Variations in the POLG1 gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma, have recently been associated with Parkinson's disease (PD), especially in patients diagnosed with progressive external ophthalmoplegia (PEO). However, the majority of the studies reporting this association mainly focused on the genetic identification of the variation in POLG1 in PD patient primary cells, and determination of mitochondrial DNA copy number, providing little information about the cellular alterations existing in patient brain cells, in particular dopaminergic neurons. Therefore, through the use of induced pluripotent stem cells (iPSCs), we assessed cellular alterations in novel p.Q811R POLG1 (POLG1Q811R) variant midbrain dopaminergic neuron-containing spheroids (MDNS) from a female patient who developed early-onset PD, and compared them to cultures derived from a healthy control of the same gender. Both POLG1 variant and control MDNS contained functional midbrain regionalized TH/FOXA2-positive dopaminergic neurons, capable of releasing dopamine. Western blot analysis identified the presence of high molecular weight oligomeric alpha-synuclein in POLG1Q811R MDNS compared to control cultures. In order to assess POLG1Q811R-related cellular alterations within the MDNS, we applied mass-spectrometry based quantitative proteomic analysis. In total, 6749 proteins were identified, with 61 significantly differentially expressed between POLG1Q811R and control samples. Pro- and anti-inflammatory signaling and pathways involved in energy metabolism were altered. Notably, increased glycolysis in POLG1Q811R MDNS was suggested by the increase in PFKM and LDHA levels and confirmed using functional analysis of glycolytic rate and oxygen consumption levels. Our results validate the use of iPSCs to assess cellular alterations in relation to PD pathogenesis, in a unique PD patient carrying a novel p.Q811R variation in POLG1, and identify several altered pathways that may be relevant to PD pathogenesis.

中文翻译：

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从多能干细胞衍生的中脑球体中鉴定出的细胞改变，该球体是由患有渐进性外部眼肌麻痹和帕金森氏症的女性患者产生的，该患者在POLG1基因中带有新型变异（p.Q811R）。

最近，编码线粒体DNA聚合酶γ催化亚基的POLG1基因变异与帕金森氏病（PD）有关，特别是在诊断为进行性眼外肌麻痹（PEO）的患者中。然而，大多数报道这种关联的研究主要集中于对PD患者原代细胞中POLG1变异的遗传鉴定，以及线粒体DNA拷贝数的确定，尤其是关于患者脑细胞中存在的细胞改变的信息很少。多巴胺能神经元。因此，通过使用诱导多能干细胞（iPSC），我们评估了来自患有早期PD的女性患者的新型p.Q811R POLG1（POLG1Q811R）变异中脑多巴胺能神经元球体（MDNS）的细胞变化，并将其与源自相同性别健康对照的文化进行比较。POLG1变体和对照MDNS都包含功能性中脑区域性TH / FOXA2阳性多巴胺能神经元，能够释放多巴胺。Western印迹分析确定与对照培养相比，POLG1Q811R MDNS中存在高分子量寡聚α-突触核蛋白。为了评估MDNS中与POLG1Q811R相关的细胞变化，我们应用了基于质谱的定量蛋白质组学分析。总共鉴定出6749种蛋白质，其中POLG1Q811R与对照样品之间有61种显着差异表达。促炎和抗炎信号传导以及参与能量代谢的途径都发生了改变。尤其，PFKM和LDHA含量的增加表明POLG1Q811R MDNS中糖酵解的增加，并通过糖酵解速率和耗氧量的功能分析得到证实。我们的结果验证了在携带POLG1中新型p.Q811R变异的独特PD患者中，使用iPSC评估与PD发病相关的细胞改变的可能性，并确定了可能与PD发病相关的几种改变的途径。