L-Proline Prevents Endoplasmic Reticulum Stress in Microglial Cells Exposed to L-azetidine-2-carboxylic Acid
Jordan Allan Piper,
Nour Al Hammouri,
Margo Iris Jansen,
Kenneth J. Rodgers,
Giuseppe Musumeci,
Amolika Dhungana,
Sahar Masoumeh Ghorbanpour,
Laura A. Bradfield,
Alessandro Castorina
Affiliations
Jordan Allan Piper
Laboratory of Cellular and Molecular Neuroscience (LCMN), School of Life Sciences, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia
Nour Al Hammouri
Laboratory of Cellular and Molecular Neuroscience (LCMN), School of Life Sciences, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia
Margo Iris Jansen
Laboratory of Cellular and Molecular Neuroscience (LCMN), School of Life Sciences, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia
Kenneth J. Rodgers
Neurotoxin Research Group, School of Life Sciences, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia
Giuseppe Musumeci
Department of Biomedical and Biotechnological Sciences, Section of Anatomy, Histology and Movement Science, School of Medicine, University of Catania, Via S. Sofia n°97, 95123 Catania, Italy
Amolika Dhungana
School of Life Sciences, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia
Sahar Masoumeh Ghorbanpour
School of Life Sciences, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia
Laura A. Bradfield
School of Life Sciences, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia
Alessandro Castorina
Laboratory of Cellular and Molecular Neuroscience (LCMN), School of Life Sciences, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia
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 µM), or co-treated with L-proline (50 µM), 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.
