Abstract
Lysosomal storage disorders are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. 573C10 is a Schwann cell line derived from a mouse model of Niemann-Pick type C disease-1, NPC (−/−). Under serum-starved conditions, NPC (−/−) cells manifested impaired autophagy accompanied by an increase in the amount of p62 and lysosome enlargement. Addition of L-leucine to serum-starved NPC (−/−) cells ameliorated the enlargement of lysosomes and the p62 accumulation. Similar autophagy defects were observed in NPC (−/−) cells even without serum starvation upon the knockdown of Spinster-like 1 (SPNS1), a putative transporter protein thought to function in lysosomal recycling. Conversely, SPNS1 overexpression impeded the enlargement of lysosomes, p62 accumulation and mislocalization of the phosphorylated form of the mechanistic Target of rapamycin in NPC (−/−) cells. In addition, we found a reduction in endogenous SPNS1 expression in fibroblasts derived from NPC-1 patients compared with normal fibroblasts. We propose that SPNS1-dependent L-leucine export across the lysosomal membrane is a key step for triggering autophagy, and that this mechanism is impaired in NPC-1.
Highlights
Autophagy is a mechanism for generating nutrients for survival under starvation conditions
When overexpressed in HEK293 cells, the human spin homolog Spinster-like 1 (SPNS1) (HSpin1) induced cell death, which was accompanied by increases in autophagic vacuoles and the production of the mature form of cathepsin D13 These seminal works that implicate Spin/SPNS1 in autophagy prompted us to examine the possible involvement of SPNS1 in lysosomal storage disorder (LSD) pathology using a Schwann cell line 573C10 derived from the NPC1 model mouse[14], which we refer to as Niemann-Pick type C disease (NPC) (−/−) cells hereafter
We show that the addition of L-leucine to the medium for NPC (−/−) culture or the overexpression of SPNS1 in NPC (−/−) cells suppresses both the enlargement of lysosomes and the p62 accumulation induced by serum starvation
Summary
NPC (−/−) cells manifest autophagy defects under serum starvation. To address whether serum starvation affects the structure of lysosomes of NPC (−/−) cells, we employed a lysotracker to label the lysosomes in these cells, which were counterstained with phalloidin for F-actin and DAPI for DNA (Fig. 1A). Addition of L-leucine to the nutrient-deprived NPC (−/−) culture led to altered positioning of phosphorylated mTOR and lysosomes: they were enriched in the periphery rather than in the perinuclear region (Fig. 2C) In this context, it is noteworthy that Leucyl-tRNA synthetase (LRS) plays a critical rule in amino acid-induced mTORC1 activation by sensing the intracellular leucine concentration[23]. To test for the possible involvement of SPNS1 in the observed rescue by L-leucine, we examined the effect of SPNS1-knockdown in NPC (−/−) cells on the subcellular localization of phosphorylated mTOR, since this likely represents a key step for the L-leucine-mediated rescue of autophagy defects in these cells. In NPC (−/−) cells with SPNS1-knockdown that were maintained in a normal medium, phosphorylated mTOR was preferentially localized to closed enlarged lysosomes in the perinuclear area (Fig. 2D). This hypothesis awaits rigorous experimental testing, our present findings implicate the importance of SPNS1 in lysosomal homeostasis, and suggest a novel target for future LSD therapy
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