Lysoglycosphingolipids have the ability to induce cell death through direct PI3K inhibition.

Sphingolipidoses are inherited metabolic disorders associated with glycosphingolipids accumulation, neurodegeneration, and neuroinflammation leading to severe neurological symptoms. Lysoglycosphingolipids (lysoGSLs), also known to accumulate in the tissues of sphingolipidosis patients, exhibit cytotoxicity. LysoGSLs are the possible pathogenic cause, but the mechanisms are still unknown in detail. Here, we first show that lysoGSLs are potential inhibitors of phosphoinositide 3-kinase (PI3K) to reduce cell survival signaling. We found that phosphorylated Akt was commonly reduced in fibroblasts from patients with sphingolipidoses, including GM1/GM2 gangliosidoses and Gaucher's disease, suggesting the contribution of lysoGSLs to the pathogenesis. LysoGSLs caused cell death and decreased the level of phosphorylated Akt as in the patient fibroblasts. Extracellularly administered lysoGM1 permeated the cell membrane to diffusely distribute in the cytoplasm. LysoGM1 and lysoGM2 also inhibited the production of phosphatidylinositol-(3,4,5)-triphosphate and the translocation of Akt from the cytoplasm to the plasma membrane. We also predicted that lysoGSLs could directly bind to the catalytic domain of PI3K by in silico docking study, suggesting that lysoGSLs could inhibit PI3K by directly interacting with PI3K in the cytoplasm. Furthermore, we revealed that the increment of lysoGSLs amounts in the brain of sphingolipidosis model mice correlated with the neurodegenerative progression. Our findings suggest that the down-regulation of PI3K/Akt signaling by direct interaction of lysoGSLs with PI3K in the brains is a neurodegenerative mechanism in sphingolipidoses. Moreover, we could propose the intracellular PI3K activation or inhibition of lysoGSLs biosynthesis as novel therapeutic approaches for sphingolipidoses because lysoGSLs should be cell death mediators by directly inhibiting PI3K, especially in neurons.

Oxidative Stress Impairs Autophagy via Inhibition of Lysosomal Transport of VAMP8.

Autophagy is a highly conserved intracellular degrading system and its dysfunction is considered related to the cause of neurodegenerative disorders. A previous study showed that the inhibition of endocytosis transport attenuates soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein transport to lysosomes and block autophagy. The other studies demonstrated oxidative stress, one of the inducers of neurodegenerative diseases inhibits endocytosis transport. Thus, we hypothesized that oxidative stress-induced endocytosis inhibition causes alteration of SNARE protein transport to lysosomes and impairs autophagy. Here, we demonstrated that oxidative stress inhibits endocytosis and decreased the lysosomal localization of VAMP8, one of the autophagy-related SNARE proteins in a human neuroblastoma cell line. Moreover, this oxidative stress induction blocked the autophagosome-lysosome fusion step. Since we also observed decreased lysosomal localization of VAMP8 and inhibition of autophagosome-lysosome fusion in endocytosis inhibitor-treated cells, oxidative stress may inhibit VAMP8 trafficking by suppressing endocytosis and impair autophagy. Our findings suggest that oxidative stress-induced inhibition of VAMP8 trafficking to lysosomes is associated with the development of neurodegenerative diseases due to the blocked autophagosome-lysosome fusion, and may provide a new therapeutic target for restoring the autophagic activity.

Aberrant autophagy in lysosomal storage disorders marked by a lysosomal SNARE protein shortage due to suppression of endocytosis.

Lysosomal storage disorders (LSDs) are inherited metabolic diseases caused by genetic defects in lysosomal enzymes or related factors. LSDs are associated with excessive accumulation of natural substrates in lysosomes leading to central nervous system and peripheral tissue damage. Abnormal autophagy is also involved in pathogenesis, although the underlying mechanisms remain unclear. We demonstrated that impairment of lysosome-autophagosome fusion is due to suppressed endocytosis in LSDs. The fusion was reduced in several LSD cells and the brains of LSD model mice, suggesting that the completion of autophagy is suppressed by the accumulation of substrates. In this brain, the expression of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, VAMP8 and Syntaxin7, was decreased on the lysosomal surface but not intracellular. This aberrant autophagy preceded the development of pathological phenotypes in LSD-model mice. Furthermore, the enzyme deficiency leading to the substrate accumulation could suppress endocytosis, and the inhibited endocytosis decreased SNARE proteins localized on lysosomes. These findings suggest that the shortage of SNARE proteins on lysosomes is one of the reasons for the impairment of lysosome-autophagosome fusion in LSD cells. Defects in lysosomal enzyme activity suppress endocytosis and decrease the supply of intracellular SNARE proteins recruited to lysosomes. This shortage of lysosomal SNARE proteins impairs lysosome-autophagosome fusion in lysosomal storage disorders.