Pathogenic missense variants in SLC2A1 (GLUT1) misroute to lysosomes in endothelial cells: insights into molecular pathology of GLUT1 deficiency

Background Glucose transporter type 1 (GLUT1), encoded by the SLC2A1 gene, mediates basal glucose uptake and is essential for brain energy homeostasis. Genetic variants in SLC2A1 cause GLUT1 deficiency syndrome (GLUT1 DS), a rare neurometabolic disorder characterized by impaired glucose transport across the blood-brain barrier and encompassing a wide and expanding phenotypic spectrum. Although numerous SLC2A1 missense variants have been identified, their functional interpretation remains challenging and many are currently classified as “variants of uncertain significance”, mainly due to our limited understanding of the underlying molecular mechanisms of pathogenesis. To date, most functional studies have focused on glucose transport activity, while the contribution of altered intracellular trafficking has remained poorly explored. Methods Eight SLC2A1 missense variants were selected and mutants were generated by site-directed mutagenesis of GLUT1-EGFP encoding plasmid. Subcellular localization and trafficking were investigated by using confocal microscopy and quantitative colocalization analysis in human brain endothelial (hCMEC/D3) and HeLa cells, also in response to changes in extracellular glucose availability; protein content in response to autophagy perturbation was evaluated by Western blotting. Results We unveiled that four of the selected variants altered GLUT1 distribution and metabolic responsiveness, promoting intracellular retention, with most of them showing marked association to lysosome-related compartments (N34S, R126L and Q283R). Conclusions These findings suggest a novel pathogenic mechanism, converging to misrouting to lysosomes, that might represent a shared feature for a subset of GLUT1 missense variants. In this scenario, trafficking-based cellular phenotyping might represent a complementary strategy to conventional transport assays for improving the interpretation of GLUT1 variants and refining genotype-phenotype correlations in the expanding spectrum of SLC2A1 -related disorders.