FAM3A Protects Against Glutamate-Induced Toxicity by Preserving Calcium Homeostasis in Differentiated PC12 Cells

Abstract

Background/Aims: Stroke is the leading cause of adult disability, and glutamate-induced dysregulation of intracellular Ca²⁺ homeostasis is a key mechanism. FAM3A is the first member of the family with sequence similarity 3 (FAM3) gene family, and its biological function remains largely unknown. We have recently reported that FAM3A exerts protective effects against oxidative stress and mitochondrial dysfunction in HT22 cells. Methods: Here, we investigated the protective effects of FAM3A using a glutamate-induced neuronal injury model in nerve growth factor (NGF)-differentiated PC12 cells. The protective effects were determined by measuring lactate dehydrogenase (LDH) release, apoptosis and mitochondrial oxidative stress. Ca²⁺ imaging was performed to detect changes in intracellular Ca²⁺ concentration in PC12 cells. The related molecular mechanisms were investigated by fluorescence staining, coimmunoprecipitation (Co-IP) and western blotting. Results: Upregulation of FAM3A by lentivirus transfection markedly decreased LDH release, inhibited apoptosis and reduced mitochondrial oxidative stress, which were accompanied by alleviated intracellular Ca²⁺ levels as measured by calcium imaging. The results of western blotting showed that FAM3A significantly decreased the surface expression of metabotropic glutamate receptor 1/5 (mGluR1/5), with no effect on the expression of N-methyl-d-aspartic acid receptor (NMDAR) or α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) subunits. FAM3A overexpression also inhibited the intracellular Ca²⁺ release mediated by mGluR1/5 and inositol 1,4,5-trisphosphate receptor (IP₃R), but not the ryanodine receptor (RyR). In addition, FAM3A significantly attenuated the store-operated calcium entry (SOCE) induced by thapsigargin (Tg), but the expression of SOCE-related proteins was not altered. The results of coimmunoprecipitation (Co-IP) showed that FAM3A disrupted the interaction of stromal interaction molecule 1 (STIM1) with Orai1 triggered by glutamate. Conclusion: These results suggest that the upregulation of FAM3A protects against glutamate-induced dysfunction of Ca²⁺ homeostasis not only by inhibiting mGluR1/5-dependent endoplasmic reticulum (ER) Ca²⁺ release, but also by attenuating SOCE mediated by the STIM1-Orai1 interaction.