Roles Of TRPC1in Proliferation Inhibition Of Adult Neural Stem Cells Induced By Glucocorticoid

Objectives: The glucocorticoid(GC) is secreted by the rodent adrenal cortex inresponse to stress.exposure to stressor activation of the hypothalamic-pituitary-adrenal(HPA) axis and elevation of glucocorticoids are involved in learning and memorydamage.Extensive research has shown that the hippocampus correlates significantly withmemory formation and is also very susceptible to a high level of glucocorticoid,GCdecrease the proliferation of adult hippocampal neural stem cells,but the mechanism needsfurther research.It is known that Ca2+is a ubiquitous second messenger that affects cellproliferation. The Ca2+influx through TRPC1contributes to proliferation of embryonic ratNSCs.If TRPC1expressed in adult hippocampal neural stem cells is unclear. proliferation ofNSCs and neurogenesis were dependent on PI3K/Akt and CREB signaling. This study hasidentified TRPC1-mediated regulatory mechanism of GC-induced neural stem cellproliferation inhibition which may have significant implications for prevention andtreatment of leaning and memory injury, disease.Methods:(1) Immunocytochemistry is used to test the self-renewal capacity and multipotency ofthe adult NPCs.(2) The BrdU incorporation assay with a labeling and detection kit and CCK8detection kit were used to assay cell proliferation In vitro.(3) Real-time RT-PCR, Western blotting were used to detect the expression ofTRPC1-7, CREB, Akt.(4) Changes of intracellular Ca2+were recorded by laser scanning confocalmicroscope..(5) siRNA and construction of shRNA lentivirus expression vectors were used todetect the proliferation and SOCE in adult hippocampal neural stem cells.(6) TRPC1-antibody-treated cells were used to detect the proliferation and SOCE in adult hippocampal neural stem cells.(7) Cell cycle analysis and Real-time RT-PCR-based gene array assay were used todetect NSCs cell cycle regulation and gene expression.(8) Immunohistochemistry was used to observe the expression of TRPC1/SOX2,TRPC1/Orai1/STIM1in adult hippocampal neural stem cells.Results:(1) GC inhibits the proliferation of adult hippocampal neural stem cells.(2) TRPC1is involved in the proliferation of adult hippocampal neural stem cells.(3) TRPC1is expressed in adult hippocampal neural stem cells.(4) TRPC1is expressed in adult hippocampal SGZ in vivo. GC inhibits geneexpression of TRPC1.(5) SOCE in proliferating aNSC,2-APB, Gd3+and SKF96365, antagonists of SOCEand TRPC, respectively, inhibited the increase in SOCE and aNSCs proliferation.(6) TRPC1expressed in aNSC.knockdown of TRPC1and using an antibody againstTRPC1markedly reduced the degree of SOCE and aNSCs proliferation.(7) Suppression of aNSC proliferation was found to be associated with cell cycle arrestin G0/G1phase. The expression level of10genes changed at least2-fold followingknockdown of TRPC1, indicating these genes may mediate the effects of TRPC1on aNSCsproliferation.(8) Knockdown of Orai1or STIM1attenuated SOCE and proliferation in aNSCs.(9) GC inhibits the SOCE of proliferating aNSCs, whereas overexpression of TRPC1reverses the reduction of SOCE induced by GC.(10) Silencing of TRPC1inhibits the protein phosphorylation of Akt and CREB ofaNSCs.GC inhibits the protein phosphorylation of Akt and CREB.Conclusions:(1) GC inhibits the proliferation of adult hippocampal neural stem cells. TRPC1isexpressed in adult hippocampal SGZ in vivo.(2) GC inhibits gene expression of TRPC1. TRPC1is involved in the proliferation ofadult hippocampal neural stem cells.(3) SOCE in proliferating aNSCss, Suppression of aNSCs proliferation was found tobe associated with cell cycle arrest in G0/G1phase. The expression level of10genes changed at least2-fold following knockdown of TRPC1, indicating these genes maymediate the effects of TRPC1on aNSCs proliferation.(4) Knockdown of Orai1or STIM1attenuated SOCE and proliferation in aNSCs,Orai1and STIM1are also required for TRPC1-SOC channels.(5) GC inhibits the SOCE of proliferating aNSCs, whereas overexpression of TRPC1reverses the reduction of SOCE induced by GC. TRPC1-mediated PI3K/Akt and CREBsignaling are involved in GC-induced neural stem cell proliferation inhibition.