BACKGROUND Mutations in PINK1 and parkin cause autosomal recessive Parkinson's disease (PD). Evidence placing PINK1 and parkin in common pathways regulating multiple aspects of mitochondrial quality control is burgeoning. However, compelling evidence to causatively link specific PINK1/parkin dependent mitochondrial pathways to dopamine neuron degeneration in PD is lacking. Although PINK1 and parkin are known to regulate mitophagy, emerging data suggest that defects in mitophagy are unlikely to be of pathological relevance. Mitochondrial functions of PINK1 and parkin are also tied to their proteasomal regulation of specific substrates. In this study, we examined how PINK1/parkin mediated regulation of the pathogenic substrate PARIS impacts dopaminergic mitochondrial network homeostasis and neuronal survival in Drosophila. METHODS The UAS-Gal4 system was employed for cell-type specific expression of the various transgenes. Effects on dopamine neuronal survival and function were assessed by anti-TH immunostaining and negative geotaxis assays. Mitochondrial effects were probed by quantitative analysis of mito-GFP labeled dopaminergic mitochondria, assessment of mitochondrial abundance in dopamine neurons isolated by Fluorescence Activated Cell Sorting (FACS) and qRT-PCR analysis of dopaminergic factors that promote mitochondrial biogenesis. Statistical analyses employed two-tailed Student's T-test, one-way or two-way ANOVA as required and data considered significant when P < 0.05. RESULTS We show that defects in mitochondrial biogenesis drive adult onset progressive loss of dopamine neurons and motor deficits in Drosophila models of PINK1 or parkin insufficiency. Such defects result from PARIS dependent repression of dopaminergic PGC-1α and its downstream transcription factors NRF1 and TFAM that cooperatively promote mitochondrial biogenesis. Dopaminergic accumulation of human or Drosophila PARIS recapitulates these neurodegenerative phenotypes that are effectively reversed by PINK1, parkin or PGC-1α overexpression in vivo. To our knowledge, PARIS is the only co-substrate of PINK1 and parkin to specifically accumulate in the DA neurons and cause neurodegeneration and locomotor defects stemming from disrupted dopamine signaling. CONCLUSIONS Our findings identify a highly conserved role for PINK1 and parkin in regulating mitochondrial biogenesis and promoting mitochondrial health via the PARIS/ PGC-1α axis. The Drosophila models described here effectively recapitulate the cardinal PD phenotypes and thus will facilitate identification of novel regulators of mitochondrial biogenesis for physiologically relevant therapeutic interventions.

中文翻译：

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PARIS 诱导的线粒体生物发生缺陷在 Parkin 或 PINK1 缺陷的情况下导致多巴胺神经元损失。

背景 PINK1 和 Parkin 突变会导致常染色体隐性遗传帕金森病 (PD)。越来越多的证据表明 PINK1 和 Parkin 处于调节线粒体质量控制多个方面的共同途径中。然而，缺乏令人信服的证据将特定的 PINK1/parkin 依赖性线粒体途径与 PD 中的多巴胺神经元变性联系起来。尽管 PINK1 和 Parkin 已知可调节线粒体自噬，但新出现的数据表明线粒体自噬的缺陷不太可能具有病理相关性。PINK1 和 Parkin 的线粒体功能也与其特定底物的蛋白酶体调节有关。在这项研究中，我们研究了 PINK1/parkin 介导的致病底物 PARIS 的调节如何影响果蝇中多巴胺能线粒体网络稳态和神经元存活。方法 UAS-Gal4 系统用于各种转基因的细胞类型特异性表达。通过抗 TH 免疫染色和阴性趋地性测定评估对多巴胺神经元存活和功能的影响。通过定量分析 mito-GFP 标记的多巴胺能线粒体、评估荧光激活细胞分选 (FACS) 分离的多巴胺神经元中的线粒体丰度以及促进线粒体生物发生的多巴胺能因子的 qRT-PCR 分析来探讨线粒体效应。统计分析根据需要采用双尾学生 T 检验、单向或双向方差分析，并且当 P < 0.05 时数据被认为是显着的。结果我们发现，在 PINK1 或 Parkin 功能不全的果蝇模型中，线粒体生物发生的缺陷导致成年后多巴胺神经元进行性丧失和运动缺陷。这种缺陷是由于 PARIS 依赖性抑制多巴胺能 PGC-1α 及其下游转录因子 NRF1 和 TFAM 共同促进线粒体生物发生。人类或果蝇 PARIS 的多巴胺能积累概括了这些神经退行性表型，这些表型可通过体内 PINK1、parkin 或 PGC-1α 过表达有效逆转。据我们所知，PARIS 是 PINK1 和 Parkin 唯一在 DA 神经元中特异性积累的共底物，并因多巴胺信号传导中断而导致神经变性和运动缺陷。结论 我们的研究结果确定了 PINK1 和 Parkin 在通过 PARIS/PGC-1α 轴调节线粒体生物合成和促进线粒体健康方面具有高度保守的作用。这里描述的果蝇模型有效地概括了主要的 PD 表型，因此将有助于识别线粒体生物发生的新调节因子，以进行生理相关的治疗干预。