Dennis W. Dickson, MD, University of Iowa - US grants

Among the histopatholigical alterations that are found at autopsy in brains of nondemented elderly humans are dystrophic axons, granular degneration of myelin and, in some cases, senile plaques (SP). Although SP are also found in Alzheimer's disease (AD), they are morphologically different fromthose in the non-demented brain. We hypothesize that SP evolve from nonfibrillar preamyloid deposits that undergo fibrillogenesis promoted by physical and biochemical changes in Abeta. The long-live amyloid deposits are progressively glycated, which elicits recruitment and activation of microglia and astrocytes and production of neurotoxins and other amyloid associated molecules (e.g., apo-E). The ensuing reaction leads to further amyloid deposition and local neuritic dystrophy. In the aged brain dystrophic neurites in SP have coarse granular ubiquitin-immunoreactivity. In contrast, paired helical filament (PHF) immunoreactivity is detected in AD. Ubiquitin is also found in dystrophic axons that are consistently found in the limbic gray matter of the elderly brain. It is our hypothesis that progressive neuroaxonal dystrophy may be responsible for age- associated cognitive impairment not due to early AD. Ubiquitin also labels granular degeneration of myelin, which is an inevitable consequence of aging, that may underlie motor and mental slowing of the elderly. To further elucidate the role of neuritic and axonal dystrophy and SP in cognitive impairment of the elderly, we will pursue three major specific aims: 1) Determine if there are qualitative differences in SP brains of people with cognitive impairment compared to those without impairment by analyzing the type of SP neurites (dystrophic vs. PHF types), apolipoprotein-E (apo-E) and advanced glycation endproducts (AGE) immunoreactivity and presence of astrocytes and microglia. These qualitative analyses will be correlated with quantitative immunoassays for apo-E and AGE, the latter with respect to brain fractions enriched in Abeta of PHF. 2) To characterize and objectively measure the amount of neuritic dystrophy and granular myelin degeneration by image analysis of immunostained sections and immunoassays (ELISA & dot blots) for ubiquitin in fresh tissue. 3) Characterize neuroaxonal dystrophy in limbic gray matter with double immunolabeling, laser confocal microscopy and immunoelectron microscopy and immunoelectron microscopy with respect to cellular and non-cellular components of the neuropil.

Neuropathology remains a useful discipline, especially in characterization of neurodegenerative diseases. In fact, it provides the "gold-standard" for diagnosis of non-Alzheimer neurodegenerative diseases, including progressive supranuclear palsy, corticobasal degeneration and the frontotemporal dementias. The broad goal of the Neuropathology Core (NP Core) is to provide neuropathologic characterization of brains of humans and animal models that are engineered in research projects that focus on the role of tau protein in neurodegeneration. In particular, the NP Core will characterize and provide postmortem tissue for genetic studies, provide electron microscopic and confocal microscopic services, and process animal brains with a complement of histopathologic methods. Among the specific activities of the NP Core are the following: 1. Perform neuropathologic evaluation of human and animal brains using standardized methods for collection and banking of fresh and fixed brain tissue; standard gross dissection and tissue sampling; histopathologic analysis with routine histological methods as well as fluorescent microscopy, immunocytochemistry, laser confocal microscopy, image analysis and electron microscopy. 2. Conduct gross and microscopic clinicopathologic conferences for discussion of cases relevant to this application. 3. Characterize brains of humans and animals with immunocytochemistry and a panel of antibodies to tau protein, as well as antibodies to phosphorylated and non-phosphorylated neurofilament, ubiquitin, alpha-synuclein, synaptophysin, glial fibrillary acidic protein and class II major histocompatibility antigen. 4.Embed and section tissue culture specimens for electron service for confocal microscopy of tissue and cultured cells. 6. Provide image analysis of animal brains to provide a measure of neuronal loss. 7. Maintain a database of pathologic parameters for cases and controls. In its functions of NP Core serves a link between the basic science projects of this grant and clinical studies of a group of uncommon, but related, neurodegenerative disorders.

neuropathology; genotype; Alzheimer's disease; biomedical facility; family genetics; tissue resource /registry; human genetic material tag; postmortem; immunoelectron microscopy; enzyme linked immunosorbent assay; immunocytochemistry; human tissue;

DESCRIPTION (provided by applicant): The goal of this project is to use quantitative neuropathologic methods to define mild cognitive impairment (MCI) in the elderly and its relationship to Alzheimer's disease (AD). The focus is on a select group of markers for different stages of a pathogenetic sequence that we hypothesize begins with deposition of amino-terminally modified or truncated AB followed by deposition of full-length Ap that leads to neuroinflammation and subsequent cellular stress that manifests in aberrant activation of the cell cycle and cytoskeletal pathology in neurons and eventual synaptic loss. We hypothesize that biochemical alterations are widespread in the brain and not only in areas with obvious structural changes. We also hypothesize that there may be regional differences with respect to significant synaptic loss. Previously, we analyzed frontal lobe, but we will analyze cortical areas theoretically more (temporal) or less (occipital) vulnerable than the frontal lobe. The specific aims are: 1) To analyze amyloid peptide heterogeneity using assays for amino-terminal modifications or truncation as well as full length A-beta, which we hypothesize deposits first as A-beta-1-42 and later as A-beta-l-40; 2) To analyze markers of neuroinflammation and astrocytosis, which we hypothesize is due to the changing nature of amyloid in the brain, by measuring microglial activation (HLA-DR and IL1a), astrocytic gliosis (GFAP) and proinflammatory cytokines (IL-l-beta, TNF-alpha, IL-6 and IL-1ra.); 3) To analyze cytoskeletal changes that we hypothesize may be the product of aberrant activation of the cell cycle due to cellular stress due to amyloid or neuroinflammation by extending tau assays to include assays for cyclin-dependent kinase-2 and downstream effects of cell cycle activation, specifically, markers of apoptosis (activated caspase-3 and caspase-3-cleaved fodrin); 4) To analyze markers indicative of neuronal structure and number, including synaptic markers (synaptophysin, SNAP25 and synuclein), NeuN and alpha-internexin; and finally, 5) To complete studies on age-related changes in white matter that may be structural correlates of cognitive and motor slowing in normal aging with immunoassays and immunocytochemistry for ubiquitin and myelin basic protein on additional normal elderly cases. The data will be analyzed across all diagnostic categories and clinical parameters to validate the proposed pathogenetic cascade. A profile of individual MCI cases will also be analyzed and compared to the average values of normals and AD to address the issue of heterogeneity of MCI and specifically to define an MCI phenotype that most closely resembles AD.

DESCRIPTION (provided by applicant): The Udall Center for Excellence in Parkinson Disease Research at the Mayo Clinic is an integrated, multi-disciplinary research program focusing on of the Genetics and Molecular Biology of Parkinsonism. The Udall Center draws upon the clinical strengths of the Mayo Clinic Movement Disorder Section with its longitudinal studies of Parkinson disease (PD) and a focus on the clinical genetics of familial Parkinsonism, a large brain bank of Parkinsonian disorders and strong institutional commitment to PD research and education. All components of the Udall Center are based in Jacksonville, Florida, although core resources in Rochester, Minnesota, particularly the Division of Biomedical Statistics will be used. Mayo Foundation educational resources, including sponsored seminar series and graduate level educational courses on PD and other neurodegenerative disorders are also utilized. The proposal is highly innovative and original in that it addresses the intriguing interaction between two molecules with surprising similarities, tau and a-synuclein, both of which are strongly implicated as critical factors in pathogenesis of parkinsonian disorders. The clinical, genetic and pathologic studies proposed capitalize on inherent strengths of the Udall Center investigators and unique clinical, genetic and pathological resources that have been built over the years due to the fervent devotion of investigators to better understanding PD and related disorders. The Center also brings established investigators in complementary areas of research (Drs. Rademakers and Petrucelli) to the task of understanding how tau is related PD and how tau-directed therapies may translate into novel treatments for PD and parkinsonian tauopathies. The proposed Center will have three projects and four cores with an overarching theme to understand the role of tau in PD and parkinsonian tauopathies. Project 1, entitled Identification of novel parkinsonian genes by whole-genome sequencing, is led by Rosa Rademakers, PhD. Project 2, entitled Genetic determinants of a-synuclein and tau pathology in Parkinsonism, is led by Dennis W. Dickson, MD. Project 3, entitled Identification and testing of novel compounds to treat Parkinsonism, is led by Leonard Petrucelli, PhD. Core A (Administration) and Core D (Neuropathology) are led by Dennis W. Dickson, MD. Core B (Clinical) is led by Zbigniew K. Wszoiek, MD. Core C (Genetics) is led by Owen A. Ross, PhD.

Identification of the earliest stage of Parkinson's disease (PD) is critical to understanding its etiology. The idea that the substantia nigra is the primary site of pathology in PD is challenged by the anatomical staging for Lewy body disease (LBD) proposed by Braak and co-workers, in which substantia nigra LBs are detected at mid-stage and neuronal loss at some undefined later stage. If the LBD staging scheme is validated, the implications are clear. Non-motor manifestations are the earliest signs of PD (i.e. preclinical PD), not the extrapyramidal signs that are currently used to diagnose PD. The aim of this proposal is to validate the LBD staging scheme using clinicopathologic approaches and the Mayo Medical Records Linkage System (MMRLS). Medical records of cases determined to have incidental LBs will be screened for clinical, demographic and environmental risk factors that have been implicated in PD and compared to matched cases without LBs. The LBD staging scheme predicts several non-motor features of preclinical PD, including anosmia, autonomic dysfunction, sleep disorders and depression. The MMRLS will be queried for cases that have come to autopsy meeting these clinical descriptors and their brains and matched controls will be studied for LBs. The proposed LBD staging scheme does not include estimates of severity or neuronal loss, but these will be assessed with quantitative methods. Specifically, in brains found to have incidental LBs and those with more advanced stages, neuronal counts and gliosis will be measured in brainstem and basal forebrain nuclei that are vulnerable at the earliest stages. In collaboration with Project by Yen, biochemical measures of abnormal alpha-synuclein species will be measured in multiple brain regions to compare biochemical with histopathologic staging. To validate LBD staging in an independent pathologic cohort, a large series of progressive supranuclear palsy (PSP) brains will be screened for LBs, and the distribution will be compared to staging in non-PSP cases. The applicability of the LBD staging scheme will also be assessed in LBD cases acquired through various resources available to the Neuropathology Core, including cases of dementia with LBs, PD with later developing dementia and familial LBD cases studied by the Clinical and Genetic Cores. Finally, the possible contribution of tau pathology to LBD will be assessed since studies by the Genetic Core implicate TAU in PD.

Previous clinicopathological studies have indicated that some elderly individuals have numerous cerebral amyloid deposits, yet remain cognitively intact, a process we call "pathological aging" (PA). It remains uncertain if PA is pre-clinical Alzheimer's disease (AD), but the proposed studies i n the application will address this issue. In particular, clinicopathologic correlations with increasingly sophisticated neuropsychologic measures, such as those developed in Project 2, will be applied to determine if there are cognitive differences between PA and brains with no amyloid deposition. If PA is pre-clinical AD, it will be important to understand the nature of the amyloid that is deposited and the mechanism of its formation, especially if it is one of the earliest markers of AD. While tau pathology in PA is minimal, it will be important to precisely define the nature of tau pathology in PA as well as in cases with cognitive impairment ranging from mild amnestic deficits (amnestic cognitive impairment - ACI) to frank dementia. In this competing renewal, Specific Aim 1 is focused on characterizing amyloid in PA compared to AD, as well as studying the mechanism of amyloid deposition. Specific aim 2 is focused on the hypothesis that progressive tau pathology underlies cognitive impairment in ACI and AD. Studies in this aim will use quantitative analyses and a panel of tau antibodies, including recently developed antibodies specific to exon 10 splice forms of tau, to study progressive abnormalities in tau. Since most clinicopathologic studies of community residing subjects indicate that mild cognitive impairment and AD is often associated with mixed degenerative and vascular pathology, Specific aim 3 is focused on efforts to better characterize vascular pathology in brains to understand the relative contribution of vascular and degenerative lesions to cognitive impairment in the elderly. It will do so through development of novel quantitative assays of white matter pathology and arteriosclerotic vascular disease. In summary, the clinicopathologic correlations that form the underpinnings of this project focus on quantitative analyses of the three major structural pathologies in the aging brain - amyloid, tau and vascular disease.

The Administration, Data Management and Statistics Core will be responsible for the (1) overall management of the Udall Center for Excellence in Parkinson's Disease Research, (2) implementation of database and biostatistical support for the Cores and Projects, (3) promotion of education and research opportunities among young PD investigators, and (4) expansion of interactions between the Mayo Clinic and other Udall Centers. More specifically, the management structure of the Administrative Core is designed to ensure consistent communication and collaboration between the Cores and Projects, as well as provide independent guidance via an External Advisory Committee. An Executive Committee will be headed by the PI and composed of the PIs of the other Cores and Projects and the chiefs of the Movement Disorder Sections, and it will meet on a bi-weekly basis. An External Advisory Committee will be recruited and convened every 12 to 18 months for review of the Center's activities. In addition, this Core will provide technical and analytical expertise for the data management and analysis requirements of the other Cores and Projects, as well as develop an integrated data warehousing system designed to enhance collaboration between the Cores and Projects. Consistent with Mayo Clinic policy, all database designs will meet or exceed HIPAA compliance standards. The Core will also support PD research through active participation in training and education programs, the annual funding of a pilot research grant, and establishment of funds to support travel for research fellows to interact with other Udall Centers. In anticipation of a national Udall Center database, this Core will coordinate data collection according to the established reporting standards. This Core will promote efforts of the Clinical and Genetic Cores to submit genetic samples, especially for familial PD, when appropriate consent is given, to the NINDS-sponsored DNA repository. The PI of the Administrative Core will communicate with the NINDS and be responsible for annual progress reports. Finally, the Udall Center will be compliant with the training requirements for key personnel as outlined by the Mayo Foundation and will be HIPAA compliant.

The Neuropathology (NP) Core will perform diagnostic evaluations and quantitative analyses on brains collected from participants in the Florida Alzheimer Disease Research Center (FADRC). The NP Core will also provide support to pilot research projects of the FADRC. The specific aims of the NP Core are: 1) To oversee performance of brain autopsies on participants of the FADRC in a timely fashion and according to protocol. 2) Provide neuropathologic evaluations and collect neuropathologic data for all brains using standardized neuropathologic methods for gross dissection and neurohistology, including traditional histological methods and thioflavin-S fluorescent microscopy to assign a Braak stage and a CERAD neuropathologic diagnosis; 3) Immunostain all cases with antibodies to alpha-synuclein, and on all cases with Lewy bodies, assign a Consortium for Dementia with Lewy Bodies diagnosis based upon counts of Lewy bodiesin cortical regions; 4) Photograph and map vascular lesions on cases with vascular pathology; 5) Postmortem biochemical analyses for correlative studies with Project 1; 6) Characterize non-Alzheimer dementias with immunocytochemistry and biochemistry and electron microscopy, as needed. A final diagnosis will be achieved by clinicopathological consensus conferences to be held at least quarterly by videoconference with the Clinical Core. Brain tissue and brain derived material will be stored and provided to approved investigators for research projects and pilot projects of the FADRC and to qualified outside investigators. The Neuropathologic data will be provided to the National Alzheimer Coordinating Center (NACC) through the Biostatistics Core.

Neurofibrillary inclusions composed of the microtubule associated protein tau (tau) are a hallmark feature ot the pathology in several neurodegenerative diseases including Alzheimer's disease, Progressive supranuclear Palsy (PSP) and Corticobasal Degeneration (CBD). A causal link between tau dysfunction and neurodegeneration was demonstrated by the idenification of mutations in tau that give rise to FTDP-17. The overall goal of this current program is to identify modifiers that influence the progression of tau pathology, in human neurodegenerative disease and to build on this information to identify therapeutic targets that will form the basis for eventual patient treatments. This goal follows on naturally from the progress made in the first period of funding in which this Program was highly successful in developing both cell culture and transgenic animal models of tauopathy as well as in characterizing the genetic causes of these diseases. The four projects, alongwith a central Neuropathology core, that make up this program will address this overall goal through different but complementary strategies. Project 1 (Dr Farrer) will utilize a genetic approach to identify tau gene variants that increase the risk for developing 4R tauopathy (PSP and CBD) and will determine the mechanism by which these variants lead to disease. This project will thus define a potential therapeutic target in these diseases. Project 2 (Dr Yen) will utilize a cell culture model of early stage tau filament formation and pathogenesis to study the impact of several factors that have been suggested as causes of tauopathy (eg oxidative stress, proteasome inhibition). This project will identify modifiers of tau pathogenesis in this cell model that can then be studied in our transgenic models. Projects 3 (Hutton) and 4 (Duff) will employ transgenic mouse models of tauopathy developed by the Program over the past 4 years to study potential targets already identified by preliminary studies. Project 3 will study the impact of the chaperone Hsp70 and its co-chaperone CHIP on tau pathogenesis whilst Project 4, will examine the impact of tau phosphorylation on pathology and neurodegeneration.

Core A (Administration) will be responsible for (1) fiscal management of the Udall Center for Excellence in Parkinson Disease Research, (2) insuring responsible conduct of research, and (3) promoting interactions between the Mayo Clinic and other Udall Centers. Moreover, it will provide scientific direction and accountability with the assistance of an External Advisory Committee, and it will be responsible for reporting to the NINDS.

The Udall Center for Excellence in Parkinson Disease Research at the Mayo Clinic is an integrated, multidisciplinary research program of neurologists, neuropsychologists, geneticists, neuropathologists and basic scientists in the study of the "Genetics and Molecular Biology of Parkinsonism." The Center draws upon the clinical strengths of the Mayo Clinic Movement Disorder Section and longitudinal studies of Parkinson disease (PD) and dementia with Lewy bodies (DLB) for the clinical material used in the research projects, as well as strong institutional commitment to PD research. The research proposed is highly synergistic despite the wide range of expertise and scientific background of the members of the Udall team. Each member of the team brings unique knowledge and skill sets to the mission, and together we are greater than the sum of the parts. The work proposed builds upon highly successful and productive cores that have been working together for over a decade. The clinical, genetic and pathologic resources are rivaled by few centers. The Mayo Udall Center is a unique complement to the Udall Center program. Success of our Udall Center has been based upon building successful collaborations and generous sharing of resources and data. This proposal has the overarching goal to better understand Lewy-related PD, which is the most common form of PD. Lewy bodies are also the hallmark of DLB and PD with dementia (PDD). How PDD and DLB relate to each other is unknown, but this non-motor complication of PD is of increasing interest to the Parkinson community. Strengths of our Udall Center are the large collection of multi-incident PD families, a proven track record of success in discovery of PD genes and a large collection of PD, PDD and DLB brains in a well annotated brain bank. The research proposal has three projects and five cores: Project 1. "Identification of genetic risk factors that predict disease onset, susceptibility and progression of PD," (PL: Matthew J. Farrer, PhD);Project 2. "Identification of candidate therapeutics for a-synuclein aggregation and cytotoxicity," (PL: Shu-Hui C. Yen, PhD);Project 3. "Molecular pathology of Lewy-related cognitive dysfunction," (PL: Dennis W. Dickson, MD);Core A. Administrative (CL: Dennis W. Dickson, MD);Core B. Clinical (CL: Zbigniew K. Wszolek, MD);Core C. Genetic (CL: Matthew J. Farrer PhD);Core D. Neuropathology (CL: Dennis W. Dickson MD) and Core E. Education and Outreach (CL: Ryan J. Uitti, MD).

Project 2 will use intemnediate pathologic phenotypes to explore associations with genetic variants in progres- sive supranuclear palsy (PSP) and Lewy body disease (LBD). The results will provide insights into the molecular underpinnings of Parkinsonian disorders. We will use MAPT, as well as non-M(4PT single nucleotide polymor- phisms (SNPs) from genome-wide association studies (GWAS). Aim 1. Generate intermediate pathologic phenotypes for PSP. We will measure burden of tau using digital imaging in superior frontal gyrus, motor cortex, amygdala, caudate nucleus, pontine base and cerebellar dentate nucleus; microgliosis with IBA-1 immunohisto- chemistry in subthalamic nucleus and substantia nigra. We will record clinical phenotypes (sex, diagnosis, age at onset, age at death, disease duration), pathologic groupings (typical PSP vs. atypical PSP; pure PSP vs. mixed PSP), semi-quantitative scores of neuronal, astrocytic and oligodendroglial lesion density, biochemical characte- rization of tau from Western blots of caudate nucleus, and estimated latent trait variables constructed from the semiquantitative lesion scores. Aim 2. Assess association of intermediate pathologic phenotypes with gene variants in PSP. We will focus on 2 AMPT SNPs and 23 non-MAPT SNPs that achieved p<1x10'^ in the PSP GWAS. Each SNP will be tested for association in more than 700 PSP cases against 5 primary pathologic phe- notypes. We hypothesize that intermediate pathologic phenotypes, a large sample size and targeted SNPs will be powerful in identifying mechanisms of genetic risk variants in PSP. Aim 3. Generate intermediate patholog- ic phenotypes for LBD. We will measure burden of a-synuclein, A3, and tau in mid-frontal gyrus, superior tem- poral gyrus, amygdala and putamen; tyrosine hydroxylase immunohistochemistry of putamen; and microgliosis in substantia nigra and basal nucleus of Meynert. We will record clinical phenotypes (as for PSP, but also dementia and/or Parkinsonism), pathological phenotypes (brainstem, transitional, or diffuse LBD; pure LBD vs. mixed LBD) as well as Lewy body counts in 5 cortical areas and the amygdala. We will estimate latent trait variables underly- ing the semiquantitative scores of LBs, plaques and tangles as in Aim 1. Aim 4. Assess association of inter- mediate pathologic phenotypes with gene variants from PD GWAS. SNPs will be identified as top hits from the autopsy PD GWAS. We will focus on 2 MAPT SNPS and 23 non-/W>!\P7SNPs that achieved p<1x10'(R). Each SNP will be tested for association with 9 intermediate phenotypes in more than 700 LBD cases. Ultimately, ge- netic and phenotypic data on a large number of PSP and LBD brains will not only provide mechanistic insight, but also be a resource for Udall Center collaborators and for others.

PROJECT SUMMARY ? CORE A The Administrative Core is crucial to the operation of the Lewy body dementia Center without Walls (LBD CWOW). It provides administrative, fiscal, and scientific oversight of all cores and projects. It is responsible for maintaining a webpage for reporting on scientific achievements of the Center and establishing a publicly- available library of all subspecies of alpha-synuclein and amyloid-beta characterized by the Center. In conjunction with the other components of the LBD CWOW, Core A assures that educational materials, data, and research resources are available for sharing with other CWOWs and with non-CWOW LBD investigators. Core A assures that the LBD CWOW will cooperate fully with NINDS initiatives and has a dedicated Center Administrator who has experience with fiscal and administrative management of large multi-institutional grants as well as expertise in navigating the Mayo Clinic Brain Bank. To accomplish these goals, Core A has the following specific aims: Specific Aim 1: Provide administrative structure and fiscal oversight for the CWOW. Specific Aim 2: Assume responsibility for the quality control of the Center?s activities by ensuring responsible conduct of research and rigor and reproducibility of research practices. Specific Aim 3: Promote the scientific direction and integration of the Center components by organizing regular Executive Steering Committee meetings and overseeing external collaborations that support the Center cores and projects. Specific Aim 4: Establish an External Advisory Committee and report progress to the NIH. Specific Aim 5: Establish and maintain a publicly-available library of all ?-syn and A? subspecies characterized by the center in the form of a LBD CWOW website. Specific Aim 6: Promote education on LBD and encourage CWOW investigators to participate in patient and caregiver support groups.

PROJECT SUMMARY/ABSTRACT The discovery of mutations in C9ORF72 as a cause of both FTLD and ALS provides a clear link between these two degenerative diseases and suggests that they form a clinicopathologic disease spectrum. Research on C9ORF72 related neurodegenerative disorders, thus, holds promise to unlock secrets related to this disease spectrum. Postmortem neuropathology plays an important role in establishing the range of pathology in both ALS and FTLD, as well as documenting other disease processes that may have contributed to the clinical syndrome. As animal models are developed to study disease pathogenesis of ALS and FTLD, neuropathologic studies are increasingly important to monitor outcomes of experimental manipulations and for comparison to human disorders. Core C participates in these activities as well as providing tissue samples to investigators obtain from postmortem studies. Core C will work closely with clinical coordinators to obtain rapid autopsies of patients enrolled in clinical studies in Core B. In addition to brain and spinal cord, tissue samples will also be obtained from skeletal muscle, peripheral nerve and the internal organs. Core C will bank these fixed and frozen tissue specimens and process them for histologic studies. Core C will provide diagnostic evaluations. Core C will provide tissue samples for genetic studies, basic research, and clinicopathologic studies in Projects 1, 2 and 3, respectively. Core C will provide technical support to Projects 2 and 3 with respect to neurohistology of human and animal brains. The specific aims of the core are as follows: Specific Aim 1. For enrolled subjects in the deeded autopsy program who die during the course of the project, conduct autopsies to harvest brain, spinal cord, skeletal muscle and peripheral nerve, as well as samples from internal organs, with a portion of each sample frozen for subsequent research and another portion preserved in fixative for diagnostic evaluation and for future research studies. A neuropathologic diagnostic evaluation will be performed on each case using a standardized protocol. Upon completion of the diagnostic evaluation, reports will be entered into the electronic medical record and copies will be sent to the next-of-kin along with an explanatory letter in lay person terms. Specific Aim 2. Process and assist in the neuropathologic characterization of mouse brains from Project 2, and assist with characterizing antibodies generated in Project 2. Specific Aim 3. Provide tissue samples to Projects 1, 2 and 3 as well as to qualified investigators at other institutions. Core C will facilitate research in the program project, but also contribute to medical education, physician quality control, and feedback to families whose relatives were engaged in research on ALS and FTLD.

PROJECT SUMMARY/ABSTRACT While C9ORF72 mutations were originally described in individuals with either amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTLD), the clinical spectrum is wider and includes patients clinically thought to have amnestic dementia compatible with Alzheimer's disease. Pathologically, while most c9FTD/ALS cases have Type B TDP-43 pathology, this is not universal and some cases also have Type A or even Type C TDP-43 pathology. The basis for this heterogeneity is unknown and will be explored in this project. Discovery of RAN translation and the generation of antibodies specific to predicted RAN translation products (C9RANT) from the GGGGCC hexanucleotide repeat in C9ORF72 is a major development in identifying disease specific biomarkers. Thus, C9RANT immunohistochemistry might prove to be a valuable tool for screening for C9ORF72 expansion carriers. The nature of the pathology related to RAN translation remains to be defined. Pathologic inclusions in c9FTD/ALS are heterogeneous and include TDP-43-positive and TDP-43-negative neuronal and glial inclusions. TDP-43 negative inclusions are detected with immunohistochemistry for ubiquitin (Ubq) and Ubq-binding proteins such as p62/sequestosome and ubiquilin2 (Ubqln2). It is uncertain how inclusions detected by C9RANT immunohistochemistry relate to those detected by Ubq, Ubqln2 and p62. The relative contribution of these various pathologic processes to the clinical phenotype is an area that remains unexplored and will be addressed in this project using qualitative and quantitative methods, including advanced digital microscopy, as well as immunogold electron microscopy. It also remains to be determined if genetic risk factors, in particular the C9ORF72 GGGGCC repeat length, influence clinical and pathologic heterogeneity of c9FTD/ALS. The following Specific Aims are proposed to address gaps in knowledge about c9FTD/ALS. Aim 1. Characterize the multiproteinopathy of c9FTD/ALS using double labeling immunohistochemistry and immunogold electron microscopy with antibodies specific to c9RAN, Ubqln2, TDP-43, p62 and to markers of proteotoxic stress. Aim 2. Map distribution of neuronal inclusions immunoreactive with antibodies to c9RAN polypeptides in all reading frames in sense and antisense direction, as well as arginine methylation, with markers of neurodegeneration using digital microscopy. Aim 3. Create a database of quantitative and semiquantitative data from Aims 1 and 2 as well as biochemical data from Project 2 for exploratory associations with clinical features as well as C9ORF72 repeat length (from Project 1) and common genetic variants in GRN and TMEM106B (from Core B).

PROJECT SUMMARY/ABSTRACT Neurodegenerative tauopathies are among the most common causes of dementia and parkinsonism in middle to late adult life. Diagnostic accuracy for tauopathies is reasonably good (over 75%) for some of these disorders, such as progressive supranuclear palsy (PSP), but poor (<50%) for others, such as corticobasal degeneration. Developing better biomarkers for these disorders would be useful for diagnosis, prognosis and future clinical trials. The genetic, cell modeling and immunoassays proposed by investigators in this CWOW address these important research objectives. The discovery of genetic variants that modulate risk and severity of tauopathy could provide significant insight into disease pathogenesis, as well as illuminate key pathways to target for potential therapeutics. Three genomic loci were reported in 2011 for the GWAS of PSP, including STX6, EIF2AK3, and MOBP, but the mechanisms by which each polymorphism impact disease risk remain unknown. Discovery of genetic factors that mediate sensitivity or resistance to tauopathy in Projects 1 and 2 will potentially necessitate development of novel resources to elucidate how each gene is functionally linked to tau deposition and toxicity in disease. Core C will generate new research tools to provide the Center with the means to uncover the underlying mechanisms for risk of disease mediated by the genetic variants, as well as identifying common pathways that potentially include multiple genetic modifiers and would thus represent ideal pathways to target therapeutically. In this Center, Core C plays an essential role through its large collection of pathologically-confirmed tauopathies and for provision of services to the research projects. In particular, we will provide biologic material from human tauopathy brains, as well as assist with the neuropathologic characterization and quantitation of tau burden in humans and mice. We will also validate the relationship between tau pathology and key discoveries by project investigators. Finally, we will establish novel assays to provide mechanistic insight into the link between genetic modifiers and disease.

PROJECT SUMMARY Human ?-Synuclein (h?S) aggregation is considered as a primary mechanism of neurodegeneration in Parkinson's Disease(PD). Accordingly, the theory of cell-to-cell propagation of h?S aggregates is well- accepted, as it can reasonably explain progressive propagation of h?S pathology and associated neuronal degeneration in PD. On the other hand, the theory does not account for trigger for h?S aggregation. Our recent study suggested that one possible explanation for initial h?S aggregation in PD and related disorders may be disruption of nuclear membrane integrity, as occurs during apoptosis. This exposes cytoplasmic h?S to proaggregant nuclear factors, which remain to be determined, but could include histones. Aggregates released in the immediate vicinity of the affected neurons may template h?S in nearby neurons that may then spread by cell-to-cell propagation, leading to more distant neurodegeneration. Conditions that limit clearance of apoptotic bodies (e.g., age-associated declines in immune competence) would be predicted to accelerate the process. Accordingly, pathological h?S might be at least, in part, a secondary phenomenon associated with loss of nuclear membrane integrity or neuronal apoptosis instead of primary event in the pathogenic cascade of PD. It remains to be seen if abnormal conformers of h?S may compromise nuclear membrane integrity. In our previous study in support of our hypothesis, we have exogenously inoculated mice brains with h?S aggregates generated from apoptotic neuronal cultures to test neuronal uptake in vivo. In this proposal, we will introduce the herpes simplex virus thymidine kinase(HSVtk)/ ganciclovir(GCV) system, a well- established strategy for drug induction of cell apoptosis in cancer therapy, to PD research for the first time and perform stereotaxic brain injection of lentivirus into bilateral substantial nigra (SN) of a mouse line to overexpress both HSVtk and Myc-h?S via lentivirus infection. The animals are from a human ?S transgenic mouse line without any observable pathology throughout the life cycle. Upon GCV administration, this mouse model is expected to self-generate transmissible filamentous Myc-h?S aggregates whose spreading can be tracked by histologically detecting Myc tag. We will investigate in vivo how the initial self-generated Myc-h?S aggregates seed subsequent aggregation of normal non-pathological h?S and whether such propagation of h?S pathology can cause further neuron loss beyond the SN regions. To our knowledge, this PD mouse model will be the first one able to conditionally generate transmissible filamentous h?S aggregates for spatio- temporally tracking propagation of h?S aggregates originated in SN regions. We expect that this study can provide more direct evidence to support our hypothesis, and Braak staging theory as well.

Progressive supranuclear palsy (PSP) is the most common frontotemporal lobar degeneration associated with tau pathology. While rare pathogenic variants, common risk factors, and ? more recently ? rare risk-associated variants have been identified in PSP, a significant proportion of the heritability for neurodegenerative tauopathies and other frontotemporal lobar degenerations remains unexplained, strongly suggesting that additional genetic risk factors await discovery. In this application, we propose to identify novel genetic variation associated with PSP using a multi-stage strategy. First, we will detect variants through whole-genome sequencing of neuropathologically characterized PSP. Second, we will prioritize pathological brain tissue samples for a multidimensional screen that includes transcriptional, proteomics, and epigenetic assays. Through recursive application of a prioritization algorithm, regions and variants most likely to have a high impact on disease risk will be identified. Finally, we will follow up on these variants using a high-throughput functional screen. This project taps unprecedented pathologic resources of PSP, leverages a pathologic and genetic infrastructure created with support from private foundations, and offers to transform our understanding of the genetic architecture of PSP and to advance towards the biology and downstream effects of this prototypical tauopathy downstream effects of this prototypical tauopathy.

The overarching goal of this proposed Lewy body dementia (LBD) center without walls (CWOW) is to understand synergistic interactions of amyloid-beta (A?) and alpha-synuclein (?-syn) and to determine how genetic risk factors, such as apolipoprotein E4, contribute to LBD pathogenesis. Using well-characterized postmortem brain tissue from the Mayo Clinic Brain Bank, CWOW investigators will probe genetics, transcriptomics, proteomics, lipidomics, structure, biochemistry, and function of A? and ?-syn species in LBD. The CWOW has an Administrative Core and a Neuropathology and Biochemistry Core, as well as four research projects. The Administrative Core will provide fiscal and scientific oversight, management, and reporting functions. The Neuropathology and Biochemistry Core will provide neuropathologically well- characterized LBD brain samples and quantitative endophenotypes to all projects. The research projects are led by highly qualified investigators who focus on complementary and fundamental aspects of LBD. Project 1 will explore genetic correlates of clinical and pathologic heterogeneity of LBD, as well as cell-specific transcript data from single nuclei sequencing to build network analyses, which will provide insights into underlying disease mechanisms. Project 2 will identify and validate proteins and low-abundant bioactive lipid species in LBD brains that may be important to the unique pathologic signature of LBD. Project 3 will use state-of-the?art cryo-EM techniques to characterize the native and molecular structures of ?-syn and A? filaments in LBD brain to generate novel insight into the etiology, toxicity and spreading of protein aggregates in this disease. Project 4 will isolate and characterize A? and ?-syn subspecies from postmortem brains and use cellular models to reveal mechanistic insight into toxic pathways contributing to disease pathogenesis. We envision that, at the conclusion of the funding period, we will have made significant progress towards understanding unique structural and molecular features of LBD and how genetics and interactions between A? and ?-syn contribute to the unique symptoms, progression, and underlying pathology of LBD.

PROJECT SUMMARY ? NEUROPATHOLOGY AND BIOCHEMISTRY CORE Neuropathologic evaluation remains an important part of clinical and translational research on dementia with Lewy bodies (DLB) and Parkinsonian disease dementia (PDD), disorders that have been grouped in the research recommendations of NIH-sponsored Alzheimer Disease and Related Dementias (ADRD) recommendations as the Lewy body dementias (LBD). The purpose of the Core B: Neuropathology is to perform quantitative postmortem neuropathologic evaluations and data collection for all LBD brains that will be distributed to the CWOW scientists. The Core B will provide pulverized frozen human brain samples, brain homogenates, purified protein preparations (A? and ?-Syn) or paraffin sections of LBD cases and matched controls to all CWOW investigators, as well as to qualified investigators at other research institutions as approved by the Executive Committee of Core A and the Mayo Clinic Biospecimen Committee. The quantitative traits generated include counts of cortical Lewy bodies, semiquantitative estimates of substantia nigra neuronal loss, dopaminergic deficiency in putamen with tyrosine hydroxylase immunohistochemistry and counts of senile plaques and neurofibrillary tangles in cortical and subcortical areas with thioflavin S fluorescent microscopy. Core B will perform digital microscopy and image analysis on sections processed for immunohistochemistry for ?-Syn, phospho-tau and A?40, A?42, as additional other quantitative endophenotypes, such as phospho-TDP-43.