| Autophagy has been linked to aging in a variety of model organisms,and is shown to suppress mitochondrial metabolism via the mitophagy pathway to preserve hematopoietic stem cells in mice.However,the mechanism by which autophagy regulates hematopoietic aging.in particular in humans,is not fully understood.Here,we demonstrate that autophagy delays hematopoietic aging by activating the downstream expression of Sirt3?a key protein in the regulation of hematopoietic aging via modulation of mitochondrial homeostasis and oxidative stress.sirt3 is more enriched in the hematopoietic stem and progenitor cells(HSPC s)than in their progeny cells and is the most abundant sirt family member in HSPCs,though it progressively decreases as the capacity for autophagy deteriorates with aging.In wild-type mice,depletion of sirt3 in HSPCs accelerates hematopoietic aging,whereas activation of autophagy up-regulates sirt3 transcription and decelerates hematopoietic aging.In contrast,in autophagy-defective mice,sirt3 expression is crippled,but overexpression of Sirt3 in HSPCs prevents accelerated hematopoietic aging.Furthermore,in an aged human population,low hematopoietic autophagy activity is associated with reduced sirt3 expression,and induction of autophagy reverses a decline in sirt3 in HSPCs.Therefore,our results identify an autophagy-Sirt3 axis in counteracting hematopoietic aging and suggest a possible means for blood rejuvenation by securing function of the axis.Objectives:1.Explore the relationship between autophagy and aging in human hematopoietic stem and progenitor cells,and determine the potential for human blood rejuvenation by intervening autophagy-aging pathway.2.Elucidate the molecular mechanism underlying anti-aging by autophagy in hematopoietic system,in paticular in hematopoietic stem and progenitor cells.Methods:1.Analysis of human hematopoietic stem progenitor cell samples.(a)We analyzed human peripheral blood from a population of 4950 individuals ranging in age from 20 to over 90 years old.(b)We purified human hematopoietic stem and progenitor cells from bone marrow samples,and divided the samples into young(<40 years old)and old(>70 years old)groups.Then we measured the mRNA levels of autophagy related genes by quantitative PCR,and the colocalization of LC3 and Lampl by ImageStream technology in human hematopoietic stem and progenitor cells.2.Identification of mouse senescence model.We analyzed the peripheral blood of atg7floxp/floxp;vav-iCre mice by blood analyser.3.Analysis of aging markers in mouse hematopoietic system.(a)We detected the differentiation in hematopoiesis in atg7floxp/floxp;vav-iCre and old wild-type mice by flow cytometry.(b)We examined aging markers in hematopoietic stem and progenitor cells of atg7floxp/floxp;vav-iCre model,including DNA damage(?-H2AX),loss of proteostasis(Ubiquitin),telomere attrition(tert,terfl,terf2)and cell cycle(7-AAD&Brdu,Hoechst&Ki67).4.RNA-sequencing analysis of mouse HSPCs.(a)We performed RNA sequencing of HSPCs from wild-type and atg7-deleted mice.(b)We validated the result of RNA sequencing by quantitative PCR,and measured the expression of Sirt3 protein in HSPCs from wild-type and atg7-deleted mice by western blotting and immunofluorescence.5.Knockdown and overexpression of sirt3 in mouse HSPCs.(a)We utilized RNAi to knock down sirt3 in mouse HSPCs.Then performed colony-forming assays in which the sirt3-depleted HSPCs.prepared by infection of the HSPCs(GFP+)with a lenti-virus vector bearing shRNA against the sirt3 gene,were cultured in methylcellulose medium supplemented with growth factors.Meanwhile,we transplanted sirt3-depleted HSPCs that were infected with the lenti-virus,together with 106 CD45.1 bone marrow supporting cells into lethally-irradiated mice.And we examined the percentage of donor-derived cells and hematopoietic differentiation in recipients at 8 weeks from transplantation.(b)We performed ectopic expression of sirt3 by lenti-virus infection in the hematopoietic atg7-depleted mice.The HSPCs infected with the sirt3 expression vector.together with 106 CD45.1 bone marrow supporting cells.were transplanted into the lethally-irradiated mice.Then we examined the percentage of donor-derived cells and hematopoietic differentiation in recipients at 8 weeks from transplantation.6.Analysis of sirt3 expression by interfering autophagy in mouse HSPCs.(a)We tracked a time course of sirt3 transcription in wild-type and atg7floxp/floxp;vav-iCre mice by quantitative PCR.(b)We interfered an ex vivo or in vivo autophagy response in wild-type mouse HSPCs with rapamycin(an autophagy inducer),starvation or Bafilomycin A1(an autophagy inhibitor),and mapped out transcription expression of sirt3 by quantitative PCR and ImageStream technology.7.Analysis of sirt3 expression by interfering autophagy in human HSPCs.(a)We measured the expression of sirt family members by quantitative PCR in human bone marrow CD45CD34 cells.(b)We measured sirt3 expression by quantitative PCR in bone marrow CD45CD34 cells from two differently aged human groups(below age of 40 years group and above age of 70 years group).(c)We treated human bone marrow CD45CD34 cells with autophagy inducers.Then we measured sirt expression by quantitative PCR in these treated cells.Results:1.Reduced autophagy is correlated with aging in human HSPCs.(a)With increase of age,there is a progressive decrease in hemoglobin levels,red blood cells and lymphocyte counts.(b)Expression of genes encoding autophagy machinary is highly reduced in the HSPCs in the aged group(over 70 years old)compared with the young group(below 40 years old).The results show that in comparison with the young group,there is a significant decrease in basal autophagy levels in both total bone marrow hematopoietic cells(CD45 positive)and hematopoietic stem and progenitor cells(CD45CD34 positive)in the aged human population.2.Autophagy defect in hematopoietic system leads to severe aging phenotypes in mice.(a)At 10 weeks old,mice carrying the atg7 deletion display a significant decrease in their RBC and lymphocyte counts,as well as in their level of HGB,comparable to that of 90-week-old mice.(b)atg7 deletion in the mouse model also accelerates biased myeloid-lymphoid differentiation.(c)Examination by flow cytometry showed that aging markers were increased in the atg7-deleted mouse HSPCs(Lin-Sca1+c-Kit+,LSK),including protein homeostasis,mitochondrial accumulation,and generation of ROS and DNA damage.And we measured the expression of three telomerase genes,namely tert,terfl.and terf2 in the HSPCs by quantitative reverse transcription PCR,and found that expression of these telomerase genes was reduced in the hematopoietic atg7-deleted 10-week-old mice,comparable to their expression in the wild-type mice at 90 weeks old.Cell proliferation assays measured by flow cytometry displayed a decreased frequency of G0 in the atg7-deleted HSPCs.3.Sirt3 is the major sirt family member in mouse HSPCs.(a)The volcano map of differentially expressed genes between atg7+/+ and atg7-/-HSPCs of the mice revealed a total of 1062 significantly up-regulated genes,with 789 down-regulated genes in the atg7-depleted HSPCs.The transcriptome profiling reveals that sirt3 is the most transcriptionally abundant member in sirt family,and deletion of atg7 only led to significant loss of sirt3 expression,among all seven of the sirt family genes.(b)Validation with Q-PCR supports the RNA sequencing data that sirt3 is the most highly transcribed among the sirt family genes expressed in HSPCs.Further validation with western blot analysis also shows reduced Sirt3 protein levels in bone marrow hematopoietic progenitor cells(Lin-)of the hematopoietic autophagy-defective mice.Confocal microscopy further revealed that in the atg7-deleted HSPCs,Sirt3 protein maintains a relatively high level in 2-week-old mice,which is similar to the wide-type HSPCs,though by 8 weeks of age the Sirt3 levels are rapidly reduced.4.Sirt3 of HSPCs effectively counteracts hematopoietic aging in mice.(a)Knockdown of sirt3 reduced the percentage of HSPCs that were sorted against scal-1+c-kit+markers in the GFP+cells of 8-week-old mice,and hence a significant increase in the number of GM colonies derived from the sirt3-depleted HSPCs.Depletion of sirt3 compromised the donor-derived hematopoiesis in the wild-type host mice,with about 60%reduction in peripheral blood.Additionally,the data shows a significantly elevated frequency of myeloid cells and a reduced frequency of B cells.(b)Ectopic expression of sirt3 by lenti-virus in the atg7-deleted mice rescued total bone marrow cellularity.Remarkably,overexpression of Sirt3 in the atg7-deleted mice normalized the generation of myeloid cells and restored the generation of lymphoid cells in the peripheral blood,effectively reversing the biased myeloid-lymphoid differentiation.5.Autophagy positively regulates sirt3 in mouse HSPCs.(a)The data shows that sirt3 increased its expression until 24 weeks,and thereafter transcription progressively decreased in the wild-type mouse HSPCs.Whereas,sirt3 expression began to dramatically decrease at 4-6 weeks in the mouse model in which hematopoietic autophagy is disrupted,a pattern almost identical to the accelerated hematopoietic aging due to the disruption of autophagy.(b)Sirt3 expression was clearly upregulated in wild-type HSPCs,but was blocked by autophagy inhibitor Bafilomycin A1;in contrast,sirt3 expression was not upregulated in response to autophagy induction in the atg7-deleted HSPCs.Furthermore,activation of autophagy by starvation,another frequently used autophagy trigger,selectively upregulated sirt3 expression.but did not alter the expression levels of the rest of sirt family members.More importantly,in vivo activation of autophagy by steady starvation with progressive calorie restriction in mice upregulated sirt3 expression.6.Activation of autophagy upregulates sirt3 in human HSPCs.(a)Compared to other family members,sirt3 is highly expressed in human HSPCs.(b)The results show that sirt3 expression was decreased in the group of over 70 years old as compared to the group of less 40 years old.(c)Induction of autophagy by different autophagy inducers all upregulated sirt3 expression in human HSPCs.Conclusions:?Sirt3 is suppressed by the deterioration of the capacity for autophagy in hematopoietic aging;?Increase of sirt3 reverses hematopoietic aging from autophagy deficiency;?Enhancement of autophagy decelerates hematopoietic aging at least in part via reversing sirt3 decline.Significances:?My study provides the first evidence of autophagy-dependent activity by Sirt3 in HSPCs in the deceleration of hematopoietic aging;?Similar to what has been observed in my mouse study,sirt3 was found to be suppressed in the HSPCs of aged humans;induction of autophagy effectively upregulates Sirt3 in hematopoietic cells and their stem cells from aged hunans,suggesting that an autophagy-Sirt3 regulatory pathway is similarly present in the human hematopoietic system.This finding provides promising translational avenues for decelerating hematopoietic aging by enhancing the autophagy-Sirt3 axis with autophagy inducers,or by enhancing sirt3 expression in the event of irreversible dcfects in autophagy in HSPCs. |
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