The Biological Effects And Underlying Mechanism Of GDF11 On Mouse Neural Stem Cells

BackgroundAround the world,advances in medicine and public health measures and improvement in living standards have greatly prolonged human life span.With the number of aged people precipitously increasing,more and more subjects suffer from age-related cognitive decline.Age-related cognitive decline,with the main symptom of context memory decline,is a common degenerative disease of the central nervous system.With the features of long disease duration,slow progress and complex etiology,age-related cognitive decline remains incurable,and even there are few drugs that can prevent or delay its progress.Age-related cognitive decline which brings heavy burdens to the patients,their families and society,has become an important social issue.A looming question is to develop new strategies for treating or delaying age-related cognitive decline.Growth differentiation factor 11(GDF11)is a member of transforming growth factor β(TGF-β)superfamily.GDF11,highly homologous with myostatin(also known as GDF8),participates in modulating proliferation and differentiation,and is recognized as a negative regulator of muscle growth and neurogenesis.Recent studies suggested that GDF11 in blood was a rejuvenation factor to repair injured skeletal muscle,increase dendritic spine density,enhance cerebral blood flow and function,improve cognitive function,etc.However,several studies questioned the positive effects of GDF11,and they implied that GDF11 was not with the effect of anti-ageing.At present,the studies of GDF11 on neural stem cells mainly focus on the role of GDF11 on proliferation and differentiation,and it is not entirely clear about the effects of GDF11 on neural cells.The studies of the underlying mechanism primarily focus on TGF-β/Smad signal pathway,however the detailed mechanism of action is still not clear.Therefore,to further study the effects and mechanism of action of GDF11 on neurogenesis is quite important to confirm GDF11 as a rejuvenation factor or a blockage target for age-related cognitive decline,and to develop new drugs for treating or delaying age-related cognitive decline.ObjectiveThis study aims to clarify the effects of GDF11 on the life activities of neural stem cells(including proliferation,differentiation,apoptosis,cell cycle,migration,etc.),and to investigate the mechanism of action.Methods1.PC12,C17.2 and HT22 neural stem cells were used to systematicly analyze the effect of GDF11 on the life activities of neural stem cells(including proliferation,differentiation,apoptosis,cell cycle,migration,etc.)via the methods of CCK-8,real time cellular analysis(RTCA),cell population doubling,flow cytometry,CalceinAM/EthD-1 staining,Annexin V-FITC/PI staining,Hoechst/PI staining,scratch wound healing assay,western blot and qRT-PCR.2.We used TMT-based quantitative proteomics analysis to reveal the changes of proteomic profiles of PC12 cells when treated with or without GDF11,and then bioinformatics analysis was applied to explore the key differentially expressed proteins(DEPs)and signal pathways.Furthermore,signaling pathway antibody microarray(CSP100 PLUS)was performed to analyze the major enriched signal pathway that GDF11 regulated the cellular behaviors of neural stem cells.3.The measures of western blot,qRT-PCR,CCK-8,flow cytometry,RTCA and scratch wound healing assay were employed to study the roles of the screened signal pathway on GDF11 regulating neural stem cells,and to explore the underlying mechanism.Results1.The results of CCK-8,RTCA and cell population doubling showed that GDF11 had no effect on the proliferation(or cell viability)of PC 12,Cl7.2 and HT22 neural stem cell lines within 48 h,however high concentrations of GDF11(50 and 100 ng/mL)significantly inhibited the proliferation of neural stem cell for long-term treatment(>70 h).By cytometry and fluorescence microscope,it revealed GDF11 substantially induced apoptosis of PC 12,C17.2 and HT22 neural stem cells with significant dose-dependent effects.And GDF11 obviously arrested the cell cycle of neural stem cells at G2/M by down-regulating Cyclin D2 and Cyclin B1.Scratch wound healing assay highlighted GDF11 suppressed the migratory potential of neural stem cells.GDF11 significantly down-regulated the protein and mRNA levels of nestin(neural stem cell biomarker)and up-regulated βⅢ-tubulin(neuronal biomarker)and GFAP(astrocytic biomarker).2.The results of TMT-based quantitative proteomics analysis displayed there were 165 proteins that were differentially altered(GDF11 group/control group>1.2 or<0.83)in PC 12 cells induced by GDF11,of which 90 proteins up-regulated and 65 down-regulated.Gene ontology(GO)enrichment analysis exhibited the DEPs were mainly involved in phosphatidylinositol phosphorylation,phosphatidylinositolmediated signaling,and so on.And PI3K-Akt pathway was significantly enriched by KEGG enrichment analysis.CSP100 PLUS microarray uncovered GDF11 remarkably altered the pathways of PI3K-Akt,apoptosis and cell cycle.3.The phosphorylation levels of Smad2/3 and Akt markedly increased in neural stem cells after treatment with GDF11,indicating that GDF11 activated TGFβ/Smad2/3 and PI3K-Akt pathways.Inhibiting ALK5 by SB431542 counteracted GDF11-mediated phosphorylation of Smad2/3 and Akt.Concomitantly,it also reversed the GDF11-mediated cellular behaviors which were inhibition of proliferation and migration,increase of apoptosis and differentiation,and cell cycle arrest,which implied GDF11 depended on ALK5 to activate the downstream pathways to regulate neural stem cells.Blocking PI3K by LY294002 resulted in down-regulation in GDF 11-induced phosphorylation of Akt with no change of GDF11-mediated Smad2/3 phosphorylation.Moreover,inhibition PI3K by LY294002 also attenuated GDF11-mediate inhibition of proliferation and migration,increase of apoptosis and differentiation,and cell cycle arrest,which suggested GDF11 activated PI3K-Akt to modulate neural stem cells.ConclusionGDF11 activated the downstream PI3K-Akt signal pathway through an ALK5dependent way to regulate the biological effects of neural stem cells,including attenuating proliferation and migration,promoting apoptosis and differentiation,and arresting cell cycle.