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Background <p>Glucose transporter type 1 (GLUT1), encoded by the <i>SLC2A1</i> gene, mediates basal glucose uptake and is essential for brain energy homeostasis. Genetic variants in <i>SLC2A1</i> 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. Despite the fact that a high proportion of reported pathogenic <i>SLC2A1</i> variants are missense, their effects on GLUT1 cellular biology remain incompletely understood, and the relative contribution of altered transport, protein stability, and intracellular trafficking has not been fully defined.</p> Methods <p>Eight <i>SLC2A1</i> 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; protein degradation rate was assessed through cycloheximide chase assays.</p> Results <p>We found that four of the selected variants were associated with altered GLUT1 distribution and responsiveness to extracellular glucose availability, promoting intracellular retention, with most of them showing marked association to lysosome-related compartments (N34S, R126L and Q283R).</p> Conclusions <p>Our study identifies variant-associated differences in GLUT1 intracellular distribution in a subset of pathogenic <i>SLC2A1</i> missense variants, with increased lysosomal targeting representing a recurrent cellular fate. These findings provide insights into variant-associated differences in GLUT1 cellular behaviour and highlight intracellular trafficking analysis as a complementary approach to transport-based functional assays for investigating GLUT1 biology. Further studies integrating cellular phenotypes with patient-derived models, biochemical and clinical data will be required to determine how the observed trafficking alterations relate to disease biology and clinical presentation.</p>

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A subset of SLC2A1 missense variants interfere with GLUT1 trafficking in human brain endothelial cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月29日

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  • Sara Lazzari,
  • Agnese Giovannetti,
  • Francesco Petrizzelli,
  • Veronica De Paolis,
  • Francesca Matarazzo,
  • Alvaro H. Crevenna,
  • Tommaso Biagini,
  • Tommaso Mazza,
  • Chiara Parisi,
  • Viviana Caputo

摘要

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. Despite the fact that a high proportion of reported pathogenic SLC2A1 variants are missense, their effects on GLUT1 cellular biology remain incompletely understood, and the relative contribution of altered transport, protein stability, and intracellular trafficking has not been fully defined.

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; protein degradation rate was assessed through cycloheximide chase assays.

Results

We found that four of the selected variants were associated with altered GLUT1 distribution and responsiveness to extracellular glucose availability, promoting intracellular retention, with most of them showing marked association to lysosome-related compartments (N34S, R126L and Q283R).

Conclusions

Our study identifies variant-associated differences in GLUT1 intracellular distribution in a subset of pathogenic SLC2A1 missense variants, with increased lysosomal targeting representing a recurrent cellular fate. These findings provide insights into variant-associated differences in GLUT1 cellular behaviour and highlight intracellular trafficking analysis as a complementary approach to transport-based functional assays for investigating GLUT1 biology. Further studies integrating cellular phenotypes with patient-derived models, biochemical and clinical data will be required to determine how the observed trafficking alterations relate to disease biology and clinical presentation.