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

亨廷顿病(HD)是一种遗传性成人起病的神经退行性疾病，由一种异常的 亨廷顿蛋白(Htt)基因CAG密码子的扩展。HD的特征是突变的HTT聚集在一起 (MHTT)蛋白与纹状体中棘神经元(MSN)选择性变性。高清建模使用 患者来源的神经元一直具有挑战性，主要是因为缺乏实验方法来获得 HD患者的成体神经元。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)获得高效率和特异性的成人皮肤成纤维细胞 使用microRNAs和转录因子。重要的是，转换后的人类MSN类似于 人类成年人，这是对迟发性疾病建模的一个重要特征。但是，直接转换为 MSNS作为成人型HD的细胞模型仍有待确定。最近，我们的前期工作 证明了MSN可以从HD患者的成纤维细胞(HD-MSN)直接转化而来， 由此产生的HD-MSN表现出HD病理的关键特征，如mHTT聚集体、DNA 损伤，以及培养中的自发性细胞死亡。在目前的拨款中，我们建议使用HD-MSN作为蜂窝 建立HD模型，定义减轻HD-MSN神经元死亡的遗传因素。在目标1中，我们重点关注 SP9，一种转录因子，我们发现在HD-MSN中显著下调 控制健康人的MSN(Ctrl-MSN)。有趣的是，据报道，SP9是 MSN的维持和存活，我们发现在HD-MSN中强制表达SP9具有保护作用 自发性细胞死亡的细胞。为了确定SP9在HD-MSN中的神经保护作用，我们将确定 指导SP9的靶基因，揭示与SP9‘S整合的基因促进HD-MSN存活的功能。在目标2中， 我们将研究灵长类动物特有的microRNA miR-663b作为神经保护miRNA在先天性巨噬细胞病中的功能。 MSNS。我们的初步工作表明miR-663b对氧化应激诱导的MSN具有保护作用 神经退行性变。鉴于HD-MSN中氧化细胞应激与神经变性之间的联系，我们 将测试增加HD-MSN中miR-663b的水平是否会提供神经保护并确定直接靶点 MiR-663b基因在HD-MSN中的作用。在目标3中，我们将识别基因 MSN不同阶段神经元死亡易感性差异的通路 疾病的发展。我们发现HD-MSN产生于临床发病前的成纤维细胞 与HD-MSNS前的症状相比，DNA损伤和细胞死亡的程度明显降低 至HD-MSNS发病后出现临床症状。我们将进行转录组分析以确定 HD-MSN前期和症状性HD-MSN的差异表达基因及其鉴定 表达了导致神经元死亡的差异易感性的基因。总体而言， 目前的提议将为使用患者来源的神经元来研究HD中的神经元死亡提供见解。

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.