Spermidine Induced ATM-mediated Mitophagy In Human Skin Fibroblasts

Spermidine, a kind of endogenous polyamines, its intracellular concentration may decrease during the ageing process of cells or organisms. Spermidine could activate certain autophagy-related genes by inhibiting histone acetyltransferase to deacetylate histone H3 of these genes, thus leading to the activation of autophagy. Mitochondrion, an important organelle for cellular oxidative phosphorylation and energy synthesis, is susceptible to oxidative stress. Dysfunctional mitochondria need to be eliminated in time to keep intracellular homeostasis. However, whether spermidine could induce mitophagy, a specific type of autophgy to eliminate damaged mitochondria, is still unknown. ATM, which loss of function could cause ataxia-telangiectasia, plays key roles in DNA damage response as a nuclear protein. However, recent reports domensrtated that a fraction of ATM localized in cytoplasmic compartments exihibiting non-nuclear functions in various cell types. Especially, a portion of ATM localizes in the mitochondria, which could be activated by mitochondrial dysfunction to regulate mitophagy, but it's detailed mechanism remains to be elucidated.To untangle these issues, immortalized human skin fibroblasts cell line GM00637 was chosen to confirm whether spermidine could induce mitophagy and explore the role of ATM in spermidine-induced mitophagy. After the cells were treated with 50 ?M spermidine for different time points (2 h,4 h,6 h,8 h,10 h,12 h), transmission electronic microscopy and confocal microscopy were employed to observe mitophagosomes and mitolysosomes. Meanwhile, immunoblotting and immunocytochemistry were used to detect the transformation of LC3 ? to LC3 ?, accumulation of mitophagy related protein PINK1 and translocation of Parkin. In addition, the specific ATM kinase inhibitor KU55933 was used to confirm the role of ATM in the process of spermidine-triggered mitophagy.The results showed that 50 ?M spermidine caused significant transformation of LC3 ? to LC3? (2.2 times) after 8 h treatment in human fibroblasts cell line GM00637. Also, spermidine could induce mitophagy by eliciting mitochondrial depolarization in 2 h, which then triggered the formation of mitophagosomes and mitolysosomes, accumulation of PINK1, and translocation of Parkin in 8 h. ATM plays an important role in regulating mitolysosomes formation and accumulation of PINK1 and translocation of Parkin. This research perfectly addressed the issues that spemidine could induce PINK 1/Parkin-mediated mitophagy and this effect was ATM dependent. These findings will deepen our insights into the molecular mechanisms of mitophagy.