Pamela J. McLean, Ph.D. - US grants

? DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) has been considered a causative factor for Parkinson's disease (PD) based in case-control studies and brain injury has been suggested to facilitate ?syn aggregation and PD development. This notion is further supported in an experimental study showing that ?syn expression and aggregation increased transiently following TBI in aged mouse brain. In addition, the exposure of mice to the pesticide paraquat, combined with TBI, resulted in triple the risk of the mice developing Parkinsonian symptoms, and ?syn has been found to be increased in the CSF of patients with severe head trauma, indicative of secondary neuropathologic events occurring after injury. Despite these intriguing data, the exact mechanism(s) underlying brain injury-induced aggregation of ?syn remain elusive. Our preliminary data suggest that extracellular ATP (eATP) is released from injured cells and can facilitate ?syn aggregation. Here, we propose to investigate the release of eATP from damaged neural tissue in vivo as a trigger for alpha-synuclein aggregation following TBI. We will explore this novel hypothesis in three coordinated aims. In aim 1 we will use an innovative eATP reporter probe coupled with real-time in vivo imaging to determine if eATP levels increase post-TBI. In aim 2 we will investigate if cellular degradation pathways are modulated by TBI using an original brain tissue recovery method. In aim 3, we will examine the contribution of P2X receptors to alpha-synuclein aggregation following TBI and eATP release in vivo. The high-risk nature of the hypothesis makes the R21 mechanism ideal for further exploration. If successful, the experiments described in this proposal will advance our knowledge of mechanisms of protein aggregation following TBI and TBI-induced PD.

ABSTRACT The overall aim of this application is to investigate mechanisms of alpha-synuclein (?syn)-induced mitochondrial dysfunction in Lewy body diseases (LBD). Despite its predominant localization in the cytosol, ?syn localizes to mitochondria in post-mortem LBD brains. Within the mitochondria, ?syn accumulation can impair complex I and IV function, decrease membrane potential, increase levels of reactive oxygen species, and increase apoptosis associated with cytochrome c release from the mitochondria. Together these data suggest an increase in mitochondrial ?syn expression and/or abnormal accumulation of toxic aggregates interferes with mitochondrial function. Maintaining mitochondrial health is essential to prevent neuronal cell death in the brain. Sirtuin 3 (SIRT3) is a NAD+-dependent protein deacetylase exclusively localized to the mitochondria where it regulates mitochondrial processes including protein deacetylation, organelle biogenesis, and oxidative stress. SIRT3 is expressed at high levels in the brain and other nervous system tissues, and can act as a pro- survival factor, playing an essential role in protecting neurons under conditions of excitotoxicity and rescuing neuronal loss in neurodegenerative models. Experimental evidence indicates that SIRT3- induced neuroprotection against oxidative stress is partially mediated by enhancement of mitochondrial biogenesis and integrity. As we consider sirtuin-based drug therapies for diseases of ageing, it is important to determine if modulating SIRT3 can protect against neurodegeneration where mitochondrial dysfunction has been demonstrated to play a role. This proposal will investigate how mitochondrial SIRT3 contributes to ?syn-induced mitochondrial dysfunction in 3 coordinated aims. In aim 1 we will perform comprehensive mitochondrial function analyses to reveal how ?syn accumulation leads to mitochondrial damage and the role of SIRT3 therein using patient-derived cells and postmortem brain. In aim 2 we will interrogate the SIRT3 regulated acetylome to identify novel targets of ?syn-associated mitochondrial dysfunction, and in aim 3 we will validate SIRT3 as a novel target for therapeutic intervention in PD in a comprehensive in vivo approach using genetic overexpression and pharmacological activation. The proposed rigorous analysis of various mitochondrial aspects will dissect causes from consequences and reveal the cross-talk between ?syn, SIRT3, and mitochondrial signaling pathways as well as oxidative and protein stress responses.

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

ABSTRACT Elucidation of underlying disease mechanisms in Lewy body dementia requires the systematic characterization of disease-specific changes in LBD brain tissue. The combination of dementia and motor symptoms at the clinical level is reflected in the neuropathology by the accumulation of alpha-synuclein (?-syn) in Lewy bodies and beta-amyloid (A?) in amyloid plaques. Both A? and ?-syn are capable of aggregating into oligomers, fibrils, and beta sheets, but the aggregates that form are extremely heterogeneous in terms of both structure and function. The identification of which specific subspecies of the proteins are neurotoxic or support aggregation is poorly understood and as a result development of diagnostics and treatments that depend on knowledge of protein structure has been slow. Recent studies have suggested a ?seeded? or ?prion-like? propagation for both ?-syn and A?, indicating that abnormal conformations may ?spread? from diseased to healthy cells. While synthetic seeds of ?-syn and A? confer toxicity in cells through various mechanisms, it is well known that the exact method of preparing these species influences their bioactivity to a high degree. Herein we take the next important step to analyze the actual subspecies from post-mortem brains, to define and compare aggregates, and identify mechanisms of toxicity and spreading underlying the selective regional vulnerabilities. We will extract soluble and insoluble ?-syn and A? species from neuropathologically confirmed LBD brains with or without genetic LBD risk factors for structural and functional characterization. We will assess load and distribution of ?-syn and A? subspecies and will detail their characteristics including size, structure and self- templating capabilities (Aim 1). The critical involvement of autophagy-lysosomal pathways in LBD is emphasized by the underlying genetic risks for LBD. Besides mutations in glucocerebrosidase (GBA) that interfere with lysosomal functions, it is well established that individuals with the APOE ?4 allele have a 6-fold greater risk for DLB. We will assess alterations in lysosome function in LBD brain and determine their response and contribution to processing, aggregation, and toxicity of ?-syn and A? subspecies (Aim 2). We will use patients? iPSC-derived neurons with different genetic risk factors to functionally validate the contribution to aggregation, toxicity and spreading of ?-syn and A? (Aim 3). The expertise of this group of investigators synergizes to identify differences in structure and function of subspecies, to investigate mechanisms of aggregation and toxicity, and to unravel the contributions of the genetic variations and alterations in selective autophagy pathways to regional vulnerabilities. This puts us in a unique position to complement the studies of Projects 1, 2, and 3 of this CWOW at multiple levels and towards the full characterization of ?-syn and A? subspecies.