Screening for Compounds that Lower Intracellular Alpha-Synuclein Levels

项目摘要/摘要 本附录的目标，与父母赠款一样，是为了确定降低水平和 α-突触核蛋白(α-Syn)的积聚是帕金森病(PD)发病机制的基础。这个 这项补充建议的前提是由来自父母赠款的初步数据和许多 包括SNCA(突触核蛋白α)基因的点突变或复制/三倍体导致 α-SYN增加导致常染色体显性早发性帕金森病。发现SNCA基因的启动子 帕金森病患者甲基化是否导致表达增加，该基因的过度表达是一个因素 帕金森病的发病机制提示降低α-Syn水平是治疗干预的一个有前景的靶点。 此外，使用α2肾上腺素受体(β2AR)激动剂等药物降低βSyn水平 据报道，在细胞系和啮齿动物模型中都具有神经保护作用。一例挪威人的流行病学分析 人群显示，使用β2AR激动剂沙丁胺醇治疗哮喘的个体患哮喘的风险较低 警察。在本补充中，作为目标1扩展的一部分，我们将评估经过验证的HITS对α系统的影响 利用双分子荧光互补技术实现齐聚反应和胞内聚集体的形成 (BIFC)测定。这类抑制剂可通过下游效应增强α同步而降低细胞内α同步水平 退化。路易相关病理(Lrp)，主要由αsyn组成，并不局限于PD， 在尸检的阿尔茨海默病(AD)脑和路易体痴呆的脑中发现 (DLB)患者。载脂蛋白E4是AD的危险因素，也是PD的危险因素，并与早期 帕金森病发作。最近的一项研究还表明，脑脊液中较高水平的α-syn与早期 阿尔茨海默病认知功能衰退的发展阶段。在本补编中，作为目标3扩展的一部分，我们将 评估IPSC来源神经元中的优先HIT对AD生物标志物的影响。帕金森病的现代治疗方法 虽然只提供症状缓解，但迫切需要开发治疗疾病的化合物。 能够减缓或阻止帕金森病的进展。为了满足PD中的这一需求以及AD和DLB中的LRP，在 家长建议我们正在对UCLA 200K化合物文库进行高通量筛选(HTS)，以 确定细胞内αSYN水平较低的命中；这些命中将根据效力、类似药物的情况进行验证和优先排序 性质，和脑渗透性，以供进一步分析。在这份针对AIM 1扩展的增刊中，确定了 降低SK-N-MC人神经上皮瘤细胞内α突触蛋白的能力 在一次和二次试验中验证的母公司赠款将在αSYN齐聚中使用以下方法进行评估 我们将在补充提案期间建立的BIFC分析。这将使进一步澄清 主动命中的作用机制(MOA)。在目标3展开中，从目标2中选择化合物将是 帕金森病患者、阿尔茨海默病患者和正常患者多巴胺能神经元的多巴胺能神经元的研究 捐献者；我们将评估αSyn水平和AD生物标记物，包括Sappα、Sappβ和Aβ42。

PROJECT SUMMARY/ABSTRACT The goal of this Supplement, as with the parent grant, is to identify agents that reduce the levels and accumulation of alpha-synuclein (αSyn) that underlies the pathogenesis of Parkinson's disease (PD). The premise of this Supplement proposal is supported by preliminary data from the parent grant and by numerous reports including point mutations or duplication/triplication of the SNCA (Synuclein Alpha) gene that results in αSyn increases leading to autosomal-dominant early-onset PD. The finding that the promoter for the SNCA gene is hypomethylated in PD resulting in increased expression and that over-expression of the gene is a factor contributing to onset of PD suggests reducing the levels of αSyn is a promising target for therapeutic intervention. Furthermore, reduction of αSyn levels using agents such as β2-adrenoreceptor (β2AR) agonists has been reported to be neuroprotective in both cell line and rodent models. An epidemiological analysis of a Norwegian population revealed individuals using β2AR agonist, salbutamol for asthma, have a reduced risk of developing PD. In this Supplement, as part of expansion of the aim 1, we will evaluate the effect of validated hits on αSyn oligomerization and formation of intracellular aggregates using the bimolecular fluorescence complementation (BiFC) assay. Such inhibitors could reduce intracellular αSyn levels by downstream effects enhancing αSyn degradation. The Lewy-related pathology (LRP), primarily comprised of αSyn, is not restricted to PD and has been found in a subset of autopsied Alzheimer's disease (AD) brains and brains of Dementia with Lewy Bodies (DLB) patients. Apolipoprotein E4, a risk factor for AD, is also a risk factor for PD and is associated with earlier onset of PD. A recent study also suggests there is a link between higher levels of αSyn in the CSF and early stages of development of cognitive decline in AD. In this Supplement as part of expansion of the aim 3 we will evaluate prioritized hits in iPSC derived neurons for effect on AD biomarkers. Current therapeutics for PD provide only symptomatic relief, there is an urgent need for the development of disease-modifying compounds capable of slowing or halting PD progression. To address this need in PD as well as LRP in AD and DLB, in the parent proposal we are performing high throughput screening (HTS) of the UCLA 200K compound library to identify hits that lower intracellular αSyn levels; these hits will be validated and prioritized by potency, drug-like properties, and brain permeability for further analysis. In this Supplement for Aim 1 Expansion, hits identified from HTS by AlphaLISA that reduce intracellular αSyn in SK-N-MC human neuroepithelioma cells and are validated in primary and secondary assays in the parent grant would be evaluated in αSyn oligomerization using the BiFC assay that we will set up during the Supplement proposal. This will enable further elucidation of the mechanism of action (MoA) of the active hits. In Aim 3 Expansion, select compounds from Aim 2 would be evaluated in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons from PD, AD, and normal donors; we will evaluate for both αSyn levels and AD biomarkers including sAPPα, sAPPβ and Aβ42.