Xiaobo Mao, Ph.D - US grants

?Influence of Aging on Pathogenic ?-Synuclein Strains and Transmission Mechanism? Project Summary: Besides ?-synuclein transmission mechanism, the role of distinct strains of ?-synuclein is compelling to be understood. Understanding the influence of aging on the pathogenesis of synucleinopathies, including Parkinson's disease (PD), PD with dementia (PDD), dementia with Lewy body (DLB) and multiple system atrophy (MSA), is crucial for developing more effective symptomatic therapies. In this K01 proposal, three specific aims are proposed for understanding the influence of aging: (1) To generate and characterize pathogenic strain-specific ?-syn from brain tissue/CSF of patients with PD/PDD/DLB/MSA to controls by the factor of aging longitudinally and cross-sectionally. (2) To understand the influence of age on distinct pathogenic ?-syn strains from PD/PDD/DLB/MSA in vitro and in vivo. (3) To uncover the transmission mechanism of age-related distinct pathogenic ?-synuclein. To achieve the 3 specific aims, 6 methods/steps are required: (i) Dr. Juan Troncoso and Dr. Liana Rosenthal will provide the training to candidate on handling human brain tissue/CSF. Drs. Ted and Valina Dawson, and Dr. Mark Mattson will train the candidate using the misfolded ?-synuclein in brain tissue/cerebrospinal fluid (CSF) as templates, and with protein misfolding cyclic amplification (PMCA) technique, to generate distinct ?-synuclein strains. (ii) Scanning tunneling microscopy (STM) will be applied for imaging molecular structures of distinct ?-synuclein strains and distinguish the differences, and the alternative training will be available from Dr. Chen Wang if the potential problems appear. (iii) Atomic force microscopy (AFM) will be applied for observing assembly morphologies of distinct ?-synuclein strains. Dr. Mingdong Dong will provide the training on studying nano-mechanical properties and dynamic growth features of distinct ?-synuclein strains. (iv) Electrophysiology study on firing experiment will be hands- on training from Dr. Antonello Bonci. This training study is to understand the intrinsic and synaptic properties affected by distinct ?-synuclein strains. (v) In vivo microscopy will be hands-on training from Dr. Da-Ting Lin. This training includes performing all necessary surgical procedure to insert gradient index (GRIN) lens into substantia nigra and two-photon microscopy. This study will allow candidate to study the dopamine neuronal circuit dysfunction affected by stereotaxically injected with distinct ?-synuclein strains in striatum region for evaluation the transmission. (vi) Lymphocyte-activation gene 3 (LAG3) has been identified as ?-synuclein preformed fibrils (PFF) receptor, so it is worth to explore whether LAG3 can mediate the transmission of distinct ?-synuclein strains. Above all, this project proposed is to develop an independent research laboratory equipped to understand the distinct strains of amyloid proteins on misfolded structures, the transmission and the toxicity in neurodegenerative disorders, and to develop a programmatic line of research by successfully competing for funding.

?-Synucleinopathies are a subset of neurodegenerative diseases, including Parkinson's disease (PD), dementia with Lewy Bodies (DLB) and multiple system atrophy (MSA) (1), which are characterized by abnormal accumulation of misfolded ?-synuclein (?-syn) protein in neurons or glial cells. Emerging evidence suggests that the cell-to-cell transmission of pathological ?-syn substantially cause neurodegeneration. However, the molecular mechanism of ?-syn transmission is poorly understood. We have identified three transmembrane proteins that strongly bind with ?-syn PFF: (i) lymphocyte activation gene-3 (LAG3), (ii) amyloid ? precursor-like protein 1 (APLP1), and (iii) neurexin 1-?. LAG3 is an essential receptor, mediating internalization of ?-syn PFF; however, substantial ?-syn PFF binds to LAG3-/- (knockout) neurons, suggesting that a unidentified candidate(s) (e.g. APLP1) binds with pathologic ?-syn, and facilitate the internalization. In this proposal, we hypothesize: (i) APLP1 is an essential receptor, that mediates ?-syn transmission; (ii) APLP1-LAG3 complex synergistically mediates ?-syn transmission; (iii) depletion of APLP1, LAG3, or APLP1- LAG3 complex, can reduce ?-syn-induced neurodegeneration of ?-syn transgenic mice. Accordingly, experiments are proposed to characterize the roles of APLP1, LAG3 and the AL complex in mediating the pathogenesis of ?-synucleinopathies, in cell-to-cell transmission models and the ?-syn transgenic mouse model. Experiments in two mice models with ?-synucleinopathies, will be complemented by studies in cells and cell-free experiments, thus allowing the deciphering of each spreading step in the interaction of APLP1, LAG3, or the AL complex with pathologic ?-syn. The successful completion of these studies will greatly enhance our understanding of the molecular mechanisms of ?-syn cell-to-cell transmission via receptors, by: (i) identifying APLP1 as a novel receptor that mediates ?-syn spreading, (ii) understanding the synergistic effect of the AL complex on mediating binding and internalization of ?-syn PFF, (iii) providing essential preliminary evaluation of anti-LAG3 antibody as a potential therapeutic agent against PD and related ?-synucleinopathies, and (iv) understanding the roles of APLP1, LAG3, and the AL complex in mediating neurodegeneration of ?-syn transgenic mice.

PROJECT SUMMARY Aging is the greatest risk factor to ?-synucleinopathy, a group of neurodegenerative diseases with severe cognitive impairmentand progressive motor dysfunction and dementia, such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and Parkinson's disease with dementia (PDD) and half of Alzheimer's disease patients (AD). Dementia is a common symptom in ?-synucleinopathies: DLB is the 2nd most common dementia after Alzheimer's disease (AD) accounting for 30% of dementia cases; Around 30% of AD cases also suffer from ?-synucleinopathy resulting in a more rapid and severe cognition decline than AD alone. PD is the 2nd most common neurodegenerative disease, and greater than 50% of PD cases develop PDD. In addition to cognitive and memory dysfunctions, patients with dementia also suffer from anxiety, depression and mood swings. Although ?-syn pathology is highly associated with dementia, the underlying aging-related mechanism driving the pathogenesis and contributing to their progression is not known and there is no available disease modifying therapy yet. Based on substantial postmortem analysis, Braak et al. demonstrated that ?-syn pathology spreads in a stereotyped fashion from the vagus to the brain, which may initiate in the gastrointestinal tract. Particularly, nearly all the DLB and PDD cases present with ?-syn pathology in the gut. Both clinical and experimental observations support that pathogenic ?-syn spreading is a master trigger that drives ?-synucleinopathy. In our gut-brain ?-synucleinopathy (GBAS) mouse model, gut-injection of pathogenic ?-synuclein (?-syn) can recapitulate ?-syn pathology gut-brain spreading and cognitive impairment. In our preliminary studies, heterochronic blood exchange (HBE) from young mice inhibited pathogenic ?-syn transmission and neuroinflammation in aged mice, suggesting an HBE-transferred phenotype that may effectively inhibit ?-synucleinopathy. We identified lymphocyte-activation gene 3 (LAG3)1, a major receptor of pathologic ?-syn transmission. To identify the mechanism underlying rejuvenation and accelerated aging event, we further identified two novel LAG3-related and aging-regulating proteins that can mediate pathogenic ?-syn transmission. Our studies support the feasibility to modulate plasma levels of FGL1 and sLAG3 in aged mice by the HBE approach. To determine the underlying mechanism if FGL1 and sLAG3 in the plasma are molecular mediators essential for the inhibitory effects of HBE on ?-synucleinopathy an d related cognitive impairment, we have established a rigorous and robust experimental system combining the HBE approach, the GBAS model, genetically engineered mice without these factors, and recombinant FGL1 and sLAG3 proteins, for comprehensive gain- and loss-of-function analysis. Our Central Hypothesis is to identify the underlying mechanism that HBE inhibits ?-synucleinopathy and related cognitive impairment through FGL1 and sLAG3. FGL1 functions as a rejuvenation factor to inhibit pathologic ?-syn spreading in the gut-brain axis and alleviate consequent neurodegeneration, neuroinflammation, and cognitive impairment. sLAG3 acts as an age-acceleration factor contributing to the pathogenesis and with antagonistic function to FGL1. Strikingly, human postmortem evidence shows that ?-syn pathology is observed first in the gastrointestinal system and then spreads to the brain in a stereotyped fashion. Recently, our collaborator Dr. Dawson developed a novel Gut-Brain ?-synucleinopathy (GBAS) model, a sporadic ?-synucleinopathy model recapitulating pathologic ?-syn spreading among multiple organs and brain regions in patients. However, it remains largely unknown how aging-associated blood-borne components modulate ?-synucleinopathy. As the foundation of this project, we identified lymphocyte-activation gene 3 (LAG3), a major receptor of pathologic ?-syn transmission. Our preliminary results showed that heterochronic blood exchange (HBE) from young mice can inhibit pathologic ?-syn transmission to cells and inflammation in aged mice. We further identified two blood-borne aging-modulated proteins that regulate LAG3-mediated pathologic ?-syn transmission. The first plasma protein fibrinogen-like protein (FGL1) as the major inhibitory ligand of LAG3, is decreased by aging and inhibits ?-syn transmission. The second plasma protein sLAG3 is the soluble form of LAG3 protein, and it is increased by aging and promotes ?-syn transmission. Our studies also support the feasibility to use HBE approach to modulate plasma levels of FGL1 and sLAG3 in aged mice by young blood. Our Central Hypothesis is that HBE with young blood inhibits ?-synucleinopathy through two aging-associated circulatory proteins (FGL1 and sLAG3) essential for LAG3-mediated pathologic ?-syn transmission. FGL1 functions as a rejuvenation factor to inhibit pathologic ?-syn spreading in the gut-brain axis and consequent neurodegeneration, neuroinflammation, and behavioral deficits associated ?-synucleinopathy. sLAG3 acts as an age-acceleration factor with antagonistic functions to FGL1. In specific aim 1, we propose to determine if HBE ameliorates ?-synucleinopathy and related cognitive impairment by increasing aging-reduced FGL1 to inhibit pathological ?-syn spreading. In specific aim 2, we propose to determine if HBE ameliorates ?-synucleinopathy and related cognitive impairment by decreasing aging-induced sLAG3 to inhibit pathological ?-syn spreading. Modulating plasma factors is a novel strategy to inhibit pathologic ?-syn spreading and treating ?-synucleinopathy and dementia. Positive results from this study will justify the development of novel ?-synucleinopathy therapies based on plasma factor modulation. Novel molecular insights from this project will lay a solid foundation for the optimization and clinical translation of ?-synucleinopathy therapies based on FGL1 and sLAG3 modulation.

Project Summary/Abstract: Neurofibrillary tangles (NFTs) as a hallmark pathology of Alzheimer's disease (AD) are widely distributed in the AD brain. The major constituent of NFTs is abnormal tau aggregates filling the intraneuronal and glial cell body. Emerging evidence indicated that pathologic tau fibrils are capable of triggering a self-propagating process in neurons and other brain cells that leads to neurodegeneration and neuroinflammation. Experimental data have shown that intracranial injection of pathologic tau fibrils extracted from AD brains results in substantial spreading of tau pathology in mouse brains and induces behavioral deficits. However, therapeutic targets to block this pathologic tau spreading have not been identified. We identified for the first time that lymphocyte-activation gene 3 (Lag3) is an essential receptor mediating the pathologic ?-synuclein transmission. Our preliminary studies further support that Lag3, as a cell surface receptor, mediates the transmission of tau fibrils and pathologic tau-induced neuronal and microglial deficits. These results suggest that Lag3 may serve as a novel target for blocking pathogenic tau spreading for therapeutic development. We have established two mouse models of pathologic tau spreading with validated neuronal and behavioral deficits as well as neuroinflammatory response. Of note, our preliminary data suggests that Lag3 protein is expressed both in neurons and microglia, and depletion of Lag3 can inhibit tau neuronal propagation and microglial activation. All these results support our central hypothesis that Lag3 is an essential receptor of pathologic tau in neurons and microglia that mediates tau internalization, transmission and tauopathy. Now, it is feasible to explore the role of Lag3 in facilitating tau pathogenesis and the therapeutic efficacy of Lag3 targeting via genetic deletion and monoclonal antibodies. Our goals are (1) to define the role of Lag3 in mediating internalization of pathologic tau and the consequent neuronal and microglial responses involved in the pathogenesis of AD and other tauopathies, and (2) to develop a clinical translatable strategy to inhibit Lag3-mediated tau pathogenesis for the treatment of tauopathies. If successful, discoveries from this study will identify a cell-surface receptor that mediates pathologic tau spreading and serve as a novel therapeutic target for therapeutic development. This project may also provide novel molecular insights into key mediators of pathologic tau spreading in neurons and other brain cells. Given the on-going clinical trials using anti-Lag3 antibodies for cancer immunotherapy, discoveries from this project will also facilitate the repurposing of these anti-Lag3 antibodies for treating AD and other tauopathies.