L-Proline Prevents Endoplasmic Reticulum Stress in Microglial Cells Exposed to L-azetidine-2-carboxylic Acid

L-Azetidine-2-carboxylic acid (AZE) is a non-protein amino acid that shares structural similarities with its proteogenic L-proline amino acid counterpart. For this reason, AZE can be misincorporated in place of L-proline, contributing to AZE toxicity. In previous work, we have shown that AZE induces both polarization and apoptosis in BV2 microglial cells. However, it is still unknown if these detrimental effects involve endoplasmic reticulum (ER) stress and whether L-proline co-administration prevents AZE-induced damage to microglia. Here, we investigated the gene expression of ER stress markers in BV2 microglial cells treated with AZE alone (1000 & mu;M), or co-treated with L-proline (50 & mu;M), for 6 or 24 h. AZE reduced cell viability, nitric oxide (NO) secretion and caused a robust activation of the unfolded protein response (UPR) genes (ATF4, ATF6, ERN1, PERK, XBP1, DDIT3, GADD34). These results were confirmed by immunofluorescence in BV2 and primary microglial cultures. AZE also altered the expression of microglial M1 phenotypic markers (increased IL-6, decreased CD206 and TREM2 expression). These effects were almost completely prevented upon L-proline co-administration. Finally, triple/quadrupole mass spectrometry demonstrated a robust increase in AZE-bound proteins after AZE treatment, which was reduced by 84% upon L-proline co-supplementation. This study identified ER stress as a pathogenic mechanism for AZE-induced microglial activation and death, which is reversed by co-administration of L-proline.

Automated summary

L-Azetidine-2-carboxylic acid (AZE) is a non-protein amino acid that can be misincorporated in place of L-proline, leading to toxicity. This study investigated the effects of AZE on BV2 microglial cells and found that it induced ER stress, M1 phenotypic marker changes, and reduced cell viability and NO secretion. Co-administration of L-proline prevented these detrimental effects. The study also showed an increase in AZE-bound proteins after AZE treatment, which was reduced by L-proline co-supplementation.