Loss-of-function mechanisms in Huntington's Disease

描述（由申请人提供）：亨廷顿舞蹈病（HD）是一种显性神经退行性疾病，由亨廷顿舞蹈病基因的蛋白产物亨廷顿舞蹈病蛋白（htt）中编码聚谷氨酰胺（polyQ）的CAG三重重复序列延伸引起。HD被认为是由与htt正常功能无关的polyQ拉伸扩展引起的有害功能获得的结果。最近的研究表明，虽然功能获得可能在HD发病机制中发挥重要作用，但相应的正常htt功能的丧失也有助于疾病进程。我们的长期目标是利用遗传学和生物化学方法来了解htt正常功能在HD发病机制中的作用，并在恢复HD患者正常htt功能的基础上发现治疗HD的新的潜在治疗策略。为了实现这一目标，我们提出了三个具体的目标，旨在帮助我们理解扩展的polyQ拉伸如何影响正常的htt功能，以及缺乏正常短拉伸的polyQ (？Q-htt)能够挽救HD小鼠模型中的HD表型。在目标1中，我们将检验假设？Q-htt能够通过增强突变体htt的自噬清除来挽救HD小鼠模型表型。我们将使用细胞培养和小鼠模型来关注可能导致？Q-htt的效果。首先,?Q-htt可能介导p62/SQSTM1对突变体htt聚集体的增强识别，p62/SQSTM1是一种多泛素结合蛋白，可以靶向这种聚集体进行自噬降解。第二,?Q-htt可能通过增强自噬体向溶酶体的逆行运输间接影响突变体htt聚集体的自噬降解。为了测试这些机制，我们将描述来自表达？Q-htt表达或不表达140Q-htt，以及缺乏神经元htt表达的条件敲除小鼠对p62/SQSTM1功能的影响。此外，逆行运输的效率将通过测量细胞器和动力蛋白复合物的运动来表征初级神经元培养。最近，我们还观察到，将小鼠htt polyQ的长度从7Q增加到正常人的平均长度20Q，可以加速正常和突变htt的相互作用。为了验证正常和突变htt之间的相互作用可以影响HD发病机制的假设，我们将在Aim 2中比较表达7Q/140Q htt的小鼠和表达20Q/140Q htt的小鼠的行为和神经病理表型。最后，在目标3中，我们将检验假设？Q-htt可能与一组新的结合伙伴相互作用和/或抵抗140Q-htt影响htt与其结合伙伴相互作用的潜力，通过使用表达表位标记的htt等位基因的小鼠来检测正常和？在htt突变体表达存在和缺失的情况下，Q-htt相互作用蛋白。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a dominant neurodegenerative disease that is caused by the expansion of a stretch of CAG triplet repeats encoding polyglutamine (polyQ) within huntingtin (htt), the protein product of the HD gene. HD is considered to be the consequence of a deleterious gain-of-function caused by the expanded polyQ stretch that is unrelated to htt's normal function. Recent work suggests that although gain-of-function may play an important role in HD pathogenesis, a corresponding loss of normal htt function also contributes to the disease process. Our long-term objective is to use genetic and biochemical approaches to understand the role of htt's normal function in HD pathogenesis, and to discover new potential therapeutic strategies for the treatment of HD based on restoring normal htt function in HD. To accomplish this objective, we propose three specific aims that are designed to help us understand how the expanded polyQ stretch can affect normal htt function, and how a version of htt that lacks its normal short stretch of polyQ (?Q-htt) is able to rescue HD phenotypes in a mouse model for HD. In Aim 1, we will test the hypothesis that ?Q-htt is able to rescue HD mouse model phenotypes by enhancing autophagic clearance of mutant htt. We will use both cell culture and mouse models to focus on two potential mechanisms that may be responsible for ?Q-htt's effects. First, ?Q-htt may mediate the enhanced recognition of mutant htt aggregates by p62/SQSTM1, a polyubiquitin binding protein that can target such aggregates for autophagic degradation. Second, ?Q-htt may affect autophagic degradation of mutant htt aggregates indirectly by enhancing retrograde transport of autophagosomes to lysosomes. To test these mechanisms, we will characterize brains and primary neurons derived from mice expressing ?Q-htt with or without 140Q-htt expression, and conditional knockout mice lacking neuronal htt expression, for p62/SQSTM1 function. In addition, the efficiency of retrograde transport will be characterized in primary neuronal cultures by measuring organelle and dynein complex movement. Recently, we have also observed that increasing the length of the mouse htt polyQ stretch from 7Q to the normal human average length of 20Q can accelerate interactions of normal and mutant htt. To test the hypothesis that an interaction between normal and mutant htt can affect HD pathogenesis, we will compare in Aim 2, behavioral and neuropathological phenotypes in mice expressing 7Q/140Q htt, and in mice expressing 20Q/140Q htt. Finally, in Aim 3, we will test the hypothesis that ?Q-htt may interact with a novel set of binding partners and/or is resistant to 140Q-htt's potential to influence the interaction of htt with its binding partners, by using mice expressing epitope-tagged htt alleles to detect differences in the repertoire of normal and ?Q-htt interacting proteins in the presence and absence of mutant htt expression.