Subcellular localization of alpha-synuclein and its impact on neurodegeneration

α-突触核蛋白是路易体的主要蛋白组分，路易体是退行性变的主要特征。 帕金森病患者的大脑。已经证明α-突触核蛋白通过介导的方式嵌入脂质膜中。 在其N-末端形成α螺旋结构。我们最近证明了孵化 重组A53 T突变体α-突触核蛋白与从永生化中脑衍生的 多巴胺能神经元或在多巴胺能细胞内表达A53 T后，无论是在培养中还是在 在转基因动物中，蛋白质定位于内部线粒体！膜（IMM）的形式 低聚物线粒体定位可能是由于A53 T不能进行丝氨酸129（ser-129） 磷酸化，因为这一事件可以由氧化应激诱导，驱动野生型蛋白质向 细胞质定位和寡聚化。A53 T定位于IMM伴随着减少 线粒体膜电位（MMP）和线粒体自噬（mitophagy）增加。减少 其他人已经表明MMP影响线粒体分裂-融合速率，驱动线粒体 走向线粒体自噬在本资助申请的目标#1中，我们建议：（1）评估脱乙酰核酸的能力， ser-129磷酸化以确定其亚细胞定位和寡聚化（自身相互作用） 在第11和第6部分的吉布森和休斯博士的协助下， (2)检查线粒体定位对线粒体分裂-融合比率和线粒体自噬的影响 在第11部分的尼科尔斯博士的协助下，（3）检查TOR变化的影响 在Kapahi博士的协助下进行相关线粒体自噬活动（组分2和co-Pi，组分 9）。' α-突触核蛋白选择性地核转位到多巴胺能中脑神经元中已经被证实。 在其过度表达或增加的氧化应激之后证实。核转位可能 也取决于蛋白质的Ser-129磷酸化状态和/或其切割以及其能力 与多巴胺能细胞质因子有关。核定位似乎通过以下途径导致神经毒性： α-突触核蛋白结合细胞核内组蛋白的能力，减少其乙酰化。还原组蛋白 乙酰化可以影响基因转录。在本申请的目的2中，我们提出评估α-asynuclein 用于翻译后修饰和与多巴胺能胞质因子相关的相互作用 在组分11和6的吉布森和休斯博士的帮助下进行核转位， 分别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， 或特异性HDAC siRNA来保护免受这些影响，Vijg和Melov博士作为 第五部分：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.