Alice Chen-Plotkin - US grants

[unreadable] DESCRIPTION (provided by applicant): Frontotemporal dementia is a fatal neurodegenerative disease that results in progressive decline in behavior, executive function, and language. Currently, there are no effective treatments. Recent advances, however, have led to a greater understanding of the most common pathological form of the disease, called frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). In 2006, the major protein in the hallmark ubiquitinated inclusions of FTLD-U was identified as TAR DMA binding protein 43 (TDP-43). The function of TDP-43 is largely unknown. However, several lines of evidence indicate a role in the regulation of gene expression. TDP-43 binds DNA and RNA, regulating through these interactions the temporal and tissue-specific expression of some genes, as well as the splicing of others. Moreover, physiologic TDP-43 is nuclear, where it associates with heterogeneous ribonucleoproteins with well-known splicing activities. This research proposal is designed around the hypothesis that through aberrant activity of TDP-43, dysregulation of gene expression is a key disease mechanism in FTLD-U. Genome-wide approaches will be employed to evaluate this hypothesis. The following are the proposal's specific aims: 1. Obtain and analyze genome-wide expression profiles of FTLD-U at the mRNA and microRNA levels. 2. Identify DNA binding partners of TDP-43 in cell culture models and in FTLD-U using chromatin immunoprecipitation of TDP-43 followed by microarray analysis (ChlP-on-chip). 3. Characterize the relationship of specific microRNAs and TDP-43 to changes in mRNA expression. 4. Extend techniques developed in the study of FTLD-U to other diseases characterized by the accumulation of pathologic TDP-43. RELEVANCE (See instructions): Frontotemporal dementia is the second-most common cause of dementia in individuals under age 65. Understanding key genes dysregulated in this disease could lead to the development of targeted therapies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The need for well-validated, easily-accessible biomarkers for Parkinson's disease (PD) and endophenotypes within PD is great. Despite this need, candidate markers reported in the literature are few, and the most well-establishd biochemical biomarkers described to date in PD are cerebrospinal fluid (CSF) markers, creating a substantial barrier to widespread use. Until very recently, the search for biomarkes in PD and many other diseases was limited by the fact that a targeted approach was needed - that is, one could not find a biomarker unless there was a priori reason to believe that a particular gene/protein would be informative in the first place. In the past 10 years, however, technological advances have allowed the advent of large, unbiased screens of hundreds, if not thousands, of potential candidates, a radical change in approach pioneered in the world of genetics/genomics. We have previously used such an unbiased approach to discover, and subsequently replicate, a novel association between plasma levels of epidermal growth factor (EGF) and cognitive impairment in PD, demonstrating that low EGF levels may both correlate with and predate the onset of dementia in PD. In parallel, we hve found that low plasma Apolipoprotein A1 (ApoA1) levels may indicate increased risk for loss of dopaminergic system integrity and uncovered five potential plasma-based biomarkers correlating with rate of decline in PD. Here, we propose to build on this hands-on experience to move our findings forward in a pipeline towards clinical translation while also conducting a de novo discovery screen of 450 plasma proteins for additional biomarkers for PD and endophenotypes within PD. Embedded in our approach is an understanding that unbiased biomarker discovery methods require replication in additional cohorts of subjects, followed by validation through across-site replication of findings and investigations into biological mechanisms. Thus, our aims are: 1) To validate two previously-replicated plasma-based biomarkers in PD -- determining whether plasma epidermal growth factor (EGF) levels are a biomarker for cognitive performance in PD, and whether plasma apolipoprotein A1 (ApoA1) levels are a biomarker for PD risk by evaluating their performance in additional cohorts of patients from independent clinical sites and exploring aspects of their biology and potential for clinical translation~ 2) To replicate five newly-discovered plasma-based biomarkers for rate of decline in PD -- determining whether levels of AXL receptor tyrosine kinase (AXL), matrix metalloproteinase-2 (MMP-2), interleukin-7 (IL-7), EGF, and C-reactive protein (CRP) correlate with rate of PD motor decline in additional cohorts of UPenn patients, using alternative platforms for measurement~ and 3) To perform an unbiased discovery screen for plasma-based biomarkers of motor and cognitive disease progression in PD using a novel, protein-DNA-aptamer-based technology for simultaneous measurement of 450 plasma proteins.

DESCRIPTION (provided by applicant): The Genetic Regulation and Disease Function of the Frontotemporal Dementia Protein TMEM106B Frontotemporal dementia (FTD) is the second-most common cause of presenile dementia, characterized clinically by deterioration in language, behavioral control, or both. There are no effective treatments, and progressive neurodegeneration causes death within an average of 6-7 years. FTD is comprised of several neuropathological subgroups, likely representing different underlying pathophysiologies. The largest subgroup (~50% of FTD cases) is characterized by pathological inclusions of the HIV TAR DNA-binding protein of 43 kD (TDP-43) and accordingly named frontotemporal lobar degeneration with TDP-43 inclusions, or FTLD-TDP. FTLD-TDP can be either sporadic or familial, with a substantial proportion of cases (~10%) attributable to mutations in the progranulin gene (GRN). Inherited in an autosomal dominant manner, GRN mutations appear to cause FTLD-TDP through haploinsufficiency of progranulin, a secreted protein with trophic effects on neurons. Recently, we performed a genomewide association study (GWAS) in FTLD-TDP, identifying a 7p21 locus containing only the uncharacterized gene TMEM106B that confers increased risk of FTLD-TDP in both non-Mendelian and GRN mutation-associated FTLD-TDP. Carriers of risk-associated genetic variants and diseased individuals independently showed increased mRNA expression of TMEM106B, strongly implicating this gene as the cause of the GWAS signal. Beyond the fact that TMEM106B is the only risk factor for non- Mendelian cases of FTLD-TDP described to date, little is known about it. Preliminary data suggest, however, that TMEM106B expression is increased in FTLD-TDP~ that TMEM106B is regulated by the microRNAs miR- 132 and miR-212, which are both decreased in FTLD-TDP~ that increased TMEM106B expression leads to endosomal-lysosomal dysfunction~ and that increased TMEM106B expression leads to abnormalities in progranulin trafficking. These data lead to a working model in which genetic variants at TMEM106B confer increased risk of FTLD-TDP by increasing TMEM106B expression. Increased TMEM106B expression, in turn, alters endosomal-lysosomal function, which influences the proper sorting, internalization, or secretion of progranulin. The Specific Aims of the project, which will test the working model, are: AIM 1: Confirm the association of TMEM106B over-expression with disease states and with TMEM106B risk genotypes~ AIM 2: Investigate the precise cis-acting mechanism(s) by which TMEM106B gene expression is regulated~ AIM 3: Elucidate the normal and pathophysiological function of TMEM106B in endosomal-lysosomal pathways as well as in progranulin trafficking. The over-arching goal of this proposal is to move from a statistical association obtained by GWAS between TMEM106B and FTLD-TDP, to a mechanistic understanding of both the genetic regulation of TMEM106B and the normal and pathophysiological function of this protein. In the process, we will identify many potential avenues for therapy not otherwise in the landscape of research and drug discovery efforts for this currently untreatable disease.

PROJECT SUMMARY: At present, Parkinson's disease (PD) affects more than 4 million people worldwide, with numbers expected to nearly double by 2030. PD is a progressive neurodegenerative disease, and there are no disease-modifying therapies. Currently, the diagnosis of PD relies almost entirely on clinical examination, with no laboratory-based confirmatory testing available. While clinical accuracy is reasonably high in longitudinally followed patients with moderate symptoms, it is much lower in earlier stages of PD. As a consequence, both for clinical care and to accelerate the development of much-needed therapeutics, a confirmatory diagnostic biomarker would be of great utility. To meet this need, we previously used a novel aptamer-based technical platform to measure nearly 1000 proteins from the plasma of 64 Parkinson's disease and 30 normal control individuals recruited at the University of Pennsylvania (UPenn). We identified candidate biomarkers differentiating the two groups, then narrowed these candidates by stability selection to an eight-protein panel. This eight-protein panel classified an independent test set of 32 PD and 15 control subjects from UPenn with 91% accuracy. Furthermore, these same eight proteins differentiated PD individuals from a cohort of 25 AD individuals with >90% accuracy as well. Together, our preliminary findings suggest that a blood test based on just eight plasma proteins has the potential to serve as a confirmatory diagnostic test for PD. While our preliminary work already contains a replication (as we have employed a training/test set design to avoid over-fitting of data), we seek further replication in national or international cohorts of patients recruited outside of our university. This will allow us to understand the generalizability of our findings and pave the way for clinical development of a blood test to confirm PD diagnosis. To do this, we propose three specific aims: (1) To evaluate the robustness of candidate proteins nominated in UPenn subjects through the Somalogic platform. We will do this through (a) the analysis of 38 quality control samples using the aptamer-based array, (b) the analysis of duplicate aliquots of 30-50 samples previously investigated on the Somalogic panel, using a mass spectrometry-based approach to quantitative proteomics. (2) To repeat the aptamer-based screen on 319 samples from the Parkinson's Disease Biomarker Program biorepository, representing PD as well as controls, with both PD and controls originating from two clinical sites. Using our existing data, as well as the PDBP data, we will determine whether our 8-marker panel can reproducibly identify PD samples. (3) To test a small panel of proteins that pass quality control in Aim 1 and are replicated in Aim 2, using alternative, low-cost assays, in 450 samples from PPMI.

PROJECT SUMMARY: At present, Alzheimer's Disease (AD) affects 5 million in the US alone, with numbers projected to double by 2050. AD is a progressive neurodegenerative disease, and there are no disease-modifying therapies. Biomarkers ? objective measures of physiological of pathophysiological states ? can accelerate therapeutic development by making clinical trials more targeted and more efficient. However, successful biomarker development requires availability of biosamples from well-characterized patient populations, attention to detail in sample collection and handling, and quality control (QC) for sources of preanalytical variability. The recently-renewed Penn ADCC follows a longstanding clinical cohort of cognitively normal subjects, subjects with mild cognitive impairment (MCI), patients with early AD, and patients with AD related diseases (ADRD), numbering 500 individuals total. We propose to form a stand-alone Biomarker Core (Core G) to augment efforts of the existing 6 Penn ADCC cores by creating a systematic resource for biofluid samples, an accompanying set of baseline biochemical data, and QC tools to enable biomarker discovery. To do this, we propose four specific aims. ? SPECIFIC AIM 1: Oversee and direct all banking and dispersal of biofluid samples from Clinical Core B. These biofluids will consist of CSF, plasma, and serum collected at each visit. ? SPECIFIC AIM 2: Characterize biochemical biomarkers previously reported in the literature to confirm diagnosis of AD, predict outcome in early (or prodromal) stages of AD, indicate target engagement with an experimental therapeutic, or monitor disease progression. Specific markers that will be assayed in all 500 ADCC Clinical Core subjects at baseline include CSF A?1-42, CSF t-tau, and CSF p-tau181, CSF neurofilament light chain (NF-L), CSF ferritin, plasma NF-L, plasma epidermal growth factor (EGF). Moreover, biochemical biomarkers emerging from early investigations in ADNI (such as CSF total and phospho-?-SYN, neurogranin, and Vilip1), may be incorporated if data from ADNI appears sufficiently promising. ? SPECIFIC AIM 3: Create and maintain reference pools of CSF, plasma, and serum samples from the ADCC Clinical Core cohort as well as cohorts of patients with ADRD from other Penn clinics, making them available to investigators within the ADCC, and throughout the wider AD research community. ? SPECIFIC AIM 4: Create biomarker readouts for quality of sample handling from protein profiles obtained from systematic sample perturbations. We will leverage separately-funded investigations in which replicate plasma aliquots are systematically perturbed (left at room temperature, subjected to freeze-thaw) prior to interrogation on an aptamer-based platform for >1000 protein analytes. Through these separately-funded investigations, we will be able to identify many protein candidates that change in a predictable way with these systematic perturbations. In this Aim, we will develop assays for these candidate proteins, creating ?readouts? for poor sample handling.

PROJECT SUMMARY/ABSTRACT: Biomarkers of Cognitive Decline in Parkinson's Disease While patients with Lewy body disorders (LBD) share the core feature of deposition of misfolded alpha-synuclein (aSyn) into neuropathological inclusions, they exhibit pronounced heterogeneity in both initial clinical phenomenology, as well as in trajectory of outcomes. Specifically, among human patients with aSyn inclusions in neurons (or neuronal synucleinopathy), some manifest predominantly with cognitive symptoms and dementia from disease onset ? resulting in a clinical diagnosis of dementia with Lewy bodies (DLB). Others manifest predominantly with motor symptoms ? resulting in a clinical diagnosis of Parkinson?s disease (PD). Among PD patients, most subsequently develop significant cognitive decline and eventual dementia (PD with dementia, or PDD), while others do not, and the time course to PDD varies widely. The reasons for these differences in phenomenology among synucleinopathy patients are not well understood. This project aims to define endophenotypes within the LBD spectrum using objectively-measured biomarker characteristics, developing predictors of cognitive decline in PD and comparing these molecular signals to those found in DLB and Alzheimer?s disease (AD) patients. We use both unbiased screening approaches and hypothesis- driven approaches to develop genetic and biochemical biomarkers in three Aims: Specific Aim 1: Develop biochemical biomarkers of differential PD cognitive progression. Through unbiased screening of >1000 plasma proteins in >300 PD patients from multiple cohorts, we have derived a candidate list of 10 plasma proteins that predict future cognitive decline. We will assay these markers in >1000 additional PD subjects, developing multi-protein classifier panels for accurate prediction of cognitive trajectory. We will characterize these proteins in comparator groups of DLB and AD patients, as well as neurologically- normal controls. Specific Aim 2: Investigate causal influences on cognitive trajectory among LBD patients using Mendelian randomization. We will use Mendelian randomization (MR) to test the hypotheses that candidate biochemical biomarkers and AD-related disease processes causally influence cognitive trajectory in LBD. To do this, we will use as instrumental variables for MR single nucleotide polymorphisms (SNPs) nominated from (1) their relationships with protein levels of candidate biochemical biomarkers or (2) their genome-wide association with AD risk. These SNPs may then be developed as genetic biomarkers predicting cognitive trajectory in LBD. Specific Aim 3: Determine whether biochemical and genetic biomarkers predictive of cognitive decline differ for PD with vs. without GBA mutations. We propose to use a unique resource in development at the University of Pennsylvania ? the Molecular Integration in Neurological Disease (MIND) Initiative ? to compare biochemical and genetic biomarkers predictive of cognitive decline in PD with vs. without GBA mutations.

PROJECT SUMMARY/ABSTRACT Project IV: Tackling Heterogeneity of Cognitive Trajectory in Lewy Body Disorders Project IV Leader: Alice Chen-Plotkin; Co-Leaders: Daniel Weintraub, Rizwan Akhtar While patients with Lewy body disorders (LBD) share the core feature of deposition of misfolded alpha-synuclein (aSyn) into neuropathological inclusions, they exhibit pronounced heterogeneity in both initial clinical phenomenology, as well as in trajectory of outcome. Specifically, among human patients with aSyn inclusions in neurons (or neuronal synucleinopathy), some manifest predominantly with cognitive symptoms and dementia from disease onset ? resulting in a clinical diagnosis of Dementia with Lewy bodies (DLB). Others manifest predominantly with motor symptoms ? resulting in a clinical diagnosis of Parkinson?s Disease (PD). Among PD patients, some subsequently develop significant cognitive decline and eventual dementia (Parkinson?s Disease with Dementia, or PDD), while others do not. The reasons for these differences in phenomenology among synucleinopathy patients are not well understood. Project IV, like Projects I, II, and III, investigates the role of aSyn in the Alzheimer?s Disease related dementias (ADRD), in the context of living patients who manifest with DLB vs. PD vs. PDD vs. AD. This project aims to define endophenotypes within the LBD vs. AD spectrum using objectively-measured biomarker characteristics. We use both unbiased screening approaches and hypothesis-driven approaches to develop genetic and biochemical biomarkers in three Aims: Specific Aim 1: Develop biochemical biomarkers of differential PD cognitive progression. Through unbiased screening of >1000 plasma proteins in >300 PD patients from multiple cohorts, we have derived a candidate list of 10 plasma proteins whose baseline levels associate with subsequent cognitive decline. We will validate these markers in >1000 additional PD subjects, developing multi-protein classifier panels for accurate prediction of cognitive trajectory. We will characterize these proteins in comparator groups of DLB and AD patients, as well as neurologically normal controls. Specific Aim 2: Investigate causal influences on cognitive trajectory among LBD patients using Mendelian randomization. We will use Mendelian randomization (MR) to test the hypotheses that candidate biochemical biomarkers and AD-related disease processes influence cognitive trajectory in LBD. To do this, we will use as instrumental variables for MR single nucleotide polymorphisms (SNPs) nominated from (1) their relationships with protein levels of candidate biochemical biomarkers or (2) their genomewide association with AD risk. These SNPs may then be developed as genetic biomarkers predicting cognitive trajectory in LBD. Specific Aim 3: Define the clinical correlates of different strains of aSyn. We will use enzyme-linked immunosorbent assays (ELISAs) developed with antibodies raised to different conformations of aSyn ? ?strains? as defined in Project I ? to test the hypothesis that different strains of aSyn result in differential development of cognitive features among the synucleinopathies PD without dementia, PDD, and DLB. We will characterize AD and neurological normal controls as comparator groups.

Clinician-scientists are uniquely positioned to ask new and insightful scientific questions inspired by patient observations, yet, they often lack the expertise to be able to translate their observations into carefully designed basic scientific and translational experiments. There are likely many reasons for this, but the most cited barriers are lack of specific training, mentoring, funding, and time. If these barriers could be removed, more clinician-scientists could pursue careers in laboratory-based translational research, thereby helping to reverse the current state of affairs in many neurological disorders, in which basic research is proceeding at an increasingly rapid pace but translational research is lagging, and most patients with neurological disorders are left without preventions, treatments or cures. Here we propose a research training program for MD-PhDs or MDs who have finished their clinical training in a neuroscience-related specialty and are highly motivated to pursue careers as physician-scientists in innovative, laboratory-based translational research in brain diseases. The ReConNecT-IT (Remapping Clinical Neurosciences through Translation and Innovation Training) program consists of intense research training under the close mentoring of 1-2 faculty mentors. Trainees design and conduct independent research projects that they can take with them when they transition to independent support, and upon which they will base their NIH K-award application. Research projects are directed toward the translation of the genetic, molecular and cellular pathophysiology of neurological diseases into strategies for prevention, treatment or cure. Trainees will be encouraged to pursue projects that are collaborative and cross-disciplinary, as this fosters their research development, and linking disciplines helps generate ideas that are novel. Trainees will have access to 20 core faculty and can collaborate with other groups. Trainees will work alongside PhD researchers and participate in journal clubs, lab meetings and basic science seminars. The curriculum includes formal training in experimental design, statistical methodology and quantitative literacy, and well as individualized training on statistical/quantitative methodology by our Director of Statistical Training. Trainees will gain an understanding of critical topics in translational research and how basic research is translated into clinical trials (patient-oriented research) via two specific, semester-long courses. They will gain professional skill and understand career opportunities by participating in workshops on developing a K-award application, grant and scientific writing, pursuing an academic career, job search skills, laboratory and project management, and responsible conduct of research. A unique feature of ReConNecT-IT is that prospective trainees can know of their acceptance before their clinical training ends, allowing them to schedule research into their remaining clinical time, thereby expanding the total amount of research experience they will have before writing a K-award application. The main expected short-term outcome for this program is application for an NIH K award or equivalent grants.

The neurodegenerative diseases ? Alzheimer?s Disease (AD), Parkinson?s Disease (PD), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and others ? together constitute one of the most significant unmet challenges in human health, affecting greater than 50 million people worldwide with no treatments to slow or stop progression. With the advent of the genomewide association study (GWAS) in 2005, and the subsequent identification of hundreds of common variant risk factors for AD, PD, FTD, and ALS, we have many loci that may translate into new targets for therapeutic intervention. To date, however, few mechanistic studies have been performed as follow-up to these GWAS-generated leads. One exception to this general rule has been with respect to the 7p21 locus we and others reported in 2010 to confer risk for the AD-related dementia FTD. In the first five years of this R01, we used a combination of computational and bench-based approaches to definitively establish the expression quantitative trait locus (eQTL) relationship between GWAS-identified single nucleotide polymorphisms (SNPs) and expression of the target gene TMEM106B. We furthermore defined the causal genetic variant at this locus, its CTCF-based mechanism for altering expression of TMEM106B, and the deleterious effects on lysosomal pathways of altering TMEM106B expression. We coupled these mechanistic experiments with investigations of the genetic modifier effects of TMEM106B genotype in FTD due to C9orf72 hexanucleotide expansions. Thus, through our work and the work of others, the field has gained an understanding of the pathways through which genetic risk at 7p21 is conferred, and the groups of patients in which targeting of TMEM106B may be viable therapeutically. In this RO1 renewal application, we propose to deepen our understanding of TMEM106B biology, investigating its influence in multiple neurodegenerative diseases, and elucidating its role in lysosomal function and cellular health. Specific Aim 1: Determine whether genetic modifier effects of the GWAS-identified FTD common variant risk factor TMEM106B extend across a spectrum of neurodegenerative diseases. We will investigate TMEM106B genotype effects in >1300 longitudinally-followed AD, PD, FTD, and ALS patients. We will determine whether TMEM106B acts as a genetic modifier in PD associated with GBA mutations. Specific Aim 2: Elucidate the mechanisms by which changes in TMEM106B expression affect lysosomal- autophagy pathway function and cellular health. We will follow-up preliminary work demonstrating that TMEM106B may affect autophagosome-lysosome fusion through a VAMP8-Syntaxin17 pathway. We will investigate TMEM106B-induced changes in lysosomal acidification through direct measurement of ion conductances across the lysosomal membrane and investigations of TMEM106B?s role in assembly of the vacuolar ATPase.

BIOMARKER CORE SUMMARY: With the increasing emphasis in the Alzheimer?s Disease (AD) field on molecular diagnosis, the Biomarker Core has two over-arching goals. First, we will continue a highly-successful tradition at the University of Pennsylvania of banking and widely dispersing biofluid samples obtained by the Clinical Core, while annotating these samples with baseline molecular measures that allow for Amyloid-Tau-Neurodegeneration (A/T/(N)) classification within the NIA-AA framework. Second, we will pave new ground by relating well-established CSF- and imaging-based A/T/(N) biomarkers to measures obtained from emerging plasma-based biomarkers, and performing measures that will allow for investigation of the contribution of vascular disease to heterogeneity of clinical phenotype. These goals will be met through activities along five Specific Aims. AIM 1: Oversee and direct all banking and dispersal of biofluid samples obtained in ADRC UDS participants (current n=537). AIM 2: Characterize established biochemical biomarkers previously reported in the literature in order to annotate all Clinical Core samples within the A/T/(N) classification scheme. A CSF biochemical biomarker profile defined by (1) CSF amyloid-beta 1-42 (CSF A?42) and (2) 1-40 (CSF A?40), (3) CSF total tau (CSF t-tau), (4) CSF tau phosphorylated at residue 181 (CSF p-tau181), (5) CSF neurofilament light chain (CSF NfL), and (6) plasma NfL measures will be established. AIM 3: Relate emerging plasma-based biomarkers to established CSF and imaging biomarkers, as well as neuropathology. We will focus on assays of plasma p-tau and plasma A?, validating measures from samples obtained antemortem against postmortem findings. For well-validated assays, we will obtain plasma measures on all ADRC UDS participants. AIM 4: Collaborate with other Cores to investigate the contribution of vascular disease to heterogeneity of clinical phenotype. We will support the collection of biomarker measures (i.e. CRP, cholesterol, hemoglobin A1c, interleukin-6 (IL-6), homocysteine) that allow for assessment of vascular risk in all ADRC UDS participants. AIM 5: Provide advice and support to investigators within and outside the Penn ADRC.