PROJECT SUMMARY/ABSTRACT Aggregation of α-Synuclein (αSyn) in brain Lewy bodies (LB) is a key pathological feature in Parkinson’s dis- ease (PD) and Alzheimer’s disease related dementias (ADRD) including Lewy body dementia (LBD). The neu- rodegeneration that leads to PD and LBD precedes diagnosis by up to a decade or more, with constipation being one of the earliest symptoms. LBs are found also in the enteric nervous system, which, unlike the brain, is readily accessible through routine screening colonoscopy. Yet, the initial events leading to αSyn aggregation remain unclear. While no single biomarker is sufficiently specific so far for routine use in the diagnostic or prognostic disease evaluation, αSyn has emerged as a leading therapeutic target because it is a central player in PD and ADRDs. αSyn binding to synaptic vesicle membranes defines its physiological and pathological roles. We found that a reduction in synaptic vesicle binding in the brain is predictive of αSyn aggregation. Yet, a detailed biochemical analysis of αSyn membrane binding in the gut has never been done. The objective in this application is to determine changes in synaptic vesicle-binding of αSyn in neurons of the ENS and the brain. The central hypothesis is that changes in synaptic vesicle-binding of αSyn can serve as an early bi- omarker of PD and ADRD such as LBD. This hypothesis will be tested in two specific aims: 1) Identify the GI area with the most prominent changes in synaptic vesicle-binding of αSyn; 2) Establish a timeline of changes in synaptic vesicle-binding of αSyn. Under the first aim, four gut areas of 10-month-old αSynBAC mice, a mild synucleinopathy model, and fresh biopsies taken from similar areas in humans during routine screening colon- oscopy will be analyzed by subcellular fractionation and quantitative immunoblotting, and immunohistochemis- try. Under the second aim, motor and GI function will be assessed in αSynBAC mice at weekly intervals from 1 month of age (before onset of symptoms) and 3 months of age (when symptoms are present). Changes will be correlated with total and membrane-bound levels of αSyn in the brain and gut of these mice, and with changes in neuron density, synapse density and gliosis. Same readouts will be applied to gut biopsies from subjects with severe and mild PD and subjects at risk for developing PD. This study is expected to show a reduction in synaptic vesicle-binding of αSyn as a function of disease severity in the gut of human subjects and our αSyn- BAC mice, and that this reduction can be utilized as an early prodromal disease biomarker. This research is in- novative because it represents the first biochemical assessment of αSyn in the neurons of the gut and because of our use of fresh biopsies taken during routine cancer screening colonoscopies, enabling biochemical frac- tionation. This work is significant because it will lead to a better understanding of pathogenic changes in αSyn, may provide a better understanding of neuronal vulnerability in the gut and brain, and may lead to the devel- opment of a new biomarker for PD and ADRDs/LBD, targeting prodromal disease.

PROJECT NARRATIVE The proposed research is relevant to public health because alpha-synuclein pathology is linked to neurological diseases including Parkinson’s disease, Alzheimer Disease related dementias such as Dementia with Lewy bodies, Multiple System Atrophy, and other synucleinopathies, and understanding early pathogenic events that trigger alpha-synuclein pathology will allow us to better understand the underlying disease mechanisms and develop novel intervention therapies aimed at prodromal disease stages. Thus, this research is relevant to the NIH’s mission that pertains to supporting the ongoing basic research, foster research that has immediate medical relevance, and encourage translational research that links the discoveries from basic research into therapeutic interventions for treating neurological disorders such as Parkinson’s disease and Dementia with Lewy bodies.