Jing Zhang, MD PhD - US grants

Parkinson's disease (PD) is an important public health problem for older Americans. Although dramatic advances have been made in the clinical management of PD, the mechanisms that underlie its neurodegeneration are incompletely understood. It is necessary to understand these mechanisms in order to design effective procedures and therapeutic approaches to prevent the development or to impede the progression of PD. The long-term objectives of this project are to determine the mechanisms by which dithiocarbamate (DTC)-based pesticides linked to PD epidemiologically contribute to dopaminergic neurodegeneration in PD. The specific aims of this project are: (1) to determine structure- activity relationships of DTCs in vitro as mitochondrial and dopaminergic neurotoxins, and in vivo as dopaminergic neurotoxins after exposure to systemically administered DTCs in young and old wild type mice as well as mice with genetically altered anti-oxidant defenses, (2) to determine structure-activity relationships of systemically administered DTCs as inducers of oxidative stress in different brain regions that have either significant or minimal neurodegeneration in PD in wild type mice and mice with genetically altered antioxidant defenses, (3) to assess mechanisms of oxidative stress and the effects of oxidative stress on excitotoxicity in different brain regions that have either significant or minimal neurodegeneration in PD in rats exposed to DTCs directly through brain microdialysis. The new information will shed more light on the mechanisms by which pesticides interact with aging and genetic vulnerability thereby contributing to development and progression of PD.

DESCRIPTION (provided by applicant): Parkinson's disease is a common age-related neurodegenerative disease characterized pathologically by a loss of dopaminergic neurons in the substantia nigra with resultant depletion of striatal dopamine and presence of Lewy bodies in the remaining neurons. Lewy body contains numerous functional and structural proteins, including alpha-synuclein and ubiquitin, and aggregation of alpha-synuclein is thought to be important in Lewy body formation as well as neurodegeneration, although the detailed mechanisms remain to be defined. Increasing evidence has suggested that mitochondrial dysfunction, increased oxidative stress and dysfunction of the ubiquitin-proteasome system may be involved in Lewy body formation as well as neurodegeneration. A few neurotoxicants, e.g. rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine, all capable of inhibiting mitochondria and enhancing oxidative stress, have been utilized widely to generate parkinsonian models in vitro and in vivo. Interestingly only rotenone produces Lewy body-like cytoplasmic inclusions. On the other hand, there are also human nigral degenerative diseases with or without Lewy body formation in the substantia nigra, including Parkinson's disease, dementia with Lewy body disease and multiple system atrophy. Our preliminary studies in cell cultures demonstrated that proteins interacting with alpha-synuclein may influence alpha-synuclein aggregation, Lewy body formation and cell death. Hence, this application proposes to advance our understating of the development of Lewy bodies as well as molecular mechanisms of development of Parkinson's disease with subtractive proteomics by looking for differences in the proteins associated with alpha-synuclein in the substantia nigra between rotenone-exposed and control rats first, and then comparing the protein profiles in rotenone-exposed rats vs. rats exposed to 1- methyl-4-phenylpyridinium, the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, or 6- hydroxydopamine. A second phase would examine tissues from patients with Parkinson's disease vs. controls as well as patients with multiple system atrophy. Finally, we will study the roles of the common and differentially displayed proteins, those related to oxidative stress, mitochondrial and proteosomal function in particular, in alpha-synuclein aggregation, Lewy body formation and neurodegeneration.

DESCRIPTION (provided by applicant): Parkinson's disease (PD), the most common movement disorder afflicting millions of Americans, is diagnosed when patients present with cardinal parkinsonian signs, e.g. bradykinesia, rigidity, and tremor, and show favorable response to levodopa or dopamine (DA) agonists. However, there are quite a few other movement disorders that mimic PD clinically including response to levodopa and DA agonists, making accurate diagnosis of PD difficult sometimes even in the best hands. In addition, the natural course of PD varies substantially, with most patients developing first the mild cognitive impairment (MCI) and then dementia as the disease progresses. While current functional neuroimaging methods to monitor PD progression with fluorodopa positron-emission tomography (F-Dopa-PET) or beta-CIT single photon emission computer tomography (Beta-CIT-SPECT) show relatively high sensitivity and specificity, they are not widely accessible, particularly in developing countries, and do not elucidate biological mechanisms of PD progression. Furthermore, there are no comparable biomarkers for monitoring MCI or even dementia in PD, which has important clinical consequences in these patients with regard to increased mortality, caregiver burden, and risk of admission to nursing home. We hypothesize that there are unique protein markers for PD, PD progression, and PD dementia in brain tissue, and some of which will be reflected in the human cerebrospinal fluid (CSF). Hence, we are proposing to use a high throughput proteomic approach to identify proteins unique to PD, PD progression, and development of cognitive deficits in PD first in pathologically involved brain tissues and then select a panel of unique proteins in CSF that could serve as the basis of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) to diagnose PD, to monitor PD progression, and to detect PD patients at risk to develop cognitive deficits.

[unreadable] DESCRIPTION (provided by applicant): The clinical diagnosis of most neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD), currently relies on the analysis of symptoms and signs, limited laboratory tests, and more recently, brain imaging. The considerable overlap in the clinical presentation of these diseases, especially in their early course, increases the difficulty of distinguishing these diseases. In general, clinical diagnostic accuracy of various neurodegenerative diseases, as determined by pathological examination, falls between 50-85%, depending on the disease involved and the experience of the physician. Therefore, alternative approaches that are complementary and/or can provide greater selectivity and accuracy for disease diagnosis, especially at early clinical stage, are urgently needed. In the past few years, we have applied mass spectrometry based quantitative proteomics to study biomarkers related to neurodegenerative diseases. Such efforts have led to the identification of a group of protein biomarker candidates associated with AD and PD in cerebrospinal fluid (CSF), an ideal source for biomarker discovery in diseases related to the central nervous system (CNS). However, CSF is a less preferred diagnostic material compared to plasma or serum. Thus, in this proposal, we will apply a mass spectrometry based targeted quantitative proteomics approach to quantitatively detect and verify CSF biomarker candidates associated with AD and PD in human plasma. Successful completion of this project may lead to the development of clinician/patient-friendly sensitive assays that can assist with clinical diagnosis of AD and PD as well as monitor the progression of diseases or effects of current therapeutic reagents. In this study, we proposed to apply a mass spectrometry based targeted quantitative proteomics approach to quantitatively detect and verify CSF biomarkers associated with AD and PD in human plasma. We hope such effort will lead to or benefit the development of a clinicians-/patients-friendly sensitive assays that can assist with clinical diagnosis of AD and PD as well as monitoring the progression of diseases or effects of current therapeutic reagents. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Parkinson's disease (PD), the most common movement disorder afflicting millions of Americans, is diagnosed when patients present with cardinal parkinsonian signs, e.g. bradykinesia, rigidity, and tremor, and show favorable response to levodopa (L-DOPA) or dopamine (DA) agonists. However, even at clinical "early" stage of the disease, PD patients have already lost more than 60-70% of their DA neurons in the nigrostriatal system. Thus, the nature course of this disease makes it very challenging for any neuroprotective medicine to be effective, simply because there are not many neurons to be protected to begin with in PD patients when the diagnosis can be made clinically. While current functional neuroimaging methods with fluorodopa positron-emission tomography (F-Dopa-PET) or beta-CIT single photon emission computer tomography (Beta-CIT-SPECT) show relatively high sensitivity and specificity in assessing nigrostriatal function even before PD patients become symptomatic, they are very expansive, meaning that they are not currently appropriate for routine diagnostic screening. In addition, these methods are not widely accessible, particularly in developing countries, and do not elucidate biological mechanisms of PD progression. We hypothesize that there are unique protein markers for PD, including preclinical PD, in brain tissue, and some of which will be reflected in the human cerebrospinal fluid (CSF). Hence, we are proposing to use a high throughput proteomic approach to identify proteins unique to preclinical PD and PD progression in two models simultaneously, i.e. a nonhuman primate treated with MPTP and familial PD patients secondary to LRRK2 mutations. This is followed by validation of candidate markers in human CSF of in a different but larger set of both sporadic and familial PD patients, including those at preclinical stage as determined by PET imaging. When completed, we anticipate having a panel of makers in CSF that could serve as the basis of a highly sensitive and specific microsphere-based Luminex xMAP assay to diagnose preclinical PD and to monitor PD progression at early stages. PUBLIC HEALTH RELEVANCE: This proposal is designated to investigate biomarkers that can detect patients with Parkinson's disease before clinical presentation. This proposal, if successful, can significantly increase the therapeutic window for Parkinson's patients. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): It has become increasingly clear that Parkinson's disease (PD) is often associated with cognitive impairment. Pathological evaluations have repeatedly demonstrated that dementia in PD patients (PD-D) is associated with either formation of Lewy bodies (LBs) or Alzheimer's changes (presence of neurofibrillary tangles (NfTs) and senile plaques (SPs)) in the cortex. It is well known that formation of LBs, NfTs and SPs are related to deposition of 1-synuclein (SNCA), tau and A?, respectively. However, the changes in these key proteins are not known in relationship to the development of PD-D. We have hypothesized that development of cognitive impairment in PD is associated with unique post-translational modifications (PTMs) of SNCA, tau and A?, and that the PTMs are isoform/species specific, as well as disease stage specific. Thus, one of the major goals of the proposal is to characterize the PTMs of various isoforms/species of SNCA, tau and A? as a function of PD- D development using various state-of-the-art proteomics techniques. Additionally, as development of PD-D likely involves cellular processes beyond just SNCA, tau and A?, for the purpose of discovering biomarkers that are clinically accessible, we will also use a high throughput proteomic technique to characterize a sub- proteome with a unique PTM (glycosylation) that is highly enriched in body fluids, i.e. proteins carrying great potentials to be biomarkers for PD-D. Both analyses (targeted and unbiased profiling) will be applied to pathologically confirmed brain tissues initially, followed by confirmation and validation in the cerebrospinal fluid (CSF) and plasma, respectively. The confirmed and validated markers, whether in CSF or plasma, can then serve as the basis of highly sensitive and specific multiplex immunoassays (xMAP) used to identify PD patients at risk for developing cognitive deficits. Four Specific Aims have been designed to accomplish these goals: 1) Identify proteins unique to the development of PD-D in pathologically involved brain regions in confirmed PD cases with and without dementia clinically. 2) Confirm and validate unique proteins, revealed in brain tissue, in lumbar CSF obtained from living subjects at different stages with and without dementia clinically. 3) Confirm and validate unique proteins, revealed in brain tissue, in plasma obtained from living subjects at different stages with and without dementia clinically. 4) Establish xMAP assays for detecting cognitive impairment in PD 7. PUBLIC HEALTH RELEVANCE: This project investigates the potential biomarkers correlating with the development of cognitive impairment in Parkinson's patients. Identification of these makers can increase therapeutic window for Parkinson's patients at risk for developing dementia that is associated with mortality, caregiver burden and risk for nursing home admission.

Environmental exposure to metals, including manganese, is an important risk factor for the development of parkinsonism (PS) in workers of welding or related industries. However, the relevance of metal-mediated PS to idiopathic Parkinson's disease (iPD) remains to be characterized. Clinically, differential diagnosis between metal-related PS and iPD is difficult, even in the best hands. This proposal is focused on discovering plasma biochemical markers unique to welders for differential diagnosis of various PS, monitoring PS progression, and identification of the population at risk for developing disabling PS. The techniques to be utilized are state-of-the-art proteomics that are actively employed currently in our laboratory in revealing biomarkers specific to iPD in both human brain tissue and cerebrospinal fluid (CSF). Three specific aims are designed for the current proposal: 1) to differentiate PS in the plasma samples of welders from those of iPD using brain/CSF specific markers identified in iPD patients, 2) to develop plasma biomarkers unique to PS progression as well as early stages in welders using targeted and nonbiased quantitative proteomics, and 3) to confirm and validate PS plasma biomarkers in welders, which is a key process of biomarker discovery. The significance of this investigation includes: 1) identification of markers unique to PS, both in symptomatic welders and those at risk for developing PS, will help diagnose and monitor these patients as well as make it possible to remove the subjects at risk from the environment, thereby preventing them from developing PS; 2) identification of protein markers unique to PS secondary to metal exposure likely suggests novel pathogenesis and therapeutic targets for the disease process; and 3) PS markers, if identified, can be widely utilized, given that plasma-based assays can be readily implemented in a clinical setting, even in developing countries or in remote areas of developed countries.

The overall goal of the Genetics, Biomarkers, and Neuropathology Core is to provide resources and expertise to PANUC investigators and other collaborators. This Core will: 1. GENETICS; Provide expert preparation of plasma and DNA from Clinical Core participant blood samples. DNA will be stored and used for genetic analysis by Project 3. Blood samples will be appropriately deposited with the NINDS Genetic Resource Center at the Coriell Institute for distribution to other investigators while insuring proper safeguards. 2. BIOMARKER; Provide analysis of CSF using validated biomarkers, and provide a platform for rapid translation of potential new biomarkers for cognitive impairment in Parkinson's disease discovered in a linked ROI AG033398 (Zhang's project) and Project 3. Plasma obtained in Aim 1 will also be stored as a resource for future biomarker studies. 3. NEUROPATHOLOGY: Provide diagnostic expertise to the Clinical Core by providing family members of the deceased and physicians involved in their care with timely autopsy reports based on the most current standardized diagnostic criteria, and optimally prepare brain donations to advance research in Project 2 and Zhang's project. This Core sen/es several initiatives in the NIH Blueprint. It uses existing NIH resources, contributes essential materials to NIH repositories, and is highly patient-oriented and translational

DESCRIPTION (provided by applicant): The role of microglial activation and neuroinflammation has recently emerged as a potential mediator and /or potentiator of Parkinson's disease (PD) pathogenesis. While the precise mechanisms and pathways responsible for neurodegeneration are relatively unclear, sufficient evidence has been put forth to suggest that PD has a multifactorial etiopathogenesis. Indeed, environmental as well as genetic factors have been demonstrated to cause activation of microglia, leading to neuroinflammation and subsequent damage to the nigrostriatal dopamine system. Furthermore, genetic and environmental insults both involve activation of NADPH oxidase (PHOX), a key mediator of the neurotoxic response following microglial activation. These data highlight the importance of PHOX and provides a common link between genetic susceptibility and environmental insult in the development of PD. Thus, this proposal will exploit the intersection of genetic and environmental factors at PHOX to gain a better understanding of the mechanisms involved in microglia- mediated neurotoxicity. Through the utilization of in vitro and in vivo models, the aims proposed will systematically elucidate the interplay between endogenous and exogenous insults and their synergistic contribution to microglial activation and neurodegeneration. Moreover, specific interacting proteins and pathways involved in these processes will be examined and these results will be further validated in human tissue from PD patients and compared to other neurodegenerative diseases. Completion of these proposed aims will provide a better understanding of the interaction of genetic and environmental factors at PHOX. Finally, the identification of specific proteins and pathways involved in microglia-mediated neurodegeneration may provide potential targets of therapeutic intervention for patients with PD. PUBLIC HEALTH RELEVANCE: This project investigates the potential interplay between genetic vulnerability and environmental exposure that likely contributes to the development of the majority of Parkinson's cases. Elucidation of the mechanisms involved in this interplay may yield new therapies for the devastating disease.

DESCRIPTION (provided by applicant): Both genetic vulnerability and environmental exposure have been linked to the development of idiopathic Parkinson's disease (PD); however, the precise mechanisms by which these two factors intercept remain elusive. This proposal is designated to explore a potential link between genetic susceptibility and environmental factors - a tight coupling between microglial Mac1 and NADPH oxidase, both are membrane proteins critically involved in microglial activation which is a hallmark of neuroinflammation. The mechanisms involved in microglial activation are not understood completely. While some components of neuroinflammation can also be beneficial to neuronal survival, pro- inflammatory factors, especially reactive oxygen species (ROS), when produced in excess, are believed to cause collateral damage to the central nervous system. The coupling between Mac1 and NADPH oxidase enzyme appears to be one of the major sources of pro-inflammatory ROS causing neural damage. More importantly, the action of many endogenous toxins and exogenous neurotoxicants seems to converge on the activation of Mac1-NADPH oxidase pathway. Thus, this proposal will be centered on the coupling of these two proteins, with the use of various in vitro and in vivo experimental systems, to examine the detailed mechanisms by which genetic susceptibility (modeled by mutations of -synuclein gene) interact with parkinsonian toxicants via Mac1-NADPH oxidase coupling. Additionally, contribution of astroglia to microglial activation will also be explored. Finally, we will investigate novel and specific inhibitors that block Mac1 and/or NADPH oxidase, thereby providing new therapies to inhibit pro-inflammatory factors specifically while sparing neuroprotective elements of neuroinflammation, to slow down the progression of PD.

MISSION AND GOALS OF THE FUNCTIONAL GENOMICS &PROTEOMICS FACILITY CORE (FGP-FC) The FGP-FC plays a crucial role in supporting the Center's mission to identify the interactions between genetic, epigenetic and environmental factors that contribute to major chronic diseases. The FGP-FC does this by providing state-of-the-art genomics and proteomics technologies to investigate gene-environment interactions in the context of environmental health sciences research and population-based studies. MISSION AND GOALS OF THE EABM-FC Exposure assessment is the process of characterizing human contact with and uptake of agents from the environment (including the occupational environment). Given a particular agent, accurate quantification of exposure is critical for epidemiological studies that seek to define dose-response relationships and for risk assessment studies that quantify the probability of a harmful effect to exposed individuals or populations. Misclassification of exposure can lead to failure to adequately protect public health if the risk of harm is underestimated. Alternatively, if the health risk posed by a specific agent is overestimated due to inaccurate measures of exposure, scarce public health dollars are wasted needlessly. The overarching mission of EABM-FC is to provide state-of-the-art exposure assessment tools to CEEH invesfigators in order to reliably quantify exposure to agents in the environmental and subsequent health consequences and understand the role that gene-environment interactions play in modulating health outcomes

DESCRIPTION (provided by applicant): Diagnosis of Parkinson's disease (PD) is complicated by the overlap of its symptoms with those of other disorders, especially at early stages. Additionally, clinical management of PD is hampered by a lack of objective assessment of disease progression. These factors make discovery of objective biomarkers an urgent priority, but a number of challenges have impeded their development. Although CSF levels of some PD-related proteins are altered in PD patients, none discovered so far are specific enough to differentiate between parkinsonian disorders, diagnose PD at early stages, or trace its progression. Profiling experiments have identified large numbers of potential candidates, but the development of protein-specific assays, which often depend on high-quality, well-characterized antibody sets that may not be available, presents a significant bottleneck in carrying these candidates through further development. Therefore, in this study, we propose a multi-pronged effort including a variety of complementary strategies to optimize the possibility of identifying P biomarkers. First, we will further explore the maximal utility of proteins previously observed to change in CSF in PD, by determining whether these proteins, or with post-translationally modified forms of them, perform well in disease diagnosis or monitoring progression. Second, we will expand the search for CSF biomarkers by using a newly developed peptide-based platform, which will allow us to perform high-throughput targeted discovery, followed by mass spectrometry-based measurement of specific peptide biomarkers in samples from human patients. Notably, we will make use of a unique combination of multiple large cohorts, including one in which samples are collected longitudinally, to allow independent validation and assessment of performance as progression markers of all promising candidates. Additionally, we will develop several novel techniques for biomarker discovery, including profiling based on aptamers, or based on RNA screening/sequencing. Further, we will attempt to extend the biomarker discovery process to a more easily collected sample type, plasma, by examining the performance of our best-performing candidates in plasma samples from the same cohorts. Finally, we will test the best candidate biomarkers, whether in plasma or CSF, in several cohorts selected to include an enriched population of subjects at risk for PD, in order to identify biomarkers capable of diagnosing PD at its earliest stages, when treatment is likely most effective. Importantly, each step of this process provides a novel step forward in biomarker research, providing the opportunity to improve the PD diagnostic process facilitate the search for better treatments.

DESCRIPTION (provided by applicant): The applicants are applying for support for years 36 to 40 of the Environmental Pathology/Toxicology (EP/T) Training Program at the University of Washington (UW). This training program has been continuously funded by the NIEHS since 1978. The long-term goal of the program remains mentoring pre- and postdoctoral trainees to become successful independent scientists who are well equipped to respond to the environmental health research needs of the US in the coming generations. This long-established program has been highly successful in this endeavor, and the current application incorporates the themes and goals of the 2012 NIEHS Strategic Plan. The Training Program is a 30-year-long collaboration between the Department of Pathology in the School of Medicine and the Department of Environmental and Occupational Health Sciences (DEOHS) in the School of Public Health. In the previous cycle, the applicants added the outstanding Department of Genome Sciences in the School of Medicine to enhance the training experience and to stay current with exciting advances in gene-environment approaches to improving environmental health research. The EP/T Training Program is directed by Dr. Thomas Montine (Program Director), Dr. Elaine Faustman (Associate Director), and Dr. David Eaton, (Associate Director). The EP/T Training Program (i) embraces virtually all NIEHS-supported investigators at UW, including the addition this cycle of several experts in exposure science, (ii) is closely linked wih NIEHS- supported centers at UW focused on ecogenetics, toxicogenomics, and risk assessment, among others, (iii) and is highly integrated with a wide array of well-funded, complementary research centers and projects. Given the excellent track record of training, as well as the outstanding and growing opportunities at UW, the applicants are requesting renewal of their training grant, so that they can continue to train future leaders in environmental health science.

ABSTRACT The goal of the Functional Genomics, Proteomics, and Metabolomics Facility Core (FGPM-FC) is to enable Center affiliates to integrate state-of-the-art genomics, transcriptomics, epigenetics, proteomics and metabolomics methods into their research in a cost effective manner. It is well established that chemical exposure of biological systems results in expression changes of numerous RNA and protein molecules and these changes are correlated with, and are indicative of toxicity. In addition, many molecular epidemiologic studies have identified correlations between genetic polymorphisms and incidence of environmental-related disease. Toxicological monitoring increasingly involves the assessment of genetic and other molecular measurements derived from individuals and sentinel animals, to detect markers of disease susceptibility and to identify early indicators of chemical effect, such as alterations in gene expression profiles due to exposure to environmental toxicants. Various targeted molecular methods as well as OMICs based approaches have been developed in recent years and continue to develop rapidly. These methods complement each other and allow for mechanistic investigations of entire biological pathways and networks, as well as their individual components. The optimal application of these state-of-the-art methodologies requires considerable expertise in a) sample preparation and processing, b) generation and quality assessment of the data, c) rigorous statistical and bioinformatics analysis, d) as well as interpretation of the complex data sets. Most investigators do not have the financial resources or specialized expertise to keep up with the rapid technological advancements. However, it is critical for investigators to have access to the latest technologies in order to be competitive and to perform cutting edge research. The Functional Genomics, Proteomics and Metabolomics Facility Core (FGPM-FC) together with the Bioinformatics and Biostatistics Unit (BBU) of the Integrative Environmental Health Sciences Facility Core (IEHS-FC) address these challenges. It provides expertise in genomics, transcriptomics, epigenetics, proteomics and metabolomics based methods that support the study of gene- environment interactions in the context of environmental health sciences research and population-based studies.

? DESCRIPTION (provided by applicant): Objective, reliable, and reproducible biomarkers are clearly needed to assist with accurate diagnosis of Parkinson disease (PD), especially at early stages, as well as for facilitating differential diagnosis and disease monitoring. The proposal is designed to meet several major challenges of current biomarker research, specifically: 1) significant variations associated with antibody-based protein assays, 2) low sensitivity and specificity of blood based markers, and 3) detection of PD at early stages. To address the problems of antibody-based assays, our strategy is development of targeted mass spectrometry-based techniques, such as selected reaction monitoring (SRM), to identify unique peptide markers derived from proteins either showing promise in previous proteomics profiling, or known to be critical to PD pathogenesis, e.g., ?-synuclein, parkin and LRRK2, in human cerebrospinal fluid (CSF). To facilitate discovery and validation of blood based biomarkers, a specific population of central nervous system derived plasma exosomes, the cargo-carrying microvesicles recognized recently to transport biomolecules among different cells or organ systems, will be isolated before SRM analysis. The unique peptide markers will be tested in several large, well-established cohorts, e.g., Udall Centers affiliated with the University of Washington and University of Pennsylvania, DATATOP (Deprenyl and tocopherol antioxidative therapy of parkinsonism) and PPMI (Parkinson Progression Marker Initiative), with cross-sectional and longitudinal samples collected, along with extensive clinical characterization. Finally, to improve early diagnosis, we will make use of two cohorts consisting of subjects at elevated risk for PD (i.e., asymptomatic subjects with LRRK2 mutations or anosmia/hyposmia), with the goal of discovering biomarkers capable of identifying subjects with early or premotor PD. The studies designed for this project, if successful, have the potential to result in a panel(s of biomarkers that are robust, with less variation than can currently be achieved, and in a body fluid that is readily accessible in a regular clinical setting. Markers for early diagnosis and progression of PD are critical in understanding how to arrest or slow PD progression.

Summary Significant progress has been made in using neuroimaging and cerebrospinal fluid (CSF) protein measurements as Alzheimer disease (AD) biomarkers. However, there is still a critical need to identify more reliable and reproducible biomarkers with further improved diagnostic accuracy, especially to differentiate AD from other dementias, to track or monitor the disease progression and to objectively evaluate drug effects. There is also a growing interest in developing novel biomarkers that could reflect different aspects of AD pathology and accurately detect pathogenic components of AD before appearance of significant cognitive decline, thereby assisting with AD diagnosis at early symptomatic and even preclinical stages. This proposal is designed to meet several major challenges of current biomarker research, specifically: 1) difficulties in development of antibody-based, quantitative protein assays for most novel candidates identified by proteomic profiling and significant variations associated with most existing immunoassays, 2) low sensitivity and specificity of blood-based markers, and 3) detection of AD at early or even preclinical stages. To address the problems of antibody-based assays, our strategy is development of targeted mass spectrometry-based techniques, such as selected reaction monitoring (SRM), to identify unique peptide markers derived from proteins either showing promise in previous proteomics profiling, or known to be critical to AD pathogenesis in human cerebrospinal fluid (CSF). To facilitate discovery and validation of blood based biomarkers, a specific population of central nervous system derived plasma exosomes, the cargo-carrying microvesicles recognized recently to transport biomolecules among different cells or organ systems, will be isolated before SRM analysis. The unique peptide markers will be tested in several large, well-established cohorts, e.g., Alzheimer's Disease Research Centers (ADRCs) affiliated with the University of Washington, Oregon Health and Science University, University of California at San Diego, and University of Pennsylvania, and ADNI (Alzheimer's Disease Neuroimaging Initiative), with cross-sectional and longitudinal samples collected, along with extensive clinical characterization. Finally, to improve early diagnosis, we will make use of a very early MCI cohort consisting of subjects at elevated risk for AD, with the goal of discovering biomarkers capable of identifying subjects with early or preclinical AD. The studies designed for this project, if successful, have the potential to result in a panel(s) of biomarkers that are robust, with less variation than can currently be achieved, and in a body fluid that is readily accessible in a regular clinical setting. Markers for early diagnosis and progression of AD are critical in understanding how to arrest or slow AD progression.

Summary Growing evidence suggests that exosomes, a class of membrane vesicles that can be secreted by most cell types to mediate intercellular communication, play important roles in the initiation and or progression of Alzheimer disease (AD). Specially, it has been demonstrated that cell-to-cell transfer of amyloid beta (A?), tau, and other proteins critically involved in AD pathogenesis, as well as the prion-like propagation of AD pathology within the central nervous system (CNS) is mediated at least in part via exosomes. Additionally, exosomes carrying unique, disease-specific, and functionally important cargo are detectable in vivo in blood, cerebrospinal fluid (CSF) and other body fluids. More recently, we and others have demonstrated not only that exosomes may cross the blood-brain barrier (BBB), though the transportation mechanism remains unclear, but also that blood-based but CNS-specific exosomal molecules can be a valuable source of biomarkers for neurodegenerative diseases, including AD. In this study, we will first use our advanced proteomics techniques to screen for exosome surface markers specific to AD-related neuronal subpopulations or brain regions to identify more CNS- and AD- specific exosome markers, and in parallel adapt our nanoparticle sorting and single-molecule quantification technologies to enable high-purity isolation of CNS-derived exosomes in plasma and high-precision quantification of proteins in such exosomes to address several major challenges in the current field. Using the currently known (e.g., L1CAM) and more CNS- and AD- specific, CNS-derived exosomal surface markers, as well as the existing and further developed exosome isolation and quantification technologies, we will then compare AD- related biomarkers in L1CAM-containing exosomes or those from AD-related neuronal subpopulations in blood plasma from human patients, focusing on the performance of classic AD proteins and known exosomal candidates, specifically, A?, tau, ?-synuclein, and their various isoforms; additional novel targets may be studied when necessary. For the verified AD-related exosomal proteins, we will further examine their longitudinal changes in animal models and explore the mechanisms by which they are transported from the brain to blood (e.g., crossing BBB) in cellular and animal models and potential ways to alter them as novel future AD treatment targets. The proposed experiments will likely establish the foundation leading to an inexpensive and widely available test to aid in AD diagnosis and/or disease tracking. Additionally, the proposed set of studies is an important initial step toward elucidating a novel potential clearance pathway for potential toxic CNS protein species and ultimately it may provide critical opportunities for therapeutically addressing the pathology associated with neurodegeneration in AD.

Abstract Although ?-synuclein (?-syn) is largely a cytosolic protein, extracellular ?-syn is believed to play an important role in the pathology of PD, contributing to key processes such as the progressive, prion-like spread of Lewy body pathology throughout the brain, and initiation of cellular pathology in neurons and glia. In neuronal systems, ?-syn was found to be exported through a non-classical pathway and secreted in extracellular vesicles (EVs). In addition to its implications for understanding the role of ?-syn in PD pathogenesis, the localization of ?-syn to EVs presents a unique strategy for utilizing peripheral ?-syn as a PD biomarker, by specifically targeting ?-syn in EVs originating in the brain. Indeed, we have recently demonstrated that ?-syn crosses from brain to blood, and that a fraction of this central nervous system (CNS)-derived ?-syn, recovered from plasma, is contained within exosomes (small EVs of endocytic origin). Further, this peripherally accessible exosomal ?-syn performed similarly to cerebrospinal fluid (CSF) total ?-syn in differentiating control from PD human subjects. Intriguingly, while CSF ?-syn is decreased in PD, CNS-derived exosomal ?-syn was increased, suggesting that clearance of ?-syn from the brain in EVs may be up-regulated in PD. However, only a small portion of intracerebroventricularly-injected ?-syn was associated with blood exosomes; the compartment in which the rest resided is not known. For example, it is unclear whether shedding microvesicles (MVs, vesicles that bud directly from the plasma membrane) are involved in ?-syn secretion, especially from the perspective of CNS-derived plasma EVs as PD biomarkers, nor is it clear what types of cells may participate in generating ?-syn-containing vesicles that cross the BBB. Finally, the role of ?-syn secretion via EVs in PD pathogenesis remains to be defined. Here, we propose to investigate whether ?-syn transported from the brain to the blood via EVs potentially provides a mechanism for clearance of toxic ?-syn species, as well as a potential cellular source of EV-contained ?-syn altered in PD patients, which may be suitable for use as diagnostic or progression biomarkers. We will first examine the total and post-translationally modified (phosphorylated and oligomeric) ?-syn in plasma EVs derived from neurons, astrocytes, and oligodendrocytes. This protein will be characterized both using sensitive, quantitative immunoassays, as well as Nanoparticle Tracking Analysis (NTA), to determine the distribution of ?-syn forms in different types of EVs. Both types of analysis will be used to compare samples from PD patients, healthy controls, and patients with multiple system atrophy, a related but distinct synucleinopathy featuring oligodendrocyte inclusions. We will then develop an animal model suitable for studying the mechanisms by which EV-contained ?-syn may cross the BBB and enter the plasma, and begin to study the potential mechanisms of ?-syn-containing EV transport across the BBB. These experiments will be valuable for future studies aimed at developing biomarkers for PD progression, as well as for seeking novel therapeutic treatments aimed at this potential clearance mechanism.

Extracellular microvesicles (EMVs) are small, membrane-bound vesicles released by most cell types, and can be found circulating in the blood and other biofluids. The proteins, miRNAs, and other molecular components they carry as cargo have become a target for the development of novel biomarkers that reflect the EMV parent cell types. In particular, a strategy of targeting cellular markers carried on their membrane surfaces has been used to probe the state of the brain by examining EMVs carrying L1CAM, a relatively CNS-specific neuronal marker. Measurement of cargo proteins in such EMVs has shown particular promise in identifying blood-based biomarkers for neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Their utility in probing the state of the brain in other pathological conditions, such as after a traumatic injury, remains to be determined. Additional targets are now being developed to identify EMVs from non-neuronal brain cell types, including GLAST and GLT-1 for astrocytes and CNPase for oligodendrocytes. Despite this progress, identification of cell-specific markers remains crude, focused only on markers that tend to be present across a broad cell type. The cells themselves, in contrast, encompass multiple sub-types with different functional niches, likely differentially affected in pathological states. Thus, it should be possible to identify sub- types of EMVs and examine their composition for more specific reflections of brain processes. However, the appropriate surface markers, or combinations of markers, to target have not yet been identified. Despite the promise of EMV-based biomarkers for CNS conditions, serious challenges to their widespread adoption for clinical usage remain. The current strategies for EMV isolation are largely centered on ultracentrifugation, yield vesicle samples with contamination by large protein aggregates, and usually require large sample volumes. The newly developed immunocapture method that has allowed specific measurement of L1CAM EMV cargos is far more specific, as it targets surface markers, but is expensive, time consuming, and tends to have poor yield. Therefore, novel technologies are needed to identify EMVs of interest, isolate them, and quantify cargos. Here, we will address these challenges by developing novel strategies and technologies to better quantify and characterize brain-derived plasma EMVs in the R21 stage, and then validating them in a large cohort of human subjects in the R33 stage. First, we will optimize two new EMV capture and sorting strategies, precipitation using Smart Beads, and sorting using a microfluidics device, to isolate specific categories of EMVs based on surface markers. Second, we will identify new, more specific targets, to isolate sub-populations of EMVs that might better represent disease-relevant cells of interest. Next, in the R33 stage, we will scale up these new techniques to examine large cohorts, and measure cargo proteins of interest within these specific EMV populations as biomarkers of brain dysfunction caused by AD, PD, and traumatic brain injury.

Summary Progressive and fatal neurodegenerative disorders, including Alzheimer disease (AD) and Parkinson disease (PD), represent a huge unmet need for treatment. The low efficacy of current treatment methods is partially due to the presence of various obstacles in the delivery routes, with the blood? brain barrier (BBB) being the main barrier of drug delivery to the brain. In order to develop effective therapies for these disorders, efficient and safe drug delivery systems to cross the BBB are needed. Promising results have been obtained by using synthetic nanoparticles or liposomes as drug carriers, but these systems still face challenges such as immune activation mediated rapid clearance of the vehicle and concomitant decrease in efficacy when re-administered. To overcome the limitations, researchers in recent studies have developed targeted systems that use self-derived exosomes, the membrane vesicles secreted by most cell types and act as carriers of biomolecules between cells, for nucleic acid or protein delivery to the brain, and demonstrated their beneficial properties and efficiency in animal models of neurodegenerative diseases, though the targeting doesn't seem to be cell type or system specific. In this study, based on our recent findings on central nervous system (CNS) cell-type specific exosomes, we propose to further develop exosome-based but more efficient and functional specific delivering systems. Specifically, our previous studies have demonstrated that red blood cell (RBC)-derived exosomes/microvesicles could cross the BBB, particularly under inflammatory conditions, and that exosomes carrying CNS cell specific surface markers (e.g., L1CAM for neurons and CNPase for oligodendrocytes) could be transported from the brain into peripheral blood and re-enter the brain readily even without peripheral inflammation. Therefore, we hypothesize that exosomes derived from hematopoietic stem cells (HSCs)/RBC progenitors, when modified to express brain cell-specific surface markers, can cross the BBB from peripheral blood more efficiently and deliver nucleic acid or protein cargos into the target brain cells more specifically. In this study, we will first develop such exosomal delivery systems by engineering cultured human or mouse HSCs to express brain cell-specific surface markers, with or without additional previously identified strategies to enhance transportation efficiency and stability, and verify the cellular uptake and BBB crossing of the modified exosomes in in vitro and in vivo models. We also plan to engineer exosomes to carry additional surface markers to target more specific neuronal subpopulations, and finally test the delivery of potential treatments in neurodegenerative disease animal models. This proposed study will likely develop a more efficient and targeted drug delivery system for neurodegenerative diseases, and provide the foundation for future studies of effective and targeted treatments.