Thomas J. Montine, MD PhD - US grants

Alzheimer's disease is a major public health problem for the United States. Up to 4 million older Americans currently suffer with Alzheimer's disease, and up to 12 million are expected to by the year 2020. Rational design of effective therapeutics will require a detailed understanding of the pathophysiology of Alzheimer's disease. The long-term objective of this project is to understand how different isoforms of apolipoprotein E specifically modify the distribution and amount of neurotoxic lipid peroxidation products in the brain, thereby contributing to the stratification of risk for Alzheimer's disease with apolipoprotein E genotype. The specific aims of this application are: 1) to determine the mechanisms of apoE isoform-specific distribution of human native central nervous system lipoproteins by quantifying cell surface receptor binding, internalization, and degradation of human central nervous system lipoproteins in cultured neurons and astrocytes; 2) to determine the pathogenicity of apolipoprotein E isoform-directed delivery of oxidized central nervous system lipoproteins by purifying oxidized central nervous system lipoproteins from Alzheimer's disease patients or oxidizing ex vivo central nervous system lipoproteins from control subjects and quantifying their neurodegenerative and cytoskeletal damaging effects in neurons and astrocytes; and 3) to quantify regional brain lipid peroxidation in vivo and examine its relationship to differences in the apolipoprotein E gene by quantifying brain regional F2-isoprostanes and F4-neuroprostanes in Alzheimer's disease patients with different apolipoprotein E genotypes and in homozygous apolipoprotein E deficient mice. This new information will aid in laying the foundation for designing rational therapeutic approaches to ameliorate brain oxidative damage in Alzheimer's disease.

Alzheimer's disease (AD) is a serious public health problem for the United States. Experimental, post mortem, and initial clinical trials all support the hypothesis that regionally increased brain lipid peroxidation may contribute to the pathogenesis of AD. Lacking, however, has been the means to objectively measure brain lipid peroxidation during life. The long-term objective of this project is to determine if isoprostanes and neuroprostanes, specific free radical-mediated products of arachidonic acid (AA) and docosahexaenoic acid (DHA) peroxidation, respectively are quantitative intra vitam biomarkers of brain lipid peroxidation that may serve as surrogate biomarkers for AD severity, progression, and response to anti-oxidant interventions. The specific aims for this project are: 1) to determine if isoprostanes and neuroprostanes are accurate intra vitam biomarkers of brain lipid peroxidation, 2) to determine if the extent of brain lipid peroxidation, quantified by isoprostanes and neuroprostanes, correlates with AD severity and progression, and 3) to determine effective concentrations and doses of anti-oxidants that suppress brain isoprostane and neuroprostane production, and 3) to determine effective concentrations and doses of anti-oxidants that suppress brain isoprostane and neuroprostane production in vitro and in vivo. This new information will establish the relationship between brain lipid peroxidation and the progression of AD, and guide future development of anti-oxidant therapy for patients with AD.

DESCRIPTION: (adapted from applicant's abstract) Parkinson's disease (PD) is a serious public health problem for the United States. Although dramatic advances have been made in the clinical management of Parkinson's disease, understanding of the mechanisms that underlie its neurodegeneration is incomplete. Such knowledge will be necessary for future development of therapeutics that impede or halt the progression of Parkinson's disease. The long-term objectives of this project are to determine the mechanisms by which oxidation products of dopamine and related catechols, catechol thioethers, may be converted to neurotoxins that contribute to nigrostriatal oxidative damage and neurodegeneration. The Specific Aims of this projects are: (1) to determine the potency of endogenous catechol thioether produced in the mercapturic acid pathway and the mechanisms by which they may contribute to dopaminergic neurodegeneration in whole mitochondria, cultured dopaminergic neurons, brain regions of aged control and genetically engineered mice with deficiencies in anti-oxidant defenses, (2) to determine the activities of mercapturic acid pathway enzymes and the concentration of catechol mercapturates in brain regions and CSF from patients with Parkinson's disease, other nigrostriatal neurodegenerative diseases, and aged-matched controls, and (3) to determine the mechanisms by which environmental toxicants epidemiological linked with an increased risk of Parkinson's disease may augment neurotoxicity form catechol thioethers. This new information will determine the extent to which catechol thioethers produced in the mercapturic acid pathway may contribute to Parkinson's disease progression, interact with aging and environmental toxicants implicated in Parkinson's disease, and serve as intra vitam biomarkers of nigrostriatal degeneration.

Description (provided by applicant): The overall hypothesis of this rograin project is that progressively severe dopaminergic deafferentation of the striatum that occurs as Parkinson's Disease (PD) advances leads to unopposed glutatmatergic excitation on the spines of medium spiny neurons, ultimately causing degeneration of these structures. Our long-term goal is to determine the mechanisms that underlie this secondary generation in MSN spines in PD, new knowledge that may be useful in understanding and ultimately effectively treating the altered pharmacologic responses that complicate management of advancing PD. In Project 4, we propose to test this hypothesis by performing a comprehensive biochemical, ultra structural, and morphologic examination of MSN spines in tissue obtained by rapid autopsy (post-mortem interval less than 5 hr) from volunteers who died with PD, Dementia, with Lewy Bodies (a similar but less severe form of mesostriatal dopaminergic deafferenation), and age-matched controls, and to correlate these biochemical and structural changes with eachother and the clinical characteristics of each patient's movement disorder and response to therapy. The specific aims for Project 4: Aim 1 will be to determine ultrastructural features of MSN spine excitatory input onto MSNs spine heads and shafts in different regions of human striatum and to compare these with rats following unilateral dopaminergic deafferentation with 6-hydroxydopamine (6-OHDA), Aim 2 will be to determine the relative concentrations of protein elements of the glutamate receptor complexes, and Aim 3 will be to determine MSN spine degeneration by measuring striatal regional MSN spine density using Golgi silver impregnation and quantitative imaging.

oxidation; lipid metabolism; Alzheimer's disease;

Patients with the pathologic diagnosis of Alzheimer's disease (AD) commonly (approximately 30 to 60%) have concomitant Lewy body (LB) formation as detected with o_-synuclein immunohistochemical analysis of extra-nigral sites. These patients with AD/LB, along with a much less common dementia characterized by LB formation alone, are currently classified as having Dementia with Lewy Bodies (DLB). There is substantial clinical and pathologic heterogeneity among patients with DLB, thwarting efforts to understand fully the significance of distinguishing AD from DLB and strongly suggesting further distinct subgroups within what is currently called DLB. Here we propose to test the hypothesis that DLB differs from AD at clinical, pathologic, molecular genetic, and biochemical levels, and that these same criteria may be used to discem multiple distinct subgroups of DLB. We will expand an already functioning cooperative study among five Alzheimer Disease Centers (ADC) across the United States: Oregon Health & Science University, University of California at San Diego, University of Pennsylvania, University of Pittsburgh, and University of Washington. We propose to collect clinical and neuropathological data as well as banked tissue from approximately 100 age-matched controls, 250 patients with AD, and 250 patients with DLB. We estimate collection of 20, 50, and 50, respectively, additional cases for each year of this project. Using the robust design of patient data and material from five separate ADCs and biostatistical support from the National Alzheimer's Coordinating Center (NACC), we will test our hypothesis by pursuing these specific aims: to distinguish controls, AD, and DLB, and as well as DLB subgroups by determining (1) clinical and pathological features, (2) characteristics of candidate genes, (3) quantitative differences in oxidative damage, and (4) alterations in both soluble and insoluble forms of tan, A[3, and (z-synuclein. Successful completion of this project will solidify our understanding of DLB and provide a foundation for future clinical and molecular studies of this second most common form of dementia. Specifically, this project will establish a National DLB Resource, including a database of clinico-pathologic data, as well as an inventory of DNA samples and frozen brain tissue. This resource will be made available for future investigations of DLB.

[unreadable] DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common dementing disorder in the United States. AD already is a major public health burden that is poised to burgeon in the next generation. There is a clear therapeutic imperative for AD and a critical need to quickly and robustly validate candidate protectants from observational studies to insure that the best possible agents advance to randomized clinical trials, and to discover new and safe neuroprotectants. The long-term objective of this project is to provide pathological and biochemical validation of candidate protectants and to aid in the discovery of new candidates for AD. The Adult Changes in Thought (ACT) study is an ongoing large population-based longitudinal study involving approximately 3,200 elderly individuals who were cognitively intact when enrolled. A unique and powerful feature of ACT is its extensive pharmacy database that extends back to 1986. We propose to use this pharmacy database to test the hypothesis that some commonly used therapeutics suppress pathological features of AD-type neurodegeneration in elderly individuals without dementia, individuals with prodromal dementia, and patients with incident AD. We will test this hypothesis by determining associations between documented exposure to therapeutics and pathologic endpoints obtained in this project. Therapeutics to be evaluated include antihypertensives, antioxidants, non-steroidal anti-inflammatory drugs, hormone replacement therapy in women, and emerging candidate protectants identified during the course of this project. Pathologic endpoints to be determined in multiple brain regions obtained by rapid autopsy protocol are: (1) magnitude of oxidative damage to neuronal membranes (2) dendritic and synaptic degeneration (3) protein insolubility (4) density and staging of histopathologic structures, viz., neuritic plaques, neurofibrillary tangles, and Lewy bodies. This study offers a unique opportunity to discover and validate candidate protectants from among commonly used therapeutics, an endeavor unlikely to be pursued by pharmaceutical industry, and will provide needed rational either for or against the advancement of candidate protectants in randomized clinical trials for prevention of AD. [unreadable] [unreadable] [unreadable]

Alzheimer's disease (AD) is the most common form of geriatric dementia; however, there are other, partially overlapping, pathogenic processes that also contribute to cognitive impairment late in life, most frequently Lewy body disease and vascular disease. Surrogate measures such as biomarkers, in concert with neurological examination and neuroimaging, will aid in the diagnosis of geriatric dementias and serve as objective measures of disease progression and response to therapeutics. Here we propose to test the hypothesis that unbiased identification and quantification of the cerebrospinal fluid (CSF) proteome can be used to assemble a roster of proteins that objectively characterizes the different facets of geriatric dementia. We will test this hypothesis by accomplishing the following Specific Aims: (1) Discovery: Using Isotope Coated Affinity Tagging coupled with 2-dimensional liquid chromatography/electrospray ionization-mass spectrometry we will determine relative quantitative differences in the CSF proteome of controls vs. patients with different types of geriatric dementia, (2) Confirmation: Using a combination of immunochemical methods we will confirm the major discoveries of Aim 1 and develop a multianalyte profile (MAP) for geriatric dementias, (3) Validation: While blinded to diagnosis, we will validate our findings in a separate set of controls and patients with mild cognitive impairment (MCI) and different forms of geriatric dementia, and (4) Data and Sample Exchange: databases and resources resulting from these analyses will be made available to other investigators using alternative methods in the investigation of biomarkers for geriatric dementias. Once completed, these studies will have discovered, confirmed, and validated a MAP for objectively assessing different facets of geriatric dementia.

The Pacific Northwest Dementia and Aging (PANDA) Neuropathology (NP) Group is a cooperative effort between the NP Cores of the Alzheimer's Disease Centers (ADCs) at Oregon Health & Science University (OHSU) and the University of Washington (UW) that was initiated in July 2003. Primary motivations for forming PANDA NP Group were the unique geographic and demographic relationship of these two ADCs, already established extensive collaboration between faculty, the complementary nature of resources within the two NP Cores, and the new flexibility permitted by National Institute on Aging in organizing the required NP function, including emphasis on cross-center standardization and resource sharing. The Specific Aims of the PANDA NP Group are: (1) provide diagnostic expertise to the diverse population of the Pacific Northwest by providing family members of the deceased and physicians involved in their care with timely autopsy reports based on the most current standardized diagnostic criteria (2) facilitate research by collecting, storing and distributing a highly accessible, but appropriately safeguarded, repository of well-prepared brain tissue and neuropathologic data from carefully and longitudinally characterized patients with mild cognitive impairment or dementia as well as non-cognitively impaired control individuals using a variety of methods that maximizes usefulness to scientists (3) teach trainees in pathology, neurology, psychology, psychiatry, and basic sciences the current methods of neuropathological classification of neurodegenerative diseases and age-related changes and mentor junior faculty in neuropathology and (4) develop innovative new approaches to maximize achievements in Aims 1 to 3.

Patients with the pathologic diagnosis of Alzheimer's disease (AD) commonly (approximately 30 to 60%) have concomitant Lewy body (LB) formation as detected with o_-synuclein immunohistochemical analysis of extra-nigral sites. These patients with AD/LB, along with a much less common dementia characterized by LB formation alone, are currently classified as having Dementia with Lewy Bodies (DLB). There is substantial clinical and pathologic heterogeneity among patients with DLB, thwarting efforts to understand fully the significance of distinguishing AD from DLB and strongly suggesting further distinct subgroups within what is currently called DLB. Here we propose to test the hypothesis that DLB differs from AD at clinical, pathologic, molecular genetic, and biochemical levels, and that these same criteria may be used to discem multiple distinct subgroups of DLB. We will expand an already functioning cooperative study among five Alzheimer Disease Centers (ADC) across the United States: Oregon Health & Science University, University of California at San Diego, University of Pennsylvania, University of Pittsburgh, and University of Washington. We propose to collect clinical and neuropathological data as well as banked tissue from approximately 100 age-matched controls, 250 patients with AD, and 250 patients with DLB. We estimate collection of 20, 50, and 50, respectively, additional cases for each year of this project. Using the robust design of patient data and material from five separate ADCs and biostatistical support from the National Alzheimer's Coordinating Center (NACC), we will test our hypothesis by pursuing these specific aims: to distinguish controls, AD, and DLB, and as well as DLB subgroups by determining (1) clinical and pathological features, (2) characteristics of candidate genes, (3) quantitative differences in oxidative damage, and (4) alterations in both soluble and insoluble forms of tan, A[3, and (z-synuclein. Successful completion of this project will solidify our understanding of DLB and provide a foundation for future clinical and molecular studies of this second most common form of dementia. Specifically, this project will establish a National DLB Resource, including a database of clinico-pathologic data, as well as an inventory of DNA samples and frozen brain tissue. This resource will be made available for future investigations of DLB.

[unreadable] DESCRIPTION (provided by applicant): Parkinson-dementia complex (PDC) of the Chamorros on Guam and surrounding Mariana Islands is a unique neurodegenerative disease that remains a significant public health burden to the older members of this indigenous ethnic minority. Current data indicate that PDC is a complex phenotype that likely derives significant contributions from advancing age, inherited susceptibilities, and as yet to be clarified environmental factors. This complex phenotype represents a severe challenge to standard genomic and epidemiologic approaches to identifying key pathogenic elements in PDC. Identification of detergent-insoluble (DI) protein has provided keen insight into the molecular pathogenesis of other complex neurodegenerative diseases like Alzheimer's disease. Here we will test the hypothesis that discovering and quantifying DI proteins specific to PDC will provide much needed new perspective on this enigmatic disease and perhaps other neurodegenerative diseases. We will test our hypothesis through specific aims that employ novel discovery tools focused at the protein level so as to have the capacity to capture pathologic changes from either genetic or environmental influences. We will: (i) discover and quantify relative amounts of DI protein specific to PDC using LC-MALDI-TOF-TOF with isobaric tagging for relative and absolute protein quantification, (ii) confirm and validate results using a series of antibody-based techniques, and (iii) localize validated proteins in tissue sections from patients with PDC, other tauopathies, and appropriate controls. Once complete, data from these experiments will provide a fresh and unique perspective on new elements in the pathogenesis of PDC and thereby serve to focus future genetic screens, searches for environmental influences, and evaluation of experimental therapeutics in model systems. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant) [unreadable] [unreadable] The investigators are applying for support for years 31 to 35 for the Environmental Pathology/ Toxicology (EP/T) Program at the University of Washington (UW), a Training Program that has been continuously been funded by the NIEHS since 1978. The long-term goal of the investigators' program remains mentoring pre- and postdoctoral trainees to become successful independent scientists who are well equipped to respond to the environmental health research needs of the US in the coming generations. The long-established program has been highly successful in this endeavor, and proposed changes ensure that it conforms closely to the NIEHS Strategic Plan 2006. The investigators also are highly responsive to the recent restructuring of T32 applications announced by the NIEHS (NOTES-06-007). The Program is a 30-year-long collaboration between the Department of Pathology (School of Medicine) and the Department of Environmental and Occupational Health Sciences (DEOHS; School of Public Health and Community Medicine). The EP/T Program is divided into four research core areas (leaders): Gastrointestinal, Liver, and Kidney (Dr. Eaton); Neurotoxicology and Reproductive Biology/Development (Dr. Costa); Cardiorespiratory (Dr. Kavanagh); and Molecular Mutagenesis and Carcinogenesis (Dr. Monnat) with 27 training faculty, an increase of 5 from the previous cycle, who are mostly from the DEOHS (11) or the Department of Pathology (10). New to this competitive renewal is the addition of pre- and post-graduate training as well as three faculty in Genome Sciences, and new opportunities for trainees to participate in translational and clinical research programs. The EP/T Program is directed by Dr. Thomas Montine (Program Director), Dr. Elaine Faustman (Deputy Director), and a Steering Committee constituted by the research core leaders. The Program includes virtually all NIEHS-supported investigators at UW, is closely linked with NIEHS-supported centers at UW focused on ecogenetics, toxicogenomics, and risk assessment, among others, and is highly integrated with a wide array of well-funded, complementary research centers and projects. Given the investigators' excellent track record of training as well as the outstanding and growing opportunities at UW, they propose to increase their Program with two additional postdoctoral training positions for a total of 8 pre-doctoral and 5 postdoctoral trainees, and thereby continue to train future leaders in environmental heath science. [unreadable] [unreadable] [unreadable] BACKGROUND [unreadable] [unreadable] The current application proposes an increase of 2 postdoctoral positions for a total of 8 pre-doctoral and 5 postdoctoral positions. The faculty will be increased to 27, an increase of 5 mentors since the previous grant application. Basic research on disease mechanisms related to environmental health was the initial scope of the program when initiated in 1978 and was expanded to include research Clusters (cores) in 1998 (Cluster 1: Gastrointestinal, Liver and Kidney; Cluster II: Neurologic, Reproductive, and Developmental; Cluster III: Cardiorespiratory Diseases; and Cluster IV: Molecular Mutagenesis & Carcinogenesis. In the upcoming application period these aspects will be maintained and added emphasis will be placed on genome sciences and new opportunities for training and participation in translational and clinical research. The added emphasis on genome sciences for pre-doctoral and postdoctoral students is also reflective of the integration of the University of Washington's Genome Sciences Graduate Program into the proposed Training Program. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Parkinson's disease (PD) derives from the confluence of advancing age, inherited susceptibility, and environmental exposures. While the mechanisms involved are not yet clear, inherited abnormalities in alpha- synuclein (SNCA) and exposure to selected toxicants can mimic some of the facets of PD in rodent models. We propose that the point of convergence for these processes is innate immune activation, especially in the substantia nigra, which may have both deleterious and beneficial effects on neuronal survival. The prostaglandin E2 pathway, a major component of activated innate immunity, is effected through a family of four G-protein coupled receptors called EP1 through EP4. We will test the hypothesis that EP receptor subtypes are fundamentally important in determining the balance of neurotrophic vs. neurotoxic effects of innate immune activation. We will test our hypothesis through Specific Aims that use a genetically altered mouse models and potentially relevant toxicant models: MPTP and cytokine cocktails that mimic the central nervous system effects of peripheral endotoxin exposure. This application is responsive to several points in the NIH Blueprint: it is a direct response to the therapeutic imperative for PD, it is highly translational, and it draws on existing NIH-funded resources. When successfully completed, our proposed work will have advanced and refined our knowledge about potentially important new therapeutic targets that complement other ongoing efforts to protect dopaminergic neurons from innate immune activation and to reduce pathologi forms of SNCA in brain. PUBLIC HEALTH RELEVANCE We and others already have shown in rodent models that activation of innate immune response with increased production of prostaglandin (PG) E2 damages the same dopaminergic neurons that degenerate in Parkinson's disease (PD). In addition, increased generation of PGE2 in diseased regions of brain is a feature of patients with PD. Here we propose to focus on the PGE2 receptors, called EP1 through EP4, with the goal of developing new therapeutic targets for PD. We will test our hypothesis using genetically altered mouse models combined with potentially relevant environmental toxicant models: MPTP and cytokine cocktails that mimic the central nervous system effects of peripheral endotoxin exposure. When successfully completed, our proposed work will have advanced and refined our knowledge about potentially important new therapeutic targets that complement other ongoing efforts to protect dopaminergic neurons from innate immune activation in patients with PD.

DESCRIPTION (provided by applicant): Vascular brain injury from small (micro) vessel dysfunction (¿VBI) and Alzheimer's disease (AD) are highly prevalent in older adults, are commonly co-morbid, are major contributors to the dementia syndrome in the elderly, and are present in over 85% of cognitively normal individuals 75 years of age or older where these diseases presumably contribute to age-related cognitive decline. Enigmatically, while tremendous effort has been invested in understanding mechanisms of gray matter damage and neuron death in neurodegenerative diseases, carefully executed studies in humans and non-human primates have shown that neuron loss is not a feature of advancing age. In contrast, numerous studies, mostly neuroimaging-based, have associated white matter (WM) changes with advancing age; however, the molecular and cellular bases of this WM injury (WMI) with advancing age remain unclear. In this renewal, we hypothesize that ¿VBI and AD cause WMI through direct and indirect mechanisms that converge on deleterious responses that impede myelin repair. Indeed, our observations from the current cycle indicate that ¿VBI and AD conspire in the adult human brain to produce WMI and perturb response and repair of WMI through cellular and molecular mechanisms that are very similar to what we and others have demonstrated previously in pediatric WMI and adult demyelinating diseases. Our highly integrated multi-component R01 will continue to pursue the following Specific Aims: (1) Developing a unique and highly complementary resource of brain tissue from a human population-based study of brain aging and incident MCI. Tissue is prepared to maximize investigation of white matter. Using tissue from this unique resource, we will continue our work determining associations magnetic resonance imaging (MRI), histological, and immunohistochemical measures of white matter damage, (3) free radical damage to myelin or axons, (4) specific subpopulations of oliogodendrocyte precursor cells (OPCs), and (5) biochemical factors that suppress appropriate maturation and function of OPCs in aged brain. This project not only will continue to develop a unique resource for the community of scientists investigating white matter injury, but also employs this resource to answer key questions about the structural, cellular, and biochemical bases of WMI associated with cognitive decline in the elderly. PUBLIC HEALTH RELEVANCE: Age-related cognitive decline and prodromal dementia are of paramount public health import given the projected demographics of the US population. While numerous studies have associated white matter changes with advancing age, the structural and cellular bases of this white matter damage remain enigmatic. Continuation of our research project not only will continue to develop a unique resource for the community of scientists investigating white matter injury, but also will employ this resource to answer key questions about the structural, cellular, and biochemical bases of white matter damage associated with cognitive decline in the elderly.

The Pacific Northwest Udall Center (PANUC) is a collaborative enterprise among physicians and scientists at Oregon Health &Science University and the University of Washington that focuses considerable experience and expertise on cognitive impairment, a relatively poorly understood but devastating consequence that is common in patients afflicted with Parkinson's disease (PD). This proposed new Udall Center would be the only one in Oregon or the WWAMl region;WWAMI is an acronym for the cooperative arrangement among states in Pacific Northwest for which the University of Washington is the only medical school and academic medical center: Washington, Wyoming, Alaska, Montana, and Idaho. Thus, PANUC would cOver an area greater than one-fourth of the entire U.S.A. where more than 13 million Americans live. PANUC will achieve several goals of the NIH and NINDS. PANUC is: eminently patient-oriented, focused on a common non-motor feature of PD that is relatively poorly understood, organized into highly inter-related cores and projects that will promote exchange of new knowledge among clinical, translational, and basic research, anxious to contribute fully to the PD Data Organizing Center (PD-DOC) and the Coriell Institute for submission to the NINDS Human Genefics Repository, and composed of investigators who have a longstanding track record of highly collaborative productivity despite being at two institutions. Our mission is twofold. First, PANUC will serve PD patients, along with their caregivers and health care providers. We will provide improved education and clinical care, as well as offer new opportunities to participate in clinical research. Second, PANUC will discover and investigate the mechanisms that underlie cognitive impairment in PD using an array of cutting-edge technologies from accomplished and collaborative laboratories, generating novel resources for the community of scientists focused on PD, and identifying potential targets for future therapeutic development.

Age-related cognitive decline (ARCD) is a complex convergent phenotype that prominenfiy involves impaired execufive funcfion (EF) likely through disrupfion of the dorsolateral prefrontal cortex (PFC) circuit;despite much speculafion, the cellular and molecular mechanisms that underiie this presumed disruption have not been demonstrated in humans. Cognifive impairment (Cl) or dementia (D) is highly prevalent among pafients with Parkinson's disease (PD) but remains an incompletely understood non- motor complication of this devastating illness. Indeed, many pafients with PD have neuropsychologically determined "Cl, no demenfia" (PD-CIND) at the time of inifial diagnosis. Like ARCD, patients with PD- CIND or PD-D display prominently impairment of EF;however, also like ARCD, the cellular and molecular bases are not clear. We hypothesize that selective regional- and neurotransmitter-specific degeneration in PFC or anterior neostriatum contributes significantly to impaired EF in ARCD, PD-CI, and PD-D. In Project 2 we will test our hypothesis through the following Specific Aims: (i) determine the magnitude and regional distribufion of dopamine (DA), norepinephrine (NE), and serotonin (5HT) degeneration in neocortex and neostriatum, and their associafions with cognitive function test results, from aged individuals without PD or demenfia but varying levels of ARCD, aged individuals with pathologic changes of PD but not a clinical diagnosis of PD and varying levels of Cl, and patients with closely related neurodegenerafive diseases, (ii) determine the magnitude, regional distribufion, and associations with cognifive test results of neostriatal medium spiny neuron spinodendritic degeneration in the same people whose fissue was invesfigated in Aim 1, and (iii) determine if selective loss of DA neurotransmission without neurodegenerafion leads to regionally restricted spinodendrific degenerafion in mice from Project 1. Complefion of these Specific Aims will provide the first integrated clinical, behavioral, morphometric, and neurochemical analysis of PFC and neostriatum in ARCD and PD with and without cognifive impairment, as well as in novel transgenic mice. This project is responsive to several points in the NIH Blueprint: it is a direct response to the therapeutic imperative for PD, it is highly translational as it will inform crifically neurolmaging and therapeufic efforts, and it draws on existing NIH funded resources. RELEVANCE (See instructions): This project will provide rafionale for new evidence-based intervenfions for cognifive impairment in PD beyond DA replacement strategies, including enhancing synaptic levels of other neurotransmitters, prevenfing death of selected neuron populafions, or ameliorafing the consequences of cell death.

The Pacific Northwest Udall Center (PANUC) is a collaborative enterprise among physicians and scienfists at Oregon Health &Science University and the University of Washington that will focus considerable experience and expertise on cognifive impairment in pafients afflicted with Parkinson's disease (PD). The Administrative and Outreach Core for PANUC has overall responsibility for the goal of producing an integrated and collaborative multidisciplinary center of excellence that promotes cutting-edge research advances in PD, and integrates effecfively with local support and advocacy groups. The Director and Administrator, in full collaborafion with the Execufive Committee, will coordinate and integrate PANUC components, activifies, and resources;solicit and review pilot project applicafions;foster producfive interactions with other scientists and communifies in the Pacific Northwest to assure progress in scientific and educational initiafives;assure compliance with human subjects, animal welfare, scientific integrity, and financial policy requirements of the NIH, UW and OHSU;provide fimely transmissions of appropriate datasets to the PD Data Organizing Center (PD- DOC);collaborate fully with the Coriell Institute for submission to the NINDS Human Genetics Repository;and help train the next generation of researchers focused on PD. RELEVANCE (See instructions): The Pacific Northwest Udall Center (PANUC) will focus considerable experience and expertise on cognitive impairment in pafients afflicted with Parkinson's disease (PD). The Administrative and Outreach Core for PANUC will facilitate this by producing and supporting an integrated and collaborafive multidisciplinary center of excellence that promotes cutting-edge research advances in PD.

Age-associated memory impairment; Alleles; Alzheimer's Disease; Apolipoprotein E; Autopsy; biomarker; Brain; Brain region; Caregivers; Cerebrospinal Fluid; Clinical; Clinical Trials; Cognitive; cognitive function; Core Facility; Data; Dementia; Diabetes Mellitus; Disasters; Disease; effective therapy; Funding; Genes; Healthcare Systems; Human; improved; Indium; insight; Insulin; Investigation; Knowledge; mild cognitive impairment; Molecular Target; Neuraxis; Neurodegenerative Disorders; neuropathology; Neurosciences Research; novel; Pathogenesis; Patients; pre-clinical; Proteins; Proteome; public health medicine (field); Publications; randomized placebo controlled trial; Resources; response; Sampling; Series; Specificity; tau Proteins; Testing; Therapeutic; Tissues; United States National Institutes of Health

DESCRIPTION (provided by applicant): This application seeks a renewal of NIA T32 AG00258 entitled Neurobehavior, Neuroendocrinology, and Genetics of AD. By the end of our second funding cycle our program will have supported 27 post-doctoral trainees (12 with M.D. degrees, 3 of whom also have PhDs, and 15 with Ph.D. degrees). Our training efforts have been successful, as evidenced by the accomplishments of our trainees, as well as by our ability to attract qualified candidates and physician trainees to our program. In the next funding cycle, we will continue to provide post-doctoral training in clinical research regarding the neurobehavior, neuroendocrinology, and neurogenetics of Alzheimer's disease (AD) and related dementias. In particular, the program will focus on training clinical researchers capable of translating critical findings from basic science into hypotheses regarding the etiology, pathophysiology, and treatment of AD. Furthermore, clinical researchers will receive specialized training in two areas of study, neuroendocrinology and neurogenetics, that hold promise for increasing our understanding of the pathogenesis of AD and for developing new therapeutic approaches. These areas are not typically emphasized in graduate training of psychiatrists, neurologists, or neuropsychologists. However, recent advances have underscored the importance of genetic factors such as amyloid precursor protein and presenilin mutations, and apolipoprotein e genotype. Neuroendocrine factors such as disruptions of lipid, insulin and glucose metabolism, inflammation, and glucocorticoid status may also play an important pathogeneticjole in modifying the effects of AD susceptibility genes, thereby affecting the neuropsychologic expression of AD. Clinical investigators capable of bridging the fields of neurogenetics, neuropsychology, and neuroendocrinology will be needed to disentangle and define these potentially critical interactions. The program is supported by the rich and interactive research environment of the University of Washington and Veterans Affairs Puget Sound Health Care System, where a critical mass of faculty conduct both basic science and clinical research in the neuroendocrinology and neurogenetics of AD. The ADRC at the UWwill also serve as a resource for faculty and trainees. Although there is a long-standing commitment to aging training at our institution, our proposed program is unique in its interdisciplinary nature and focus on clinical translational research in AD and related conditions. As such, it will provide a much-needed approach to the training of clinical research scientists whose work will address these complex disorders. RELEVANCE: Our program trains researchers who are able to translate findings from basic science into clinical models of Alzheimer's disease and other dementias, and thereby facilitate the development of new treatments for these challenging diseases. In particular, the program focuses on the interactive role played by genetic and metabolic factors such as diabetes and stress in increasing the risk of dementia. These factors are highly prevalent in our society, and a better understanding of their role may lead to new treatments and strategies for preventing dementia or delaying its onset.

DESCRIPTION (provided by applicant): The overall goal of the University of Washington (UW) Alzheimer's Disease Research Center (ADRC) is to continue to provide a research milieu that facilitates the acquisition of new knowledge about the pathobiology of AD and related neurodegenerative dementing disorders and applies this knowledge to the development of experimental therapeutics. An interdisciplinary collaborative team of basic and clinical investigators, health care professionals, and administrative personnel supports and carries out an array of productive dementia research and career development programs. Although ADRC affiliated research addressing the genetics of AD and other dementing disorders will continue, the ADRC theme has refocused on biomarkers and experimental therapeutics. The ADRC supports productive research within the ADRC, in the greater UW research community and nationally through direct collaborations and formal multi-center programs such as the National Alzheimer's Coordinating Center (NACC), the Alzheimer's Disease Cooperative Study (ADCS), the integrated UW ADRC/Oregon Health and Science University ADC Neuropathology Core, and the NIA genetics of late-onset familial AD initiative. A career development environment in which fellows and junior faculty acquire research and clinical skills is supported by a productive pilot grant program. Previous accomplishments have generated basic, clinical and behavioral contributions to knowledge about AD. The ADRC proposes five ADRC cores and three research projects: Project 1: CSF binding profiling of drug- induced increased CNS insulin activity, Dr. Thomas J. Montine; Project 2: Therapeutic Effects of Intra-Nasal Insulin Detemir, Dr. Suzanne Craft; Project 3: Modulation of A¿ peptide accumulation and neuron damage in vivo with adult bone marrow transplants, Dr. C. Dirk Keene.

7. PROJECT SUMMARY [UNCHANGED] The development of strategies to prevent Alzheimer's disease (AD) and related dementing illnesses will depend on an understanding of the underlying pathologic processes. In recent years it has become apparent that in older persons, these illnesses are usually the result of two or more fundamental pathogenic processes, often interacting additively. This complexity has been recognized largely as a result of neuropathological research in the context of longitudinal epidemiologic projects such as the Nun Study and the Honolulu-Asia Aging Study (HAAS), both now completed. The proposed project will compile accrued data and images from 854 HAAS autopsies and approximately 500 Nun Study autopsies, develop a common dataset and archive of photographic images of brain sections, and will be employed in parallel assessments of newly revised neuropathologic criteria for the identification and measurement of the AD disease process, which will be compared to previous criteria. In addition, these same data will be utilized for in an in-depth analysis of the interdependent and independent roles of AD brain lesions and brain atrophy as proximate causal factors responsible for dementia. These efforts are expected to: (1) provide a foundation for future analytic use of the accrued resources of the two projects, (2) examine the likely impact and utility of the revised neuropathologic AD assessment criteria for future research addressing the dementing illnesses of late life, and (3) facilitate a conceptual convergence of our understanding of the causes and importance of brain atrophy from neuroimaging and neuropathological perspectives.

DESCRIPTION (provided by applicant): The preclinical development of Alzheimer's disease (AD) begins decades prior to onset of cognitive decline. Epidemiologic studies demonstrate that in cognitively normal aging populations, non-steroidal anti-inflammatory drugs (NSAIDs), which block cyclooxygenase (COX-1/COX-2) activity and PGE2 production, prevent development of AD. Given that the prevalence of AD doubles every 5 years in persons above the age of 65, a fundamental challenge in the AD field will be to stem the projected exponential increase in new AD diagnoses and the significant societal and economic costs that this will cause. A compelling clue to a mechanism underlying NSAID prevention has emerged from our recent studies modeling the preventive effects of NSAIDs in wild type and mutant APP mice, wherein we identified suppressive effects of ibuprofen on expression of enzymes involved in tryptophan metabolism. The enzymes TDO2 and IDO1 metabolize the essential amino acid tryptophan to kynurenine, itself a substrate of the neuroactive molecules quinolinic acid and kynurenic acid; moreover, in metabolizing tryptophan, the substrate for serotonin synthesis, TDO2 and IDO1 will negatively influence levels of serotonin. Importantly, recent biomarker studies in human serum report a significant increase of tryptophan metabolism in AD patients. Thus, in this proposal, we will test whether the enzymes TDO2 and IDO1 contribute to early and late development of AD pathology and cognitive decline using genetic and pharmacologic strategies in AD model mice. We will also test whether levels of tryptophan metabolites in cerebrospinal fluid and serum will correlate with measures of cognition, Aß42/tau ratios, and/or diagnosis in control, mild cognitive impairment (MCI), and AD subjects from the ADRC at the University of Washington. Our proposed studies will determine whether increased tryptophan metabolism is mechanistically linked to development of AD and whether TDO2/IDO1 tryptophan metabolism can be targeted in prevention and treatment of AD.

DESCRIPTION (provided by applicant): This proposal uses proteomics to better understand Alzheimer's disease pathogenesis with a large-scale, unbiased, and direct approach to discover and validate novel disease processes in postmortem AD brain, and to prioritize new targets for early stage therapeutic intervention. The AD proteome mediates the effects of aging, genetics and other risk factors and contains unidentified protein targets for therapies. The approach leverages the strengths of a national team of collaborating AD Centers and associated studies of aging, an innovative proteomics platform, advanced systems biology, and model systems to produce new treatment targets. The first aim will identify novel proteomic targets selectively altered in asymptomatic AD brain. Brains will be analyzed by mass spectrometry (MS), yielding discovery proteomes to compare 1) controls free of AD and other pathologies; 2) asymptomatic controls with AD pathology; 3) non-demented mildly impaired cases with AD pathology, 4) definite AD, and 5) other neurodegenerative diseases. Protein changes in synapses, insoluble aggregates, glial and neuron-specific nuclei, and select posttranslational modifications will be determined. Bioinformatics will be used with available large-scale data to identify potentially druggable targets in key networks and cellular processes. The second aim will validate candidate proteomic targets in postmortem brains from independent community and clinic-based cohorts and determine relationships with clinicopathological features, including cognition. Absolute levels of candidate proteins will be quantified using selected reaction monitoring MS. The third aim will establish links between the validated proteome and AD pathogenesis and druggability. The most promising candidates will be studied for effects on neuronal viability and interactions with Ass and tau using cell culture and drosophila models. These results and other data will drive selection of the most promising candidates to advance to mouse models to assess therapeutic potential.

The Pacific Northwest Udall Center (PANUC) is a collaborative enterprise among physicians and scientists at Oregon Health & Science University and the University of Washington that focuses considerable experience and expertise on cognitive impairment, a relatively poorly understood but devastating consequence that is common in patients afflicted with Parkinson's disease (PD). This proposed new Udall Center would be the only one in Oregon or the WWAMl region; WWAMI is an acronym for the cooperative arrangement among states in Pacific Northwest for which the University of Washington is the only medical school and academic medical center: Washington, Wyoming, Alaska, Montana, and Idaho. Thus, PANUC would cOver an area greater than one-fourth of the entire U.S.A. where more than 13 million Americans live. PANUC will achieve several goals of the NIH and NINDS. PANUC is: eminently patient-oriented, focused on a common non-motor feature of PD that is relatively poorly understood, organized into highly inter-related cores and projects that will promote exchange of new knowledge among clinical, translational, and basic research, anxious to contribute fully to the PD Data Organizing Center (PD-DOC) and the Coriell Institute for submission to the NINDS Human Genefics Repository, and composed of investigators who have a longstanding track record of highly collaborative productivity despite being at two institutions. Our mission is twofold. First, PANUC will serve PD patients, along with their caregivers and health care providers. We will provide improved education and clinical care, as well as offer new opportunities to participate in clinical research. Second, PANUC will discover and investigate the mechanisms that underlie cognitive impairment in PD using an array of cutting-edge technologies from accomplished and collaborative laboratories, generating novel resources for the community of scientists focused on PD, and identifying potential targets for future therapeutic development.

CORE A: ADMINISTRATIVE - ABSTRACT The Administrative Core provides leadership and expert support to ensure that the University of Washington (UW) Alzheimer's Disease Research Center (ADRC) is successful in pursuing its vision of precision medicine for Alzheimer's disease (AD). The Administrative Core's Specific Aims align with UW ADRC's Overall Specific Aims to build a research infrastructure, promote discovery, share with the community of physicians and scientist dedicated to developing solutions for AD, and propel advancement through commitment and collaboration. The Administrative Core's goals are (i) Provide scientific leadership and administrative direction, (ii) Organize regular administrative meetings, (iii) Recruit outstanding new faculty, (iv) Run a vibrant Pilot Award program for AD research across UW Medicine, (v) Provide expert grants management, fiscal support, and appropriate regulatory oversight for the Center and related activities, (vi) Promote seamless interactions among Projects and Cores, (vii) Facilitate collaborations with the national Alzheimer's Disease Center (ADC) Program and other researchers focused on AD, (viii) Ensure effective interactions with national and international efforts in AD research, and (ix) Maximize the impact of UW ADRC in our community, our university, and nationally. The Administrative Core integrates UW ADRC components and promotes synergy with the national ADC Program.

PROJECT 1 (MONTINE): Abstract Genetic risk for PD-related cognitive impairment and its disease mechanisms The inaugural PANUC award was funded to pursue the association of three candidate genes with cognitive impairment and dementia in PD: APOE ?4 allele and variants in SNCA and MAPT; to these we added GBA and LRRK2 mutations. With our collaborators, we demonstrated that cognitive impairment and dementia are significantly more common among PD patients who carry APOE ?4 or GBA variants, less common in patients with LRRK2 mutations, and not associated with variants in MAPT or SNCA. In this project our approach will be to bring insight to disease mechanisms by determining the corresponding molecular pathology. During the current PANUC award, we and others used standard histopathologic criteria for Lewy bodies (LB) or Alzheimer's disease (AD). Unfortunately, this approach has led to unexpected and conflicting results for GBA variants and APOE ?4 in PDD in part because some studies were limited by low number of cases or inconsistent attention to genetic risk. Specific Aim 1 proposes to fill these gaps in knowledge by regional quantification of neurotoxic proteins and synaptic degeneration using a novel technique developed by our laboratory coupled with a large, multisite autopsy cohort assembled by PANUC. Specific Aim 2 reflects that although successful with our candidate gene approach during the inaugural PANUC award, we propose to expand gene discovery for cognitive impairment and dementia in PD by evaluating additional candidate genomic regions recently identified by the PDCGC, and by determining genetic influences on longitudinal change in cognition measured prospectively using consensus assessments.

PROJECT SUMMARY/ABSTRACT Autopsy research has demonstrated that some individual who appeared to be cognitively normal in their final years had sufficient brain lesions at death to support a diagnosis of Alzheimer's disease (AD). Clearly the clinical and pathologic markers of the disease are not perfectly concordant. This phenomenon has been interpreted as implying that some individuals possess a special resilience to the cognitive decline commonly caused by the brain disease process. It suggests that clinical dementia might be prevented, delayed, or ameliorated even after the brain lesions have developed, if we could identify and cultivate those characteristics responsible for resilience. The basis of resilience could be one or a combination of several factors, including: (i) greater numbers of neurons and more extensive connectivity, (ii) a superior system of cognitive resources, including learned skills, (iii) having avoided certain aspects of the AD process, or of other structural brain disease distinct from AD that independently lead to cognitive decline and dementia. The proposed research will examine each of these alternatives in depth, employing a wealth of already available information accrued over the past 25 years of Nun Study and the Honolulu-Asia Aging Study research, plus new data to be generated with further detailed examination of Nun Study brains. Results are expected to inform the development of new and novel strategies for dementia prevention and illness amelioration.

Neuropathology Core (Core D) ? Project Summary The Neuropathology Core will provide state-of-the-art collection, storage, and distribution of brain and other biospecimens, establish neuropathological diagnoses of decedent Clinical Core participants, and provide resources and expertise for nonhuman primate (NHP) models for pivotal mechanistic and therapeutic AD research. The Core will oversee repositories derived from longitudinal collection of biospecimens obtained from adults at high risk for AD due to genetic and metabolic risk factors, as well as low-risk adults, and adults with MCI and AD, and from analogous studies in NHPs. These repositories will provide unique opportunities to identify novel biomarkers, neurodegenerative antecedents, and therapeutic targets, and will be made available to NCRAD and to ADC investigators. The Core utilizes an innovative inter-institutional leadership model in which a team of neuropathologists from Wake Forest and the University of Washington will employ state-of- the-art virtual microscopy, webconferencing, and biospecimen sharing to accelerate the development of Neuropathology operations at the newly established Wake Forest ADCC. Interinstitutional publication has already resulted, and an NHP study of age-associated AD-like neuropathology completed which provide proof of concept for this approach. Thus, we will accomplish the following Specific Aims: 1) To oversee a repository of brain tissue, CSF, DNA, and blood from participants enrolled in the Clinical and MESA Cores of the Wake Forest ADCC, using state-of-the-art methods for specimen collection, processing, storage, and distribution; 2) To facilitate distribution of data and specimens in the repository to Wake ADCC and ADC network investigators, as well as to NCRAD and AD researchers world-wide; 3) To conduct rigorous neuropathological diagnostic evaluations and clinical-pathological investigations of decedent Clinical and MESA Core participants, using an innovative inter-institutional model that incorporates the latest technology in virtual microscopy and web conferencing; 4) To facilitate the measurement of key biomarkers of AD pathology and innovative markers of metabolic/vascular function; and 5): To establish preclinical models of AD and pathological brain aging in NHPs using procedures analogous to human protocols, and in doing so (a) create a repository of brain tissue, CSF, DNA, biospecimens, and neuroimaging data, and (b) NHP cohorts and rodent models, that can be used for pivotal mechanistic and therapeutic studies.

ABSTRACT Genetic risk for AD now clearly highlights the potential for multiple molecular drivers and perhaps multiple pathogenic pathways, including forms of AD that derive from disease causing mutations in PSEN1 or PSEN2, increased risk from APOE ?4, and sporadic disease that does not have identified genetic risk. Regardless of genetic risk, AD is a chronic illness whose ultimate clinical expression as dementia follows years if not decades of injury, response to injury, consumption of reserve, and compensation. Moreover, as highlighted at the 2013 AD Related Dementias summit, longitudinal population-based cohort studies have repeatedly observed that AD most commonly is co-morbid with vascular brain injury (VBI) and less commonly with Lewy body disease (LBD). Finally, these same longitudinal cohort studies have revealed individuals who had high levels of AD neuropathologic change but no significant clinical expression ? a state of apparent resilience to AD. Here we propose to enable progress in precision medicine for AD by vastly improving the molecular characterization of disease and sharing this unique resource with the community of scientists. Indeed, much of our knowledge about injury/response to injury in AD is based on histopathologic assessments rooted in technology that is about 140 years old. Emerging technologies now permit a depth of molecular phenotyping that until recently was difficult even to imagine. We hypothesize that determining quantitative, high dimensional protein phenotypes from carefully clinically characterized individuals from longitudinal cohorts who have donated their brains for research will illuminate components of AD that currently are obscured by limited standard neuropathologic assessments. We will use this novel approach to test our hypothesis through three Specific Aims. In Aim 1, we will collect proteomics data on post-mortem brain samples from the University of Washington Alzheimer's Disease Research Center and the Adult Changes in Thought Study using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy known as data independent acquisition (DIA). DIA enables the comprehensive and systematic sampling of protein digests. This data acquisition will create a permanent digital molecular archive of this unique and highly valuable sample collection. In Aim 2, we will analyze the molecular phenotype of three groups that will be critical to precision medicine for AD: (i) different genetic risk, (ii) common co-morbidities, and (iii) resilience to AD neuropathologic change. We anticipate identifying a molecular signature that is predictive of cognitive impairment as a replacement for traditional histopathological assessment. In Aim 3, we will make our data available through a novel cloud based solution, called the Chorus Project (http://chorusproject.org), engineered to enable big data reanalysis by the community of scientists. We will develop a novel query engine that will enable informatics experts without knowledge of the complexities of mass spectrometry signal processing to perform reanalysis of our data.

Abstract/Summary The proposed work will investigate the pathogeneses of late life cognitive and motor impairments attributed to Alzheimer's disease, closely allied cerebrovascular and neurodegenerative diseases, brain aging, and their common patterns of co-occurrence in three existing cohorts: the Honolulu-Asia Aging Study, the Nun Study, and the 90+ Study. Our focus will be on relationships among objective, longitudinal measures of motor function and cognition?with the last obtained proximate to death?and a defined set of neuropathologic substrates identified at autopsy. Parallel and merged analyses will be carried out using existing resources provided from these three large population-based longitudinal studies composed of participants with distinctly different gender, age, and ethnicity. We will leverage these substantial existing resources to test the hypothesis that cognitive and motor impairments that occur with advancing age are syndromic and derive from a partially overlapping mix of diseases that vary among individuals in part by age, sex, and ethnicity, and that can be identified best at the current time by their neuropathologic features. This will allow the identification of common patterns for the singular, concurrent, or sequential development of cognitive and/or motor impairments in persons with and without dementia. The informational wealth of these three studies (all established and supported with NIA funding) will be further expanded and available to teams of collaborating researchers for continuing analyses.

DESCRIPTION (provided by applicant): This proposal uses proteomics to better understand Alzheimer's disease pathogenesis with a large-scale, unbiased, and direct approach to discover and validate novel disease processes in postmortem AD brain, and to prioritize new targets for early stage therapeutic intervention. The AD proteome mediates the effects of aging, genetics and other risk factors and contains unidentified protein targets for therapies. The approach leverages the strengths of a national team of collaborating AD Centers and associated studies of aging, an innovative proteomics platform, advanced systems biology, and model systems to produce new treatment targets. The first aim will identify novel proteomic targets selectively altered in asymptomatic AD brain. Brains will be analyzed by mass spectrometry (MS), yielding discovery proteomes to compare 1) controls free of AD and other pathologies; 2) asymptomatic controls with AD pathology; 3) non-demented mildly impaired cases with AD pathology, 4) definite AD, and 5) other neurodegenerative diseases. Protein changes in synapses, insoluble aggregates, glial and neuron-specific nuclei, and select posttranslational modifications will be determined. Bioinformatics will be used with available large-scale data to identify potentially druggable targets in key networks and cellular processes. The second aim will validate candidate proteomic targets in postmortem brains from independent community and clinic-based cohorts and determine relationships with clinicopathological features, including cognition. Absolute levels of candidate proteins will be quantified using selected reaction monitoring MS. The third aim will establish links between the validated proteome and AD pathogenesis and druggability. The most promising candidates will be studied for effects on neuronal viability and interactions with Ass and tau using cell culture and drosophila models. These results and other data will drive selection of the most promising candidates to advance to mouse models to assess therapeutic potential.

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) is a leading cause of disability and death in the US and a major global public health problem. Time is running short if we wish to avert a global public health disaster with untold suffering, disruption of families, and severe challenges to health care systems and economies. Solutions will come only from innovative research. Genomic studies for late-onset AD (LOAD) have identified over 20 risk loci; however, translating the relevant molecules identified by genomics to AD-specific mechanistic pathways has been challenging. Our application, entitled ?The Phenotypic Landscape of Cognitive Decline Revealed by Next- Generation Multiplexed Ion Beam Imaging,? is highly responsive to this urgent scientific need by proposing a uniquely innovative molecular imaging platform called multiplexed ion beam imaging (MIBI) that will determine high dimensional protein interactions for AD-relevant molecules identified by genomics studies in normal and pathological states. Our proposal has three Specific Aims. In Aim 1, we propose to analyze four regions in healthy aged brains: two sectors of the hippocampal pyramidal layer and from two isocortical regions. Next- generation MIBI instrumentation will be used to image simultaneously 30+ proteins that mark subtypes of neurons, synapses, and non-neuronal cells, while covering regulatory signaling, neuro-inflammatory components, and AD risk gene protein products with subcellular resolution. In Aim 2, we propose to use the same multiplexed imaging methods in AD brain stratified by APOE genotype. We will analyze these data with statistical machine learning methods already established in our laboratory, similar to what we have done previously with different cancers and the immune system. In Aim 3, using infrastructure we have already have established, we will create a web-based portal where all of the images from this study can be accessed, multi- color overlays generated ad hoc, and all the features we derive shared freely. We propose a transformative, collaborative approach to AD that leverages a long-standing NIA-funded longitudinal cohort and is highly responsive to the National Alzheimer's Plan. These novel insights will illuminate pathways that hold potential for new therapeutic targets and will create a shared research resource and analysis platform for the community of scientists committed to developing solutions for AD.

ABSTRACT Alzheimer's disease (AD) remains a leading cause of disability and death in the US and major global public health problem due to rise in aging population resulting in untold suffering, and severe challenges to health care systems and economies is certain. Solutions will come only from innovative research. Our application is highly responsive to this urgent scientific need by proposing to leverage an innovative molecular imaging platform invented at Stanford, multiplexed ion beam imaging (MIBI). We will determine the high dimensional cellular and subcellular protein-level expression, interaction, and localization for AD-relevant molecules identified by genomic and proteomic studies in resilience and pathologic states using human brain sections from an ongoing proteomic investigation of AD. We hypothesize that in-depth, stage-specific phenotypic signatures extracted from these unique groups will provide insight into the modifiable factors that endorse the protective mechanisms of brain reserve. We will test our hypothesis through three Specific Aims: (1) Establish a subcellular, phenotypic framework of vulnerable and resistant brain areas. Next-generation MIBI equipment will be used to rigorously image 30+ proteins simultaneously; targets marking subtypes of neurons, synapses, non-neuronal cells, neuro-inflammatory, and vascular components will be concurrently measured. (2) Reveal cognitive resilience multiplexed phenotypes. Using approaches we have successfully applied in other single cell studies, we will analyze multiplexed imaging data with statistical deep learning methods already established in our lab to identify topological, cellular, and molecular phenotypic differences among resilient, co- morbid, and AD dementia. (3) Implement a shared data repository. The power of multiplexed imaging in biology is its ability to reveal co-localization or mutual exclusivity to infer regulatory roles and gain mechanistic insight. To disseminate hundreds of these images from our proposed highly multiplexed study, we will create a web-based portal, using already established infrastructure, where all of the images from this study can be accessed, multi-color overlays generated ad hoc, and all the features will be shared freely.

? DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a growing global health problem that causes untold suffering for patients and their loved ones, and challenges health care systems. Motor symptoms are the classic features for which there are effective interventions; however, non-motor symptoms, especially cognitive impairment, are very common, may precede motor symptoms, and do not respond well to existing therapies. Solutions will come only from research. The Pacific Northwest Udall Center (PANUC), one of nine NINDS-funded Morris K. Udall Centers of Excellence for Parkinson's Disease Research, is our response to this research imperative. PANUC is committed to contributing to the long term, ambitious goal of developing the knowledge and tools for optimally targeted and timed interventions for cognitive impairment in PD. Our innovative three Cores and three Projects are structured around genetic risk for different types of cognitive impairment in PD. Our research focuses on molecular pathology and mechanisms of disease, investigation of neural systems using novel brain imaging, and mechanisms of balance and gait problems, in part an expression of cognitive impairment, in genetically-defined subgroups of patients with PD. Each of our research goals is ranked among the highest priority recommendations of two recent NINDS Conferences: Parkinson's Disease 2014: Advancing Research, Improving Lives and 2013 Alzheimer's Disease-Related Dementias: Research Challenges and Opportunities (which included PD dementia).

OVERALL ABSTRACT American Indians/Alaska Natives (AI/ANs) and Native Hawaiians/Pacific Islanders (NHPIs) are increasingly concerned about Alzheimer?s disease and related dementias (ADRD), as these conditions will have a major impact on their communities. Although AI/ANs and NHPIs are culturally diverse and dispersed throughout the US, they share a high prevalence of ADRD risk factors. In addition, life expectancies for AI/ANs and NHPIs have improved over the last 50 years by as much as 30 years, resulting in a tripling of their population share of people aged 65 and older. The confluence of perceived need, high risk, and limited data motivates us to propose a Native Alzheimer?s Disease-related Resource Center in Minority Aging Research (NAD-RCMAR). Our research team offers unique scientific resources and opportunities as well as an extensive network of academic and community partnerships. Based at Washington State University (WSU), with collaborators at Stanford University and the University of Colorado, we will create an innovative program to understand, intervene on, and mitigate the ADRD-related health disparities experienced by AI/ANs and NHPIs. The Center?s theme will emphasize AI/AN and NHPI health disparities and aging from the individual to the population level. Our Research Education Component will fund meritorious Pilot Studies led by early-career investigators known as RCMAR Scientists. RCMAR Scientists will receive mentorship and training in methods, measurements, and study designs that meet high scientific standards and are culturally appropriate. They also will be mentored in the development of successful funding proposals so that they can pursue independent careers in ADRD research. Notably, 13 professionals who are either AI/AN or NHPI will participate as Co-Investigators, Consultants, or RCMAR Scientists. Our Specific Aims are to: 1) Build infrastructure, assemble resources, and provide scientific expertise to enhance the diversity of the aging research workforce by mentoring promising junior and mid-level underrepresented minority scientists for sustained careers in ADRD research, focusing on AI/AN and NHPI populations; 2) Recruit RCMAR Scientists, especially those of AI/AN, NHPI, and other URM backgrounds, to conduct Pilot Studies in the social, behavioral, epidemiological, and clinical sciences on ADRD, and use these Pilot Studies as a vehicle for delivering mentorship and career guidance; and 3)Promote advances in ADRD research and increase the number of independent underrepresented minority researchers, with an emphasis on AI/AN and NHPI health faculty, who can address the health and well-being of AI/AN and NHPI elders, health disparities, and aging from the individual to the societal level. NAD-RCMAR will also capitalize on the national network of Satellite Centers created by Drs. Buchwald and Manson and led by AI/AN researchers. These Satellite Centers will ensure that our efforts are national in scope, especially our recruitment of RCMAR Scientists, our identification of datasets relevant to ADRD in AI/ANs and NHPIs, and our dissemination of results. This work will help address NIA?s research priorities for ADRD among underrepresented minorities, as articulated in the 2012 National Plan to Address Alzheimer's Disease. Our Center responds to NIA?s interest in diversifying the scientific workforce.

Mass spectrometry is an extraordinarily powerful bio-analytical technique that has had a profound impact on our molecular understanding of human health and disease. Major advances in mass analyzer technology, gas-phase separations, dissociation techniques, and ionization methods are largely attributed to the central role that mass spectrometry plays in the field of systems biology. While mass spectrometry has evolved over the last century into a highly effective analytical tool, there are still opportunities for new advances, allowing an even more diverse array of biological questions to be addressed. This proposal is centered on the development and characterization of novel, multi-functional, glycan- specific chemical tags for increased detection by spectroscopic (i.e., absolute quantification) and biological mass spectrometry (i.e., relative quantification) methods. Moreover, the establishment of gas- phase separations of glycans to overcome the isomer barrier challenge is essential to fully understanding the underlying biology. The short-term objective of this proposal is to develop and validate a library of novel multi-functional tags and ion mobility (IM) measurements to resolve glycan isomers, with the long-term goal being their application in an Alzheimer?s disease human tissue repository. This project will also develop open-source software for automated glycan analysis to facilitate accurate and high-throughput analysis, including the support of IM data. These N-glycan results will be combined with data on the glycoproteome and proteome, contributing to the ultimate goal elucidating glycans and glycoproteins for a deeper understanding of the role a key protein modification has in the development of AD. Public Description of Proposed Research Mass spectrometry (MS), the science related to the ?weighing of molecules?, has had a profound impact on the study of human health and disease including cancer, heart disease, neural development, and auto-immune diseases. However, this research will leverage front-end chemistries and gas-phase separations for MS-based glycomics/glycoproteomics to dramatically improve the ability of MS to detect, quantify, and resolve glycan isomers extracted from brain tissue proteins. This will address a more diverse array of contemporary biomedical questions including the quantification of diagnostic and prognostic biomarkers. This proposal is centered on the elucidation of a glycan-specific biomarkers and which proteins are modified for understanding the diagnosis and progression of AD. Another major benefit of this research will be to provide new drug targets (e.g., glycoproteins).

PROJECT SUMMARY Multiple population and community based studies conducted across the US, Europe, Japan, and Australia all have repeatedly observed complex neuropathologic changes in individuals with a clinical diagnosis of Alzheimer's disease (AD) dementia. Although usually including neuritic plaques (NP) and neurofibrillary tangles (NFT), the regional levels and extent of distribution of these hallmark lesions are variable. Additionally, more than half of individuals with AD dementia have other comorbid lesions in brain that when present in isolation can be diagnostic of dementia?the AD-Related Dementias (ADRDs). These include cerebral amyloid angiopathy (CAA), vascular brain injury (VBI), Lewy body disease (LBD), and hippocampal sclerosis of the elderly (HS), among others. Indeed, individuals with a clinical diagnosis of AD dementia frequently show a complex mix of AD lesions and comorbid lesions, making it unclear the extent to which each contributed to cognitive decline and dementia in that person. We hypothesize that the mechanisms of injury and response to injury that produce these different disease-specific brain lesions are influenced by differing genetic factors. With few exceptions, genetic studies for AD have associated genetic variants with a clinical diagnosis of AD dementia. The comorbid complexity described above is a serious limitation to interpreting these data. Are the associations with AD dementia related to the hallmark lesions of AD (common assumption), the variably present comorbid lesions, or both? Only two studies have attempted to address this limitation. As a core analysis of the Alzheimer Disease Genetics Consortium (U01AG032984), our study of AD neuropathologic changes was the larger of these studies with approximately 4900 brain autopsies. However, even this initial study was limited by sample size, platform, and less sophisticated analysis tools. To address these limitations and to advance our knowledge of the full spectrum of dementia neuropathology, we propose a genomics study of hallmark AD lesions together with comorbid lesions associated with ADRDs. This study will expand the sample size of neuropathology subjects, will expand efforts to include next-generation sequence data, and will implement more advanced statistical techniques to better understand the relationships between traits. When successfully completed, our results will point to novel, relevant molecular contributors for each of the pathologic lesions of AD or ADRDs, either alone or in combination. To accomplish these goals we propose four Specific Aims. SA1: Identify genetic variants associated with hallmark AD lesions by whole genome sequencing and genome-wide genotyping; SA2: Identify genetic variants associated with comorbid lesions commonly present in brains of older individuals; SA3: Determine the inter-trait genetic landscape by assessing confounding and genetic correlations across traits; and, SA4: Determine regional, cellular, and lesion distribution of protein products of selected genes identified in SA1-2.

PROJECT SUMMARY/ABSTRACT (OVERALL) We propose here to continue our work to create the world?s best community resource for the curation and dissemination of knowledge on genetic variations relevant to clinical care through the development of the Clinical Genome (ClinGen) Resource. ClinGen?s goals can be summarized by answering questions related to the evidence that variation in a gene causes disease (gene validity), specific variants within a disease gene are associated with disease (variant classification) and whether there is evidence for specific clinical actions if such variants are found (actionability). As part of the three grants submitting U41 applications, our team will have a particular focus on implementation of ClinGen processes for gene and variant curation across non-classic Mendelian disorders including hereditary cancer, somatic variation in cancer, pharmacogenomics and complex inheritance including non-coding variation in common disease. We are also proposing to further enrich the analysis of these variants across populations by the introduction of the ever increasing sequence and genotyping datasets from diverse populations. We will also explore the complex issues around reporting of ancestry in clinical genomics particularly as alleles in different settings may have different disease impact. During the first phase of ClinGen funding the Stanford/Baylor informatics and computational biology teams have built a number of curation interfaces for gene, variant and actionability curation. In this application we plan to expand the suite of online resources that seamlessly aggregates, normalizes and presents disparate sources of evidence to curators. Our goal is to enable consistent curation and improve the clinical application of genomic data in medicine through this informatics infrastructure. We will provide training for the community in the use of these tools by facilitating online learning courses, support of the clinical domain working groups and creation of helpdesks. The clinicalgenome.org online public portal will provide the outcome of these analyses for the community to utilize for clinical interpretation and development of clinical practice guidelines that are based on genetic results.

Abstract Alzheimer's disease (AD) is a major threat to health of older individuals and is a looming public health disaster. Promised solutions in diagnostics and therapeutics will come only through research. Project 2 proposes to gain biological and medical insights into the proteins discovered in the Center by multiplex mapping to specific CSF extracellular vesicles (EV) and lipoproteins (LP), and to specific cells and subcellular structures in specific regions of brain. Proteins in CSF remain the best performing biomarkers for AD. CSF proteins exist in various forms: free in solution, on a unique class of LP, and on EV that include exosomes, microvesicles, and apoptotic bodies. LP and EV have varying mechanisms of production and biological functions. Localization and relative quantification of proteins of interest discovered in Project 1 to each class of CSF particles will provide powerful insight into cell of origin, cellular biology, and potential medical meaning. Further biological and medical insights can be gained by carefully mapping proteins in human brain. Indeed, the power of multiplexed imaging is its ability to reveal co-localization or mutual exclusivity, and to infer regulatory roles and gain mechanistic insight. For example, we think very differently about the biological roles of proteins restricted in their expression to synapses vs. mitochondria, glutamatergic vs. GABA-ergic neurons, or hippocampal pyramidal neurons vs. striatal medium spiny neurons. In pathologic states, co-localization with hallmark pathologic structures (major protein) highlights pathways of injury and response to injury: senile plaques (amyloid beta peptides), neurofibrillary degeneration (paired helical filament-tau), Lewy bodies and neurites (phospho-alpha-synuclein), and phospho-TDP-43 inclusions. We will test the hypothesis that multiplex analysis of CSF particles as well as subcellular, cellular, and regional mapping will provide key biological and medical insights into CSF proteins discovered by de novo proteomic analysis of CSF in Project 1. The same probes will be applied to CSF LP and EV using flow cytometry, and to brain regions using Multiplexed Ion Beam Imaging (MIBI).

Abstract Alzheimer's disease (AD) is a major, growing global public health problem. This is a daunting scientific challenge and solutions will come only from innovative research. We have brought together a unique interdisciplinary team of investigators with the goal of bridging the divide between state-of-the-art technologies and translational applications. To monitor AD disease progression or response to treatment, cerebrospinal fluid (CSF) represents the preferred fluid to reflect brain pathophysiology. The brain interstitial fluid is in direct contact with the CSF by unrestricted bidirectional flow of proteins and the CSF is protected from the peripheral system because of the ? restricted transportation of molecules and proteins by the blood-brain barrier. Known CSF biomarkers have been demonstrated to reflect the three main pathological changes that occur in the AD brain -- amyloid- deposition, neurofibrillary degeneration, and neuronal injury. Therefore, analysis of molecules and particles in CSF holds the greatest potential to improve our diagnosis and characterization of neurodegenerative diseases in vivo. We propose to enable precision medicine for AD by developing a cooperative research program that will unite a unique research team with the specific goal of vastly improving the molecular characterization of CSF as predictors of cognitive decline and AD pathophysiology. The efforts of this program will complement existing efforts to accelerate the identification and validation of clinically relevant therapeutic targets. We will work in parallel with programs like Accelerating Medicines Partnership (AMP) and the AD Centers Program, to provide unique diagnostic capabilities in support of the process of bringing new medicines to patients. Our U19 research program will consist of four projects and four cores that are synergistic to our mission. Moreover, our research team is uniquely suited to the development, validation and translational application of new biomolecular assays to reflect AD pathophysiology. This U19 will harness the collective expertise of multiple fields, combined with the financial resources sufficient to extend and apply next generation proteomics technologies to clinical specimens that have been extensively annotated with longitudinal, consensus clinical diagnostic and neuropsychological test data. We will create new assays with the goal of deployment to the clinical lab. We will create unique and sustainable reagents that will facilitate dissemination and deployment of these methods worldwide. Our program will expand existing infrastructure, developed by our labs, for sharing and disseminating these data and protocols. The application of new technologies, development of novel reagents, creation of new clinical assays, and dissemination of data and protocols will accelerate neuroscience in a way not feasible under traditional NIH mechanisms.

1. SUMMARY (Neuropathology Core) The Neuropathology Core supports the vision of the Stanford Alzheimer?s Disease Center (ADRC) to serve as a shared resource to facilitate and enhance multidisciplinary and interdisciplinary research in Alzheimer?s disease (AD) and related disorders. The Core is structured to continue its track record of enabling and applying innovative cutting-edge technologies with emphasis on brain aging and the diversity of pathologic processes that underlie cognitive impairment and dementia caused by AD and related diseases. The critical functions of the Neuropathology Core include diagnostic expertise, serving as a biorepository, facilitating innovation, and training. Over the past five years, the Neuropathology Core has completed over 50 research brain autopsies and held a leadership position in setting and evaluating national research guidelines for neuropathologic evaluation of AD. We have banked over 800 tissue specimens, contributed data and biospecimens to local, regional, national, and international research studies and consortia, and fostered development and implementation of innovative technologies in translational research. Through the Core, we trained clinicians and scientists from different disciplines in neuropathology and the molecular pathology of dementia-related neurodegenerative diseases. The Neuropathology Core will pursue its mission through four Specific Aims. The Core will provide comprehensive neuropathologic evaluations for physicians and researchers with timely and comprehensive autopsy reports that describe the neuropathologic features of AD and related diseases according to the most current guidelines and consensus diagnostic criteria. It will maintain and continue to grow its highly accessible, but appropriately safeguarded, repository of brain tissue and biospecimens from carefully and longitudinally characterized patients with cognitive impairment or dementia, as well as cognitively normal individuals, using methods that increase and maximize tissue and data quality. In doing so, the Core will empower innovative high-content imaging approaches for both formalin-fixed, paraffin-embedded tissue and for frozen brain regions to maximize the research value of the Stanford ADRC repository and other brain banks around the country. Finally, we will maintain a rich training environment for medical and graduate students, residents, fellows, and junior faculty across departments to teach concepts and state-of-the-art techniques that advance current interdisciplinary research and foster future advances and insights in brain aging and neurodegeneration. The Core is organized to maximize integration with all other Cores in the Stanford ADRC. It also will continue to serve as a hub of support for affiliated ADRCs and independent research awards through diagnostic practices, molecular assays, and tissue sharing. In doing so, the Core and Center will continue to contribute nationally to advance our understanding of the pathologic features and pathophysiologic processes of brain aging, all stages of AD, and related dementias.

PROJECT SUMMARY / ABSTRACT Through decades of research, genome-wide association studies (GWAS) have identified heritable coding and noncoding single-nucleotide polymorphisms (SNPs) that lead to an increased risk of developing Alzheimer's disease (AD). However, the vast majority of these SNPs remain largely under-characterized, and their contribution to AD pathogenesis remains unclear, marking a critical roadblock to our understanding of AD genetics and pathogenesis. While SNPs within the APOE and TREM2 genes have identified vital nodes in AD biology, most AD-related SNPs reside within the noncoding genome, making their functional roles in the disease less clear. Co-inheritance of nearby SNPs (linkage disequilibrium) and the cell type-specificity of noncoding regulatory elements further complicate functional annotation of noncoding SNPs in AD. As part of the Alzheimer's Disease Sequencing Project Functional Genomics Consortium (ADSP FGC), this project will provide a robust and conclusive functional characterization of AD-related noncoding SNPs. To do this, we will first create a comprehensive single-cell atlas of gene expression and chromatin accessibility across a cohort of diverse clinico-pathologic states related to AD (Aim 1). Using these cell type-specific gene regulatory landscapes, we will develop and implement innovative machine learning and statistical genomics methods to predict functional noncoding, splicing, and coding SNPs (Aim 2). We will then validate these predictions using massively parallel reporter assays (MPRAs) and large-scale, scarless, single-base CRISPR editing of iPSCs followed by cell type-specific differentiations (Aim 3). Taken together (Aim 4), this project will pinpoint the functional SNPs and target cell types for dozens of AD-related risk loci and provide an unprecedented picture of the gene regulatory landscape of AD. This work will be performed as a joint collaboration between Stanford University and the Gladstone Institutes at UCSF. Our team, with many long-standing collaborations, has extensive experience in consortium science with long-term involvement in the Encyclopedia of DNA Elements, The Cancer Genome Atlas, and The Genotype-Tissue Expression Project. The proposed project is thus well- positioned to integrate into the highly collaborative ADSP Functional Genomics Consortium.

Neuropathology Core ? Project Summary The Neuropathology (NP) Core will provide unique resources to support the Wake Forest Alzheimer?s Disease Research Center (ADRC) focus on transitions from normal aging to MCI, AD, and related disorders, and the role of metabolic and vascular risk pathways in these transitions. The Core will foster innovative research in all areas relevant to AD. To accomplish these goals, the Core will provide state-of-the-art collection, storage, and distribution of brain and other biospecimens, establish neuropathological diagnoses of decedent Clinical Core (CC) participants, and provide resources and expertise for non-human primate (NHP) models for pivotal mechanistic and therapeutic research. The NP Core will also contribute to the ADRC theme of health disparities with a unique program focused on increasing brain donation from underrepresented groups (URGs). Through these activities, the NP Core will make impactful contributions to many National Alzheimer?s Project Act (NAPA) Research Milestones, such as supporting deep, longitudinal molecular endophenotyping of cohorts that include URGs (1A), data and sample sharing (3A, 4D), and development of the next generation of animal models (4A, B, C). The NP Core currently maintains a biospecimen repository of DNA, blood, and cerebrospinal fluid (CSF) from >1,100 well-characterized participants from the ADRC and its affiliated studies. In the coming cycle, we will continue to archive biospecimens from ADRC CC participants who have received careful metabolic and vascular characterization. DNA, CSF and blood will be provided to NCRAD, other consortia, and individual investigators. AD CSF biomarkers (Ab40, Ab42, tau, and p-tau181) are rigorously measured on all participants who undergo lumbar puncture (LP), and special expertise is available for blood and CSF biomarkers of metabolic and vascular disease. We continually assess scientific advances in identifying novel blood and CSF biomarkers of ADRD and will implement these as the field evolves. Further, human protocols for brain, blood and CSF collection have been applied to NHP models, creating a unique resource with which the AD research community can conduct pivotal translational research. In the past cycle, we demonstrated that our NHP model has neuropathologic changes similar to early AD such as amyloid plaques, AD-like CSF Ab42 profiles, cerebral hypometabolism, and reduced brain volumes. Consultation about NHP and other models (rodent, organoid) will be available. In summary, through careful clinico-pathologic analysis of well characterized cohorts, unique biospecimen repositories, and novel translational models, the NP Core will promote the WF ADRC themes, and provide an exceptional resource to investigate underlying mechanisms, novel biomarkers, and therapeutic targets that impact progression from normal aging to MCI and ADRD.