Mechanism of Fast Axonal Transport Inhibition by PolyQ-Expanded Huntingtin

描述(申请人提供)：亨廷顿氏病(HD)是由多聚谷氨酰胺(PolyQ)区域扩张超过临界数量引起的，这会导致纹状体和其他大脑区域随着疾病的进展而出现中年神经变性。这种单基因疾病表现出显性遗传，被认为涉及亨廷顿蛋白(Htt)的有毒功能获得。我们实验室的工作表明，多聚Q扩展亨廷顿(Htt)显著抑制了分离的鱿鱼轴浆中的快速轴突运输(FAT)。主要目标是了解多聚Q扩展的Htt是如何抑制脂肪的。目的1：探讨PolyQ扩展的Htt抑制脂肪的分子机制。A)确定Htt和马达蛋白之间的潜在相互作用。为了解决多聚Q Htt聚集体隔离马达蛋白的可能性，Htt和Kinesin的免疫共沉淀将揭示Htt和马达之间任何可能的直接相互作用。此外，多聚Q Htt和WT Htt的共分级将揭示Kinesin是以聚集体形式分配还是保留在可溶部分中。这些研究将在已建立的Htt细胞系和HD的小鼠模型中进行。B)确定脂肪调节通路中潜在的激酶变化。为了解决PolyQ Htt可能激活错误调节脂肪的激酶通路的可能性，在分离的鱿鱼轴浆中，将PolyQ Htt与特定激酶的药物抑制剂共灌流，将揭示任何涉及的激酶通路，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分析的后续研究可以证实在鱿鱼轴浆中获得的结果。目的2：确定PolyQ Htt中与抑制脂肪有关的基序。在Htt蛋白中有几个蛋白质-蛋白质相互作用基序，包括一个多脯氨酸结构域和一个热重复序列。将带有特定基序缺失的多Q Htt灌流到鱿鱼轴浆中，将揭示多Q Htt抑制脂肪所需的多Q束外的哪个结构域(如果有的话)。相关性：HD是一种毁灭性的疾病，没有有效的治疗方法；患者面临中年神经变性的发作，并不断下降，直到死亡。最近的研究表明，马达蛋白的突变会导致神经退化，而且脂肪受到酶信号通路的高度调控。了解多聚Q亨廷顿抑制快速轴突运输的机制可能会带来新的治疗靶点。

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.