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

描述(申请人提供)：帕金森氏病，第二常见的神经退行性疾病，以进行性功能障碍和黑质多巴胺能神经元丢失为特征。伴随这一基本特征的是多巴胺能神经元及其突起(路易小体和突起)中蛋白质包涵体的积聚，其主要成分是α-突触核蛋白(AS)。AS有7个类似于载脂蛋白脂结合α-螺旋结构域的重复序列，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)发现AS与脂肪酸(FA)结合蛋白(FABP)家族的同源性；b)发现纯AS可逆地与游离FA结合；c)在多巴胺能细胞、正常小鼠和人脑中检测到一组高度可溶性的、与脂相关的AS寡聚体，并在PD和DLB脑中检测到高水平的AS寡聚体；d)显示活的中脑神经元暴露于多不饱和FA可增强~并抑制饱和FA的形成；以及e)研究发现，在AS过表达的神经细胞中，内源性PUFA水平和膜流动性增加，而在AS敲除小鼠中，情况正好相反。基于这些发现，我们假设AS通常与细胞质的水和膜-磷脂之间的FA相互作用，有助于调节脂质成分(特别是PUFA含量)的某些方面，从而有助于调节膜的性质，AS-FA的相互作用有助于调节AS的齐聚，从而可以启动AS首先组装成可溶的寡聚体，然后再组装成不溶的寡聚体。为了探索AS功能和功能障碍的分子假说，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多不饱和脂肪酸水平(例如，在用A6去饱和酶抑制剂处理的细胞中降低或在建立齐薇格综合征模型的小鼠中升高)会导致脑细胞中内源性AS寡聚体的相应减少或增加。2)。研究AS-FA相互作用对活细胞膜泡的形成、超微结构和生物物理性质的影响。3)为了确定PUFA-CTS的相互作用是否以及如何影响迄今已有文献记载的一项离散的生化功能：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来自于两位主要研究人员的一系列新观察。此外，最近在表达AS的酵母或果蝇中进行的无偏见的遗传筛选有力地支持了这一点，这意味着AS在脂质调节和膜运输中的功能。来自这项资助的新发现应该同时阐明AS的生理学和其病理性寡聚的最早步骤，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.