Dynamin-related protein 1 (Drp1) plays a critical role in mitochondrial dynamics. Partial inhibition of this protein is protective in experimental models of neurological disorders such as Parkinson’s disease and Alzheimer’s disease. The protective mechanism has been attributed primarily to improved mitochondrial function. However, the observations that Drp1 inhibition reduces protein aggregation in such neurological disorders suggest the involvement of autophagy. To investigate this potential novel protective mechanism of Drp1 inhibition, a model with impaired autophagy without mitochondrial involvement is needed. We characterized the effects of manganese (Mn), which causes parkinsonian-like symptoms in humans, on autophagy and mitochondria by performing dose-response studies in two cell culture models (stable autophagy HeLa reporter cells and N27 rat immortalized dopamine neuronal cells). Mitochondrial function was assessed using the Seahorse Flux Analyzer. Autophagy flux was monitored by quantifying the number of autophagosomes and autolysosomes, as well as the levels of other autophagy proteins. To strengthen the in vitro data, multiple mouse models (autophagy reporter mice and mutant Drp1+/− mice and their wild-type littermates) were orally treated with a low chronic Mn regimen that was previously reported to increase α-synuclein aggregation and transmission via exosomes. RNAseq, laser captured microdissection, immunofluorescence, immunoblotting, stereological cell counting, and behavioural studies were used. data demonstrate that at low non-toxic concentrations, Mn impaired autophagy flux but not mitochondrial function and morphology. In the mouse midbrain, RNAseq data further confirmed autophagy pathways were dysregulated but not mitochondrial related genes. Additionally, Mn selectively impaired autophagy in the nigral dopamine neurons but not the nearby nigral GABA neurons. In cells with a partial Drp1-knockdown and Drp1+/− mice, Mn induced autophagic impairment was significantly prevented. Consistent with these observations, Mn increased the levels of proteinase-K resistant α-synuclein and Drp1-knockdown protected against this pathology. This study demonstrates that improved autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of its role in mitochondrial fission. Given that impaired autophagy and mitochondrial dysfunction are two prominent features of neurodegenerative diseases, the combined protective mechanisms targeting these two pathways conferred by Drp1 inhibition make this protein an attractive therapeutic target.

中文翻译：

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部分 Drp1 敲除可改善自噬通量，与线粒体功能无关

Dynamin 相关蛋白 1 (Drp1) 在线粒体动力学中发挥着关键作用。部分抑制该蛋白在帕金森病和阿尔茨海默病等神经系统疾病的实验模型中具有保护作用。保护机制主要归因于线粒体功能的改善。然而，Drp1 抑制可减少此类神经系统疾病中蛋白质聚集的观察结果表明自噬的参与。为了研究 Drp1 抑制的这种潜在的新型保护机制，需要一种自噬受损但不涉及线粒体的模型。我们通过在两种细胞培养模型（稳定自噬 HeLa 报告细胞和 N27 大鼠永生化多巴胺神经元细胞）中进行剂量反应研究，描述了锰（Mn）对自噬和线粒体的影响，锰（Mn）会导致人类帕金森病样症状。使用 Seahorse Flux 分析仪评估线粒体功能。通过量化自噬体和自溶酶体的数量以及其他自噬蛋白的水平来监测自噬通量。为了加强体外数据，多个小鼠模型（自噬报告小鼠和突变型 Drp1+/- 小鼠及其野生型同窝小鼠）接受了低慢性 Mn 方案的口服治疗，先前报道该方案可增加 α-突触核蛋白通过外泌体的聚集和传输。使用RNAseq、激光捕获显微切割、免疫荧光、免疫印迹、体视细胞计数和行为研究。数据表明，在低无毒浓度下，锰会损害自噬通量，但不会损害线粒体功能和形态。在小鼠中脑中，RNAseq 数据进一步证实自噬途径失调，但线粒体相关基因并未失调。此外，Mn 选择性地损害黑质多巴胺神经元的自噬，但不损害附近的黑质 GABA 神经元。在部分 Drp1 敲低的细胞和 Drp1+/- 小鼠中，Mn 诱导的自噬损伤被显着预防。与这些观察结果一致，Mn 增加了蛋白酶 K 抗性 α-突触核蛋白的水平，并且 Drp1 敲低可防止这种病理。这项研究表明，自噬流的改善是 Drp1 抑制所赋予的一种独立机制，与其在线粒体裂变中的作用无关。鉴于自噬受损和线粒体功能障碍是神经退行性疾病的两个显着特征，Drp1 抑制赋予的针对这两种途径的联合保护机制使该蛋白成为有吸引力的治疗靶点。