Mechanistic insights into neurodegeneration in Huntington's disease using patient-derived neurons through direct conversion of fibroblasts

亨廷顿氏病（HD）是一种遗传性成人发病的神经退行性疾病， 亨廷顿蛋白（HTT）基因中CAG密码子的扩增。HD的特征在于突变HTT的聚集 （mHTT）蛋白和选择性变性的纹状体中型棘神经元（MSN）。HD建模使用 患者来源的神经元一直具有挑战性，主要是由于缺乏实验方法来获得 HD患者的成年神经元。我们之前的工作表明，人类MSN可以通过 通过直接细胞命运转换（重编程）从成人皮肤成纤维细胞获得高效率和特异性 使用microRNA和转录因子。重要的是，转化的人类MSN类似于神经元。 成年人，这是建模迟发性疾病的重要特征。然而，直接转换的效用 MSN作为成人发病HD的细胞模型仍有待确定。最近，我们的前期工作 证明MSN可以由HD患者的直接转化成纤维细胞（HD-MSN）产生， 由此产生的HD-MSN表现出HD病理学的关键标志，如mHTT聚集体、DNA 损伤和培养物中的自发细胞死亡。在目前的授权中，我们建议使用HD-MSN作为蜂窝网络。 模型的HD和定义的遗传因素，减轻神经元死亡的HD-MSN。在目标1中，我们专注于 SP 9是一种转录因子，我们发现它在HD-MSNs中的表达显著下调， 来自健康个体的对照MSN（Ctrl-MSN）。有趣的是，据报道，SP 9是 维持和生存的MSN，我们发现，加强SP 9表达在HD-MSN保护 防止细胞自发死亡为了确定SP 9在HD-MSN中的神经保护作用，我们将确定 指导SP 9的靶基因，并揭示SP 9促进HD-MSN存活功能的基因。在目标2中， 我们将研究灵长类特异性microRNA，miR-663 b作为HD中神经保护性miRNA的功能。 MSN。我们的初步工作表明，miR-663 b可以保护MSN免受氧化应激诱导的损伤。 神经变性考虑到HD-MSN中氧化细胞应激和神经变性之间的联系，我们 将测试增加HD-MSN中的miR-663 b水平是否会赋予神经保护作用，并确定直接靶点。 miR-663 b的基因以描绘miR-663 b在HD-MSN中的功能。在目标3中，我们将确定遗传 负责不同阶段的MSN之间对神经元死亡的差异脆弱性的途径 疾病进展。我们发现，HD-MSNs产生于临床发病前取样的成纤维细胞， 症状（前HD-MSN）显示出显着较低程度的DNA损伤和细胞死亡相比， 到临床症状发作后衍生的HD-MSN。我们将进行转录组分析， HD前MSNs和有症状的HD-MSNs之间差异表达的基因， 表达的基因负责对神经元死亡的差异脆弱性。总的来说， 目前的建议将提供使用患者衍生的神经元的HD中的神经元死亡的见解。

Huntington’s disease (HD) is an inherited adult-onset neurodegenerative disorder caused by an abnormal expansion of CAG codons in the huntingtin (HTT) gene. HD is characterized by the aggregation of mutant HTT (mHTT) protein and selective degeneration of striatal medium spiny neurons (MSNs). Modeling HD using patient-derived neurons has been challenging mainly due to the lack of experimental approaches to obtain adult neurons from HD patients. Our previous work demonstrated that human MSNs could be generated with high efficiency and specificity from adult skin fibroblasts through direct cell fate conversion (reprogramming) using microRNAs and transcription factors. Importantly, the converted human MSNs resembled the neurons of human adults, an important feature for modeling late-onset diseases. However, the utility of directly converted MSNs as a cellular model of adult-onset HD remained to be determined. Recently, our preliminary work demonstrated that MSNs could be generated from directly converting fibroblasts of HD patients (HD-MSNs), and the resulting HD-MSNs manifested key hallmarks of HD pathology such as mHTT aggregates, DNA damage, and spontaneous cell death in culture. In the current grant, we propose to use HD-MSNs as a cellular model of HD and define genetic factors that alleviate the neuronal death of HD-MSNs. In Aim 1, we focus on SP9, a transcription factor that we found to be significantly downregulated in HD-MSNs in comparison to control MSNs from healthy individuals (Ctrl-MSNs). Interestingly, SP9 has been reported to be required for the maintenance and survival of MSNs, and we discovered that enforcing SP9 expression in HD-MSNs protected the cells from spontaneous cell death. To define the neuroprotective role of SP9 in HD-MSNs, we will identify direct target genes of SP9 and reveal genes integral to SP9’s function to promote HD-MSN survival. In Aim 2, we will investigate the function a primate-specific microRNA, miR-663b as a neuroprotective miRNA in HD- MSNs. Our preliminary work indicated that miR-663b protected MSNs from oxidative stress-induced neurodegeneration. Given the link between oxidative cellular stress and neurodegeneration in HD-MSNs, we will test if increasing the miR-663b level in HD-MSNs would confer a neuroprotection and identify direct target genes of miR-663b to delineate the function of miR-663b in HD-MSNs. In Aim 3, we will identify genetic pathways responsible for differential vulnerability to neuronal death between MSNs at different stages of disease progression. We found that HD-MSNs generated from fibroblasts sampled before the onset of clinical symptoms (pre-HD-MSNs) displayed significantly lower degrees of DNA damage and cell death in comparison to HD-MSNs derived after the onset of clinical symptoms. We will conduct transcriptome analysis to identify differentially expressed genes between pre-HD-MSNs and symptomatic HD-MSNs and identify differentially expressed genes responsible for the differential vulnerability to neuronal death. Overall, results from the current proposal will provide insights to neuronal death in HD using patient-derived neurons.