The in vivo Role of Serine 421 in Huntington's Disease

亨廷顿舞蹈病（HD）是一种进行性的，成人发病的神经退行性疾病，由蛋白质亨廷顿蛋白（htt） n端区域的聚谷氨酰胺（polyQ）扩增引起。HD的病理特征包括细胞核和细胞质聚集和显著的选择性神经元丢失。目前还没有已知的HD的治愈方法或改善疾病的治疗方法，病理和选择性易感性背后的具体机制仍不清楚。Htt在丝氨酸421处有一个高度保守的磷酸化位点。先前对细胞系和原代神经元的研究发现，显著增加该位点的htt磷酸化可消除突变体htt毒性；相反，降低磷酸化会增强HTT毒性。此外，磷酸化水平在小脑中最高，在皮层中较低，在纹状体中更低-与HD的神经变性模式成反比。尽管有这些有趣的发现和缺乏有效的治疗方法，S421磷酸化与HD脑发病机制的相关性尚不清楚。我们研究的目的是确定体内S421磷酸化位点在突变型http诱导的神经变性中的作用。我们产生了新的转基因小鼠系，表达在S421位点突变的htt，以模仿补补性磷酸化（S421D）或防止磷酸化（S421A）。在Aim 1中，我们将描述这些小鼠产生的任何行为缺陷和神经病理学，并将其与未修饰的HD小鼠和野生型对照进行比较。我们的初步结果表明，强直性磷酸化显著改善突变体http诱发的行为功能障碍。从机制上讲，磷酸化通常在蛋白质中起调节作用，S421位于几个报道的htt切割位点附近。这是潜在的重要意义，因为htt的切割通常通过产生n端片段增加其毒性，这些片段通常在细胞核和聚集体中发现。因此，我们假设S421磷酸化改变了特别有毒的n端片段的产生或积累。事实上，我的初步数据表明，年轻小鼠中htt的切割模式被S421修饰改变了。然而，各种n端片段在体内的意义是有争议的。在Aim 2中，我们将对每个S421突变系产生的片段进行详细的比较。具体来说，我们将比较易受和不受htt毒性影响的组织的细胞核和细胞质中片段的产生和积累。此外，我们将在小鼠衰老和获得HD表型的选择时间点进行这些实验。通过这种方式，我们将确定哪些独特的片段与不同组织的行为缺陷、聚集形成和/或神经退行性变的发病有关。该项目完成后，我们将验证S421作为HD的治疗靶点。我们还将确定磷酸化是否与HD的选择性神经病理有关，以及特定的n端片段是否与病理和/或突变蛋白聚集最相关。

Huntington's disease (HD) is a progressive, adult-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminal region of the protein huntingtin (htt). Pathological hallmarks of HD include nuclear and cytosolic aggregates and significant and selective neurons loss. There is no known cure or disease-modifying treatment for HD, and the specific mechanisms behind the pathology and selective vulnerability remain unclear. Htt has a highly conserved phosphorylation site at serine 421. Prior work in cell lines and primary neurons discovered that significantly increasing phosphorylation of htt at this site eliminates mutant htt toxicity; conversely, decreasing phosphorylation enhances htt toxicity. Furthermore, phosphorylation levels are highest in the cerebellum, lower in the cortex, and lower still in the striatum-inversely proportional to the pattern of neurodegeneration in HD. Despite these intriguing findings and the lack of effective therapies, the relevance of S421 phosphorylation to HD pathogenesis in the brain is unknown. The goal of our study is to determine in vivo the role of the S421 phosphorylation site in mutant htt-induced neurodegeneration. We generated new lines of transgenic mice that express htt mutated at S421 either to mimic tonic phosphorylation (S421D) or to prevent phosphorylation (S421A). In Aim 1, we will characterize any behavioral defects and neuropathology developed by these mice and compares them to unmodified HD mice and wildtype controls. Our preliminary results indicate that tonic phosphorylation significantly ameliorates mutant htt-evoked behavioral dysfunction. Mechanistically, phosphorylation often plays a regulatory role in proteins, and S421 is located in close proximity to several reported htt cleavage sites. This is potentially significat because cleavage of htt generally increases its toxicity by creating N-terminal fragments that are commonly found in the nucleus and in aggregates. Accordingly, we hypothesize that S421 phosphorylation alters the generation or accumulation of particularly toxic N-terminal fragments. Indeed, my preliminary data show that the cleavage pattern of htt in young mice is altered by S421 modification. The in-vivo significance of various N-terminal fragments, however, is controversial. In Aim 2, we will make a detailed comparison of the fragments generated by each S421 mutant line. Specifically, we will compare fragment generation and accumulation in the nucleus and cytoplasm of tissues that are and are not susceptible to htt toxicity. Additionally, we will perform these experiments at select time points as mice age and acquire the HD phenotype. In this manner, we will determine which unique fragments correlate with the onset of behavioral deficits, aggregate formation, and/or neurodegeneration across different tissues. Upon completion of this project, we will have validated S421 as a therapeutic target for HD. We also will have determined whether phosphorylation is related to the selective neuropathology in HD and whether particular N-terminal fragments are most associated with pathology and/or mutant protein aggregation.