Charles Glabe, PhD - US grants

Sperm adhesion in sea urchins is mediated by bindin, a protein located in the acrosomal granule at the apex of the sperm head. The long term objectives of my research program are to understand the detailed structure, organization and function of fertilization of bindin. I propose to investigate the molecular basis of adhesive specificity and the underlying mechanisms of adhesive force generation. My working hypothesis is that bindin specifically interacts with a fucose-containing glycoconjugate on the egg surface. I propose experiments that will further characterize the structure of the egg surface glycoconjugate and the polysaccharide binding domain of the bindin polypeptide as well as the molecular nature of their specific interaction. These experiments should provide a significant advancement of our understanding of the general problem of the molecular basis of cellular recognition and adhesion. I also propose to investigate the formation of membrane-like vesicles by bindin and the possible significance of this phenomenon to the biochemistry of plasma membrane proteins in general and sperm-egg plasma membrane fusion in particular. Preliminary observations suggest that a conformation change occurs in the bindin polypeptide resulting in the formation of protein-rich vesicles which resemble lipid bilayers in the electron microscope. In contrast to lipid vesicles, these bindin vesicles are stable in non-ionic detergents, but are disrupted by proteolysis or other treatments which solubilize proteins. I propose to further characterize this apparent conformation change as well as the processes which regulate this transformation. In addition, I hope to exploit this membrane-like vesicle forming property of bindin to determine the structure and organization of the bindin polypeptide in these vesicles at a high level of resolution. Knowledge gained through these investigations may ultimately be useful for our understanding of the causes of infertility in man and domestic animals as well as the development of nonhormonal methods of contraception based on the inhibition of gamete interaction.

Sperm adhesion in sea urchins is mediated by bindin, a protein that coats the acrosomal process at the apex of the sperm head. The long term objectives of this project are to provide an understanding of the structure of bindin and its function in fertilization. Bindin specifically associates with gel-phase phospholipid vesicles and can induce the fusion of vesicles in vitro. We propose to examine whether these properties of bindin are important of maintaining the association of bindin with the acrosomal process and for catalyzing the fusion of the sperm and egg plasma membranes in vivo. We will investigate the manner in which the bindin polypeptide associates with the phospholipid bilayer and how this association is regulated by the gel/fluid state of the bilayer. The ability of bindin to specifically associate with gel-phase bilayers may be useful for studying gel-phase domains in biological membranes and suggests a novel regulatory mechanism whereby the physical state of the membrane can modulate the function of the membrane by controlling the proteins associated with the membrane. We also propose to investigate whether the ability of bindin to induce the fusion of vesicles in vitro is related to the fusagenic properties of the acrosomal process in vivo. It is well established the electrical potential across the egg plasma membrane regulates the ability of the sperm to fuse with the egg membrane. In this context it is interesting to note that bindin shares considerable sequence homology with a region of the sodium channel (including a unique segment of 8 contiguous glutamic residues) which has been suggested to be a voltage sensing element. We plan to examine the effect of altering the electrical potential across the vesicle membrane on the ability of bindin to induce the fusion of phospholipid vesicles with cellular plasma membranes. This could be potentially very useful for delivery of drugs and macromolecules such as nucleic acids into the cytoplasm of cells. We propose to investigate the molecular basis for the specific interaction of sulfated, fucoe-containing glycoconjugates with bindin determine whether these glycoconjugates serve as species-specific egg surface receptors for bindin during sperm adhesion. These investigations should advance our understanding of the molecular basis of gamete interaction which could be useful for understanding the causes of infertility and serve as the basis for the development of novel contraceptives.

This proposal requests funding for instrumentation to further develop a comprehensive and integrated core facility for the structural characterization of peptides, proteins and nucleic acids and their organic synthesis for use as reagents. The PIs propose to purchase an automated DNA sequencing instrument to facilitate the analysis of DNA sequences. In addition to the major users of DNA sequencing, the PIs have a relatively large number of users who sequence a few KB of DNA per year that could benefit from this instrument. Currently one of the major services of the existing core facilities is in the synthesis of oligopeptides and oligonucleotides. Demand for synthetic peptides is increasing for use as models of protein structure and function and the production of anti-peptide antibodies. Anti-peptide antibodies are useful reagents for identifying gene products and probing the relationships between protein structure and function. The rate limiting step in the production of synthetic peptides has become the purification of the desired product by HPLC as most of the users do not have their own chromatography instrumentation. Funding for critical supporting instruments such as liquid chromatographs to purify synthetic peptides and capillary electrophoresis equipment to assess purity is also requested. Chromatography equipment is requested for the isolation of proteins prior to sequencing.

The long term objective of this proposal is to elucidate some of the fundamental mechanisms of the accumulation of amyloid deposits in Alzheimer's disease. We plan to investigate how the structure of the amyloid A4/beta protein, the major protein component of senile plaque amyloid deposits in Alzheimer's brain tissue, is related to the intrinsic biochemical properties of the protein in terms of its solubility properties and ability to assemble into amyloid-like filaments. We also plan to investigate how the biochemical properties of the A4/beta protein are related to the mechanism of amyloid deposition in terms of the proteolytic processing of the amyloid precursor protein and its resistance to degradation by cells. Key amino acid residues will be identified which are necessary for the unique fibril assembly and aggregation properties of the A4/beta protein, by synthesizing A4/beta analogs and characterizing the effects of amino acid replacements on assembly and aggregation. The basis for the differences in the solubility properties of senile plaque amyloid and the properties of the A4/beta protein in vitro will be examined. The role of other components that have ben identified in senile plaque in promoting amyloid fibril formation and stabilizing the aggregated state of the synthetic A4/beta protein will be investigated. We hope to establish some of the molecular details for the aggregation and fibril assembly properties of the peptide by 2-D NMR and chemical crosslinking studies on the wild type and mutant peptides. We will screen for compounds and treatments that interfere with aggregation and fibril formation using a rapid and facile assay. A major focus of this proposal is to determine the mechanism for the intracellular accumulation of the A4/beta protein and its resistance to degradation. The relevance of degradation resistance to the mechanisms of amyloid deposition and the molecular pathogenesis of Alzheimer's disease will be investigated. The structure and aggregation state of the accumulated intracellular fraction of the A4/beta protein will be established. The subcellular localization of the intracellular peptide will be determined. We will determine whether the assembled or aggregated states of the peptide are intrinsically resistant to proteolysis or whether the A4/beta protein escapes degradation by some other mechanism. The hypothesis that the segment of the unprocessed amyloid precursor protein that contains the A4/beta protein is selectively resistant to degradation will be tested to determine whether lysosomal processing of the precursor protein is a plausible mechanism for the production of amyloid. Potential differences in the fate of the intracellular A4/beta protein in normal fibroblasts, fibroblasts from Alzheimer's disease patients and cultured neurons will be explored. The fate of the intracellular peptide and long term effects of intracellular accumulation of the A4/beta protein on cultured cells will be explored.

The goal of this research project is to examine the biochemical properties and biological effects of the major beta-amyloid (Abeta) isoforms in cell culture models and to explore potential mechanisms of amyloid deposition in Alzheimer's disease and normal aging. Recent work suggests that the longer major Abeta isoform, Abeta1-42, may be more intimately associated with AD pathology than the shorter form, Abeta1-40. Our investigations of the biochemical properties of these peptides has established that Abeta1-42 is significantly less soluble than Abeta1-40 and shorter isoforms. Unexpectedly, we also discovered a major biological difference in the capacity of cells to catabolize Abeta1-42 compared to Abeta1-40 and the shorter Abeta- peptides. In the previous award period, we have extended these observations to the interaction of Abeta with differentiated PC12 cells. We found that Abeta1-42 is internalized by PC12 cells in vitro and accumulates intracellularly in late endosomes or lysosomes. Although the shorter Abeta analogs are also internalized by PC12 cells, they are degraded or eliminated and do not accumulate. This specific accumulation of Abeta1-42 is largely due to the resistance of the internalized Abeta to degradation. Unlike our results with human fibroblasts, we found that a significantly larger amount of Abeta1-42 relative to Abeta1-40 is adsorbed to the surface of PC12 cells. We have also examined the effect of Abeta1-42 on the processing and catabolism of APP. The results of these investigations demonstrate that Abeta1-42 dramatically stimulates the accumulation of amyloidogenic fragments of APP, particularly a 16 kDa fragment, in an insoluble fraction of the cell. The specific aims of this proposal are designed to compare the biological properties of Abeta1-42 and Abeta1-40 and more completely characterize the effects of Abeta1-42 on APP catabolism and Abeta secretion and to test whether these effects may be relevant to the accumulation of insoluble amyloid deposits in AD. We propose to develop a facile and quantitative assay to distinguish Abeta1-42 and Abeta1-40 in biological samples which takes advantage of the unique biochemical properties of the longer isoform. We will characterize differences in the adsorption, internalization and catabolism of Abeta1-42 and Abeta1-40 in cultured PC12 cells. We will extend our preliminary observations on the effects of Abeta1-42 on the processing and catabolism of APP in transfected 293 cells to cultured PC12 cells. We will also determine whether there is a precursor-product relationship between the 16 kDa amyloidogenic APP fragment and Abeta in the insoluble fraction of cells containing intracellular Abeta1-42 aggregates. We will determine whether the 4 kDa Abeta product is Abeta1-42. We will determine whether the internalization and accumulation of Abeta1-42 impairs endocytic trafficking and normal lysosomal function.

high performance liquid chromatography

DESCRIPTION (Adapted from Applicant's Abstract): The overall goals of this proposal are to understand the structure of the Amyloid Ab peptide and the dynamics of its assembly into amyloid fibrils that are characteristic of Alzheimer's disease (AD). The mechanisms of action of other macromolecules that may promote or inhibit assembly and deposition in amyloid plaque will be determined. Studies on the effects of mutations associated with familial forms of AD on the production of Ab implicated amyloid as a causative agent in the etiology of AD. Results from studies on Ab toxicity in cell culture systems suggest that the aggregation and self assembly properties of Ab may also play a critical role in the biological activity of Ab. The first specific aims is to determine the structure of Ab in solution and when it is assembled into amyloid fibrils. Distances between different points along the Ab sequence will be measured in the oligomeric and fibrillar state by resonance energy transfer and chemical crosslinking. We have developed a set of fluorescent Ab analogs and amino acid substitution analogs that display the same assembly properties as wild type Ab for use in these studies. The location of solvent exposed amino acid residues and position residing in polar and non-polar environments will be probed by fluorescence spectroscopy, quenching analysis and reactivity with polar thiol-reactive agents analogous to the strategy to map the polar surface of acetylcholine receptor channel. These studies should provide information about the spatial relationship between different points along the peptide backbone and will be critical to validate or rule out structures obtained by molecular modeling of NMR analysis of smaller fragments of Ab. This information may ultimately be useful in conceptualizing strategies and therapeutic agents to interfere with Ab aggregation and understanding the relationship between Ab structure and cytotoxic activity. In the second aim, we will characterize oligomeric aggregates that may represent intermediates in the fibril assembly reaction and determine which steps of the assembly process are rate limiting. We will examine whether the fibril elongates equally at both ends. We will investigate whether the dynamics of assembly are altered under different conditions that may be physiologically relevant to amyloid accumulation. In the third specific aims, we will characterize the mechanism of action of a number of potential inhibitors and effectors of Ab assembly that have been proposed to play a role in the deposition of amyloid in AD. We hope to identify classes of molecules that specifically inhibit or promote several different, critical steps in Ab fibril assembly such as nucleation of fiber assembly. Knowing the specific effects of the various molecules and the concentration dependence will provide a means of evaluating the potential relevance in amyloid deposition; strengthening the relevance of molecules that are effective under conditions that are believed to prevail in vivo and weakening the relevance of molecules that would not be expected to function under physiological conditions.

proteins; lasers; biomedical resource; biological products;

DESCRIPTION (from abstract) The overall goals of the proposal are to elucidate some of the fundamental pathways and basic mechanisms for the turnover and degradation of the transmembrane domains (TMD) of type I membrane spanning proteins in the endoplasmic reticulum. Because the gamma- secretase processing of the TMD of the amyloid precursor protein (APP) is critical for the production of the amyloid Abeta peptide and determining the length of the carboxyl terminus of Abeta, the applicants are especially interested in examining pathways that may contribute to the production of the more pathologically relevant Abeta42 in Alzheimer's disease (AD). In order to accomplish these goals, they propose three specific aims that explore the basic mechanisms for TMD proteolysis and degradation, examine their relationship to gamma secretase processing and define the pathways that contribute to Abeta production. In the first specific aim, they will exploit a novel probe that they have designed to specifically examine cleavage events within the membrane. The applicants propose five sub-aims designed to increase our understanding of the basic mechanisms and pathways for the degradation and turnover of the TMD of membrane proteins and to investigate how these pathways may contribute to gamma secretase processing. There are two potential ways in which TMD degradation and gamma secretase processing may be linked: TMD degradation may prevent amyloid production by destroying mis-folded APP substrates that would otherwise give rise to Abeta and perhaps preferentially the more pathological Abeta1-42 form of amyloid. Alternatively, the degradative pathways may give rise to Abeta as a result of partial or incomplete degradation. In the second specific aim, they will analyze the potential role of site 2 protease (S2P) in TMD turnover and APP processing. This protease has recently been identified as the enzyme that cleaves the sterol response element binding protein within the transmembrane domain and preliminary data show that cells deficient in this activity are also deficient in the turnover of the TMD probe. The third specific aim is to characterize the pathways of APP processing and Abeta production in a unique cell line that secretes predominantly (80% of the total Abeta) the more pathological Abeta1-42 form of amyloid. The identification and characterization of the pathways that give rise preferentially to Abeta1-42 may provide insight into the pathological pathways of amyloid production in Alzheimer's disease.

DESCRIPTION (provided by applicant): The overall goals of this proposal are to understand the three dimensional structures of the amyloid Abeta peptide as soluble low molecular weight species, oligomeric intermediates and when it is polymerized into amyloid fibrils. The goal is also to understand the dynamics of Abeta assembly into amyloid fibrils and the mechanisms of action of inhibitors of amyloid assembly. Previous work on the effects of mutations associated with familial forms of AD on the production of Abeta implicates amyloid as a causative agent in the etiology of AD. Results from studies on Abeta toxicity in cell culture systems suggest that the structure and assembly state of Abeta also plays a critical role in the biological activity of Abeta. The first specific aim is to determine the three dimensional structures of Abeta in solution and when it is assembled into amyloid fibrils. Distances between different points along the Abeta sequence will be measured in the soluble, oligomeric and fibrillar state by site-directed spin labeling and electron paramagnetic resonance (SDSL/EPR) spectroscopy and solid state nuclear magnetic resonance SS-(NMR). The conformation and location of solvent exposed amino acid residues and positions residing in polar and non-polar environments will be probed by SDSL/EPR. This information will be used to construct three-dimensional models of Abeta. We will examine the structural basis for potential alternative structures in "nonfibrillar" insoluble Abeta that are believed to be less pathologically significant than fibrillar Abeta. In the second specific aim, we will characterize soluble oligomeric aggregates that may represent key nucleation centers or intermediates in the fibril, assembly reaction and determine which steps are rate limiting. These results will also establish the stabilities of the intermediates and identify rate-limiting steps in the assembly, process. The issue of whether fibril assembly is bi-directional will provide an independent means of evaluating models of fibril structure. In the third aim, we will examine the mechanism of action of a number of different inhibitors of amyloid aggregation. We propose to characterize how these molecules interact with Abeta and determine which step(s) of assembly these molecules inhibit. This information may ultimately be useful in conceptualizing strategies and therapeutic agents to interfere with amyloid assembly and deposition in vivo and in understanding the relationship between Abeta structure and cytotoxic activity.

Abstract

The goals of this proposal are to understand the molecular mechanisms of species-specific sperm egg adhesion in sea urchins and the evolution of genes encoding the macromolecules that mediate adhesion during fertilization. Sperm adhesion is one of many specific cell adhesion and recognition events that occur during development, but the molecular basis for the specific recognition and adhesion remains poorly understood. After 30 years of intensive investigation, we have finally succeeded in cloning and sequencing the egg surface receptor (Egg Bindin Receptor, EBR1) for sperm adhesion from two species of urchins, Strongylocentrotus purpuratus and S. franciscanus (S. p. and S. f.). EBR1 from S. f. and S. p. encode a novel subclass of ADAMTS proteins that contain a number of different functional modules, including a metalloprotease domain and thrombospondin type 1 repeats (TSR) core region, but have entirely different species-specific domains. ADAMTS molecules are widely expressed and are believed to play important roles in cell and matrix recognition and adhesion events during development.
Dr. Glabe will investigate the molecular basis for the species-specific interaction between the sperm adhesive protein, bindin and EBR1. He will determine which of the repeat domains (alone or in combination) in the EBR1 protein are important for mediating the species-specificity of the interaction of EBR1with bindin and we will analyze the molecular basis for specific adhesion dissected by mapping key residues that differ between closely related species and testing their contribution to the specificity. EBR1s contain a reprolysin type zinc metalloprotease domain that contains a complete catalytic active site, thus the hypothesis that this protease domain is catalytically active will be tested. He will examine several potential functions of protease activity during sperm-egg interaction, including sperm penetration of the vitelline envelope (VE), the elevation of the VE from the egg surface, and the release or destruction of the sperm receptor on the VE.
Because of the fact that EBR1 genes from other closely related species of sea urchins have species-specific adhesive function, we will sequence EBR1s from other species in order to determine their sequence divergence, molecular phylogeny, and whether they display evidence of positive Darwinian selection. Regions of sequence divergence that are important for determining species-specificity will be identified. Positive selection for sequence divergence is often associated with molecules that play a role in speciation.

Broader impact: ADAMTS proteins are a family of proteins that play important roles in cell recognition, adhesion and morphogenesis in fertilization and early development. Further characterization of EBR1s and the sperm protein bindin may provide more general insights into the mechanisms of adhesive specificity and the functions of ADAMTS family proteins. How new species arise and become reproductively isolated is an important and fascinating problem in evolution. Understanding the evolution of EBR1 proteins will lead to greater knowledge of the molecular mechanisms associated with reproductive isolation and speciation.

3xTg-AD mice; 3xTg-AD mouse; Address; Age; Age of Onset; Aging; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer's; Alzheimer's Disease; Alzheimers Dementia; Alzheimers disease; Amyloid; Amyloid Fibrils; Amyloid Substance; Amyloid deposition; Antibodies; Antigenic Determinants; Assay; Award; Behavioral; Binding Determinants; Bioassay; Biochemical; Biologic Assays; Biological Assay; Body Tissues; Brain; Brain Diseases; Brain Disorders; Cell model; Cells; Cellular model; Cessation of life; Cognitive; Cognitive Disturbance; Cognitive Impairment; Cognitive decline; Cognitive function abnormal; Collaborations; Condition; Conflict; Conflict (Psychology); Data; Death; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Deposit; Deposition; Disease; Disease Progression; Disorder; Disturbance in cognition; Dot Immunoblotting; Drugs, Nonproprietary; ELISA; Encephalon; Encephalon Diseases; Encephalons; Enzyme-Linked Immunosorbent Assay; Epitopes; Fibrils, Amyloid; Generic Drugs; Genetic Polymorphism; Goals; Head; Hortega cell; Human; Human, General; Immune Precipitation; Immune response; Immunoblotting, Dot; Immunoprecipitation; Impaired cognition; In Vitro; Inflammatory; Intracranial CNS Disorders; Intracranial Central Nervous System Disorders; Investigators; Localized; Mammals, Mice; Man (Taxonomy); Man, Modern; Mass Spectrum; Mass Spectrum Analysis; Measures; Methods; Mice; Microglia; Moab, Clinical Treatment; Molecular Configuration; Molecular Conformation; Molecular Stereochemistry; Monoclonal Antibodies; Murine; Mus; Nerve Cells; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neuronal Dysfunction; Neurons; Numbers; Pathogenesis; Pathology; Peptides; Photometry/Spectrum Analysis, Mass; Play; Polymorphism (Genetics); Polymorphism, Genetic; Population; Primary Senile Degenerative Dementia; Principal Investigator; Programs (PT); Programs [Publication Type]; Protein Precursors; Publishing; R01 Mechanism; R01 Program; RPG; Research Grants; Research Personnel; Research Project Grants; Research Projects; Research Projects, R-Series; Research Resources; Researchers; Resources; Role; Sampling; Science; Senescence; Severity of illness; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Staging; Testing; Tg2576; Tissues; Toxic effect; Toxicities; Transgenic Animals; Transgenic Organisms; Vaccination; Work; age effect; aging brain; aging effect; amyloid assembly; amyloid formation; brain control; cognitive dysfunction; cognitive function; cognitive loss; cognitively impaired; conformation; conformational state; conformer; dementia of the Alzheimer type; disease severity; disease/disorder; dot blotting; experiment; experimental research; experimental study; generic; gitter cell; host response; immunoresponse; mesoglia; microglial cell; microgliocyte; mind control; monomer; mouse model; neuronal; novel; perivascular glial cell; polymorphism; primary degenerative dementia; programs; research study; senescent; senile dementia of the Alzheimer type; size; social role; tissue resource; transgenic

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Study the different conformations of amyloid aggregates and their recognition by conformation-specific antibodies both in vitro and in vivo, using the FCS approach.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aggregation of Abeta peptides is a major contributor to Alzheimer's disease. Oligomeric forms of these peptides are especially interesting due to their high neurotoxicity and potentially important role in disease progression. However, very little structural information is currently available on Abeta oligomers. We have previously established that Abeta oligomers can be divided into at least two structural classes (prefibrillar and fibrillar oligomers) based on their reactivity with conformational antibodies. Here we compared both site-specific and global conformational stability of Abeta 40 fibrils and both classes of oligomers. To measure the site-specific stability, we introduced cysteine residues throughout the sequence of Abeta 40, labeled the new cysteines with the fluorescent dye acrylodan, and investigated their environment within the aggregates in guanidine thiocyanate-induced denaturation experiments. We found that Abeta 40 fibrils show high stability towards denaturation and moderate hydrophobicity in the 15-35 region with the exception of residues 25-30. This pattern is consistent with previously published structures of Abeta 40 fibrils. Fibrillar oligomers show similar pattern consistent with our hypothesis that they represent fragments of protofibrils. However, prefibrillar oligomers show lower stability towards denaturation and more hydrophobic environment throughout the 15-35 region of the peptide. Distinct conformational properties of prefibrillar oligomers may explain their differential recognition by conformational antibodies and serve as a basis for their neurotoxicity.

DESCRIPTION (provided by applicant): The goal of this research project is to explore the structural heterogeneity of amyloid aggregates and the relationships of this conformational variation to the toxicity or pathogenic activities of amyloid oligomers. Several distinct types of amyloid deposits accumulate in disease brain and current evidence suggests that soluble, oligomeric forms of Ass may play primary role in pathogenesis. Recent results indicate that conformation-dependent monoclonal antibodies can distinguish between different types soluble Ass oligomers. These antibodies also distinguish other conformations of Ass, including monomers, fibrils and natively-folded APP. We have recently prepared two additional conformation-dependent antisera that recognize generic epitopes associated with amyloid fibrils and pore-like annular protofibrils that are formed from many different types of amyloids (see Preliminary Data, below). These antibodies are complementary to the anti-oligomer antibody, A11 and recognize epitopes that are specific to fibrils and annular protofibril aggregates. We hypothesize that these conformationally distinct assembly states of Ass are differentially associated with AD pathogenesis. We anticipate that these results will help clarify some apparent inconsistencies and conflicting data, such as the observations that the total Ass amyloid deposited correlates poorly with disease and some people have large amounts of amyloid and are cognitive normal, while other brain samples that have little observable amyloid deposits are associated with cognitive dysfunction. We hypothesize that the distinct types of soluble oligomeric or annular protofibril forms of Ass have distinct toxicities. Conformation-dependent antibodies hold the potential of identifying and distinguishing specific assembly states because they only recognize a specific misfolded state and do not react with the natively folded precursor protein. The specific aims of this project will address the following questions: What is the conformational diversity of amyloid aggregates? What is the structural basis of conformation dependent antibody specificity and amyloid oligomer structural? What are the relationships between different conformational states of amyloids? What is the pathological significance of the different amyloid conformational states? The answers to these questions should provide insight into the range of amyloid oligomer conformational diversity and monoclonal antibody reagents that distinguish different conformations of oligomers. This may provide a more rational structural basis for the classification of oligomers and provide insight into the variability in oligomer preparations reported by different laboratories. Determining the 3 dimensional structure of the monoclonal Fabs bound to amyloid oligomers may provide unprecedented insight into the structure of amyloid oligomers and the mechanism of specific antibody recognition that may be useful for development of immunological therapeutics that target oligomer formation or prevent their interaction with cellular targets. The identification of small molecules that specifically inhibit the formation of different types of oligomers should help to clarify whether the oligomers are intermediates in the formation of higher order structures, like fibrils or whether they represent stable alternative end products. This aim may also provide small molecule lead compounds that specifically inhibit amyloid oligomer formation for therapeutic development. The characterization of which types of amyloid oligomers are more closely related to pathogenesis in human and transgenic mouse brain may help to identify targets for therapeutic development. PHS 398/2590 (Rev. 04/06) Page Continuation Format Page

DESCRIPTION (provided by applicant): This proposal seeks to transform our understanding of the causes and mechanisms of Alzheimer's disease. Alzheimer's disease is one of the most serious and economically important diseases for which there is no disease modifying therapy. The problem for therapeutic development in AD is that there is no commonly understood mechanism for AD pathogenesis. Human genetics implicates the APP and its proteolytic processing and the production of the amyloid A? peptide as a mediator of pathogenesis, but human clinical trials targeting the secretion of A? or the removal of plaques have demonstrated little or no effectiveness or have actually accelerated cognitive decline. Recent trials of gamma secretase inhibitors that prevent the secretion of A? reported that treated patients were cognitively worse than controls, suggesting that out understanding of the disease mechanisms is flawed or incomplete. The goal of this proposal is to identify and characterize the proteins and protein fragments that accumulate in and around the nuclei that are reactive to the fibril specific monoclonal antibody M78. We will test the hypothesis that APP and its amyloidogenic fragments misfold and aggregate into insoluble species prior to their proteolytic conversion to A? and accumulate intracellularly due to the intrinsic resistance of the aggregated A? domain to proteolysis. In preliminary studies we have discovered a novel amyloid fibril immunoreactivity in and around nuclei using a fibril specific monoclonal antibody, M78, which recognizes a discontinuous A? epitope in A? fibrils. Our studies suggest that M78 immunoreactivity colocalizes with both intracellular perinuclear APP and early extracellular plaque stages of AD prior to significant cognitive dysfunction and at intermediate times of pathogenesis in 3xTg-Ad mice. Some of the nuclear M78 immunoreactivity colocalizes with APP and A? as visualized with APP and A? specific antibodies suggesting that M78 immunoreactivity may represent misfolded, aggregated APP or APP fragments accumulating as intracellular amyloid. In 3xTg-AD mice, M78 positive plaques accumulate at later times that are not stained with 6E10 or 4G8, suggesting that a unique type of plaque is also identified by this monoclonal antibody. We also observed that the same spatial distribution of M78 and APP immunoreactivity in neuritic plaques is colocalized with DNA visualized with the fluorescent dye DAPI located in the central core of the neuritic plaque surrounded by a halo of APP immunoreactivity in dystrophic neurites. Together, our observations suggest a previously unidentified nuclear related intracellular pathway for amyloid pathogenesis and plaque biogenesis in AD. If this novel pathogenesis is a key event in AD, it will dramatically change our way of thinking about this disease, refocus the resources for therapeutic development and enable early clinical trials to alter the course of the disease.

Project 1: Intracellular amyloid accumulation, innate immunity and pathogenesis. Project 1 proposes that the accumulation of intracellular amyloid contributes to pathogenesis by activating an innate inflammatory cascade that represents a futile attempt to degrade or eliminate this amyloid by activating autophagy and ultimately leading to neuronal death and the initiation of a neuritic plaque. Project 1 examines the contribution of two unique types of amyloid immunoreactivity to aging and AD through this inflammatory mechanism and it explores the potential role of late onset AD genes in this process. One type of unique amyloid involves the dramatic accumulation of amyloid fibril monoclonal (M78) immunoreactivity in nuclei in human and Tg mouse brain, which is correlated with intraneuronal nuclear accumulation of APP immunoreactivity. The nuclear amyloid immunoreactivity is specifically associated with eariy plaque pathology (stage A or B) in humans and at 12 months in 3xTg-AD transgenic mice. The staining is reduced or absent in late stage AD and at later times in transgenic mice. The other type of unique amyloid is specifically associated with subset of vascular amyloid. Confocal immunofluorescence indicates that the M31 immunoreactive vascular amyloid accumulates at least partially intracellularly in vascular smooth muscle cells. The goals of project 1 are to test the hypothesis that the accumulation of the amyloids represent an infection of the cell with a prion like agent that is associated with activation of the innate immune response. Specific aim 1 will establish the fundamental characteristics of the accumulation of these unique types of amyloid and test the hypothesis that their accumulation is associated with leakage of lysosomal cathepsins and cystatin c into the cytosol and activation of the NLR infiammasome, resulting in the activation of caspase 1 leading to neuronal death by a mechanism related to NETosis. Specific aim 2 will examine whether LOAD genes, PICALM, clusterin and TREM2, recentiy identified by GWAS play a role in the intracellular accumulation of the unique amyloids. Specific aim 3 will explore whether these unique types of amyloid display fundamental properties of infectious agents, like prions and whether they can be propagated by seeding to cells. We will determine whether the the phenotype of the infected cells matches the intracellular amyloid pathology in human brain. Dr. Glabe's project 1 will provide Dr. Cotman with oligomers. Project 1 will interact with Dr. LaFeria on intracellular APP and Aß in the 3XTg-AD mouse and AAV vectors and Dr. Cribbs on the accumulation of vascular amyloid. It will interact with Dr. Tenner on the role of Clq in neuritic plaque formation and the relationship of intraneuronal CRIto intraneuronal amyloid.

Project Summary We seek to understand how structural polymorphisms in amyloid aggregates are associated with Alzheimer's disease (AD) pathogenesis, whether they contribute to disease heterogeneity analogous to strain variation in prion diseases and whether endogenous human antibodies to these different structures may be useful therapeutics and biomarkers for disease subtypes. We have raised 28 monoclonal antibodies that recognize different epitopes and distinguish the structural polymorphisms, so we want to test the hypothesis that antibodies that recognize some structures may be more effective than others in protecting against AD and may be useful biomarkers for AD and its subtypes. These insights have the potential to provide a much finer and detailed view of amyloid pathology and the significance of different types of amyloid deposits and reveal specific therapeutic targets. Advances in our understanding of Aß amyloid structure have shown that peptide can adopt multiple alternative amyloid structures or polymorphs raising the question of whether this polymorphism is meaningful for pathogenesis and a contributor to disease polymorphisms. So far, at least 6 different Aß fibril structures have been determined by solid state NMR and different oligomeric structures have been distinguished based on spectroscopy. Moreover, different fibril structures have been found in human brain with different AD clinical histories suggesting that polymorphisms may be associated with disease subtypes. Consistent with the structural polymorphisms reported, we have found that conformation dependent monoclonal antibodies can distinguish 9 immunologically and morphologically distinct types of amyloid deposits in human and transgenic mouse brain. We will establish the time course of the temporal evolution of the 9 specific amyloid types in transgenic mouse and determine how the 9 different amyloid types are associated with AD and its subtypes. Aducanumab is a human monoclonal antibody that is the first disease modifying therapy to prevent or slow cognitive decline in Alzheimer disease. Aducanumab was isolated from normal human blood, indicating that disease modifying antibodies are expressed in humans. This is an important result because it suggests that there may be other antibodies or combinations of antibodies that confer a more effective neuroprotective activity in humans. Using a random sequence, phage display library, we cloned and sequenced more than 75,000 random 12 mer sequences that interact with our 28 monoclonals. We discovered that there are unique patterns of amino acid sequence recognized by each of the antibodies, indicating that we can use this signature of immunoreactivity to identify individual antibodies reactive with different amyloid polymorphs in human serum or plasma. The goal of this proposal is to use this microarray to search for antibodies protective for AD and endogenous antibodies that are biomarkers of disease and disease subtype.

Our strategic goal is to validate a new paradigm for the age-related causative mechanisms of Alzheimer's disease (AD) by interventions in cellular and amyloid metabolism with small molecules. Specifically, we hypothesize that age-related metabolic changes alter the processing and accumulation of amyloid aggregates inside neurons leading to neurodegeneration. This hypothesis is the inverse of the common formulation of the amyloid hypothesis, which proposes that soluble Aß is secreted from neurons, aggregates in the extracellular space and causes neurodegeneration from the outside. Based on new preliminary data, we propose two alternatives that a) age-related metabolic shifts lead to increased iAß aggregation and death, or b) age-related intraneuronal amyloid (iAß) aggregation leads to neuronal metabolic dysfunction and death. Recent clinical trials of drugs that inhibit secretion of Aß and decrease the concentration of Aß in the interstitial fluid exacerbate cognitive dysfunction in humans and have no effect on plaque accumulation. These results encourage the alternative amyloid hypothesis and suggest that an age-related intracellular retention and accumulation of insoluble long Aßs may initiate pathogenesis leading to neurodegeneration. Our proposal combines 26 years of Charles Glabe's expertise on amyloid aggregation and conformation specific antibodies with Greg Brewer's 30 years of experience in culturing brain neurons and their age-related metabolic changes. Brewer's studies suggest that an oxidative shift in the brain NADH/NAD redox state is upstream of age-related deficits in glucose uptake, mitochondrial deficits and defenses against oxyradicals. Here, we will test the hypothesis that this age-related oxidative shift directs amyloid processing toward a more pathogenic aggregation-prone state that overwhelms proteostasis or conversely, that age-related changes in iAß proteostasis combine with and worsen age-related metabolic changes to promote neurodegeneration. We propose interventions to test key predictions of our hypothesis at three levels: a) in primary AD-transgenic (AD- Tg) neuron cultures from aged compared to those from younger mice and aged non-Tg mice, b) in AD-Tg mouse brain and c) iAß levels in human AD cases. In aim 1, we will evaluate metabolic interventions for downstream effects on iAß processing. In aim 2, we will use gamma secretase inhibitors and modulators for their downstream impact on neurodegenerative metabolism. In aim 3, based on our new findings, we will demonstrate target validity by using safe small molecule precursors to NADH and a Nrf2 inducer or gamma secretase modulator in combination to determine their efficacy to decrease iAß levels and improve metabolic function. Together with our unique tools of end-specific and conformation-dependent antibodies and culture of neurons from adult and aged mice, we will determine the mechanistic basis for an age-related metabolic shift in Aß processing. The completion of this project may initiate a paradigm shift for an age-related metabolic basis of iAß processing as a mechanism of neuronal degeneration underlying cognitive dysfunction in AD. An accurate understanding of the disease mechanisms will provide a better focus for development of drugs that prevent or reverse cognitive decline.