Screening for Compounds that Lower Intracellular Alpha-Synuclein Levels

筛选降低细胞内 α-突触核蛋白水平的化合物

• 批准号: 10524695
• 负责人: Varghese John
• 金额: $ 12.99万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10524695
• 关键词: Address; Agonist; Albuterol; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease risk; Alzheimer’s disease biomarker; Amyloid beta-42; Applications Grants; Asthma; Autopsy; Biological Assay; Brain; Cell Line; Cells; Complement; Data; Dementia; Dementia with Lewy Bodies; Development; Disease; Disease Progression; Epidemiology; Fluorescence; Genes; Goals; Human; Impaired cognition; Individual; Induced pluripotent stem cell derived neurons; Libraries; Link; Neuroepithelioma; Neurons; Parents; Parkinson Disease; Pathogenesis; Pathology; Patients; Permeability; Pharmaceutical Preparations; Point Mutation; Population; Property; Proteins; Public Health; Reducing Agents; Reporting; Risk; Risk Factors; Rodent Model; Therapeutic; Therapeutic Intervention; age related; alpha synuclein; alpha synuclein gene; apolipoprotein E-4; cognitive development; dopaminergic neuron; early onset; efficacy testing; high throughput screening; in vivo; induced pluripotent stem cell; inhibitor; lead candidate; motor disorder; overexpression; parent grant; promoter; screening; targeted treatment

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.

项目概要/摘要 与家长补助金一样，本补充的目标是确定可降低水平和 α-突触核蛋白 (αSyn) 的积累是帕金森病 (PD) 发病机制的基础。这 本补充提案的前提得到母基金的初步数据和大量数据的支持 报告包括 SNCA（突触核蛋白 Alpha）基因的点突变或重复/三倍体，导致 αSyn 增加导致常染色体显性早发性 PD。研究发现 SNCA 基因的启动子 PD 中甲基化程度低，导致表达增加，并且该基因的过度表达是一个因素 促进 PD 的发生表明降低 αSyn 水平是治疗干预的一个有希望的目标。 此外，使用β2-肾上腺素受体（β2AR）激动剂等药物降低αSyn水平已被证实 据报道在细胞系和啮齿动物模型中具有神经保护作用。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降低 PD。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命中对 αSyn 的影响 使用双分子荧光互补进行寡聚化和细胞内聚集体的形成 （BiFC）测定。此类抑制剂可以通过增强 αSyn 的下游效应来降低细胞内 αSyn 水平 降解。路易相关病理 (LRP) 主要由 αSyn 组成，不限于 PD，并且具有 在尸检的阿尔茨海默病 (AD) 大脑和路易体痴呆症大脑的子集中发现了 （DLB）患者。载脂蛋白 E4 是 AD 的危险因素，也是 PD 的危险因素，并且与早期症状相关。 PD 发作。最近的一项研究还表明，脑脊液中较高水平的 αSyn 与早期 AD 认知能力下降的发展阶段。在本补充文件中，作为目标 3 扩展的一部分，我们将 评估 iPSC 衍生神经元中的优先命中对 AD 生物标志物的影响。目前帕金森病的治疗方法 仅提供症状缓解，迫切需要开发缓解疾病的化合物 能够减缓或阻止 PD 进展。为了满足 PD 以及 AD 和 DLB 中的 LRP 的这一需求， 家长提案我们正在对 UCLA 200K 化合物库进行高通量筛选 (HTS)，以 识别降低细胞内 αSyn 水平的命中；这些命中将根据效力、类药物进行验证和优先排序 特性和大脑通透性以供进一步分析。在本目标 1 扩展的补充中，确定了命中 来自 AlphaLISA 的 HTS，可减少 SK-N-MC 人神经上皮瘤细胞中的细胞内 αSyn，并且 在母体资助的主要和次要测定中验证的将在 αSyn 寡聚化中使用进行评估 我们将在补充提案中设置的 BiFC 检测。这将有助于进一步阐明 主动命中的作用机制（MoA）。在目标 3 扩展中，从目标 2 中选择的化合物将是 在 PD、AD 和正常人的诱导多能干细胞 (iPSC) 衍生的多巴胺能神经元中进行评估 捐助者；我们将评估 αSyn 水平和 AD 生物标志物，包括 sAPPα、sAPPβ 和 Aβ42。