Subcellular localization of alpha-synuclein and its impact on neurodegeneration

α-突触核蛋白是路易小体的主要蛋白质成分，是退行性变的主要特征 帕金森氏症患者的大脑。α-突触核蛋白已被证实通过 在其N-末端形成α螺旋结构。我们最近证明，无论是孵化 永生化中脑线粒体重组A53T突变型α-突触核蛋白的研究 多巴胺能神经元或跟随A53T在多巴胺能细胞中的表达 转基因动物，这种蛋白质定位于内部线粒体！膜(IMM)的形式 齐聚物。线粒体定位可能是由于A53T不能通过丝氨酸129(Ser-129) 作为这一事件的磷酸化，可以由氧化应激诱导，驱动野生型蛋白走向 胞质定位和寡聚化。A53T对IMM的本地化伴随着减少 线粒体膜电位(Mp)和线粒体自噬增加(有丝分裂)。减少了 其他研究表明，线粒体膜蛋白可以影响线粒体的分裂-融合率，从而驱动线粒体 朝向有丝分裂。在这项拨款申请的目标1中，我们建议：(1)评估α-核蛋白的能力 Ser-129的磷酸化决定其亚细胞定位和寡聚化(自身相互作用) 分别在组件11和6的Gibson博士和Hughes博士的协助下， (2)检测线粒体定位对线粒体分裂融合率和有丝分裂的影响 在组件11的Nicholls博士的帮助下，以及(3)检查TOR的改变的影响 在Kapahi博士的协助下对相关有丝分裂的活性(成分2和co-Pi，成分 9)。‘ α-突触核蛋白选择性地核移位到多巴胺能中脑神经元已经被认为是 在其过度表达或氧化应激增加后表现出来。核移位可能 也取决于蛋白质的Ser-129磷酸化状态和/或它的切割以及它的能力 与多巴胺能细胞质因子相关。核定位似乎通过以下途径导致神经毒性 α-突触核蛋白与细胞核内的组蛋白结合的能力，减少了它们的乙酰化。还原的组蛋白 乙酰化可影响基因转录。在应用程序的目标2中，我们建议评估α-核蛋白 翻译后修饰以及与相关的多巴胺能胞浆因子的相互作用 在11号部件和6号部件的吉布森和休斯博士的帮助下进行了核移位， 分别进行了分析。Ellerby博士作为组件7的一部分，我们还将检查HDAC抑制剂的能力 或特定的HDAC siRNA来保护免受这些影响，Vijg和Melov博士是 第五部分：α突触核蛋白的核定位对基因转录的影响。

Alpha-synuclein is a major protein component of Lewy bodies, a cardinal feature of the degenerating Parkinsonian brain. Alpha-synuclein has been demonstrated to intercalate into lipid membranes via formation of an alpha helical structure in its N-terminal end. We recently demonstrated that either incubation of recombinant A53T mutant alpha-synuclein protein with mitochondria isolated from immortalized midbrainderived dopaminergic neurons or following A53T expression within dopaminergic cells either in culture or in transgenic animals, the protein localizes to the inner mitochondria! membrane (IMM) in the form of oligomers. Mitochondrial localization may be due to A53T's inability to undergo serine 129 (ser-129) phosphorylation as this event, which can be induced by oxidative stress, drives the wildtype protein towards cytoplasmic localization and oligomerization. Localization of A53T to the IMM is accompanied by decreased mitochondrial membrane potential (MMP) and increased mitochondrial autophagy (mitophagy). Decreases in MMP has been shown by others to influence the mitochondrial fission-fusion rate, driving mitochondria towards mitophagy. In aim #1 of this grant application, we propose to: (1) Assess the ability of alphasynuclein ser-129 phosphorylation to determine its subcellular localization and oligomerization (selfinteraction) state with the assistance of Drs. Gibson and Hughes of Components 11 and 6, respectively, (2) Examine the impact of mitochondrial localization on mitochondrial fission-fusion ratios and mitophagy with the assistance of Dr. Nicholls of Component 11, and (3) Examine the effects of alterations in TOR activity on associated mitophagy with the assistance of Dr. Kapahi (Component 2 and co-Pi, Component 9). ' Nuclear translocation of alpha-synuclein selectively into dopaminergic midbrain neurons has been demonstrated following either its overexpression or increased oxidative stress. Nuclear translocation may also be dependent upon the protein's ser-129 phosphorylation state and/or its cleavage as well as its ability to associate with dopaminergic cytoplasmic factors. Nuclear localization appears to result in neurotoxicity via alpha-synuclein's ability to bind histones within the nucleus reducing their acetylation. Reduced histone acetylation could impact on gene transcription. In aim 2 of the application, we propose to assess alphasynuclein for post-translational modifications and interactions with dopaminergic cytosolic factors associated with its nuclear translocation with the assistance of Drs. Gibson and Hughes of Components 11 and 6, respectively. With Dr. Ellerby as part of Component 7, we will also examine the ability of HDAC inhibitors or specific HDAC siRNAs to protect against these effects and with Drs. Vijg and Melov as part of Component 5 the impact of nuclear localization of alpha synuclein on gene transcription.