Cell Biology of alpha syn and Parkin and the Creation of Dynamic Cellular Models

α syn 和 Parkin 的细胞生物学以及动态细胞模型的创建

• 批准号: 7312748
• 负责人: MICHAEL GEBHARD SCHLOSSMACHER
• 金额: $ 39.43万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7312748
• 关键词: Parkinson' s disease; alpha synuclein; fatty acid metabolism; gene mutation; gene targeting; genetically modified animals; human tissue; laboratory mouse; neurogenetics; parkin gene /protein; posttranslational modifications; protein binding; protein protein interaction; protein structure function; ubiquitin; unsaturated fatty acids

Evidence from many laboratories indicates that alpha-synuclein is implicated at the levels of both genetics and biochemical pathology in Parkinson's disease, dementia with Lewy bodies, multiple systems atroohy and other alpha-synucleinopathies. In Project 2, two investigators who have collaborated productively during the current Udall grant period will pursue the central hypothesis that certain post-translational modifications -- including glycan-association, ubiquitination and oligomerization -- of alpha-synuclein (alphaS) may represent key pathogenic steps in both familial and "sporadic" forms of PD. Based on substantial progress on this hypothesis during the current grant period and extensive preliminary data, we propose two broad, interrelated Specific Aims that apply a range of methods in cell biology, biochemistry and animal modeling to the questions of how the normally soluble alphaS protein is converted into metastable oligomers and higher polymers and whether this pathological conversion arises from its normal properties or via a distinct toxic pathway. Our Specific Aims include: (1) to pursue our novel findings that physiological alterations in neuronal fatty acid metabolism, particularly in the levels of long-chain PUFAs, help regulate the oligomerization state of aS, and that this effect involves an interaction between PUFAs and alphaS that occurs normally but is exaggerated in PD, DLB and other disorders in which alphaS oligomerizes progressively; and (2) to characterize two distinct events in alphaS processing that we have identified in human tissue and model them in dopaminergic cells: a) the glycan association of alphaS that enables its binding to Parkin, and b) the exocytosis of alphaS. We are strongly committed to completing the structural and glycobiological analysis of aSp22 (which we have now purified to homogeneity by lectin and antibody affinity steps) and fully characterizing the non-covalent association of alphaS with complex glycans that we have found. As part of this second Aim, we will also study the potentially related mechanism of alphaS exocytotic release into extracellular fluids that we have detected in humans in vivo. These and related experiments detailed herein should provide information about distinct post-translational modifications that may enhance the accumulation and aggregation of human alphaS into potentially neurotoxic oligomers in PD and other synucleinopathies.

许多实验室的证据表明，α-突触核蛋白与帕金森病、路易体痴呆、多系统萎缩和其他 α-突触核蛋白病的遗传学和生化病理学水平有关。在项目 2 中，在当前 Udall 资助期间进行富有成效合作的两名研究人员将追求一个中心假设，即 α-突触核蛋白 (alphaS) 的某些翻译后修饰（包括聚糖缔合、泛素化和寡聚化）可能代表家族性和“散发性”形式 PD 的关键致病步骤。基于当前资助期内这一假设的实质性进展和广泛的初步数据，我们提出了两个广泛的、相互关联的建议 具体目标是应用细胞生物学、生物化学和动物模型中的一系列方法来解决通常可溶的 αS 蛋白如何转化为亚稳态低聚物和高级聚合物以及这种病理转化是否源于其正常特性或通过独特的毒性途径的问题。我们的具体目标包括：(1) 追求我们的新发现，即神经元脂肪酸代谢的生理改变，特别是长链 PUFA 水平的改变，有助于调节 aS 的寡聚状态，并且这种效应涉及 PUFA 和 alphaS 之间的相互作用，这种相互作用正常发生，但在 PD、DLB 和其他 alphaS 逐渐寡聚的疾病中被夸大； (2) 表征我们在人体组织中发现的 αS 处理中的两个不同事件，并在多巴胺能细胞中对它们进行建模：a) αS 的聚糖缔合使其能够与 Parkin 结合，b) αS 的胞吐作用。我们坚定地致力于完成 aSp22 的结构和糖生物学分析（我们现已通过凝集素和抗体亲和步骤将其纯化至同质），并充分表征 alphaS 与我们发现的复杂聚糖的非共价关联​​。作为第二个目标的一部分，我们还将研究 我们在人体体内检测到的αS胞吐释放到细胞外液的潜在相关机制。本文详述的这些实验和相关实验应提供有关不同翻译后修饰的信息，这些修饰可能会增强人 αS 的积累和聚集，形成 PD 和其他突触核蛋白病中潜在的神经毒性寡聚物。