SYNUCLEOPATHIES IN NEURODEGENERATION

描述(申请人提供)：突触蛋白家族在突触前神经元中大量存在，并与许多神经退行性疾病状态有关，统称为突触核病症。这个家族的一个成员(-突触核蛋白)是路易小体[Lb]的主要成分，在阿尔茨海默病、LBS痴呆、散发性帕金森病、多系统萎缩和伴有脑铁积聚的神经退行性变中存在。α-突触核蛋白的突变体，即A30P和A53Tα-突触核蛋白，存在于某些遗传形式的PD的LB中。然而，α-突触核蛋白的主要正常功能及其致病作用模式尚不清楚。了解α-突触核蛋白的分子和功能相关性将有助于理解这种蛋白的正常和异常活性，这些活性导致纤维聚集和LBS的形成，这一过程因老年人疾病中发现的氧化应激和炎症增加而加速。 我们提供的初步证据表明，(-突触核蛋白)的一个可能的新作用是调节突触前多巴胺转运体[DAT]的活性。DAT活性的这种调节是双峰的，导致转运蛋白活性的增加和正常转运蛋白活性的减弱，正如[31H]多巴胺摄取试验所指示的那样。DAT功能的调节是通过转运蛋白快速运输到质膜和从质膜中转移来进行的。A30P和A53T在DAT活性的这种双重调节中的作用方式不同于(-突触核蛋白)，它们之间也有非常显著的不同。 在这项建议中，我们将使用与α-突触核蛋白及其亚型共转染的细胞和DAT基因，以及在原代培养的神经元中，详细研究这种对转运蛋白活性的双峰调节的机制。将通过包括免疫学、转运蛋白分析、免疫细胞化学和FRET在内的一系列研究，在正常生长条件下彻底分析α-突触核蛋白及其A30P和A53T突变体的能力，以及参与直接蛋白质：蛋白质复合体形成的结构成分的同一性。我们将研究氧化应激在导致α-突触核蛋白三种变种功能障碍中的作用。将进行研究，以确定α-突触核蛋白/DAT相互作用的变化是否会减少氧化应激和细胞死亡。

DESCRIPTION (provided by applicant): The family of synucleins is abundantly present in presynaptic neurons and is associated with numerous neurodegenerative disease states, collectively termed synucleinopathies. One member of this family, (-synuclein, is present as the major component of Lewy bodies [LB] in LB variant of Alzheimer's disease, dementia with LBs, sporadic Parkinson's disease, multiple system atrophy and neurodegeneration with brain iron accumulation. Mutants of alpha-synuclein, the A30P and A53T alpha-synucleins, are present in LBs of certain genetic forms of PD. However, neither the primary normal function of alpha-synuclein nor its mode of disease inducing action is known. Understanding the molecular and functional correlates of alpha-synuclein would help in the understanding of both the normative, and aberrant activity of this protein that give rise to the formation of fibrillary aggregates and LBs, in a process that is accelerated by increased oxidative stress and inflammation found in diseases of the aged. We provide preliminary evidence to suggest that one possible novel role for (-synuclein is to regulate the activity of the presynaptic dopamine transporter [DAT]. This regulation of DAT activity is bimodal, causing both the increased activity of the transporter and attenuation of normal transporter activity, as is indexed by [31H] dopamine uptake assays. The modulation of DAT function proceeds through rapid trafficking of the transporter to and from the plasma membrane. The mode of action of the A30P and A53T in such dual regulation of DAT activity differs from that of (-synuclein, and they also differ from one another in a highly prominent manner. In this proposal we will study in detail the mechanisms which underline such bimodal regulation of transporter activity, using cells co-transfected with alpha-synuclein and its subtypes, and the DAT cDNA, as well as in primary cultured neurons. The ability of the alpha-synuclein and its A30P and A53T mutants, and the identity of the structural components, which participate in direct protein:protein complex formation will be analyzed thoroughly in normal growth conditional states, through a battery of studies to include, immunology, transporter assays, immunocytochemistry and FRET. We will examine the role of oxidative stress in causing dysfunction of the three variants of alpha-synuclein. Studies will be conducted to determine if changes in alpha-synuclein/DAT interactions will reduce oxidative stress and cell death.