Synuclein Function in the Synaptic Vesicle Cycle

突触小泡周期中的突触核蛋白功能

• 批准号: 6478475
• 负责人: Vincent A Pieribone
• 金额: $ 14.73万
• 依托单位: JOHN B. PIERCE LABORATORY, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-15 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6478475
• 关键词: Agnatha; Lewy body; Parkinson's disease; alpha synuclein; axon; electron microscopy; electrophysiology; fluorescence microscopy; immunocytochemistry; motor neurons; nerve endings; neural degeneration; neuropathology; neurophysiology; neurotransmitter transport; polymerase chain reaction; protein protein interaction; protein structure function; synaptic vesicles; voltage /patch clamp

Several independent lines of evidence have linked the nerve terminal- enriched synuclein family of proteins to Parkinson's disease. Genetic studies have linked mutations in the alpha-synuclein gene to the disease in several families. Synucleins are a major constituent of Lewy bodies, a hallmark histopathology of Parkinson's disease. Over-expression of synucleins are a major constituent of Lewy bodies, a hallmark histopathology of Parkinson's disease. Over-expression of synucleins in mice causes Lewy body-like neuropathology and motor deficits. While there is a wealth of information on the genetic and histological features of synucleins, very little information exists on the function of the proteins in normally functioning nerve terminals. We propose to study the function of these proteins in nerve terminals using classic neurophysiologic experiments in an isolated nerve terminals using classic neurophysiologic experiments in an isolated nerve terminal. Pre-synaptic injection of agents that will modify synuclein function will be made into the giant pre-terminal of a primitive vertebrate (lamprey). The effects of pre-terminal injections of several synuclein affecting agents will be analyzed with electrophysiologic and ultrastructural methods. Inj3ected agents will include: full length and fragments of lamprey synuclein, recombinant human alpha-synuclein containing Parkinson's disease associated mutations (A53T and A30P). Mock casein kinase I and src kinase phosphorylated (S129E/S87E) human alpha-synuclein and antibodies against lamprey synuclein. Using paired intracellular recordings between the pre- and post-synaptic elements we will examine the effects of injections on the ESPS generated by intraoxonal stimulation. Many parameters of synaptic vesicle release kinetics will be examined. In addition, giant pre-synaptic elements receiving injections of agents followed by varying levels of stimulation will be examined under the electron microscope. Effects of injections on the synaptic vesicle cycle can be established by characteristic changes in nerve terminal morphology. Establishing the function of the synucleins in normal neurotransmitter release will shed light on the function and dysfunction of the protein in Parkinson's disease.

一些独立的证据表明，神经末梢富集的突触核蛋白家族与帕金森病有关。遗传学研究已经将几个家族的α -突触核蛋白基因突变与该病联系起来。突触核蛋白是路易体的主要成分，路易体是帕金森病的标志性组织病理学。突触核蛋白的过度表达是路易小体的主要组成部分，路易小体是帕金森病的标志组织病理学。突触核蛋白在小鼠中的过度表达导致路易体样神经病理和运动缺陷。虽然关于突触核蛋白的遗传和组织学特征的信息非常丰富，但关于突触核蛋白在正常功能的神经末梢中的功能的信息却很少。我们建议在离体神经末梢进行经典神经生理学实验，研究这些蛋白在神经末梢中的功能。在原始脊椎动物（七鳃鳗）的巨大的突触前端注射改变突触核蛋白功能的药物。用电生理和超微结构方法分析几种突触核蛋白影响剂的终末注射效果。注射制剂将包括：全长和片段的七鳐突触核蛋白，重组人α -突触核蛋白含有帕金森病相关突变（A53T和A30P）。模拟酪蛋白激酶I和src激酶磷酸化（S129E/S87E）人α -突触核蛋白和抗七鳃鳗突触核蛋白抗体。通过对突触前和突触后的细胞内记录，我们将研究注射对腔内刺激产生的ESPS的影响。将检查突触囊泡释放动力学的许多参数。此外，巨大的突触前元件接受药物注射，随后是不同程度的刺激，将在电子显微镜下检查。注射对突触囊泡周期的影响可以通过神经末梢形态的特征性变化来确定。建立突触核蛋白在正常神经递质释放中的功能，将有助于揭示该蛋白在帕金森病中的功能和功能障碍。