Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure

ABSTRACT

Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes.

KEYWORDS:

• acid sphingomyelinase
• ATG9A
• autophagy
• ischemia-reperfusion injury
• kidney
• lysosome
• Niemann Pick disease
• sphingomyelin
• trafficking

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank D. Skousborg Larsen, K. Grøn Henriksen, M. Mari, S. Mathieu and S. Iversen for excellent technical assistance, H. Leffler, Y. Luo, P. Ross-Macdonald, K. Sandhoff and T. Yoshimori for valuable reagents, M. Høyer-Hansen and J. Nylandsted for helpful discussions, and the Developmental Studies Hybridoma Bank for the LAMP2 antibody. We acknowledge L. Plantard and the Core Facility for Integrated Microscopy, University of Copenhagen for the help in 3D-SIM analyses.

Additional information

Funding

This work was supported by grants from the Danish Cancer Society, the Danish National Research Foundation, the European Research Council (LYSOSOME), the Danish Medical Research Council, the Alfred Benzon Foundation, the Lundbeck Foundation, the Association for International Cancer Research and the Novo Nordisk Foundation (M.J.), and Deutsche Forschungsgemeinschaft SFB 877 (D.A.) as well as postdoctoral fellowships from the Federation of European Biochemical Societies (FEBS) and the Danish Medical Research Council (E.F.).