Depletion of Bcl-xL Impairs Mitochondrial Motility in Primary Hippocampal Neurons

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ObjectivesB-cell lymphoma-extra large (Bcl-xL) is a pro-survival protein localized to mitochondria and is also reported to support brain function by enhancing neuronal energy metabolism and synapse formation. We have previously shown that Bcl-xL is required for neurite outgrowth, and neurons lacking Bcl-xL were susceptible against neurotoxic challenges. In this study, we hypothesized that Bcl-xL supports maintaining neurite ATP by regulating mitochondrial motility. We thus tested if Bcl-xL depletion altered normal mitochondrial dynamics, neuronal energy retention, and neurite morphology. MethodsPrimary hippocampal neurons were transduced with either Bcl-xL shRNA or scrambled shRNA for 3 weeks. Mitochondria were labeled using mito-RFP BacMam2.0 and image sequences were obtained. Mitochondria motility parameters were quantified using KymoAnalyzer. Local ATP/ADP ratio was analyzed applying PercevalHR fluorescence biosensor, and neurite branches were quantified using Sholl analysis. We further tested viability of neurons against excitotoxicity applying calcein and propioduim iodin staining. ResultsPrimary hippocampal neurons transduced with Bcl-xL shRNA decreased antero- and retrograde movement of mitochondria, lowered ATP/ADP ratio in neurites, and decreased length of neurites and number of branching points. Failure of achieving neurite complexity increased susceptibility of neurons to glutamate-induced excitotoxicity. ConclusionsPrimary hippocampal neurons transduced with Bcl-xL shRNA decreased antero- and retrograde movement of mitochondria, lowered ATP/ADP ratio in neurites, and decreased length of neurites and number of branching points. Failure of achieving neurite complexity increased susceptibility of neurons to glutamate-induced excitotoxicity. Funding SourcesRGC Program (University of Alabama) Crenshaw Research Fund (University of Alabama) Sigma Xi Grants in Aid of Research (The National Academy of Sciences).