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Loss of ZIP12 Leads to Mitochondrial Dysfunction in Neuro-2A Neuroblastoma Cells
Intro: Zinc is an essential micronutrient needed for proper brain function. Previous studies on the zinc transporter gene, ZIP12, have shown that it is necessary for brain development. However, it is not known what cellular processes require ZIP12 for neuronal function.
Methods: Loss of zinc transporter ZIP12 function in Neuro-2A cells was achieved by ZIP12-targeted shRNA depletion via plasmid transfection or the generation of a ZIP12 knockout (KO) cell line by Cas9/CRISPR-mediated genome editing. ZIP12-depleted or ZIP12 KO cells were differentiated in medium containing retinoic acid (2% fetal bovine serum in DMEM + 20 uM retinoic acid) for 48 hours. ZIP12-depleted cells were assayed for cellular oxygen respiration, a measure of mitochondrial function, using the Seahorse XFe96 flux analyzer. Different measures of mitochondrial function include maximal respiration, proton leak and ATP production capacity were assessed by the Seahorse flux analyzer. The effect of ZIP12 KO on mitochondrial abundance during differentiation was quantified by Mitotracker Green fluorescence staining as measured using the BioTek Synergy HT plate reader and fluorescence microscopy.
Results: ZIP12-targeted shRNA depletion resulted in reduced ATP production, proton leak and maximal respiration of mitochondria compared to control shRNA transfected cells, as measured by the Seahorse flux analyzer. In addition, mitochondrial abundance of ZIP12 KO cells during differentiation was also significantly decreased. Furthermore, Mitotracker Green intensity is lower in ZIP12 KO cells as imaged by fluorescence microscopy.
Conclusion: Loss of function of ZIP12 impairs mitochondrial function in Neuro-2A cells during differentiation. ZIP12 may play an important role in the regulation of mitochondria, an organelle implicated in neurodegeneration and cognitive function.