Neuronal DNA repair pathways in Huntington's disease pathophysiology

亨廷顿病(HD)是一种神经退行性疾病，由体内CAG重复束扩张引起 亨廷顿蛋白(HTT)基因，导致神经元死亡，主要在纹状体和皮质。CAG重复序列是 高度不稳定，在世代间和体细胞中都是如此。HD患者表现出高度的年龄- 纹状体中CAG重复的依赖性躯体扩张，尽管这种扩张也发生在其他 纸巾。最近对HD患者的全基因组关联研究揭示了基因的存在 疾病发病年龄的修饰物；其中包括几个与DNA修复有关的基因，尤其是 DNA错配修复(MSH3、MLH1、PMS2、PMS1)。此外，对多发性硬化症小鼠模型的研究表明 DNA错配修复基因Msh2、Msh3、MLH1或Mlh3的基因敲除减少了体细胞的不稳定性 在纹状体内重复CAG。这些数据表明，CAG重复序列的躯体扩张可能与 HD患者的纹状体神经元丢失和疾病症状的出现。因此，在目标1中，我们建议 描述和比较小鼠纹状体和其他识别小鼠大脑区域的蛋白质组合 和加工CAG挤出。我们预计，这些蛋白质复合体可能有助于阐明 CAG的扩张，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HEK293细胞中建立基于邻近生物素化(TurboID)的检测来评估 在含有CAG挤出物的DNA上瞬时和稳定的蛋白质组装，以期最终应用于 这种方法适用于神经元起源的细胞。MSH3等关键错配修复基因的敲除效应 或FAN1对此类DNA损伤的处理也将进行评估。这些方法的目标是 确定决定CAG挤出产物是由FAN1还是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.