Elucidating the Role of Store Operated Calcium Entry in Human Oligodendrocyte Progenitor Cells
MetadataShow full item record
Multiple sclerosis is a chronic inflammatory demyelinating disease characterized by limited neuronal remyelination, which results in impaired saltatory conduction of signals, axonal atrophy and eventually irreversible neurodegeneration. Our previous studies showed that muscarinic receptor (M1/3R) antagonists induced human OPC (hOPC) differentiation and accelerated myelination in an in vivo model of hypomyelination. Furthermore, lentiviral knock down of M3R in hOPCs increased oligodendrocyte differentiation and, in mice, conditional knock out of M3R in OPCs led to enhanced remyelination thus demonstrating an important regulatory role of M3R in OPC differentiation. We found that muscarinic agonist treatment induces intracellular Ca2+ store depletion and subsequent induction of store operated calcium entry (SOCE). SOCE is known to mediate signaling events and regulate gene expression downstream of Gq-coupled receptors such as M3R. In this study, we hypothesized that the SOCE Ca2+ response is necessary for the anti- differentiative effect of M3R and that antagonism of SOCE would block the effect of muscarinic agonist on hOPC differentiation. SOCE is mediated by stromal interaction molecule (STIM1&2), the ER Ca2+ sensor which interacts directly with Orai1, the pore forming element to form a Ca2+ specific channel, CRAC channel. STIM is also reported to activate transient receptor potential canonical 1 (TRPC1) channel which contributes to SOCE currents in many cells. We first assessed the expression of SOCE machinery in hOPCs and the pattern of expression during mouse development and in MS patient lesions. Moreover, we investigated the physiological role of TRPC1, STIM 1 and 2 proteins by blocking their function in hOPCs. TRPC1 was blocked using TRPC1 specific antibody and siRNA against STIM1 and 2 genes in hOPCs, the effect on differentiation was analyzed in vitro. Blocking the function of TRPC1 channel with a specific antibody resulted in reduction in Calcium influx currents post store depletion induced by muscarinic activation in hOPCs. Moreover, TRPC1 channel blockade and gene downregulation reduced hOPCs differentiation in vitro. Using a pool siRNA against STIM1 and STIM2, revealed the importance of STIM2 in driving hOPCs differentiation where downregulation of STIM2 resulted in significant inhibition of differentiation. Whereas knocking down STIM1 had no influence on hOPCs differentiation. In addition, we assessed the functional role of STIM1 and STIM2 in hOPCs utilizing individual siRNA but we detected variability in the phenotype exhibited by using individual siRNA against STIM1 and STIM2 of comparable knock down efficiency. Therefore, we developed lentivirus for overexpressing STIM1 and STIM2 in hOPCs to study the influence of the gain of function on hOPCs differentiation. When STIM1 gene was upregulated in hOPCs, we observed no significant change on hOPCs differentiation while overexpressing STIM2 in our preliminary studies resulted in enhancement of hOPCs differentiation, these results were in consistence with down regulation of STIM1 and STIM2 in hOPCs using pool siRNA. Furthermore, we used STIM1/2 floxed mice to study the role of SOCE in adult murine NG2+ OPCs during remyelination in vivo. Interestingly, our pilot study indicated enhancement in OPCs differentiation evident by increase in CC1+ oligodendrocytes in mice with double deletion of STIM1 and STIM2 in the lysolecithin induced hypomyelinated lesion area in comparison with the wild type animals. Together these studies will provide additional understanding of and mechanistic insight into M1/3R signaling in the regulation of OPC fate. The functional importance of Ca2+ influx via SOCE may represent a final common pathway for failed OPC differentiation and represents an intriguing target for future therapeutic intervention.