol reduction and (4) A p immunization are under development A s a target to alter A P metabolism, inhibition of both P- and y-secretases are being pursued. Treatment of mice with y-secretase inhibitors has resulted in a significant reduction in brain A p levels and an attenuation o f A p deposition  164  '. Inhibition o f p-  secretase is also under study and is thought to be a better target than y-secretase inhibitors since y-secretase cleavage is associated with the cleavage o f other proteins, such as N O T C H , which is important for development. Studies on B A C E 1 ~'~ mice demonstrate an absence in brain A p and no other obvious n o n - A D related pathologies  1 6 5  . Moreover, in  B A C E 1 7 A P P mice there is an absence o f amyloid plaques, microgliosis and dystrophic 7  41  neuritis in the brain. A t present, inhibitors for y-secretase are in phase 1 clinical trials; however, more research is needed before such inhibitors are readily available for treatment. In vivo, in vitro and epidemiological studies suggest that cholesterol may play a role in the generation o f amyloid and its subsequent accumulation by its role in lipid metabolism.  Retrospective  analysis  o f people  taking (3-hydroxy-P-methylglutaryl-  coenzyme A reductase inhibitors, or statins, show that there was up to a 70% reduced risk for developing A D , whereas individuals with high cholesterol levels had a higher risk for AD  1 6 6 , 1 6 7  .  In vitro studies on primary neuronal cultures have shown that membrane  cholesterol affects the cleavage sites for both P- and y-secretase, favouring the production of APi_42, and that the removal of membrane cholesterol caused a decrease in A p production  1 6 8 1 6 9  .  Other studies, in which A P P mice were fed high cholesterol diets,  found that there was a decrease in s A P P a and an increased the A P P cytosolic fragment A I C D and plaque burden in high cholesterol diet mice compared to mice on a control diet 1 7 0  '  1 7 1  . Furthermore, feeding mice with a statin called atorvostation, commercially known  as Lipitor, resulted in a 50% decrease in plaque load and an increase in the fonnation of s A P P a in A P P transgenic mice  1 7 2  . Similar results are seen in humans, where statin use  resulted in an approximate 40% decrease in A p serum levels  1 7 3  . Taken together, these  data suggest that cholesterol modulating drugs may have a significant clinical benefit to the treatment and prevention o f A D . Over the past few years the role of inflammatory mechanisms in A D pathogenesis is becoming more evident.  N S A I D s , such as ibuprofen (Ibu), indomethacin  and  nimesulide (Nim), have been used as therapeutics for the treatment o f A D . These drugs  42  have been shown to have anti-inflammatory properties by reducing the enzymatic activity Of  of C O X enzymes and thus the production o f prostaglandins been demonstrated to have the capability of altering  . These drugs have also production, presumably by  A(3M2  acting on the y-secretase complex and shifting cleavage towards the shorter less toxic forms of A P  1 7 4  . Treatment o f neuroblastoma cells with the above N S A I D s resulted in  the stimulation o f a-secretase and secretion o f s A P P a  3 3  .  Recent clinical studies with  N S A I D s are inconclusive due to small sample sizes and a large drop out rates.  Thus,  while the exact targets o f anti-inflammatories remain to be clarified, new clinical studies, with larger cohorts and more extensive research into new drugs are ongoing and w i l l hopefully illuminate the practice o f anti-inflammatory therapeutics for A D . The most recently developed a n t i - A P therapy is aimed at the reversal and/or clearance of A P aggregates and employs immunization with either A P peptides or antiA P antibodies. A P immunization appears to be effective in reducing amyloid deposition and plaque formation, neuritic dystrophy, astro- and microgliosis, memory and cognitive 175 176 177 178 179 180 181  deficits and early Tau pathology " >" >'">"°J  D  H  q  w  i  m  m  u  n  i  z  a  t  i  o  n  reverses or attenuates disease pathology remains to be elucidated.  It is thought that A p or anti-Ap  antibodies act by eliciting one or more of the following: 1) preventing fibril formation, 2) blocking the toxic effects o f soluble and aggregated A p , 3) disrupting A p fibrils, and 4) enhancing the clearance of A P by microglia  1 6 4  .  Clinical trials of active A p peptide  immunization were undertaken but were suddenly halted in phase II when 5% o f the patients exhibited meningio-encephalitis. N e w administration techniques, such as type o f adjuvant and dose of A p peptide/anti-Ap antibody, have been explored and currently new clinical trials, involving both active and passive immunization, are being pursued. 43  1.7 Project rationale and general approach The focus of this thesis was to add to the basic understanding of the relationship between microglia and amyloid beta and to the various aspects of A D pathology. Both in vitro and in vivo experiments were performed in order to examine the role of microglial gene expression in response to Ap stimulation as well as the role of microglia in plaque formation.  It was hypothesized that in response to general activation, microglia  upregulate the expression of specific genes, in particular, p97, and that these changes in gene/protein expression may be used as a means to determine anti-inflammatory drug efficacy.  Moreover, it was hypothesized that activated microglia exacerbate the  pathology of A D and contribute to plaque formation. Finally, it was hypothesized that the damage that activated microglia and Ap incur on the B B B can be reversed by A p immunotherapy. Taken together, the studies presented here provide new ways by which A D progression and treatment can be monitored, as well as providing a new therapeutic target for A D treatments and disease prevention.  44  Chapter 2: Materials and Methods 2.1 Mice 2.1.1 Tg2576 AD model mice The transgenic mice used in this study are the Tg2576 A D model mouse (Tg/+), which expresses the Swedish mutant o f the amyloid precursor protein ( K 6 7 0 N / M 6 7 1 L )  8 4  under control o f the hamster prion protein promoter. M i c e were maintained by mating Tg2576 males to C 5 7 B 6 / S J L F I females. This strategy was undertaken since Tg2576 mice are heterozygous for the h A P P gene. Tg2576 mice were distinguished from control littermates by P C R at 2 weeks o f age as previously described  8 4 , 8 5  . Briefly, primers  specific to the Hamster prion promoter and human A P P were used along with an internal control (1501, 1502, 1503; Table 2) to determine i f mice contains the human A P P gene. Animals were fed lab chow and water ad libitum and kept under a 12 hr light/dark cycle. M i c e were group housed where possible, although the occasional male mouse had to be housed alone due to aggression. W i l d type (+/+) littermates were used as controls. For the vaccination studies described below, 6 week and 11 month old mice were used.  2.1.2 Colony stimulating factor-1 deficient mice (op/op) Colony stimulating factor -1 (CSF-1) deficient mice, referred to as op/op mice, are osteopetrotic model mice. These mice have a spontaneous mutation resulting in a base pair insertion approximately 280 base pairs into the coding region o f the CSF-1 gene generating a stop codon and a nonfunctional CSF-1 > > _ CSF-1 is a major growth I82  183  184  factor for macrophages in vivo controlling survival, proliferation and differentiation  1 85  45  The osteopetrosis phenotype is characterized by the lack o f osteoclasts, thus impairing bone remodeling accompanied by retarded skeletal growth, excessive accumulation o f bone, and the absence o f incisors. macrophage.  In addition there is an absence o f monocyte derived  Mature macrophages are produced from other precursor cells by the  influence o f granulocytes and macrophage colony stimulating factor.  There is also a  reduction in the number and an alteration in the morphology and function o f microglia in the brain  1 8 6  '  1 8 7  .  These animals also have a reduced viability and poor breeding  performance, op/op mice were fed with powdered chow in infant milk formula and water ad libitum and kept under a 12 hr light/dark cycle.  Homozygous op/op mice were  distinguished phenotypically from normal littermates by the absence o f incisors and by a domed skull at day 10  1 8 8  .  2.1.3 Generation of Tg/+;op/op mice • .  '  Tg/+;op/op mice were first generated by crossing female Tg2576 A D model mouse, described above, to male op/op mice. The Tg/+;op/+ offspring were interbred to produce double mutant  Tg/+;op/op mice.  Littermates Tg/+;+/+, +/+;op/op, +/+;+/+  served as controls. A l l procedures were conducted with approval by the University o f British Columbia A n i m a l ethics committee.  2.2 Preparation of reagents The amyloid beta peptides ( A p  M  0  and A p  4 0  ) used in the present study were  }  synthetic peptides o f human A p (Bachem, Torrance, C A ) . The peptides were dissolved in double distilled water at 100 p M concentration and incubated at 37°C with 5% C 0  46  2  for 5 days t o allow for fibrils-to form. The final concentration of Ap for cell stimulation was 10 uM. This concentration was chosen as a result of a dose response experiment which showed optimal activation at 10 uM Ap. Flourescent-labelled A p  M Q  (Calbiochem,  La Jolla, CA), used for BBB studies, was resuspended in distilled water and used immediately after reconstitution. LPS from  Salmonella typhimurium  and mouse IFN-y  (Sigma-Aldrich Canada Ltd, Oakville, ON) was resuspended in sterile phosphate buffered saline (PBS) as 1 pg/ml stocks. For NSAID studies, Ibu and Nim (Sigma) were dissolved in dimethylsulfoxide (DMSO) at a ImM concentration. Inhibitors of MAPK pathways, SB203580 (SB; p38 MAPK inhibitor) and PD98059 (PD; MEK1/2 inhibitor) (Calbiochem, La Jolla, CA) were made as 20 mM stocks in DMSO. All chemicals were of analytical grade. All primers used in this study were synthesized by Sigma Genosis and are listed in Table 2. Primer name  Primer sequence  Product size (bp)  1501 (mouse APP)  5'-AAGCGGCCAAAGCCTGGAGGGTGGAACA-3'  -760  1502 (PrP promoter)  5'-GTGGATAACCCCTCCCCCAGCCTAGACCA-3'  1503 (hAPP)  5 '-CTG ACC ACTCG ACC AGGTTCTGGGT-3'  -420  P97+1  5 '-G AGGGTG ACTTTTTGGCTACT-3'  -450  P97-1  5'-AACGGA AGGCTCC ACTGAGC-3'  SI5 sense  5'-TTCCGCAAGTTCACCTACC-3'  SI5 antisense  5'-CGGGCCGGCCATGCTTTACG-3'  P1800 FWD  5 '-GGCACGGGTAGTAGTAGG GAA-3'  P1800 REV  5'-GGCAACGTTGGGTTGGCT-3'  -360 - 1800  Table 2. List of primer sequences and product Primers were custom synthesized by Sigma genosis, made up in distilled water at a concentration of 20 pM, aliquoted and kept at -20°C. 47  2.3 Cell  culture  The murine microglial cell line, B V - 2 , provided by Dr. Michael M c K i n n e y (Mayo Clinic, Jacksonville, F L ) , was derived from vraf/v-myc-transfected primary murine microglia. These cells exhibit morphological and functional features similar to primary microglia, such as cytokine secretion and phagocytosis  1 8 9  .  It has been reported that  phenotype changes can occur in B V - 2 cells after multiple passages  1 9 0  . To avoid this, all  cells used were passaged no more than 3 times. B V - 2 cells were cultured in Dulbecco's modified Eagle's medium ( D M E M ) (Invitrogen Life Technologies, Burlington, O N ) supplemented with 10% (v/v) fetal bovine serum (FBS) (Medicrop, Montreal, PQ.), 2 m M L-glutamine (Sigma) and 20 m M Hepes (Sigma). J B / M S , a murine melanoma cell line which expresses high levels o f p97, were provided by D r . Vincent J. Hearing (National Institute o f Health, Bathesda, M D ) and bEnd.3, a murine brain endothelial cell line, obtained from American Tissue Type Culture Collection ( A T C C ) , were maintained in D M E M supplemented with 10% (v/v) F B S , 2 m M glutamine and 20 m M Hepes. 3T3, a murine fibroblast cell line was obtained from A T C C and maintained in D M E M supplemented with 10 % (v/v) bovine calf serum, 4 m M L-glutamine and 20 m M Hepes. A l l cells were cultured at 37°C in a 5% CO2 humidified incubator.  2.4 Cell  stimulation  A l l cell lines described above were maintained and prepared the same way. Twenty-four hours before treatment, cells were washed with P B S , plated at a density o f 5x10 cells/ml in serum free D M E M and incubated overnight at 37°C. For determining 5  48  the effect o f microglial activation on p97 expression, the cells were then washed twice with serum free D M E M and treated with either 10 u M A ( 3 IFN-y, 10 u M A | 3  4 0 }  M 0  , 50 ng/ml L P S , 5 ng/ml  or P B S for 24 hours. The supernatant was harvested and stored at  -80°C for T N F - a E L I S A . The cells were then washed with ice cold P B S and R N A was extracted for R T - P C R as described below. For N S A I D studies, cells were treated with 10 uM  Ap  M Q  or 50 ng/ml L P S in the presence or absence o f 10 u M drug or left untreated.  The N S A I D s used in this study were Ibu, a non-selective inhibitor o f the C O X enzyme family, and N i m , a C O X - 2 specific inhibitor. After 24 hours o f treatment, the supernatant was harvested and stored at -80°C for T N F - a E L I S A and the cells were washed with ice cold P B S . m R N A was then extracted for R T - P C R or cell lysates were harvested for Western blot analysis.  2.5 Creation of stable BV-2 transfectant cell lines The Balb/c mouse brain genomic D N A was used to clone the promoter region o f mouse p97. The p97 promoter, spanning approximately 1800bp from nucleotides -1641 to +59 (nucleotides numbered according to GenBank accession N M 013900 from the National Centre for Biotechnology Information database), was cloned by P C R using the sense p i 8 0 0 F W D primer and antisense p i 8 0 0 R E V primer (see Table 2 for primer sequences). The P C R product was subcloned into pCR2.1 - T O P O vector (Invitrogen Life Technologies  Burlington,  ON)  and  sequenced  using  standard  ml3R  (5'-  C A G G A A A C A G C T A T G A C C - 3 ' ) and T7 primers ( 5 ' - T A A T A C G A C T C A C T A T A G G G 3') at the N A P S Facility (Nucleic acid-Protein Service Unit, University of British  49  Columbia, Vancouver, B C ) . After digestion with A p a l  and HindiII (New England  BioLabs), the p97 promoter was subcloned in-frame into the p E G F P - 1 expression vector (Figure 2.1) (Clonetech, Palo Alto, C A ) . p E G F P - 1 was chosen as a reporter system, since it lacks its own promoter and produces green fluorescence upon E G F P gene expression initiated by a ligated promoter. sequenced  with  p i 800  FWD  and  The resulting p l 8 0 0 - G F P construct was EGFP-N  sequencing  primer  (5'-  C G T C G C C G T C C A G C T C G A C C A G - 3 ' ) and digested with A p a I and Hind III to ensure correct D N A sequence and orientation (Figure 2.2).  50  MCS  fco0109 I  Sful  TAGCGCTACCG G AC TCAGATCTC GAGCTC AAG CTT CG A ATT CTC CAG TCG ACG GTA CCG CGG GcC CBS GAT CCA CCG GTC GCC ACC ATS GTS teMU Be/tl JfA-Ti V U l fool I M l fell i f I "1 *\"~§»™H I V I Stc\ Aec\ 4io7t* I \ Si»139 I Xmi I  Figure 2 . 1 . p E G F P - 1 vector and multiple cloning site This vector encodes a red-shifted variant o f wild-type green fluorescent protein G F P which has been optimized for brighter fluorescence and higher expression in mammalian cells. pEGFP-1 encodes the G F P m u t l variant which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. pEGFP-1 is a promoterless E G F P vector which can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the M C S located upstream o f the E G F P coding sequence. The coding sequence o f the E G F P gene contains more than 190 silent base changes which correspond to human codon-usage preferences. Sequences flanking E G F P have been converted to a Kozak consensus translation initiation site to further increase the translation efficiency in eukaryotic cells. S V 4 0 polyadenylation signals downstream of the E G F P gene direct proper processing o f the 3' end o f the E G F P m R N A . A neomycin-resistance cassette (Neo ) allows stably transfected cells to be selected using G418. A bacterial promoter upstream of this cassette confers kanamycin resistance in E. coli. (Adapted from Clontec). 1  51  Figure 2.2. G e l of digested p E G F P and p97 promoter construct. Each construct, p E G F P - p l 8 0 0 and p E G P F , were digested with A p a l and H i n d l l l for 3 hours at 37°C. The digested products were analyzed on a 1% agarose gel and visualized. The 4200 bp fragment indicates the pEGFP-1 vector while the smaller 1800 bp product indicates the promoter region o f p97.  B V - 2 cells, at approximately 70% confluency, were transfected with the 5 pg o f the p l 8 0 0 - G F P construct or the promoter-less pEGFP-1 vector by using Lipofectamine™ 2000 reagent (Life Technologies, Inc., Gaithersburg, M D ) under serum free conditions. Cells containing either the p l 8 0 0 - G F P construct or the promoter-less pEGFP-1 vector were selected by adding 500 pg/ml G418 (Invitrogen). The transfectants continued to grow for approximately 3 weeks with 500 pg/ml G418, and then were sorted using the F A C S D i v a ( B D Pharmingen, San Jose, C A ) into single cell clones.  Clonal cell lines  continued to grow until confluency under selective conditions and were then analyzed for G F P expression. Since G F P expression is under control o f the p97 promoter, cells were treated with 10 p M  APMO  for 24 hours and then sorted for high G F P expressing cells.  Cells transfected with pEGFP-1 vector alone were sorted for low G F P expression. Three days later, cells were sorted again for low expressing G F P to isolate cells in an inactive,  52  resting state.  These cell lines continued to grow for until confluency under selective  conditions and were then used for experiments described.  2.6 RNA Isolation m R N A from B V - 2 , J B / M S , 3T3 and b.End3 cells, as well as from mouse brain regions was extracted using the RNeasy mini K i t (Qiagen Inc., Mississauga, O N ) according to the manufacturer's instructions. Cells were washed once in cold P B S , lysed with lysis buffer containing P-mercaptoethanol and spun down in a spin column. The cells were then washed with an appropriate buffer and R N A was isolated.  RNA  concentrations were determined using spectrophotometry at U V wavelengths o f 260 and 280. Final R N A concentration was obtained form the following calculation O D o X 40 = [ R N A ] pg/ml 26  2.7 Reverse transcriptase and Polymerase Chain Reaction Reverse Transcriptase and Polymerase Chain Reaction ( R T - P C R ) was performed with oligonucleotide primers custom synthesized by Sigma Genosis listed in Table 2. The expression o f p97 and S I 5 (loading control) was examined. To make the c D N A , 1 p g o f R N A was used along with 1 p l o f deoxyribonucleotide triphosphate (dNTPs) m i x (lOpl each o f d A T P , d C T P , d G T P and dTTP) (Invitrogen), l u l o f Oligo dT . (Invitrogen) and Rnase free water to a total volume o f 12 p l . The mixture was incubated at 65°C for 5 minutes followed by a quick chill on ice. Next, 4 p l o f 5x first strand buffer, 2 p l o f 0 . 1 M dithiothreitol ( D T T ) and 1 p l o f RNase O U T (Invitrogen) was added and incubated 53  at 4 2 ° C for 2 minutes followed by the addition o f 1 p l o f Superscript II (Invitrogen). The solution was mixed by gentle pipetting and incubated at 4 2 ° C for 50 minutes.  After  incubating, the reaction was then inactivated by heating the solution at 70°C for 15 minutes, followed by the addition o f 1 p l of Rnase H (Invitrogen) and incubation at 37°C for 20 minutes. P C R amplification was performed through 35 cycles at 94°C for 30 sec, 56°C for 30 sec, and 72°C for 45 sec. The P C R was stopped with a final extension for 10 min at 72°C. Samples were then loaded into a 1% agarose gel and images were digitally captured and analyzed.  2.8 Real-time Polymerase Chain Reaction R T - P C R results were confirmed by semi-quantitative real-time P C R for p97 and for S15 m R N A (used as internal control).  R N A ( l p g ) from each sample was reverse  transcribed using Superscript'" II Reverse Transcriptase (Invitrogen).  Real-time P C R  was performed using the Roche Light Cycler. In brief, 1 p l of c D N A and gene specific primers were added to SYBR® Green Taq ReadyMix™  ( S Y B R Green dye, Taq D N A  polymerase, JumpStart Taq antibody, d N T P mix and optimal buffer S I G M A ) and subjected to P C R amplification.  components;  The following protocol was used:  denaturation (95°C for 5 minutes), amplification and quantification (40 cycles at 95°C for 5 seconds, 56°C for 5 seconds and 72°C for 30 seconds), melting curve program (6595°C with a heating rate o f 0.2°C per second and a continuous fluorescent measurement) and a final cooling step to 40°C. The primers used for p97 and SI5 were synthesized by Sigma Genosis and are shown in Table 2. The amplified transcripts were quantified using  54  the comparative C T method. Briefly, fold increase was calculated by determining the cycle at which the fluorescence passes the fixed threshold (CT) for each sample. Below are formulas used to calculate the fold induction for each sample (as presented by Applied Biosystems).  A C T = C T (gene o f interest) - C T (endogenous control) A A C T - A C T (sample X ) - A C T (control sample) Fold Induction = 2 "  A A C |  2.9 TNF-a ELISA assay T N F - a produced by B V - 2 microglial cells was determined in culture supernatants using a specific murine T N F - a E L I S A kit capable of detecting levels as low as 5.1 pg/ml ( R & D Systems, Inc., Minneapolis, M N ) according to the manufacturer's protocol. T N F a was chosen as a measurement o f microglial activation since it has been established that microglia upregulate the production and secretion o f T N F - a upon activation . After 24 2 6  hours o f treatment the cell culture supernatant was removed and stored at -80°C until assayed for T N F - a .  E L I S A measurements were performed using the Spectra M a x 190;  (Molecular Devices) using the standard and instructions supplied by the manufacturer.  2.10 Western blot analysis Antibodies The phosphorylated forms of E R K and p38 M A P K were visualized using rabbit polyclonal primary antibodies raised against phosphor-(Thr202/Tyr204) E R K (1:1000) and phosphor-(Thrl80/Thr182) p38 M A P K (1:1000), (Cell Signaling,  55  Beverly, M A ) . G F P was visualized using a mouse monoclonal raised against G F P (1:1000) (Santa Cruz Biotechnology, Santa Cruz, C A ) .  Loading controls for E R K , p38  M A P K and G F P were determined by using extracellular regulated kinase 1/2 ( E r k l / 2 ) (1:1000) polyclonal antibody, extracellular regulated p38 antibody (1:1000) and G A P D H (1:10000) (Santa Cruz Biotechnology) respectively. Secondary goat anti-rabbit (1:10000) and goat anti-mouse (1:10000) antibodies conjugated to horse radish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, P A ) were used. B V - 2 cells transfected with the p i 8 0 0 promoter region of p97 fused to G F P were washed, resuspended at 5 x 10 cells/ml in D M E M containing G418 and incubated 5  overnight at 37 °C. Cells were then washes 3 times in serum free media and incubated with 10 ( i M Api-40 or 50 ng/ml L P S for 24 hrs. For signal transduction studies, cells were treated with 20 u M S B or 50 U.M P D for 30 min and then with either 10 u M A p i ^ o or 50 ng/ml L P S for 6 hrs.  Following treatment, cells were lysed in 50 p i o f lysis buffer  containing 50 m M T r i s - H C l (pH 7.5), 150 m M N a C l , 10% glycerol, 1% Nonidet P-40, 5 m M E D T A , 1 m M sodium vandadate, 5 m M sodium fluoride, 1 m M sodium molybdate, 5 m M P-glycerol phosphate, in the presence of 10 pg/ml soybean trypsin inhibitor, pepstatin, and 40 pg/ml phenylmethylsulfonyl fluoride. Protein concentrations were determined using the B C A protein assay (Pierce, Rockford, IL).  Equal amounts o f  proteins were denatured by boiling in S D S sample buffer (0.16% (w/v) S D S , 0.002% (w/v) bromophenol blue and 1% (w/v) D T T . ) run on 12% S D S - P A G E gel along prestained broad range standard (Bio-rad, Hercules, C A ) and transferred to nitrocellulose membrane.  Membranes were blocked for non-specific binding in 5% non-fat dry skim  milk in P B S and 0.1% (v/v) Tween 20 detergent (Bio-rad).  Primary antibodies to  56  phospho-p38 and phosphor-p44/42 were incubated in P B S / 0 . 1 % Tween 20 and the G F P primary antibody was incubated in blocking buffer. with membranes  overnight at 4°C.  Primary antibodies were incubated  After incubation with HRP-linked  secondary  antibodies, proteins were detected using E C L detection reagent (Amersham Biosciences, Piscataway, NJ). After development, the blots were stripped in 62.5 m M T r i s - H C l (pH 7.5), 0.2% S D S , and 100 m M 2-mercaptoethanol for 30 min at 50 °C and then reprobed with either a monoclonal antibody to G A P D H , extracellular regulated kinasel/2 ( E r k l / 2 ) (K-23) polyclonal  antibody  and  extracellular regulated  p38  (C-20) (Santa  Cruz  Biotechnology) as a protein-loading control.  2.11 Immunohistochemistry Adjacent tissue sections were immunostained for the detection of amyloid deposits (4G8) and activated microglia (F4/80).  Brains were fixed in 4% paraformaldehyde,  paraffin embedded and cut into 8 p M sections.  Sections were then deparaffinized,  hydrated and incubated in D A K O Target retrieval Solution (DakoCytomatiom) in a steamer for 20 minutes to ensure proper unmasking of the antigen. The samples were then cooled at room temperature for 30 minutes and washed in water 3 times for 5 minutes each. Slides were then incubated in 3% H 0 for 30 minutes, rinsed 3 times in 2  2  water and blocked in the appropriate blocking solution for 40 minutes. Slides were then incubated in either 4G8 (1:500, Signet Labs Inc., Dedham, M A ) or F4/80 (1:10, Serotec, Oxford, U K ) overnight at 4°C, washed and incubated with a secondary biotinylated antimouse  antibody for 4G8 or a biotinylated anti-rat  LSAB+system, DakoCytomation) for 25 minutes.  antibody for F4/80  (DAKO  Sections were developed by D A B  57  (Vector Laboratories Inc., Burlington, O N ) , counterstained with Mayer's hematoxylin, dehydrated and mounted.  Slides were examined under a Zeiss microscope and images  were captured using OpenLab software. The number of plaques per cortical brain section per mouse was counted and analyzed.  Data were collected from 4 equally spaced  sections. The values for all sections from one mouse were averaged to obtain a single sample for statistical analysis.  2.12 Ap and antibody injection Fluorescent-labelled A|3MO, P B S , bovine serum albumin ( B S A ) conjugated to Texas red (Sigma), anti-human A(3 antibody (4G8), Biotin labeled anti-human A p antibody (4G8) (Signet Labs Inc.) and biotin (Sigma) were injected intravenously (i.v.) into the tail veins o f mice. For injections, a total volume of 200 ul was injected into each mouse containing 200 ug o f protein. One hour after injection, the.mice were anesthetized with ketamine (25 mg/kg i.p.) and xylazine (5 mg/kg i.p.) and perfused with 1 X P B S followed by 4% P F A .  Following perfusion, the brains were dissected, embedded into  paraffin and sectioned into 8 u M sections.  Brain sections from mice injected with  fluorescent A P , P B S and Texas-red conjugated B S A were examined directly by confocal microscopy without any processing.  Brain sections for mice injected with anti-Ap  antibodies (both unlabelled and biotin labeled) were stained with secondary antibodies or processed with D A B respectively. Briefly, slides were deparaffinized and hydrated as described above. For mice injected with anti-Ap antibodies, slides were blocked in 10% skin milk in P B S for 30 minutes, washed 2 times for 5 minutes in water and then incubated in A l e x a 488 nm conjugated goat-anti mouse IgG secondary antibody (1:500)  58  (Molecular probes, Eugene, O R ) diluted in 2 % B S A / P B S for one hour. The slides were then washed 3 times for 5 minutes.  The coverslip was mounted with 5 pl Antifade  (Molecular probes) before sealing with nail polish. For mice injected with biotin labeled antibody or biotin alone, slides were developed with D A B (Vector laboratory) washed 3 times for 5 minutes in water, dehydrated in 3 washes o f xylene and mounted with a coverslip using permount. The slides were then examined using a confocal microscope or a light microscope. A l l mice used for these studies were 6 weeks old and o f similar weight.  2.13 Vaccination protocol Prior to immunization each mouse was bled and serum collected. Two groups o f mice were vaccinated beginning at 6 weeks and 11 months of age and sacrificed at 12 months and 15 months respectively. A(3 peptide was freshly prepared from lyophilized powder for each set o f injections.  For immunizations, 2 mg o f A(3 (human A f $  M 0  ;  Bachem) was added to 0.9 m l o f deionized water and mixed until a solution o f uniform suspension was obtained. Then 100 p l o f 10X P B S was added to obtain a final I X P B S concentration. The solution was vortexed and placed at 37°C overnight until use the next day.  AP  (100 pg antigen per injection) or P B S (control) was mixed 1:1 (v/v) with  complete Freund's adjuvant ( C F A ) for the first immunization. This was followed by a boost with A P  M Q  (100 pg) or P B S mixed 1:1 (v/v) with incomplete Freund's adjuvant  ( I C F A ) at two weeks and monthly thereafter. The 6 week old mice were vaccinated for a total o f 11 months, and the 11 month old mice were vaccinated for a total o f 4 months.  59  A p or PBS alone were injected from the fifth immunization onward. Antibody titres for anti-Ap antibodies was assayed after the second immunization. Anti-AP antibody titres were determined by serial dilutions of sera against aggregated A p , which had been coated in microtitre wells.  Detection was done by using goat anti-mouse immunoglobulin  conjugated to horseradish peroxidase and ABTS (2'2-AZINO-bis (3-ethylbenzthiazoline6-sulfonic acid; Sigma) as substrate. A fluorescence plate reader (Spectra Max 190; Molecular Devices) then measured fluorescence at 405 nm.  2.14 Evans blue assay Quantitative Evans blue analysis was performed as previously described by Ujiie et al.  149  In brief, Tg2576 mice vaccinated with either A P or PBS alone and their age-  matched controls were weighed and injected intra-peritoneal (i.p.) with 50 u.g/g Evans blue dye (Sigma) in 1XPBS. Three hours after injection, the mice were anesthetized with ketamine (25 mg/kg i.p.) and xylazine (5 mg/kg i.p.) and perfused with 1XPBS for 5 minutes.  Following perfusion, the brains were dissected, olfactory and cerebellum  removed, weighed and dounce homogenized in 0.5 ml of 50% trichloroacetic acid. The sample was then centrifuged at 10000 rpm for 10 minutes. The supernatant was collected and diluted 1:4 in 100% ethanol. An EL1SA plate reader (Spectra Max 190; Molecular Devices) then measured Evans blue fluorescence at 620 nm. Values are graphed as OD62o/brain weight, and the data were statistically analyzed with the student t-test. As a control for Evan blue distribution throughout the mouse,  OD620  values for the liver, a  60  tissue known to be highly permeable, were also evaluated.  A l l mice used in this study  exhibited high levels o f Evans blue dye in the liver.  2.15 Statistical  analysis  A l l analyses were performed using the GraphPad Prism software.  TNF-a ELISA  data and Real-time P C R data were analyzed by performing A N O V A tests followed by Bonferroni analysis. A l l experiments were performed at least 3 times and in triplicate within each experiment.  Statistical significance was established at a level o f P < 0.05.  A P vaccination studies were analyzed by comparing A p vaccinated transgenic mice and P B S vaccinated transgenic mice within each group by the student t-test. Transgenic and non-transgenic mice injected with A p and transgenic and non-transgenic mice injected with P B S were also compared to one another respectively. Statistical significance was established at a level o f P < 0.05.  61  Chapter 3 : P 9 7 expression in activated microglia 3.1  Rationale  It is now becoming widely accepted that inflammatory processes are involved in the pathogenesis o f AD.  There have been extensive studies over the past 10 years which  have characterized the increased expression o f many pro-inflammatory cytokines, cell surface markers and various neurotoxins corresponding to the accumulation o f AP and disease pathology.  The findings that amyloid plaques are surrounded by activated  microglia also provide evidence for the contribution of inflammation in A D .  These  observations provide the rationale for developing therapeutics for A D targeted against inflammation and thus activated microglia. Further support of such therapies came from epidemiological evidence from arthritic patients who were on long term N S A I D s in which N S A 1 D use correlated with a significant decreased risk for A D by approximately 60-80%  1 0 2  . Furthermore, N S A I D treatment appears to slow down disease progression,  delay onset and diminish symptom severity  1 0 3  .  While it has been established that the  target o f N S A I D s are microglia, it is unclear what intracellular process in microglia mediate the response elicited by N S A I D s . It has been suggested that either the C O X - 1 / 2 enzymes or the transcription factor, P P A R - y , are involved ' . Finding a specific marker for inflammation resulting from Ap accumulation would aid in the diagnosis of A D as well as in evaluating the efficacy o f new anti-inflammatory therapeutics. There have been many biomarkers suggested to aid in the diagnosis o f A D . These are mostly limited to the ratio between phosphorylated Tau levels in the C S F .  Ap42 and Ap4o or total Tau or  There have also been some markers o f  62  inflammation that have been investigated in AD, such as levels of 1L-6  191  and TNF  192  however; these markers are general for all forms of CNS inflammation and not specific for AD related inflammation. An alternative candidate that is believed to be specific for AD is a protein called melanotransferrin. Melanotransferrin, also known as the human melanoma associated antigen p97, was originally identified as a cell surface marker associated with human skin cancer  193  .  Woodbury et al. analyzed the expression of p97 in normal adult, fetal, and neoplastic tissues and found that the protein was highly expressed in fetal colon, lung and umbilical cord as compared to adult tissues  194  . Further studies revealed that p97 is also highly  expressed in fetal liver, placenta and sweat glands  193  . Due to high p97 expression in  fetal tissues, it has been suggested that p97 may play a role in fetal development. In addition to its expression in human tissues, p97 has been found to be expressed in many cultures of normal cells including liver, intestinal epithelial cells and fetal intestinal cells 195,196,197,198  e x  i t i two forms: one is a glycosylphosphatidylinositol (GPI)-linked s  s  n  cell surface form and the other is a secreted soluble form generated by alternative splicing 199,200,201  j  s a  m e r n D e r  of a group of iron binding proteins that include serum  transferrin, lactoferrin and ovotransferrin and maps to the same region of chromosome 3q in humans as the genes of other proteins involved in iron transport  202  . Human transferrin  and p97 share 40% sequence identity and p97 is able to bind one iron molecule . Thus, 203  p97 may be one of the many uncharacterized iron transport molecules that operate in a transferrin independent fashion. The relationship between p97 and AD remains to be elucidated in molecular detail. The distribution of p97 in the brains of AD and non-AD subjects was studied to identify  63  locations where p97 is expressed  2 0 4 , 1 1 0  .  In previous studies comparing A D brains with  brains o f patients who died from neurodegenerative diseases other than A D , such as Huntington's disease or Parkinson's disease (PD), the distribution of p97 in the n o n - A D brains appeared to be limited to the endothelial cells, which make up the B B B , with occasional positive staining for astrocytes and oligodendrocytes. Interestingly, in the A D brain, p97 was also detected in reactive microglia associated with senile plaques. Reactive microglia that stained positive with H L A - D R antibody but were not closely positioned near senile plaques did not express p97 in situ hybridization studies  1 1 1  n 0  . Further immunohistochemical and  revealed a high expression o f p97 m R N A in the reactive  microglia associated with senile plaques and lower levels o f p97 around endothelial cells. In the n o n - A D brain, endothelial cells stained weakly for p97 m R N A and there was no p97 m R N A or protein detected in microglia.  It is possible that there is an increased  requirement for the utilization and/or scavenging o f iron by reactive microglia associated with senile plaques since, in addition to p97, increased concentrations o f iron, transferrin and ferritin  '  have been noted in the region.  With the finding that p97 is detected on the B B B and in the reactive microglia associated with the senile plaques o f A D patients  1 1 0  it was postulated that p97 may be  used as a biological marker for A D . Initial studies by Kennard et al. investigated this putative relationship and found evidence that the soluble form of p97 is elevated, up to 6 fold, in the serum taken from A D patients as compared to controls (n=17)  m  .  Further  regression analysis of these data revealed that there was no correlation between p97 levels and age. To eliminate the possibility that environmental factors may result in the increased levels observed, serum samples from A D patients and their cognitively normal  64  spouses (n=10) were obtained  . Again it was found that the levels of p97 in AD  patients compared to their spouses were significantly elevated thereby suggesting that environmental factors have no influence on serum p97 levels. A subsequent study by Feldman et al, involving a larger cohort of subjects, showed that there is a 2 fold significant increase in serum p97 levels from patients with AD compared to non-AD controls (41 ng/ml versus 20 ng/ml, p < 0 . 0 0 1 ) . 209  were corroborated by an independent group of investigators  2 1 0  These results  who reported that there  was a 3 to 5 fold significant increase in the serum p97 levels in AD cases compared to non-AD cases of dementia and controls. In addition, there was no correlation between p97 serum levels and age or severity of disease. Collectively these are compelling findings which independently support the potential of serum p97 levels as a biomarker of AD. The role of p97 as an iron transporter in AD pathology is unclear. It has been established that in many neurodegenerative diseases, such as AD and PD, there is a 211  dysfunction in iron metabolism and hence homeostasis (reviewed in Qian, 1998) How exactly iron gets into the brain remains unresolved. It is thought that most iron transport in the body is mediated by the classical transferrin-transferrin receptor (TfR) pathway. However, in regards to iron transport in the brain, it is possible that other proteins may be involved. This is supported by looking at TfR, transferrin and iron distribution in the brain. There appears to very little overlap between TfR and iron distribution and between TfR and transferrin distribution  212  .  In addition, there is a  difference in the rate of transfer of iron and transferrin across the BBB indicating that iron transport across the BBB is likely to be facilitated by other means. It has been 65  previously established that p97 is able to bind and internalize iron into cells independent of transferrin and TfR  1 9 9  '  2 0 0  . Furthermore, the rate o f iron uptake by p97 was equivalent  to the rate of iron uptake seen with transferrin/TfR.  These data suggest that p97 may  transport iron across the B B B in a manner similar to the TfR. Dysregulation of p97 may be one of the causes of iron deposition in A D . Studies have confirmed the presence o f an iron-responsive element in the 5'UTR o f the A P P and that at the biochemical level, copper, zinc and iron are shown to accelerate the aggregation o f the A p peptide and amyloid plaque formation . Microglia may express p97 in response to A P as a means to 4 5  help clear A p plaques by binding to iron deposited in the plaques and destabilizing it. Alternatively, p97 expression in microglia may be a harmful response.  If there is an  initial dysregulation o f iron in the brain causing a decrease in the concentration o f free iron, (for example sequestration in plaques) p97 may cross the B B B into the periphery, bind iron and cross back into the brain. Recently, it has been demonstrated that p97 may participate in the vascularization o f solid tumors and promote endothelial cell migration 2 I 3  .  This angiogenic activity may depend on activation o f endogenous  vascular  endothelial growth factor ( V E G F ) expression ' . We have established that the B B B is 2  compromised in A D model mice expression in A D > > 214  215  216  1 4 9  3  and it is known that there is an increase in V E G F  Thus p97 may contribute to the increased permeability o f the  B B B in A D . More studies are needed in order to determine i f an increase in brain iron levels is associated with an increase in p97 expression. Also, the distribution o f p97 in different brain regions and different cells types may help elucidate the role o f p97 in brain iron homeostasis and possibly A D pathology.  66  The aims of this study were to determine if microglia upregulate the expression of p97 upon general activation or whether this occurs in response to AD specific stresses, and to investigate which signal transduction pathway is responsible for this up-regulation. Moreover, if p97 could be used as a marker for inflammation, it may be possible to use it as an indicator of anti-inflammatory drug efficacy. To further investigate this possibility, p97 expression was examined in cells treated with NSAIDs.  3.2 Results 3.2.1 Microglial activation As a prerequisite for analysis of levels of p97 in activated microglia upregulate the expression of p97, it is important to confirm the state of microglia after treatment with known stimulants was confirmed. It has been well established that once microglia become activated they secrete many pro-inflammatory cytokines including IL-1 P and TNF-a, as well as reactive oxygen and nitrogen radicals . Therefore to determine if 26  microglia were indeed in an activated state, TNF-a levels were measured by a sandwich ELISA with antibodies specific for murine TNF-a. Cultured BV-2 cells were incubated for 24 hours with known activators of microglia including 50 ng/ml LPS, 5 ng/ml IFN- y or 10 uM fibrillar Api_ o, which is a stress specifically associated with AD. As negative 4  controls, cells were treated with 10 U.M of the reverse Ap peptide (Ap ) or PBS. 40 }  Stimulation of BV-2 cells with A P  M Q  , LPS and IFN-y lead to an increase in the  67  production o f T N F - a (Figure 3.1). Treatment with the A p  4 Q }  had some effect on T N F - a  production likely due to non-specific effects of addition o f peptides to the media..  NT  L P S IFN-y  ApY4 A p 0  4 ( M  PBS  Treatment * P < 0.001; ** P < 0.01  Figure 3.1. T N F - a production increased in BV-2 cells treated with various known activators. Cells were plated at a density of 5 x l 0 cells/ml and treated for 24 hours with 50 ng/ml L P S , 5 ng/ml IFN-y, l O u M A p , 10 p M A p - i and P B S . C e l l culture supernatant was collected and T N F - a levels were measured using an E L I S A specific for murine T N F - a . There was a significant increase in T N F - a production in cells treated with L P S , IFN-y and A p compared to no treatment ( N T ) ( A N O V A , * P < 0.001, ** P < 0.01). Treatment with the reverse A p peptide had some effect, likely due to nonspecific effects o f added peptides while P B S had no significant effect on T N F - a production. A l l experiments were performed at least 3 times in triplicate. Error bars represent ± S D . The results above are representative o f all replicates. 5  M 0  4 0  M 0  68  3.2.2 P97 expression in BV-2 cells The expression o f p97 was determined by R T - P C R in B V - 2 cells under nonstimulating conditions and following treatment with fibrillar A ( 3  M 0  , L P S and IFN-y. The  results show that expression o f p97 was largely increased in B V - 2 cells in response to treatment with A[3 (Figure 3.2 a). Treatment with other known activators o f microglia, such as IFN-y had no significant effect on the expression o f p97. There was an increase in treatment with L P S ; however, not as large a magnitude as with treatment with A p . There was no effect on the expression o f S I 5 , a ribosomal subunit m R N A used as a loading control. In addition, there seemed to be no change in the expression o f p97 in murine melanoma cells ( J B / M S ) , murine brain endothelial cells (bEnd.3), nor in murine fibroblasts (3T3) in response to these stimuli (Figure 3.2 a). Real-time P C R analysis o f p97 expression in B V - 2 cells was performed in order to quantitate the change in m R N A expression.  Treatment with A p resulted in an approximate 6 fold increase in p97  expression.  The 6 fold increase corresponds to the increased protein serum levels 208 209 210  previously reported in humans  '  * . Treatment with L P S exhibited an approximate 4  fold increase in p97 expression (Figure 3.2 b). Since there is no monoclonal antibody to murine p97, B V - 2 cells were transfected with the p E G F P - 1 promoterless vector where the expression o f G F P is under control o f the p97 promoter (pl800-GFP). To investigate i f protein levels o f murine p97 corresponded to the increase in m R N A expression, Western blot analysis was performed on p l 8 0 0 - G F P transfected B V - 2 cells and blotted for G F P expression. Treatment with A P and to a lesser extent L P S resulted in an increase in G F P expression compared no treatment (Figure 3.2 c).  69  Figure 3.2. P97 expression in treated cells. p97 expression in B V - 2 cells was upregulated ~6 fold in response to l O p M A p compared to no treatment and treatment with other known activators (IFN-y) o f microglia. Treatment with 50 ng/ml L P S resulted in an -3.5 fold increase in p97 expression, (a-b). In addition, treatment o f endothelial cells (bEnd.3), melanoma cells ( J B / M S ) , and fibroblast cells (3T3) with 10 u M A p , 50 ng/ml L P S and 5 ng/ml IFN-y had no effect on p97 expression. This increase in p97 expression was also observed at the protein level in response to 10 p M A P and 50 ng/ml L P S (c). S I 5 m R N A was used as a loading control for R T - P C R and G A P D H for western blot, (a) R T - P C R of p97 expression in B V - 2 , b.End3, J B / M S and 3T3 cells (b) P97 expression as determined by real-time P C R . Relative gene expression was determined by calculating the cycle threshold (CT) for p97 as well as for S I 5 . Treated sample thresholds were then compared to non-treated and fold induction o f gene expression determined. Error bar represent ± S D for C T values, (c) Western blot analysis o f G F P expression in p l 8 0 0 - G F P transfected B V - 2 cells after treatment with A P and L P S . A l l experiments were performed at least three times and data indicated here are representative of all trials.  70  GFP GAPDH  3.2.3 P97 expression in Tg2576 AD model mice Finally, the m R N A expression levels of p97 in different brain regions o f Tg2576 A D model mice was examined. Sixteen month old Tg2576 A D model mice, where there is a substantial presence o f amyloid plaques and activated microglia, and their control littermates, were perfused with P B S and the brains were dissected into 6 different regions; frontal, hippocampal, occipital, olfactory, parietal, and temporal. The brains were then processed for total R N A extraction and subjected to R T - P C R to assess for p97 expression. Results illustrate that p97 expression is increased in the Tg2576 A D mouse in the brain regions associated with heavy plaque burden and activated microglia such as the temporal cortex and hippocampus (Figure 3.3).  72  F R HI O C O L P A T E Tg2576  SJL/BI6  Figure 3.3. p97 expression is increased in affected b r a i n regions in Tg2576 mice. Brains were dissected into 6 regions, R N A extracted and R T - P C R performed for p97 gene expression. Areas o f the brain known to have significant amyloid deposition and activated microglia, F R , H I , P A and T E , also have an increase in p97 gene expression. Non-transgenic mice exhibit no change in the expression o f p97. There was also no change in S15 m R N A levels in all brain regions in both transgenic and non-transgenic mice. FR-frontal; Hl-hippocampal; OC-occipital; OL-olfactory; PA-parietal; T E temporal. A l l experiments were performed at least three times and data indicated here are representative o f all trials.  73  3.2.4 MAPK pathways control the expression of p97 To seek further insight in what signal transduction pathway is involved in p97 expression, the promoter region o f p97 was studied to identify possible transcription factor binding sites. Within this region there are 3 A P - 1 transcription factor binding sites, indicating a possible regulation by MAPK-dependent pathways. Moreover, Roze-Heusse et al.  found that increased m R N A levels of Jun/Fos, the transcription factors which 217  constitute A P - 1 , correlated with m R N A levels o f p97 in human melanoma cells  . To  directly test which M A P K pathway is involved in the expression o f p97, p l 8 0 0 - G F P transfected B V - 2 cells were stimulated with 10 u M A(3MO or 50 ng/ml L P S with and without the addition of selective inhibitors of the p38 M A P K and E R K 1 / 2 pathways. SB203580 (SB) is a selective inhibitor o f p38 M A P K and has no effect on E R K 1 / 2 or J N K while PD98059 (PD) is a selective inhibitor o f upstream kinases, M E K 1/2, in the E R K pathway. Cells were incubated with each o f the M A P K inhibitors for 30 minutes and then stimulated with either 10 p:M A p ^ o or 50 ng/ml L P S for 6 hours:  N o treatment and  treatment with inhibitor alone were used as controls. G F P protein levels were measured by Western blot. Treatment o f p l 8 0 0 - G F P B V - 2 cells with the inhibitor o f p 3 8 M A P K (SB, 20 u M ) resulted in a decrease in G F P expression in both A P and L P S treated samples (Figure 3.4 a). Treatment with inhibitors to E R K 1 / 2 ( P D ; 50 u M ) had no effect on G F P expression in stimulated cells (Figure 3.4 b). Cells transfected with vector alone, p E G F P - 1 , showed no change in G F P expression in all cases, but did show a difference in phospho-p38 and phospho-ERK expression upon treatment with A p or L P S with and  74  without respective inhibitors (Figure 3.4 b,d). This suggests that the p97 promoter region is regulated by the p38 M A P K pathway.  NT  NT  Ap  Ap  LPS  NT  AP  LPS  20 p M S B  20 p M S B  GFP  GFP  p-38  p-38  anti-p38  anti-p38  LPS  NT  Ap  LPS  50 p M P D  50 p M P D  GFP  GFP  p-ERK  pERK  ERK  anti-ERK  Figure 3.4. The p97 promoter appears to be regulated by the p38 M A P K pathway. B V - 2 cells transfected with p l 8 0 0 - G F P were stimulated with 10 p M A P i ^ and 50 ng/ml L P S as well as with inhibitors for p38 (20 p M ) and p - E R K (50 p M ) for 6 hours. G F P expression was measured by Western Blots of cell lysates with antibodies against G F P and the activated (phosphorylated) forms of p38 and p - E R K . Levels o f extracellular regulated M A P K s were used as loading controls. G F P expression was decreased when treated with A p or L P S in conjunction with S B implying that the p38 M A P K pathway controls the regulation o f the p97 promoter region. There was no change in G F P levels in cells treated with P D or in cells transfected with an empty vector, (a) G F P expression in p l 8 0 0 - G F P cells treated with p38 M A P K inhibitor, S B , (b) G F P expression in pEGFP-1 cells treated with p38 M A P K inhibitor, S B , (c) G F P expression in p l 8 0 0 - G F P cells treated with E R K 1/2 inhibitor, P D and (d) G F P expression in p E G F P - 1 cells treated with E R K 1/2 inhibitor, P D . Each gel is a representative gel from three separate experiments. 0  75  3.2.5 P97 expression in BV-2 cells after treatment with NSAIDs Next, the expression o f p97 in microglia in the presence o f N S A I D s , was tested. To assess i f N S A I D treatment reduced the activation of microglia, T N F - a production was again measured in cells either treated with stimulant alone or stimulant and N S A I D . Treatment with two N S A I D s , 10 p M Ibu and 10 p M N i m , appeared to reduce the production o f T N F - a from cells treated with both stimulant and drug (Figure 3.5 a-b). When examining the gene expression o f p97, it appears that the expression was decreased in cells treated with both Ibu and N i m , where R T - P C R and real-time P C R show that the expression o f p97 in N S A I D treated cells was reduced close to the levels o f non-treated cells (Figure 3.6 a-b and 3.7 a-b). Since previous studies have demonstrated that serum levels o f p97 are elevated in A D , protein levels were also examined.  Western blot  analysis with p l 8 0 0 - G F P transfected B V - 2 cells also shows a decrease, similar to the levels o f m R N A . G F P levels were also decreased in cells treated with drug compared to treatment with A P and L P S alone (Figure 3.6 c and 3.7 c).  76  A.  Figure 3.5. NSAID treatment decreased TNF-a production in activated BV-2 cells. Cells were plated at a density of 5 x 10 cells/ml and treated for 24 hrs with 10 U.M A ^ M O or 50 ng/ml LPS with or without the addition of 10 p.M Ibu or 10 uJVl Nim. There was a significant decrease in T N F - a production in cells treated with drug compared to treatment with A p or LPS alone ( A N O V A , *, ** P < 0.001). (a) T N F - a production in B V - 2 cells after treatment with 10 (iM Ibu and (b) T N F - a production in B V - 2 cells after treatment with 10 | i M Nim. A l l experiments were performed at least 3 times in triplicate. Error bars represent ± SD. The results illustrated above are representative of all replicates. 5  M  0  77  NT  Aft  LPS  1-40  O CM  X Wf.  p97  :'**i*»Wv :.%HM»"  VHBP  ri — ***  10 u M I B U  S15  PT?  O w/out IBU • w/IBU  AB1-40  Treatment  NT  ApV40  LPS +  10uMIBU  »«» G F P GAPDH  F i g u r e 3.6. p97 expression is decreased in BV-2 cells treated with Ibuprofen. Cells were plated at a density of 5 x 10 cells/ml and treated for 24 hrs with 10 | i M A|3MO or 50 ng/ml L P S with and without the addition of 10 urn Ibu. There was a significant decrease in p97 expression in cells treated with A|3 or L P S in conjunction with Ibu compared to A p and L P S treatment alone, (a) R T - P C R o f B V - 2 cells treated with A p Y 40, L P S and Ibu. (b) p97 expression as determined by real-time P C R . Relative gene expression was determined by calculating the cycle threshold (CT) for p97 as well as for SI5. Treated sample thresholds were then compared to non-treated and fold induction o f gene expression determined. Error bar represent ± S D for C T values, (c) Western blot o f G F P protein levels in p l 8 0 0 - G F P transfected B V - 2 cells after treatment with 10 | i M A p Y 40 and 50 ng/ml L P S with and without the presence o f 10 u M Ibu. The levels o f the G A P D H loading control are also shown. A l l experiments were performed at least three times and data indicated here are representative of all trials. 5  M 0  78  NT  A(3i-40  LPS  o  + CxN 10 p M NIM  p97 S15  AB1-40 Treatment  C.  NT  Api-40  LPS + m*  «•  10 uM NIM GFP GAPDH  F i g u r e 3.7. p97 expression is decreased in B V - 2 cells treated w i t h Nimesulide. Cells were plated at a density of 5 x 10 cells/ml and treated for 24 hrs with 10 p M A p ^ o or 50 ng/ml L P S with and without the addition o f 10 p m N i m . There was a significant decrease in p97 expression in cells treated with A p or L P S in conjunction with N i m compared to A p and L P S treatment alone, (a) R T - P C R o f B V - 2 cells treated with Ap,_ 40, L P S and N i m . (b) p97 expression as determined by real-time P C R . Relative gene expression was determined by calculating the cycle threshold (CT) for p97 as well as for SI5. Treated sample thresholds were then compared to non-treated and fold induction o f gene expression determined. Error bar represent ± S D for C T values, (c) Western blot o f G F P protein levels in p l 8 0 0 - G F P transfected B V - 2 cells after treatment with 10 p M Ap,_ 40 and 50 ng/ml L P S with and without the presence of 10 p M N i m . The levels o f the G A P D H loading control are also shown. A l l experiments were performed at least three times and data indicated here are representative of all trials. 5  M 0  79  3.3 Discussion In this study, it was demonstrated that fibrillar A(3 significantly promoted the upregulation o f p97 at both the m R N A and protein level in microglia. Furthermore, the signal transduction pathway involving p38 M A P K appears to be important in p97 production by stimulated microglia whereas the pathway involving E R K is not.  It was  also shown that the expression o f p97 can be down-regulated with the use of N S A I D s . There have been a few studies that have proposed that p97 may be a putative biomarker for A D , with an ~4 fold increase in serum levels o f p97 in A D patients  208  > > 209  210  \  n  addition, p97 appears to be expressed in the active microglia closely associated with amyloid plaques in the A D brain ' " .  However, there was no direct evidence that  microglia upregulate p97 expression as a result o f activation. In this regard, the results from this study are very intriguing. Not only is it demonstrated that p97 is upregulated in activated microglia, but the upregulation is largely specific to activation via A p \ a stress specific to A D .  It has been previously established by many groups, that in response to  stimulation from molecules such as L P S and IFN-y, microglia upregluate the expression and secretion o f many cytokines and other proteins such as I L - i p , T N F - a and urokinase plasminogen-activator receptor  2 , 8 , 1 1 3  .  Therefore, it is possible that p97 is one o f few  proteins whose expression may be regulated by A(3 in A D .  It has been shown here by  real-time P C R that the expression o f p97 increased approximately 6 fold after treatment of microglia with 10 p M fibrillized A p . A previous gene expression study  2 1 9  using gene  array chips supports these results and demonstrated that the expression o f p97 increased 2 fold after 24 hrs of treatment with 2.5 p M A p .  80  A number o f different parameters of microglial activation in A D have been defined both in vivo and in vitro. In particular, the phosphorylation o f various proteins involved in the M A P K . signal transduction pathway, such as p38 and E R K , are increased in activated microglia. The pathways downstream o f these proteins share a common transcription factor, A P - 1 , which has 3 potential binding sites in the promoter region o f 220  p97, suggesting a regulatory role for this transcription factor  Further evidence in  human melanoma cells support this and show that the R N A levels of Jun and Fos, which make up A P - 1 , is correlated to the expression o f p97  2 X 1  . It is known that in microglia,  L P S and A P stimulate the M A P K pathways and we have shown that A p and L P S treatment cause an increase in p97 expression. The present study also examined the expression o f p97 in stimulated cells with and without the addition o f p38 and E R K specific inhibitors.  Inhibition o f the p38 pathway resulted in a decrease o f G F P  expression in p1800-GFP transfected B V - 2 cells. G F P expression.  Inhibition o f E R K had no effect on  Thus, it would appear that the expression o f p97 in A p and L P S  stimulated microglia may be under control o f the p38 M A P K signal transduction pathway.  Since p97 expression was not completely abolished by S B , it is possible that  p97 expression may also be controlled by other mediators or pathways. The role o f inflammation is becoming more evident in A D pathogenesis and it has been  recently  hypothesized  that the neurodegeneration  observed  in A D is the  consequence of an inflammatory response to A p and N F T s rather than as a result o f these hallmarks  2 6  . Early epidemiological studies offer support for the role o f inflammation in  A D and have demonstrated that the use o f N S A I D s confers protection against A D and appears to slow down disease progression  ' '. In vitro experiments have shown that  81  N S A I D s , such as Ibu and indomethacin inhibit the gene expression o f nitric oxide 221 222  synthase in macrophage and decrease cytokine production in neuronal cells  '  . In  addition, N S A I D s including Ibu, indomethacin and flurbiprophen, have been shown to reduce the amounts o f shorter derivatives  2 2 3  APi_4  2  in human glioma cells by shifting the cleavage of A P to its  . In animal models, it has been established that oral administration  of Ibu approximately around the time o f initial disease pathology, attenuated plaque pathology resulting in the decrease in both the size and number of plaques as well as a decrease in the number o f dystrophic neuritis and activated microglia  2 2 4  . Moreover, A p i .  42 brain levels and the presence o f activated microglia in N S A I D treated mice were 22 S 223  significantly lowered  "' ' .  Conversely, recent clinical trials examining a variety o f  N S A I D s failed to report any beneficial effects o f N S A I D treatment. confounded by their small size and large withdrawal rates  2 2 6  .  These trials are  The findings from the  many in vitro, in vivo and clinical studies suggest that the molecular targets of N S A I D s play a key role in the development o f brain amyloidosis, however, what the exact targets are remain unclear.  It is evident that more clinical trials are needed that focus on  determining i f anti-inflammatory drugs can delay and/or prevent disease onset. To date, there are no end-point criteria that can be used as a means to assess drug efficacy. Plasma levels of A p are not correlated with A P brain levels indicating that measuring A p plasma levels as an indicator o f drug efficacy is inconclusive and erroneous  2 2 3  . Here it is  shown that the expression o f p97, at both the m R N A and protein level, is affected by the presence of Ibu and N i m , potent anti-inflammatory drugs, after 24 hours o f treatment. Thus it is possible that p97 may be a potential marker to determine drug efficacy.  It is  still unclear as to what aspect o f inflammation in A D N S A I D s target or i f they target  82  inflammatory cells at all. Mechanistically, it has been shown that the beneficial effects o f N S A I D s are a result o f the drugs' ability to reduce or attenuate the activity o f C O X enzymes thereby precluding the synthesis of prostaglandins and thromboxanes from arachidonic acid  2 6  . There is still much debate as to the pathological role o f each o f the  C O X enzymes, C O X - 1 and C O X - 2 . Alternatively, N S A I D s have been shown to work in a C O X independent manner reducing the amounts o f Api.49 by targeting the y-secretase complex  227  > . in this study, the effects o f both a non-selective C O X inhibitor, Ibu, and 223  a C O X - 2 specific inhibitor, N i m , were examined. In both instances, p97 expression was decreased.  This suggests that p97 expression may be dependent or related to the  upregulation o f the both the C O X - 1 and C O X - 2 enzymes. Both E R K and p38 M A P K pathways have been demonstrated to increase the transcription and to regulate the 228  stabilization C O X - 2  . In contrast, Ibu has also been shown to reduce  little inhibition o f C O X enzymes  174  AP1.42  levels with  > . i order to resolve this, treatment with N S A I D s 223  n  that are C O X - 1 specific inhibitors and have no effect on the activity of C O X enzymes, such as Thalidomide which inhibits T N F - a , needs to be examined. Nonetheless, these data indicate that expression o f p97 is upregulated in response to microglia activation and that treatment with N S A I D s causes a down regulation o f m R N A expression. Therefore, it is possible that serum levels o f p97 may be used in future N S A I D clinical trials as a criterion to resolve whether a certain drug is beneficial. In summary, these data establish that p97 m R N A is upregulated in microglia in response to treatment with A p and that the expression o f p97 is regulated by the A P - 1 transcription factor downstream of the p38 M A P K pathway, which has also been shown to be highly active in microglia. Moreover, the expression o f p97 can be modified by  83  anti-inflammatory drug treatment. plausible biomarker for A D .  It has been previously, proposed that p97 may be a  These studies show that in addition to being a diagnostic  biomarker for A D , p97 may be used as an indicator o f A P specific mediated inflammation/microglial activation. P97 may also be used as an aid in determining the efficacy o f new and already established anti-inflammatory therapies for. A D .  84  Chapter 4: The role of microglia in amyloid deposition 4.1 Rationale Activation of microglia is among the first cellular changes in the injured CNS. In an acute inflammatory response activated microglia produce many beneficial neurotrophic and neuro-regenerative factors, in addition to pro-inflammatory mediators and potential neurotoxins.  This response is short-lived and only the injured neurons are  regenerated or removed. Alternatively, in a chronic inflammatory response, neurons are exposed to large amounts of neurotoxins for a prolonged period of time and this exposure ultimately leads to their demise. neurodegenerative  It has been proposed that microglia bring about the  changes in A D by eliciting an inflammatory response and by  contributing to amyloid deposition.  However, the decisive role of microglia in the  accumulation of A p and the subsequent propagation of amyloid plaques remain to be clearly elucidated. It has been demonstrated in A D , that activated microglia co-localize with and infiltrate amyloid plaques in both humans and APP transgenic mice  ll6  - > > ll7  n8  90  ultra-  structural studies by Wisniewski et al. have found that plaque associated microglia displayed intracellular channels containing amyloid fibrils  2 2 9  . A subsequent study also  found A P to be present in the secondary lysosomes of macrophage-like cells suggesting 230  that microglia are involved in both the production and phagocytosis  of A p  Furthermore, microglia may be able to drive the fibrillization of A P monomers/oligomers into fibrils and plaques  1 1 9 , 2 3 1  . It is also been established that microglia are able to rapidly  internalize and degrade microaggregates of A p by scavenger receptors or by activation of  85  complement pathways  ' ' ' "".  Regardless o f the mechanism of internalization, the  degradation of amyloid appears to be the main issue in A p accumulation. The ultimate fate o f phagocytosed A P is still unclear.  It has been shown that A p is degraded by  microglia; however, this degradation is slow and can lead to the accumulation o f A P inside the cell. Therefore, in the case o f AD, the overproduction and thus the persistence of A p may become too overwhelming for the microglia thereby disrupting the dynamic balance between A P deposition and removal. Regardless o f the mechanism it would appear that activated microglia contribute to the formation o f amyloid plaques. M a n y studies have focused on the role o f complement in amyloid deposition and have shown that the activation o f complement, both the classical and alternative pathways, results in the production o f many complement opsonin proteins that bind to A p 116,234  j/hese opsonins promote microglial phagocytosis o f A p , thereby implying that 23 ^  microglial activation promotes amyloid removal, precluding plaque formation " .  In a  study by Wyss-Coray et al. it was found that in mice which overexpress astroglial T G F pi  and human  APP695,751,770,  there was a 3 fold reduction in the number o f A p  parenchymal plaques and an overall 50% reduction in amyloid burden  2 3 6  . These mice  also exhibited an increase in complement protein C3 as well as activated microglia suggesting the T G F - p i promote the degradation o f A p by microglia via complement activation  2 3 6  . Further studies involving the inhibition o f C3 activation in A D model mice  by expression o f the soluble complement receptor-related protein, found that in C3 inhibited mice there were significant increases in amyloid plaque burden, the levels o f APi^2,  and the number o f degenerating neurons  2 3 7  .  Overall, these data suggest that  certain inflammatory defense mechanisms are indeed neuroprotective. In contrast, other  86  studies involving complement C l q deficient h A P P mice showed that the absence o f C l q resulted in a decrease in the degree o f degenerating neurons compared to C l q sufficient mice, with no difference in amyloid burden  2 3 8  . This implies that complement plays a  detrimental role in disease pathology. A better understanding o f the mechanisms that regulate amyloid accumulation and degradation may help facilitate the generation o f therapeutics that w i l l prevent the accumulation or enhance the degradation of A p and ameliorate A D treatments. It is hypothesized that microglia are activated in response to A p and phagocytose it removing it from the parenchyma. In time, the activated microglia may become overburdened and unable to remove the amyloid thereby facilitating its aggregation and subsequent plaque formation.  This study addresses the role o f microglia in plaque deposition using the  Osteopetrotic (CSF-1 deficient; op/op) mouse. A s a model for osteopetrosis, this mouse has a spontaneous frameshift mutation that results in a complete deficiency o f the CSF-1 factor, an important mitogen for brain microglia promoting survival, proliferation and 182 183  differentiation  '  . A s a result, these animals possess a reduced number of mature,  functioning microglia in their brains  1 8 7  '  1 8 6  .  In culture, microglia isolated from op/op  mice can be restored to full functionality with the addition o f CSF-1  . Moreover, daily  CSF-1 administration, before B B B formation, can largely restore microglial function  2 3 9  .  A n i m a l models with a defective microglial response, such as osteopetrosis, provide an approach to explore the many relationships fostered by microglia in the C N S .  To  explore the course o f A P plaque development, the op/op mouse was crossed with the Tg2576 A D model mouse to generate an A D mouse with reduced microglial capabilities. This new mouse model w i l l hopefully help resolve the contribution o f microglia to A D  87  pathogenesis. In this study, the role o f microglia in amyloid deposition was investigated. In accordance  with previous data on complement  and amyloid pathology,  it is  hypothesized that in A D mice with reduced microglial function, there w i l l be an increase in amyloid burden in the brain.  4.2 Results 4.2.1 Characterization of Tg/+;op/op mice T g 2 5 7 6 ; C S F - l deficient (Tg/+;op/op) mice were generated by initially crossing Tg2576 (Tg/+;+/+) mice by CSF-1 deficient (+/+;op/op) mice.  The F i generation o f  these mice (Tg/+;op/+) were crossed to get Tg/+;op/op mice. To distinguish between Tg/+ mice and wild-type (+/+;+/+), all mice were genotyped for the h A P P transgene (Figure 4.1). +/+;op/op mice were distinguished from +/+;+/+ mice by the absence o f incisors and a domed skull 10 days after birth.  Tg/+;op/op and +/+;op/op mice were  separated from Tg/+;+/+ and +/+;+/+ mice and fed on wet chow. Tg/+;op/op mice were significantly smaller than their control littermates (Figure 4.2). Mouse growth and body weight was followed for a 3 month period. A t 3 months, op/op mice were approximately 33% smaller from +/+ littermates. B o d y weight was also evaluated at 6 and 9 months o f age at time o f sacrifice. A s depicted in Figure 4.2, Tg/+;op/op mice had a significantly lower body weight that control mice. In accordance with the phenotype o f the op/op mice, Tg/+;op/op mice appeared to be infertile. In addition, the viability o f the Tg/+;op/op mice was reduced, as many mice died before the 9 month time point. The average life span o f the op/op mouse is reported  88  to be 7 months o f age (Jackson Laboratories); however, we have maintained this mouse for periods exceeding 12 months.  Figure 4 . 1 . P C R genotyping of Tg2576 A D model mice. Tg2576 h A P P transgenic mice and littermate controls were genotyped from ear punch D N A by P C R as previously described. The P C R products were analyzed on a 1% agarose gel and visualized. Tg2576 mice amplify a single 420 base pair (bp) product while control mice do not amplify any product. Each P C R was performed twice. Lane 1, Tg2576 founder control; Lane 2, C 5 7 / S J L non-transgenic control; Lane 3-6 genotyped mice. The gel shown is representative o f all P C R reactions.  89  35-i  Tg/+;op/op ' Tg/+;+/+ +/+;op/op 1  30-|  ra 3  I £  +/+;+/+  \  20  15-  10-j  o4 0.0  —f2.5  5.0  -i— 7.5  10.0  Time (months)  Figure 4.2. Tg/+;op/op mice are smaller than control littermates. M i c e were weighed beginning at one month o f age until 9 months o f age. Tg/+;op/op were significantly smaller than control littermates ;Tg/+;+/+ and +/+;+/+ at every age (P < 0.01, two-way A N O V A followed by Bonferroni analysis). Tg/+;op/op mice were smaller than +/+;op/op mice at all time points, however the difference was not significant (P > 0.05; two-way A N O V A followed by Bonferroni analysis).  4.2.2 A m y l o i d burden in Tg/+;op/op mice A m y l o i d plaques are first seen in Tg2576 mice at approximately 9 months o f age, therefore this time point was used to evaluate plaque burden.  In addition, Tg/+;op/op  mice have a high mortality and generally die at 10 months o f age. A t 9 months o f age, Tg/+;op/op mice exhibited similar amounts o f A p immunostaining as Tg/+;+/+ mice (Figure 4.3 a-c. P = 0.1; t-test). There was no presence o f A P in +/+;op/op and +/+;+/+ mice (Figure 4.3 d and e). A t 6 months and 3 months there was no A P immunostaining present in all animal brains.  Interestingly, there appeared to be A p deposition in cerebral  blood vessels o f Tg/+;op/op mice compared to Tg/+;+/+ mice (Figure 4.4). Therefore, it is possible that the absence o f microglia increases vascular angiopathy.  90  Figure 4.3. Amyloid plaque burden in 9 month Tg/+;op/op mice compared to controls. A m y l o i d plaques in cortical sections from mice were visualized with an antihuman AP antibody.  There was no significant difference in the total number o f  Ap plaques in Tg/+;op/op mice compared to Tg/+;+/+ controls. There was no detection of AP plaques in +/+;op/op and +/+;+/+ mice (a) Quantitative assessment o f plaque burden in Tg/+;op/op mice and Tg/+;+/+ mice, T-test; P = 0.1. Qualitative assessment o f A P in (b) Tg/+;op/op mice, (c) Tg/+;+/+ mice, (d) +/+;op/op mice, and (e) +/+;+/+ mice. Brain sections shown are representative o f their respective groups (n=3 for Tg/+;op/op, n=4 for Tg/+;+/+, +/+';op/op and +/+;+/+).  91  92  Figure 4.4. Amyloid accumulation in cerebral blood vessels of Tg/+;op/op mice. A m y l o i d accumulation in cerebral blood vessels (thin arrow) in the brains of Tg/+;op/op mice compared to Tg/+;+/+ controls as visualized with an anti-human A B antibody, (a) Tg/+;op/op mice (b) Tg/+;+/+ mice. Brain sections shown are representative o f their respective groups (n=3 for Tg/+;op/op, n=4 for Tg/+;+/+).  93  94  4.2.3 Microgliosis in Tg/+;op/op mice M a n y studies examining the number and morphology o f microglia in op/op mice are controversial  183  ,  186  ,  187  >  240  .  This study demonstrates that there is a decrease in the  number of activated microglia in the Tg/+;op/op mice compared to the Tg/+;+/+ mice. In addition, the majority o f the microglia in the Tg/+;op/op mice exhibited a resting morphology with small cell bodies and thin ramified processes whereas the microglia in the Tg/+ mice had a more activated morphology with more condensed cell bodies and thicker processes (Figure 4.5). These results are supported by previous studies indicating a reduced number and altered morphology o f microglia in the op/op mouse  I 8 7  .  95  Figure 4.5. Reduced number and altered morphology of microglia in Tg/+;op/op mice. Brain sections were stained with F4/80 to reveal the presence o f microglia. Tg/+;op/op mice exhibited an overall decrease in the number o f microglia than Tg/+;+/+ mice and fewer activated microglia than Tg/+;+/+ mice. +/+;op/op mice and +/+;+/+ mice exhibited resting microglia, with +/+;op/op mice having a reduced number i f cells. Activated microglia can be distinguished from resting microglia by the presence o f ramified processes and condensed cell bodies (black arrow) compared to small cell bodies and thin ramified processes (white arrow). Control littermates exhibited no microgliosis (a) Tg/+;op/op mice, (b) Tg/+;+/+ mice, (c) +/+;op/op mice, and (d) +/+;+/+ mice. Brain sections shown are representative o f their respective groups (n=3 for Tg/+;op/op, n=4 for Tg/+;+/+, +/+;op/op and +/+;+/+).  96  • '•  v  V-•  t V  .  Y  - ** • \ ' * *  *•  -. *  .»- . •  V  I-  Y  **Y* >  -1  >v  .100|iM  97  4.3 Discussion It has been hypothesized that microglia play an integral role in the formation o f amyloid plaques and results from many in vitro and in vivo studies support this premise. These previous studies established that activated microglia are attracted to and surround amyloid plaques  116  - > > ll7  118  90  However, whether microglia promote plaque formation by  converting monomeric/oligomeric A P to fibrillized A p or whether microglia prevent A p deposition by complement mediated phagocytosis in vivo remains unresolved.  The  present study uses a CSF-1 deficient h A P P transgenic model to assess the implication o f activated microglia in A D pathogenesis. Tg2576 were bred with mice deficient for C S F 1 and hence lacked mature, differentiated microglia. The results obtained here indicated that the Tg/+;op/op mouse was smaller in weight than control littermates and had a higher mortality rate. This was in accordance with previous studies on op/op mice, which found that op/op mice were smaller than +/op and +/+ littermates, had a decreased life-span and poor reproductive performance  188  > . 182  in this study, the oldest age group was 9 months  of age, the age o f initial plaque formation in the Tg2576 A D mouse. Older age groups were not examined due to the fact that no Tg/+;op/op mice survived past 10 months o f age. The amount of plaque pathology has been investigated in many mouse models o f A D . In the mouse model created in this study, levels o f fibrillar A P in Tg/+;op/op mice appeared to accumulate at rates comparable to Tg/+;+/+ mice since there was no significant difference in plaque burden in the cortices o f 9 month old mice. N o plaques were observed in the brains of 6 month old animals. It was also observed that there was more A p accumulated in the cerebral blood vessels o f the Tg/+;op/op mice than in the  98  Tg/+;+/+ mice. With the high incidence of C A A in A D such deposits are likely to be found. Since there were few Tg/+;op/op mice in the study, it was difficult to clearly determine a difference in plaque deposition as well as establish an explanation in the shift of A p deposition patterns. It is possible that with a decrease in the amount of activated microglia in the brains of these animals, other cells, such as astrocytes and pericytes, compensated and upregulated the production of certain cytokines and other important factors. For instance, TGF-P, a known immunosuppressive cytokine, is upregulated in A D and has been implicated in A D in astrocytosis, microglial activation, and 26  accumulation and regional distribution of A p  . Recently, TGF-pl was implicated as an  inducer of vascular amyloid deposition > . i addition, Lesne et al. demonstrated that 236  241  n  TGF-P 1 potentiated A p production in human astrocytes and, as a result, may enhance the 242  formation of amyloid deposits  . However, the role of TGF-P, beneficial or detrimental,  in the CNS is still unresolved. It is also feasible that with the functional deficiency of microglia in the op/op mouse the number or proportions of factors produced by activated microglia is skewed. This, in turn, may be amplifying certain pathological changes such as amyloidogenesis and amyloid accumulation in the blood vessels. A more detailed analysis on brain A p levels and vascular A p levels is required in order to determine if there is indeed a shift towards vascular amyloid deposition and what cellular and molecular processes are involved. There is still great controversy as to the state of microglia in the op/op mouse. Initial studies by Blevins and Fedoroff found that microglia were not affected by the mutation in  CSF-1,  in that they have normal morphology and are present at normal  186  frequency  . They did find that the microglia isolated from op/op mice needed CSF-1 in  99  order to differentiate and proliferate in culture, implying a possible deficiency in function 1 8 6  .  Other studies established that there was indeed a reduction in the number o f  microglia in the brains o f op/op mice; 47% in the corpus callosum, 37% in the parietal cortex and 34% in the parietal cortex compared to +/+ and +/op controls  243  > > 244  187  Morphological differences, such as, smaller size and shorter cytoplasmic processes were 187  also found and limited to the frontal cortex.  .  There have also been some studies  focused on the response o f microglia to acute C N S injury in the op/op mouse.  In the  facial motor nucleus paradigm, there was a lack of cell proliferation, mobilization and change in morphology in op/op mice compared to controls  2 4 5  .  Microglia in the op/op  mouse responded to neuronal signals but there was a decrease in neuronal regeneration due to the lack o f proliferation and mobilization. Further studies examining the role o f microglial mediated neurodegeneration found similar results.  Here, endotoxins were  injected directly into the brains o f animals and microglial activation and neuronal degeneration were assessed. It was found that microglia in the op/op mouse have normal activated morphological phenotype, however, only 30-40% o f activated microglia were observed in the brains o f op/op mice compared to controls a were increased in response to injury  2 4 6  2 4 0  . Moreover, levels of T N F -  and were at. par with the amount o f activated  cells with op/op mice having 48% of levels to that o f controls  2 4 0  .  In regards to  neurodegeneration, it appears that microglia in the op/op mice were able to promote neurotoxicity potentially by T N F - a mediated expression o f I L - i p , IL-6 and other proinflammatory cytokines  2 4 6  '  2 4 0  .  Therefore, it is now evident that the microglia in op/op  mice are deficient in their proliferative ability but are able to elicit neurodegeneration and removal o f debris. It should be noted that all the aforementioned experiments focused on  100  acute C N S injury.  The response o f microglia in op/op mice in chronic inflammatory  reactions is not well documented. This is where the experiment outlined in this study is unique. From the data presented in the literature one can speculate that, with respect to the CSF-1 deficient h A P P , there may be more AP deposition at a later age due to the inability of microglia to proliferate.  The microglia present in the brain may be able to  accommodate and degrade accumulated A p at the initial stages o f the disease, but as the amount o f A p increases there is not enough activated microglia to remove it. This also is in agreement with the complement experiments by Wyss-Coray et al. where inhibition o f complement (which results from microglial activation) results in an increase in plaque development  2 3 1  .  With fewer cells able to express complement proteins and activate  complement pathways there may be a reduction in the effective removal o f A p . According to the "microglial dysfunction" hypothesis  1 0 9  , one can also anticipate an  accelerated accumulation o f A P since with age it is thought that microglia become senescent and/or dysfunctional therefore their ability to support neurons and to act as phagocytic macrophage would be diminished. With fewer microglia present in the brain, the degree of senescence would be greater in Tg/+;op/op mice than Tg/+;+/+ mice perhaps hastening A D pathology. Moreover, the extent o f neurodegeneration, like A p deposition, may start at the same rate in Tg/+;op/op and Tg/+;+/+ mice.  The A P  deposition may then accelerate in Tg/+;op/op mice due to the increase in amyloid and its toxic effects on neurons, as well as, the presence of microglial neurotoxins.  Also, it is  possible that microglia in op/op mice have a diminished ability to undergo phagocytosis and support neuronal survival due to the lack o f CSF-1 which may result in an alteration  101  in the expression and response of many cytokines and other neurotrophic factors that microglia provide to the C N S . The number and ages o f mice used in this study were limited due to the high mortality o f the Tg/+;op/op mouse. It is possible that with a larger subject cohort and/or at later age, there could be differences in plaque burden between the different mice. Moreover, differences in other pathological hallmarks o f A D , such as neurodegeneration, may be evident at later ages. In a study by Fonseca et al, C l q deficient h A P P transgenic mice had similar plaque burden to h A P P mice at 9 and 16 months o f age where older C l q deficient h A P P mice exhibited less neuropathology than controls.  2 3 8  . Studies by Wyss-  Coray et al. on T G F - p i overexpression h A P P mice and on C3 inhibited h A P P mice, demonstrated brain pathology at 10-12 months of age after significant plaque deposition 236 217  occurs  ' ".  In addition, the h A P P mice used in these studies express mutated  hAPP695,751,770 under control o f the platelet derived growth factor p chain promoter and develop plaques at approximately 6 months of age  2 4 1  . Therefore, since the op/op mouse  has a shortened life span it would be beneficial to use an A D mouse that develops significant A p deposition prior to 9 months of age to evaluate the relationship between microglial activation and plaque formation. Many studies have demonstrated that genetic background regulates the extent o f A P P processing, A p deposition and mouse mortality. Initial attempts by Hsiao et al. to 84  create transgenic mice failed in part due to host strain effect on transgene expression  .  These mice were generated in the F V B / N inbred strain. The second attempt utilized a C 5 7 B L / 6 x C 5 7 B 6 / S J L hybrid proved more successful and is the basis for the Tg2576 A D transgenics used in this study. These mice also had some initial complicating factors. 102  The life span o f the Tg2576 mouse on the inbred C 5 7 B L / 6 background is approximately 6 months whereas on a mixed background o f C 5 7 B L / 6 ; S J L mice can survive for over 2 years (Younkin, personal communication). Gene dosage also has an effect on mouse mortality. For example, the Tg2576 A D mouse exists only as a hemizygote and is lethal in a homozygous state.  It is possible that the Tg/+;op/op generated in this study  contained more of the C 5 7 B L / 6 background which impacted its life span, since very few of these mice survived past 9 months of age. Recently studies on A P P proteolysis, A p metabolism and A P deposition in transgenic mice have demonstrated that these processes are regulated by genetic background  2 4 8  .  The C 5 7 B L / 6 background exhibits greater  plaque burden, earlier age of A p deposition, and increased levels o f brain and plasma levels o f A P M O and A P i ^ , compared to D B A / 2 J and 129Sl/SvImJ strains  2 4 S  .  These  results suggest that certain gene alleles in some mouse strains, such as C 5 7 B L / 6 , are dominant over alleles present in other strains, such as D B A / 2 J , and alter AP-related pathologies in A D .  A s a consequence o f host effects, most transgenic mice used in A D  research have hybrid backgrounds. The diversity o f the response seen from this study and many others examining microglia and their induction o f inflammatory and complement pathways, indicates the complexity o f microglial responses and the multiplicity o f microglial activation states in A D . A considerable amount of investigation is required and necessary to elucidate these interactions.  103  Chapter 5: Ap immunization and the blood-brain barrier 5.1 Rationale Conflicting with the concept that  ABMO  and AB1-42 are cytotoxic are noteworthy  advances in recent A D research which advocated the use o f peripherally administered A p peptides as a vaccine in an effort to reduce senile plaque loads o f A D model mice While the exact relationship between A p production and deposition and its role in A D neurodegeneration remains unclear, one cannot dispute that treatments aimed at reducing the amyloid burden have resulted in reduction in senile plaques and behavioral benefits. Recent vaccination studies aimed at reducing the burden of all A p types in the brain have proven successful in transgenic mice. The first o f such studies by Schenk et al. injected fibrillar Api.42 into six week old P D A P P mice, prior to A D symptom onset, and in 11 month old P D A P P mice, after noticeable A P deposition has occurred in the brain  175  > . 249  After eleven months, mice which had been vaccinated since they were six weeks old showed an almost total absence o f amyloid plaques, and a reduced amount of dystrophic neuritis, astrogliosis and microgliosis in the brain compared to P B S injected controls. After five months, mice which had been vaccinated since they were 11 months old showed a significant decrease in plaque burden and in the amount o f diffuse A p , as well as a reduction in dystrophic neuritis and astro- and microghosis  1 7 5  .  Many other  immunization studies, utilizing both active and passive immunization protocols, have been performed in the same and different A D model mice and have found similar results 177 250 251  in regard to the pathological features o f A D  ' ~'  . O n the other hand, some studies in  different A D mouse models have found that immunization had little effect on plaque 104  burden i f dense plaques were pre-existing " . Studies focused on memory impairment have found that immunization with A(3 or passive transfer o f antibodies against A p protected mice from learning and age-related memory deficits behavioral impairment 178,179 a  g  w e  jj  a g  |  n  a  s  ig if n  i c a n  t i y delay the onset o f memory deficits  2 5 3  -  2 5 4  . Recently, it  was determined that A P immunotherapy can clear early Tau pathology but not late hyperphosphorylated Tau aggregates  1 8 1  . With the success o f amyloid immunization in  mice, clinical trails in humans were undertaken.  These trials were halted when  approximately 5% of the test subjects showed signs of inflammation o f the C N S , clinically described as aseptic meningoencephalitis  2 5 5 , 2 5 6  .  Subsequent studies in A P P 2 3  mice found the same results as in the human trials. In these mice A p immunization did reduce plaque burden, as in other studies; however, there was also an increase in cerebral microhemorrhages associated with amyloid-laden vessels  2 5 1  .  Regardless o f the early success in animals, it remains unclear how peripheral immunization can affect plaque burden in 'immunoprivileged' tissues such as the brain that possess vasculature that limit immune surveillance. This vasculature, also known as the B B B , is formed by a continuous layer o f capillary endothelium joined by tight junctions that are generally impermeable, except by active transport, to most large molecules including antibodies and other proteins.  With this in mind, three possible  mechanisms have been proposed to explain the effects of vaccination in A D . The first suggests that the antibodies against A p , normally excluded from the C N S , enter the brain by an obscure mechanism involving receptor mediated transport and then proceed to bind to A p . Entry into the brain may be facilitated by binding o f the antibody to A P in the periphery creating an Ap-antibody complex which is then transported in the brain via  105  receptor mediated transcytosis by receptors that recognize A p  " .  The A p - a n t i b o d y  complex is then degraded by the microglia via Fc receptor (FcR) mediated phagocytosis 259 176 260 261  " '  '  '  .  However, when fragments of antibodies were administered there was  reduction in A P load in the brain  '  . This indicates that non-FcR mediated clearance  of A P is also occurring. A second possible mechanism suggests that antibodies against A p are actively transported into the brain, again by an unknown mechanism, and bind directly to A p fibrils resulting in plaque disaggregation. In this instance, the antibodies sequester the A p  and prevent further fibril/plaque formation  2 6 1  .  Finally, it is also  postulated that there is an equilibrium formed by active transport o f A P into and out o f the brain by receptor mediated transcytosis and by antibodies against A p , binding A p in the periphery removing it from circulation. This then causes a shift in the equilibrium between A p in the plasma and C N S , resulting in a suppression o f A P deposition in the 1 77  brain  OK/l  '  9 / ^ 9/^/i  '  '  . Studies involving intravenous administration o f A p specific antibodies  demonstrated an efflux o f A P from the brain to the plasma of a  l25  2 6 4  . In addition, administration  I - A P - a n t i b o d y complex into the periphery inhibited the movement o f  the brain  2 6 6  l 2 5  l - A p into  .  The permeability o f the B B B to A P peptides and antibodies to A P is addressed in this study.  In addition, the effect o f A P immunization on the integrity of the B B B is also  examined. It is hypothesized that, as with previous immunization studies where there was a reversal o f pathology, amyloid immunization w i l l restore B B B integrity in Tg2576 mice. These results w i l l aid in further substantiating this form of therapy in A D research and will also serve as a means to explain the mechanism by which immunization reverses plaque pathology. 106  5.2  Results  5.2.1 Ap peptide and anti-Ap antibodies and their ability to cross the BBB To investigate the possible mechanisms involved in Ap vaccination, the ability of both the APi^o peptide and antibodies against Ap to cross from the periphery into the brain was examined. First, fluorescent labeled Api_4o or PBS were injected i.v. into 6 week Tg2576 mice and control littermates. The fluorescent Ap was detected in the brain parenchyma of both groups of animals (Figure 5.1 a-d). Since it has been previously shown that AP can gain entry into the brain via receptor mediated transcytosis, this data also supports this hypothesis. Other studies focused on the movement of Ap into the brain support these results. Yan et al. found that the endothelial cells which make up the B B B express the R A G E receptor . This receptor has been shown to bind to Ap with 68  high affinity and transport AP into the brain. As a control for B B B permeability, BSA, a protein known not to be able to cross the B B B , was conjugated to Texas Red and injected into mice. As seen in Figure 5.1 (e and f), the B S A was primarily localized in the vessels indicating an intact B B B ; however, there is some B S A found in the brain parenchyma of transgenic mice. The presence of B S A in the brain may be an indicator of the beginning of stages of increased B B B permeability in the Tg2576 mouse. Next an anti-human Ap antibody, clone 4G8, was injected into 6 week old transgenic and non-transgenic mice. In contrast to the Ap peptide, there was no presence of anti-Ap antibodies in the brain parenchyma (Figure 5.2 a-d). This experiment was repeated using a biotin-labeled antiAP antibody. In this instance either the labeled antibody or biotin alone was injected. As with the above experiment, biotin conjugated anti-Ap antibodies were only present in the 107  vessels and not in brain parenchyma (Figure 5.2 e-h). This would indicate that anti-AP antibodies cannot gain access to the brain from the periphery. However, recent studies using iodinated antibodies against A P indicated that antibodies can cross the B B B but with very low efficiency  2 5 8  .  In the study by Poduslo et al. the mice used were  significantly older than the ones used here  2 5 8  and it has been established that the B B B in  Tg2576 A D model mice is compromised by 4 months of age  1 4 9  . Thus at 6 weeks o f age,  there is little damage to the B B B and it is possible that antibodies cannot gain access to the brain.  108  Figure 5.1. A p peptides can cross the BBB in both transgenic and wild-type mice. Fluorescent labeled A p i ^ o was injected i.v. into 6 week old transgenic and control littermates. A p was able to cross the B B B and enter the brain parenchyma in both transgenic and non-transgenic mice thereby suggesting a receptor mediated transport across the B B B . (a) cortical section of a Tg2576 mouse injected with A P , (b) cortical section of a control littermate injected with A p , (c) cortical section o f a Tg2576 mouse injected with P B S control and (d) cortical section o f a control littermate injected with P B S . A s a control for B B B integrity, Texas red conjugated B S A was injected into both mice. A s seen in (e) Tg2576 mouse and (f) non transgenic littermate, the B B B is relatively intact. There appears to be a small amount o f B S A in the brain parenchyma o f the transgenic mouse indicating the beginning o f the B B B breakdown. *, brain parenchyma with A p peptide, arrow, brain vasculature.  109  Figure 5.2. anti-Ap antibodies cannot cross the B B B in both transgenic and wildtype mice. Anti-AP antibodies, clone 4 G 8 against human APi_4 , were injected i.v. into 6 2  week old transgenic and control littermates. Both unlabeled antibodies (visualized by immunofluorescence) and biotin labeled antibodies (visualized by light microscopy) were used. In both cases anti-Ap antibodies were localized to the vasculature (white and black arrows) and were unable to cross the B B B and access the brain in both Tg2576 and control littermates. (a) cortical section o f a Tg2576 mouse injected with anti-AP antibodies, (b) cortical section o f a control littermate injected with anti-AP antibodies, (c) cortical section o f a Tg2576 mouse injected with P B S control and (d) cortical section o f a control littermate injected with P B S . (e) cortical section o f a Tg2576 mouse injected with biotin labeled anti-Ap antibodies, (f) cortical section o f a control littermate injected with biotin labeled anti-Ap antibodies , (g) cortical section o f a Tg2576 mouse injected with biotin (control) and (h) cortical section of a control littermate injected with biotin (control).  Ill  4G8  PBS  biotin-4G8  5.2.2 A n t i - A p antibody titres in immunized animals To assess the immune response of the mice to immunization with A P , antibody titres for antibodies against A p were measured. Serological analysis o f serum samples collected from transgenic and non-transgenic mice vaccinated with A p and P B S were analyzed for the titres o f anti-Ap antibodies by E L I S A using synthetic  AP1.40  peptide.  Serum samples from mice in all groups were collected after the second injection and results indicated that transgenic and non-transgenic mice vaccinated with A P produced a high IgG response to  APi^o-  N o detectable antibodies were detected in transgenic and  non-transgenic mice vaccinated with P B S (Figure 5.3).  M i c e vaccinated with A p , which  did not exhibit high a n t i - A P antibody titres, were not used in the study.  113  1000001 10000'  .1  1000H  o o  100'  o cu  10H  Tg/+  +/+  Tg/+  +/+  PBS  B.  10000n  1000'  « o o  100H  o  10H  0£  Tg/+  +/+  T*  Tg/+ PBS  1  +/+  100001  •co  •  § 1000'  CO  100H  o  ioi  o o  a:  •'A  Tg/+  MA.  +/+  Tg/+ +/+ PBS  Figure 5.3. Antibody titre in serum of transgenic and non-transgenic mice  immunized with either Ap or PBS. Serum antibody titres were measured after the second vaccination by ELISA. In all cases, mice (transgenic and non-transgenic) immunized with fibrillar APi_4o exhibited a high anti-Ap antibody titre against Ap peptide. No anti-AP antibodies were detected in mice immunized with PBS. A. 15 month age group, B. 12 month age group, C. 6 month age group.  114  5.2.3 Amyloid plaque burden in immunized animals Next, amyloid plaque burden was assessed in both A p and P B S immunized transgenic studies  175  and '  176,267  '  non-transgenic 178  '  177  '  180  '  181  animals.  As  noted  in  other  immunization  there was a significant reduction in the plaque burden in the  cortex and hippocampus o f transgenic mice immunized with A p compared to those immunized with P B S . In 15 month old mice, which were immunized with A p after disease onset, there was a decrease in plaque burden with a reduction in both the size and number o f plaques, whereas brain sections from PBS-treated transgenic mice contained numerous amyloid deposits (Figure 5.4 a-b; Figure 5.5 a).  These data agree with  previous studies where there was not a total elimination or prevention o f plaques in these animals.  In mice immunized with A p prior to disease onset (12 month) there was an  almost complete prevention o f A p deposition. In A P immunized transgenic mice, 4 out of 6 had no detectable amyloid deposits.  Two mice from this treatment group had a  single isolated plaque in the 4 brain sections examined. A s with the 15 month group o f mice, 12 month old, PBS-treated transgenic mice exhibited numerous amyloid deposits in their cortical and hippocampal regions (Figure 5.4 e-f; Figure 5.5 b).  There were no  detectable plaques in non-transgenic controls vaccinated with either A p or P B S (Figure 5.4 c-d, g-h). N o amyloid plaques were found in 6 month old mice since amyloid plaques do not manifest in Tg2576 mice until 9 months o f age.  Non-transgenic mice injected  with either A P and P B S exhibited no plaque burden (Figure 5.5 c-d, g-h). This confirms that immunization with A P may prevent plaque formation and thereby protect against disease.  115  F i g u r e 5.4. A m y l o i d Pathology in Tg2576 M i c e I m m u n i z e d with Ap or P B S . A m y l o i d plaques in cortical sections from mice were visualized with 4 G 8 , an antibody against human A p . There was a significant reduction in the total number of A P plaques in Tg2576 mice vaccinated with A p compared to those vaccinated with P B S at 11 months of age and at 6 weeks o f age. Very few 4G8 positive plaques were found in brains o f mice vaccinated at 6 weeks o f age with A p . (a) 15 month Tg2576 mice vaccinated with P B S , (b) 15 month Tg2576 mice vaccinated with A p , (c) 15 month wild-type controls vaccinated with P B S and (d) 15 month wild-type controls vaccinated with A p , (e) 12 month Tg2576 mice vaccinated with P B S , (f) 12 month Tg2576 mice vaccinated with A p , (g) 12 month wild-type controls vaccinated with P B S and (h) 12 month wildtype controls vaccinated with A p . N o plaques were present in all 6 month old mice. Brain sections shown are representative o f their respective treatment groups.  116  117  A.  16 14  I  12 10  o  (/> CD  C  CD  go  Q) cr  *  _CU CL  ,  J  ,  A B  PBS  Treatment  Figure 5.5. Cerebral amyloid levels are reduced in Tg2576 mice following A p immunization. Plaques were detected using an anti-human A p antibody, 4 G 8 , on sequential brain sections. The presence o f discrete plaques made it feasible to count the number of plaques in the entire section. Plaques were counted by visual inspection under the microscope for each o f 4 sections at equal plane for each mouse. Total averaged number o f plaques is presented. There was a significant reduction in the total number o f A p plaques in Tg2576 mice vaccinated with A p compared to those vaccinated with P B S for both the 15 moth and 12 moth age groups. No plaques were seen in all 6 month old mice, (t-test * P < 0.05).  118  5.2.4 M i c r o g l i o s i s in immunized animals The presence o f activated microglia in immunized and non-immunized animals was also analyzed. Activated microglia are usually found associated with the amyloid plaques and  can be distinguished from resting microglia by the expression o f specific  proteins, such as IL-1 p, C D 1 l b and major histocompatibility class II, and by distinct morphology  2 6  .  Activated microglia exhibit an altered morphology from  resting  microglia first by the presence o f thickened ramified processes and larger cell bodies which progresses to a final amoeboid/phagocytic state  1 0 4 , 1 0 5  .  Sections of mouse brains  were reacted with an antibody specific for the F4/80 antigen.  The F4/80 antigen is 268  expressed by a majority o f mature macrophages, including microglia with previous studies  1 7 5  . In agreement  , there appeared to be a reduction in the presence o f activated,  microglia in the brains of A p immunized mice compared to P B S controls suggesting a dampening of the inflammatory response.  In 15 month transgenic mice immunized with  P B S there are more plaque infiltrating microglia present compared to those immunized with A p (Figure 5.6 a,b). There were more densely stained F4/80 positive microglia with swelled cell bodies and thickened processes in PBS-treated transgenic mice compared to Ap-treated mice.  Similarly, in 12 month old transgenic mice immunized with P B S there  were more amoeboid shaped and thick ramified microglia, whereas there microglia in the A P immunized mice have smaller cell bodies and more extensively ramified processes (Figure 5.6 e,h).  The microglia present in non-transgenic mice displayed largely a non-  activated morphology with small cell bodies and thin, highly branched processes (Figure 5.6 c-d, g-h).  119  Figure 5.6. Microgliosis in I m m u n i z e d M i c e . Brain sections were stained with F4/80 to reveal the presence of microglia. Activated microglia can be distinguished from resting microglia by the presence of ramified processes and condensed cell bodies. Cortical sections from Tg2576 mice immunized with P B S or with A p , both at 11 months and at 6 weeks, show that A p immunized mice have reduction o f activated, plaque associated microglia (black arrow). Control littermates exhibited no microgliosis (a) 15 month Tg2576 mice vaccinated with P B S , (b) 15 month Tg2576 mice vaccinated with A p , (c) 15 month non-transgenic controls vaccinated with P B S and (d) 15 month non-transgenic controls vaccinated with A P , (e) 12 month Tg2576 mice vaccinated with P B S , (f) 12 month Tg2576 mice vaccinated with A P , (g) 12 month non-transgenic controls vaccinated with P B S and (h) 12 month nontransgenic controls vaccinated with A p . Brain sections shown are representative o f their respective treatment groups.  120  5.2.5 BBB permeability in immunized animals To test the effect o f vaccination on the B B B , B B B permeability was assessed in mice at various time points. The first group was immunized with either A p peptide or P B S at 11 months of age, after disease onset, and immunized for a 4 month period (15 month old mice). The second group was immunized beginning at 6 weeks o f age, well before any pathological disease symptoms, for an 11 month period (12 month old mice), similar to previous studies by Schenk et al.  .  115  The final group was immunized beginning at 6  weeks o f age for a 4 month period (6 month old mice).  This last group was only  immunized for a short duration since it has been demonstrated that B B B permeability can be compromised as early as 4 months o f age. B B B permeability was assessed using the quantitative Evans blue assay  '  . Organs with a selective barrier, such as the brain,  only take up a small amount o f the dye. If there is a breach in the B B B then Evans blue w i l l enter the parenchyma, whereas it is excluded from the brain with an intact B B B . In all groups o f mice (15 month, 12 month and 6 month mice), the integrity o f the B B B of transgenic mice was compromised compared with that of non-transgenic controls (** p < 0.05). These data concur with the study by Ujiie et al. where B B B integrity was compromised in 4 month and 10 month old Tg2576 mice  1 4 9  . Upon immunization with  A(3, the A D transgenic mice displayed a significant decrease in B B B permeability, indicating a possible restoration o f the B B B .  Transgenic mice injected with A(3 had a  significantly lower amount o f Evans blue dye in their brain parenchyma compared to A D transgenic mice injected with P B S alone (Figure 5.7 a,b; * P < 0.05). This was true for both 15 month old mice and 12 month old mice immunized with A p . In the 12 month old mice, it is possible that vaccination with A P maintained an intact B B B , with little to no  122  compromise in B B B function.  There was no change in B B B permeability in non-  transgenic mice injected with A p or P B S . This latter observation is interesting as A P has been shown to be cytotoxic to endothelial cells and neuron in-vitro by eliciting apoptosis 27115154  , ",  272  and angiogenesis  , yet the low amounts used in this study do not appear to  affect B B B integrity in normal mice. Finally, results from the 6 month old group are intriguing. In this instance, transgenic mice vaccinated with A p appeared to exhibit a breach in B B B integrity, similar to that o f transgenic mice immunized with P B S (Figure 5.7 c). There was no effect elicited by A P immunization on the B B B as seen with the other groups.  A s demonstrated above,  transgenic mice vaccinated with P B S had a compromised B B B in comparison to nontransgenic mice and no changes in B B B integrity were evident in non-transgenic mice injected with either A p or P B S . There was in an increase in the permeability o f the B B B between transgenic and non-transgenic mice (Figure 5.7 c; ** P < 0.05).  These data  agree with previous data demonstrating that B B B integrity can become compromised as early as 4 month of age  149  '.  123  Figure 5.7. BBB permeability as determined by Evans Blue in cortical regions of and PBS immunized mice. In all groups the permeability o f the B B B in Tg2576 is greater than non-transgenic controls (t-test, **P < 0.05) (A) 15 months Tg2576 mice immunized with A(3 (n=5) show a decrease in the permeability o f the B B B compared to P B S Controls (n=4) (t-test, *P < 0.05). The level o f B B B permeability in A p immunized mice is similar to the permeability of non-transgenic littermates. (B) 12 months Tg2576 mice immunized with A P (n=6) show a decrease in the permeability o f the B B B compared to P B S controls (n=6) (t-test, *P < 0.05). The level o f B B B permeability in A p immunized mice is similar to the permeability o f non-transgenic littermates. (C) 6 month Tg2576 mice immunized with A p (n=9) show no difference in B B B permeability compared to P B S controls (N=9). Tg2676 mice (both A p and P B S vaccinated) do exhibit an increase in B B B permeability compared to control littermates.  124  5.3 Discussion Research focused on systemic AP-lowering strategies is becoming more important in the  development  of therapeutics  for  AD.  Recent  advances  have  focused  on  immunotherapy using amyloid. These studies have proven successful in mice and have reached clinical trials in humans.  What remains unclear is the mechanism by which A p  immunotherapy alleviates the pathology seen in A D . This issue was first addressed by examining the ability o f A p peptides to pass through the B B B and demonstrated that A p was able to access the brain in both transgenic and non-transgenic mice. This suggests that A P can gain entry into the brain via receptor mediated transcytosis  2 7 3  ,  2 7 4  , . 7 0  Ap  transport across the B B B has been investigated by many groups and all results indicate an active transport o f A P across the B B B .  In a recent study it was shown that soluble A P  can be transported from the C N S to the plasma since direct injection o f radiolabeled A p into the brain was recovered in the plasma.  Moreover, when radiolabeled A P was  injected into the periphery, radioactive counts were found in the brain examined the influx o f both models, C D - I and S A M P 8  A P M 2  2 7 5  and  APMO  7 0  . Banks et al.  (mouse and human) in the different mouse  . They found that all forms of A p were transported into the In addition, all forms o f A p  brain, with mouse 1-42 and human 1-40 being the fastest.  were also transported out o f the C N S . When examining the permeability coefficient x surface area product (PS) o f proteins known to cross the B B B v i a receptor-mediated transcytosis (i.e. insulin) and of proteins known to have limited entry into the brain (i.e. albumin) it was shown that the PS value of A p was similar to that of insulin  '  \  Further studies have suggested that the R A G E receptor is a receptor on the B B B that is responsible for the influx o f A P from the blood to the brain > . R A G E , is a multiligand 68  273  126  receptor in the immunoglobulin superfamily, binding ligands such as Ap, the SlOO/calgranulin family of pro-inflammatory cytokine-like mediators, the high mobility group 1DNA binding protein amphoterin and products of nonenzymatic glycoxidation . 61  The expression of RAGE is ligand dependant. In transgenic mice, RAGE expression correlates with AP deposition and is localized to affected cerebral vessels, neurons and activated microglia. The increase in expression may facilitate an increase in Ap transport 67  into the brain thereby exacerbating cellular dysfunction and disease progression  . Other  studies in animal models have shown that RAGE mediates the transport of Ap from the blood to the brain and deletion of RAGE results in the inhibition of Ap transport thus protecting the CNS from the accumulation of peripheral AP pools . 69  When an antibody against human Ap, was injected into mice there was no apparent presence of antibody in the brain parenchyma. Whether antibodies can cross into the brain from the periphery is under debate. It is possible that in this study, the amount of antibody used was insufficient to cross the BBB. Antibody injected into the periphery could have bound to circulating peripheral amyloid thereby preventing its movement into the brain. In this study the mice used were 6 weeks old. In other studies, where there was a presence of antibody in the brain, the mice were significantly older. It has been previously shown that the BBB is compromised in AD mice as early as 4 months of age. Thus, it is possible that at 6 weeks of age antibodies cannot gain access into the brain whereas in older animals the BBB is damaged thereby facilitating the entrance of antibodies to the brain. When evaluating the PS values of antibodies across the BBB, Podsulo et al. found that the PS value of non-specific IgG and AP specific antibodies were similar, 0.5 to 1.1 X 10" ml/g/s and 0.6 to 1.4 X10" ml/g/s respectively, and less 6  6  127  than that for albumin  147  . This indicates that the passage of antibodies across the BBB  occurs at a very low efficiency  258  . Furthermore, Demattos et al. were unable to identify  any plaque associated anti-Ap antibodies after immunization  177  . Conversely, there is  abundant evidence which illustrates that anti-AP antibodies can cross the BBB.  In a  study using active immunization with A p peptides there was the appearance of anti-Ap antibodies associated with microglia localized with plaques  175  . In addition, antibodies to  AP may be able to enter the brain once in complex with A p . 258  It has also been  demonstrated that anti-Ap antibodies can enter the brain at a rate similar to that of albumin; however, due to the long half-life of the antibody in the blood there was a longer clearance time from the brain . Taken together, the disruption of the BBB likely 276  facilitates an increase of antibodies across the BBB efficiently enough to explain the action of the vaccine. The most significant finding in this study demonstrates that immunization with A p tends to repair the damage found in BBB in Tg2576 AD mice. In 15 month old transgenic mice, which have been inoculated for 4 months, there is a decrease in Evans blue uptake compared to PBS-treated transgenic mice. The significant decrease in Evans blue uptake observed in A p immunized transgenic mice compared to the PBS mice suggest that AP immunization results in the restoration of the BBB. In 12 month old mice inoculated for 11 months starting at 6 weeks of age, A p immunization may be able to actually prevent further disease progression. In these mice, Evans blue uptake is comparable to non-transgenic mice, where there is no measurable decreased integrity of the BBB. One possible explanation of the restoration of the BBB in older mice is that the vaccination leads to the decrease in the amount of circulating A p that could directly or  128  indirectly affect the function o f endothelium in the B B B . A s demonstrated in this study 17^17X250 180 251 181  and in many other studies  '  '"'  '  '  a  , there is a significant decrease in A p deposits  and microgliosis following immunization. With the removal o f A p from the brain and the subsequent deactivation o f microglia, there w i l l be a decrease in the brain in the amount  of reactive oxygen and nitrogen compounds  and inflammatory cytokines  produced by these cells which have been shown to activate vascular endothelial cells 277 153  '  . Studies focused on vasculature activation found that in response to systemic  inflammation from either the periphery or brain parenchyma, endothelial cells upregulate the expression o f molecules that are generally associated with inflammatory processes such as prostaglandin E 2 , nitric oxide, C D 4 0 and C O X - 2  153  - > 154  155  These then  can act agonistically on microglia and neuronal cells to further intensify inflammation and disease pathology. A(3 has also been demonstrated to elicit many pro-apoptotic and pro-angiogenic responses in the endothelial cells that make up the B B B  152  > ; 151  150  These effects are  dependent on the amount of A P present. It has been exhibited in vitro that exposure o f 272 271 151  endothelial cells to u M concentrations (5-25 u M ) elicits pro-apoptotic signals  '  '  whereas treatment with n M concentrations (50-250 n M ) of A p elicits pro-inflammatory signals and increased monocyte migration with minimal disruptions to the endothelial monolayer  '  Treatment o f primary cerebral mouse endothelial cells with AP25-35  resulted in the activation o f AP-1 and the subsequent expression o f B i m , a member o f the B H 3 only family o f proapoptotic proteins  1 5 1  . Moreover, A p treatment also resulted in  the translocation o f second-mitochondria derived activator o f caspase (Smac), a regulator of apoptosis, from the mitochondria to the cytosol where it can bind to the X 129  chromosome linked inhibitor o f apoptosis protein ( X I A P ) , resulting in cell death  1 5 1  .  Cytochrome C release from the mitochondria and the subsequent activation o f several caspases, such as caspase 8 and caspase 3, all important events on the apoptotic cascade, have also been reported  1 5  ° . Several studies have also shown that exposure of endothelial  cells to A p results in cytotoxic damage elicited from the generation o f reactive oxygen species and increased calcium levels causing alterations in endothelial cell structure and function  '  . Angiogenesis is also observed in endothelial cells in A D .  mediators  such as T N F - a ,  IL-lp,  and IL-6 stimulate  angiogenesis  Inflammatory 2 8 1  .  These  inflammatory mediators, as well as C O X - 2 and amyloid, have been found to cause an increase in the expression o f may angiogenic factors including V E G F , T G F - P and T N F - a 215 282  '  . Moreover, in A D patients there is an increase in V E G F expression in the bram as  well as an increase in the serum and C S F  2 1 4  '  2 1 5  . Recently, it was discovered that V E G F  binds to A P with high affinity and specificity and is thus co-localized to the plaques. Therefore, it is possible that in response to pro-angiogenic and pro-apoptotic signals from A p and activated microglia, tight junctions disappear, cells round up creating a leaky barrier  2 1 6  . The endothelial cells in turn release neurotoxins, free oxygen radicals, A P P  and more pro-angiogenic factors such as V E G F and T G F - p . With the removal o f signal provided by A p and inflammation, it is possible that the endothelial cells quiesce, terminate the release of toxic substances, reform tight junctions reforming an intact, tight barrier. In regards to the mechanism by with A P immunization effectively reduces A D pathology; it is possible that the debate as to whether antibodies can cross the B B B is resolved. With a leaky B B B , antibodies are able to gain access into the brain, bind to A p 130  and are either degraded by microglia v i a F c R mediated phagocytosis or the AP-antibody complex can leave the brain. Alternatively, with a leaky B B B the efflux o f A P from the brain can occur at a faster rate than by active transport by specific receptors, such as L R P 7 0  , thereby re-establishing equilibrium between plasma and C N S A p pools  2 5 8  .  Once the  excess amyloid is removed, by microglia or by efflux into the periphery, amyloid levels return to tolerable a threshold, microglia become deactivated, the B B B reforms and the brain becomes  an 'immunoprivileged' site once again.  In summary, these new  observations provide an intellectual framework for understanding the efficiency o f vaccination to modify disease progression. In addition, resealing o f the B B B provides a previously  undescribed  intervention  point  for  modifying  disease  outcome  in  amyliodopathies such as A D .  131  Chapter 6: Concluding remarks and future directions Since the eponym "Alzheimer's disease" was coined, there has been much debate as to the exact etiology and pathology o f the disease. M a n y view that A D is not a single, uniform disease.  Rather, A D is more likely a heterogeneous and multifactorial disease  influenced by the interactions o f various susceptibility genes and environmental factors. Intriguingly,  as  with  other  medical conditions, A D represents  a  disease  with  heterogeneity in its origins ( E O F A D compared to L O A D ) and homogeneity in its clinical appearance and pathology. Therefore, it is hypothesized that A D results from a complex sequence o f steps involving multiple factors that extend well beyond the accumulation o f Ap.  This thesis concentrates on many facets o f one o f the main foundation of A D , the  contribution o f microglia in disease progression. In particular the relationship o f A p to the activation o f microglia, subsequent signal transduction pathways and gene expression and the impact of microglia on A P deposition and clearance and its effect on another pathological hallmark, the B B B , were addressed. According to the "amyloid cascade hypothesis" it is thought that AP accumulation is the principle event in A D .  A possible discrepancy with this theory is that there is no  direct evidence linking A p accumulation to neuronal death.  Neuroinflammatory  processes may be this missing link and have been suggested to exacerbate neuronal damage/death microglia  in  seen in A D . With the current understanding o f the role of activated disease  progression  many  inflammatory processes have been pursued.  therapeutic  strategies  directed  against  Epidemiological studies on N S A I D use 102 103  demonstrated a reduced risk and decrease in the rate of cognitive decline  '  and  treatment o f h A P P transgenic mice with anti-inflammatories decreased plaque size and  132  slowed down cognitive deficits  . However, the use o f N S A I D s appears to be effective  only when administered prior to disease onset and not once symptoms are manifest, as indicated from current clinical trials where no beneficial effect o f N S A I D use is reported 2 2 6  .  The development of new anti-inflammatory therapies that interfere with one or more  specific steps in the inflammatory cascade is underway.  Nevertheless, it is essential to  establish a specific, reliable and non-invasive biomarker to assess the efficacy o f new drug treatments. Results from the studies performed in this thesis support the use o f p97 as a marker of inflammation. The levels o f p97, m R N A and protein, were upregulated in activated microglia.  Interestingly, the increase in p97 expression occurred largely in  response to A P stimulation and not in response to other known A D stresses such as I F N y. Further investigation into the expression of p97 found that p97 production appears to be controlled by the p38 M A P K signal transduction pathway.  This is intriguing since  many studies have demonstrated that A P is able to induce the activation o f p38 M A P K in vitro and that p38 M A P K phosphorylation is increased in affected brain regions in the 57 128 129  A D brain  '  '  . In addition, this thesis has demonstrated that p97 levels can be altered  with treatment o f activated microglia with N S A I D s , Ibu and N i m , promoting its potential role as an A D inflammatory biomarker. Understanding  the mechanism  of amyloid aggregation  ultimately lead to new advances in therapeutic development.  and clearance  will  Clearance o f A p through  microglial activation, chemotaxis, proliferation and phagocytosis has received a lot o f interest over the years, in particular from the recent immunization studies.  Activated  microglia displaying a variety o f cell surface markers that distinguish them from resting microglia are often found within and immediately surrounding maturing amyloid plaques  133  ' '  . Whether activated microglia contribute to or aid in the clearance o f plaques is  controversial. Using a h A P P A D mouse model which lacked functional microglia it was found that, at the early stages o f the disease, there was no significant difference in plaque burden in Tg/+;op/op and Tg/+;+/+ mice. Interestingly there were deposits o f cerebral amyloid in the Tg/+;op/op mice implying a possible shift in amyloid deposition patterns. These results are intriguing but more mice need to be examined in order to resolve the complex relationship between activated microglia and A p aggregation and clearance. One o f the latest therapies to combat A D employs active or passive immunization with A p peptide or antibodies against A p . This method was successful in transgenic mice, but failed during clinical trials due to the incidence o f meningio-encephalitis  256  > . 255  It has previously been established that there is increased permeability in the B B B o f Tg2576 A D mice compared to age match controls at 10 months o f age, as the signs o f the disease become manifest, and as early as 4 months of age, prior to disease onset and plaque deposition  1 4 9  .  Therefore, it was hypothesized that disruption of the B B B is  another pathological hallmark o f A D that may explain the mechanism by which immunization therapies have proven successful.  Specifically', peripheral antibodies are  able to pass into the brain via a breached barrier and bind to and sequester brain amyloid. The antibody-amyloid complex is then degraded by F c R mediated phagocytosis by activated microglia. The present study addressed the effect o f A p immunization on the integrity o f the B B B and revealed that immunization with A P , prior to and after disease onset, resulted in a decrease in permeability and a restoration o f B B B integrity.  These  observations provide an intellectual framework for understanding the efficiency o f  134  vaccination to modify disease progression as well as providing new possible therapeutic interventions. It is clear that there are several directions this project could take in the future. F o r instance: studies focused on further establishing p97 as a biomarker for A D and as an indicator for drug efficacy in an in vitro and in vivo system could be addressed. Measuring p97 levels in primary mouse and/or human microglia with or without exposure to A p and in A D model mice treated with various putative drug treatments would clarify i f indeed p97 can be used as a monitoring system. If successful, this assay could serve as a means to test new drug development initially in an in vitro system and, i f efficacious, move into an in vivo system before clinical trials.  In order to do these  experiments monoclonal antibodies to mouse p97 need to be generated,  l n addition, it is  possible that, since p97 is an iron transport molecule, it may play a role in disease progression.  Therefore, determining i f p97 contributes to disease pathology could be  investigated. One way to address this hypothesis would be by creating transgenic mice overexpressing p97 and breeding these mice to h A P P mice and examining the brains for signs o f A D pathology. In regards to the role o f activated microglia in A p accumulation and plaque formation, it was found that the amount o f A P plaques in h A P P mice with dysfunctional microglia was similar to the amount in h A P P mice. However, the number o f animals used in this study was small and since there is variability between mice, a larger cohort is needed in order to draw a more definite conclusion.  Moreover, since CSF-1 deficient  mice have a short life span it would be beneficial to use an A D mouse model that develops pathological symptoms at an earlier age than the Tg2576 mouse model. A s  135  well, it would be interesting to assess the other hallmarks o f A D in these mice, namely neurodegeneration,  vascular amyloid burden  and B B B permeability.  Furthermore,  measuring the levels of certain complement proteins and other indicators o f microglial activation would be of interest to see i f A P had a direct or indirect effect on microglial maturation and function. It was demonstrated in this thesis that A p immunization restores B B B integrity in mice immunized after disease onset and possibly prevents B B B deterioration when immunized prior to disease onset. investigated on a global level.  In this thesis, the permeability o f the B B B was  It would be interesting to see i f there is a difference in  permeability in specific brain regions, in particular, regions of the brain more severely affected in A D and i f the decrease in B B B permeability coincides with the decrease in A p burden.  Some studies have shown the occurrence o f micro-hemorrhages in the brain  after immunization. However, these micro-hemorrhages may have been due to the type of anti-Ap antibody used as an immunogen. It is possible that immunization could result in small hemorrhages in specific areas o f the brain but an overall restoration o f B B B integrity.  This needs to be investigated more thoroughly. Dissecting the brains o f  immunized mice into various regions and performing the Evans blue assay is a quantitative way of assessing regional permeability.  Alternatively, qualitative analysis  using sections from Evans blue perfused animals can be done since Evans blue fluoresces under specific light wavelengths.  Recently, Ujiie, et al. developed a new technique for  assessing B B B permeability using succinimidyl ester o f carboxyfluorescein diacetate  1 4 9  .  Perfusing immunized mice with succinimidyl ester o f carboxyfluorescein diacetate is another way to assess regional permeability. Moreover, it w i l l allow one to determine i f  136  the B B B is more permeable in plaque laden regions. Finally, it would be interesting to examine which cells in the brain are responsible for the damage and re-establishment on the B B B . Overall, there is an important  link between  microglial activation, plaque  formation and degradation and the progression of other pathological hallmarks, which needs to be clearly elucidated.  Determining the mechanism o f A p clearance in the  normal brain and upon A p immunization is required in order to facilitate the design o f specific treatment regimens, allowing exclusive targeting o f plaques without inducing detrimental side effects.  Such experiments are difficult since transgenic mice have less  genetic variability than humans, and their plaques have a different chemical composition, making them far more soluble and easier to remove. Furthermore, there is no transgenic mouse to date that can clearly mimic all the hallmarks of A D . immunization regimes, the consequences  In regards to  are different between human and mouse.  Vaccination of transgenic mice removes human A p while leaving endogenous mouse A p intact, whereas in humans the immune response is directed against an endogenous target that occurs naturally and plays an essential role in maintaining healthy brain tissue.  In  conclusion the results from this thesis shed significant new light on activated microglial gene expression in A D and also on the role o f microglia in plaque development and clearance possibly due to alterations in B B B integrity.  137  Appendix I: D o m a i n  S t r u c t u r e of A P P  signal peptide Cys-och  KPl domain  W|  1  |  acidic domain  l i f e  CuBDZnBD  • Thr-rich  -secretase  • OX-2  Schematic representation of the domain structure of APP. Full length A P P contains a signal peptide at the N-terminal end; a cysteine residue-rich region (Cys-rich) with copper binding ( C u B D ) and zinc binding sites ( Z n B D ) ; an acidic domain; a threonine residue-rich domain; Kinitz protease inhibitor domain (KPI); and an O X - 2 homology domain ( O X 2 ) . The A p fragment is flanked by the P- and y-secretase cleavage sites and contains the a-sectretase cleavage site.  138  Appendix II: R e g i o n a l  d i a g r a m of t h e brain  Parietal Lobe  Frontal Lobe  Occipital Lobe  Hippodampus  CA1 Region  Dentate G y r u s  Region  References 1.  2. 3. 4.  5. 6. 7. 8. 9. 10. 11.  12. 13. 14.  15. 16. 17.  18.  Turner, P. R., O'Connor, K . , Tate, W . P. & Abraham, W . C . Roles o f amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory. Prog Neurobiol 70, 1 -32 (2003). Verbeek, M . M . , Ruiter, D . J. & de Waal, R. M . The role o f amyloid in the pathogenesis o f Alzheimer's disease. Biol Chem 378, 937-50 (1997). Selkoe, D . J. & Schenk, D . Alzheimer's disease: molecular understanding predicts amyloid-based therapeutics. Annu Rev Pharmacol Toxicol 43, 545-84 (2003). Small, D . H . The role o f the amyloid protein precursor ( A P P ) in Alzheimer's disease: does the normal function o f A P P explain the topography o f neurodegeneration? Neurochem Res 23, 795-806 (1998). Price, D . L., Tanzi, R. E . , Borchelt, D . R. & Sisodia, S. S. Alzheimer's disease: genetic studies and transgenic models. Annu Rev Genet 32, 461-93 (1998). Nunan, J. & Small, D . H . Regulation o f A P P cleavage by alpha-, beta- and . gamma-secretases. FEBS Lett 483, 6-10 (2000). Allsop, D . , Landon, M . & K i d d , M . The isolation and amino acid composition o f senile plaque core protein. Brain Res 259, 348-52 (1983). Hardy, J. A . & Higgins, G . A . Alzheimer's disease: the amyloid cascade hypothesis. Science 256, 184-5 (1992). Tanzi, R. E . A genetic dichotomy model for the inheritance of Alzheimer's disease and common age-related disorders. J Clin Invest 104, 1175-9 (1999). Tanzi, R. E . & Bertram, L . N e w frontiers in Alzheimer's disease genetics. Neuron 32, 181-4 (2001). Wisniewski, K . E . , Wisniewski, H . M . & Wen, G . Y . Occurrence o f neuropathological changes and dementia o f Alzheimer's disease in Down's syndrome. Ann Neurol 17, 278-82 (1985). Rumble, B . et al. A m y l o i d A 4 protein and its precursor in Down's syndrome and Alzheimer's disease. NEnglJMed320, 1446-52 (1989). Tanzi, R. E . Neuropathology in the Down's syndrome brain. Nat Med 2, 31 -2 (1996). Suzuki, N . et al. A n increased percentage o f long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants. Science 264, 1336-40(1994). Cai, X . D . , Golde, T. E . & Younkin, S. G . Release o f excess amyloid beta protein from a mutant amyloid beta protein precursor. Science 259, 514-6 (1993). Selkoe, D . J. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 81, 741-66(2001). Wolfe, M . S. et al. Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398, 513-7 (1999). Luo, W . J. et al. P E N - 2 and A P H - 1 coordinately regulate proteolytic processing of presenilin \ .JBiol Chem 278, 7850-4 (2003).  140  19.  Gu, Y. et al. APH-1 interacts with mature and immature forms of presenilins and nicastrin and may play a role in maturation of presenilin.nicastrin complexes. J Biol Chem 278, 7374-80 (2003).  20. 21. 22. 23.  Kimberly, W. T. et al. Gamma-secretase is a membrane protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2. Proc Natl Acad Sci U S A 100, 6382-7 (2003). Selkoe, D. J. The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer's disease. Trends Cell Biol 8, 447-53 (1998). Russo, C. et al. Presenilin-1 mutations in Alzheimer's disease. Nature 405, 531-2 (2000). Strittmatter, W. J. et al. 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 USA 90, 1977-81 (1993).  24. 25.  Mahley, R- W. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 240, 622-30 (1988). LaDu, M. J. et al. Isoform-specific binding of apolipoprotein E to beta-amyloid. J Biol Chem 269, 23403-6 (1994).  26. 27. 28. 29. 30. 31. 32. 33. 34.  Akiyama, H. et al. Inflammation and Alzheimer's disease. Neurobiol Aging 21, 383-421 (2000). Selkoe, D. J. Clearing the brain's amyloid cobwebs. Neuron 32, 177-80 (2001). McGeer, P. L. & McGeer, E. G. Inflammation, autotoxicity and Alzheimer disease. Neurobiol Aging 22, 799-809 (2001). Yoshikai, S., Sasaki, H., Doh-ura, K., Furuya, H. & Sakaki, Y. Genomic organization of the human amyloid beta-protein precursor gene. Gene 87, 257-63 (1990). Tanaka, S. et al. 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, 1406-14 (1989). Storey, E., Beyreuther, K. & Masters, C. L. Alzheimer's disease amyloid precursor protein on the surface of cortical neurons in primary culture co-localizes with adhesion patch components. Brain Res 735, 217-31 (1996). Annaert, W. G. et al. Interaction with telencephalin and the amyloid precursor protein predicts a ring structure for presenilins. Neuron 32, 579-89 (2001). Allinson, T. M., Parkin, E. T., Turner, A. J. & Hooper, N. M. ADAMs family members as amyloid precursor protein alpha-secretases. J Neurosci Res 74, 34252 (2003). Ling, Y., Morgan, K. & Kalsheker, N. Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer's disease. Int J Biochem Cell Biol 35, 1505-35 (2003).  35. 36. 37.  Vassar, R. et al. Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286, 735-41 (1999). Cai, H. et al. BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci 4, 233-4 (2001). Acquati, F. et al. 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 (2000).  141  38. 39.  40. 41. 42.  43.  44.  45.  46. 47. 48.  49.  50.  51.  52.  53.  Duff, K. et al. Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1. Nature 383, 710-3 (1996). LaVoie, M . J. et al. Assembly of the gamma-secretase complex involves early formation of an intermediate subcomplex of Aph-1 and nicastrin. J Biol Chem 278, 37213-22 (2003). Roher, A. E. et al. Oligomerizaiton and fibril assembly of the amyloid-beta protein. Biochim Biophys Acta 1502, 31-43 (2000). Small, D. H. & McLean, C. A. Alzheimer's disease and the amyloid beta protein: What is the role of amyloid? J Neurochem 73, 443-9 (1999). Braak, H. & Braak, E. Development of Alzheimer-related neurofibrillary changes in the neocortex inversely recapitulates cortical myelogenesis. Acta Neuropathol (Berl) 92, 197-201 (1996). Gabuzda, D., Busciglio, J., Chen, L. B., Matsudaira, P. & Yankner, B. A. Inhibition of energy metabolism alters the processing of amyloid precursor protein and induces a potentially amyloidogenic derivative. J Biol Chem 269, 13623-8 (1994). Mattson, M . P. & Pedersen, W. A. Effects of amyloid precursor protein derivatives and oxidative stress on basal forebrain cholinergic systems in Alzheimer's disease. Int J Dev Neurosci 16, 737-53 (1998). Atwood, C. S. et al. Amyloid-beta: a chameleon walking in two worlds: a review of the trophic and toxic properties of amyloid-beta. Brain Res Brain Res Rev 43, 1-16 (2003). Cherny, R. A. et al. Aqueous dissolution of Alzheimer's disease Abeta amyloid deposits by biometal depletion. J Biol Chem 214, 23223-8 (1999). Querfurth, H. W. & Selkoe, D. J. Calcium ionophore increases amyloid beta peptide production by cultured cells. Biochemistry 33, 4550-61 (1994). Ueda, K., Shinohara, S., Yagami, T., Asakura, K. & Kawasaki, K. Amyloid beta protein potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels: a possible involvement of free radicals. J Neurochem 68, 265-71 (1997). Pierrot, N . , Ghisdal, P., Caumont, A. S. & Octave, J. N. Intraneuronal amyloidbeta 1-42 production triggered by sustained increase of cytosolic calcium concentration induces neuronal death. J Neurochem 88,1140-50 (2004). Wertkin, A. M . et al. Human neurons derived from a teratocarcinoma cell line express solely the 695-amino acid amyloid precursor protein and produce intracellular beta-amyloid or A4 peptides. Proc Natl Acad Sci USA 90, 9513-7 (1993). Takahashi, R. H. et al. Intraneuronal Alzheimer abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology. Am J Pathol 161, 1869-79 (2002). Echeverria, V. et al. Rat transgenic models with a phenotype of intracellular Abeta accumulation in hippocampus and cortex. JAlzheimers Dis 6, 209-19 (2004). Takahashi, R. H. et al. Oligomerization of Alzheimer's beta-amyloid within processes and synapses of cultured neurons and brain. J Neurosci 24, 3592-9 (2004).  142  54. 55. 56. 57.  58.  59.  60.  61.  62.  63.  64.  65.  66.  67. 68. 69. 70.  Dickson, D . W . Apoptotic mechanisms in Alzheimer neurofibrillary degeneration: cause or effect? J Clin Invest 114, 23-7 (2004). Schultz, D . R. & Harrington, W . J., Jr. Apoptosis: programmed cell death at a molecular level. Semin Arthritis Rheum 32, 345-69 (2003). Lustbader, J. W . et al. A B A D directly links Abeta to mitochondrial toxicity in Alzheimer's disease. Science 304, 448-52 (2004). McDonald, D . R., Bamberger, M . E . , Combs, C . K . & Landreth, G . E . betaA m y l o i d fibrils activate parallel mitogen-activated protein kinase pathways in microglia and THP1 monocytes. J Neurosci 18, 4451-60 (1998). McDonald, D . R., Brunden, K . R. & Landreth, G . E . A m y l o i d fibrils activate tyrosine kinase-dependent signaling and superoxide production in microglia. J Neurosci 17, 2284-94 (1997). Verdier, Y . , Zarandi, M . & Penke, B . A m y l o i d beta-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer's disease. J'Pept Sci 10, 229-48 (2004). Paresce, D . M . , Ghosh, R. N . & Maxfield, F. R. Microglial cells internalize aggregates o f the Alzheimer's disease amyloid beta-protein via a scavenger receptor. Neuron 17, 553-65 (1996). Husemann, j . , Loike, J. D . , Anankov, R., Febbraio, M . & Silverstein, S. C . Scavenger receptors in neurobiology and neuropathology: their role on microglia and other cells o f the nervous system. Glia 40, 195-205 (2002). Grewal, R. P., Yoshida, T., Finch, C . E . & Morgan, T. E . Scavenger receptor m R N A s in rat brain microglia are induced by kainic acid lesioning and by cytokines. Neuroreport 8, 1077-81 (1997). Bamberger, M . E., Harris, M . E . , McDonald,"D. R., Husemann, J. & Landreth, G . E . A cell surface receptor complex for fibrillar beta-amyloid mediates microglial activation. J Neurosci 23, 2665-74 (2003). Christie, R. H . , Freeman, M . & Hyman, B . T. Expression of the macrophage scavenger receptor, a multifunctional lipoprotein receptor, in microglia associated with senile plaques in Alzheimer's disease. Am J Pathol 148, 399-403 (1996). C u i , Y . , Le, Y . , Yazawa, H . , Gong, W . & Wang, J. M . Potential role o f the formyl peptide receptor-like 1 ( F P R L 1 ) in inflammatory aspects o f Alzheimer's disease. J Leukoc Biol 72, 628-35 (2002). Yazawa, H . et al. Beta amyloid peptide (Abeta42) is internalized via the G .protein-coupled receptor F P R L 1 and forms fibrillar aggregates in macrophages. Faseb J15, 2454-62 (2001). Y a n , S. D . et al. Receptor-dependent cell stress and amyloid accumulation in systemic amyloidosis. Nat Med 6, 643-51 (2000). Y a n , S. D . et al. R A G E and amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature 382, 685-91 (1996). Deane, R. et al. R A G E mediates amyloid-beta peptide transport across the bloodbrain barrier and accumulation in brain. Nat Med 9, 907-13 (2003). Shibata, M . et al. Clearance of Alzheimer's amyloid-ss(l-40) peptide from brain by L D L receptor-related protein-1 at the blood-brain barrier. J Clin Invest 106, 1489-99(2000).  143  71. 72.  73.  74. 75. 76. 77. 78.  79. 80.  81. 82. 83.  84.  85. 86.  87. 88.  Zlokovic, B . V . Clearing amyloid through the blood-brain barrier. J Neurochem 89,807-11 (2004). Kang, D . E . et al. Modulation o f amyloid beta-protein clearance and Alzheimer's disease susceptibility by the L D L receptor-related protein pathway. J Clin Invest 106, 1159-66 (2000). V a n Uden, E . et al. Increased extracellular amyloid deposition and neurodegeneration in human amyloid precursor protein transgenic mice deficient in receptor-associated protein. J Neurosci 22, 9298-304 (2002). Marx, J. Major setback for Alzheimer's models. Science 255, 1200-2 (1992). Quon, D . et al. Formation o f beta-amyloid protein deposits in brains o f transgenic mice. Nature 352, 239-41 (1991). Andra, K . et al. Expression of A P P in transgenic mice: a comparison of neuronspecific promoters. Neurobiol Aging 17, 183-90 (1996). Borchelt, D . R. et al. A vector for expressing foreign genes in the brains and hearts o f transgenic mice. Genet Anal 13, 159-63 (1996). Kulnane, L . S. & Lamb, B . T. Neuropathological characterization o f mutant amyloid precursor protein yeast artificial chromosome transgenic mice. Neurobiol Dis 8, 982-92 (2001). Games, D . et al. Alzheimer-type neuropathology in transgenic mice overexpressing V 7 1 7 F beta-amyloid precursor protein. Nature 373, 523-7 (1995). Irizarry, M . C. et al. Abeta deposition is associated with neuropil changes, but not with overt neuronal loss in the human amyloid precursor protein V 7 1 7 F ( P D A P P ) transgenic mouse. J Neurosci 17, 7053-9 (1997). Dodart, J. C. et al. Behavioral disturbances in transgenic mice overexpressing the V717F beta-amyloid precursor protein. Behav Neurosci 113, 982-90 (1999). Chen, G . et al. A learning deficit related to age and beta-amyloid plaques in a mouse model o f Alzheimer's disease. Nature 408, 975-9 (2000). Irizarry, M . C , McNamara, M . , Fedorchak, K . , Hsiao, K . & Hyman, B . T. A P P S w transgenic mice develop age-related A beta deposits and neuropil abnormalities, but no neuronal loss in C A 1 . JNeuropathol Exp Neurol 56, 965-73 (1997). Hsiao, K . K . et al. Age-related C N S disorder and early death in transgenic F V B / N mice overexpressing Alzheimer amyloid precursor proteins. Neuron 15, 1203-18 (1995). Hsiao, K . et al. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 274, 99-102 (1996). M c G o w a n , E . et al. A m y l o i d phenotype characterization o f transgenic mice overexpressing both mutant amyloid precursor protein and mutant presenilin 1 transgenes. Neurobiol Dis 6, 231-44 (1999). Chapman, P. F. et al. Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice. Nat Neurosci 2, 271-6 (1999). Schauwecker, P. E . & Steward, O. Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches. Proc Natl Acad Sci USA 94, 4103-8 (1997).  144  89.  90. 91. 92. 93.  94.  95.  96.  97.  98.  99. 100. 101. 102.  103. 104. 105. 106.  Sturchler-Pierrat, C . et al. Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. Proc Natl Acad Sci USA 94, 13287-92(1997). Stalder, M . et al. Association o f microglia with amyloid plaques in brains o f A P P 2 3 transgenic mice. Am J Pathol 154,1673-84 (1999). Kelly, P. H . et al. Progressive age-related impairment of cognitive behavior in A P P 2 3 transgenic mice. Neurobiol Aging 24, 365-78 (2003). Calhoun, M . E . et al. Neuron loss in A P P transgenic mice. Nature 395, 755-6 (1998) . Calhoun, M . E . et al. Neuronal overexpression o f mutant amyloid precursor protein results in prominent deposition o f cerebrovascular amyloid. Proc Natl Acad Sci USA 96, 14088-93 (1999). Holcomb, L . et al. Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nat Med 4, 97-100(1998). Carlson, G . A . et al. Genetic modification o f the phenotypes produced by amyloid precursor protein overexpression in transgenic mice. Hum Mol Genet 6, 1951-9 (1997). Moechars, D . , Lorent, K , D e Strooper, B . , Dewachter, I. & Van Leuven, F. Expression in brain of amyloid precursor protein mutated in the alpha-secretase site causes disturbed behavior, neuronal degeneration and premature death in transgenic mice. Embo J15, 1265-74.(1996). Moechars, D . , Gilis, M . , Kuiperi, C , Laenen, I. & V a n Leuven, F. Aggressive behaviour in transgenic mice expressing A P P is alleviated by serotonergic drugs. Neuroreport 9,3561-4 (1998). Moechars, D . et al. Early phenotypic changes in transgenic mice that overexpress different mutants o f amyloid precursor protein in brain. J Biol Chem 274, 6483-92 (1999) . Janus, C , Chishti, M . A . & Westaway, D . Transgenic mouse models o f Alzheimer's disease. Biochim Biophys Acta 1502, 63-75 (2000). Janus, C . & Westaway, D . Transgenic mouse models o f Alzheimer's disease. Physiol Behav 73, 873-86 (2001). Gotz, J. et al. Transgenic animal models of Alzheimer's disease and related disorders: histopathology, behavior and therapy. Mol Psychiatry (2004). McGeer, P. L . , Schulzer, M . & McGeer, E . G . Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer's disease: a review o f 17 epidemiologic studies. Neurology 47, 425-32 (1996). Stewart, W . F., Kawas, C , Corrada, M . & Metter, E . J. Risk o f Alzheimer's disease and duration o f N S A I D use. Neurology 48, 626-32 (1997). Streit, W . J., Walter, S. A . & Pennell, N . A . Reactive microgliosis. Prog Neurobiol 57, 563-81 (1999). Nelson, P. T., Soma, L. A . & Lavi, E . Microglia in diseases o f the central nervous system. Ann Med 34, 491-500 (2002). Perry, V . H . & Gordon, S. Macrophages and the nervous system. Int Rev Cytol 125, 203-44(1991).  145  107. 108.  109. 110.  111. 112. 113.  114.  115.  116. 117.  118. 119.  120. 121.  122.  123.  Kaur, C , Hao, A . J., W u , C . H . & Ling, E . A . Origin o f microglia. Microsc Res Tech 54, 2-9 (2001). Streit, W . J. & Graeber, M . B . Heterogeneity o f microglial and perivascular cell populations: insights gained from the facial nucleus paradigm. Glia 7, 68-74 (1993). Streit, W . J. Microglia as neuroprotective, immunocompetent cells o f the C N S . Glia 40, 133-9 (2002). Jefferies, W . A . et al. Reactive microglia specifically associated with amyloid plaques in Alzheimer's disease brain tissue express melanotransferrin. Brain Res 712,122-6(1996). Yamada, T. et al. Melanotransferrin is produced by senile plaque-associated reactive microglia in Alzheimer's disease. Brain Res 845, 1-5 (1999). Schipper, H . M . et al. Evaluation o f heme oxygenase-1 as a systemic biological marker o f sporadic A D . Neurology 54, 1297-304 (2000). Walker, D . G . , Lue, L . F. & Beach, T. G . Increased expression o f the urokinase plasminogen-activator receptor in amyloid beta peptide-treated human brain microglia and in A D brains. Brain Res 926, 69-79 (2002). Espey, M . G . , Chernyshev, O. N . , Reinhard, J. F., Jr., Namboodiri, M . A . & Colton, C . A . Activated human microglia produce the excitotoxin quinolinic acid. Neuroreport 8, 431-4 (1997). Piani, D . , Spranger, M . , Frei, K . , Schaffner, A . & Fontana, A . Macrophageinduced cytotoxicity of N-methyl-D-aspartate receptor positive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines. Eur J Immunol 22, 2429-36 (1992). McGeer, P. L., Akiyama, H . , Itagaki, S. & McGeer, E . G . Immune system response in Alzheimer's disease. Can J Neurol Sci 16, 516-27 (1989). Styren, S. D . , C i v i n , W . H . & Rogers, J. Molecular, cellular, and pathologic characterization o f H L A - D R immunoreactivity in normal elderly and Alzheimer's disease brain. Exp Neurol 110, 93-104 (1990). Frautschy, S. A . et al. Microglial response to amyloid plaques in A P P s w transgenic mice. Am J Pathol 152, 307-17 (1998). Itagaki, S., McGeer, P. L , Akiyama, H . , Zhu, S. & Selkoe, D . Relationship o f microglia and astrocytes to amyloid deposits o f Alzheimer disease. J Neuroimmunol 24, 173-82(1989). Mackenzie, I. R., Hao, C . & Munoz, D . G . Role o f microglia in senile plaque formation. Neurobiol Aging 16, 797-804 (1995). Sasaki, A . , Yamaguchi, H . , Ogawa, A . , Sugihara, S. & Nakazato, Y . Microglial activation in early stages o f amyloid beta protein deposition. Acta Neuropathol (Berl) 94, 316-22 (1997). A r d , M . D . , Cole, G . M . , W e i , J., Mehrle, A . P. & Fratkin, J. D . Scavenging o f Alzheimer's amyloid beta-protein by microglia in culture. J Neurosci Res 43, 190202(1996). Paresce, D . M . , Chung, H . & Maxfield, F. R. Slow degradation o f aggregates o f the Alzheimer's disease amyloid beta-protein by microglial cells. J Biol Chem 272, 29390-7 (1997).  146  124.  125.  126. 127.  128.  129.  130. 131. 132. 133.  134. 135. 136. 137. 138. 139.  140. 141. 142.  Frackowiak, J. et al. Ultrastructure o f the microglia that phagocytose amyloid and the microglia that produce beta-amyloid fibrils. Acta Neuropathol (Bed) 84, 22533 (1992). Akiyama, Ff. et al. Granules in glial cells o f patients with Alzheimer's disease are immunopositive for C-terminal sequences o f beta-amyloid protein. Neurosci Lett 206, 169-72(1996). Rogers, J. et al. Elucidating molecular mechanisms o f Alzheimer's disease in microglial cultures. Ernst Schering Res Found Workshop, 25-44 (2002). Chung, H . , Brazil, M . I., Soe, T. T. & Maxfield, F. R. Uptake, degradation, and release o f fibrillar and soluble forms of Alzheimer's amyloid beta-peptide by microglial cells. J Biol Chem 274, 32301-8 (1999). Combs, C . K , Johnson, D . E . , Cannady, S. B . , Lehman, T. M . & Landreth, G . E . Identification o f microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins. J Neurosci 19, 928-39 (1999). Savage, M . J., L i n , Y . G . , Ciallella, J. R., Flood, D . G . & Scott, R. W . Activation of c-Jun N-terminal kinase and p38 in an Alzheimer's disease model is associated with amyloid deposition. J Neurosci 22, 3376-85 (2002). Koistinaho, M . & Koistinaho, J. Role of p38 and p44/42 mitogen-activated protein kinases in microglia. Glia 40, 175-83 (2002). Hensley, K . et al. p38 kinase is activated in the Alzheimer's disease brain. J Neurochem 72, 2053-8 (1999). X i e , Z., Smith, C . J. & V a n Eldik, L . J. Activated glia induce neuron death via M A P kinase signaling pathways involving J1MK and p38. Glia 45, 170-9 (2004). Pyo, H . , Jou, I., Jung, S., Hong, S. & Joe, E . H . Mitogen-activated protein kinases activated by lipopolysaccharide and beta-amyloid in cultured rat microglia. Neuroreport 9, 871-4 (1998). Ballabh, P., Braun, A . & Nedergaard, M . The blood-brain barrier: an overview: structure, regulation, and clinical implications. Neurobiol Dis 16, 1-13 (2004). Hirase, T. et al. Occludin as a possible determinant o f tight junction permeability in endothelial cells. J Cell Sci 110 ( Pt 14), 1603-13 (1997). Prat, A . , Biernacki, K . , Wosik, K . & Antel, J. P. Glial cell influence on the human blood-brain barrier. Glia 36, 145-55 (2001). Janzer, R. C . & Raff, M . C . Astrocytes induce blood-brain barrier properties in endothelial cells. Nature 325, 253-7 (1987). Ramsauer, M . , Krause, D . & Dermietzel, R. Angiogenesis o f the blood-brain barrier in vitro and the function o f cerebral pericytes. Faseb J16, 1274-6 (2002). Kern, T. S. & Engerman, R. L . Capillary lesions develop in retina rather than cerebral cortex in diabetes and experimental galactosemia. Arch Ophthalmol 114, 306-10(1996). Lindahl, P., Johansson, B . R., Leveen, P. & Betsholtz, C . Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 211, 242-5 (1997). Saunders, N . R., Habgood, M . D . & Dziegielewska, K . M . Barrier mechanisms in the brain, I. Adult brain. Clin Exp Pharmacol Physiol 26, 11-9 (1999). Kastin, A . J., Pan, W . , Maness, L . M . & Banks, W . A . Peptides crossing the blood-brain barrier: some unusual observations. Brain Res 848, 96-100 (1999).  147  143. 144. 145. 146. 147.  148. 149.  Jolliet-Riant, P. & Tillement, J. P. Drug transfer across the blood-brain barrier and improvement of brain delivery. Fundam Clin Pharmacol 13, 16-26 (1999). Rothenberger, S. et al. Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium. Brain Res 712, 117-21 (1996). Claudio, L. Ultrastructural features of the blood-brain barrier in biopsy tissue from Alzheimer's disease patients. Acta Neuropathol (Bed) 91, 6-14 (1996). Caserta, M. T., Caccioppo, D., Lapin, G. D., Ragin, A. & Groothuis, D. R. Bloodbrain barrier integrity in Alzheimer's disease patients and elderly control subjects. J Neuropsychiatry Clin Neurosci 10, 78-84 (1998). Poduslo, J. F., Curran, G. L., Wengenack, T. M., Malester, B. & Duff, K. Permeability of proteins at the blood-brain barrier in the normal adult mouse and double transgenic mouse model of Alzheimer's disease. Neurobiol Dis 8, 555-67 (2001). Perry, V. H., Anthony, D. C , Bolton, S. J. & Brown, H. C. The blood-brain barrier and the inflammatory response. Mol Med Today 3, 335-41 (1997). Ujiie, M., Dickstein, D. L., Carlow, D. A. & Jefferies, W. A. Blood-brain barrier permeability precedes senile plaque formation in an Alzheimer disease model. Microcirculation 10, 463-70 (2003).  150. 151. 152. 153. 154.  Xu, J. et al. Amyloid beta peptide-induced cerebral endothelial cell death involves mitochondrial dysfunction and caspase activation. J Cereb Blood Flow Metab 21, 702-10(2001). Yin, K. J., Lee, J. M., Chen, S. D., Xu, J. & Hsu, C. Y. Amyloid-beta induces Smac release via AP-l/Bim activation in cerebral endothelial cells. J Neurosci 22, 9764-70 (2002). Vagnucci, A. H., Jr. & Li, W. W. Alzheimer's disease and angiogenesis. Lancet 361, 605-8 (2003). Wong, M. L. et al. Inducible nitric oxide synthase gene expression in the brain during systemic inflammation. Nat Med 2, 581-4 (1996). Ek, M. et al. Inflammatory response: pathway across the blood-brain barrier. Nature 4W, 430-1 (2001).  155. 156. 157. 158. 159.  Uchikado, H. et al. Activation of vascular endothelial cells and perivascular cells by systemic inflammation-an immunohistochemical study of postmortem human brain tissues. Acta Neuropathol (Berl) 107, 341-51 (2004). Attems, J., Lintner, F. & Jellinger, K. A. Amyloid beta peptide 1-42 highly correlates with capillary cerebral amyloid angiopathy and Alzheimer disease pathology. Acta Neuropathol (Berl) 107, 283-91 (2004). Weller, R. O., Massey, A., Kuo, Y. M. & Roher, A. E. Cerebral amyloid angiopathy: accumulation of A beta in interstitial fluid drainage pathways in Alzheimer's disease. Ann N YAcad Sci 903, 110-7 (2000). Vinters, H. V. Cerebral amyloid angiopathy. A critical review. Stroke 18, 311-24 (1987). Bergeron, C , Ranalli, P. J. & Miceli, P. N. Amyloid angiopathy in Alzheimer's disease. Can J Neurol Sci 14, 564-9 (1987).  148  160.  161.  162. 163.  164. 165.  166.  167. 168. 169.  170.  171.  172.  173.  174. 175. 176.  Prelli, F., Castano, E . , Glenner, G . G . & Frangione, B . Differences between vascular and plaque core amyloid in Alzheimer's disease. J Neurochem 51, 64851 (1988). Plassman, B . L. & Breitner, J. C . Recent advances in the genetics o f Alzheimer's disease and vascular dementia with an emphasis on gene-environment interactions. J Am Geriatr Soc 44, 1242-50 (1996). Munch, G . & Robinson, S. R. Potential neurotoxic inflammatory responses to Abeta vaccination in humans. J Neural Transm 109, 1081-7 (2002). N i c o l l , J. A . et al. Neuropathology o f human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med 9, 448-52 (2003). Golde, T. E . Alzheimer disease therapy: can the amyloid cascade be halted? J Clin Invest 111, 11-8 (2003). Luo, Y . et al. Mice deficient in B A C E 1 , the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci 4, 231-2 (2001). Wolozin, B . , Kellman, W . , Ruosseau, P., Celesia, G . G . & Siegel, G . Decreased prevalence o f Alzheimer disease associated with 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors. Arch Neurol 57, 1439-43 (2000). Notkola, 1. L . et al. Serum total cholesterol, apolipoprotein E epsilon 4 allele, and Alzheimer's disease. Neuroepidemiology 17, 14-20 (1998). Simons, M . et al. Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons. Proc Natl Acad Sci U S A 95, 6460-4 (1998). Fassbender, K . et al. Simvastatin strongly reduces levels o f Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo. Proc Natl Acad Sci US A 98, 5856-61 (2001). Shie, F. S., Jin, L . W . , Cook, D . G . , Leverenz, J. B . & LeBoeuf, R. C . Dietinduced hypercholesterolemia enhances brain A beta accumulation in transgenic mice. Neuroreport 13, 455-9 (2002). George, A . J. et al. A P P intracellular domain is increased and soluble Abeta is reduced with diet-induced hypercholesterolemia in a transgenic mouse model o f Alzheimer disease. Neurobiol Dis 16, 124-32 (2004). Refolo, L . M . et al. A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model o f Alzheimer's disease. Neurobiol Dis 8, 890-9 (2001). Friedhoff, L . T., Cullen, E . I., Geoghagen, N . S. & Buxbaum, J. D . Treatment with controlled-release lovastatin decreases serum concentrations o f human betaamyloid ( A beta) peptide. Int J Neuropsychopharmacol 4, 127-30 (2001). Weggen, S. et al. A subset of N S A I D s lower amyloidogenic Abeta42 independently o f cyclooxygenase activity. Nature 414, 212-6 (2001). Schenk, D . et al. Immunization with amyloid-beta attenuates Alzheimer-diseaselike pathology in the P D A P P mouse. Nature 400, 173-7 (1999). Bard, F. et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model o f Alzheimer disease. Nat Med 6, 916-9 (2000).  149  177.  178. 179. 180.  181.  182. 183.  184.  185. 186. 187.  188. 189.  190.  191. 192. 193.  DeMattos, R. B . et al. Peripheral anti-A beta antibody alters C N S and plasma A beta clearance and decreases brain A beta burden in a mouse model o f Alzheimer's disease. Proc Natl Acad Sci USA 98, 8850-5 (2001). Morgan, D . et al. A beta peptide vaccination prevents memory loss in an animal model o f Alzheimer's disease. Nature 408, 982-5 (2000). Janus, C . et al. A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease. Nature 408, 979-82 (2000). Lemere, C . A . et al. Evidence for peripheral clearance o f cerebral Abeta protein following chronic, active Abeta immunization in P S A P P mice. Neurobiol Dis 14, 10-8 (2003). Oddo, S., Billings, L . , Kesslak, J. P., Cribbs, D . H . & LaFerla, F. M . Abeta Immunotherapy Leads to Clearance o f Early, but Not Late, Hyperphosphorylated Tau Aggregates via the Proteasome. Neuron 43, 321-32 (2004). Yoshida, H . et al. The murine mutation osteopetrosis is in the coding region o f the macrophage colony stimulating factor gene. Nature 345, 442-4 (1990). Wiktor-Jedrzejczak, W . et al. Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. Proc Natl Acad Sci USA 87, 4828-32 (1990). Pollard, J. W . , Morgan, C . J., Dello Sbarba, P., Cheers, C . & Stanley, E . R . Independently arising macrophage mutants dissociate growth factor-regulated survival and proliferation. Proc Natl Acad Sci U S A 88, 1474-8 (1991). Stanley, E . R. et al. Biology and action o f colony—stimulating factor-1. Mol ReprodDev 46, 4-10 (1997). Blevins, G . & Fedoroff, S. Microglia in colony-stimulating factor 1-deficient op/op mice. J Neurosci Res 40, 535-44 (1995). Wegiel, J. et al. Reduced number and altered morphology o f microglial cells in colony stimulating factor-1-deficient osteopetrotic op/op mice. Brain Res 804, 135-9(1998). Marks, S. C , Jr. & Lane, P. W . Osteopetrosis, a new recessive skeletal mutation on chromosome 12 of the mouse. J Hered 67, 11-18 (1976). Blasi, E . , Barluzzi, R., Bocchini, V . , Mazzolla, R. & Bistoni, F. Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J Neuroimmunol 27, 229-37 (1990). L i , M . , Pisalyaput, K . , Galvan, M . & Tenner, A . J. Macrophage colony stimulatory factor and interferon-gamma trigger distinct mechanisms for augmentation o f beta-amyloid-induced microglia-mediated neurotoxicity. J Neurochem 91, 623-33 (2004). Hampel, H . et al. Decreased soluble interleukin-6 receptor in cerebrospinal fluid of patients with Alzheimer's disease. Brain Res 780, 356-9 (1998). Fillit, H . et al. Elevated circulating tumor necrosis factor levels in Alzheimer's disease. Neurosci Lett 129, 318-20 (1991). Brown, J. P., Woodbury, R. G . , Hart, C. E . , Hellstrom, I. & Hellstrom, K . E . Quantitative analysis o f melanoma-associated antigen p97 in normal and neoplastic tissues. Proc Natl Acad Sci USA 78, 539-43 (1981).  150  194.  195. 196.  197.  198.  199.  200.  201.  202. 203. 204. 205. 206. 207.  208.  209. 210. 211.  Woodbury, R. G . , Brown, J. P., Loop, S. M . , Hellstrom, K . E . & Hellstrom, I. Analysis o f normal neoplastic human tissues for the tumor-associated protein p97. Int J Cancer 27, 145-9(1981). Brown, J. P. et al. Human melanoma-associated antigen p97 is structurally and functionally related to transferrin. Nature 296, 171-3 (1982). Real, F. X . et al. Class 1 (unique) tumor antigens o f human melanoma: identification o f unique and common epitopes on a 90-kDa glycoprotein. Proc Natl Acad Sci USAS5, 3965-9 (1988). Sciot, R. et al. In situ localization o f melanotransferrin (melanoma-associated antigen P97) in human liver. A light- and electronmicroscopic immunohistochemical study. Liver 9, 110-9 (1989). Alemany, R. et al. Glycosyl phosphatidylinositol membrane anchoring of melanotransferrin (p97): apical compartmentalization in intestinal epithelial cells. J Cell Sci 104 ( Pt 4), 1155-62 (1993). Food, M . R. et al. Transport and expression in human melanomas o f a transferrinlike glycosylphosphatidylinositol-anchored protein. J Biol Chem 269, 3034-40 (1994). Kennard, M . L., Richardson, D . R., Gabathuler, R., Ponka, P. & Jefferies, W . A . A novel iron uptake mechanism mediated by GPI-anchored human p97. Embo J 14,4178-86(1995). McNagny, K . M . , Rossi, F., Smith, G . & Graf, T. The eosinophil-specific cell surface antigen, E O S 4 7 , is a chicken homologue o f the oncofetal antigen melanotransferrin. Blood 87, 1343-52 (1996). Plowman, G . D . et al. Assignment o f the gene for human melanoma-associated antigen p97 to chromosome 3. Nature 303, 70-2 (1983). Baker, E . N . et al: Human melanotransferrin (p97) has only one functional ironbinding site. FEBS Lett 298, 215-8 (1992). Jefferies, W . A . et al. Transferrin receptor on endothelium of brain capillaries. Nature 312, 162-3 (1984). Loeffler, D . A . et al. Transferrin and iron in normal, Alzheimer's disease, and Parkinson's disease brain regions. J Neurochem 65, 710-24 (1995). Grundke-Iqbal, I. et al. Ferritin is a component of the neuritic (senile) plaque in Alzheimer dementia. Acta Neuropathol (Berl) 81, 105-10 (1990). Kaneko, Y . , Kitamoto, T., Tateishi, J. & Yamaguchi, K . Ferritin immunohistochemistry as a marker for microglia. Acta Neuropathol (Berl) 79, 129-36(1989). Kennard, M . L., Feldman, H . , Yamada, T. & Jefferies, W . A . Serum levels o f the iron binding protein p97 are elevated in Alzheimer's disease. Nat Med 2, 1230-5 (1996). Feldman, H . et al. Serum p97 levels as an aid to identifying Alzheimer's disease. J Alzheimers Dis 3, 507-516 (2001). K i m , D . K . et al. Serum melanotransferrin, p97 as a biochemical marker o f Alzheimer's disease. Neuropsychopharmacology 25, 84-90 (2001). Qian, Z . M . & Wang, Q. Expression of iron transport proteins and excessive iron accumulation in the brain in neurodegenerative disorders. Brain Res Brain Res Rev 27, 257-67(1998).  151  212. 213.  214. 215.  216.  217.  218. 219.  220. 221.  222. 223. 224. 225.  226.  227.  228.  Dwork, A . J., Schon, E . A . & Herbert, j. Nonidentical distribution of transferrin and ferric iron in human brain. Neuroscience 27, 333-45 (1988). Sala, R. et al. The human melanoma associated protein melanotransferrin promotes endothelial cell migration and angiogenesis i n vivo. Eur J Cell Biol 81, 599-607 (2002). Kalaria, R. N . et al. Vascular endothelial growth factor in Alzheimer's disease and experimental cerebral ischemia. Brain Res Mol Brain Res 62, 101-5 (1998). Tarkowski, E . et al. Increased intrathecal levels o f the angiogenic factors V E G F and TGF-beta in Alzheimer's disease and vascular dementia. Neurobiol Aging 23, 237-43 (2002). Yang, S. P. et al. Co-accumulation o f vascular endothelial growth factor with beta-amyloid in the brain of patients with Alzheimer's disease. Neurobiol Aging 25, 283-90 (2004). Roze-Heusse, A . , Houbiguian, M . L., Debacker, C , Zakin, M . M . & Duchange, N . Melanotransferrin gene expression in melanoma cells is correlated with high levels o f Jun/Fos family transcripts and with the presence of a specific A P 1 dependent ternary complex. Biochem 7318 ( Pt 3), 883-8 (1996). McGeer, E . G . & McGeer, P. L . Inflammatory processes in Alzheimer's disease. Prog Neuropsychopharmacol Biol Psychiatry 27, 741-9 (2003). Walker, D . G . , Lue, L. F. & Beach, T. G . Gene expression profiling of amyloid beta peptide-stimulated human post-mortem brain microglia. Neurobiol Aging 22, 957-66 (2001). Nakamasu, K . et al. Structure and promoter analysis o f the mouse membranebound transferrin-like protein (MTf) gene. Eur J Biochem 268, 1468-76 (2001). Ogawa, O. et al. Inhibition o f inducible nitric oxide synthase gene expression by indomethacin or ibuprofen in beta-amyloid protein-stimulated J774 cells. Eur J Pharmacol 408, 137-41 (2000). Blasko, I. et al. Ibuprofen decreases cytokine-induced amyloid beta production in neuronal cells. Neurobiol Dis 8, 1094-101 (2001). Eriksen, J. L . et al. N S A I D s and enantiomers o f flurbiprofen target gammasecretase and lower Abeta 42 in vivo. J Clin Invest 112, 440-9 (2003). L i m , G . P. et al. Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer's disease. J Neurosci 20, 5709-14 (2000). Netland, E . E . , Newton, J. L., Majocha, R. E . & Tate, B . A . Indomethacin reverses the microglial response to amyloid beta-protein. Neurobiol Aging 19, 201-4(1998). Etminan, M . , G i l l , S. & Samii, A . Effect of non-steroidal anti-inflammatory drugs on risk o f Alzheimer's disease: systematic review and meta-analysis o f observational studies. Bmj 327, 128 (2003). Weggen, S. et al. Evidence that nonsteroidal anti-inflammatory drugs decrease amyloid beta 42 production by direct modulation of gamma-secretase activity. J Biol Chem 278, 31831-7 (2003). Matsuura, H . et al. Regulation o f cyclooxygenase-2 by interferon gamma and transforming growth factor alpha in normal human epidermal keratinocytes and squamous carcinoma cells. Role o f mitogen-activated protein kinases. J Biol Chem 274, 29138-48 (1999).  152  229: 230. 231. 232.  233. 234.  Wisniewski, H. M., Wegiel, J., Wang, K. C , Kujawa, M. & Lach, B. Ultrastructural studies of the cells forming amyloid fibers in classical plaques. Can J Neurol Sci 16, 535-42 (1989). Wisniewski, H. M., Barcikowska, M. & Kida, E. Phagocytosis of beta/A4 amyloid fibrils of the neuritic neocortical plaques. Acta Neuropathol (Berl) 81, 588-90(1991). Wegiel, J., Wang, K. C , Tarnawski, M. & Lach, B. Microglia cells are the driving force in fibrillar plaque formation, whereas astrocytes are a leading factor in plague degradation. Acta Neuropathol (Berl) 100, 356-64 (2000). Brazil, M. I., Chung, H. & Maxfield, F. R. Effects of incorporation of immunoglobulin G and complement component Clq on uptake and degradation of Alzheimer's disease amyloid fibrils by microglia. J Biol Chem 275, 16941-7 (2000). Webster, S. D. et al. Antibody-mediated phagocytosis of the amyloid beta-peptide in microglia is differentially modulated by Clq. J Immunol 166, 7496-503 (2001). Rogers, J. et al. Complement activation by beta-amyloid in Alzheimer disease. Proc Natl Acad Sci USA 89, 10016-20 (1992).  235. 236. 237. 238. 239. 240. 241. 242. 243. 244.  Bradt, B. M., Kolb, W. P. & Cooper, N. R. Complement-dependent proinflammatory properties of the Alzheimer's disease beta-peptide. J Exp Med 188,431-8 (1998). Wyss-Coray, T. et al. Alzheimer's disease-like cerebrovascular pathology in transforming growth factor-beta 1 transgenic mice and functional metabolic correlates. Ann N Y Acad Sci 903, 317-23 (2000). Wyss-Coray, T. et al. Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer's mice. Proc Natl Acad Sci USA 99, 10837-42 (2002). Fonseca, M. I., Zhou, J., Botto, M. & Tenner, A. J. Absence of C l q leads to less neuropathology in transgenic mouse models of Alzheimer's disease. J Neurosci 24, 6457-65 (2004). Wiktor-Jedrzejczak, W. et al. Correction by CSF-1 of defects in the osteopetrotic op/op mouse suggests local, developmental, and humoral requirements for this growth factor. Exp Hematol 19, 1049-54 (1991). Rogove, A. D., Lu, W. & Tsirka, S. E. Microglial activation and recruitment, but not proliferation, suffice to mediate neurodegeneration. Cell Death Differ 9, 801-6 (2002) . Wyss-Coray, T. et al. Amyloidogenic role of cytokine TGF-betal in transgenic mice and in Alzheimer's disease. Nature 389, 603-6 (1997). Lesne, S. et al. Transforming growth factor-beta 1 potentiates amyloid-beta generation in astrocytes and in transgenic mice. J Biol Chem 278, 18408-18 (2003) . Naito, M. et al. Abnormal differentiation of tissue macrophage populations in 'osteopetrosis' (op) mice defective in the production of macrophage colonystimulating factor. Am J Pathol 139, 657-67 (1991). Witmer-Pack, M. D. et al. Identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse. J Cell Sci 104 ( Pt 4), 1021-9 (1993).  153  245.  246.  247.  248.  249.  250.  251. 252.  253. 254. 255. 256. 257. 258.  259.  260.  261.  Raivich, G . , Moreno-Flores, M . T., Moller, J. C . & Kreutzberg, G . W . Inhibition of posttraumatic microglial proliferation in a genetic model of macrophage colony-stimulating factor deficiency in the mouse. Eur J Neurosci 6, 1615-8 (1994). Bruccoleri, A . & Harry, G . J. Chemical-induced hippocampal neurodegeneration and elevations in TNFalpha, TNFbeta, IL-1 alpha, IP-10, and M C P - 1 m R N A in osteopetrotic (op/op) mice. J Neurosci Res 62, 146-55 (2000). Mucke, L . et al. High-level neuronal expression o f abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation. J Neurosci 20, 4050-8 (2000). Lehman, E . J. et al. Genetic background regulates beta-amyloid precursor protein processing and beta-amyloid deposition in the mouse. Hum Mol Genet 12, 294956 (2003). Masliah, E . et al. Comparison of neurodegenerative pathology in transgenic mice overexpressing V 7 1 7 F beta-amyloid precursor protein and Alzheimer's disease. J Neurosci 16, 5795-811 (1996). Mohajeri, M . H . et al. Passive immunization against beta-amyloid peptide protects central nervous system ( C N S ) neurons from increased vulnerability associated with an Alzheimer's disease-causing mutation. J Biol Chem 211, 33012-7 (2002). Lemere, C . A . et al. Amyloid-beta immunization in Alzheimer's disease transgenic mouse models and wildtype mice. Neurochem Res 28, 1017-27 (2003). Das, P., Murphy, M . P., Younkin, L . H . , Younkin, S. G . & Golde, T. E . Reduced effectiveness o f Abetal-42 immunization in A P P transgenic mice with significant amyloid deposition. Neurobiol Aging 22, 721-7 (2001). Dodart, J. C . et al. Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model. Nat Neurosci 5, 452-7 (2002). Hock, C . et al. Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease. Neuron 38, 547-54 (2003). Orgogozo, J. M . et al. Subacute meningoencephalitis in a subset o f patients with A D after Abeta42 immunization. Neurology 61, 46-54 (2003). Check, E . Nerve inflammation halts trial for Alzheimer's drug. Nature 415, 462 (2002). Pfeifer, M . et al. Cerebral hemorrhage after passive anti-Abeta immunotherapy. Science 298, 1379 (2002). Poduslo, J. F. & Curran, G . L . A m y l o i d beta peptide as a vaccine for Alzheimer's disease involves receptor-mediated transport at the blood-brain barrier. Neuroreport 12, 3197-200 (2001). Bacskai, B . J. et al. Imaging of amyloid-beta deposits in brains o f living mice permits direct observation o f clearance o f plaques with immunotherapy. Nat Med 1, 369-72 (2001). Solomon, B . , Koppel, R., Hanan, E . & Katzav, T. Monoclonal antibodies inhibit in vitro fibrillar aggregation o f the Alzheimer beta-amyloid peptide. Proc Natl Acad Sci US An, 452-5 (1996). Solomon, B . , Koppel, R., Frankel, D . & Hanan-Aharon, E . Disaggregation o f Alzheimer beta-amyloid by site-directed m A b . Proc Natl Acad Sci USA 94, 4109-12 (1997).  154  262. 263.  264.  265.  266.  267. 268. 269.  270. 271.  272.  273.  274.  275.  276.  277.  Bacskai, B . J. et al. Non-Fc-mediated mechanisms are involved in clearance o f amyloid-beta in vivo by immunotherapy. J Neurosci 22, 7873-8 (2002). Wilcock, D . M . et al. Intracranially administered anti-Abeta antibodies reduce beta-amyloid deposition by mechanisms both independent o f and associated with microglial activation. J Neurosci 23, 3745-51 (2003). DeMattos, R. B . , Bales, K . R., Cummins, D . J., Paul, S. M . & Holtzman, D . M . Brain to plasma amyloid-beta efflux: a measure o f brain amyloid burden in a mouse model o f Alzheimer's disease. Science 295, 2264-7 (2002). DeMattos, R. B . et al. Plaque-associated disruption o f C S F and plasma amyloidbeta (Abeta) equilibrium in a mouse model of Alzheimer's disease. J Neurochem 81, 229-36 (2002). Pan, W . , Solomon, B . , Maness, L . M . & Kastin, A . J. Antibodies to beta-amyloid decrease the blood-to-brain transfer o f beta-amyloid peptide. Exp Biol Med (Maywood) 221, 609-15 (2002). Games, D . et al. Prevention and reduction o f AD-type pathology in P D A P P mice immunized with A beta 1-42. Ann N YAcad Sci 920, 274-84 (2000). Austyn, J. M . & Gordon, S. F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur J Immunol 11, 805-15 (1981). Uyama, O. et al. Quantitative evaluation o f vascular permeability in the gerbil brain after transient ischemia using Evans blue fluorescence. J Cereb Blood Flow Metab 8, 282-4 (1988). Methia, N . et al. A p o E deficiency compromises the blood brain barrier especially after injury. Mol Med 1, 810-5 (2001). Blanc, E . M . , Toborek, M . , Mark, R. J., Hennig, B . & Mattson, M . P. A m y l o i d beta-peptide induces cell monolayer albumin permeability, impairs glucose transport, and induces apoptosis in vascular endothelial cells. J Neurochem 68, 1870-81 (1997). Thomas, T., Thomas, G . , McLendon, C , Sutton, T. & Mullan, M . beta-Amyloidmediated vasoactivity and vascular endothelial damage. Nature 380, 168-71 (1996). Mackie, J. B . et al. Human blood-brain barrier receptors for Alzheimer's amyloidbeta 1- 40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer. J Clin Invest 102, 734-43 (1998). Poduslo, J. F., Curran, G . L., Sanyal, B . & Selkoe, D . J. Receptor-mediated transport o f human amyloid beta-protein 1-40 and 1-42 at the blood-brain barrier. Neurobiol Dis 6, 190-9 (1999). Banks, W . A . , Robinson, S. M . , Verma, S. & Morley, J. E . Efflux o f human and mouse amyloid beta proteins 1-40 and 1-42 from brain: impairment in a mouse model o f Alzheimer's disease. Neuroscience 121, 487-92 (2003). Banks, W . A . et al. Passage of amyloid beta protein antibody across the bloodbrain barrier in a mouse model o f Alzheimer's disease. Peptides 23, 2223-6 (2002). Guo, J. T., Y u , J., Grass, D . , de Beer, F. C . & Kindy, M . S. Inflammationdependent cerebral deposition o f serum amyloid a protein in a mouse model o f amyloidosis. J Neurosci 22, 5900-9 (2002).  155  278. 279.  280.  281. 282.  Paris, D . et al. Soluble beta-amyloid peptides mediate vasoactivity v i a activation of a pro-inflammatory pathway. Neurobiol Aging 21, 183-97 (2000). G i r i , R. et al. beta-amyloid-induced migration o f monocytes across human brain endothelial cells involves R A G E and P E C A M - 1 . Am J Physiol Cell Physiol 279, C l 772-81 (2000). Suo, Z . , Fang, C , Crawford, F. & Mullan, M . Superoxide free radical and intracellular calcium mediate A beta(l-42) induced endothelial toxicity. Brain Res 762, 144-52 (1997). Grammas, P. & Ovase, R. Inflammatory factors are elevated in brain microvessels in Alzheimer's disease. Neurobiol Aging 22, 837-42 (2001). Pogue, A . I. & L u k i w , W . J. Angiogenic signaling in Alzheimer's disease. Neuroreport 15, 1507-1510(2004). Bamberger, M . E . & Landreth, G . E . Microglial interaction with beta-amyloid: implications for the pathogenesis o f Alzheimer's disease. Microsc Res Tech 54, 59-70(2001). 4  283.  156  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0092329/manifest

Comment

Related Items