Determination of the clinical relevance of Parkinson disease-associated intronic enhancer of the alpha-synuclein gene, in a novel mouse deletion model

我们将评估SNCA内含子增强子的生物学必要性，并确定其与 帕金森病(PD)。SNCA在家族性帕金森病中经常发生突变；它编码α-突触核蛋白(α-SYN)， 路易体的主要成分(Lb)。Lb的形成/堆积是PD和IS的病理标志 由SNCA结构突变、基因扩增或遗传/环境促进的蛋白质错误折叠驱动 提高α-SYN水平的侮辱。预测影响SNCA转录调控的非编码变体 也是散发性帕金森病的危险因素。我们最近在SNCA内含子4中发现了与PD相关的变异。 变异体位于多巴胺(DA)能神经元的开放染色质区域(OCR)，我们已经证明与该区域相互作用 SNCA启动子是儿茶酚胺能神经元的顺式调节增强子。我们预测这一点 序列及其变异体影响SNCA转录调控并调节帕金森病风险。 假设缺失小鼠SNCA增强子将降低PD相关细胞的SNCA转录 对其活性/分布以及对运动/非运动PD表型的潜在影响(目标1)；删除将 减少DA神经元对PD相关侮辱的易感性，并改善疾病的发病和严重程度(AIM2)。 有几种高效的策略可以在小鼠中诱导帕金森病的病理。四环素(TET)依赖 PD突变型和野生型SNCA在DA神经元中的表达导致显著和进行性 腹侧中脑DA神经元群丢失，与帕金森病病理一致。同样，纹状体内注射 α-SYN预形成纤维(PFF)也导致小鼠模型表现出进行性DA神经元丢失。 与SNCA水平影响PD风险和进展的已知作用一致，α-SYN缺陷小鼠 防止PFF诱导的神经变性和PD的tet依赖表达(通过病毒载体) 错义突变体(HA53T)或WT SNCA。α-SYN缺失的小鼠也受到保护，不受其他 促进PD(和提升SNCA)的侮辱，如MPTP、6-OHDA和LPS。我们将测试细胞依赖 同样，在一种新的小鼠模型中，SNCA水平的滴定可以改善风险和进展。 我们已经培育出缺乏这种增强子(SNCA Enhdel)的小鼠品系，并提供了初步证据 该序列影响SNCA转录。使用已建立的技术，我们建议分析SNCA的效果 关于前驱、行为和运动表型(目标1a)以及帕金森病相关神经元的Enhdel 中脑和嗅球的存活/分布和小胶质细胞激活(目标1b) 免疫组织化学和单分子荧光原位杂交。同样，我们将确定 SNCA Enhdel是否减少前驱/运动/非运动疾病表型的发病和进展(目的 2a)，并影响DA神经元脆弱性(目标2b)，当暴露于与PD相关的侮辱时(纹状体内注射 SNCA pFF和腺病毒介导的SNCA hA53T)。我们将确定调制SNCA的程度 转录影响小鼠帕金森病相关病理的风险，及其作为一种新的治疗途径的潜力。

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 adenoviral-delivery of SNCA hA53T). We will establish the extent to which modulating Snca transcription impacts risk of PD relevant pathology in mice, and its potential as a novel therapeutic avenue.