Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) produce profound motor and cognitive impairment associated with aggregation of α-synuclein (αS). Female sex is commonly reported to delay the age of onset and to produce milder phenotypes, but the reasons are unknown. Accumulation of αS is increasingly implicated in familial and sporadic forms of these synucleinopathies. Our lab has discovered that female sex preserved a part of physiologically folded, aggregate-resistant tetrameric αS in PD-type mice, supporting published evidence for the existence of normal multimeric αS forms in healthy brain. During the first grant period, we have analyzed female sex and elevation of brain estradiol (by the estradiol prodrug DHED) and showed it can normalize the αS tetramer- monomer ratio and decrease early LB-type aggregates. Given this progress, we now wish to extend this novel hypothesis to answer certain key questions about protective estrogen pathways that could be used as disease modifying treatments. Our proposed new experiments will be enabled by our early-onset, progressive mouse models in which the E46K-like tetramer abrogating αS mutation (3K) caused sex dimorphism early in the PD-type neuropathology development. In aim 1, we will determine the age- and sex dependencies of αS pathological aggregation for WT, 1K and 3K in premature and mature mice. The studies will be controlled in mice with depleted estrogen. In aim 2, we will investigate the role of extranuclear and synaptic estrogen receptor on the temporal relationship between changes in synaptic function (electrophysiological studies) and alterations in αS pathologic aggregation (histopathology and biochemical studies) in genetic model mice with membrane-only estrogen receptor alpha. We will further examine the effectiveness of a novel therapy that increases palmitoylation (using a small molecule inhibitor for acyl-protein thioesterase 1) and thereby the level of synaptic estrogen receptors. In aim 3, we will determine, whether the shared αS tetramer-abrogation by PD risk factors (GBA1-L444P, GBA1-E326K) known for pathologic excess of soluble wildtype αS monomer accumulation and that produces more severity by male sex, are also responsive to DHED treatment. We will compare these with our novel familial G51D-and amplified (3D) mice with excess soluble mutant αS. The results of these studies will be far reaching as they will provide the foundation to understanding mechanisms through which estrogen prevents αS dyshomeostasis in the brain of robust PD-type mice in vivo, to improve and create neuroprotective treatments in PD, DLB and Gaucher’s PD.