No abstract

Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress. In this system, although autophagic structures increased, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal dysfunction. The influence of autophagy activity on senescence development was confirmed by both positive and negative autophagy modulators; and MTOR-dependent autophagy activators, rapamycin and PP242, efficiently suppressed cellular senescence through a mechanism relevant to restoring autophagic flux. By time-phased treatment of cells with the antioxidant N-acetylcysteine (NAC), the mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation, we found that mitochondrial dysfunction plays an initiating role, while lysosomal dysfunction is more directly responsible for autophagy impairment and senescence. Interestingly, the effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both mitochondrial and lysosomal functions. Together, this study demonstrates that autophagy impairment is crucial for oxidative stressinduced cell senescence, thus restoring autophagy activity could be a promising way to retard senescence.

Summary AMPK activation is beneficial for cellular homeostasis and senescence prevention. However, the molecular events involved in AMPK activation are not well defined. In this study, we addressed the mechanism underlying the protective effect of AMPK on oxidative stress‐induced senescence. The results showed that AMPK was inactivated in senescent cells. However, pharmacological activation of AMPK by metformin and berberine significantly prevented the development of senescence and, accordingly, inhibition of AMPK by Compound C was accelerated. Importantly, AMPK activation prevented hydrogen peroxide‐induced impairment of the autophagic flux in senescent cells, evidenced by the decreased p62 degradation, GFP‐RFP‐LC3 cancellation, and activity of lysosomal hydrolases. We also found that AMPK activation restored the NAD + levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD + synthesis. In addition, the mechanistic relationship of autophagic flux and NAD + synthesis and the involvement of mTOR and Sirt1 activities were assessed. In summary, our results suggest that AMPK prevents oxidative stress‐induced senescence by improving autophagic flux and NAD + homeostasis. This study provides a new insight for exploring the mechanisms of aging, autophagy and NAD + homeostasis, and it is also valuable in the development of innovative strategies to combat aging.

Global germ line loss of fat mass- and obesity-associated (FTO) gene results in both the reduction of fat mass and lean mass in mice. The role of FTO in adipogenesis has been proposed, however, that in myogenesis has not. Skeletal muscle is the main component of body lean mass, so its connection with FTO physiologic significance need to be clarified. Here, we assessed the impact of FTO on murine skeletal muscle differentiation by in vitro and in vivo experiments. We found that FTO expression increased during myoblasts differentiation, while the silence of FTO inhibited the differentiation; in addition, skeletal muscle development was impaired in skeletal muscle FTO-deficient mice. Significantly, FTO-promoted myogenic differentiation was dependent on its m6A demethylase activity. Mechanically, we found that FTO downregulation suppressed mitochondria biogenesis and energy production, showing as the decreased mitochondria mass and mitochondrial DNA (mtDNA) content, the downregulated expression of mtDNA-encoding genes and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) gene, together with declined ATP level. Moreover, the involvement of mTOR-PGC-1α pathway in the connection between FTO and muscle differentiation is displayed, since the expression of FTO affected the activity of mTOR and rapamycin blocked FTO-induced PGC-1α transcription, along with the parallel alteration pattern of FTO expression and mTOR phosphorylation during myoblasts differentiation. Summarily, our findings provide the first evidence for the contribution of FTO for skeletal muscle differentiation and a new insight to study the physiologic significance of RNA methylation.

Oxidative stress-induced DNA damage, the senescence-associated secretory phenotype (SASP), and impaired autophagy all are general features of senescent cells. However, the cross-talk among these events and processes is not fully understood. Here, using NIH3T3 cells exposed to hydrogen peroxide stress, we show that stress-induced DNA damage provokes the SASP largely via cytosolic chromatin fragment (CCF) formation, which activates a cascade comprising cGMP-AMP synthase (cGAS), stimulator of interferon genes protein (STING), NF-κB, and SASP, and that autolysosomal function inhibits this cascade. We found that CCFs accumulate in senescent cells with activated cGAS-STING-NF-κB signaling, promoting SASP and cellular senescence. We also present evidence that the persistent accumulation of CCFs in prematurely senescent cells is partially associated with a defect in DNA-degrading activity in autolysosomes and reduced abundance of activated DNase 2α. Intriguingly, we found that metformin- or rapamycin-induced activation of autophagy significantly lessened the size and levels of CCFs and repressed the activation of the cGAS-STING-NF-κB-SASP cascade and cellular senescence. These effects of autophagy activators indicated that autolysosomal function contributes to CCF clearance and SASP suppression, further supported by the fact that the lysosome inhibitor bafilomycin A1 blocked the role of autophagy-mediated CCF clearance and senescence repression.

We propose that the FTO-dependent control of cholesterol deposition may provide avenues for the treatment of atherosclerosis.

Nicotinamide adenine dinucleotide (NAD+) is indispensable for the anti-aging activity of the sirtuin (SIRT) family enzymes. AMP-activated protein kinase (AMPK) upregulates NAD+ synthesis and SIRT activity in a nicotinamide phosphoribosyltransferase (NAMPT)-dependent manner. However, the molecular mechanisms that affect AMPK-driven NAMPT expression and NAD+/SIRT activation remain unclear. In this study, we tried to identify senescence-associated microRNAs (miRNAs) that negatively regulate the cascade linking AMPK and NAMPT expression. miRNA-screening experiments showed that the expression of miR-146a increased in senescent cells but decreased following AMPK activation. Additionally, miR-146a overexpression weakened the metformin-mediated upregulation of NAMPT expression, NAD+ synthesis, SIRT activity, and senescence protection, whereas treatment with the miR-146a inhibitor reversed this effect. Importantly, these findings were observed both in vitro and in vivo. Mechanistically, miR-146a directly targeted the 3′-UTR of Nampt mRNA to reduce the expression of NAMPT. AMPK activators metformin and 5-aminoimidazole-4-carboxamide (AICAR) hindered miR-146a expression at the transcriptional level by promoting IκB kinase (IKK) phosphorylation to attenuate nuclear factor-kappaB (NF-κB) activity. These findings identified a novel cascade that negatively regulates the NAD+/SIRT pathway by suppressing miR-146a-mediated NAMPT downregulation. Furthermore, our results showed that miR-146a impedes the anti-aging effect of AMPK. This mutual inhibitory relationship between miR-146a and AMPK enriches our understanding of the molecular connections between AMPK and SIRT and provides new insight into miRNA-mediated NAD+/SIRT regulation and an intervention point for the prevention of aging and age-related diseases.

The G protein-coupled receptor APJ/Aplnr has been widely reported to be involved in heart and vascular development and disease, but whether it contributes to organ left-right patterning is largely unknown. Here, we show that in zebrafish, aplnra/b coordinates organ LR patterning in an apela/apln ligand-dependent manner using distinct mechanisms at different stages. During gastrulation and early somitogenesis, aplnra/b loss of function results in heart and liver LR asymmetry defects, accompanied by disturbed KV/cilia morphogenesis and disrupted left-sided Nodal/spaw expression in the LPM. In this process, only aplnra loss of function results in KV/cilia morphogenesis defect. In addition, only apela works as the early endogenous ligand to regulate KV morphogenesis, which then contributes to left-sided Nodal/spaw expression and subsequent organ LR patterning. The aplnra-apela cascade regulates KV morphogenesis by enhancing the expression of foxj1a , but not fgf8 or dnh9 , during KV development. At the late somite stage, both aplnra and aplnrb contribute to the expression of lft1 in the trunk midline but do not regulate KV formation, and this role is possibly mediated by both endogenous ligands, apela and apln . In conclusion, our study is the first to identify a role for aplnra/b and their endogenous ligands apela/apln in LR patterning, and it clarifies the distinct roles of aplnra-apela and aplnra/b-apela/apln in orchestrating organ LR patterning.