Abstract
Autophagy is essential for optimal cell function and survival, and the entire process accompanies membrane dynamics. Ceramides are produced by different enzymes at different cellular membrane sites and mediate differential signaling. However, it remains unclear which ceramide-producing pathways/enzymes participate in autophagy regulation under physiological conditions such as nutrient starvation, and what the underlying mechanisms are. In this study, we demonstrate that among ceramide-producing enzymes, neutral sphingomyelinase 2 (nSMase2) plays a key role in autophagy during nutrient starvation. nSMase2 was rapidly and stably activated upon starvation, and the enzymatic reaction in the Golgi apparatus facilitated autophagy through the activation of p38 MAPK and inhibition of mTOR. Moreover, nSMase2 played a protective role against cellular damage depending on autophagy. These findings suggest that nSMase2 is a novel regulator of autophagy and provide evidence that Golgi-localized ceramides participate in cytoprotective autophagy against starvation.
Highlights
Autophagy is a highly conserved intracellular catabolic process that degrades cytoplasmic components within lysosomes[1]
To investigate the ceramide-producing enzyme or pathway involved in the regulation of autophagy, autophagic flux was analyzed in the presence of various inhibitors of ceramide-generating enzymes (Fig. 1a) in rat neuroblastoma PC12 cells, which feature a well-conserved sphingolipid metabolism (Supplementary Figure 1A)
To explore the downstream pathways of nSMase[2] involved in autophagy regulation, we examined the effects of nSMase[2] on phosphorylation of p38 mitogen-activated protein kinase (MAPK), mechanistic target of rapamycin, Akt, c-Jun N-terminal kinase (JNK) 1/2, AMPactivated protein kinase (AMPK), and Unc-51 like autophagy activating kinase (ULK1)
Summary
Autophagy is a highly conserved intracellular catabolic process that degrades cytoplasmic components within lysosomes[1]. Overexpression of V5-tagged nSMase[2] induced LC3 puncta formation, LC3 turnover, and degradation of the autophagy substrate p62 in V5-positive cells (Fig. 2a). To evaluate if localization of ceramides to the Golgi complex is required to trigger autophagy, we examined the effects of a CERT inhibitor (HPA-12) and an SMS inhibitor (D609) on starvation-induced autophagic flux.
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