John Q. Trojanowski, MD,Tufts University, PhD, Tufts University 1976 - US grants

Intermediate filaments (IF) are the 10-12 nm in diameter cytoskeletal structures of nucleated mammalian cells. Five different classes are distinguished, and three are present in cells of the central (CNS) and peripheral (PNS) nervous system: neurofilaments (IF of neurons), glial filaments (IF of CNS glia), and vimentin (IF of CNS and PNS glia as well as of mesenchymal and other cells). The two other classes of IF are desmin (IF of muscle cells) and keratin (IF of epithelial cells). Thus, except for vimentin, IF proteins are cell specific and can be considered cell type-specific markers in oncological pathology. Antisera (AS) against different IF classes have been used with some success in diagnostic pathology. However, the use of anti-IF AS has resulted in conflicting reports on the cellular distribution of certain IF classes. Recent studies, using anti-IF monoclonal antibodies (MAs), suggest that this may be due to the fact that the different IF classes have both unique and shared epitopes. This potential obstacle to the use of anti-IF antibodies in diagnostic pathology can be overcome through the use of MAs specific for individual IF classes. Studies will carefully evaluate the hypothesis that anti-IF class-specific MAs are useful reagents for the evaluation of human CNS and PNS tumors (including tumors of the diffuse neuroendocrine system or apudomas). IF proteins of each class will be purified and used to produce class-specific MAs, which will be characterized by immunochemical and immunohistochemical methods. The distribution of each IF class will be determined using these MAs by immunohistochemistry in normal and neoplastic CNS and PNS tissues. Pending these results, spinal fluid cytology specimens will also be examined. In addition, electron microscopy and immunochemical methods will be used to further characterize the IF of selected tumors. These studies will lead to improvements in tumor diagnosis, patient care, and the understanding of tumor biology. (4)

The proposed studies utilize molecular probes to neurofilament (NF) proteins to characterize the full nature of NF protein alterations which occur during the transformation of the neuronal genotype in neoplastic conditions of the human nervous system. Biochemical methods will be combined with immunocytochemical applications of these probes. Comparisons will be made to determine the extent to which NF triplet proteins of transformed neurons are similar to or different from the NF protein subunits of immature, developing and mature neurons. Studies will be conducted to elucidate the mechanisms underlying NF abnormalities in transformed neurons using two neoplastic human cell lines derived from an embryonal carcinoma and a medulloblastoma. The replicating cells of the embryonal carcinoma cell line express keratin rather than NF proteins, but they can be induced to differentiate into non-dividing, neurons that express normal NF triplet proteins and no longer contain keratin proteins. In contrast, cells in the medulloblastoma cell line divide rapidly, are undifferentiated, and express all three NF subunits. However, the NF in these cells are abnormal. The molecular probes we will use to accomplish our objectives will be selected from our large and well-characterized library of monoclonal antibodies to human NF proteins. They recognize immunologically distinct, posttranslationally modified variants of each NF subunit that result from the differential phosphorylation of "nascent" NF polypeptides. The complement our analysis of epigenetic mechanisms leading to NF abnormalities in transformed neurons, cDNA probes to human NF triplet protein mRNAs will be used to evaluate genetic mechanisms which may also account for these derangements. The accomplishment of these specific aims will: 1) Establish molecular criteria for the diagnostic and prognostic assessment of neuronal tumors; 2) Define the extent to which neoplastic NF proteins are deranged relative to those in normal mature and immature human neurons; 3) Elucidate mechanisms leading to abnormal NF expression in neoplastic neurons. This work has important implications for understanding the multi-step process of transformation and tumor progression, and it will lead to improvements in the management of patients with neuron derived malignancies.

The mechanisms that underlie the behavioral and molecular abnormalities of Alzheimer's disease (AD) remain poorly understood. Although the etiology of AD may be multifactorial, and several pathways may lead to the massive loss of neurons in AD, characterization of the molecular substrates of diminished cognition and the selective degeneration of vulnerable neurons in AD is critical for elucidating the pathobiology of this disorder as well as for the design of strategies for the early diagnosis, prevention and/or amelioration of AD. As a result of the recruitment of new faculty and the development of newly funded AD research programs, investigators at the University of Pennsylvania have formed an informal, multidisciplinary consortium to increase basic and clinical understanding of AD. This ADCC will formalize existing links between this group of behavioral and molecular neuroscientists from the Departments of Otorhinolaryngology, Pathology and Laboratory Medicine, Pharmacology, Psychiatry and Neurology at the University of Pennsylvania. By extending these links to education specialists at the Penn Center for the Study of Aging and the Nursing School, this ADCC will significantly augment a vigorous developing AD research program at the University of Pennsylvania in order to: 1) Increase understanding of AD; 2) Promote interactions between this ADCC and ongoing AD research programs; 3) Develop new, extramurally funded research initiatives. To accomplish this, the Penn ADCC will support: 1) An Administrative Core (Core A) to oversee the direction and activities of the ADCC and related AD research programs; 2) A Clinical Core (Core A) to unify and coordinate the recruitment and continued assessment of AD and control subjects in the Departments of Neurology and Psychiatry; 3) A Neuropathology Core (Core C) to obtain postmortem tissues from deceased Core B subjects for diagnostic evaluation and research; 4) An Education and Information Transfer Core (Core D) to foster interest in and awareness of AD and the ADCC, as well as to develop the skills of clinical and basic scientists interested in AD research. Finally, Pilots will be recruited and supported by this ADCC to stimulate novel lines of investigation that are likely to develop into extramurally funded and independent research projects. In sum, this ADCC will foster the expansion of an interactive and cohesive cadre of Penn investigators from different disciplines who will focus their efforts on increasing understanding of the clinical and molecular basis of AD, as well as the broad effects of this late-life dementia on our society.

Advances in understanding Alzheimer's disease (AD) have partially clarified the composition of 2 major lesions in the AD brain, i.e. amyloid rich senile plaques (Sps) and neurofibrillary abnormalities including neurofibrillary tangles (NFTs), dystrophic plaque-associated neurites and neuropil threads (NTs). Specifically, the filamentous components in Sps assemble from incompletely degraded Beta-amyloid or A4 peptides (BetaA4) derived from 1 or more of 3 major amyloid precusor proteins (APPs) in brain (i.e. APP695, APP751 and APP770) while the paired helical filaments (PHFs) in NFTs, dystrophic plaque neurites and Nts are abnormally phosphorylated tau derivatives (known as PHFtau or A680. Although recently identified mutations in the APP gene may play a role in the etiology of AD in a small subset of patients with familial AD (FAD), increasing evidence suggests that AD results from the abnormal metabolism of APPs and tau. This has prompted efforts to establish models of AD pathology in mice bearing BetaA4 containing partial or complete APP transgenes, and in rats injected with BetaA4 or SP cores. However, the development of additional animal models that more closely mimick AD is critical for elucidating the etiology and pathogenesis of AD. To this end, the goal of Project #4 is to establish alternative models of AD pathology in rodents. To probe the biology of PHFs and their interactions with BetaA4 in vivo, we will extend recent work from our lab in which A68 proteins were injected into rat brains and induced long lived co-deposits of rodent BetaA4 at the injections site. Additionally, we will probe the in vivo generation of BetaA4 in rodents transplanted with cultured human neurons (NT2N cells derived from the NT2 cell line) that constitutively secrete BetaA4 and express APP695 almost to the exclusion of APP751/770. Studies also will be conducted on transplants of transfected NT2N cells that over express APP695 or APP751 with and without a FAD mutation. The injected A68 proteins and grafts of the NT2N human neurons will be introduced into regions of the rodent brain that are homologous with regions of the human AD brain that accumulate abundant (hippocampus) or negligible (cerebellum) amyloid and neurofibrillary lesions. The analysis of injected A68 and NT2N grafts will be carried out at post- operation survival times of days to 24 months using biochemical methods as well as by immunohistochemistry in conjunction with light, confocal an electron microscopy. These strategies to model major AD lesions in the rodent brain should lead to significant new insights into the pathogenesis of AD.

Although a defined clinical phenotype is used to distinguish Alzheimer's disease (AD) from other late-life dementias, the accuracy of antemortem tests for the diagnosis of AD are imperfect. Ultimately, AD is defined most securely when the clinical phenotype is coupled with the postmortem detection of specific brain lesions considered to be "pathological signatures" of AD. Uncertainty about the antemortem diagnosis of AD is likely to impede efforts to develop and test potentially useful therapies. Indeed, as many as 20% of elderly patients with a dementia ascribed to AD will fail to exhibit postmortem evidence of the neurofibrillary tangles (NFTs) and senile plaques (SPs) identified as hallmarks of AD. This subset of elderly patients with an AD-like dementia, but no AD pathology, may exhibit lesions characteristic of Pick's disease, diffuse Lewy body (LB) disease (DLBD) or other neurodegenerative diseases associated with profound cognitive dysfunction. The consistent assignment of elderly demented patients followed by the Clinical Core (Core B) of this ADCC to 1 of several diagnostic categories (e.g. AD, DLBD) is central to the mission of this ADCC. Hence, the goal of the Neuropathology Core (Core C) of this ADCC is to obtain and thoroughly characterize postmortem tissues from all deceased ADCC patients for whom permission for an autopsy is obtained. Complete autopsies will be performed and objective criteria will be used to sort the cases into well defined diagnostic categories. Further, all of the vital information on this case material (e.g. age of the patient, diagnosis, postmortem interval, means of tissue denaturation, tissue recipients, etc.) will be recorded in an electronic data base for correlation with clinical data obtained on these patients in Core B. Finally, Core C will serve as a resource to other investigators in this ADCC, a related Program Project ("Molecular Substrates Of Aging & Neuron Death", P01 AG-09215) on AD and idiopathic Parkinson's disease (PD), and new research projects on AD stimulated by the presence of this ADCC at the University of Pennsylvania.

The brains of demented Alzheimer's disease (AD) patients are characterized by abundant senile plaques (Sps) and neurofibrillary tangles (NFTs). Further, the quantity of NFTs correlates with the dementia in AD, and cerebrospinal fluid (CSF) tau levels are abnormally high in most AD patients consistent with the fact that the subunits of paired helical filaments (PHFs) in AD NFTs are altered forms of tau (PHFtau). Despite the presence of small numbers of NFTs in the brains of non-demented elderly subjects, the molecular and cellular substrates of age-related, mild cognitive impairments in otherwise normal elderly individuals are not known. Although PHFtau is abnormally phosphorylated, we showed that biopsy derived normal adult human tau is phosphorylated at nearly all of the sites previously identified in PHFtau, albeit to a lesser extent. Further, biopsy derived tau proteins are rapidly dephosphorylated at sites that are retained in PHFtau. Thus, differences in the phosphorylation sites and/or state of normal human tau versus PHFtau may reflect the differential activities of phosphatases in the control versus AD brain. Indeed, deficient protein phosphatase 2A (PP2A) activity has been implicated in the formation of PHFtau, and alterations in the accessory, catalytic and regulatory subunits of PP2A could be part of a "final common neurodegenerative pathway" leading to the generation of PHFtau, NFTs and the death of neurons in AD. Since similar mechanisms also might account for milder cognitive impairments in otherwise normal elderly individuals, we will test the hypothesis that alterations in PP2A subunits may play a role in the pathogenesis of neurofibrillary lesions in normal aging, and that low levels of neurofibrillary lesions may contribute to mild cognitive impairments in the elderly. These studies will be conducted in well characterized elderly members of a "religious orders study cohort" with no or mild cognitive impairments versus dementia due to AD.

neuropathology; neurogenetics; tau proteins; model design /development; laboratory mouse; genetically modified animals; disease /disorder model; cerebral degeneration; gene targeting; pathologic process; genetic promoter element; genetic models; gene mutation; transfection /expression vector; Adenoviridae; animal breeding;

DESCRIPTION (provided by applicant): Understanding of the behavioral, cognitive, neurological and biological underpinnings of frontotemporal 1obar degeneration (FTLD) remains fragmentary, but a dramatic paradigm shift in basic and clinical research into the etiology and neuropathology of FTLDs occurred in 1998 with the discoveries of tau gene mutations that were shown to be pathogenic for familial frontotemporal dementia linked to chromosome 17 (FTDP-17) in multiple kindreds, including those with diverse syndromes known descriptively as pallido-ponto-nigral degeneration, Pick's disease, and dementia-disinhibition-parkinsonism-amyotrophy complex. While some of these kindreds were previously thought to represent familial Alzheimer's disease (AD), the hallmark neuropathology of these syndromes and several other sporadic FTLDs was demonstrated to be characterized by prominent accumulations of abnormal tau proteins, and these disorders are collectively known as tauopathies. Prompted by these remarkable advances, the goals of this Program Project Grant (PPG) are to determine the genetic, clinical and imaging features of FTLDs, especially those features that distinguish FTLDs from AD, other dementias, and normal aging. Thus, to support accomplishment of these goals, the key functions of the Neuropathology Core in this PPG are to provide diagnostic histopathological and biochemical evaluations of postmortem brains from subjects followed by PPG investigators, enter all information obtained from these analyses in a secure and confidential database, and provide data from the Neuropathology Core to PPG investigators to facilitate elucidation of the defining genotypic and phenotypic features of hereditary and sporadic FTLDs. Thus, the Neuropathology Core performs critical functions in support of the mission of this PPG to increase understanding of FTLD and related tauopathies.

[unreadable] DESCRIPTION (provided by applicant): This grant application is a supplement to the University of Pennsylvania (PENN) Alzheimer's Disease (AD) Core Center (ADCC; P30 AG 10124) which proposes to revise, improve, and substantially expand the biostatistics and data management activities in the PENN ADCC in response to comments in the Summary Statement to the previous review of this ADCC and the growing demands for these services by the ADCC investigators and their collaborators who are working with the ADCC. It proposes the establishment of a Data Management and Biostatistics Core (Core F) to support the ADCC investigators for data, database, statistical, and computing related work. These services include: (a) providing needed support for data form/questionnaire design and development, Access database development and management, data entry, database audit trail, database security, database backup, and stringent data quality control procedures, (b) development of study design, including performing sample size and power calculations, and randomization schemes, (c) performing analyses of PENN ADCC data and the development of new statistical methodology where needed for data analysis, (d) providing computing and programming support for all of the PENN ADCC activities, including implementation and integration of hardware and software upgrades necessary for data management and research, routine and archival off-site backup of computing systems central to the ADCC, and support of the PENN ADCC website, (e) promoting an effective working relationship between the PENN ADCC and the National Alzheimer's Coordinating Center (NACC). Thus, Core F will play an important and significantly larger role that is critical to the progress of research and the conduct of studies in the PENN ADCC, and it will foster collaborative interactions between the PENN ADCC and other AD Centers (ADCs) through a close and effective working relationship with the NACC. The requested supplemental funding will be used to cover the above increased and improved biostatistics and data management activities that were not budgeted in the 2001 ADCC competing renewal. [unreadable] [unreadable]

PROJECT SUMMARY: Neurodegenerative diseases are characterized by CNS accumulations of misfolded protein aggregates that define the neuropathology of each disorder, including frontotemporal dementia (FTD) or frontotemporal lobar degeneration (FTLD), the second most common cause of dementia in patients under the age of 65. There have been dramatic advances in research on FTLD that have revolutionized concepts about FTLD since this Program Project Grant (PPG) was renewed in 2005. Thus, tau, TDP-43 or FUS inclusions now are recognized as the defining CNS lesions in ~45%, ~50% and ~5% of FTLD, respectively, and these FTLD variants are designated FTLD-Tau, FTLD-TDP and FTLD-FUS, respectively. FTD may be sporadic or familial, and hereditary FTD with parkinsonism linked to chromosome 17q21-22 is caused by >30 M>APT mutations while >30 TARDBP and FUS mutations were discovered in the last 3 years that associate with amyotrophic lateral sclerosis (ALS), but TARDBP mutations also associate with FTLD plus ALS. Notably, although FTLD-Tau, FTLD-TDP and FTLD-FUS account for nearly all FTLD cases, Alzheimer's disease (AD) is the cause of FTD in ~25% of patients. Moreover, TDP-43 pathology co-occurs in 30-50% of patients with AD, Parkinson's disease and dementia with Lewy bodies. Given the heterogeneity of FTD and the clinical overlap of FTD with AD and other disorders, a thorough postmortem examination of FTD patients followed in Clinical Core of this PPG is essential to define the clinicopathologic phenotypes of FTD and link them to genetic, imaging and biomarker findings as well as to provide CNS samples for research. Further, the collection of biofluids is essential to identify FTD biomarkers for the early and reliable diagnosis of FTD. Accordingly, Core D acquires, characterizes, banks, distributes and studies CNS and biofluid samples from FTD patients and control subjects followed in the Clinical Core of this PPG.

This new application from the University of Pennsylvania School of Medicine (PENN) for a National Institute of Neurological Disorders and Stroke (NINDS) Morris K. Udall Parkinson's Disease Research Center of Excellence (Udall Center) builds on the continuing momentum of a team of PENN clinical and basic scientists to accomplish the overarching goals of this new multidisciplinary Parkinson's disease (PD) research program. Briefly, the goals of this Udall Center are to elucidate mechanisms of brain degeneration in patients with PD, especially those underlying poorly understood neuropsychiatric impairments. PD with dementia (PDD), which may be pathologically and clinically indistinguishable from dementia with Lewy bodies (DLB) frequently co-occurs with Alzheimer's disease (AD), and the Lewy body (LB) variant of AD is the most common subtype of AD. However, the reasons for the convergence of IBs and AD pathologies remain enigmatic. Here, we hypothesize that accumulations of oligomeric/fibrillar species of a-synuclein lead to neurodegeneration and neuropsychiatric deficits in addition to parkinsonism, which may be compounded by tau and Abeta pathologies seeded by alpha-synuclein oligomers/fibrils. To accomplish the goals of the PENN Udall Center, Projects 1 and 2 are patient-oriented studies designed to develop: 1) a disease-specific rating scale to assess the impact of cognitive impairment on daily function in PDD;and 2) a functional imaging study to better define the anatomic substrate of cognitive impairment in PD and PDD. Projects 3 and 4 bridge patient oriented studies and research on novel animal models of PD/PDD to clarify how the misfolding, fibrillization and aggregation of a-synuclein, tau and Abeta contribute to the neuron dysfunction and degeneration that result in cognitive impairments in PDD. These 4 highly integrated and synergistic Projects are supported by an Administrative Core (A), a Clinical and Education Core (B), a Neuropathology and Genetics (C) and a Data Management and Biostatistics Core (D). The PENN Udall Center investigators will accomplish the goals outlined above by working in a seamlessly interdisciplinary manner as well as by collaborating with other Udall Centers. Further, data, reagents, and resources generated by the PENN Udall Center will be shared with researchers at other Udall Centers. Thus, the PENN Udall Center team will contribute to improving the diagnosis and management of patients with PD, PDD, DLB and related disorders.

PROJECT suiViiViARY: The goal ofthe Education, Recruitment and Retention Core ofthe Penn ADCC ~ Core E - - is to generate and properiy use resources and personnel to (1) develop, implement, and monitor recruitment and retention programs that assure research proceeds in a time- and cost-efificient manner; (2) train researchers and staff in skills in state-of-the-art techniques in clinical assessment, and recruitment and retention; and (3) give researchers, staff, patients, and families relevant knowledge on mild cognitive impairment (MCI), Alzheimer's disease (AD) and related diseases. Core E has developed a multi-disciplinary, highly collaborative, and productive team that has in-house talent and resources to design, produce, and disseminate novel materials for recruitment, retention, and education. The Core is well integrated into the ADCC cores and leadership, as well as the national network of ADC's, ADEAR, and the ADCS. The crux of the Education Core's significance is persuasive communication. The core's key resources are its five personnel led by Jason Kariawish (who is also associate director ofthe Clinical Core), and its materials and methods: the center's web page, bilingual resource center, the ADCC newsletter (InSight), and bilingual informational and support materials. To achieve its goal, Core E has the following three specific aims: #1: To assist investigators in developing research strategies to recruit and retain subjects for research protocols and clinical trials on AD and related disorders, with a special emphasis on the Latino community of North Philadelphia and the African- American community of West Philadelphia. #2: To support the development of health care professionals' clinical and clinical research skills related to AD, other neurodegenerative diseases and their prodromal phases (e.g. MCI). #3: To spearhead effective outreach programs that will publicize the ADCC and the PMC, educate families and caregivers, and build and maintain collaborative and trusting relationships with patients and families who participate in the ADCC longitudinal cohort, especially those in the Latino community of North Philadelphia and the African-American community of West Philadelphia.

PROJECT SUMMARY: Frontotemporal lobar degeneration (FTLD) is the second most common cause of dementia after Alzheimer's disease (AD) in patients <65 years of age. Tau and TDP-43 pathology variants of FTLD (FTLD-Tau and FTLD-TDP, respectively) account for -90 of FTLD cases, but TDP-43 pathology occurs in >50% of patients with AD, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and Guam amyotrophic lateral sclerosis (ALS)/Parkinson Dementia Complex (ALS/PDC). Despite the fact that this neuropathology overlap is well known, it is unclear how comorbid Ap, tau and alpha-synuclein pathology modify TDP-43 mediated neurodegeneration in patients with frontotemporal dementia (FTD). Conversely, it is unknown how TDP-43 modifies Ap, tau and alpha-synuclein pathologies, but TDP-43 pathology is known to independently contribute to behavioral impairments in AD. Since these issues are tractable to investigate experimentally in transgenic (Tg) mouse models of TDP-43, tau, Ap and alpha-synuclein pathology. Project 4 tests the hypothesis that comorbid tau, Ap and alpha-synuclein pathologies in Tg mice independently modify TDP-43 mediated neurodegeneration and wee versa. This will be done by studying TDP-43 Tg mice which recapitulate the hallmark features of FTLD-TDP that we cross with our previously characterized mutant P301S tau Tg mice which show tau mediated neurodegeneration, behavioral impairments and premature death, Tg2576 Tg mice that model AD-like Ap pathology and our extensively studied M83 alpha-synuclien Tg mice that develop Lewy body pathology, motor impairments and lethal neurodegeneration. Implementing these Aims will elucidate how TDP-43 mediated neurodegenerative disease is modified by comorbid tau, Ap and alpha-synuclien pathologies and vice versa. These studies are highly significant because they will clarify mechanisms of TDP-43 proteinopathy and they have translational potential to improve both the diagnosis and the treatment of patients with TDP-43 proteinopathy.

PROJECT SUMMARY: Neurodegenerative disorders are characterized by abundant protein aggregates in brain and spinal cord (CNS) that are the defining neuropathology (NP) of these disorders as exemplified by senile plaques (SPs) and neurofibrillary tangles (NFTs), the diagnostic signatures of Alzheimer's disease (AD). However, AD is associated with Lewy bodies (LBs) and TDP-43 pathologies in >50% of patients while mild cognitive impairment (MCI) often shows abundant SPs and NFTs at autopsy consistent with AD. Further, the NP in ~25% or more of frontotemporal lobar degeneration (FTLD) patients is AD, while the remaining FTLD cases are non-AD tauopathies (FTLD-Tau), TDP-43 proteinopathy (FTLD-TDP) or, rarely, FUS proteinopathy (FTLD-FUS). Thus, a definitive diagnosis of AD and related dementias is established definitively only by postmortem NP examination, and an accurate NP diagnosis is essential for informative clinicopathologic correlations to elucidate molecular mechanisms of MCI, AD, FTLD and other dementias such as Parkinson's disease with dementia and dementia with LBs. Since multiple genetic factors contribute to the risk for AD and biomarkers signal disease onset/progression, DNA and biofluid banking is critical for genetic and biomarker studies. Hence, the University of Pennsylvania (Penn) AD Core Center (ADCC) characterizes and banks CNS tissues, DNA and biofluids from well-characterized patients followed in Clinical Core B with AD and related disorders as well as normal control subjects. This is essential for research conducted in ADCC Pilots and other grants that utilize Penn ADCC resources. Accordingly, Core D is re-named the Neuropathology, Genetics and Biomarker Core to reflect the full scope of its current activities. Core D also distributes tissue, DNA and biofluids to investigators at and beyond Penn for research. Finally, Core D works with the Data Management and Statistics Core C to enter all information into a database, maintain data confidentiality, and provide these data to NACC. In summary, Core D performs critical functions to support the mission of the Penn ADCC.

Project IV is new and based on novel cell and transgenic (Tg) mouse models of alpha-synuclein (a-syn) Lewy bodies (LBs) and neurites (LNs) which we hypothesize mediate neurodegeneration in Parkinson's disease without (PD) and with dementia (PDD) as well as in dementia with LBs (DLB). Project IV examines mechanisms of pathological a-syn transmission in the human A53T a-syn Tg mice (M83 line) that model LB disease (LBD)-like a-syn pathology, and assesses the efficacy of passive immunotherapy to abrogate transmission of a-syn pathologies. Project III has defined two stains (A and B) of a-syn pre-formed fibrils (PFFs), and injections of strain A a-syn PFFs into striatum and cortex of young human M83 mice accelerated the widespread formation of LBs/LNs as well as onset of neurological symptoms, and this was associated with a dramatic reduction in the lifespan of injected M83 mice. The propagation of pathologic a-syn to widespread central nervous system (CNS) regions is consistent with transynaptic cell-to-cell passage of a-syn pathology. Injections of brain lysates containing pathological a-syn from 12-14 month old diseased M83 mice into young disease free M83 mice also had the same effects as synthetic a-syn PFFs indicating that a-syn PFFs alone induce PD-like pathology and transmit disease. These findings open up new avenues for understanding the progression of PD/PDD/DLB and provide fresh opportunities to develop novel therapies for LBD. Project IV aligns with the overall theme of the Penn Udall Center and collaborates with all Cores/Projects to elucidate mechanisms underlying the progression of LBD to dementia. The overarching hypothesis to be tested here is that different pathological species or strains of a-syn generated synthetically or isolated from different postmortem brain regions of PD/PDD/DLB patients transmit disease in distinct ways that reflect intrinsic properties of the different pathological a-syn strains. To that end, the Specific Aims of Project IV are: Aim 1: To determine if synthetic a-syn PFF strains A and B characterized by Project III differentially transmit LBD following injections into the brains of M83 mice using methods to assess the behavior, neuropathology and cerebrospinal fluid (CSF) levels of a-syn; Aim 2: To determine if lysates enriched in LBs/LNs from PD substantia nigra versus PDD/DLB cingulate cortex contain different a-syn strains that differentially transmit LBD following injections into the brains of M83 mice using the same methods as in Aim 1; Aim 3: To conduct proof of concept (POC) studies in M83 mice injected with pathological a-syn to determine if passive immunization with epitope specific anti-a-syn monoclonal antibodies identified by Project III to neutralize a-syn transmission, will abrogate induction and spread of a-syn pathology in vivo.

Neuropathology, Genetics & Biomarker Core D Core Leader: John Q. Trojanowski, Core Co-Leader: Eddie Lee, Co-Investigator: Vivianna Van Deerlin Summary/Abstract: Neuropathology, Genetics & Biomarker Core D Nearly all neurodegenerative disorders are characterized by progressive accumulations of pathological deposits of disease protein aggregates within cells, blood vessels or in the neuropil and these deposits are the signature CNS lesions that define these disorders as exemplified by the senile plaques and neurofibrillary tangles required for the postmortem diagnosis of Alzheimer's disease (AD). However, Lewy bodies (LBs) and TDP-43 inclusions occur in >50% of AD patients, and mixed pathologies including hippocampal sclerosis and cerebrovascular disease are features of AD including prodromal stages of disease as in mild cognitive impairment (MCI) which shows AD and additional pathologies associated with other forms of dementia at autopsy. Further, 20-30% of patients with clinical AD have another neurodegenerative dementia other than AD and a similar percentage of patients diagnosed with clinical frontotemporal degeneration (FTD) have AD pathology while the remaining patients have autopsy confirmed frontotemporal lobar degeneration (FTLD) with tau, TDP-43 or FUS aggregates. Thus, a definite diagnosis of AD or related dementias is established only by postmortem neuropathology studies as performed in Core D, and an accurate neuropathology diagnosis is essential to elucidate mechanisms of AD and related dementias. Since multiple genetic factors contribute to the risk for AD and biomarkers signal disease onset/progression, DNA and biofluids are critical for clinical genetic and biomarker studies of AD. Hence, the University of Pennsylvania (Penn) AD Core Center (ADCC) collects, characterizes and banks CNS tissues, DNA and biofluids from well-characterized patients with MCI, AD and related disorders as well as normal controls followed in Clinical Core B. This approach enables a more comprehensive understanding of an individual patient's genetic risks, clinical manifestations, disease progression and neuropathology which are essential for conducting ADCC related research using Penn ADCC resources. Accordingly, Core D is the ?Neuropathology, Genetics and Biomarker Core? of this ADCC and it supports the mission of the Penn ADCC by working seamlessly with all Cores to accomplish its goals.

? DESCRIPTION (provided by applicant): Although the term chronic traumatic encephalopathy (CTE) has only recently entered the research and common vernacular, the neuropsychiatric sequelae have been recognized for decades as dementia pugilistica (DP) in some individuals with careers in boxing. Yet, despite the intense new interest in `CTE', the number of reported cases has been surprisingly limited and there are no validated neuropathological diagnostic criteria to define it as a distinct disease entity. Notwithstanding this, there is a widely held, abeit unfounded, perception that repetitive TBI (rTBI) alone culminates in `CTE' and that `CTE' is primarily characterized by a unique tau pathology. However, multiple neuropathological features have been reported for DP/CTE beyond tau pathologies, including brain atrophy, amyloid-? plaques, TDP-43 pathologies and neuroinflammation. Furthermore, it is now recognized that almost all of these pathologies are observed in a proportion of cases years after just one moderate to severe single TBI (sTBI). As TBI-associated neurodegeneration has become a major health concern, there is a clear and pressing need to develop robust operational neuropathological criteria for diagnosis, which will, in turn, be critical to the success of all fuure mechanistic, diagnostic and interventional studies. Towards this end, we have assembled an international, multidisciplinary team of experts who provide unparalleled experience in both the study of long-term neurodegeneration after TBI, and the development of optimized assessment of common neuropathological diseases including Alzheimer's disease (AD). Through our team members, we have custodial access to the only comprehensive TBI brain archives in the US and UK that include both sTBI and rTBI cases as well as ~1500 longitudinally followed and extensively characterized autopsy confirmed cases of AD and related disorders for comparison. For our primary Aim 1, we will use a standardized approach successfully applied to AD, to generate baseline diagnostic neuropathological criteria using existing post-mortem material of sTBI and rTBI. In Aim 2 we will pursue biochemical, and genetic studies to explore potential associations with neuropathological outcomes from chronic TBI. In Aim 3 we will explore the temporal course of neuroinflammation to examine its potential role in progressive neurodegeneration following TBI. Finally, in Aim 4 will secure a networked archive of the extensive resources of data and biospecimens generated in Aims 1-3 for wider access across the research community.

Core A: Administrative Core Project Summary Alzheimer's disease (AD) and related disorders (RD) are characterized by progressive cognitive impairments that correlate with increasing accumulations of CNS deposits of disease proteins as exemplified by the senile plaques and neurofibrillary tangles required for the postmortem diagnosis of AD. In the competing renewal of the University of Pennsylvania (Penn) AD Core Center (ADCC), our focus is on AD and RD in keeping with the most recent Request For Applications (RFA-AG-16-018) and because the diagnostic pathologies of selected RD are common co-morbid abnormalities in most AD patients that contribute to clinical deficits in patients living with AD. The Penn ADCC includes 6 Cores, and Core A is the Administrative Core. Accordingly, Core A supports the mission of the Penn ADCC by facilitating efforts to increase research and understanding of AD and RD as well as mild cognitive impairment (MCI) and healthy brain aging in and beyond the Penn ADCC. To that end, Core A sets the direction and guides development of ADCC programs as well as promotes interactions of ADCC investigators with key stakeholders. This includes NIH/NIA funded AD Centers (ADCs), the National AD Coordinating Center (NACC), the AD Cooperative Study (ADCS), the AD Education and Referral (ADEAR) Center, the AD Neuroimaging Initiative (ADNI), the National Cell Repository for AD (NCRAD), the AD Genetics Consortium (ADGC) and other NIH/NIA and public/private AD and RD initiatives. Core A accomplishes these goals through well established and highly effective mechanisms by: providing fiscal/administrative oversight of this Center; organizing regular ADCC Executive Committee (EC) meetings to review/guide ADCC activities; soliciting ADCC Pilot grant applications and reviewing these grants with an expert committee to identify/fund 2 meritorious Pilots/year, organizing annual Penn ADCC reviews by the ADCC External Advisory Committee (EAC), facilitating ADCC participation in annual Penn retreats sponsored by the ADCC and related Penn centers/institutes for students, postdocs and faculty from diverse disciplines in addition to promoting interactions of the Penn ADCC with NACC, ADCS, ADEAR, ADNI, ADGC and other ADCs in education and research initiatives, as well as in semiannual ADC meetings, and promoting ADCC data/reagent/resource sharing in accordance with NIH/NIA policies and ensuring compliance with all NIH/NIA regulatory requirements for human subject research. Thus, Core A provides a clearly delineated administrative structure that enables the Penn ADCC to increase the quality and quantity of clinical and basic research in addition to educational activities on AD, RD, MCI and normal aging at and beyond Penn. In so doing, Core A and the entire Penn ADCC seek to implement recommendations to transform AD research as outlined by NIH Director Francis Collins (http://www.nih.gov/news/health/may2015/nia-01.htm) and NIA Director Richard Hodes (https://www.nia.nih.gov/research/blog/2015/08/special-budget-alzheimers-and-related-dementias).

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

PROJECT/CORE: Administrative Core A Project/Core Leader Name: John Q. Trojanowski, MD, PhD Project Summary/Abstract Core A is the Administrative Core of this re-submitted application for a U19 ?Center On Alpha-synuclein Strains In Alzheimer Disease & Related Dementias? at the University of Pennsylvania (Penn) Perelman School of Medicine (PSOM). It facilitates accomplishing the goals of this multidisciplinary research program to elucidate mechanisms of progressive neurodegeneration mediated by different strains of pathological alpha- synuclein (aSyn) underlying cognitive impairments in Alzheimer's disease (AD) and related dementias or ADRD including Lewy body (LB) diseases (LBD) such as dementia with LBs (DLB) and Parkinson's disease without (PD) or with dementia (PDD). Among ADRD, AD with abundant LB co-pathology is the most common subtype of AD. Hence, AD with aSyn LB (AD+LB), PDD and DLB together represent the most common forms of aging related dementias. Thus, the overall goals of this new Penn AD and LBD U19 Center are to elucidate mechanisms of pathological aSyn mediated progressive neurodegeneration in AD+LB versus pure AD (AD-LB) compared with LBD as a function of aging and the heterogeneous accumulations of aSyn, tau and A? pathologies that influence different clinical manifestations of these disorders. We hypothesize that accumulations of pathological aSyn lead to neuron dysfunction and death due to misfolding and transmission of different strains of pathological aSyn to form LBs and LNs compared to those aSyn strains underlying multiple system atrophy (MSA) characterized by glial cytoplasmic inclusions (GCI) since the MSA aSyn strain rarely induces comorbid AD pathology and dementia rarely occurs in MSA patients. Specifically, we pursue the cross-Center shared goal to correlate deep phenotypic data from Projects III/IV directly with strain data generated in Projects I/II in collaboration with the Cores to determine for the first time the correspondence of clinicopathological phenotypes with the novel aSyn strains defined by our preliminary data summarized in each Project (see also Fig. 1 and 2 in the ?Overall Component?). To accomplish its goals, Core A will implement the following Aims: oversee budgetary and fiscal aspects of this U19 Center and guide the progress of the Cores and Projects; promote/foster interactions between Cores and Projects, as well as interactions of Penn U19 investigators with scientists outside the Penn U19 Center at and beyond Penn; serve as an information resource for the patient community and general public regarding LBD and MSA; facilitate the sharing of data, reagents, and resources generated by the U19 Center with other researchers in partnership with the NIA; train the next generation of AD/LBD investigators. In this manner, Core A plays a key role in the Penn U19 Center to foster accomplishment of its mission.

PROJECT II: Pathologic Alpha-synuclein Strains & Diverse Synucleinopathies Project II Leader: J.Q. Trojanowski; Co-Investigators: V.M.-Y. Lee & K. Luk Project II Summary/Abstract Project II (formerly Project IV and renumbered II to improve the flow of the research) in this new U19 ?Center On Alpha-synuclein Strains In Alzheimer Disease & Related Dementias? at the University of Pennsylvania (Penn) Perelman School of Medicine (PSOM) tests the hypothesis that heterogeneity and progression of alpha-synuclein (aSyn) pathology in Lewy bodies (LBs) and neurites (LNs) of Parkinson's disease without (PD) or with dementia (PDD) and dementia with LBs (DLB), as well as Alzheimer's disease (AD) with aSyn (AD+aSyn) LBs and LNs co-pathologies represent the spread of different neuronal aSyn strains.1 We compare these strains to each other and with aSyn strains from multiple system atrophy (MSA) glial cytoplasmic inclusions (GCI) which rarely appear in AD or in combination with LBs/LNs. This will advance insights into how distinct aSyn strains in AD+aSyn vs PD vs PDD vs DLB vs MSA drive clinical and pathological heterogeneity of these disorders. Since dementia in PDD and DLB, referred to as LB dementia (LBD), frequently is accompanied by AD co-pathologies, including A? amyloid plaques and neurofibrillary tau inclusions, and 50% of AD patients have LBs, we also test the hypothesis that aSyn strains in PDD vs DLB vs AD+aSyn brains induce A? and tau pathologies whereas aSyn strains in PD vs MSA brains lacking AD pathology might not. Project II collaborates with Project I, which performs in vitro aSyn strain studies and provides Project II with highly characterized and validated aSyn strains from postmortem AD+aSyn, PD, LBD and MSA brains as well as in vitro amplified LB and GCI aSyn strains. For these studies, living subjects are studied in Core B and Projects III/IV and their brains are obtained through Core C while Core D provides data management, biostatistics and bioinformatics support. The extent to which the models of synucleinopathies induced by intracerebral injections of LB and GCI aSyn strains in Project II correspond to authentic human AD+aSyn vs PD vs LBD vs MSA will be assessed. Thus, Project II works closely with all U19 Center Cores/Projects to determine if the LB aSyn strain (aSyn-LB) from PD vs AD+aSyn vs LBD brains compared to the MSA GCI aSyn strain (aSyn-GCI) differentially induce pathological aSyn in neurons versus glia as well as recruit AD-like A? and tau deposits or other neurodegenerative disease co-pathologies such as TDP-43. This will be done following intracerebral injections of these aSyn strains into wild type (WT) mice, human WT aSyn transgenic (Tg) mice (line 61) on a mouse aSyn knock out (KO) background (KO61) and CNP-aSyn (M2) Tg mice with an aSyn KO background (KOM2) that model GCIs compared to Tg mouse models of AD-like A? plaques (5xFAD, Tg2576, APP knock in mice) and a Tg mouse model of AD-like tau pathologies (PS19 line). The hypothesis tested in Project II emerged from preliminary studies of intracerebral injections of synthetic aSyn preformed fibrils (PFF) into some of these models, and we innovate now by injecting different authentic human brain derived aSyn-LB and aSyn-GCI (including recently developed more potent aSyn-LB strains developed by Project I) into our models to elucidate the basis for distinct aSyn strain mediated clinicopathological heterogeneity in AD+aSyn vs PD vs LBD compared to MSA lacking AD pathology as control. Specifically, we test the novel hypothesis that AD+aSyn, PD, LBD and MSA result from different aSyn strains as well as that aSyn-LB strains from AD+aSyn and LBD brains, but not aSyn-LB strains from PD brains and the aSyn-GCI strains from MSA brains, mediate development of AD co-pathologies.

National Institute on Aging (NIA) Penn U19 ?Center On Alpha-synuclein Strains In Alzheimer Disease & Related Dementias?. Principal Investigator: John Q. Trojanowski Center Summary/Abstract: This U19 Center pursues research priorities in the ?Recommendations of the Alzheimer's disease-related dementias conference?.13 It especially focuses on priorities that address Alzheimer's disease (AD) and related dementias (ADRD) in topic areas of multiple etiology dementias, Lewy body (LB) dementias (LBD), including dementia with LBs (DLB) and Parkinson's disease without (PD) and with dementia (PDD), new guidelines on the biological definition of AD14-17 and reflects recommendations from a recent NIA meeting on ?Neurodegenerative Disease Transmissibility: Current Science and Recommendations for Future Research? (Frosch M et al, in preparation, 2018). Among ADRD, AD with abundant LB co-pathology is the most common subtype of AD. We hypothesize that accumulations of pathological aSyn lead to neuron dysfunction and death due to misfolding and transmission of different strains of pathological aSyn to form LBs and LNs and that aSyn and AD pathology interact to modify the distribution of each other and contribute to behavioral impairments. To accomplish these goals, Projects I and II complement each other and Projects III and IV by seeking to elucidate aSyn strains underlying AD+aSyn/LBD compared to PD, Multiple system atrophy (MSA) is studied as a control because it is characterized by aSyn glial cytoplasmic inclusions (GCIs) that are comprised of a distinct aSyn strain which is more potent than LBD or AD+aSyn strains of pathological aSyn. In parallel, Project III analyzes regional AD/LBD neuropathology with novel monoclonal antibodies (mAbs) to correlate these data with diverse cognitive difficulties and structural imaging. A 2X2 design is used that contrasts clinical AD phenotypes and primary AD pathologies. This is complemented by Project IV which measures cognition, blood and cerebrospinal (CSF) biomarkers, including aSyn, as well as SNP arrays, to better understand, diagnose and manage diverse clinical manifestations of AD/LBD in order to advance towards a precision medicine approach for care and disease management. The landmark discoveries of aSyn gene (SNCA) alterations pathogenic for LBD and that pathological aSyn is the disease protein in synucleinopathies, in addition to the cell-to-cell spread of aSyn strains, places aSyn at center stage for understanding mechanisms of AD+aSyn and LBD. This Penn U19 Center addresses these key issues in four Projects supported by four Cores. Moreover, this Center also will work with NIA to provide biofluids, DNA/RNA, autopsy tissue and data collected from ADRD patients over the past 20 years at Penn in addition to aSyn strains to qualified investigators. By addressing the hypothesis that distinct aSyn strains underlie AD+aSyn/LBD, we will clarify the molecular basis of heterogeneity in AD+aSyn/LBD while opening up new targets for drug discovery and biomarker development in at the Penn U19 Center in collaboration with NIA program.