Effects Of Autophagic Activator / Inhibitor On Proliferation And Differentiation Of Mouse Neural Stem Cells

Objective:To observe the effect of autophagy on proliferation and differentiation of newborn mouse and adult mouse neural stem cells(NSCs),so as to reveal the protective effect of activated autophagy of adult NSCs,which would further looking out a feasible strategy to maintain NSCs' viability.Method:1.NSCs cultured in vitro were dissociated from mice subventricular zone(SVZ)at postnatal day 0(P0).P0 NSCs were treated with autophagic inhibitor Ly294002,and then LC3 immunofluorescence staining was performed to detect the changes of the autophagosomes.P0 NSCs' vitality was analyzed through Brd U incorporation assay.In the process of differentiation of P0 NSCs,the level of autophagy-related proteins LC3 II and p62 expression were analyzed by western blot.The differentiation of P0 NSCs were treated with autophagic inhibitor Ly294002,immunofluorescence staining was performed to detect the changes of the Tuj1 positive cells.2.NSCs cultured in vitro were dissociated from mice SVZ at postnatal days 90(P90).P90 NSCs were treated with autophagic activator rapamycin,and LC3 immunofluorescence staining was performed to detect the changes of the autophagosomes.P90 NSCs' proliferation and differentiation capacity were analyzed through Brd U incorporation assay and Tuj1 immunofluorescence staining.3.The adult mice were injected intraperitoneally with rapamycin at dose of 4mg / kg(one injection once a day for 4 days).Extract the adult mice SVZ protein and analysis the level of p-m TOR expression.The effects of autophagy on the proliferation and differentiation were observed by Ki67 and DCX immunofluorescence staining.Result:1.Autophagy inhibition with Ly294002 applied on P0 NSCs for 3 days in vitro.LC3 immunofluorescence staining results showed that Ly294002 could reduce the autophagy levels of P0 NSCs.But no differences were detected between the positive rate of Brd U in the control group and Ly294002 group.The level of LC3 II expression was increased 1.0± 0.5 times than before differentiation(p < 0.05).In addition,the level of p62 expression was reduced by 0.5 ± 0.1 compared with the undifferentiated group by western blot(p <0.01).So indicating that autophagy may participate in NSCs ' differentiation ability.Furthermore,The differentiation of P0 NSCs were treated with 5?M and 10?M Ly294002,Tuj1 positive rate was 0.6 and 0.7 times significantly lower than the control group,respectively.These data demonstrated that P0 NSCs were inhibited to differentiate into neurons by reducing autophagy levels.2.Autophagy activation with rapamycin applied on P90 NSCs in vitro.LC3 immunofluorescence staining results showed that rapmaycin could improve the autophagy levels of P90 NSCs.At the same time,the positive rate of Brd U was 25.4% ± 6.0% and 26.2% ± 6.7% in rapamycin group,respectively,which was significantly higher than that in control group(12.0% ± 0.8%).Those date showed that P90 NSCs' proliferation capacity was enhanced by improving autophagy levels.Furthermore,the differentiation of P90 NSCs were treated with 20 n M and 50 n M rapamycin,the Tuj1 positive rate of rapamycin group was increased 1.5 and 1.3 times compared with control group.Which showed that autophagy could promote the differentiation of P90 NSCs into neurons.3.The adult mice were injected intraperitoneally with rapamycin,the level of p-m TOR expression was decreased 34.0%±8.6% compared with control group(p < 0.05),which rapamycinc could increase the autophagy levels in mice of SVZ region.More importantly,immunofluorescence staining results showed that the number of Ki67 positive cells were 98.9 ± 8.1,which was significantly higher than that of DMSO group(61.2 ± 8.6)(p<0.05),and the positive number of DCX was 1.5 times compared with control group.These result revealed that autophagy may promote NSCs' the potential of proliferation and differentiation in vivo.Conclusions:Autophagy can help maintain mouse NSCs' vitality,and the changes in autophagy levels can affect the proliferation and differentiation of NSCs.Application of autophagic activator can effectively improve the vitality of adult mouse NSCs and promote the neurogenesis.