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.

Sphingolipid metabolism in cancer signalling and therapy

“…Accumulation of sphingomyelin (by silencing of SMPD1 ) in cancer cells led to the accumulation of elongated and unclosed autophagosomal membranes with reduced association of autophagy-related protein 9A (ATG9A) (FIG. 2), suggesting a negative role for sphingomyelin in the maturation of early autophagosomal membranes through alterations in ATG9A trafficking 95 .…”

“…Accumulation of dihydroceramide and/or ceramide in the ER leads to permeabilization of the autophagosomal and lysosomal membranes, resulting in cathepsin release and apoptotic cell death 94 . Moreover, hydrolysis of sphingomyelin (SM) to generate ceramide via acid sphingomyelinase (ASMase) is important to promote the elongation and maturation of autophagosomal membranes via recruitment of autophagy-related protein 9A (ATG9A), suggesting a novel role for ceramide in the maturation of early autophagosomal membranes 95 .…”

SNX 18 regulates ATG 9A trafficking from recycling endosomes by recruiting Dynamin‐2

“…The PI3P‐binding protein WIPI2 is recruited to the forming phagophore 3 where it further interacts with the ATG12–5‐16L1 complex to allow conjugation of Atg8 homologues of the LC3 and GABARAP subfamilies to phosphatidylethanolamine (PE) in the autophagic membrane 4, 5. Mammalian ATG9A is the only conserved transmembrane core ATG protein and has been found to localise to the trans‐Golgi network (TGN), plasma membrane and endosomes, including recycling endosomes 6, 7, 8, 9, 10. ATG9A cycles between these compartments and the continuous trafficking of ATG9A are thought to generate an ATG9A reservoir ready to contribute to autophagosome biogenesis upon induction of autophagy 10, 11, 12.…”

“…The phagophore expands by receiving membrane input through vesicle transport or membrane contact sites from different membrane sources, including ER‐exit sites (ERES) and the ER–Golgi intermediate compartment (ERGIC) 18, 19, 20, the Golgi apparatus 21, 22, mitochondria 23, 24 and the plasma membrane 21, 22, 25. Recently, it has become evident that recycling endosomes also play an important role during autophagosome biogenesis and both ATG16L1 and ATG9A traffic from the plasma membrane to recycling endosomes on their way to sites of autophagosome formation 7, 8, 9, 10, 26. We recently identified the PX‐BAR‐containing protein sorting nexin 18 (SNX18) as a positive regulator of autophagy 26.…”

Defective Autophagy, Mitochondrial Clearance and Lipophagy in Niemann-Pick Type B Lymphocytes

“…An alteration in autophagy has been shown in many LSDs, including Niemann-Pick disease type A [5], Niemann-Pick type C (NP-C), Mucopolysaccharidosis type IIIA (MPS-IIIA), Multiple Sulphatase Deficiency (MSD) and Danon disease [6]. In particular, a marked accumulation of autophagosomes and ubiquitinated proteins occurs in the brain of Niemann-Pick type A mice and in fibroblasts from NP-A patients [5], and an amassing of elongated and unclosed autophagic membranes has been found in NP-A fibroblasts [7]. In LSDs, multiple mechanisms, resulting in dysregulated or imbalanced induction, maturation, or degradation, converge to create the pathologic condition of “autophagic stress” [8], characterized by sustained increases in autophagic vacuoles (AVs).…”