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Loss of ZIP12 Leads to Mitochondrial Dysfunction and Reduced Neurite Outgrowth in Neuro‐2A Neuroblastoma Cells
Author(s) -
Tang Tony Z.,
Hart Matthew D.,
Nacke Mariah R.,
Wu Lei,
Fiddler Joanna L.,
Clarke Stephen L.,
Smith Brenda J.,
Lin Dingbo,
Chowanadisai Winyoo
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.637.8
Subject(s) - neurite , microbiology and biotechnology , small hairpin rna , transfection , mitochondrion , rotenone , biology , chemistry , biochemistry , gene knockdown , apoptosis , in vitro , gene
Zinc is an essential micronutrient needed for proper neuronal development and brain function. Previous studies on the zinc transporter gene, ZIP12, have shown that it is necessary for neuronal development. However, it is not known what cellular processes require ZIP12 for neuronal function. Methods Loss of 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 cells were differentiated in medium containing retinoic acid (2% fetal bovine serum in DMEM + 20 uM retinoic acid) for 48 hours and assayed for cellular oxygen respiration, a measure of mitochondrial function, using the Seahorse XFe96 flux analyzer or neurite length by microscopy using ImageJ software and the NeuronJ plugin. Different measures of mitochondrial function include maximal respiration, proton leak and ATP production capacity were assessed by the Seahorse flux analyzer. Sensitivity of ZIP12‐depleted cells to a sublethal dose of rotenone, a mitochondrial inhibitor, was measured by incubating ZIP12‐depleted cells in 2.5 nM rotenone for 48 hours in differentiating media, followed by neurite imaging. The effect of ZIP12 KO on mitochondrial abundance during differentiation was quantified by Mitotracker Green fluorescence staining relative to Hoescht 33342 nuclear dye intensity, as measured using the BioTek Synergy HT plate reader. The effect of ZIP12 KO on neurite length during differentiation was also determined. 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. Rotenone exposure during differentiation resulted in significantly shorter neurites in ZIP12 shRNA transfected cells. Similarly, the ZIP12 KO cells displayed blunted neurite extension during differentiation compared to uncut control cells with functional ZIP12. Furthermore, mitochondrial abundance of ZIP12 KO cells during differentiation was also significantly decreased. Conclusions Loss of function of ZIP12 impairs neuronal development and mitochondrial function in Neuro‐2A cells during differentiation. ZIP12 may play an important role in the regulation of mitochondria which subsequently affects proper neuronal development. Support or Funding Information This work was supported by Faculty Startup funds from the Oklahoma State University Office of Research and a grant from the Oklahoma Center for the Advancement of Science and Technology (HR16‐060).