Loss-of-function mechanisms in Huntington's Disease

描述(申请人提供)：亨廷顿病(HD)是一种显性神经退行性疾病，由HD基因的蛋白质产物Huntingtin(HTT)内编码多谷氨酰胺(PolyQ)的CAG三联体重复序列的扩展引起。HD被认为是与HTT的正常功能无关的扩展多Q伸展引起的有害的功能增益的结果。最近的工作表明，虽然功能获得可能在HD的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的发展。我们的长期目标是利用遗传学和生化方法了解HTT的正常功能在HD发病机制中的作用，并在恢复HD患者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，我们提出了三个特定的目标，旨在帮助我们了解扩展的PolyQ伸展如何影响正常的HTT功能，以及缺乏正常的PolyQ短伸展的HTT版本如何能够挽救HD小鼠模型中的HD表型。在目标1中，我们将检验Q-HTT能够通过增强突变型HTT的自噬清除来挽救HD小鼠模型表型的假设。我们将同时使用细胞培养和小鼠模型来关注可能导致Q-HTT效应的两种潜在机制。首先，？q-HTT可能通过p62/SQSTM1介导对突变的HTT聚集体的增强识别，p62/SQSTM1是一种多泛素结合蛋白，可以靶向这些聚集体进行自噬降解。第二，Q-HTT可能通过促进自噬小体向溶酶体的逆行运输，间接影响突变的HTT聚集体的自噬降解。为了测试这些机制，我们将对表达Q-HTT并有或不有140Q-HTT表达的小鼠和缺乏神经元HTT表达的条件性基因敲除小鼠的大脑和初级神经元进行特征分析，以了解p62/SQSTM1的功能。此外，在原代神经元培养中，通过测量细胞器和动力蛋白复合体的运动来表征逆行运输的效率。最近，我们还观察到，将小鼠HTT多聚Q延伸的长度从7Q增加到正常人类平均20Q的长度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，我们将在目标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，我们将测试这一假设，即？Q-HTT可能与一组新的结合伙伴相互作用和/或抵抗140Q-HTT潜在地影响HTT与其结合伙伴的相互作用，方法是使用表达表位标记的HTT等位基因的小鼠来检测在存在和不存在突变HTT表达的情况下正常和？Q-HTT相互作用蛋白谱系的差异。 公共卫生意义：亨廷顿病(HD)是一种遗传性神经退行性疾病，每10,000人中就有1人受到影响，该疾病是由亨廷顿蛋白(HTT)突变引起的。目前还没有治愈这种疾病的方法，一旦检测到症状，这种疾病会在10-20年内发展，不可避免地以死亡告终。我们提出的实验将帮助我们了解突变是如何影响HTT正常功能的，这样我们就可以发现基于恢复HD患者HTT功能的新的治疗策略。

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.