Mechanism of Fast Axonal Transport Inhibition by PolyQ-Expanded Huntingtin

点击翻译按钮获取中文摘要

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.