A partial Drp1 knockout improves autophagy flux independent of mitochondrial function.
A partial Drp1 knockout improves autophagy flux independent of mitochondrial function.
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部分 Drp1 敲除可改善自噬通量，而与线粒体功能无关。
DOI：
10.1101/2023.06.29.547095
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Tieu,Kim
中科院分区：
文献类型：
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作者：
Fan,RebeccaZ;Sportelli,Carolina;Lai,Yanhao;Salehe,Said;Pinnell,JenniferR;Richardson,JasonR;Luo,Shouqing;Tieu,Kim
动力蛋白相关蛋白1 （Drp1）在线粒体动力学中起关键作用。该蛋白的部分抑制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。这种保护机制主要归因于线粒体功能的改善。然而，观察到Drp1抑制降低了这类神经系统疾病中的蛋白质聚集，表明自噬参与其中。为了研究Drp1抑制的这种潜在的新保护机制，需要一个没有线粒体参与的自噬受损模型。方法通过在两种细胞培养模型（稳定自噬的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，表征锰（Mn）对自噬和线粒体的影响，锰可引起人类帕金森样症状。使用海马通量分析仪评估线粒体功能。通过量化自噬体和自噬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，多种小鼠模型（自噬报告小鼠和突变型Drp1+/−小鼠及其野生型幼崽）口服低慢性Mn方案，该方案先前报道可增加α-突触核蛋白在外泌体中的聚集和传递。采用了RNAseq、激光捕获显微解剖、免疫荧光、免疫印迹、体视细胞计数和行为研究。结果表明，在低无毒浓度下，Mn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数据进一步证实了自噬通路的异常，但线粒体相关基因没有异常。此外，锰选择性地损害了神经多巴胺神经元的自噬，而不影响附近的神经GABA神经元。在Drp1部分下调的细胞和Drp1+/−小鼠中，Mn诱导的自噬损伤被显著阻止。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，而drp1敲低可以防止这种病理。结论本研究表明，自噬通量的改善是Drp1抑制所赋予的独立机制，独立于其在线粒体分裂中的作用。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突出特征，Drp1抑制所赋予的针对这两种途径的联合保护机制使该蛋白成为一个有吸引力的治疗靶点。图形抽象
BackgroundDynamin-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.MethodsWe 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 transmissionviaexosomes. RNAseq, laser captured microdissection, immunofluorescence, immunoblotting, stereological cell counting, and behavioural studies were used.Results in vitrodata 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.ConclusionsThis 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.Graphical Abstract