Potential Role Of20S Proteasome In Regulation Of Neural Stem Cells Aging

Objective:In vivo and in vitro, to observe the aging markers and proliferation potention of NSCs (Neural Stem Cells) in different age stages, so as to reveal the prosses of cell aging. Based on above mentioned study, in this experiment, we deeply explored the regulation effects of20S proteasome and its core subunit PSMB1and PSMB5in NSCs aging process, confirming the effects of the activity changes of20S proteasome on the NSCs proliferation capacity, unfolding the aging mechanism, providing a new strategy for recovering stem cells vitality.Method:E14(Embryonic days14)、P0(Postnatal day0)、P90(Postnatal days90) and P540(Postnatal days540) mices for the research object.1.The number of aging cell in the brain of mice were observed using the SA-β-gal(senescence-associated β-galactosidase) staining method, deciding the degree of brain aging; At the same time the NSCs potential proliferation in the SVZ (subventricular zone) were tested by the BrdU marking method for intraperitoneal injection BrdU.2. Different aging NSCs of mice for cultruing in vitro, the proliferation capacity and expression changes of aging marker in the different aging NSCs were further detected by the BrdU mixed and SA-β-gal dyeing method.3. The expression of.20S proteasome in NSCs is tested by20S/nestin immunofluorescence double-labeling.4.20S proteasome activity were tested using fluorescence spectroscopy method in different aging NSCs.5. The expressions of20S proteasome and subunit PSMB1and PSMB5in the gene level were analysed by the SYBR Green Real-time PCR.6.Protease body inhibitors MG132applied in the cultured NSCs in vitro,so as to detect the effects of different concentrations of MG132on the proliferation potention of NSCs using the BrdU mixed experiment and CCK-8method.Results:1. SA-β-gal staining showed that the SVZ zone of mouse brain is around large blue colored cytoplasmic which is the positive cells of SA-β-gal in P90and P540mice, While it is occasionally in the SVZ zone of E14and PO mice. Tips to increase with age in the brain of SA-β-gal activity enhanced, aging cells gradually increased. With the SA-β-gal activity is upregulated in P90and P540mice, the BrdU+cells of SVZ zone decreased dramatically, compared with E14mice decreased92%and95%, BrdU+cells scattered in P90and P540mice dorsal lateral horn lateral ventricle and lateral wall. The results showed reduction of adulthood and old age NSCs proliferative potential.2. The neurosphere forming cycle of P90and P540 mice is longer than E14and PO mice in vitro. With E14and PO mice (38.0%±2.0%,20.8%±1.24%,p<0.01), the BrdU positive rates of P90and P540mice (4.1%±0.4%,8.4%±0.9%, p<0.01) were significantly reduced. SA-β-gal staining showed that P90and P540positive cells increased significantly, SA-β-gal positive rates were26%±0.8%and27.6%±1.2%(p<0.01), the in vitro findings further indicate that adulthood proliferative capacity of NSCs decreased, NSCs appear to the aging phenomenon.3.20S/nestin immunofluorescence staining showed that the NSCs express20S proteasome,20S proteasome is distributed in NSCs cytoplasm and nucleus.4. We detected20S proteasome activity from different age stages of NSCs with uorescence spectrophotometric. The result shows that the P90group NSCs20S proteasome activity is0.8±0.2times than the E14group (p<0.01), P540group NSCs20S proteasome activity is0.6±0.1times than the E14group (p<0.01), suggesting that adulthood and old NSCs20S proteasome activity is downregulated.5. Real-time PCR quantitative analysis20S proteasome core subunits PSMBland PSMB5expression of mRNA from different age stages of NSCs, In P90group, the PSMB1mRNA expression level is35%±1%(p<0.001) of the E14group, and the PSMB5mRNA expression level is41%±1%(p<0.001) of the E14group. In P540group, the PSMB1mRNA expression level is34%±8%(p<0.001) of the E14group, and the PSMB5mRNA expression level is66%±1%(p<0.001) of the E14group, suggesting that the expression level of20S proteasome core subunits PSMBland PSMB5mRNA in adulthood and old age is decreased.6. Proteasome inhibitor MG132effects on NSCs for5h in vitro. BrdU incorporation experiments results showed that0.8M MG132could obviously inhibit the proliferative capacity of NSCs, the positive rate of BrdU with DMSO solvent control group decreased by22.7%(p<0.05), and this inhibition showed a dose dependent. CCK-8detection results show that NSCs viability gradually decreased with increasing MG132concentration.20μM MG132can make NSCs proliferative capacity reduced by44%p<0.05), suggesting that proteasome activity down-regulation may be the important factors of NSCs proliferative potential reduction in the process of aging.Conclusion:1.With the increasing age, NSCs proliferative potential is decreased, and the senescence markers expression level is increased. NSCs appear to the aging phenomenon.2. In adult and aging,20S proteasome activity and its core subunits PMSB1and PMSB5mRNA expression level decreased. e may be involved in the regulation of the NSCs aging process.3. Proteasome inhibitors can significantly reduce the NSCs proliferation capacity, suggesting that the20S proteasom may maintain the proliferative potential of NSCs.