The Role Of Accumulation Of Prelamin A In Premature Aging And Related Mechanisms

Objective:Hutchinson-Gilford Progeria Syndrome（HGPS）is caused by the accumulation of progerin,which is a truncated form of farnesylated prelamin A.Progerin is often found in physiological aging.HGPS patients have a normal phenotype at birth,but show hair loss,growth retardation,skeletal deformities,osteoporosis,and scleroderma at the age of one year.Due to severe involvement of the cardiovascular system in HGPS patients,children usually die of myocardial infarction or stroke in their teens.The most common HGPS is caused by a synonymous mutation（GGC→GGT）of G608G in exon 11 of the LMNA gene,which results in activation of a hidden splice site in the precursor RNA,resulting in a 150 base deletion of mRNA,the encoded prelamin A protein has 50 amino acids deleted internally and is called progerin.Under normal condition,prelamin A undergoes farnesylation and methylation modification.Finally,the C-terminus is cut off by the metalloproteinase Zmpste24 to release mature lamin A,which is co-localized to the cytoplasmic side of the inner membrane with other lamins,form the skeleton of the nuclear structure together.In HGPS patients,progerin undergoes farnesylation and methylation modification and is bound to the junction of the endoplasmic reticulum membrane and nuclear membrane.However,due to the lack of the Zmpste24 restriction site,it cannot be further processed into lamin A,the wrong accumulation of progerin on the nuclear membrane.As mitosis progresses,the nuclear membrane skeleton dissolves and reorganizes,and progerin is highly destructive.Autophagy is a cell biological behavior in which cells transport cytosolic components to lysosomes through membrane transport mechanisms in order to degrade macromolecules.Under stress such as hypoxia,starvation,and amino acid deficiency,mammalian target of rapamycin（mTOR）activity is inhibited,autophagy is enhanced.And cells degrade cytoplasm such as mitochondria and large proteins through autophagy in order to provide essential amino acids and energy for cell survival.Under physiological conditions,autophagy can also maintain cell homeostasis by eliminating aging organelles or misfolded and damaged proteins.Previous reports have shown that autophagy could maintain the survival of newborn mice during the starvation stage,participate in the remodeling of cells during multi-organ development and the clearance of mitochondria during the development of red blood cell lines.Loss of autophagy leads to neurodegenerative changes,suggesting that autophagy is critical to the survival of neuronal cells.Disruption of autophagy in hematopoietic stem cells,and researchers found that functional loss of normal hematopoietic stem cells,severe bone marrow hyperplasia,and mouse death within several week,these show that autophagy plays a key role in maintaining the survival of adult hematopoietic stem cells.Previous studies on autophagy function were based on the conditional knock-out of autophagy key proteins Atg7,Atg5,LC3,etc.,and researchers observed changes in animal phenotype through tissue-specific autophagy destruction.mTOR is regulated by insulin and growth factors in the PI3K-Akt-mTOR signaling pathway,and it can also be activated by amino acids and ATP.mTOR can inhibit autophagy by directly regulating the ULK1-Atg13-FIP200complex.In cell biology experiments,rapamycin（RAPA）is usually used to inhibit mTOR to achieve the purpose of activating autophagy.A variety of abnormalities occur in HGPS fibroblasts,including abnormal nuclear shape and defective DNA damage response.Progerin accumulated in HGPS also exists in physiological aging.By inhibiting the production of progerin,aging-related nuclear malformations can be reversed,which suggests that make sure how to treat HGPS has a great scientific significance for anti-physiological aging.In this study,we will explore how mTOR and autophagy are involved in the premature aging of Zmpste24^-/-mice.Therefore,we extracted tissues of Zmpste24^-/-mice and examined the effects of accumulation of prelamin A on mTOR and autophagy.The flag-progerin plasmid was constructed and the effect of progerin on mTOR and autophagy was tested in vitro.This study examined the effect of mTOR on the premature aging phenotype by genetically reducing mTOR in mice with a background of Zmpste24^-/-.It provides scientific basis for clinical treatment of premature aging,and also provides a new perspective for studying the mechanism of mTOR involved in physiological aging.Methods:1、Zmpste24^+/-mice were hybridized with mTOR sub-allele mice(mTOR^△/+)or autophagy-related gene 7^+/-(Atg7^+/-)mice,respectively.Double-hybrid Zmpste24^+/-mTOR^△/+(Zmpste24^+/-Atg7^+/-)mice were used as parents to obtain Zmpste24^-/-mTOR^+/+(Zmpste24^-/-Atg7^+/+)mice and Zmpste24^-/-mTOR^△/+(Zmpste24^-/-Atg7^+/-)mice in the littermates.After weaning,the number of deaths of the mice was counted every 20 days,and the survival curves of mice in control and experimental groups were drawn by GraphPad Prism 5 software.Myocardial,liver,and brain tissue of neonatal wild-type mice and Zmpste24^-/-mice were extracted,and the expression of p-mTOR and total-mTOR was detected by Western blot.2、Myocardial,liver,and brain tissue of neonatal wild-type mice and Zmpste24^-/-mice were extracted,and autophagy-related indicators,such as p62 and LC3 were detected by Western blot.Zmpste 24^+/+and Zmpste 24^-/-MEFs were established from Zmpste24^+/-heterozygous embryos（E12.5 to 14.5）,Western blot was used to detect the expression of p62 and LC3.3、Myocardial and liver tissue of wild-type mice,Zmpste24^-/-mTOR^+/+mice and Zmpste24^-/-mTOR^△/+mice in the littermates were collected,and the autophagy level indicators of p62 and LC3 were detected by Western blot.Zmpste 24^+/+and Zmpste 24^-/-MEFs were established from Zmpste 24^+/-heterozygous embryos（E12.5 to 14.5）,we divided them into three groups:（1）Zmpste 24^+/+MEFs,（2）Zmpste 24^-/-MEFs,（3） Zmpste 24^-/-MEFs were treated with 1μM RAPA for 6 hours,and the expression of p-mTOR,total-mTOR,p62 and LC3 was detected by Western blot.Myocardial tissue of wild-type mice,Zmpste24^-/-mTOR^+/+mice and Zmpste24^-/-mTOR^△/+in the littermates were extracted,Western blot was used to detect the expression of p-mTOR,total-mTOR,p-S6K1（Thr389）,and total-S6K1.4、Myocardial,liver,and brain tissue of neonatal wild-type mice and Zmpste24^-/-mice were extracted,and Western blot was used to detect the expression of p-Akt（Thr308）,p-Akt（Ser473）,and total-Akt.5、Zmpste24^+/-mice were hybridized with mTOR^△/+mice,and double-hybrid Zmpste24^+/-mTOR^△/+mice were used as parents to obtain litter offspring Zmpste24^-/-mTOR^+/+mice and Zmpste24^-/-mTOR^△/+mice.After weaning,the males and females were kept separately.The number of the death of mice was counted.The mice were weighed.The muscle weakness of the hind limb was examined.The survival curve,weight gain curve,and normal grip curve of mice in the control and the experimental group were plotted by GraphPad Prism 5.6、Zmpste24^+/-mice were hybridized with mTOR^△/+mice or Atg7^+/-mice,respectively.The number of heterozygous offspring of Zmpste24^+/-mTOR^△/+(Zmpste24^+/-Atg7^+/-)mice was predicted and observed by genotype identification.7、The eukaryotic expression plasmid of pcDNA4.0-flag-progerin was constructed by molecular clone.8、Vector and flag-progerin plasmids were transfected into cells,respectively.48hours after transfection,cells were collected,and the expression of p-mTOR,total-mTOR,p62,LC3,p-Akt（Thr308）,p-Akt（Ser473）,and total-Akt was detected by Western blot.9、Vector and flag-progerin plasmids were transfected into HEK293T cells respectively,and the Co-IP testing was used to detect the binding of progerin to Akt.The GST-pull down testing was used to detect whether progerin and Akt were bind directly.Results:1、The median lifespan of Zmpste24^-/-mTOR^△/+mice is 32.8%longer than Zmpste24^-/-mTOR^+/+mice,while there was little difference in median lifespan between Zmpste24^-/-Atg7^+/-mice and Zmpste24^-/-Atg7^+/+mice in the same litter.Rather than that in wild-type littermates,the expression of p-mTOR and total mTOR in Zmpste24^-/-mice was higher.2、Compared to which in wild-type mice,p62 was accumulated and the conversion of LC3-I to LC3-II were inhibited in Zmpste24^-/-mice,impaired basal autophagy was observed.The inhibition of autophagy in Zmpste24^-/-MEFs was determined.3、In Zmpste24^-/-mice,standardized basal autophagy was observed through a genetic reduction of mTOR.The treatment of Zmpste24^-/-MEFs with RAPA showed that the level of exptession of p62 was decreased and the conversion of autophagy flow was increased.Due to the reduction in mTOR,the over-activated S6K1 activity was reduced to normal.4、The expression of p-Akt（Thr308）and p-Akt（Ser473）was elevated in Zmpste24^-/-mutant mice.5、Compared to Zmpste24^-/-mTOR^+/+mice,the median lifespan of male and female Zmpste24^-/-mTOR^△/+mice was extended by 25%and 35.6%,respectively.Besides the increase in median survival,the reduction of mTOR raised the maximum lifespan of Zmpste24^-/-mice,from 162 days to 199 days,a rise of 22.8%.The bodyweight curve showed a gender-dependent effect.Whereas mTOR knockdown remarkably increased the bodyweight of male Zmpste24^-/-mice,it did not have the same effect in female cohorts.Zmpste24^-/-mTOR^△/+mice grow to a larger size than Zmpste24^-/-in the littermates.Compared to Zmpste24^-/-mTOR^+/+mice,the median onset of abnormal grip strength of Zmpste24^-/-mTOR^△/+mice was delayed to an extention of 61%.6、In the context of Zmpste24^-/-,79.5%of mTOR^△/+mice failed to develop normally in the uterus,while Atg7 heterozygous mice survived completely.7、The pcDNA4.0-flag-progerin eukaryotic expression plasmid was successfully constructed.8、Human progerin was overexpressed in HELA and MCF-7 cell lines,and then we found that mTOR was activated,p62 was accumulated and the conversion of LC3-I to LC3-II was inhibited.In addition,human progerin activated Akt.9、Co-IP analysis revealed that the interaction of progerin with Akt.The result of GST pull-down testing showed that progerin did not bind to Akt directly.Conclusion:1、Hybridization of mTOR sub-allele mice and Zmpste24^-/-mice was used to genetic reduction of mTOR,and then cell autophagy was enhanced,and the lifespan of Zmpste24^-/-mice was extended significantly.Genetic reduction of mTOR delays premature aging of Zmpste24^-/-mice,but at the cost of frequent embryonic lethality.2、Accumulation of prelamin A induces mTOR overactivation and impaired autophagy in newborn Zmpste24^-/-mice.3、Progerin induces mTOR overactivation and impaired autophagy in vitro.