Dennis J. Selkoe, MD - US grants

The purpose of this application remains the study of the macromolecular pathology of age-related neuronal attrition in the human brain. We seek to understand the origin and pathogenesis of the intraneuronal paired helical filaments (PHF) and the extraneuronal amyloid fibers that form in the brain during normal aging and in Alzheimer's disease (AD). This laboratory developed techniques for isolating PHF from human brain and discovered unusual and unexpected molecular properties of these fibers. The observation that PHF are highly insoluble, protease resistant, high M.W. polymers has led directly to: (1) a method for preparing enriched PHF fractions in yields suitable for further biochemical study; (2) the hypothesis that PHF result from abnormal crosslinking of neuronal proteins by nondisulfide covalent bonds; (3) the production of PHF-specific polyclonal antibodies that show no reaction with normal brain proteins; and (4) the production of a library of monoclonal antibodies to PHF. On the basis of this progress, we propose a series of molecular studies aimed at the full characterization of the protein composition of PHF and senile plaque core amyloid. Highly enriched fractions of neurofibrillary tangles (NFT) or amyloid cores prepared by our published method (1) will be labeled with our PHF or amyloid antibodies and purified by fluorescence-activated cell sorting. EM analyses of preliminary FACS trials clearly demonstrate the feasibility of this new method. Using purified NFT or amyloid cores, several protein chemical strategies will be carried out: (a) amino acid analyses; (b) N-terminal analyses; (c) modification of noncovalent bonds by chaotropic solvents (e.g., GuSCN) followed by sequential enzymatic hydrolyses; (d) CNBr cleavage; (e) partial acid hydrolysis; (f) HPLC separation of peptides derived from (c)-(e), followed by automated sequencing and computer comparison to known proteins (including other amyloids, neurofilaments and viral proteins); (g) identification of any acid-labile or -stable crosslinks; and (h) analysis of nonprotein constituents. Detailed comparisons of PHF and amyloid purified from sporadic AD, familial AD and Down's syndrome/AD will be undertaken. PHF-specific antibodies will also be used to quantitate (in an RIA) the degree of SDS insolubility of PHF in various cases and to search for related soluble proteins in brain, CSF and serum. These studies should yield new information about the origin of PHF and plaque amyloid and thus provide clues to more fundamental molecular changes that precede their formation.

The accumulation of abnormal fibrous organelles comprised of paired, helically wound intermediate filaments (PHF) represents the principal structural alteration of neuronal cell bodies and neurites during aging of the human brain and particularly in presenile and senile dementia of the Alzheimer type (Alzheimer's disease, AD). This laboratory has recently developed techniques for isolating PHF from human brain and has discovered unusual and previously unexpected molecular properties of these neuronal fibers. Our finding that PHF are highly insoluble, rigid, high molecular weight polymers has led directly to: (a) a method for preparing relatively pure PHF fractions in yields suitable for further studies; (b) the hypothesis that PHF result from abnormal crosslinking of neuronal proteins by nondisulfide covalent bonds; and (c) the production of a polyclonal antiserum which is sensitive and highly specific for PHF and fails to react with normal fibrous proteins. Based on these new findings, we now propose an integrated series of molecular studies which has as its goal the full characterization of the origin and composition of PHF. First, we shall use biochemically purified PHF as immunogen to raise monoclonal antibodies. These and the polyclonal AlphaPHF antibodies already raised will be used as specific ligands in immunoaffinity chromatography to purify PHF to homogeneity (judged by EM), a prerequisite for accurate compositional analysis of an insoluble organelle. Using purified PHF, several protein chemical strategies for structural characterization will be carried out: (a) total amino acid analyses; (b) modification of non-covalent interactions by chaotropic solvents (e.g., guanidine SCN) followed by enzymatic hydrolyses (e.g., trypsin, bacterial proteases); (c) cyanogen bromide cleavage; (e) a solid phase automated sequence analysis as well as HPLC mapping of the peptides derived from (b)-(d); (f) isolation and characterization of any acid-labile or stable crosslinks (including glu-lys dipeptide) after appropriate enzymatic or acid hydrolyses; (g) analysis of non-protein constituents of PHF. Monoclonals to PHF will also be employed in immunohistochemistry (light and EM) and immunoblotting to delineate further the distribution of PHF in AD tissue and within neurons and their processes and to identify neuronal and non-neuronal proteins with shared determinants. These various studies should provide new information about the origin of PHF and the preceding abnormal biochemical events in Alzheimer neurons that allow their formation.

This LEAD proposal will enable an interdisciplinary group of four investigators who have collaborated productively on macromolecular studies of Alzheimer's disease (AD) to expand their work into new aspects of the protein chemistry and molecular biology of regionally selective cellular dysfunction in AD brain. We will use biochemical, cell biological and molecular genetic techniques to elucidate further the complex age-related alterations of certain normal gene products (viz., the cytoskeletal protein, tau, and the precursor of the beta-amyloid protein (beta- AP) that invariably accompany progressive neuronal dysfunction in AD. Our experiments are based on specific hypotheses (described herein) about the origin of neuritic plaques and the reorganization of the neuronal cytoskeleton in AD. They seek answers to questions arising directly from recent progress in AD research. (1) How does the beta-AP precursor, whose gene is not known to be altered in AD and whose mRNAs are widely expressed in neural and non-neural tissues, undergo selective processing into amyloidogenic fragments only in certain brain regions, especially cortex and its microvasculature? (2) Whatever the mechanism of local amyloid formation, does the beta-AP itself (or associated macromolecules) exert any toxic and/or trophic effects on cortical cells, or is it inert? Can such effects be detected and studied in an in vitro or in vivo model? (3) In view of the widespread neuritic and perikaryal accumulation of altered, aggregated tau protein in AD, can we build on our recent progress in sequencing full-length cDNAs for both mouse and human tau to determine which epitopes of tau are deposited in AD, which kinases phosphorylate normal or AD tau at these epitopes and whether the critical microtubule binding domain is incorporated into PHF and thus dysfunctional in AD? Our experiments include: (a) detection of brain-region-specific fragments of the amyloid precursor that precede the formation of the final beta-AP subunit and a search for local proteases and their inhibitors that could effect this progressive processing; (b) a dynamic study of the assembly properties of fetal, adult and AD microtubule-associated proteins; and (c) analyses of the effects of beta-AP or adherent proteins on cortical neurons in vitro (humans) and in vivo (primates and rodents). We believe our new research program as well as our prior productivity and strong commitment to molecular research on AD will allow us to use the singular opportunities provided by the LEAD Award to full advantage.

DESCRIPTION (provided by applicant): Research focused on disease has often identified genes whose existence and physiology were previously unknown. In the example of Alzheimer's disease (AD), the genes encoding APP, Presenilin and Nicastrin were all discovered because of interest in the protein biology of the disorder. APP and its homologues stand out as highly conserved in metazoan evolution, ubiquitously expressed and abundant in neurons. APP mutations or duplications cause rare but mechanistically important forms of AD, and its Ass product accumulates in all AD patients. Although APP is among the most studied gene products in biology, most work has centered on its role in AD; its normal physiology has been less studied and has led to an array of complex, sometimes conflicting findings. The advent of human trials of agents that chronically inhibit APP processing makes it even more urgent to clarify its normal functions. Here, we propose new concepts and approaches regarding APP physiology that are based on strong productivity in the current MERIT Award period and preliminary data which make our multifaceted aims both compelling and technically feasible. A key approach to gene function is to understand its role during early development and then search for related activities in adults. Under this grant, we uncovered a critical function of APP n the development of the mammalian cerebral cortex. To pursue this and other discoveries, we propose three interrelated Aims. First, we seek to determine the molecular mechanism by which holoAPP is required for correct migration of neuronal precursors into the cortical plate and how DISC1 (which we found to interact with the APP cytoplasmic domain) works with DAB1, Fe65 and other factors in this migratory function. We will then ask whether APP plays a related role in neuronal or glial migration in the adult brain. Second, we will use primary neuronal co-culture assays to systematically confirm or deny numerous reported ligands of the APP ectodomain as well as certain novel ligands, e.g., the Pancortins, that have emerged from an unbiased screen. We will also search for cooperative binding of APP to proteoglycans and protein ligands (e.g., Reelin) as regards the regulation of its ectodomain shedding. Third, we will validate a new cell biological model of APP secretase processing, based on our recent identification of a multi-protein complex that appears to contain ¿-, ¿- and ?-secretases, enabling rapid and efficient substrate processing. We will search for and analyze protein partners of this putative sheddasome, particularly the tetraspanins, and learn if this model applies broadly to other intramembrane proteases and their sheddases: S1P and S2P, and SP and SPP. Together, our aims address a working hypothesis of APP function in neurons: that holoAPP interacts with extracellular factors on neurons, glia and the matrix to activate intracellular signaling pathways (via DAB1, DISC1 and Fe65), and that a spatially and temporally integrated ¿/?-secretase complex terminates holoAPP function and initiates alternate signaling by APPs¿.

Progressive cerebral deposition of the amyloid beta-protein (Abeta) is an early and invariant feature of Alzheimer's disease (AD) which appears to precede dementia by many years. In at least a few families, betaAPP mismetabolism and Abeta deposition are believed to represent the molecular basis of the disease. Recently, Abeta was discovered to be continuously produced and secreted as a soluble peptide by cultured cells during normal metabolism. As a result, the mechanism of Abeta production can now be studied dynamically in vitro and the effects of pharmacological modulation readily assessed. Here, we propose an integrated series of molecular and cell biological experiments which build on this discovery by examining the physiological control of Abeta production. Our Specific Aims are: 1) To examine the mechanism and regulation (Abeta secretion using both cDNA- transfected human cell liens and primary human donor cells (neurons, fibroblasts, endothelial cells). We will search for cell-type specific differences, effects of donor age, effects of basal and activated betaAPP phosphorylation state, and effects of several cellular stressors (serum deprivation; excess calcium; oxidative injury; etc.) on the production, release and stability of Abeta. 2) To establish the structural requirements for cellular generation of Abeta from betaAPP using site- directed mutagenesis around the N- and C-termini of Abeta as well as deletions of specific functional domains or consensus sequences within betaAPP [e.g., NPXY internalization signal; KPI domain; growth promoting motifs (e.g., RERMS); acidic domain]. Some of these mutated betaAPP constructs should provide critical substrates for the confirmation or exclusion of any candidate proteolytic enzymes which putatively release the Abeta fragment. 3) To define the molecular mechanisms of increased Abeta accumulation in various genetic forms of FAD, both those on chromosome 21 (betaAPP missense mutations) and those on chromosome 14, using primary donor cells (fibroblasts). This approach should provide information about betaAPP metabolism and Abeta production in endogenous cells from chromosome 14 donors even before the defective gene is identified. 40 To search for evidence of polarized secretion and/or transcytotic transport of Abeta in epithelial and endothelial monolayers, mimicking the microvessel wall. This approach seeks to provide experimental evidence for the longstanding but untested hypothesis that beta-amyloid, particularly in blood vessels, could arise from a vascular or circulating source. Taken together, these experiments address central questions about the mechanism of Abeta generation, its alteration in familial Alzheimer's disease and its regulation by a variety of physiological and pharmacological modulators. As such, this research should provide insights not only into the normal metabolism of betaAPP and Abeta but also about how one might control Abeta production by a variety of pharmacological strategies. The work addresses one of the most promising pathogenetic and therapeutic hypotheses of Alzheimer's disease.

This LEAD proposal will enable an interdisciplinary group of four investigators who have collaborated productively on macromolecular studies of Alzheimer's disease (AD) to expand their work into new aspects of the protein chemistry and molecular biology of regionally selective cellular dysfunction in AD brain. We will use biochemical, cell biological and molecular genetic techniques to elucidate further the complex age-related alterations of certain normal gene products (viz., the cytoskeletal protein, tau, and the precursor of the beta-amyloid protein (beta- AP) that invariably accompany progressive neuronal dysfunction in AD. Our experiments are based on specific hypotheses (described herein) about the origin of neuritic plaques and the reorganization of the neuronal cytoskeleton in AD. They seek answers to questions arising directly from recent progress in AD research. (1) How does the beta-AP precursor, whose gene is not known to be altered in AD and whose mRNAs are widely expressed in neural and non-neural tissues, undergo selective processing into amyloidogenic fragments only in certain brain regions, especially cortex and its microvasculature? (2) Whatever the mechanism of local amyloid formation, does the beta-AP itself (or associated macromolecules) exert any toxic and/or trophic effects on cortical cells, or is it inert? Can such effects be detected and studied in an in vitro or in vivo model? (3) In view of the widespread neuritic and perikaryal accumulation of altered, aggregated tau protein in AD, can we build on our recent progress in sequencing full-length cDNAs for both mouse and human tau to determine which epitopes of tau are deposited in AD, which kinases phosphorylate normal or AD tau at these epitopes and whether the critical microtubule binding domain is incorporated into PHF and thus dysfunctional in AD? Our experiments include: (a) detection of brain-region-specific fragments of the amyloid precursor that precede the formation of the final beta-AP subunit and a search for local proteases and their inhibitors that could effect this progressive processing; (b) a dynamic study of the assembly properties of fetal, adult and AD microtubule-associated proteins; and (c) analyses of the effects of beta-AP or adherent proteins on cortical neurons in vitro (humans) and in vivo (primates and rodents). We believe our new research program as well as our prior productivity and strong commitment to molecular research on AD will allow us to use the singular opportunities provided by the LEAD Award to full advantage.

DESCRIPTION: (adapted from applicant's abstract) Beta-amyloid precursor protein (BetaAPP) is an integral membrane protein that is ubiquitously expressed in mammalian cells, with highest levels in the brain. BetaAPP was identified and cloned because a 39-43 residue proteolytic fragment (Abeta) is the subunit of the amyloid filaments which accumulate as myriad extracellular ('senile') plaques in the brains of humans with Alzheimer's disease (AD). Subsequent studies of normal BetaAPP structure and function have revealed several interesting and unusual properties that have relevance to other single membrane-spanning proteins: a) betaAPP functions both as an apparent cell-anchored receptor and as a secreted derivative that acts upon other cells; b) proteolysis of betaAPP leads to regulated release of several different soluble derivatives, including hydrophobic peptides that include much of the transmembrane domain; c) betaAPP is phosphorylated solely on its ectodomain, leading to secretion of phosphorylated betaAPP; d) the large betaAPP ectodomain contains diverse structural motifs that give rise to several putative functions of the molecule; and e) betaAPP is a member of a highly conserved gene family, including homologs in Drosophila and C.elegans. Based on these findings and on extensive preliminary data, a series of integrated biochemical, cell biological and molecular biological experiments which address both the normal structure and function of betaAPP and the mechanisms of cerebral accumulation of its Abeta fragment is proposed. Specific Aims are to: 1) Identify and clone the physiological receptor and/or counter-receptor for APPs/betaAPP and characterize its role in betaAPP function and the endocytosis and processing of betaAPP. Preliminary work has established assays for betaAPP functional activity in the picomolar range and the reagents needed for expression cloning (or purification) of its receptor, 2) Understand the mechanism of the unusual ectodomain phosphorylation of betaAPP and the role of phosphorylation/dephosphorylation in both the functions of the surface and secreted isoforms and the proteolytic processing of betaAPP. This work postulates that betaAPP is one of a very few known surface proteins whose multiple extracellular functions are modulated by their state of ectodomain phosphorylation (cf, osteopontin; CD36). 3) Analyze a potential mechanism for the clearance of secreted Abeta from extracellular fluid: examine physiologically in living cells the hypothesis that apolipoprotein E mediates the clearance of Abeta from extracellular fluids via LRP or another receptor. 4) Characterize the formation and fate of natural multimers of endogenously secreted Abeta which have been recently detected in cell culture. Growing evidence suggests that Abeta polymerization is a critical determinant of its cytotoxicity, but prior studies have used high doses of synthetic Abeta in non-physiological conditions. Taken together, these Aims build on extensive published and unpublished studies of betaAPP form this lab and others to address new questions about this complexly processed, multi-functional molecule. The results should provide insights into the normal biology of this class of receptor/secretory protein and the role of betaAPP in AD.

Progress in deciphering the genotype to phenotype relationship of the presenilin form of the familial AD provides a unique opportunity to elucidate a major unresolved issue in AD in general: how gamma-secretase generates the Abeta42 peptide which accumulates early in the course of all forms of he disease. Mutations in PS1 and PS2 selective enhance Abeta42 production in cultured cells, transgenic mice and human brain tissue, and this increase has been documented presymptomatically in plasma and primary cells from mutant gene carriers. We recently found that both PS1 and PS2 form stable complexes with APP in the ER and early Golgi and that mutant PS increases Abeta42 in these sites. Therefore, our central hypothesis of this project is that PS mutations physically alter the interaction of PS with APP in a way that allows increased proteolysis of the latter by a gamma-secretase cleaving specifically at residue 42, and that understanding how this occurs will shed light on an early and invariant feature (Abeta42 deposition) of the common "sporadic" form of AD. To address this hypothesis, we will carry out 4 Aims: 1) using stable cell lines and PS transgenic mice from Cores B and C, define the regions of APP and PS that interact, how mutations change the interaction and whether additional proteins (e.g., delta-catenin) participate in the complexes; 2) use cell biological and pharmacological methods to define all of the principal subcellular loci for Abeta40 and Abeta42 production and how PS mutations differentially effect these; 3) establish the structural requirements within APP for the gamma secretase cleavages and whether the proteases act by direct intramembranous endoproteolysis at 40 (and 42) or by a "2 hit" mechanism analogous to that recently described for SREBP; and 4) identify 42- and 40-specific gamma-secretase inhibitors and use them to characterize and ultimately purify the cognitive protease(s). All 4 Aims are based on strong preliminary data, and they will involve extensive collaborations with the other 3 PI's of this Program. The results should provide information relevant in general to the proteolytic processing of transmembrane proteins and in particular to the mechanism of Abeta42 generation, an emerging therapeutic target in Alzheimer's disease.

The interplay of normal and pathological biology that characterizes research on Alzheimer's disease is particularly well illustrated by studies of two proteins that have been directly implicated in the genetic mechanisms of AD: the presenilins (PS) and the beta-amyloid precursor protein (APP). In this Program Project grant, four independent laboratories that have each contributed productively over many years to the elucidation of the mechanisms of AD are joining forces to apply a wide range of techniques in a molecular and cell biology, neuropathology and animal modeling to address key unresolved questions about the presenilins and their role in AD pathogenesis. The central vision of our Program is to use the combined expertise of these four well-established laboratories and their extensive array of techniques and reagents they possess to examine in detail the biology of the presenilins, their interactions with other functionally important neuronal proteins (including APP, Notch and the catenins and their pathogenic role in the most common and aggressive form of genetically based AD. The principal investigators, who have collaborated on numerous occasions in the past, have been meeting together regularly for many months to discuss scientific questions of mutual interest, share unpublished data, exchange reagents, cross-validate findings and design new collaborative experiments, the most compelling of which have been incorporated into this Program. Among our numerous Specific Aims (organized into 4 projects), we will: 1) characterize in detail cellular and subcellular anatomy of PS1 in our transgenic mice expressing wt versus mutant PS1, using in situ hybridization and confocal microscopy with newly developed reagents; 2) assess AD-like pathology in these mice and new mice resulting from crossing our mice with PDAPP V717F transgenic mice, using modern quantitative stereology; 3) examine the complex endoproteolysis of PS, including an exciting novel apoptotic pathway we have recently identified, and how this is changed by PS mutations, but in cells and in transgenic mice; 4) use the PS mutations as a route to defining the elusive mechanism of gamma-secretase processing of APP, in view of the highly selective effect of mutant PS on Abeta42 production and our recent demonstration of a direct interaction of APP with both PS1 and PS2 in the ER and Golgi; and 5) characterize the cell biology of a novel member of the catenin family we recently cloned as a PS-interaction in vivo and assess how it functions in cell signaling and whether it participates in the PS-APP complexes. These are but a few of the unanswered questions about the structure and function of the presenilins we will approach. Our experiments will be supported by 3 Cores, including one for breeding and maintaining transgenic mice, and one that will characterize and distribute a very large array of DNA constructs, stable cell lines, probes and antibodies and will conduct sensitive Abeta ELISAs. Our proposed experiments are hypothesis-driven and, in each case, based on strong preliminary data. We believe our combined experiences and our committed group of senior and junior scientists will enable us to successfully execute a highly integrated program of basic and applied molecular neurobiology that will have direct implications for understanding the mechanism and treatment of AD.

The central hypothesis underlying our research is that Alzheimer's disease is a multi-gene syndrome in which numerous distinct gene defects (and perhaps certain environmental factors) result in a chronic imbalance between Abeta production and Abeta clearance that leads to the microglial, astrocytic, neuronal and synaptic pathology which produce the symptoms of dementia. Based on this hypothesis, many labs including ours have focused substantial attention on the cell biology of APP and the mechanisms of Abeta production, both normally and in AD. To date, molecular causes of Abeta overproduction have explained only a fraction of AD, in all cases of which Abeta accumulates excessively. Yet the nature of Abeta degradation and clearance has hardly been studied. We have chosen to direct our further experiments in this renewal to this largely unexplored topic because of compelling data generated in the current period that demonstrate a time-dependent loss of Abeta40 and 42 peptides in several cell types, particularly microglia, and implicate the thiol metalloendopeptidase, insulin degrading enzyme (IDE), as the principal mediator of this loss. Our Specific Aims are: 1) to establish quantitatively (Km, kcat, etc.) the role of IDE (and other possible Abeta-degrading proteases) in the proteolysis of extra- and intracellular Abeta in neural and non-neural cultured cells and define where in the cell IDE contacts Abeta; 2) to assess the extent to which IDE (and other proteases identified in Aim l) is expressed and actually degrades Abeta in brain regions prone vs not prone to Abeta build-up, and whether this catabolism is altered with age or in AD in humans and APP tg mice; 3) to prove that IDE can regulate Abeta levels and deposition in vivo by creating PDGF-IDE tg mice, crossing them with PDGF-APP tg mice and assessing the progeny biochemically and pathologically; and 4) to extend our intriguing-data that IDE, besides cleaving Abeta, can mediate its conversion to stable oligomers at physiological levels. In short, we will systematically explore how Abeta is degraded extra- and intracellularly, whether IDE plays a key role in this, and how extracellular levels of Abeta monomer are regulated simultaneously by cleavage and oligomerization. Our plan utilizes the 3 complementary approaches of cell culture, in situ analyses in human and mouse brain tissues, and in vivo modelling in IDE/APP transgenic mice to decipher how Abeta proteolysis and aggregation occur under physiological conditions. The results should help open up a new area of AD pathobiology, with attendant therapeutic implications.

Progress in deciphering the genotype to phenotype relationship of the presenilin form of the familial AD provides a unique opportunity to elucidate a major unresolved issue in AD in general: how gamma-secretase generates the Abeta42 peptide which accumulates early in the course of all forms of he disease. Mutations in PS1 and PS2 selective enhance Abeta42 production in cultured cells, transgenic mice and human brain tissue, and this increase has been documented presymptomatically in plasma and primary cells from mutant gene carriers. We recently found that both PS1 and PS2 form stable complexes with APP in the ER and early Golgi and that mutant PS increases Abeta42 in these sites. Therefore, our central hypothesis of this project is that PS mutations physically alter the interaction of PS with APP in a way that allows increased proteolysis of the latter by a gamma-secretase cleaving specifically at residue 42, and that understanding how this occurs will shed light on an early and invariant feature (Abeta42 deposition) of the common "sporadic" form of AD. To address this hypothesis, we will carry out 4 Aims: 1) using stable cell lines and PS transgenic mice from Cores B and C, define the regions of APP and PS that interact, how mutations change the interaction and whether additional proteins (e.g., delta-catenin) participate in the complexes; 2) use cell biological and pharmacological methods to define all of the principal subcellular loci for Abeta40 and Abeta42 production and how PS mutations differentially effect these; 3) establish the structural requirements within APP for the gamma secretase cleavages and whether the proteases act by direct intramembranous endoproteolysis at 40 (and 42) or by a "2 hit" mechanism analogous to that recently described for SREBP; and 4) identify 42- and 40-specific gamma-secretase inhibitors and use them to characterize and ultimately purify the cognitive protease(s). All 4 Aims are based on strong preliminary data, and they will involve extensive collaborations with the other 3 PI's of this Program. The results should provide information relevant in general to the proteolytic processing of transmembrane proteins and in particular to the mechanism of Abeta42 generation, an emerging therapeutic target in Alzheimer's disease.

chemical aggregate; enzyme linked immunosorbent assay; intracellular; ion exchange chromatography; molecular assembly /self assembly; tissue /cell culture

DESCRIPTION (provided by applicant): Intensive research by many laboratories, including work performed under this grant, has advanced the hypothesis that altered processing of the B-amyloid precursor protein (APP) or decreased clearance of its amyloid B-peptide (AB) fragment are critical pathogenic events in Alzheimer' s disease. Nevertheless, despite a wealth of reports about in vitro and in vivo activities of the precursor and its derivatives, the fundamental physiological function of APP has not been established. Based on recent studies in our and other laboratories and extensive preliminary data, we propose to elucidate systematically the function of APP and the molecular mechanism by which it accomplishes this function. The central hypothesis motivating this application is that the remarkably similar proteolytic processing of APP and the Notch receptors suggests that APP is itself a cell-surface receptor with a cognate ligand, the binding of which enables the release of the cytoplasmic domain (AICD) to the nucleus to activate specific genes. Based on substantial experience in APP processing and membrane protein biology obtained during the past 24 years of this grant, we will carry out 4 interrelated Aims: 1. to purify, identify and characterize a specific protein ligand(s) for the APP ectodomain; 2. to localize the APP intracellular domain (AICD) to the nucleus and study its stabilization and activites there; 3. to assess the phenotypic consequences, including for APP processing and intracellular AB generation, of signaling by the physiological ligand(s) in intact neurons; and 4. to establish the in vivo biochemical and electrophysiological effects of APP signaling in living animals. We will compare the ligand binding and potential signaling properties of APP to those of APLP-1 and APLP-2 and also explore whether APP processing and signaling alter those of Notch. Intriguing preliminary findings about AICD nuclear localization and stabilization by Fe65 and about transcriptional changes in the brains of APP knockout mice encourage us to pursue these aims. The results of the multifaceted experimental approach described herein should be revealing not only for the normal biology of APP but also for how its regulated signaling contributes to the pathogenesis of Alzheimer's disease and to the progressive accumulation of AB that accompanies normal brain aging in humans and other primates.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Studies of the molecular basis of Alzheimer's disease (AD) exemplify the increasingly blurred distinction between basic and applied biomedical research. Genetic, biochemical, cell biological and animal modeling studies all support the hypothesis that accumulation of amyloid beta-protein (AB) causes AD, but it is not known why cerebral AB levels are elevated in the vast majority of AD patients. While great attention has been focused on the mechanisms of AB production, only a small number of cases is known to involve heightened production. Therefore, many cases of typical (late-onset) AD could be caused by faulty clearance of a peptide that is made at normal levels throughout life. In this competing renewal application, we wish to continue to pursue two hypotheses: (a) that defects in proteases which normally degrade AB may underlie some or many cases of familial and "sporadic" late-onset AD; and (b) that whether or not this turns out to be true, subtly activating or disinhibiting such proteases could lower brain AB levels therapeutically. Addressing this previously understudied topic requires a thorough understanding of which proteases regulate steady-state AB levels in vivo and how they do so. To this end, we propose four interrelated Specific Aims: 1. To test whether chronic dysfunction of insulin-degrading enzyme ODE) leads to decreased AB degradation and cerebral AB accumulation in vivo in two compelling models: the GK rat, which develops type II diabetes caused by missense mutations in IDE, and a newly generated IDE knock-out mouse; 2. To quantify the relative roles of the 3 best characterized AB-degrading proteases -- neprilysin, IDE and uPA/plasminogen -- in cerebral AB economy by comparing their effects in the progeny of the relevant transgenic and knock-out mice bred to APP transgenic mice; 3. To elucidate the normal cell biology and membrane trafficking of lDE that enables it to mediate the degradation of both extracellular/intraluminal substrates (e.g., insulin, amylin, AB) and cytosolic substrates (e.g., the APP intracellular domain); and 4. To use a chemical biology approach to identify small-molecule activators and inhibitors of IDE and neprilysin as mechanistic and therapeutic tools. The proposed experiments are based on extensive preliminary data, including the establishment of the necessary rodent models, and will allow us to extend a new line of investigation in the AD field that has emanated in part from this grant. We believe the results could have broad implications for the pathogenesis and treatment of AD as well as for peptide turnover in the brain and the fundamental cell biology of proteases. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The interplay of normal and pathological biology that characterizes research on Alzheimer's disease (AD) is well illustrated by the study of a gene product implicated in the fundamental mechanisms of life: presenilin. PS 1 missense mutations cause familial AD, but research during the past 4 years, some of it under this grant, has revealed that PS1 is also required for the genesis of all forms of AD and for the normal processing of diverse receptors in all metazoans. In this first competitive renewal, 7 labs that have each contributed productively to elucidating AD mechanisms are joining forces to apply techniques in molecular and cell biology, genetics, chemistry and animal modeling to answer unresolved questions about the normal and abnormal biology of PS. Our 3 original PIs (Selkoe, Tanzi and Hyman) have joined with 4 talented new investigators (Wolfe, Kovacs, Wasco and Xia) to develop and execute 6 research projects that build on the extensive array of methods and reagents that our labs possess. Our Specific Aims include: 1) to attempt to prove the hypothesis (first developed in this grant) that PS is an intramembrane-cleaving aspartyl protease, by carrying out step-wise addition of the 4 limiting components (PS, nicastrin, aph-1, pen-2) to reconstitute APP and Notch cleavage in mammalian and yeast cells and in pure phospholipid vesicles; 2) to conduct detailed genetic (SNP) association analyses in AD by examining positional candidate genes encoding proteins implicated in PS-related pathways, including PS interactors, gamma-secretase substrates and components, AICD interactors and targets, and PS homologs; 3) to study the interactions of the gamma-secretase components with its substrates by developing novel morphological techniques using advanced fluorescent microscopy methods (FRET and FLIM) that measure protein-protein proximity in living neurons; 4) to characterize the nature of "presenilinase", the unusual activity that converts holoPS into active heterodimers, i.e., how the putative autoproteolysis of PS differs from its gamma-secretase activity and where in the cell "presenilinase" exists; 5) to build upon our recent discovery of a novel gamma-secretase substrate, nectin-1, a synaptic adherens junction protein, to ask whether various PS 1 mutations alter nectin-1 processing, cell-cell aggregation and synapse formation, and whether modulating cell-cell adhesion alters gamma-secretase activity on nectin-1; and 6) to assess the potential signaling function of a key family of PS substrates, APLP1 and APLP2, and see if their intracellular domains differ from APP as regards nuclear translocation, Fe65 binding and patterns of gene regulation. Taken together, these Aims comprise an integrated and potentially far-reaching examination of the biology of the PS/ gamma-secretase complex, its substrates and their role in the mechanisms and treatment of AD.

amyloid proteins; protein structure function; blood proteins; Alzheimer's disease; molecular pathology; patient oriented research; human subject; clinical research;

An enduring principle for successful intervention in human disease is to identify - andthen prevent - the earliest steps in pathogenesis. In the case of Alzheimer's disease and its harbinger, mild cognitive impairment (MCI), studies from many labs support the still unproven hypothesis that the gradual accumulation and oligomerization of amyloid p-protein (A(3)in brain regions serving memory and cognition initiates this complex syndrome. Given the enormous resources being expended by academic and pharmaceutical scientists to identify anti-amyloid therapies and bring them to human trials, it is crucial to understand precisely how soluble A|3begins to oligomerize and whether this process actually induces the subtle compromise of synaptic function seen in MCI and early AD. In this new RO1 application, investigators who have collaborated productively to discover the natural secretion of low-n A|3oligomers in cell culture and then demonstrate their ability to inhibit long-term potentiation and disrupt memory in living animals now propose to rigorously define at the molecular level these earliest A(3assembly forms and elucidate their mechanisms of action on neuronal function. Based on extensive preliminary data and sensitive biochemical methods we have developed to isolate and study natural oligomers, we propose 4 interrelated Specific Aims. 1. Determine the precise molecular composition of naturally secretedA|3 oligomers by mass spectrometry and search for covalent crosslinks, associated small molecules and/or binding proteins that may contribute to their potent neuronal activity. 2. Characterize the effects of the natural oligomers on synaptic form and function, including in organotypic hippocampal cultures, and assess whether they can induce AD-type tau phosphorylation and altered transmitter release in vivo, 3. Purify the natural oligomers to homogeneity, intrinsically label them and identify their cognate molecular and cellular targets in living brain. 4. Assess 3 specific therapeutic strategies to decrease the production of cell-secreted oligomers and thereby abrogate their synaptotoxicity: (3- or y-secretase inhibitors;certain anti-aggregation compounds;and chaperone expression. Our extensive experience in studying this unlimited cellular source of physiological amounts of human A(3 oligomers should enable us to exploit this unique experimental paradigm to elucidate both the nature and the neuronal effects of the earliest A(3 assemblies, with attendant therapeutic implications. Relevance to Public Health: Because our central hypothesis is that the earliest-forming "oligomers" (doublets, triplets, etc.) of amyloid |3-protein underlie the subtle and progressive impairment of memory that is the hallmark of incipient AD, we will use a unique experimental system in which cultured cells naturally produce such early forms in order to decipher the precise nature of these pathogenic assemblies, identify their mechanism of injury on the neurons and synapses required for memory, and then block this process with novel drugs.

[unreadable] DESCRIPTION (provided by applicant): Parkinson's disease, the second most common neurodegenerative disorder, is marked by progressive dysfunction and loss of nigral dopaminergic neurons. This cardinal feature is accompanied by the accumulation of protein inclusions in dopaminergic neurons and their processes (Lewy bodies and neurites), the major constituent of which is a-synuclein (aS). aS has 7 repeats resembling the lipid-binding a-helical domains of apolipoproteins, and its binding to phospholipid membranes markedly alters its secondary structure. We have recently: a) discovered homologies of aS with the fatty acid (FA) binding protein (FABP) family; b) found that pure aS binds free FAs reversibly; c) detected a pool of highly soluble, lipid-associated aS oligomers in dopaminergic cells, normal mouse and human brains and, at elevated levels, in PD and DLB brains; d) shown that exposure of living mesencephalic neurons to polyunsaturated FAs enhances ~ and to saturated FAs retards ~ the formation of soluble aS oligomers; and e) documented increased endogenous PUFA levels and membrane fluidity in aS-overexpressing neurons, and the opposite in aS knock-out mice. Based on these findings, we hypothesize that aS normally interacts with FAs in both the aqueous and membrane-phospholipid compartments of the cytoplasm and helps regulate aspects of lipid composition (particularly PUFA content) and thus membrane properties, and that aS-FA interactions help regulate the oligomerization of aS and can thus initiate aS assembly into first soluble and then insoluble oligomers. To pursue this molecular hypothesis about aS function and dysfunction, we now propose a series of interrelated goals. 1) To attempt to prove that altering endogenous PUFA levels (e.g., lowered in cells treated with a A6 desaturase inhibitor or elevated in mice modeling Zellweger's syndrome) induces corresponding decreases or increases in endogenous aS oligomers in brain cells. 2). To examine the effects of aS-FA interactions on the formation, ultrastructure and biophysical properties of membrane vesicles in living cells. 3) To ascertain whether and how PUFA-ctS interactions affect the one discrete biochemical function of aS documented to date: inhibiting Phospholipase D. Our focus on a key role for aS in lipid metabolism and membrane vesicle formation/stability derives from a novel set of observations made by the two principal investigators. Moreover, it is strongly supported by recent unbiased genetic screens in aS-expressing yeast or Drosophila that implicated a function of aS in lipid regulation and membrane trafficking. New findings emanating from this grant should simultaneously shed light on the physiology of aS and the earliest steps in its pathological oligomerization, with attendant therapeutic insights. [unreadable] [unreadable]

Gamma-Secretase is an unusual enzyme that catalyzes the intramembrane proteolysis of numerous type I integral membrane proteins. These include the amyloid-beta protein precursor (APP), which releases the amyloid-beta peptide (Abeta) implicated in Alzheimer's disease (AD). For this reason, gamma-secretase is a prime target for the development of therapeutic and preventative agents for AD. Pharmacological, genetic, and biochemical evidence generated under this project strongly supports the hypothesis that the polytopic membrane protein, presenilin (PS), is the catalytic component of a larger multi-protein gamma-secretase complex. PS is normally cleaved into two fragments that remain associated. These heterodimers are metabolically stable, and their formation is tightly regulated by limiting cellular factors. During the current project period, we discovered that both PS endoproteolysis and gamma-secretase activity require two conserved transmembrane aspartates, consistent with our provocative hypothesis that presenilin undergoes autoproteolysis to become the catalytic component of a novel aspartyl protease. Moreover, using affinity isolation on a designed inhibitor, we showed that the PS-associated protein, nicastrin, is another member of the complex. Recently, our laboratory has found that the co-expression of PS1, nicastrin, and two proteins identified in C. elegans, aph-1 and pen-2, results in increased PS heterodimer formation, full nicastrin maturation and enhanced gamma-secretase activity. All 4 proteins co-immunoprecipitate and bind to a gamma-secretase affinity matrix. Taken together, these new finding suggest that PS, nicastrin, aph-1, and pen-2 assemble as a complex that leads to the formation of active gamma-secretase. In light of these discoveries, we now propose to purify, fully characterize and then reconstitute active gamma-secretase. Specifically, we will: 1) scale up and refine the multi-step purification protocols for gamma-secretase established by our labs in order to unequivocally confirm the presence of these 4 key components and search for any additional protein factors by mass spectrometry; 2) systematically explore phospholipid, detergent, ionic, energy, pH and other requirements to optimize our established in vitro cleavage assay (which can generate Abeta, AICD and NICD) and characterize the kinetic properties of the protease; and 3) using this new knowledge, carry out the step-wise addition of recombinantly expressed PS, nicastrin, aph-1, and pen-2 in a pure detergent/phospholipid environment to reconstitute proteolysis. The purification, detailed characterization, and reconstitution of gamma-secretase will aid the understanding of this essential protease in health and disease and the search for effective and safe AD therapeutics. Moreover, this work will advance our fundamental knowledge of a unique new class of intramembrane-cleaving proteases.

Affect; Anabolism; Apolipoproteins; Assay; Bilayer Fluidity; Binding; Binding (Molecular Function); Bioassay; Biochemical; Biologic Assays; Biological Assay; Biological Models; Brain; Brain region; Cells; Cerebro-Hepato-Renal Syndrome; Cerebrohepatorenal Syndrome; Coloring Agents; Corpus Striatum; Corpus striatum structure; Cytoplasm; DA Neuron; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Dopamine neuron; Dopaminergic Cell; Drosophila; Drosophila genus; Dyes; Dysfunction; Encephalon; Encephalons; Enzymes; Exposure to; FM 1-43; FM1 43; Family; Fatty Acids; Fruit Fly, Drosophila; Functional disorder; Genetic Condition; Genetic Diseases; Genetic Screening; Goals; Grant; Health; Hereditary Disease; Human; Human, General; Idiopathic Parkinson Disease; Knockout Mice; Lecithinase D; Lewy Bodies; Lewy Body Parkinson Disease; Life; Light; Lipid Binding; Lipids; Mammals, Mice; Man (Taxonomy); Man, Modern; Membrane; Membrane Fluidity; Membrane Protein Traffic; Membrane Structure and Function; Membrane Traffic; Metabolism, Lipids/Lipoproteins/Membrane Constituents; Mice; Mice, Knock-out; Mice, Knockout; Model System; Models, Biologic; Molecular; Molecular Disease; Molecular Interaction; Murine; Mus; NAC precursor; Nerve Cells; Nerve Unit; Nervous System, Brain; Neural Cell; Neurites; Neurocyte; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neurons; Null Mouse; Overexpression; PARK1 protein; PARK4 protein; Paralysis Agitans; Parkinson; Parkinson Disease; Parkinson's; Parkinson's disease; Parkinsons disease; Phosphatides; Phosphatidylcholine Phosphohydrolase; Phospholipase D; Phospholipids; Photoradiation; Physiology; Physiopathology; Population; Primary Parkinsonism; Principal Investigator; Process; Property; Property, LOINC Axis 2; Protein Overexpression; Proteins; Rate; Regulation; Role; SNCA; SNCA protein; Series; Solubility; Striate Body; Striatum; Structure; Synaptosomes; Therapeutic; VESCL; Vesicle; Yeasts; Zellweger Disease; Zellweger Syndrome; alpha synuclein; alphaSP22; aqueous; base; biosynthesis; brain cell; congenital iron overload; dimer; dopaminergic neuron; fat metabolism; fatty acid binding protein; fatty acid oxidation; fatty acid-binding proteins; fruit fly; gene product; genetic disorder; hereditary disorder; in vivo; inhibitor; inhibitor/antagonist; insight; lipid metabolism; lipophosphodiesterase II; membrane structure; monomer; mouse model; neurodegenerative illness; neuronal; non A-beta component of AD amyloid; non A4 component of amyloid precursor; novel; overexpress; pathophysiology; phosphatidylcholine phosphatidohydrolase; social role; striatal; synaptoneurosome; synuclein

Intensive research by many laboratories, including work performed under this grant, has advanced the hypothesis that altered processing of the 6-amyloid precursor protein (APP) or decreased clearance of its amyloid beta-peptide (AlphaBeta) fragment are critical pathogenic events in Alzheimer's disease. Nevertheless, despite a wealth of reports about in vitro and in vivo activities of the precursor and its derivitives, the fundamental physiological function of APP has not been established. Based on recent studies in our and other laboratories and extensive preliminary data, we propose to elucidate systematically the function of APP and the molecular mechanism by which it accomplishes this function. The central hypothesis motivating this proposal is that the remarkably similar proteolytic processing of APP and the Notch receptors suggests that APP is itself a cell-surface receptor with a cognate ligand, the binding of which enables the release of the cytoplasmic domain (AICD) to the nucleus to activate specific genes. Based on substantial experience in APP processing and membrane protein biology obtained during the past 24 years of this grant, we will carry out 4 interrelated Aims: 1. to purify, identify and characterize a specific protein ligand(s) for the APP ectodomain;2. to localize the APP intracellular domain (AICD) to the nucleus and study its stabilization and activites there;3. to assess the phenotypic consequences, including for APP processing and intracellular AlphaBeta generation, of signaling by the physiological ligand(s) in intact neurons;and 4. to establish the in vivo biochemical and electrophysiological effects of APP signaling in living animals. We will compare the ligand binding and potential signaling properties of APP to those of APLP-1 and APLP-2 and also explore whether APP processing and signaling alter those of Notch. Intriguing preliminary findings about AICD nuclear localization and stabilization by Fe65 and about transcriptional changes in the brains of APP knockout mice encourage us to pursue these aims. The results of the multifaceted experimental approach described herein should be revealing not only for the normal biology of APP but also for how its regulated signaling contributes to the pathogenesis of Alzheimer's disease and to the progressive accumulation of A6 that accompanies normal brain aging in humans and other primates.

y-Secretase cleaves within the transmembrane region of a growing list of type I integral membrane proteins, including the amyloid (3-protein (Ap) precursor (APR), Notch, Erb-B4 and N-cadherin, mediating events in cell and developmental biology and the pathogenesis of Alzheimer's disease (AD) and cancers. In the past five years, much progress has been made toward elucidating the biochemistry and biology of this protease. These advances include the validation of y-secretase as a novel aspartyl protease complex, with the multi-pass Presenilins (PS), mutated in familial AD, as the catalytic component. Purification to homogeneity demonstrated that PS and membrane protein cofactors Nicastrin (NCT), Aph-1, and Pen-2, are sufficient for y-secretase activity. More recently, electron microscopy (EM) coupled with single particle analysis revealed that the complex apparently contains a large interior cavity and two small ports that could allow entry of water. Despite these advances and the protease's growing biological importance and medical relevance, much remains unknown about g-secretase. The overarching goals of this project are to understand how this membrane-embedded enzyme complex carries out intramembrane proteolysis, determine how it can be biochemically and pharmacologically regulated, and appreciate the scope of its substrates. With these goals in mind, four specific aims are proposed: (1) Determine the detailed structure of the g-secretase complex through crystallographic analysis of individual members and cryoelectron microscopy of the full complex. (2) Develop small molecules that modulate g-secretase substrate selectivity to lower Ab production without affecting proteolysis of the Notch receptor, which is implicated in the toxicity of gsecretase inhibitors. (3) Determine the effects of lipids and other membrane components on the activity of this membrane-embedded protease complex. (4) Identify novel substrates for g-secretase, the proteasome of the membrane, and understand the competition between substrates in cells. LAY SUMMARY: We have discovered an enzyme that plays a key role in Alzheimer's disease and that is widely considered an important drug target. This enzyme is highly complex, being composed of four different proteins, and carries out an unusual water-requiring reaction in the water-repelling environment of cell membranes. Our research plan aims to understand critical details about how this enzyme works, how it can be controlled by lipids and drug prototypes, and the range of its roles in biology.

The Administrative and Educational Core plays the key role of managing and integrating all the activities of our highly interactive research group. The aims of Core A (below) are designed to effectively administer the scientific planning, data and reagent sharing, education, consultation and administrative functions of our program, and they reflect the commitment of the 5 principal investigators to their continued scientific development as well as that of the program as a whole. Expenses for all non-experimental parts of this Program are included in the Core A budget. The Aims of Core A are: 1) To enhance the scientific exchange, collaboration and communication among all the co-investigators and their respective scientific personnel across the 6 labs of the program (4 at MGH and 2 at BWH), including inter-institutional personnel and reagent exchanges, assisting with joint publications, regular exchange of all publications emanating from the grant, and internal sharing of mentoring responsibilities. 2) To contribute to the continuing education of the program's members and the Boston scientific community at large about new developments in basic and applied biology related to neurodegeneration, via our highly successful seminar series. 3) To efficiently manage the program's business activities, including regular income/expense reports, preparation of progress reports each year, organization of this competitive renewal application, and communications with the NIA. The Administrative Coordinator, under the guidance of the Program Director, functions as the point person for all the business and administrative tasks related to the above 3 aims as they arise from the following specific responsibilities. These are: the coordination of meetings of the Working Group and the Scientific Advisory Board (SAB) and the preparation of meeting materials and any travel-related issues for SAB members; organizing, scheduling and publicizing our annual seminar series and handling all speaker-related issues, including scheduling meetings with program scientists, coordinating travel needs, processing reimbursements, etc.; assisting with distribution of publications arising from the program among our personnel as well as dealing with shipments or licensing issues related to the resources and reagents generated by the program; managing all processes related to the timely submission of reports, renewal applications or any other communications with the NIA. In short, the Administrative Coordinator ensures the smooth and timely functioning of all non-scientific aspects of the program project on a daily basis and is an indispensible part of the Core.

DESCRIPTION (provided by applicant): Studies from many laboratories now generally support the amyloid (or Abeta) hypothesis of Alzheimer's disease (AD), and therapeutic trails based on this concept have begun. Nevertheless, we are left with many unresolved and fascinating biological questions about how a small hydrophobic peptide slowly accumulates in the brain with age and can apparently initiate cytopathology. Perhaps foremost among these questions is exactly why cerebral Abeta levels are elevated in humans who develop AD. Increased Abeta production appears to explain only a small minority of cases, principally those inheriting mutations in APP or the presenilins. This realization suggests that many, perhaps most, cases of AD are caused by faulty clearance of a peptide that is produced at normal levels throughout life. Our proposed work here is based on 3 related hypotheses: 1) defects in proteases which degrade Abeta may underlie some or many cases of familial and sporadic late-onset AD;2) non-proteolytic clearance mechanisms (e.g., receptor-mediated efflux through the blood brain barrier) could explain the rise in cerebral Abeta in other cases;and 3) whether or not these two mechanisms are actually causative, subtly activating such proteases or else stimulating protein transport mechanisms could lower cerebral Abeta levels therapeutically. To extend our productive work under this grant on both the basic and applied aspects of this understudied subject, we propose 3 aims: Aim 1: To determine whether central or peripheral expression of engineered versions of two well-characterized Abeta degrading proteases - neprilysin and insulin degrading enzyme (IDE) -can safely and effectively decrease cerebral Abeta burden in mouse models of AD. Aim 2: In view of the recent unequivocal demonstration that IDE exists normally on the plasma membrane (namely, as the receptor for varicella-zoster virus), to further ellucidate the unusual cell biology and membrane trafficking of IDE that enables it to be involved in the degradation of both extracellular/intraluminal substrates (e.g., Abeta, insulin, amylin) and cytoplasmic substrates (e.g., AICD). Aim 3: To pursue a recently initiated project that addresses an even less well-studied aspect of Abeta clearance than proteolysis, namely, to rigorously identify and validate the cell- surface receptors capable of mediating the efflux of soluble Abeta across the BBB, using both in vitro and in vivo models. Understanding how both proteolysis and non-proteolytic clearance regulate Abeta levels in the brain has major implications for both the genesis and treatment of this complex disorder. At the same time, the proposed experiments have fundamental implications for peptide turnover in the mammalian brain and for the cell biology of metalloproteases. Many scientists now support the idea that the gradual buildup of a small protein, amyloid beta-protein (Abeta), in brain regions serving memory and thinking causes Alzheimer's disease. This grant will examine exactly why this buildup occurs over time, focusing on two potential problems: 1) faulty cutting up of the Abeta protein in the brain;or 2) faulty transport of the Abeta protein from the brain into the circulation. We will also explore new ways to increase the cutting up or the transport of Abeta as future therapeutic approaches for preventing Alzheimer's disease.

DESCRIPTION (provided by applicant): The discovery of presenilins as ubiquitous intramembrane proteases in metazoans exemplifies an emerging trend in biology. The traditional distinction between basic and applied research has become increasingly blurred, as studies initiated with a strictly disease-oriented focus uncover fundamental biological mechanisms. In this renewal application, ten investigators who have collaborated successfully during the last decade on the normal and pathological biology of presenilin (PS) wish to extend their productive interactions into new experiments that will further illuminate the structure-function relationships of PS/ ? -secretase in normal biology and the role of this unusual multi-protein complex and various related gene products in the genesis of Alzheimer's disease. Based on extensive preliminary data in each of our four interrelated projects and supported by three vital cores, we will pursue numerous Specific Aims that include: 1. attempting to determine the structure of PS/ ?-secretase by performing cryo-electron microscopy of the purified, active complex as well as x-ray crystallography of the individual components;2. extending our recent discovery that cholesterol dramatically enhances the catalytic activity of purified ? -secretase and that membrane lipids in general appear to represent the most potent regulators of both A[unreadable] production and Notch cleavage;3. performing extensive genomic, functional genetic and protein analyses searching for novel lateonset AD candidate genes potentially implicated in presenilin-related pathways;4. using advanced microscopy approaches in living cells (bimolecular fluorescence complementation;FLIM) to image and quantify the conformations and interactions of presenilin with APP, with new candidate genes emerging from (2), and with lipids and ? -modulating compounds identified herein;5. identifying common binding partners and common subcellular processing pathways for the (32 subunit of NaJ, APP and APLP2, three proteins that are processed identically by both (3- and y-secretase. 6. performing an unbiased proteomics screen to more fully define the " ? -secretome", i.e., the extent of unknown ?-substrates. These and numerous additional aims and sub-aims will be pursued collaboratively across our 4 projects, with progress in one project modifying experimental plans in another. In short, we are anxious to continue our close interaction to bring greater clarity to exactly how PS and its associated proteins and lipids process a host of substrates within membranes in health and disease. Lay summary: Ten experienced scientists who have collaborated productively for the past decade wish to address the structure and the functions of an unusual protein-cutting enzyme that is required for life in all multi-cellular animals and implicated in the causation of Alzheimer's disease.

Since its cloning in 1995 and its identification (under this grant) as an unprecedented intramembrane aspartyl protease in 1999, Presenilin has been implicated in a remarkable array of signaling and regulatory events in all metazoans. PS was discovered through research on Alzheimer's disease, but it was soon shown to confer functions necessary for life, especially as the protease that enables Notch nuclear signaling. Thus, continuing to decipher the structure, functions, and protein and lipid regulators of PS is a priority for basic cell biology. At the same time, the invariant cerebral accumulation of amyloid beta-protein (AU) decades before symptoms of dementia has made PS/gamma-secretase a key target for mechanistic and therapeutic study in AD. Despite its pleiotropic role in biology, the protease's structure has only been resolved at 12 A (under this grant), and small molecules that can selectively inhibit its processing of APP are not yet validated. For all these reasons, six collaborators with deep experience in the study of Presenilin wish to apply a range of techniques in cell biology, genetics, chemistry, structural biology and animal modeling to tackle some of the thorniest questions in PS/gamma-secretase biology. Can one derive an atomic resolution structure of this 19- transmembrane complex? What is the cell biological mechanism of coordinated alpha-, beta- and gamma-secretase processing? How do certain synaptic proteins and membrane lipids regulate PS activity in neurons, affecting the crucial A(l42/4o ratio? Can one design drugs that are sufficiently potent yet selective to chronically inhibit gamma-secretase? Our group has carefully revised this application to address all of the thoughtful critiques the SEP offered. We propose numerous interrelated aims that incorporate three cross-cutting themes which unite our work. First, we will further confirm and extend our recent discovery of an endogenous complex of the alpha/beta/gamma-secretases (a sheddasome) that may mediate the efficient, sequential processing of APP - and presumably all gamma-substrates. Second, we will apply a unique FRET-based probe developed here to measure PS conformation in living neurons and learn if certain synaptotagmins we recently identified by proteomics as novel interactors of both PS1 and APP enable PS to change its conformation rapidly and reversibly in response to Ca2+ influx at the synapse, explaining the enhancement of AB production by neural activity. Third, we'll study novel gamma-secretase modulators and Notch-sparing inhibitors we've developed to define SARs for APP vs. Notch cleavage, identify the cognate binding sites, and assess their actions on other substrates, all with the goal of advancing one or more into preclinical development. In short, we are committed to applying novel approaches to elucidate the structure and function of gamma-secretase in health and disease.

The discovery of presenilin (PS) as the first intramembrane aspartyl protease and the catalytic center of gamma-secretase occurred in Project 1. Since then, >100 substrates have been identified. PS/gamma-secretase mediates critical signaling pathways necessary for life in all metazoans, and its cleavage of APP releases the amyloid B-protein that accumulates in all patients with AD. After identifying PS as a protease, Project 1 has continued to contribute actively to PS biology. We proposed - and provided the first evidence - that holoPS undergoes autocatalytic endoproteolysis to generate the active heterodimer, first reconstituted PS and its 3 cofactors in mammalian cells, purified the protease to homogeneity, obtained the first 3D structure of the ycomplex by EM, conducted SILAC screens to identify several new substrates, and designed many gamma-secretase inhibitors, some of which are potent and much more APP-selective than compounds tried in humans. The Project is now revised to respond to all of the SEP's helpful critiques. We propose to study 4 related topics in the biochemistry of gamma-secretase. 1: A new cell biological model of secretase processing We have discovered that contrary to current concepts, alpha- (ADAM 10), beta- and gamma-secretases exist in part in a large protein complex that can mediate efficient sequential processing of substrates. Our extensive supporting data include robust co-IP of endogenous a- and gamma-secretases from wt brain and the sequential alpha/gamma processing of an APP substrate. We propose to fully confirm this new model of regulated intramembrane proteolysis and ask if it generalizes to another membrane protease pair: S1P/S2P. 2: The complex regulation of gamma-secretase by membrane lipids. We will extend our recent evidence that certain head groups and fatty acyl side chains of membrane lipids potently up- and down-regulate gamma-cleavages, including the key A(i42/40 ratio. We'll seek to validate robust in vitro effects of certain lipids by manipulating their cognate biosynthetic and catabolic enzymes in vivo. 3: Toward greater structural resolution of the gamma-secretase complex Working with leading structural biologists, we will pursue the technically challenging but essential quest for the structure of gamma-secretase via: a) further cryo-EM analyses of 2D crystals; b) 3D x-ray crystallography of individual gamma-components (PS, Net, Pen-2); and c) attempted 3D x-ray crystallography of the purified holo-enzyme. 4: Refining potent and selective Notch-sparing gamma-inhibitors and defining their mechanism. Building on more than 1,600 compounds we've synthesized, we will develop SARs for inhibiting APP vs. Notch, and for the most selective compounds, assess cleavage of other gamma-substrates and test them in mice. These aims build on our experience to tackle some of the thorniest problems in gamma-secretase biology.

DESCRIPTION (provided by applicant): Pathogenic aggregation of alpha-synuclein (alphaSyn) is increasingly implicated in familial and sporadic Parkinson's disease (PD) and other human synucleinopathies. Based largely on studies of the recombinant protein, alphaSyn has long been defined as a natively unfolded monomer of 14 kD that is believed to acquire secondary (alpha- helical) structure only upon binding to certain lipid vesicles. In contrast, our lab discovered in 2011 that endogenous alphaSyn isolated under non-denaturing conditions from living human cells and neuronal lines occurs principally as a helically-folded tetramer of ~60 kD. Multiple methods, including sedimentation equilibrium analysis by analytical ultracentrifugation, scanning transmission electron microscopy, mass spectrometry and circular dichroism, established the existence of an alpha-helically folded tetramer. Not unexpectedly, this discovery engendered controversy, but we have recently completed an extensive analysis of living cells (including neurons) using in vivo crosslinking that has confirmed that the principa form of native alphaSyn in intact cells is a 60 kD tetramer. Also, 3 other labs recently provided evidence for the occurrence of alpha-helical oligomeric structures based on certain methods of preparing alphaSyn. If this finding that endogenous alphaSyn exists as a helically-folded tetramer can be further extended, it will have major implications for the biology of alphaSyn in health and disease, as the reviewers of our first version of this application recognized. A central concept of human neurodegenerative diseases -- that normally soluble proteins (alphaSyn, tau, Abeta, etc.) can misfold and aggregate into neurotoxic species - depends on understanding the normal state of the protein and what misfolding and aggregation actually mean. Accordingly, we propose an integrated series of entirely novel Specific Aims to characterize the dynamic relationship of the metastable alphaSyn tetramers to the unfolded monomer believed to be the native structure of alphaSyn since its description 20 years ago. Aim 1 Raise conformation-specific monoclonal antibodies to purified alphaSyn tetramers as key tools for all Aims. Aim 2 Examine the effects of four PD-causing missense mutations and certain structure-altering artificial mutations on the kinetic equilibrium of tetramers and monomers in intact cells. Aim 3 Study the biochemical mechanism and dynamics of the assembly of freshly synthesized monomers into tetramers (and other oligomers), their stability in the cell, and their subsequent disassembly. Aim 4 Purify endogenous human ?Syn from normal and diseased (DLB) brains to establish its structure and assembly state in the most disease-relevant organ; then, systematically analyze its biochemical properties. We have made major progress towards these Aims since our first submission (see Preliminary Data). Our work elucidates the dynamic relationship between metastable physiological (alpha-helical) oligomers, unfolded monomers, and abnormally folded (beta-sheet-rich) oligomers of ?Syn in human cells and brain, with attendant mechanistic and therapeutic implications for PD.

Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) produce profound motor and cognitive impairment associated with aggregation of ?-synuclein (?Syn). In these diseases, ?Syn undergoes complex post-translational modifications and fragmentation. In particular, many reports show increasing levels of C- terminally truncated (?C) ?Syn in brains of familial PD ?Syn mutation carriers and as a function of disease severity. Notably, truncated ?C-?Syn can induce soluble full-length ?Syn to co-assemble and thus accelerate, or even initiate, its aggregation. Limited research has focused on the proteases mediating ?C-?Syn truncation, their potential activation pathways, and the specific forms of the ?Syn substrate. Known caspase and calpain recognition sites occur in the ?Syn C-terminus, and inhibiting these proteases lowers ?C-?Syn and subsequent ?Syn aggregation. Moreover, the PI's recent studies of ?Syn in rodents exposed to paraquat, a herbicide linked to sporadic PD risk, showed elevated ?C-?Syn due to activation of calpain-1. However, these and other studies examining ?Syn in neurons or brain tissue have used denaturing conditions that preclude discriminating between monomers and the physiological ?Syn tetramer/multimers that our and other labs recently discovered. Using an intact-cell crosslinking assay that preserves the lysis-sensitive tetramers/multimers, we have shown that all fPD missense mutants shift normal tetramers to monomers. Further, we reported that mutating the N-terminal KTKEGV repeat motif, namely expressing E35K+E46K (?2K?) and E35K+E46K+E61K (?3K?) that mimic a doubling or tripling of the fPD E46K mutation, decreases the propensity of ?Syn to form normal tetramers/multimers. The resultant excess monomers associate with membranes, form ?Syn-rich vesicular inclusions, cause neurotoxicity, and could be subject to ?C truncation. Hence our central hypothesis is that excess ?Syn monomers arising under both familial and `sporadic' PD conditions provide the substrate for adverse proteolytic truncation. To pursue this hypothesis, we propose (Aim 1) to phenotype in detail our novel ?2K? and ?3K? tetramer-abrogating mice that develop neuronal aggregates in nigra and cortex containing ?C-?Syn and show progressive motor deficits with prominent tremor. As a distinct but complementary approach (Aim 2), we will apply paraquat to activate calpain-1 and to see if the resultant increase ?C-?Syn alters the multimer-to-monomer ratio by sequestering monomers into aggregates. Mechanistically, cholesterol-rich caveolar membranes regulate Ca2+ entry and calpain-1 activity, so we will ask if the calpain-mediated ?C truncation induced by paraquat or the fPD mutations is associated with changes of caveolar structure/structural proteins. In sum, we will generate new tools including novel tetramer-abrogating ?Syn tg mice and paraquat-treated neurons to identify ?Syn forms and examine proteases underlying the adverse ?C truncation that contributes to the progressive aggregation centrally implicated in PD and DLB.  

SUMMARY Genetic and cell biological evidence from many laboratories implicates both the normal function and the dysfunction of the presenilins in the fundamental mechanism of Alzheimer's disease. Progressive accumulation of A? appears to begin years before other important pathogenic features of AD such as neuroinflammation and tau tangle formation, and the presenilin/?-secretase complex mediates the final cleavages of APP which control the A?42/43 to 40 ratios that help dictate a person's lifelong propensity to AD. A failed clinical trial of a non-selective ?-secretase inhibitor (semagacestat) has led some to suggest that ?-secretase may no longer be a worthy target for therapeutic development, but we and others (e.g., DeStrooper, Cell, 2014) believe otherwise and seek much deeper knowledge of the presenilin cleavage mechanism -- in order to renew interest in targeting ?-secretase to prevent AD. Indeed, several classes of ?-secretase modulators (as opposed to inhibitors) have been described, and just a few have begun to enter clinical trials. Project 1's long-standing interest in presenilin biology has led us to publish during the current grant period several novel findings about ?-secretase: a) the existence of a physiological ?/?-secretase complex in cells; b) pinpointing the function of Nicastrin as a gatekeeper -- sterically hindering the processing of long substrates; and c) a detailed analysis of presenilin/APP TMD interactions and ?-processivity that explains the reason for the tri-peptide cleavages made by PS. Now, we will build on these advances to delve in further molecular detail into the basic mechanisms of wild-type and FAD mutant presenilin, into how and where certain ?-modulators allosterically influence their processivity, and into the cell biology of a normal ?/?-secretase complex we recently discovered that could be central to AD pathogenesis. We will pursue 3 interrelated but non-overlapping Specific Aims to gather this new knowledge. First, we will use a novel strategy (emerging from our most recent paper ? Bolduc et al, eLife, 2016) to analyze systematically many but not all familial AD mutations in PS1 to learn which PS1 amino acids contribute to the unusual active site that dictates the canonical tripeptide cleavage mechanism of ?- secretase. Second, we will use these FAD mutants to examine both the mechanisms and the PS1 binding sites of some of the most promising GSMs (?-secretase modulators), which are highly attractive candidates for slowing or preventing AD. Third, we will confirm and then functionally analyze an unexpected complex we recently discovered between the two key enzymes that make A? peptides: ?- and ?-secretase. Based on progress in the current grant period and extensive Preliminary Data herein, we are committed to using advanced cell biological and biochemical methods which we are deeply familiar with to elucidate the normal physiology and pathobiological role in AD of one of the most unusual and fascinating protein machines in metazoans: the presenilin/?-secretase complex.

! Aggregates of alpha-synuclein (?Syn) are a pathological hallmark of Parkinson's disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). Moreover, genetic increases in ?Syn expression and point mutations in the ?Syn gene are an increasingly well-documented precipitant of early- onset familial PD (fPD). Pathological oligomers have been extensively studied, but only detailed analyses of synthetic aggregates from recombinant sources are available. Therefore, we wish to characterize pathological ?Syn species we are isolating from PD/DLB and MSA brains. We plan here an extensive characterization of toxic and self-permissive aggregated forms of ?Syn, isolated from human patient brain. Characterization of the isolated species will be both functional (toxicity) and structural for a correlative analysis of structure-function relationship. Additional analysis of different brains will allow a comparison between structure specificity of the different diseases (disease ?strains?). The isolation procedure will focus on gentle, non-denaturing methods targeting 3 different sub-cellular locales (cytosol, membrane- associated, insoluble). Results from our novel concept could point to exciting possibilities in rational drug design based on exact structural details of pathogenic strains. Detection and quantification the pathogenic ?Syn oligomers may be used as a biomarker for diagnosis of synucleinopathies and will provide novel reagents to the community along with valuable platforms (strain specific PMCA assays) for therapeutic compound screening. To move our hypotheses forward, we propose to gather data in two major directions: Aim 1: Search for the existence of natively soluble `pathological seeds' of ?Syn in human brain homogenates of PD, DLB and MSA patients under non-denaturing conditions and quantify their bioactivity in dynamic assays of pathogenic aggregation and toxicity. Aim 2: Characterize the unique structural features (?strains?) of the brain isolated insoluble aggregates from each disease subgroup (PD/DLB/MSA) and their amplification products to contrast their biochemical and biophysical properties in relation to disease. Results from our novel concept could point to exciting possibilities in rational PD drug design based on exact structural details of pathogenic strains. In addition, detection and quantification the pathogenic ?Syn oligomers may be used as a biomarker for diagnosis of synucleinopathies and will provide novel reagents (synthetic human brain derived strains) to the community along with valuable platforms (strain specific detection assays) for therapeutic compound screening. ! !

A blood test to identify cognitively unimpaired individuals at risk for Alzheimer's disease (AD) and the emergence of AD in Down syndrome (DS) is desperately needed. Here, we propose experiments to address this unmet need. Specifically, we will measure (in plasma) levels of proteins (tau and A?) implicated in the etiology of AD, and naturally occurring autoantibodies (NAbs) against these proteins. In blood tau and A? are present in distinct pools, e.g. free floating, bound to other proteins, and inside exosomes. Numerous prior studies have measured A? in plasma, but few took account of the distinct pools and many were confounded by imperfect assays and/or patient specimens. Only a handful of studies have looked at tau in blood, none accounted for the molecular heterogeneity of tau or its occurrence in different pools. In contrast, we will be careful to use assays capable of detecting distinct forms of tau and to assess the contributions of different pools and how they may change with disease. Since tau is present in blood at very low levels and measurement of A? in plasma requires dilution to overcome matrix effects, we will use state-of-the-art in-house ultra-sensitive assays developed using the Simoa (tau) and Erenna (A?) platforms. NAbs-bound tau and A? will be liberated from antigen-antibody complexes and then measured, and free NAbs will be detected by quantifying binding to plate-immobilized antigen. Great care will be taken to include internal standards so as to monitor the sensitivity and reproducibility of our assays. Most prior studies evaluated analytes at only a single time point and there have been few longitudinal studies. Considering the long duration of both the pre-clinical and clinical phases of AD, and how distinct forms of tau, A? and NAbs may change during these protracted periods, it is not surprising that cross-sectional studies have, thus far, yielded conflicting and variable results. Mindful of these pitfalls, we propose for the first time to measure anti-tau and anti-A? NAbs and their cognate antigens in the same plasma samples using specimens from carefully characterized study subjects. To gain insight on how tau, A? and NAbs change throughout the course of the disease, we will apply our well- characterized assays to plasma samples from 3 distinct cohorts. These will include samples that have been collected prospectively before and just after clinical onset from individuals of whom a detailed set of clinical, genetic, and histopathology data exist, and plasma from 0-65 years old DS subjects. DS is the most common genetic cause of early-onset AD and most DS adults become demented before the age of 60. Thus, studying samples from different-aged DS subjects provides a window on different stages of AD, and should also identify the optimal therapeutic interval to treat AD in DS. Combining this systematic approach with careful statistical analysis we expect to identify one, or possibly a selection of analytes, that can: (i) accurately detect subjects at risk of developing AD, and (ii) determine an appropriate age range in which to treat AD in DS.