Mechanism of Fast Axonal Transport Inhibition by PolyQ-Expanded Huntingtin

PolyQ 扩展亨廷顿蛋白抑制快速轴突运输的机制

• 批准号: 7677914
• 负责人: Sarah Lynn Pollema
• 金额: $ 3.31万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2010-05-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7677914
• 关键词: Address; Area; Biological Assay; Brain; Cell Line; Cessation of life; Co-Immunoprecipitations; Corpus striatum structure; Disease; Face; Follow-Up Studies; Fractionation; Genes; Heating; Huntington Disease; Kinesin; Laboratories; Lead; Mediating; Molecular; Molecular Motors; Motor; Mutation; Nerve Degeneration; Neurons; Pathway interactions; Patients; Perfusion; Phosphorylation; Phosphotransferases; Proteins; Recombinants; Regulatory Pathway; Signal Pathway; Squid; System; Work; axoplasm; effective therapy; fast axonal transport; gain of function; human Huntingtin protein; inhibitor/antagonist; middle age; mouse model; new therapeutic target; polyglutamine; polyproline; protein protein interaction; research study; stress activated protein kinase; therapeutic target

DESCRIPTION (provided by applicant): Huntington's Disease (HD) is caused by expansion of a polyglutamine (polyQ) tract past a critical number, which results in midlife onset of neurodegeneration of the striatum, as well as other brain areas as the disease progresses. This single gene disease displays dominant inheritance, and is thought to involve a toxic gain of function in the Huntingtin (Htt) protein. Work from our laboratory has shown polyQ expanded Huntingtin (Htt) significanty inhibited fast axonal transport (FAT) in isolated squid axoplasm. The primary objective is to understand how polyQ expanded Htt inhibits FAT. Aim 1: To evaluate molecular mechanisms by which polyQ expanded Htt inhibits FAT. a)To determine potential interactions between Htt and motor proteins. To address the possibility of polyQ Htt aggregates sequestering motor proteins, co-immunoprecipitation of Htt and kinesin will reveal any possible direct interactions between Htt and motors. Also, co-fractionation of polyQ Htt and WT Htt will reveal if kinesin partitions with aggregates or remains in the soluble fraction. These studies will be done in an established Htt cell line as well as a mouse model of HD. b)To determine potential kinase alterations in regulatory pathways for FAT. To address the possibility that polyQ Htt may activate kinase pathways that misregulate FAT, co-perfusion of polyQ Htt with pharmacological inhibitors of specific kinases in isolated squid axoplasm will reveal any kinase pathways involved and may provide potential therapeutic targets. Follow up studies of kinase assays in a mammalian Htt cell line can confirm results obtained in squid axoplasm. Aim 2: To identify motifs in polyQ Htt that are critical for inhibition of FAT. There are several protein-protein interaction motifs in the Htt protein, including a polyproline domain and a HEAT repeat. Perfusing polyQ Htt with deletion of specific motifs into squid axoplasm will reveal which, if any, domain outside of the polyQ tract is required for FAT inhibition by polyQ Htt. Relevance: HD is a devastating illness with a no effective treatment; patients face midlife onset of neurodegeneration with a continuous decline until death. Recent studies have shown mutations in motor proteins can lead to neurodegeneration, and also that FAT is highly regulated by enzymatic signaling pathways. Understanding the mechanism by which polyQ Huntingtin inhibits fast axonal transport may lead to new therapeutic targets.

描述（由申请人提供）：亨廷顿氏病（HD）是由聚谷氨酰胺（polyQ）束扩张超过一个临界数量引起的，导致中年纹状体神经退行性变，以及随着疾病的进展，其他大脑区域也会发生神经退行性变。这种单基因疾病表现出显性遗传，并且被认为与亨廷顿蛋白（Htt）功能的毒性增益有关。我们实验室的工作表明，polyQ扩展亨廷顿蛋白（Htt）显著抑制离体鱿鱼轴质中的快速轴突运输（FAT）。主要目的是了解polyQ如何扩展Htt抑制FAT。目的1：探讨polyQ扩展Htt抑制FAT的分子机制。a)确定Htt与运动蛋白之间潜在的相互作用。为了解决polyQ Htt聚集体隔离马达蛋白的可能性，Htt和激酶的共免疫沉淀将揭示Htt和马达之间可能的直接相互作用。此外，polyQ Htt和WT Htt的共分馏将揭示运动蛋白是与聚集体分割还是留在可溶部分。这些研究将在已建立的Htt细胞系以及HD小鼠模型中进行。b)确定FAT调控通路中潜在的激酶改变。为了解决polyQ Htt可能激活错误调节脂肪的激酶途径的可能性，在分离的鱿鱼轴质中，将polyQ Htt与特定激酶的药理抑制剂共灌注将揭示任何涉及的激酶途径，并可能提供潜在的治疗靶点。在哺乳动物Htt细胞系中进行的激酶测定的后续研究可以证实在鱿鱼轴质中获得的结果。目的2：确定polyQ Htt中对抑制FAT至关重要的基序。在Htt蛋白中有几个蛋白-蛋白相互作用基序，包括一个聚脯氨酸结构域和一个HEAT重复序列。将删除特定基元的polyQ Htt灌注到鱿鱼的轴质中，将揭示polyQ Htt抑制脂肪所需要的polyQ通道外的结构域。相关性：HD是一种毁灭性的疾病，没有有效的治疗方法；患者面临中年发病的神经退行性疾病，并持续衰退直至死亡。最近的研究表明，运动蛋白的突变可导致神经退行性变，而且脂肪受到酶信号通路的高度调节。了解polyQ亨廷顿蛋白抑制快速轴突转运的机制可能会带来新的治疗靶点。