Loss-of-function mechanisms in Huntington's Disease

描述（由申请人提供）：亨廷顿氏病（HD）是一种显性神经退行性疾病，其由亨廷顿蛋白（htt）内编码多聚谷氨酰胺（polyQ）的CAG三联体重复序列的扩展引起，亨廷顿蛋白是HD基因的蛋白产物。HD被认为是由与htt的正常功能无关的扩展的polyQ拉伸引起的有害的功能获得的结果。最近的研究表明，虽然获得的功能可能发挥重要作用，在HD发病机制，相应的损失正常htt功能也有助于疾病的过程。我们的长期目标是利用遗传和生化方法来了解htt的正常功能在HD发病机制中的作用，并发现新的潜在的治疗策略，治疗HD的基础上恢复正常htt功能的HD。为了实现这一目标，我们提出了三个具体的目标，旨在帮助我们了解扩展的polyQ拉伸如何影响正常的htt功能，以及缺乏polyQ正常短拉伸的htt版本如何（？Q-htt）能够挽救HD小鼠模型中的HD表型。在目标1中，我们将测试假设，？Q-htt能够通过增强突变型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，重点放在两个潜在的机制，可能是负责？Q-HTT的影响。首先，？Q-htt可以介导p62/SQSTM 1对突变htt聚集体的增强识别，p62/SQSTM 1是一种多聚泛素结合蛋白，可以靶向这种聚集体进行自噬降解。其次，？Q-htt可能通过增强自噬体向溶酶体的逆向转运而间接影响突变htt聚集体的自噬降解。为了测试这些机制，我们将表征大脑和初级神经元来自小鼠表达？有或没有140 Q-htt表达的Q-htt和缺乏神经元htt表达的条件性敲除小鼠，用于p62/SQSTM 1功能。此外，逆行运输的效率将通过测量细胞器和动力蛋白复合体运动在原代神经元培养物中表征。最近，我们还观察到，将小鼠htt polyQ延伸的长度从7 Q增加到正常人平均长度20 Q可以加速正常和突变htt的相互作用。为了验证正常htt和突变htt之间的相互作用可以影响HD发病机制的假设，我们将在目标2中比较表达7 Q/140 Q htt的小鼠和表达20 Q/140 Q htt的小鼠的行为和神经病理学表型。最后，在目标3中，我们将测试假设，？Q-htt可能与一组新的结合伙伴和/或耐140 Q-htt的潜力，影响htt与其结合伙伴的相互作用，通过使用小鼠表达表位标记的htt等位基因检测正常和？突变型htt表达存在和不存在时Q-htt相互作用蛋白。 公共卫生相关性：亨廷顿氏病（HD）是一种遗传性神经退行性疾病，影响约1/10，000的人，由亨廷顿蛋白（htt）突变引起。目前还没有治愈这种疾病的方法，一旦发现症状，这种疾病会持续10-20年，最终不可避免地死亡。我们提出的实验将帮助我们了解突变如何影响htt的正常功能，以便我们可以发现新的治疗策略的基础上恢复htt功能的人受HD。

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. PUBLIC HEALTH RELEVANCE: Huntington's disease (HD) is a hereditary neurodegenerative disease affecting ~1 in 10,000 people that is caused by a mutation in the protein huntingtin (htt). There is currently no cure for this disorder, and once symptoms are detected, the disease progresses over 10-20 years and ends inevitably in death. We propose experiments that will help us to understand how the mutation affects htt normal function so that we can discover new therapeutic strategies based on restoring htt function in people affected with HD.