Neuronal DNA repair pathways in Huntington's disease pathophysiology

亨廷顿氏病（HD）是一种神经退行性疾病，由脑内CAG重复序列的扩张引起。 亨廷顿蛋白（HTT）基因，导致主要在纹状体和皮质的神经元死亡。CAG重复是 高度不稳定，无论是在代际和体细胞。HD患者显示出较高的年龄- 纹状体中CAG重复序列的依赖性体细胞扩增，尽管这种扩增也发生在其他细胞中， 组织中最近对HD患者的全基因组关联研究揭示了存在遗传性 疾病发病年龄的调节剂;这些包括参与DNA修复的几个基因，特别是 DNA错配修复（MSH 3、MLH 1、PMS 2、PMS 1）。此外，对HD小鼠模型的研究表明， DNA错配修复基因Msh 2、Msh 3、Mlh 1或Mlh 3的基因敲除降低了体细胞的不稳定性， 在纹状体中的CAG重复序列。这些数据表明，CAG重复序列的体细胞扩增可能与 纹状体神经元损失和HD患者的疾病症状发作。因此，在目标1中，我们建议 表征和比较小鼠纹状体和其他小鼠大脑区域的蛋白质组装， 并加工CAG挤出物。我们预计，这些蛋白质复合物可能揭示的机制， CAG扩增，同时也可能发现与重复不稳定无关的新的病理过程。 在目标2中，我们将在人HEK 293细胞中开发基于邻近生物素化（TurboID）的测定以评估 含有CAG挤出物的DNA上的瞬时和稳定的蛋白质组装体，以期最终应用于 这是一种神经元细胞的方法。敲除关键错配修复基因如MSH 3的影响 或FAN 1对这种DNA损伤的处理也将被评估。这些方法的目的是 鉴定决定CAG挤出是否由FAN 1或错配修复途径处理的蛋白质。

Huntington’s disease (HD) is a neurodegenerative disorder caused by an expansion of a CAG repeat tract within the huntingtin (HTT) gene, leading to neuronal death primarily in the striatum and the cortex. The CAG repeat is highly unstable, both intergenerationally and in somatic cells. HD patients display a high degree of age- dependent somatic expansion of the CAG repeat in the striatum, although such expansions also occur in other tissues. Recent genome-wide association studies of HD patients have revealed the existence of genetic modifiers of the age of onset of the disease; these include several genes involved in DNA repair, and in particular DNA mismatch repair (MSH3, MLH1, PMS2, PMS1). In addition, studies in mouse models of HD have revealed that genetic knockout of the DNA mismatch repair genes, Msh2, Msh3, Mlh1, or Mlh3 reduces somatic instability of CAG repeats in the striatum. These data suggest that somatic expansion of CAG repeats is likely linked to striatal neuronal loss, and onset of disease symptoms in HD patients. Therefore, in Aim 1, we propose to characterize and compare protein assemblies in mouse striatum and other mouse brain regions that recognize and process CAG extrusions. We anticipate that these protein complexes may shed light on the mechanism of CAG expansions, while also potentially uncovering novel pathological processes unrelated to repeat instability. In Aim 2, we will develop proximity biotinylation (TurboID)-based assay in human HEK293 cells to evaluate transient and stable protein assemblies on DNA containing CAG extrusions, with a view to eventually applying this approach to cells of neuronal origin. The effects of knockdown of critical mismatch repair genes like MSH3 or FAN1 on the processing of such DNA lesions will also be evaluated. The objective of these approaches is to identify proteins that determine whether CAG extrusions are processed by FAN1 or the mismatch repair pathway.