Calcineurin Controls Expression of EAAT1/GLAST in Mouse and Human Cultured Astrocytes through Dynamic Regulation of Protein Synthesis and Degradation

Int J Mol Sci. 2020 Mar 23;21(6):2213. doi: 10.3390/ijms21062213.

Abstract

Alterations in the expression of glutamate/aspartate transporter (GLAST) have been associated with several neuropathological conditions including Alzheimer's disease and epilepsy. However, the mechanisms by which GLAST expression is altered are poorly understood. Here we used a combination of pharmacological and genetic approaches coupled with quantitative PCR and Western blot to investigate the mechanism of the regulation of GLAST expression by a Ca2+/calmodulin-activated phosphatase calcineurin (CaN). We show that treatment of cultured hippocampal mouse and fetal human astrocytes with a CaN inhibitor FK506 resulted in a dynamic modulation of GLAST protein expression, being downregulated after 24-48 h, but upregulated after 7 days of continuous FK506 (200 nM) treatment. Protein synthesis, as assessed by puromycin incorporation in neo-synthesized polypeptides, was inhibited already after 1 h of FK506 treatment, while the use of a proteasome inhibitor MG132 (1 μM) shows that GLAST protein degradation was only suppressed after 7 days of FK506 treatment. In astrocytes with constitutive genetic ablation of CaN both protein synthesis and degradation were significantly inhibited. Taken together, our data suggest that, in cultured astrocytes, CaN controls GLAST expression at a posttranscriptional level through regulation of GLAST protein synthesis and degradation.

Keywords: GLAST; astrocytes; calcineurin; protein degradation; protein synthesis; proteostasis.

MeSH terms

  • Animals
  • Astrocytes / metabolism*
  • Calcineurin / metabolism*
  • Calcineurin / pharmacology
  • Calcineurin Inhibitors
  • Cells, Cultured
  • Excitatory Amino Acid Transporter 1 / genetics*
  • Excitatory Amino Acid Transporter 1 / metabolism
  • Fluorescent Antibody Technique
  • Gene Expression Regulation* / drug effects
  • Gene Knockout Techniques
  • Hippocampus / metabolism
  • Humans
  • Mice
  • Mice, Knockout
  • Models, Molecular
  • Protein Biosynthesis
  • Proteolysis

Substances

  • Calcineurin Inhibitors
  • Excitatory Amino Acid Transporter 1
  • SLC1A3 protein, human
  • Calcineurin