Loss of voltage dependent anion channel 1 affects mitochondrial bioenergetics in rat dopamine cells

Voltage dependent anion channel 1 (VDAC1) is a multi-functional protein that regulates mitochondrial membrane potential, calcium regulation, and apoptosis. VDAC1 also interacts with a number of signaling pathways important for energy homeostasis and proteins involved in neurodegenerative diseases such as alpha-synuclein. To identify novel signaling pathways dependent upon VDAC1 protein in dopamine cells, we used CRISPR-Cas9 gene editing in rat immortalized dopaminergic N27 cells. Western blot confirmed that VDAC1 protein levels were reduced ~90%. Mitochondrial bioenergetics was assessed to determine if there was functional loss of mitochondrial function without VDAC1. Loss of VDAC1 resulted in lower ATP-linked and maximum respiration, and spare respiratory capacity. Transcriptomics was conducted in these cells to identify the pathways perturbed by loss of VDAC. This study sheds novel insight into the different regulatory roles mediated by VDAC1 in dopamine cells.电压依赖性阴离子通道1（Voltage dependent anion channel 1, VDAC1）是一种多功能蛋白质，可调控线粒体膜电位、钙稳态与细胞凋亡。VDAC1还可与多条对能量稳态至关重要的信号通路，以及参与α-突触核蛋白（alpha-synuclein）相关神经退行性疾病的蛋白质发生相互作用。为鉴定多巴胺能细胞中依赖VDAC1蛋白的新型信号通路，我们在大鼠永生化多巴胺能N27细胞中采用了CRISPR-Cas9基因编辑技术。经蛋白质免疫印迹（Western Blot）验证，VDAC1的蛋白表达水平被下调约90%。我们通过检测线粒体生物能学，以评估敲除VDAC1后线粒体功能是否出现功能性损伤。结果显示，VDAC1缺失会导致ATP关联呼吸速率、最大呼吸速率及储备呼吸能力均显著下降。我们对这些细胞开展了转录组学（transcriptomics）分析，以鉴定因VDAC1缺失而发生扰动的信号通路。本研究为揭示VDAC1在多巴胺能细胞中介导的多种调控作用提供了全新的研究视角。