Explore The Mechanism Of Progerin-induced Aging And The Therapeutic Effect Of HTERT By Using HGPS Model

Background:Sarcopenia is a progressive,generalized skeletal muscle disorder,characterized by accelerated loss of muscle mass and function.It occurs commonly as an age-related process in older people,undermining the health of the elderly.Currently,there is no specific drug therapy for sarcopenia,and sarcopenia is associated with a series of adverse health consequences,placing a heavy economic burden on increasingly aging society.Progerin is considered as one of the biomarkers of aging,and its expression in skeletal muscle increases with age.Study showed that inducing progerin in myogenic cells induced senescence phenotype,suggesting progerin plays an important role in inducing skeletal muscle aging,and can be a potential therapeutic target for sarcopenia.Progerin,the protein responsible for the Hutchinson Gilford Progeria Syndrome(HGPS),accumulates in different tissues and cells causing accelerated aging and premature senescence.Due to the shared molecular and cellular mechanisms of HGPS and aging-associated diseases with respect to oxidative stress,cellular senescence,stem cell exhaustion,and high expression of progerin,HGPS provides a model for studying the mechanism of progerin-induced aging,and this model can be used to detect the therapeutic effect of drugs.Although HGPS cell and mouse models can induce senescence phenotypes by over-expressing progerin,these models cannot recapitulate all the features of human disease.Therefore,human induced pluripotent stem cells(iPSCs)disease model provides a feasible method to study the progerin-induced aging.The iPSCs derived from fibroblasts of HGPS patients did not express progerin,but re-expressed progerin after being differentiated into different cell types,and reproduced the senescence phenotypes.HGPS-iPSCs have been differentiated into vascular cells,fibroblasts,adipocytes and other cells,and the expression level of progerin was high in vascular cells,which was consistent with HGPS mouse models.Study also showed that the expression level of progerin in skeletal muscle was relatively low,and the differentiation from iPSCs to skeletal muscle cells have not been unified,and the efficacy and expansion were limited,rendering it difficult to directly differentiate HGPS-iPSCs into skeletal muscle cells and study the mechanism of progerin-induced aging.Therefore,in order to better observe the aging effects induced by progerin and remission effect after treatment,we used progerin highly-expressed vascular cells of HGPS as the model to study progerin-induced aging.Endothelial cells(ECs)and vascular smooth muscle cells(VSMCs)are two major components of blood vessels.High expression of progerin in both HGPS-ECs and HGPS-VSMCs has been reported to induced cell senescence phenotype,making them good cell models for studying the mechanism of progerin-induced aging.In this study,we differentiated HGPS-iPSCs into HGPS-ECs and HGPS-VSMCs to explore the mechanism of progerin-induced aging.Targeting the cell type with more significant senescent effect,we treated them with human telomerase reverse transcriptase(hTERT),to investigate the telomere elongating effect and senescence remission effect,and underlying mechanism.Aims:(1)To investigate the mechanism underlying progerin-induced aging by using HGPS model.(2)To explore the therapeutic effect of hTERT on progerin-induced aging.Methods:(1)Differentiate isogenic ECs and VSMCs from control-and HGPS-iPSCs.(2)Detect progerin levels in HGPS-ECs and HGPS-VSMCs.Immunofluorescence(IF)was used to detect the effect of progerin on nuclear morphology.Monochromatic multiple quantitative PCR(MMqPCR)and quantitative fluorescence in situ hybridization(qFISH)were used to detect telomere length.Cell proliferation,DNA damage and DNA accessibility,inflammatory cytokines,β-galactosidase and other senescent markers were detected.(3)Detect progerin expression level,β-galactosidase positive cells,and telomere length of HGPS-ECs and HGPS-VSMCs within 28 days after differentiation to compare the senescence severity.(4)Detect the protein interaction of shelterin protein complex(SPC)by mass spectrometry,and verified by immunoprecipitation to explore the molecular mechanism of progerin-induced aging.(5)HGPS-ECs,with more prominent senescence,were treated with hTERT.Detect telomerase activity by Telomerase Repeated Amplification Protocol(TRAP)assay,and telomere lengthening effect by qFISH,and detect other senescent markers.Then RNA sequencing and GO pathway analysis were used to explore the potential molecular mechanism of anti-aging effects of hTERT therapy.Results:(1)Progerin expression in HGPS-ECs and HGPS-VSMCs induced senescence phenotypes.HGPS-ECs,with lower level of progerin,manifested more severe signs of senescence,as indicated in part by shorter telomere length,more DNA damage signals,and more senescence markers expression.In addition,HGPS-ECs also showed accelerated senescence compared with HGPS-VSMCs by time-course experiments within 28 days.(2)MS and IP showed that the RAP1-TIN2 interaction in shelterin protein complex(SPC)was disrupted.In addition,yH2A.X binding to RAP1 was increased and TRF2 binding to Lamin A was reduced in HGPS-ECs,but not in HGPS-VSMCs,suggesting that telomere shortening may be caused by increased DNA damage and loss of telomere protection,which were more prominent in HGPS-ECs.(3)hTERT treatment extended telomere length,reduced progerin expression,significantly reduced DNA damage,partially restored gene expression,and upregulated SIRT1 expression,which improved cell function and alleviated senescence phenotype of HGPS-ECs.Conclusion:(1)Progerin induced cell senescence by mediating telomere shortening,DNA damage increasing,SPC protein interactions disruption,and senescence markers expression.(2)hTERT treatment downregulated progerin expression,extended telomere length and reduced DNA damage and negative aging effects caused by progerin,which greatly improved cell function and alleviated the aging phenotype,providing a new target and a new therapeutic strategy for sarcopenia.