Loss-of-function mechanisms in Huntington's disease

亨廷顿氏病（HD）是一种显性神经退行性疾病， 在亨廷顿蛋白（htt）内编码多聚谷氨酰胺（polyQ）的CAG三联体重复序列的延伸，亨廷顿蛋白（htt）是 HD基因HD被认为是由以下因素引起的有害功能获得的结果： 与htt正常功能无关的扩展polyQ拉伸。最近的研究表明，虽然增益功能 可能在HD发病机制中起重要作用，正常htt功能的相应丧失也 有助于疾病的发展。我们的长期目标是利用遗传和生物化学方法， 了解htt的正常功能在HD发病机制中的作用，并发现新的潜在治疗方法 基于恢复HD中正常htt功能的HD治疗策略。为了实现这一目标， 我们提出了三个具体的目标，旨在帮助我们了解扩展的polyQ拉伸如何 影响正常的htt功能，以及缺乏polyQ（？Q-htt）能够 在HD小鼠模型中挽救HD表型。在目标1中，我们将测试假设，？Q-HTT能够 通过增强突变体htt的自噬清除拯救HD小鼠模型表型。我们将重点关注两个 潜在的机制，可能负责？Q-htt的效果。首先，？Q-htt可能介导了增强的 p62/SQSTM 1识别突变的htt聚集体，p62/SQSTM 1是一种多聚泛素结合蛋白，可以靶向这种聚集体， 用于自噬降解的聚集体。其次，？Q-HTT可能影响突变HTT的自噬降解 通过增强自噬体向溶酶体的逆行转运而间接聚集。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？Q-htt与或 对于p62/SQSTM 1，无140 Q-htt表达的小鼠和缺乏神经元htt表达的条件性敲除小鼠， 功能此外，在原代神经元培养物中，逆行转运的效率将通过以下方式表征： 测量细胞器和动力蛋白复合体的运动。最近，我们还观察到， 小鼠htt polyQ的长度从7 Q拉伸到正常人的平均长度20 Q可以加速 正常和突变HTT的相互作用。为了验证正常和突变htt之间的相互作用 可以影响HD发病机制，我们将在目的2，行为和神经病理表型在小鼠中进行比较 在表达7 Q/140 Q htt的小鼠中，以及在表达20 Q/140 Q htt的小鼠中。最后，在目标3中，我们将检验假设 突变体htt的表达可以影响野生型或？Q-htt与它们的结合伴侣， 使用小鼠表达表位标记的HTT等位基因，以检测正常和？Q-htt 在存在和不存在突变体HTT表达的情况下相互作用的蛋白质。

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-offunction 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 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 mutant htt expression can influence the interaction of wild-type or ?Q-htt with their 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.