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

亨廷顿氏病（HD）是一种进行性成人发病的神经退行性疾病，由亨廷顿蛋白（htt）N-末端区域的多聚谷氨酰胺（polyQ）扩增引起。HD的病理学特征包括核和胞质聚集以及显著和选择性的神经元损失。目前尚无已知的HD治愈或改善疾病的治疗方法，病理学和选择性脆弱性背后的具体机制仍不清楚。Htt在丝氨酸421处具有高度保守的磷酸化位点。先前在细胞系和原代神经元中的研究发现，显著增加该位点htt的磷酸化可以消除突变htt的毒性;相反，减少磷酸化可以增强htt的毒性。此外，磷酸化水平在小脑中最高，在皮质中较低，在纹状体中更低-与HD中神经变性的模式成反比。尽管有这些有趣的发现和缺乏有效的治疗方法，S421磷酸化与脑中HD发病机制的相关性尚不清楚。我们研究的目的是确定在体内的作用的S421磷酸化位点突变的ht诱导的神经变性。我们产生了新的转基因小鼠品系，其表达在S421处突变的htt以模拟紧张性磷酸化（S421 D）或防止磷酸化（S421 A）。在目标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，紧张性磷酸化显着改善突变ht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。这是潜在的意义，因为htt的切割通常通过产生通常在细胞核和聚集体中发现的N-末端片段来增加其毒性。因此，我们假设S421磷酸化改变了特别有毒的N-末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。然而，各种N-末端片段的体内意义是有争议的。在目标2中，我们将对每个S421突变株系产生的片段进行详细比较。具体来说，我们将比较片段的产生和积累在细胞核和细胞质的组织是和不容易htt毒性。此外，我们将在选定的时间点进行这些实验，因为小鼠年龄和获得HD表型。通过这种方式，我们将确定哪些独特的片段与不同组织中的行为缺陷、聚集体形成和/或神经变性的发生相关。在这个项目完成后，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中的选择性神经病理学有关，以及特定的N-末端片段是否与病理学和/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.