We will evaluate the biological necessity of an intronic enhancer of SNCA and determine its relevance to Parkinson disease (PD). SNCA is frequently mutated in familial PD; it encodes alpha-synuclein (α-syn), the primary constituent of Lewy bodies (LB). LB formation/accumulation is a pathological hallmark of PD and is driven by protein misfolding promoted by SNCA structural mutation, gene amplification, or genetic/environmental insults that elevate α-syn levels. Noncoding variants predicted to impact SNCA transcriptional regulatory control are also risk factors for sporadic PD. We recently identified PD-associated variants in SNCA intron 4. These variants lie within a dopaminergic (DA) neuron open chromatin region (OCR) that we have shown interacts with the SNCA promoter and is a cis-regulatory enhancer in catecholaminergic neurons. We predict that this sequence, and the variants therein, impact SNCA transcriptional control and modulate PD risk. Hypothesis - Deletion of the Snca enhancer in mice will reduce Snca transcription in PD-relevant cells with potential impact on their viability/distribution, and on motor/non-motor PD phenotypes (Aim 1); deletion will reduce DA neuron vulnerability to PD-relevant insults and ameliorate the onset and severity of disease (Aim2). Several highly effective strategies exist to elicit Parkinsonian pathology in mice. Tetracycline (tet)-dependent expression of PD mutant and wild-type (WT) forms of SNCA in DA neurons result in marked and progressive loss of ventral midbrain DA neuron populations, consistent with PD pathology. Likewise, intrastriatal injection of α-syn pre-formed fibrils (PFF) also result in a mouse model that exhibits progressive DA neuronal loss. Consistent with the known role of Snca levels impacting PD risk and progression, α-syn deficient mice are protected from PFF-induced neurodegeneration and from tet-dependent expression (via viral vector) of the PD missense mutant (hA53T) or WT SNCA. α-syn null mice are also protected from the neurotoxic effects of other PD promoting (and Snca-elevating) insults, like MPTP, 6-OHDA, and LPS. We will test whether cell-dependent titration of Snca levels, similarly, ameliorates risk and progression in a new mouse model. We have engineered mouse lines lacking this enhancer (Snca Enhdel) and provide preliminary evidence that this sequence impacts Snca transcription. Using established techniques, we propose to assay the effect of Snca Enhdel on prodromal, behavioral, and motor phenotypes (Aim 1a), as well as on PD-relevant neuron viability/distribution and microglial activation in the midbrain and olfactory bulb (Aim 1b) via immunohistochemistry and single molecule fluorescence in situ hybridization. Similarly, we will determine whether Snca Enhdel reduces onset and progression of prodromal/motor/non-motor disease phenotypes (Aim 2a) and impacts DA neuron vulnerability (Aim 2b) when exposed to PD-relevant insults (intrastriatal injection of SNCA PFF and adenovi...