N-terminal huntingtin and Huntington disease neuropathology

亨廷顿病(HD)是一种毁灭性的神经退行性疾病，由 多聚谷氨酰胺(PolyQ)结构域在不同的蛋白质中具有不同的功能。在HD中，多Q域位于 亨廷顿蛋白的N-末端区域(Htt)。这个N-末端区域在许多物种中都很保守， 但多聚Q的扩展会导致Htt的N-末端片段的错误折叠和随后的毒性。因为一个 缺乏Htt会导致小鼠胚胎死亡，Htt也被认为是动物发育和 生死存亡。减少突变Htt的表达被广泛认为是治疗HD的重要策略，因此 已经做出了相当大的努力来开发siRNA和反义寡核苷酸来抑制 突变体Htt的表达。这些方法也引发了人们的担忧，即显著压制Htt Htt的表达可能通过削弱Htt的正常功能而导致副作用；然而，Htt是否可以 在没有包含聚Q结构域的N-末端结构域的情况下保留关键功能仍然未知。 如果我们要开发一种治疗HD的新策略，解决这个问题是很重要的：如果N端多聚Q 基本的Htt功能不需要结构域，可以移除，完全消除N-末端 由于最近开发了基因组编辑工具CRISPR/CAS9，现在可以进行Htt区域的研究。在这 竞争性更新应用，我们将使用CRISPR/Cas9来研究N端突变体Htt的毒性 通过去除含有多聚Q的N-末端区域的片段和治疗效果。在目标1中，我们将使用 CRISPR/Cas9将在HD 140Q Ki小鼠的胚胎中引入小鼠Htt基因突变以产生 表达不同N-末端Htt片段并可传递给后代的截短突变Htt基因 通过生殖系。利用新建立的表达不同N-末端mHTT片段的HD-Ki小鼠 在内源水平上包含相同的多聚Q重复序列(140Q)，我们将检查 N-末端突变型Htt片段的长度及其在纹状体神经元中的核积累和毒性。在……里面 目的2，我们将使用CRISPR/Cas9去除N-末端多Q结构域作为一种治疗策略。我们会 探讨去除Htt中N-末端多聚Q结构域是否可以消除神经病理而不影响 成年小鼠的神经元存活和功能。这些研究将使用HD敲入小鼠，其中突变的Htt是 与HD患者的内源性表达水平相同。我们希望这些研究不仅将提供新的 深入了解N端突变的Htt片段的发病机制，也使我们能够开发出一种新的 治疗亨廷顿病和其他多Q疾病的治疗策略。

Huntington's disease (HD) is a devastating neurodegenerative disease caused by expansion of a polyglutamine (polyQ) domain in distinct proteins with different functions. In HD, the polyQ domain is located in the N-terminal region of huntingtin (Htt). This N-terminal region is well conserved in a wide range of species, but polyQ expansion can lead to misfolding and subsequent toxicity of N-terminal fragments of Htt. Since a lack of Htt causes embryonic lethality in mice, Htt is also thought to be essential for animal development and survival. Reducing the expression of mutant Htt is widely accepted as an important strategy for treating HD, so considerable efforts have gone into developing siRNA and anti-sense oligonucleotides to suppress the expression of mutant Htt. These approaches have also raised concerns that markedly suppressing Htt expression could lead to side effects by diminishing the normal function of Htt; however, whether Htt can preserve critical functions without the N-terminal domain that contains the polyQ domain remains unknown. Addressing this issue is important if we are to develop a new strategy to treat HD: if the N-terminal polyQ domain is not required for essential Htt functions and can be removed, complete elimination of the N-terminal region of Htt is now possible since the recent development of the genomic editing tool, CRISPR/Cas9. In this competitive renewal application, we will use CRISPR/Cas9 to investigate the toxicity of N-terminal mutant Htt fragments and therapeutic effects by removing the polyQ-containing N-terminal region. In Aim 1, we will use CRISPR/Cas9 to introduce mutations in the mouse Htt gene in embryos from HD 140Q KI mice to generate truncated mutant Htt genes that express different N-terminal Htt fragments and can be transmitted to offspring via the germline. Using the newly established HD KI mice that express different N-terminal mHtt fragments containing the same polyQ repeat (140Q) at the endogenous level, we will examine the relationship between the length of N-terminal mutant Htt fragments and their nuclear accumulation and toxicity in striatal neurons. In Aim 2, we will use CRISPR/Cas9 to remove the N-terminal polyQ domain as a therapeutic strategy. We will explore whether removing the N-terminal polyQ domain in Htt can eliminate neuropathology without affecting neuronal survival and function in adult mice. These studies will use HD knock-in mice in which mutant Htt is expressed at the same endogenous level as in HD patients. We hope these studies will not only provide new insight into the pathogenesis of N-terminal mutant Htt fragments, but also allow us to develop a novel therapeutic strategy to treat Huntington's disease and other polyQ diseases.