Linda J. Van Eldik, Ph.D. - US grants

Our long term goal is to understand how effector proteins are involved in normal cellular processes and in mechanisms of pathophysiology. The aim of this proposal is to examine the characteristics and mechanism of action of a set of relatively tissue-specific calcium modulated proteins (S100) that may function as intracellular effector proteins. Only some of the S100 proteins have been isolated and characterized, and the relative amounts of each S100 protein in a given species or tissue have not been determined. The physiological functions of S100 proteins are not known and there are specific assays for individual S100 components. The proposed studies are a purification and characterization of rat S100 proteins and analyses of the levels, subcellular distribution, regulation and mechanism of action of these proteins using quantitative cell biological and biochemical approaches. Emphasis is on the development and refinement of methods for study of S100 Beta to allow unequivocal interpretation of biological studies. The biological systems are rat brain and the C6 cell line derived from a rat glial tumor induced by the carcinogen, N-nitrosomethylurea. We will purify S100 proteins to homogeneity from rat brain, determine their biochemical and immunochemical properties, and compare them to the analogous proteins purified from C6 cells. We will develop specific immunochemical assays for each S100 protein by various approaches. These approaches have resulted in the recent production of antibodies specific for the S100 Beta polypeptide of brain S100 fractions. Using biochemical cytology methods, we will determine the levels and subcellular distribution of S100 proteins in C6 cells under various physiological states and culture conditions. By a direct mechanistic approach, we have detected molecular targets of S100 Beta. We will further investigate these targets, including subcellular distribution and possible biochemical activities. These studies might yield insight into common and unique mechanisms of regulation by calcium modulated proteins and will provide the necessary methods and rigorous foundation for future studies on molecular mechanisms of action of these proteins and for current studies of human disease states.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

DESCRIPTION: (adapted from applicant abstract) The overall hypothesis is that the cellular responses to amyloid beta (A beta ) that culminate in the cell dysfunction and degeneration characteristic of Alzheimer disease (AD) are dependent on the specific structure of the supramolecular aggregates formed by the A beta peptides. The applicants propose that part of the process of maturation of the predominantly diffuse, non-neuritic amyloid plaques of "normal" aging into the neuritic plaques associated with dementia involves the generation of these specific A beta structures. Extending this hypothesis, the applicants further propose that the generation of "toxic" supramolecular structures of A beta aggregates can be influenced by the presence of other plaque components, particularly those components derived from glia, and that responses of glial cells contribute to an environment that facilitates and enhances the formation of these bioactive A beta forms. It is their contention that glia are not just passive bystanders, but are a part of the pathogenesis process. They postulate that A beta toxicity can be mediated through its effects on glia, that glia-derived proteins can influence the structure of A beta and its effects on neurons, and that these processes are relevant to the actual neuropathology seen in the AD brain. The questions being addressed in these studies are: What does A beta do to glia? What do glia do to A beta structure and activity? Are glia relevant to AD neuropathology? Three aims are proposed to address these questions. 1) They will evaluate the effects of A beta on glia. Specific A beta aggregates will be prepared, characterized by atomic force microscopy (AFM), and tested for activity on cultures of rat primary astrocytes. The investigators will examine the effects of A beta on the levels and activity of six relevant glial proteins: a 1-antichymotrypsin (ACT), apolipoprotein E (apoE), apoJ, butyrylcholinesterase (BChE), interleukin-1 (IL-1), and S100 beta . 2) They will evaluate the effects of glia on A beta structure and neurotoxicity. In co-aggregation experiments, they will evaluate AFM structure of A beta aggregates formed in the presence of the six glial proteins studied in aim 1. The investigators will also test A beta -evoked neurotoxicity in the presence of these six glial proteins and in glial-neuronal co-cultures. 3) They will correlate the presence and distribution of glial proteins with plaque type in AD brain. They will determine the regional distribution and immunoreactive density of the six glial proteins in AD and control brain tissue, and establish correlations with the distribution of non-neuritic vs neuritic plaques. This system examination should provide insight into how A beta affects glia, and how glia participate in amyloid plaque progression and development of neurotoxicity.

The overall hypothesis addressed by this program is that non-fibrillar aggregated assemblies of amyloid beta (Abeta) which we refer to as Abeta-derived active ligands (ADALs) are active in triggering Alzheimer's disease (AD)-specific cellular responses leading to glial activation, neuronal plasticity malfunction, degeneration and ultimately cell death. We further propose that the generation of these active supramolecular structures of Abeta aggregates can be influenced by the presence of other plaque components, particularly those components derived from glia, and that responses of glial cells contribute to an environment that facilitates and enhances the formationof these bioactive ADALs. Inherent to our hypothesis is that glia are not just appsive bystanders, but are a part of the pathogenesis process. We postulate that Abeta toxicity can be mediated through its effects on glia, that glia-derived proteins can influence the structure of Abeta and its effects on neurons, and that these processes are relevant to the actual neuropathology seen in the AD brain. The questions being addressed in this subproject are: What are the responses of glial cells to ADALs? How do glial proteins affect the formation of ADALs and their bioactivity? Are glia relevant to AD neuropathology? Three aims are proposed to address these questions. 1) The responses of glial cells to ADALs will be evaluated. Specific Abeta aggregates will be prepared and characterized by project 1. We will test the activity of these ADALs on cultures of rat primary astrocytes, by examining the levels and activity of six relevant glial proteins: alpha1-antichymotrypsin (ACT), apolipoprotein E (apoE), apoJ, butyrylcholinesterase (BchE), interleukin-1 (IL-1) and S100beta. 2) The effects of glia on ADAL formation and neurotoxicity will be evaluated. In co-aggregation experiments, we will evaluate the kinetics of aggregate formation and the structure of Abeta aggregates formed in the presence of these six glial proteins willb e correlated with plaque type in AD brain. We willdetermine the regional distribution and immunoreactive density of specific flial proteins in AD and control brain tissue, and establish correlations with the distribution of non-neuritic vs neuritic plaques. This systematic examination should provide insight into how Abeta affects glia, and how glia participate in amyloid plaque progression and development of neurotoxicity.

drug discovery /isolation;

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The goal of this research is to elucidate the molecular basis of S100B protein's involvement in the neuroinflammatory responses of activated glia (astrocytes and microglia). S100B is a glial-derived cytokine that is significantly elevated in the brains of Alzheimer's disease (AD) patients. S100B induces pro- inflammatory cytokines (e.g., IL-1beta and TNFalpha) and oxidative stress related enzymes (e.g. iNOS) in activated glia, and enhances the ability of other stimuli, such as beta-amyloid, to activate glia. These neuroinflammatory responses, in turn, can lead to neuronal dysfunction and further glial activation. Thus, S100B contributes to a vicious cycle of glial activation/ neuroinflammation/neuronal dysfunction that provides the potential for self-propagation of neurodegenerative events and chronic glial activation seen in AD. However, very little is known about the molecular mechanisms by which S100B induces iNOS and pro-inflammatory cytokines, how modulation of these specific molecular pathways in activated glia affects neuronal viability, or how modulation of S100B itself influences glial and neuronal responses. The hypothesis is that responses of glial cells to S100B contribute to and environment that enhances the progression of neuropathology, and that knowledge of the molecular basis of S100B action on glia will provide the potential for blocking specific glial activation pathways that have neurotoxic consequences. Specific aim 1 will address the mechanisms by which S100B activates glia. Aim 1A will examine if S100B stimulates iNOS through a cytokine-dependent, NFkappaB regulated pathway. Aim 1B will determine if p38 and ERK MAP kinases are important for S100B-induced IL-1beta production in activated microglia. Aim 1C will elucidate the potential role of RAGE, a new receptor that can interact with S100B, in mediating the S100B-induced neuroinflammatory responses. Aim 2 will determine the contribution of specific signal transduction pathways leading to iNOS induction on neuronal dysfunction, by utilizing a defined co-culture system of primary astrocytes and neurons where NO- dependent neuronal death occurs. Pathways will be modulated by selective pharmacological agents or molecular constructs, and effects on neuronal viability determined. Astrocytes from genetically engineered mice with overexpression or knockout of specific components of glial signaling pathways will also be used to ascertain how an in vivo alteration in a specific pathway affects neuronal function. Aim 3 will use a discovery approach employing cell-based screens of chemical libraries and recursive synthetic chemistry refinement to discover ligands that modulate S100B production in activated astrocytes. These studies will provide new insight into glial-neuronal interactions and the knowledge base necessary for pursuit of novel strategies to block glial activation and its neurotoxic consequences.

DESCRIPTION (provided by applicant): The long-term goal of this Program is to elucidate how chronic activation of glia (astrocytes and microglia) is linked to neuronal degeneration. We are testing the following hypothesis: Chronic glial activation, with its aberrant regulation of key proteins and signaling pathways, and the consequent dysregulation of normal glialneuronal interactions, promotes neuronal cell death. An increased understanding of glial activation responses will allow new approaches to modulate glial responses that influence neurodegenerative disease progression, while maintaining the important beneficial glial responses necessary for normal brain function. Our focus is on key proteins and pathways that have a documented relationship to end points of Alzheimer's disease (AD) pathology. They have been found to be abnormally regulated in human neurodegenerative disease or in animal models of neurodegenerative disease. W e will determine mechanisms of regulation of the proteins and pathways in activated glia, the relationship between glial protein expression and neuronal function, and the discovery of ligand modulators. Project by Van Eldik will examine the link between selected glial neuroinflammatory responses and neuronal cell death by defining glial cytokine contributions to neuronal death, and using chemical genomics approaches to discover small molecule modulators of neuronal death and death kinases. Project by Binder will focus on the role of abnormal tau in astrocytes and its relationship to their cytoskeletal integrity and ability to undergo the inflammatory response in vitro and in neurodegenerative disease. Project by LaDu will determine how AP induces activation of astrocytes and increases in astrocytic apoE, the role of the apoE receptors LRP and LDLR in these processes, and the extent and mechanism of apoE's anti-inflammatory activity. Project Vassar will test the hypothesis that activated astrocytes surrounding amyloid plaques exacerbate plaque development through BACE1 cleavage of APP to generate Ap during AD inflammation. Two cores have been established to provide services in grant administration and management (Core A: Statistical Core) and to perform histological services (Core B: Histochemistry Core). The projects will utilize a variety of experimental models (cell culture models of primary glia and neurons; novel transgenic and knockout mouse models of AD and related neurodegenerative diseases; and human autopsy tissue) and disciplines (synthetic chemistry, protein biochemistry, cell and molecular biology, physiology and neuropathology). The cooperative and synergistic interactions among investigators with complementary expertises provide a broad-based, yet focused, approach to addressing fundamental questions about the role of activated glia and glial-neuronal interactions in neurodegeneration.

DESCRIPTION (provided by applicant): This proposal is for funding of a new summer research and training program, called the Drug Discovery Academic Research Experience (DARE) program, that will be targeted to undergraduates from the quantitative and physical sciences. The DARE program will be a unique program that is not duplicated by existing summer research programs. Each student selected to participate in the DARE program will work on a research project with a faculty mentor. The program will have a structured curriculum that includes the research experience, an orientation week with lectures and lab practicums, weekly lectures and research discussions, a choice of specialty group meetings and seminars or journal clubs, and a final presentation of research results and educational accomplishments. This is a novel yet feasible proposal based on our documented experience in the recruitment of ethnically diverse undergraduates from all major geographical regions of the United States as well as Canada and Europe, our track record of exposure of undergraduates to biomedical research with subsequent entry into graduate and professional degree programs or careers in biomedical sciences, and the presence of the relevant infrastructure with unique capabilities. We anticipate that the DARE program will enhance the ability to attract undergraduates majoring in mathematics, engineering, chemistry, or physics into biomedical research careers. The motivated faculty with documented records of cooperation in educational endeavors complements the structured curriculum and the formal evaluation systems that are in place. The program attributes and our prior success in the administration of summer undergraduate research experiences assure a high probability of the DARE program making an impact on the recruitment of undergraduates with backgrounds in the quantitative and physical sciences into biomedical research careers.

[unreadable] DESCRIPTION (provided by applicant): Upregulation of iNOS and the consequent oxidative nitration of protein tyrosines by its product, nitric oxide, is a key feature of most neurodegenerative diseases, although the role of these alterations in tau pathogenesis is not understood. Since submission of the original proposal, we have significantly advanced our understanding of the effect of oxidative stress on tau and its potential role in the neurodegenerative process, making this a prime focus of the present proposal. Over the past two years, we have determined that there is a hierarchical nitration of the five tyrosines of tau and we have begun to study the effects of nitration at these sites on tau's ability to aggregate into filaments. We now propose to continue these ongoing studies with the goals of further understanding the role of site-specific tau nitration on its self-association into pathological inclusions and on its association with its natural binding target, the microtubule. We propose to study the appearance of nitrated tau in both neurons and glia in the brains of Alzheimer's disease, Progressive [unreadable] Supranuclear Palsy, Corticobasal Degeneration and Pick Disease patients immunohistochemically by [unreadable] producing novel antibodies that recognize only site-specific nitration of tau. Based on results gathered over the past two years, we hypothesize that certain tau nitration events prevent aggregation and cause tau to bind more tightly to microtubules while nitration at other sites are somewhat neutral in their effects. We will test these hypotheses by accomplishing the following specific aims: 1. We will continue to determine the effects of site-specific nitration on the aggregation of tau into filaments using individual tau isoforms nitrated at each of the relevant tyrosine residues. 2. We will determine the effect of nitration at each relevant tyrosine residue on the binding of the six tau isoforms to pre-formed, taxol-stabilized microtubules. This will determine which nitrotyrosine residues are involved in mediating tau's affinity for microtubules. 3. We will produce novel monoclonal antibodies specific for nitrated peptides of tau beginning with those tyrosine residues most readily nitrated. 4. We will stain human brain sections with our novel site-specific nitrotyrosine antibodies to determine when during the course of NFT evolution in AD neuronal tau is nitrated at specific tyrosines. Although it is not possible to do progression studies in PSP, CBD, and PiD, we will also check the glial and neuronal inclusions in these diseases to determine whether similar site-specific alterations occur. [unreadable] [unreadable] [unreadable]

The Statistical and Administrative Core's overall objective is to coordinate, integrate and expedite the scientific and administrative matters of the Program. Towards this end, the Core will pursue the following specific responsibilities. 1) To provide administrative support and fiscal management for the Program. Responsibilities will include: to coordinate, record, and monitor expenditures; to ensure that appropriate university forms are on file and updated as required; to assist with preparation of progress reports; to act as liaison for efficient communication between the investigators and the administration; and to coordinate administrative and budgetary matters pertaining to the ENHRI subcontract. 2) To provide a coherent infrastructure that enhances scientific activities of the Program. Responsibilities will include: to catalyze and facilitate scientific interactions among the investigators; to encourage and coordinate the flow of information generated by the projects and cores for efficient communication to relevant personnel; to organize the meetings of the investigators and the External Advisory Committee, and to provide biostatistical support for the projects. These duties will be coordinated by Dr. Linda Van Eldik, with the assistance of an administrative manager and accounting supervisor. In addition, statistical expertise for the design and interpretation of experiments will be provided by Dr. Alfred Rademaker, with the assistance of a statistical analyst. Efficient functioning of a Program Project requires leadership, administrative organization, managerial skills, and fiscal expertise. The Administrative Core will facilitate internal cohesion and communication among the members of the Program, create an infrastructure that encourages scientific productivity, and maintain a system for sound fiscal management.

This Project will examine the link between selected glial neuroinflammatory responses and neuronal cell death. The hypothesis to be tested is: Overproduction or sustained production of potentially detrimental biomolecules (NO and pro-inflammatory cytokines) by activated glia contribute to neuronal death. Insight into which of these pathways can be readily modulated by endogenous factors or synthetic ligands will provide a firm foundation for future drug discovery efforts towards developing new therapeutics for Alzheimer's Disease (AD). We will address several fundamental questions about glial cytokine production and its linkage to neuronal cell death. These include: what glial cytokines contribute to neuronal death and what pathways are potential targets for inhibition of cytokine-dependent neuronal cell death? The focus will be on: (i) beta-amyloid (Abeta)-induced upregulation of inducible nitric oxide synthase (iNOS), and its potentially neurotoxic product, nitric oxide (NO) and NO metabolites such as peroxynitrite; (ii) the Abeta-induced increases in proinflammatory cytokines interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha, and S 100B; (iii) comparison of the neurotoxic effects of these glial derived products to those of direct Abeta effects on neurons; and (iv) discovery of ligand modulators of signaling pathways key to neuronal cell death. In aim 1, we will define at the molecular level what is meant by glial cytokine-dependent neuronal cell death. Glial/neuronal co-culture models, in vivo administration of cytokines, and animal models of neuroinflammation and disease will be utilized. We will determine the potential common and distinct elements between cytokine-dependent and -independent neuronal death by focusing on key elements of standard themes that have been implicated previously in cell death mechanisms, particularly MAP kinase (MAPK)-regulated pathways and death domain signaling pathways involving death receptors like TNF receptorl (TNFR1) and death kinases like death associated protein kinase (DAPK). In aim 2, we will discover ligand modulators of cytokine-dependent neuronal cell death. Chemical genomics approaches will allow the discovery of small molecule modulators of neuronal cell death and death kinases. Pathway selective inhibitors will allow probing of the relative contribution of distinct signaling pathways to common biological end points. Potentially efficacious synthetic compounds will be tested in animal models of neuroinflammation and disease. Our results will have a potential broad impact on basic restarch in areas such as signal transduction and glialneuronal interactions as well as provide insight into possible new therapeutic approaches to neurodegenerative disorders such as Alzheimer's disease.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is one of the major health problems in this country and affects over 4.5 million persons. The key to the disease is understanding the pathogenesis of neuron degeneration in specific brain regions so that treatment and prevention can be based on solid data. This application is a resubmission of a competitive renewal of the program project, Beta-Amyloid and Oxidative Stress in AD (P01 AG05119-20) by the Sanders-Brown Center on Aging at the University of Kentucky (UK). Abundant evidence indicates that free radical mediated oxidative damage in the brain is prominent in AD, especially in the earliest detectable clinical phase of AD - mild cognitive impairment (MCI). The major hypothesis of this application is that beta amyloid peptide (AB) is one of the pivotal mechanisms in oxidative stress/damage to neurons in AD. The proposal consists of three cores including 1) an Administrative Core that provides supervision, fiscal management, biostatistical support and database management, clinical and neuropathological data, and brain specimens from the UK AD Center, 2) a unique Beta-Amyloid Core that will provide the AB index (total AB{1-40} and AB{1-42}, AB solubility, oligomeric and fibrillar AB quantitation and plaque quantitation) from human subjects and transgenic mice and also will provide toxic assembled AB and non-toxic scrambled AB for cell culture experiments, and 3) an Animal Core that will supply APP/PS1 knock-in mutant mice and WT mice, and primary rodent cortical neuron cultures for projects. Each project uses short postmortem brain specimens from longitudinally followed preclinical AD (PCAD), MCI, late AD, normal controls and frontotemporal dementia (as a disease control) subjects, APP/PS1 transgenic mice and cell cultures, and each has a therapeutic component and excellent preliminary data to support their hypotheses. Project 1 examines the hypothesis that brain RNA oxidation is an early event in AD and is mediated, at least in part, by Ap and that the RNA oxidation impairs protein synthesis and contributes to neuron dysfunction. Project 2 examines the hypothesis that AB-mediated oxidative modifications of and subsequent dysfunction of Pin1 are fundamental to the genesis of specific pathological features in AD. Project 3 will examine the hypothesis that elevations in RAGE-ligand interactions occur in AD, with soluble RAGE modulating Ap assembly and toxicity. Project 4 examines the hypothesis that sustained activation of NADPH oxidase by toxic forms of AB triggers a cascade linking increased ROS to altered redox-based signaling and neurotoxic oxidative damage. This well integrated program project will move the field of AB and oxidative stress in AD forward and gain insights into mechanisms of neuron degeneration so that successful interventions can be developed to treat or prevent the most dehumanizing disorder to affect humans.

DESCRIPTION (provided by applicant): This is an application for a 5-year Alzheimer's Disease Core Center (ADCC) at the University of Kentucky's Sanders-Brown Center on Aging. The application consists of the Clinical, Biostatistics and Data Management, Neuropathology, Education and Information Transfer Cores. The UK ADC has developed a strong program in the clinical, neuropathological, educational and research aspects of AD that serves as a resource for the university, community, state, and region for the past twenty years. The major goals of the UK ADC are to: a) provide clinical data, serum, plasma, buffy coats and CSF from thoroughly evaluated, longitudinally followed, normal control subjects, AD and non-AD dementing disorders, and postmortem material from these subjects to investigators of AD and aging at UK and to national and international investigators, b) continue longitudinal investigation of presymptomatic AD, mild cognitive impairment, early AD, mixed dementias and other dementias, and normal brain aging with autopsy as an end point;c) develop new research initiatives and attract new investigators to research in neurodegeneration and aging;d) place emphasis on serving minorities and involving them in research;and e) participate in multicenter efforts, including NACC, ADCS, ADNI, and others. The Clinical Core will evaluate and longitudinally follow 500 subjects in the Normal Control (BRAINS) Clinic and 200 patients with AD and other dementias in the Dementia Research Clinic - all with prearranged autopsies. This core will maintain a satellite clinic for African Americans in north Lexington in conjunction with an Administration on Aging grant for education of minorities about dementing disorders. The Neuropathology Core provides short postmortem interval brain specimens, CSF, and synaptosomes from longitudinally followed normal, MCI, early and late AD patients, and non-AD dementias to investigators and maintains a tissue bank. The Biostatistics and Data Management Core manages data sets from the other Cores, prepares the Uniform Data Set and the Minimum Data Set for submission to the NACC, and provides statistical design and analysis. The Education and Information Transfer Core assists with recruiting control and dementia subjects, provides research and clinical training for investigators and healthcare professionals, and disseminates information to the lay public.

DESCRIPTION (provided by applicant): The serine/threonine protein kinase p381 MAPK is an established therapeutic target for a number of peripheral inflammatory diseases where increased proinflammatory cytokine production contributes to pathology. In contrast, much less is known about the in vivo role of p381 MAPK in CNS dysfunction and its potential as a therapeutic target. Specifically, little information is available on the quantitative contribution of microglial p381 MAPK signaling in vivo to up-regulation of proinflammatory molecules that lead to disease- relevant neuropathology, the importance of p381 MAPK in the beneficial reparative and remodeling responses of activated microglia, or the role of neuronal p381 MAPK in CNS dysfunction responses. We hypothesize that p381 (and not the closely related p382 MAPK isoform) is a key in vivo contributor to microglial inflammatory activation cascades that culminate in detrimental proinflammatory cytokine overproduction and subsequent neuronal/synaptic damage, and that suppression of p38 MAPK signaling in the microglia and/or neuron can lead to selective, beneficial outcomes. The field has been limited in its ability to pursue these questions because of the lack of CNS-penetrant, selective, small molecule p381 MAPK inhibitors. Our development of a novel, orally bioavailable, brain-penetrant, selective, small molecule p381 MAPK inhibitor (compound 069A) that attenuates hippocampal proinflammatory cytokine overproduction and leads to improved neurologic outcomes in a mouse CNS injury model now provides the opportunity to address these critical questions about p38 MAPK and CNS dysfunction in vivo as well as in vitro or in situ. We will use this unique chemical biology tool and novel knock-out (KO) and drug-resistant knock-in (KI) mice to pursue several important mechanistic investigations. First, we will test the importance of p381 MAPK and p382 MAPK in vivo through the use of microglial p381 conditional KO and p382 global KO mouse models subjected to stressor stimuli. The temporal onset and profile of microglial activation and synaptic dysfunction responses will be determined. Second, we will complement these in vivo studies by using cell culture models in order to explore in more detail the relative contributions of p38 MAPK isoforms in microglia and neurons. We will utilize microglial-neuronal co-cultures combined with pharmacological and genetic knock-down approaches to determine the importance of microglial and neuronal p38 MAPK isoforms to stressor-induced responses. Successful completion of this project will provide mechanistic insight into the role of the key regulatory protein, p381 MAPK, in microglial activation and neuronal damage caused by stressors that induce CNS pathophysiology, and the contribution of microglial vs. neuronal p381 MAPK to the CNS dysfunction responses. In addition, the studies will elucidate the potential role of the highly related p38 MAPK isoform, p382, in the microglial and neuronal responses. Longer term, the knowledge generated by the proposed studies will provide a firmer foundation for future development of new classes of disease-modifying therapeutics and fuller interpretation of disease progression investigations. PUBLIC HEALTH RELEVANCE: Successful completion of this project will provide mechanistic insight into how the key regulatory protein, p381 MAPK, is involved in brain pro-inflammatory responses and CNS dysfunction caused by disease- relevant stressors. In addition, the results will delineate the relative importance of microglial versus neuronal p381 MAPK and p382 MAPK to the disease-relevant pathological responses. Longer term, the insights and knowledge generated by the proposed studies will provide a firmer foundation for future development of new classes of disease-modifying therapeutics and fuller interpretation of disease progression investigations.

PROJECT SUMMARY (See instructions): The overall objective of the Neuropathology Core of the University of Kentucky Alzheimer's Disease Center (UK-ADC) is to support research on normal brain aging, presymptomatic Alzheimer's disease (pAD), mild cognitive impairment (MCI), early and late AD, mixed dementia syndromes, and other dementing disorders. Autopsies will be performed by our Rapid Autopsy Team on longitudinally followed subjects from our Clinical Core. We will perform short post-mortem interval autopsies in relation to our clinical cohort, and we will maintain a high autopsy rate. This Core is optimally tailored to help address important research questions. The Core will provide brain tissue specimens, CSF and synaptosomes for investigators at UK, other ADCs, and outside investigators. The Core will also provide consensus conference determined diagnoses, quantitation of neurofibrillary tangles (NFT), neuritic plaques, and diffuse plaques from 8 brain regions, AB 1-40 and 1-42 quantitation, Braak staging, CERAD, and NIA-Reagan Institute staging on all autopsied cases to investigators. Since the brain bank has been operating continuously for over two decades with a strong track record, special care will be taken to ensure diagnostic excellence, consistency, and continuity. The Core will maintain a tissue bank of the above specimens and frozen serum, plasma, buffy coats and CSF from living patients. Special emphasis will be placed on defining the neuropathological findings in the brains of the oldest old (>85 years), and providing investigators with specimens from cognitively intact control subjects with no AB deposition and sparse tau pathology (successful cerebral aging) and also cognitively intact subjects with abundant plaques and neurofibrillary tangles. Providing these samples will contribute to clinical-pathological correlation studies and cutting-edge research that include sponsored studies related to AD genomics, oxidative stress, hippocampal sclerosis, dementia with Lewy bodies, amyloid precursor protein processing, Down syndrome, and neuroinflammation. Frequent consensus conferences will be held with the Clinical Core and Biostatistics Core to help define clinicalpathological diagnoses on all autopsied subjects. This Core is strongly integrated with other Cores of the UK-ADC, and exploits unique opportunities to conduct clinical-pathological correlative studies on longitudinally followed subjects. Through these methods we will better understand normal brain aging and the transition to dementia with the focused goal of contributing to therapeutic or preventive measures.

PROJECT SUMMARY (See instructions): The Education and Information Transfer Core (EITC) functions as a vital link between the UK-ADC and the local and state communities. EITC programs and activities have created and will maintain an educated local community eager to receive updated information on AD and to participate in our Center's research. Through EITC efforts, the UK-ADC will continue to work with both the Commonwealth of Kentucky and UK in directing state and institutional response to meeting Kentucky's dementia health care needs. Over the past five years, the EITC has taken a lead in developing innovative products to support research participation by African Americans, the largest minority group in Kentucky. The EITC will continue strong relationships with the Council of Ministers and the Community Action Council so that activities and products developed, such as the Book of Alzheimer's for African-American Churches and Granny Pearl, a film to educate African- American adolescents about AD, are responsive to local needs. EITC recruitment and outreach activities will be closely coordinated with the Clinical Core and the Gateway Satellite Clinic to ensure that appropriate participants are identified for ADC cohorts, clinical trials and other research studies. Work in the EITC forthe five-year grant period will be designed to meet three specific aims. Aim 1 is to support recruitment and retention activities for our Center's research protocols, clinical trials, and NIH/NIA initiatives. The core places a special emphasis on minority recruitment and retention strategies, and will continue its innovative and successful strategies for African American participants. An expanded goal is to increase research participation, including brain donation, in this population. Aim 2 is to increase community awareness of AD with an emphasis on maintaining a strong presence in the African-American community. Awareness efforts will be focused on the importance of early disease identification to support our Center theme of transitions and translation. Aim 3 is to expand UK, community and regional partnerships to meet the educational needs of community and professional audiences and UK researchers. Strong collaborations with the local Alzheimer's Association and Geriatric Education Center will continue. Based on needs assessment and survey data, our current and future professional educational efforts will focus on the following three areas: caregiver education, medical student education, and scientific conferences, including a new William R. Markesbery scientific symposium in honor of our former director.

PROJECT SUMMARY (See instructions): The primary mission of Core A is to provide leadership, coordination and integration of all the components of the UK-ADC, so that our programmatic and scientific goals are achieved. Our overall scientific objective is to support and facilitate research aimed at elucidating the pathogenic mechanisms underlying the transitions from normal cognitive aging to the development of AD, with a long-term goal of enabling more effective translation of this mechanistic knowledge to intervention strategies. The UK-ADC has historically been at the forefront of supporting research in identification of the early manifestations of disease and the pathogenic mechanisms underlying the transitions from normal cognitive aging to the development of AD. In the renewal period, we will build upon this highly successful focus, as well as leverage several recent positive developments at UK that will expand our translational research capability, and thereby help to increase the rate of progress toward new therapies to delay or prevent AD. The UK-ADC functions in a multidisciplinary, integrated, and supportive environment that catalyzes high-quality AD research within the UK community and throughout Kentucky and beyond. The ADC also enhances education and outreach within UK and in the community, and provides clinical diagnoses and care of patients with cognitive impairment. The ADC serves as the focal point of all AD-related activities in Kentucky, providing the infrastructure and resources to enhance scientific interactions and develop new collaborations. The ADC also reaches beyond Kentucky to enhance AD research, education, and clinical practice on a national level through its interactions with other ADCs, as well as other regional and national institutions, including the Alzheimer's Association, NIA and industry. An effective Administrative Core is essential for transforming ADC goals into accomplishments. Therefore, Core A will continue to support the ability of our UK-ADC to serve as a critical resource for the university, community, state, region and nation through a set of integrated and intertwined specific aims. Aim 1: Provide leadership and an organizational framework for management and coordination of all ADC activities to ensure optimal resource utilization to meet the overall goals of the ADC. Aim 2: Enable the ADC to serve as the focal point of AD-related activities in Kentucky by continuing to coordinate, facilitate, and promote scientific interactions. Aim 3: Contribute to and interact with other ADCs and other national collaborative activities and the lay community, to increase visibility of the ADC and maximize progress in AD research throughout the country.

PROJECT SUMMARY (See instructions): Central to the success of the UK-ADC over the past 25 years is Clinical Core productivity that has allowed the development and perpetuation of a large longitudinally-followed, well-characterized, continuously-replenishing cohort focused on normal aging and early transitional disease states. All subjects undergo autopsy at death supporting clinical-pathologic research in aging and dementia. This practice has allowed major contributions in the area of clinical-pathological correlative studies and our understanding of early preclinical and predementia disease states such as mild cognitive impairment (MCI). UK-ADC productivity in terms of contributions to national collaborative initiatives such as NACC (2nd in overall productivity), ADCS (2nd in overall productivity), ADNI (2nd in overall productivity), NCRAD (597 normal control cell lines), and GWAS/ADGC is well recognized by involved researchers participating in these initiatives. The Clinical Core of the UK-ADC has contributed to the success of 31 independent research studies using living subjects (in addition to the many that rely on our data for work in the area of biospecimens and neuropathology), and has led to an impressive 159 publications supported by this core over the last funding cycle. In the upcoming cycle, under the leadership of Dr. Greg Jicha, we will focus our efforts on supporting investigations in the detection and treatment of preclinical AD and MCI of the AD-type as outlined in the new proposed NIA criteria released at ICAD 2010. As such, our center is in critical transition, developing and expanding biomarker capabilities (target normal to MCI/dementia transitions, n=270 over the next funding period, and n=50 high risk normals for the development of preclinical AD) while maintaining the size of our existing cohort to ensure sufficient transitions from normal to MCI and dementia (n=500 normals and n=300 impaired), continuing to work towards the development of more sensitive cognitive measures recommended by the NIA proposed criteria for preclinical AD, and fully supporting our continued leadership position in the area of clinical-pathological correlative studies under the NR Core leadership of Dr. Peter Nelson. The clinical core fully supports the leadership role of Dr. Linda Van Eldik as center director, after the loss of Dr. Markesbery earlier this year. We are committed to remaining one of the top ADCs in the nation and across the globe. We have further strengthened our resolve and commitment to advancing research and moving diagnostic strategies and therapeutic development forward as we transition into a new era of research with the NIA..

Project Summary/Abstract: Overall This application seeks to renew the University of Kentucky Alzheimer?s Disease Core Center (UK-ADC), an experienced and collaborative ADC originally funded in 1985. Our principal mission is to serve as the focal point for all AD-related activities at UK and this region of the United States, by providing an environment and core resources that catalyze innovative research, outreach, education, and clinical programs. Our signature resources include: 1) a cognitively normal group of ~500 subjects followed longitudinally, with all committed to brain autopsy upon death; 2) a strong program in clinical-neuropathological correlations and short postmortem interval autopsies; 3) a maturing program studying the early preclinical biological emergence of pathology that may lead to dementia states, with an increasing focus on antemortem imaging and CSF biomarker collection; 4) an integrated centralized database and innovative biostatistical expertise to characterize clinical and biological transitions; and 5) a successful and close partnership with the African-American community and increased participation of underrepresented individuals in our longitudinal cohort and ADC-affiliated research studies and clinical trials. The overall scientific emphasis of the UK-ADC continues to be on our interrelated themes: Transitions & Translation. Our well-characterized, longitudinal cohort and historically strong neuropathology program focused on normal aging, preclinical disease states and early cognitive transitions have been central to our success in defining early pathogenic mechanisms underlying the transitions from normal cognitive aging to impairment. The depth of expertise and collaborative nature of our investigators have also resulted in substantial progress on translation of that knowledge into new targets and novel therapeutic strategies. The UK-ADC will continue to provide the infrastructure and resources to focus on these integrated themes and advance innovative AD research through the pursuit of four overarching specific aims. Aim 1. Facilitate and enhance basic, translational and clinical research in AD and related dementias. Aim 2. Provide the necessary resources and interactive environment to support and create new opportunities for innovative research. Aim 3. Maintain and grow educational opportunities and community partnerships to promote awareness, increase participation in research, and provide an innovative and interdisciplinary training environment. Aim 4. Contribute to the national efforts to advance AD research, education, and care. The UK-ADC provides an infrastructure and environment that focuses on these integrated themes and advances AD research, education, outreach, and clinical programs through six highly interactive and effective components: Administrative Core, Clinical Core, Data Management and Statistics Core, Neuropathology Core, Outreach and Recruitment Core, and Research Education Component.

PROJECT SUMMARY (See instructions): The Biostatistics and Data Management Core (BDMC) can be conceptualized as the lynchpin for integration of the ADC Cores and is vital to the success of the UK-ADC. Its critical roles include managing a large centralized database, consulting with ADC-affiliated researchers, and working to develop and apply innovative statistical methodology for data analysis. Data management efforts focus on collecting and storing high quality data. This focus begins with the leadership and vision and attention to detail provided by the BDMC. This core has an enviable track record of timely and accurate reporting of a high volume of data to NACC. Further, weekly Core meetings are popular with UK-ADC investigators who find that the expert advice provided by our seasoned investigators improves their success in pilot studies, grant applications, and publications. This core also participates as a full partner to the research mission of the UK-ADC emphasizing transitions and translations. One such partnership with the Clinical and Neuropathology Cores relates to clinico-pathological models of mixed dementias. A key element of this BDMC is the well established track record of developing novel methodology to analyze data collected at the UK-ADC and from other cohorts with a focus on elderly subjects' transitions to MCI and eventually dementia. The BDMC also provides training for students enrolled in the graduate programs in Gerontology, Public Health, Epidemiology and Biostatistics, Psychology, and Statistics. In keeping with the mission of the UK-ADC, faculty in this core also contribute to the dementia research community at large through service on external advisory committees, study sections, manuscript reviews, and data safety monitoring boards. The BDMC will continue these critical responsibilities through the following specific aims. 1. Maintain a centralized database of the information collected by all ADC Cores and affiliated research projects in an integrated manner. 2. Provide expertise on experimental design and statistical analysis (and developing new analytical approaches). 3. Interact dynamically with other Cores to contribute to the clinical, neuropathological, and educational/outreach missions of the ADC.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is one of the largest global public health crises facing us today, and is predicted to increase dramatically over the next decades as the world population ages. There are no effective therapies available to prevent, cure, or slow the progression of disease, and new therapeutic strategies are urgently needed. Our strategy is to target the abnormal glial activation and neuroinflammation that arises early in AD progression and that is becoming of increased interest as a therapeutic target. Dysregulated neuroinflammation responses such as proinflammatory cytokine overproduction from abnormally activated glia are seen early in AD, and are thought to be a key contributor to downstream synaptic dysfunction and cognitive deficits. This raises the possibility that selective targeting of the dysregulated cytokine response may be a useful therapeutic approach. This application seeks three years of support to bring to IND status a novel experimental therapeutic (termed MW151) whose pharmacological mechanism of action is to attenuate disease- and injury-induced proinflammatory cytokine overproduction, thereby reducing downstream synaptic and cognitive dysfunction in multiple animal models of CNS disorders. MW151 is a water-soluble, orally bioavailable, CNS-penetrant, small molecule with outstanding chemical and metabolic stability. MW151 has been extensively de-risked through several non-GLP pharmacological and toxicology screens, and a GMP compatible production scheme has been developed. However, MW151 has not progressed into final GLP preclinical development. Our hypothesis is that MW151 will be a successful candidate for development as an oral formulation for the future treatment of individuals with mild cognitive impairment due to AD. With U01 funding, we will pursue three aims that are milestone-driven with clear Go/No Go decision criteria. Aim 1: Perform preclinical pharmacokinetics and safety assessment of MW151. Aim 2: Produce GMP drug substance (API) and oral drug product. Aim 3: Obtain an IND for future first-in-human clinical investigations. This project will advance development of a promising drug candidate with the potential to prevent the transition to or slow the progression of AD. In addition, successful development of MW151 could have broad clinical applications not only to AD but to a number of other CNS disorders where proinflammatory cytokine dysregulation is part of the pathophysiology progression mechanism.

? DESCRIPTION (provided by applicant): Microglia are the resident tissue myeloid cell in the brain. Acute isolation and molecular profiling of adult microglia have clearly identified a microglia-specific gene expression profile not found in other macrophage populations, or other cells in the CNS. As a molecular and functionally unique population of cells, microglia exhibit a remarkable ability to survey the brain and rapidly undergo a spectrum of responses to insults or tissue damage. These coordinated responses normally result in protection against injury, but can be dysfunctional and exacerbate or even cause neurological disease. An added layer of complexity occurs when monocytes are recruited to the brain following disease or injury. As a functionally distinct population, newly recruited monocytes/macrophages make different contributions to disease progression compared to microglia. One of the top canonical pathways in microglia/macrophages is the p38 MAPK pathway, a well-established signaling pathway important in stress responses. In the last funding period, we documented the importance of the p38alpha isoform in microglia proinflammatory cytokine production, and found that suppression of p38alpha can prevent the inflammatory response that causes subsequent neuronal/synaptic damage. In two mouse models of diffuse traumatic brain injury (TBI), we found that deletion of p38alpha in myeloid cells was associated with decreased microglia activation, and a rescue of vestibulomotor deficits, cognitive impairments and synaptic protein loss. Our data demonstrating the importance of p38alpha in regulating key functions of macrophages/microglia that are altered by a brain injury provides a strong precedent for further mechanistic and translational studies. We have now developed an exceptionally specific small molecule p38alpha inhibitor (termed MW150), that is CNS- penetrant, has drug-like properties, and no adverse effects at very high doses. MW150 is a novel reagent with high translational relevance for future therapeutic development. As a foundation for future development, we now propose to address mechanistically how to target p38alpha to improve outcomes following a TBI. To this end we need to answer: 1) When can p38alpha be targeted; i.e., what is the optimal therapeutic window after TBI? 2) Where and how can p38alpha be targeted; i.e., does p38alpha in other inflammatory cell types besides microglia (infiltrating macrophages, astrocytes, endothelial cells) contribute to neuroinflammatory responses after TBI? Our studies will provide key mechanistic information about how injury-induced, p38alpha-dependent inflammatory responses in multiple cell types synergize and cross-talk to drive neurologic impairments. We will test the hypothesis that suppression of p38alpha signaling in appropriate therapeutic windows and appropriate cell types after CNS injury can lead to selective, beneficial outcomes. Our aims are: Aim 1. Establish the post-injury therapeutic window for efficacy of our small molecule p38a inhibitor. Aim 2. Define the contribution of p38a in resident microglia versus infiltrating macrophages to TBI-induced impairments. Aim 3. Determine how p38alpha in other CNS inflammatory cell types (astrocytes and endothelial cells) contributes to injury-induced responses.

Project Summary/Abstract: Research Education Component The Research Education Component (REC) of the UK-ADC offers a variety of educational and training programs aimed at enhancing the training of a workforce to meet the nation?s biomedical, behavioral and clinical needs in Aging and Alzheimer?s disease (AD)-related research. The overall goal of all the educational efforts of the REC is to build a trained workforce with the knowledge and skills to: ? accelerate the pace of scientific research and translate basic science discoveries more rapidly into information and interventions that address specific aspects of AD, with the aim of minimizing its symptoms or delaying its progression (researchers) ? provide high-quality care and information to individuals treating the normal aging population, and those with cognitive impairment or more advanced dementia (healthcare professionals). Consistent with the Transitions and Translation theme of our ADC, we place a major emphasis on the promotion and education of translational researchers, and provision of a collaborative environment and training opportunities designed to facilitate their career growth. Over the next funding cycle, we will continue to offer multi-faceted learning formats and programs to educate healthcare professionals and to promote the development of future leaders in AD research. These include our CME educational series on dementia, telemedicine caregiver training programs for healthcare professionals, the annual Markesbery Symposium on Aging and Dementia, Sanders-Brown Center on Aging (SBCoA) monthly seminar program, Medical Student Geriatrics Enrichment Program, Geriatrics Clinical Selective for medical students, Neurodegenerative Disorders Selective for Neurology residents, formal didactic course education, and one-on-one mentoring of students, fellows, residents, and faculty as well as extramural researchers in the region. Our different learning formats provide opportunities for trainees to: attain information, ideas or concepts; learn through demonstration and observation; practice skills, techniques, and thinking processes; and increase in-depth understanding. In addition to continuing these existing highly successful educational programs, we propose to expand our training opportunities through two new initiatives. First, we plan to develop a training program called ?AD101: From Bench to Bedside?, aimed at enhancing the clinical research expertise of basic translational scientists. Second, we plan to pilot a new primary care provider training program for dementia screening that can be used in the setting of the Medicare Annual Wellness visit for detection of possible cognitive impairment. Our program includes highly motivated ADC Core faculty who bring a variety of specialized expertise to our educational programs, strong interactions with educational partners to provide multiple learning opportunities, and a highly collaborative environment and strong institutional support. These substantial program strengths will support the REC in attainment of its goals.

Project Summary/Abstract: Administrative Core The ultimate goal of the UK-ADC is to catalyze innovative and outstanding AD research while ensuring a more rapid rate of progress toward new therapies to delay or prevent AD, so that our human volunteers, patients and caregivers become the beneficiaries of our advances in knowledge. The Administrative Core provides the foundation, infrastructure, leadership, overall direction, strategic planning, and integration of all ADC components to more rapidly achieve this goal. We support and facilitate research aimed at elucidating the pathogenic mechanisms underlying the transitions from normal cognitive aging to the development of cognitive impairment, with a long-term goal of enabling more effective translation of this mechanistic knowledge to intervention strategies. The UK-ADC functions in a multidisciplinary, integrated, collaborative and supportive environment that catalyzes high-quality AD research within the UK community, region, nation, and beyond. Our major emphases continue to support our traditional strengths in neuropathologic discoveries and provision of normal and MCI autopsy tissue to support basic translational research, as well as our maturing program in antemortem biomarker assessment of predementia-stage subjects. The UK-ADC also enhances education and outreach within UK and in the community. We have developed a trusting partnership with the African-American community that has resulted in successful outreach programs and educational products, as well as increasing minority participation in research studies. We have innovative programs focused on quality of care and support for people with dementia and their caregivers. We offer multi-faceted educational and training programs to healthcare professionals and researchers that are aimed at enhancing the training of a workforce to meet the nation?s biomedical, behavioral and clinical needs in AD-related research. Our successful Pilot Project program leveraged support from university partners to be able to fund 6 additional pilot projects beyond the 10 funded by the ADC grant. The UK-ADC also reaches beyond Kentucky to enhance AD research, education, and clinical practice on a national level through its interactions with other ADCs, as well as other regional and national institutions, including the Alzheimer?s Association, NIA and industry. The Administrative Core will continue to support the ability of our UK-ADC to serve as a vital resource for the university, community, state, region and nation through the following specific aims. Aim 1: Provide leadership and an organizational framework for management and coordination of all ADC activities to ensure optimal resource utilization to meet the overall goals of the ADC. Aim 2: Promote our translational mission and enhance research, education and scientific interactions. Aim 3: Contribute to and interact with other ADCs and other national collaborative activities and increase visibility in the local and national community.

7. PROJECT SUMMARY/ABSTRACT This is an application for a T32 training grant titled ?Training in Translational Research in Alzheimer's Disease and Related Dementias (TRIAD)? to support 4 predoctoral and 4 postdoctoral fellows. The overall goal of the proposed program is to provide cross-disciplinary training from bench to bedside, to produce a new translational workforce that is critically needed to develop and advance effective treatments for Alzheimer's disease and related dementias. Trainees will receive rigorous and innovative translational research education and training through a 3-cluster multidisciplinary and integrative mentoring program that spans the discovery continuum from molecular/biochemical methods, preclinical translational approaches and clinical research. The thematic focus is on risk factors for Alzheimer's disease and related dementias; these risk factors include cerebrovascular disease, neuroinflammation, Down syndrome, and traumatic brain injury. The training program has 18 faculty mentors with established and extensive interactions and collaborations, and successful track records in training. The faculty mentors encompass diverse areas of expertise from cell & molecular biology, genetics, and data science, through preclinical model systems, therapeutic development, neuroimaging, neuropathology, clinical trials, and longitudinal clinical studies. The majority (13) of these mentors are housed within the University of Kentucky Sanders Brown Center on Aging, which includes a long-standing NIA-funded Alzheimer's Disease Center. All faculty with primary appointments in a Center also have academic appointments in other basic science or clinical departments at the University of Kentucky. Predoctoral trainees will be recruited from our Integrated Biomedical Sciences graduate program and from the MD/PhD program in the College of Medicine, and postdoctoral trainees through targeted advertisement and recommendations from colleagues and other Alzheimer's Disease Centers. In addition to their individualized research program and career development plan, each group of trainees will participate in a series of educational experiences that include a dedicated AD101 course in Alzheimer's disease and related dementias, seminars describing research and clinic ethics, monthly research and career development seminars, journal clubs, Training the Trainers workshops, and grant writing workshops. Each year trainees will be expected to present their research at the annual Markesbery Symposium on Aging and Dementia. Postdoctoral trainees will be provided with additional training opportunities, including: 1) a short off-site externship at another laboratory that conducts Alzheimer's disease research and 2) presentations at the annual College of Medicine Postdoctoral Poster Session. Quantitative and qualitative metrics for evaluation of trainees and the training program will assure a high-quality and effective training experience. The outcomes of our program are intended to nurture highly innovative, well-trained scholars who are dedicated and passionate about finding new approaches to prevent or slow Alzheimer's disease and related dementias.

ABSTRACT Cerebrovascular pathology is a very common comorbidity with Alzheimer's pathology. However, there is limited knowledge about the molecular mechanisms underlying the vascular contributions to cognitive impairment and dementia (VCID), the mechanisms that link VCID and Alzheimer progression, or how to intervene effectively in a complex multi-pathology environment. A key mechanism that drives pathophysiology progression in many CNS disorders is dysregulated neuroinflammatory responses from innate immune cells in the brain, and chronic neuroinflammation is a common feature seen early in both VCID and Alzheimer's disease (AD) progression. An important cell signaling pathway that contributes to neuroinflammatory responses in many CNS disorders is the p38 MAPK pathway, especially the p38? isoform. Activation of this important stress- regulated protein kinase occurs early in AD, p38? activation can lead to chronic neuroinflammation that has neurodegenerative consequences, and selective p38? inhibition is beneficial in AD animal models. However, there is no information on whether p38? is important in the detrimental inflammatory responses and subsequent neurodegenerative sequelae seen in VCID or comorbid VCID/AD pathology. We hypothesize that vascular dysfunction in the context of Alzheimer pathology creates an enhanced microglia inflammatory state, driven by p38? MAPK, that worsens the pathologic outcomes. We further postulate that the pathogenic events induced by comorbid VCID/AD will be modifiable by selective inhibition of p38?. To test our hypotheses, we will use the established hyperhomocysteinemia mouse model of cerebral small vessel disease to induce vascular injury in the APP/PS1 transgenic mouse. We will also use our recently developed, exceptionally specific, small molecule p38? inhibitor MW150, which is CNS-penetrant, safe, and efficacious in mouse models of AD pathology. The goal is to determine the therapeutic efficacy of MW150, in order to extend its potential for disease modification to a complex comorbid brain environment exhibiting both VCID and AD pathologies. Aim 1 will determine the minimum dose of MW150 that mitigates VCID-induced functional deficits in AD transgenic mice in a preventative treatment paradigm. Aim 2 will determine the mechanisms underlying MW150's actions in mitigating degenerative sequelae, by testing its effects on neuroinflammatory, vascular and synaptic dysfunction endpoints. Aim 3 will test MW150 in a therapeutic treatment paradigm, to determine efficacy in older mice when pathology is already present, mimicking what we see in the clinic with AD patients exhibiting comorbid vascular pathology. Because MW150 is a drug candidate in early stage clinical development, successful completion of these mechanistic and highly translational studies will extend the potential clinical utility of MW150 to a disease indication with complex comorbid pathologies. Given the urgent unmet medical need and MW150's unique potential, success will offer the potential for a new chemical entity and therapeutic intervention for VCID/AD, either as a mono-therapy or as part of a multi-drug armamentarium.

ABSTRACT There are no approved disease-modifying drugs to prevent, delay, or slow disease progression of Alzheimer's disease (AD) and related dementias. The overwhelming majority of AD clinical trials targeted the beta-amyloid pathway with disappointingly ineffective outcomes in >99% of the studies. Therefore, there is an urgent need to diversify the portfolio of disease modifying approaches that are distinct from prior art. Our unbiased discovery strategy for the campaign described here focused on targeting a particular form of dysregulated inflammation, injurious proinflammatory cytokine overproduction in the brain, that is a key contributor to synaptic dysfunction, neurodegeneration and cognitive decline in diverse neurodegenerative diseases. We seek funding for the required phase 1 clinical safety studies of MW01-2-151SRM (=MW151). MW151 is a novel, CNS-penetrant, orally bioavailable, small molecule candidate that selectively suppresses stressor-induced proinflammatory cytokine overproduction. MW151 ameliorates synaptic damage and cognitive impairment at low doses in diverse animal models where proinflammatory cytokine dysregulation is established as a contributor to disease progression. MW151 has no liabilities in investigational new drug (IND)-enabling safety pharmacology and toxicology tests such as respiratory and cardiovascular safety pharmacology, rat and dog 28-day repeat administration toxicology, and genotoxicity. The low risk aspect of MW151 safety is reinforced by an analog developed for the demanding intravenous route of administration and its progression through phase 1a and promising status in phase 1b. We hypothesize that MW151 will be a successful oral candidate for future treatment of individuals with MCI/AD. This application seeks to progress through the required first-in-human (FIH) safety clinical trials. Our specific aims are: Aim 1: Conduct a first-in-human (FIH) phase 1a single ascending dose (SAD) study. This study will determine safety and tolerability, maximum tolerated dose, and pharmacokinetics (PK) of MW151 in healthy adult volunteers. Plasma cytokine levels will be measured to provide baseline data for a future exploratory pharmacodynamic (PD) endpoint in phase 2a clinical trials. Aim 2: Conduct a phase 1b multiple ascending dose (MAD) study of MW151. This study will determine safety and tolerability, maximum tolerated dose, and PK of MW151 in healthy adult volunteers. In addition, a cohort of elderly healthy subjects and exploratory PD inflammatory cytokine endpoints in CSF will be included. Aim 3: Prepare clinical protocol and investigators brochure for a future phase 2a clinical trial of MW151 in early AD. Based on the outcomes of the proposed phase 1 studies, we will design a phase 2a trial and prepare the documents required. This will allow immediate progress to future FIP studies for AD. Overall, MW151 represents a unique opportunity for development as a first-in-class disease-modifying therapeutic.

PROJECT SUMMARY/ABSTRACT ? OVERALL (Adapted from Abstract of parent grant, UK-ADC) This is a proposal for a University of Kentucky Alzheimer?s Disease Research Center (UK-ADRC) neuroimaging supplement. This purpose of the supplement is to provide neuroimaging data on the UK-ADRC?s deeply phenotyped clinical cohort to the NIH?s recently funded initiative, Standardized Centralized Alzheimer's and Related Dementias Neuroimaging (SCAN). In addition, a secondary purpose of this supplement is to identify neuroimaging correlates of two AD-mimics: limbic-predominant age-related TDP-43 encephalopathy (LATE) and primary age-related tauopathy (PART). The UK-ADRC is an experienced and collaborative ADRC originally funded in 1985. Our principal mission is to serve as the focal point for all AD-related activities at UK and this region of the United States, by providing an environment and core resources that catalyze innovative research, outreach, education, and clinical programs. Our signature resources include: 1) a cognitively normal group of ~500 subjects followed longitudinally, with all committed to brain autopsy upon death; 2) a strong program in clinical-neuropathological correlations and short postmortem interval autopsies; 3) an established program studying preclinical biomarkers of AD using neuroimaging and biofluids; 4) an integrated centralized database and innovative biostatistical expertise to characterize clinical and biological transitions; and 5) a successful and close partnership with the African-American community and increased participation of underrepresented individuals in our longitudinal cohort and ADRC-affiliated research studies and clinical trials. The overall scientific emphasis of the UK-ADRC continues to be on our interrelated themes: Transitions & Translation. Our well-characterized, longitudinal cohort and historically strong neuropathology program focused on normal aging, preclinical disease states and early cognitive transitions have been central to our success in defining early pathogenic mechanisms underlying the transitions from normal cognitive aging to impairment. The depth of expertise and collaborative nature of our investigators have also resulted in substantial progress on translation of that knowledge into new targets and novel therapeutic strategies. The UK-ADRC provides an infrastructure and environment that focuses on these integrated themes and advances AD research, education, outreach, and clinical programs through highly interactive and effective components: Administrative Core, Clinical Core, Data Management and Statistics Core, Neuropathology Core, Outreach and Recruitment Core, Biomarker Core, and Research Education Component. The UK-ADRC will make resources from these Cores available to support the success of this ADRC neuroimaging supplement.

Project Summary/Abstract: Research Education Component The Research Education Component (REC) of the UK-ADRC offers a variety of research educational activities and comprehensive, mentored research skills development programs that will enhance the development of the future workforce to meet the nation?s biomedical, behavioral, and clinical needs in Alzheimer?s disease and related dementias (ADRD) research. The REC will leverage resources within and outside the UK-ADRC to create an outstanding, multidisciplinary, and collaborative environment for training the next generation of researchers in scientific areas that parallel the theme of our ADRC (Transitions from Normal to Late-Life, Multi- Etiology Dementias), and with an emphasis on increasing representation of researchers from diverse backgrounds. We will continue to build on a long history of collaborative and dedicated faculty, a strong foundation in multidisciplinary team science, and an outstanding track record of success in mentoring and education of researchers across the training spectrum. We will also establish a new REC Scholar program aimed at junior investigators (Assistant Professors, senior research associates, or clinical fellows). This program will support junior investigators from diverse, multidisciplinary fields to pursue mentored basic, translational, or clinical research in ADRD, and follow a customized training plan to help the scholars move toward their career development goals. This training will be done in a supportive and collaborative academic environment with substantial training resources and an experienced and dedicated training faculty. Scholars will have the opportunity to learn state-of-the-art research skills in disciplines that have been identified as critically needed areas for the ADRC workforce of the future. Examples of training areas where the UK-ADRC has strong expertise include analysis of longitudinal clinical outcomes, clinical trial design, MRI imaging and novel fluid biomarkers, quantitative neuropathology and AD mimics, data analysis/ study design in mixed dementias, and health literacy and inequities. REC goals will involve interactions with all the UK-ADRC Cores and will be achieved through the following specific aims. Aim 1. Provide education programs and research skills development opportunities for investigators at UK. Aim 2. Identify, select, support, and retain REC scholars from diverse backgrounds and disciplines to engage in ADRD research. Aim 3. Develop an individualized mentoring plan and educational program for each REC scholar to enable advancement to independent research and funding in ADRD research. Aim 4. Evaluate progress of the REC scholars, address barriers towards the goal of independence in ADRD research and refine delivery of the program?s educational structure and processes. Following completion of the Program, participants will have the knowledge, expertise, and confidence to successfully pursue a long-term career in dementia research.

Project Summary/Abstract: Administrative Core The ultimate goal of the UK-ADRC is to catalyze innovative and outstanding AD research while ensuring a more rapid rate of progress toward new therapies to delay or prevent AD, so that our human volunteers, patients and caregivers become the beneficiaries of our advances in knowledge. The Administrative Core provides the foundation, infrastructure, leadership, overall direction, strategic planning, and integration of all ADRC components to more rapidly achieve this goal and advance our overall theme: ?Transitions from Normal to Late-Life Multi-Etiology Dementia?. This theme builds on our traditional strengths in defining mechanisms underlying the early changes that occur in individuals as they transition through the cognitive continuum, risk factors associated with these cognitive states, and how mixed pathology and co-morbidities influence risk and progression. Two historically outstanding resources of the UK-ADRC that enable research relevant to this theme are: 1) a strong autopsy program providing clinical-neuropathological correlation and very short (2-4 hrs) postmortem interval (PMI) research material, and 2) a unique, continuously replenished group of ~500 cognitively intact subjects followed longitudinally, together with another ~300 initially normal individuals who transition to pre-MCI, MCI or dementia, and all committed to brain autopsy upon death. These signature resources have contributed to our becoming one of the premier centers defining pathogenic mechanisms underlying the transitions from normal cognitive aging to the earliest stages of cognitive impairment. The UK- ADRC functions in a multidisciplinary, integrated, collaborative and supportive team science environment that catalyzes high-quality AD research within the UK community, region, nation, and beyond. We will continue to provide the infrastructure, resources and intellectual capital to enhance transdisciplinary research and scientific interactions, develop new collaborations and partnerships, contribute to national initiatives, and offer innovative education and training opportunities in the basic and clinical neurosciences. An effective Administrative Core is essential for transforming our goals into accomplishments. Therefore, the Core will continue to support the ability of our UK-ADRC to accomplish its goals, and to promote the NAPA research implementation milestones and goals, through the following specific aims. Aim 1: Provide leadership, administrative structure, coordination, and financial management to ensure optimal resource utilization to meet the overall goals of the UK-ADRC. Aim 2: Promote and catalyze clinical/translational research activities, maximize interactions with other departments and centers, and foster new avenues of innovative research. Aim 3: Solicit, review, select, and monitor the Developmental Projects. Aim 4: Contribute to and interact with other ADRCs and other national collaborative activities and increase visibility in the local and national community.

PROJECT SUMMARY Acute brain injuries resulting from cerebrovascular injury or trauma, such as intracerebral hemorrhage (ICH) or traumatic brain injury, are major medical problems that cause considerable mortality and morbidity in older Americans. Secondary neuroinflammatory events after ICH can further damage the brain and lead to increased risk of neurologic complications including Alzheimer?s disease (AD) and related dementias. Despite significant advances in the medical management of these patients, there is a clear and urgent need for interventions that improve neurologic recovery and outcomes. To address this unmet need, the clinical candidate, MW189, is a CNS-penetrant, small molecule that selectively attenuates injury- and disease-induced proinflammatory cytokine overproduction. Proinflammatory cytokine overproduction from abnormally activated glia contributes to cerebral edema, long-term neurological damage, and cognitive deficits following acute brain injuries. This mechanistic linkage of the acute cytokine surge to progression of injury, plus the attractive therapeutic time window of hours to days post-insult, provide a rational therapeutic target for intervention in the acute care setting. The Biomarker and Edema Attenuation in IntraCerebral Hemorrhage (BEACH) trial is a first-in-patient phase 2a, proof-of-concept study of MW189 in patients with ICH. The study aims are to: (1) Prepare, recruit, and conduct the phase 2a clinical study of MW189, and (2) Evaluate safety, pharmacokinetics (PK), inflammatory biomarkers, and clinical outcomes. This multicenter, prospective, randomized, double-blind controlled trial will enroll 120 non-traumatic ICH participants, with an anticipated average age in their mid-60s and substantial numbers of individuals with cerebral small vessel disease and cerebral amyloid angiopathy. Patients will be randomized to MW189 or placebo in a 1:1 ratio, with the first dose initiated within 24 hours of symptoms, then dosing every 12 hours for 5 days (or until discharge, whichever is first). Safety and tolerability of MW189 compared to placebo, and PK profiles of MW189 will be determined. Exploratory outcomes will include radiographic and clinical endpoints and measurement of plasma levels of brain-derived inflammatory and neuronal injury biomarkers to demonstrate engagement of pharmacological mechanism. Success with MW189 in ICH patients will further de-risk the compound for subsequent larger trials of acute CNS injury and/or to develop the drug for AD and other age-related dementias. The study will also generate important information about the utility of targeting the acute proinflammatory cytokine aspects of neuroinflammation in older Americans with vascular disease.

Project Summary/Abstract: Overall The University of Kentucky Alzheimer?s Disease Research Center (UK-ADRC) is an experienced and collaborative center that has facilitated pioneering research in AD and related dementias (ADRD) since its inception in 1985. Our principal mission is to serve as the focal point for all AD-related activities at UK and this region of the United States, by providing an environment and core resources that catalyze innovative research, outreach, education, and clinical programs. Our signature resources include: 1) a cognitively normal group of ~500 subjects followed longitudinally, together with ~300 additional subjects who transitioned to MCI or dementia, and all committed to brain autopsy upon death; 2) a strong autopsy program with clinical- neuropathological correlations and short postmortem interval research material; 3) a maturing program studying the early preclinical biological emergence of mixed pathologies and how they contribute to late life dementia states, with an increasing focus on antemortem biomarker collection; 4) an integrated centralized database and innovative biostatistical expertise to characterize clinical and biological transitions; 5) a successful and close partnership with the African-American community and increased participation of underrepresented individuals in our longitudinal cohort and ADRC-affiliated research studies and clinical trials; and 6) a rich, interdisciplinary training environment that provides multi-faceted educational opportunities for researchers, healthcare providers, and our community partners. The overarching theme of the UK-ADRC is: Transitions from Normal to Late-Life Multi-Etiology Dementia. Our well-characterized, longitudinal cohort and historically strong neuropathology program focused on normal aging, preclinical disease states and early cognitive transitions have been central to our success in defining early pathogenic mechanisms underlying the transitions from normal cognitive aging to impairment. In addition, these efforts have been a driving force in our recognition of the heterogeneity and multiple pathologies that characterize late-life dementia. The UK-ADRC will continue to leverage our strengths to enhance our impact and ?Centerness? by our focus on this overarching theme, and the pursuit of four overall specific aims. Aim 1. Facilitate and enhance basic, translational and clinical research in AD and related dementias. Aim 2. Provide the necessary resources and interactive environment to support and create new opportunities for innovative research. Aim 3. Maintain and grow educational opportunities and community partnerships to promote awareness, increase participation in research, and provide an innovative and interdisciplinary training environment. Aim 4. Contribute to the national efforts and collaborative activities with other centers, programs and groups to advance AD/ADRD research, education, and care.