The Function And Mechanisms Of ESC-sEVs In Improving Cognitive Dysfunction By Rejuvenation Of Senescent Hippocampal NSCs

BACKGROUND AND OBJECTIVE:The number of patients with cognitive impairment gradually increased with the aging of the population.Cognitive dysfunction seriously affects quality of life in patients and brings severe burden to the family and society.However,the effective therapeutic strategy is still lacking.Hippocampus plays a key role in maintaining cognitive function,and the hippocampal neural stem cells(H-NSCs)in the subgranular zone(SGZ)are critical for maintaining structural and functional integrity in hippocampus.It has been reported that cell senescence occurs during the process of natural aging or after injury,causing tissue or organ dysfunction or impairment of recovery after injury.Therapeutic approaches targeting senescent cells have been demonstrated to be an effective method to prevent or ameliorate aging related diseases and promote tissue or organ recovery after injury.Small extracellular vesicles(sEVs)derived from stem cells,as a kind of biological nano vesicles,possess parental cell-like therapeutic functions against multiple diseases,and have the advantages including no risk of tumor formation and low possibility of immune rejection.In consideration of Embryonic stem cells(ESCs)have infinite proliferation ability and multiple differentiation potential,as well as the intrinsic youthful barrier to aging,suggesting that the ESCs derived sEVs(ESC-sEVs)may have some therapeutic effects on anti-aging.As no study have reported the relationship between H-NSCs senescence and cognitive dysfunction,and the therapeutic potential of ESC-sEVs in rejuvenating stem cell senescence.Therefore,we applied the age-related cognitive dysfunction model,as well as vascular dementia(VD)model,to explore H-NSCs senescence in the progress of cognitive dysfunction,and evaluate the effects of ESC-sEVs in rejuvenating H-NSCs senescence.METHODS:1.To explore the role of H-NSCs aging in the course of age-related cognitive dysfunction,we firstly applied Morris water maze to measure the cognitive function of SAMP8 mice at different ages,and then Immunofluorescence staining,SA-?-gal staining and western blot were performed to assess dynamic changes of number and senescence phenotype in H-NSCs.In order to clarify that ESC-sEVs can restore agerelated cognitive impairment by reducing H-NSCs senescent and promoting neurogenesis,we firstly treated 6-month old SAMP8 mice with ESC-sEVs to 12-months old,and 12-month old C57/BL mice to 20-months old.The changes of cognitive function,H-NSCs senescence phenotype,H-NSCs number and neurogenesis were detected in different intervention groups.Lastly,in vitro H-NSCs replicative senescence model was employed to evaluate the anti-aging function of ESC-sEVs.2.To explore the role of H-NSCs aging in the course of VD,we firstly constructed rats VD model,and then detected the cognitive function,the number and senescence phenotype in H-NSCs of different groups.In order to clarify that ESC-sEVs can restore cognitive impairment in VD by reducing H-NSCs senescent and promoting neurogenesis,we treated VD rats with ESC-sEVs and evaluate the changes of cognitive function,H-NSCs senescence phenotype,H-NSCs number and neurogenesis in different intervention groups.Lastly,in vitro D-galactose(D-Gal)induced H-NSCs senescence model was used to evaluate the anti-aging function of ESC-sEVs.3.To identify the mechanism of ESC-sEVs in rejuvenating H-NSCs senescence,we constructed in vitro H-NSCs replicative senescence model.RNA-seq was carried out to investigate the key target gene in regulating H-NSCs senescence and ESC-sEVs rejuvenate H-NSCs senescence.RT-q PCR and Western blot were applied to verify the expression level of target gene and protein.Sh RNA knockdown was used to investigate the function of target gene in regulating H-NSCs senescence.Proteomics analysis,RTq PCR,and Western blot were applied to detect the molecular mechanism of ESC-sEVs in rejuvenating H-NSCs senescence.RESULTS:1.ESC-sEVs improve cognitive dysfunction by rejuvenating senescent H-NSCs during aging:(1)The results from Morris water maze and western blot showed an age-related decline of cognitive functions in mice.Immunofluorescence staining,SA-?-gal staining and western blot demonstrated an age-related H-NSCs loss and senescence in mice.(2)ESC-sEVs rejuvenated the senescence of H-NSCs in aged mice,promoted neurogenesis,and restored cognitive function in aged mice.(3)H-NSCs replicative senescence model showed H-NSCs senescence impaired their proliferation and neuronal-differentiation capacities;ESC-sEVs can rejuvenate senescent H-NSCs to restore their self-renewal and neurogenesis function.2.ESC-sEVs improve cognitive dysfunction via rejuvenation of senescent H-NSCs in VD rats:(1)Morris water maze and western blot showed an incr emental reduction of cognitive function with the increase of ischemic duration in VD rats.Immunofluorescence staining,SA-?-gal staining,and western blot demonstrated an ischemic duration related H-NSCs loss and H-NSCs senescence in VD rats.(2)ESC-sEVs could rejuvenate the senescence of H-NSCs in VD rats,increased neurogenesis,and restored cognitive function in VD rats.(3)D-Gal induced H-NSCs senescence model showed H-NSCs senescence impaired their proliferation and neuronal-differentiation capacities;ESC-sEVs can rejuvenate senescent H-NSCs to restore their self-renewal and neurogenesis function.3.ESC-sEVs transfer SMADs to regulate MYT1-Egln3-Sirt1 axis in senescent H-NSCs:(1)RNA-seq,RT-q PCR,and western blot showed MYT1 was down-regulated in aged H-NSCs,which caused up-regulation of Egln3,followed by the down-regulation of HIF-2?,NAMPT,and Sirt1.(2)Proteomics analysis and western blot showed ESC-sEVs can transfer SMAD4 and SMAD5 to activate MYT1 in aged H-NSCs,which led to the inactivation of Egln3 and the activation of HIF-2?,NAMPT,and Sirt1.CONCLUTIONS:1.H-NSCs senescence impair their self-renewal and neurogenesis,rejuvenation of senescent H-NSCs improves their proliferation and neural-differentiation abilities.2.H-NSCs gradually senescence with the progression of aging,which cause age-related cognitive dysfunction;ESC-sEVs can ameliorate the senescence of H-NSCs to recover age-related cognitive dysfunction.3.H-NSCs gradually senescence with the progression of VD,which cause time-related cognitive impairment in VD;ESC-sEVs can rejuvenate H-NSCs senescence to restore cognitive function in VD.4.The decreasing of MYT1 in H-NSCs led to the up-regulation of Egln3,which in turn inhibite the expression of HIF-2?,NAMPT and Sirt1,and cause H-NSCs senescence;ESC-sEVs rejuvenate senescent H-NSCs by transferring SMAD4 and SMAD5 to activate MYT1,inhibit Egln3,and up-regulate HIF-2?,NAMPT,and Sirt1.5.ESC-sEVs may be a novel cell-free therapeutic tool for cognitive-dysfunction diseases and other aging-related diseases.