Alfy-mediated selective macroautophagy and the pathogenesis of Huntington???s Dis

描述(申请人提供)：亨廷顿病(HD)是一种神经退行性疾病，由HD基因内遗传的CAG扩展突变引起，导致突变的亨廷顿蛋白(Htt)含有扩展的聚谷氨酰胺(PolyQ)途径。纹状体的退化导致患者逐渐失去自主运动控制，精神障碍，以及衰弱的认知能力下降。HD的一个病理特征是突变的Htt和泛素在整个大脑的细胞内沉积。Htt聚集体和疾病症状之间的联系尚不清楚，但在沉默Htt转基因的研究中，已经观察到细胞和行为病理学的改善。在可诱导的HD小鼠模型中，转基因沉默导致聚集体的消除和运动缺陷的改善。这些发现表明，集合移除有可能缓解与HD相关的潜在细胞功能障碍。了解神经元处理聚集蛋白的分子机制是开发各种神经退行性疾病治疗方法的关键一步。细胞模型系统表明，Htt聚集体通过一种称为宏自噬的降解过程来分解，在这种过程中，胞浆蛋白被隔离到一个双层膜结构中，该结构与溶酶体融合，以降解其货物。巨型自噬机制由一组与自噬相关的核心蛋白组成，如ATG5、ATG7和LC3。虽然最初发现这一过程会大量降解蛋白质以应对饥饿，但它也可以选择性地发生在特定的底物上。我们的实验室已经鉴定出一种名为Alfy(自噬连接的FYVE蛋白)的蛋白质，它介导聚集蛋白的选择性巨型自噬。在表达突变型Htt的细胞中，Alfy是清除聚集体所必需的。此外，在果蝇和多聚Q扩展的初级神经元模型中，c-末端Alfy的过表达减少了包涵体，并防止了神经退化。这项提案试图解决Alfy介导的选择性巨细胞自噬在大脑老化和HD中的作用。从发育中的大脑中消除ATG7等核心自噬蛋白会导致神经退化和蛋白质积累，但选择性和非选择性自噬在衰老过程中的作用在很大程度上仍未在体内得到探索。该项目将通过在衰老小鼠中消除Alfy或ATG7来研究这一点，方法是使用他莫昔芬诱导的Cre来驱动条件等位基因的切除(目标1)。这将使您能够深入了解 巨噬细胞在脑老化中的重要性。此外，该项目将通过在可诱导的HD小鼠模型中消除Alfy，在体内研究Alfy介导的Htt聚集体的宏自噬(Aim 2)。这将解决Alfy的移除如何影响突变Htt的清除，以及疾病逆转是否依赖于聚集体的清除。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a neurodegenerative disorder arising from an inherited CAG expansion mutation within the HD gene, resulting in mutant huntingtin (Htt) protein containing an expanded polyglutamine (polyQ) tract. Degeneration of the striatum causes patients to suffer from progressive loss of voluntary motor control, psychiatric disturbances, and debilitating cognitive decline. A pathological hallmark of HD is the accumulation of mutant Htt and ubiquitin in intracellular deposits throughout the brain. The link between Htt aggregates and disease symptoms remains unclear, but improvements in both cellular and behavioral pathology have been observed in studies that silence the Htt transgene. In an inducible HD mouse model, transgene silencing leads to the elimination of aggregates in conjunction with amelioration of motor deficits. These findings imply that aggregate removal has the potential to alleviate the underlying cellular dysfunction associated with HD. Understanding the molecular mechanisms by which neurons dispose of aggregated proteins is a critical step towards the development of therapeutics for a variety of neurodegenerative disorders. Cellular model systems have suggested that Htt aggregates are broken down by a degradation process known as macroautophagy, in which cytosolic proteins are sequestered into a double-membrane structure that fuses with a lysosome to degrade its cargo. The macroautophagic machinery comprises a core group of autophagy-related proteins, such as Atg5, Atg7, and LC3. While this process was initially found to degrade proteins in bulk in response to starvation, it can also occur selectively for particular substrates. Our lab has identified a protein called Alfy (autophagy-linked FYVE protein) that mediates selective macroautophagy of aggregated proteins. In cells expressing mutant Htt, Alfy is essential for the clearance of aggregates. Additionally, overexpression of c-terminal Alfy decreases inclusions and protects against neurodegeneration in drosophila and primary neuronal models of polyQ expansion. This proposal seeks to address the role of Alfy-mediated selective macroautophagy in aging brain and in HD. Eliminating core macroautophagic proteins such as Atg7 from developing brain results in neurodegeneration and protein accumulation, but the role of selective and nonselective macroautophagy during aging remains largely unexplored in vivo. This project will examine this by eliminating Alfy or Atg7 in aging mice using a tamoxifen-inducible Cre to drive excision of a conditional allele (Aim 1). This will provide insight into the importance of macroautophagy in aging brain. Furthermore, this project will investigate Alfy- mediated macroautophagy of Htt aggregates in vivo, by eliminating Alfy in an inducible HD mouse model (Aim 2). This will address how removal of Alfy affects clearance of mutant Htt, and whether disease reversal is dependent upon clearance of aggregates.