Alpha-Synuclein, PUFA and Membrane Vesicles in Health and Parkinson's Disease

描述（由申请人提供）：帕金森病是第二常见的神经退行性疾病，其特征是进行性功能障碍和黑质多巴胺能神经元的丧失。这一主要特征伴随着多巴胺能神经元及其过程（路易体和神经突）中蛋白质包涵体的积累，其主要成分是a-突触核蛋白（aS）。aS有7个重复序列，类似于载脂蛋白的脂质结合a-螺旋结构域，其与磷脂膜的结合显著改变了其二级结构。我们最近发现：a) aS与脂肪酸结合蛋白（FABP）家族具有同源性；b)发现纯aS可与游离FAs可逆结合；c)在多巴胺能细胞、正常小鼠和人脑以及PD和DLB大脑中检测到高可溶性脂质相关aS低聚物；d)表明活的中脑神经元暴露于多不饱和脂肪酸中可增强，而暴露于饱和脂肪酸中可延缓可溶性aS低聚物的形成；e) aS过表达神经元内源性PUFA水平和膜流动性增加，而aS敲除小鼠则相反。基于这些发现，我们假设aS通常在细胞质的水质区室和膜磷脂区室中与FAs相互作用，并有助于调节脂质组成（特别是PUFA含量）和膜特性，aS- fa相互作用有助于调节aS的寡聚化，从而启动aS首先组装成可溶的，然后组装成不可溶的低聚物。为了追求这个关于aS功能和功能障碍的分子假设，我们现在提出了一系列相互关联的目标。1)试图证明改变内源性PUFA水平（例如，在A6去饱和酶抑制剂处理的细胞中降低或在齐韦格综合征模型小鼠中升高）诱导脑细胞中内源性aS低聚物相应减少或增加。2）. 探讨aS-FA相互作用对活细胞膜泡形成、超微结构及生物物理性质的影响。3)为了确定PUFA-ctS相互作用是否以及如何影响迄今为止记录的aS的一个离散生化功能：抑制磷脂酶d。我们对aS在脂质代谢和膜泡形成/稳定性中的关键作用的关注源于两位主要研究者的一组新的观察结果。此外，最近在表达aS的酵母或果蝇中进行的无偏倚基因筛选也有力地支持了这一观点，表明aS在脂质调节和膜运输中具有功能。从这项资助中产生的新发现应该同时阐明aS的生理学及其病理寡聚化的最早步骤，以及随之而来的治疗见解。

DESCRIPTION (provided by applicant): Parkinson's disease, the second most common neurodegenerative disorder, is marked by progressive dysfunction and loss of nigral dopaminergic neurons. This cardinal feature is accompanied by the accumulation of protein inclusions in dopaminergic neurons and their processes (Lewy bodies and neurites), the major constituent of which is a-synuclein (aS). aS has 7 repeats resembling the lipid-binding a-helical domains of apolipoproteins, and its binding to phospholipid membranes markedly alters its secondary structure. We have recently: a) discovered homologies of aS with the fatty acid (FA) binding protein (FABP) family; b) found that pure aS binds free FAs reversibly; c) detected a pool of highly soluble, lipid-associated aS oligomers in dopaminergic cells, normal mouse and human brains and, at elevated levels, in PD and DLB brains; d) shown that exposure of living mesencephalic neurons to polyunsaturated FAs enhances ~ and to saturated FAs retards ~ the formation of soluble aS oligomers; and e) documented increased endogenous PUFA levels and membrane fluidity in aS-overexpressing neurons, and the opposite in aS knock-out mice. Based on these findings, we hypothesize that aS normally interacts with FAs in both the aqueous and membrane-phospholipid compartments of the cytoplasm and helps regulate aspects of lipid composition (particularly PUFA content) and thus membrane properties, and that aS-FA interactions help regulate the oligomerization of aS and can thus initiate aS assembly into first soluble and then insoluble oligomers. To pursue this molecular hypothesis about aS function and dysfunction, we now propose a series of interrelated goals. 1) To attempt to prove that altering endogenous PUFA levels (e.g., lowered in cells treated with a A6 desaturase inhibitor or elevated in mice modeling Zellweger's syndrome) induces corresponding decreases or increases in endogenous aS oligomers in brain cells. 2). To examine the effects of aS-FA interactions on the formation, ultrastructure and biophysical properties of membrane vesicles in living cells. 3) To ascertain whether and how PUFA-ctS interactions affect the one discrete biochemical function of aS documented to date: inhibiting Phospholipase D. Our focus on a key role for aS in lipid metabolism and membrane vesicle formation/stability derives from a novel set of observations made by the two principal investigators. Moreover, it is strongly supported by recent unbiased genetic screens in aS-expressing yeast or Drosophila that implicated a function of aS in lipid regulation and membrane trafficking. New findings emanating from this grant should simultaneously shed light on the physiology of aS and the earliest steps in its pathological oligomerization, with attendant therapeutic insights.