SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20

Abstract

Mutations in SNX14 cause the autosomal recessive cerebellar ataxia 20 (SCAR20). Mutations generally result in loss of protein although several coding region deletions have also been reported. Patient-derived fibroblasts show disrupted autophagy, but the precise function of SNX14 is unknown. The yeast homolog, Mdm1, functions in endoplasmic reticulum (ER)-lysosome/vacuole inter-organelle tethering, but functional conservation in mammals is still required. Here, we show that loss of SNX14 alters but does not block autophagic flux. In addition, we find that SNX14 is an ER-associated protein that functions in neutral lipid homeostasis and inter-organelle crosstalk. SNX14 requires its N-terminal transmembrane helices for ER localization, while the Phox homology (PX) domain is dispensable for subcellular localization. Both SNX14-mutant fibroblasts and SNX14KO HEK293 cells accumulate aberrant cytoplasmic vacuoles, suggesting defects in endolysosomal homeostasis. However, ER-late endosome/lysosome contact sites are maintained in SNX14KO cells, indicating that it is not a prerequisite for ER-endolysosomal tethering. Further investigation of SNX14- deficiency indicates general defects in neutral lipid metabolism. SNX14KO cells display distinct perinuclear accumulation of filipin in LAMP1-positive lysosomal structures indicating cholesterol accumulation. Consistent with this, SNX14KO cells display a slight but detectable decrease in cholesterol ester levels, which is exacerbated with U18666A. Finally, SNX14 associates with ER-derived lipid droplets (LD) following oleate treatment, indicating a role in ER-LD crosstalk. We therefore identify an important role for SNX14 in neutral lipid homeostasis between the ER, lysosomes and LDs that may provide an early intervention target to alleviate the clinical symptoms of SCAR20.

The assembly of lipid droplets and their roles in challenged cells

“…Several NVJ tethers including Nvj1p are not conserved in metazoans, but Mdm1p exhibits clear homologs in mammals. Consistent with this, Mdm1 homolog SNX14, which is associated with pediatric cerebellar ataxia, was recently shown to associate with droplets formed in oleate‐treated mammalian cells, suggesting a conserved role for this protein in droplet dynamics (Bryant et al , ).…”

Diverse Species-Specific Phenotypic Consequences of Loss of Function Sorting Nexin 14 Mutations

“…Mutations usually result in complete loss of SNX14 protein, however truncation or microdeletions have also been investigated in SCAR20 patient derived tissues (1,9).…”

“…This does not appear to affect viability but does manifest through its impact on lipid metabolism. SNX14 has previously been associated with a role in endocytic/lysosomal/endoplasmic reticulum associated processes (1, 2, 9, 10) and mutations result in disruption to normal lipid metabolism (9,10). Interestingly, both SNX13 and SNX14 are homologues of the single Drosophila gene snz and the yeast gene Mdm1, both of which have also been demonstrated to have a role in lipid metabolism in Drosophila (11,14) and yeast (12) respectively.…”

“…Inside the cell, SNX14 mutations impact both autophagy and lipid metabolism (1, 2,9). The most apparent subcellular phenotype is the accumulation of autolysosomes containing lipids (1,9).…”

“…Inside the cell, SNX14 mutations impact both autophagy and lipid metabolism (1, 2,9). The most apparent subcellular phenotype is the accumulation of autolysosomes containing lipids (1,9). SNX14 is localised to the endoplasmic reticulum membrane via its N-terminal transmembrane domain where it is enriched in proximity to lipid droplets (9) and loss of SNX14 disrupts lipid droplet morphology and proper lipid droplet growth following the addition of the exogenous fatty acid oleate (10).…”