2. Academic Validation
  3. Pro-Inflammatory and Pro-Apoptotic Effects of the Non-Protein Amino Acid L-Azetidine-2-Carboxylic Acid in BV2 Microglial Cells

Pro-Inflammatory and Pro-Apoptotic Effects of the Non-Protein Amino Acid L-Azetidine-2-Carboxylic Acid in BV2 Microglial Cells

  • Curr Issues Mol Biol. 2022 Sep 28;44(10):4500-4516. doi: 10.3390/cimb44100308.
Jordan Allan Piper 1,  Margo Iris Jansen 1,  Sarah Thomas Broome 1,  Kenneth J Rodgers 2,  Giuseppe Musumeci 3 4,  Alessandro Castorina 1
Affiliations

Affiliations

  • 1 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.
  • 2 Neurotoxin Research Group, School of Life Sciences, Faculty of Science, University of Technology Sydney, P.O. Box 123, Broadway, Sydney, NSW 2007, Australia.
  • 3 Department of Biomedical and Biotechnological Sciences, Anatomy, Histology and Movement Science Section, School of Medicine, University of Catania, Via S. Sofia No. 87, 95123 Catania, Italy.
  • 4 Research Center on Motor Activities (CRAM), University of Catania, Via S. Sofia No. 97, 95123 Catania, Italy.
PMID: 36286023 DOI: 10.3390/cimb44100308
Abstract

L-Azetidine-2-carboxylic acid (AZE) is a toxic non-protein coding amino acid (npAA) that is highly abundant in sugar and table beets. Due to its structural similarity with the amino acid L-proline, AZE can evade the editing process during protein assembly in eukaryotic cells and be misincorporated into L-proline-rich proteins, potentially causing protein misfolding and Other detrimental effects to cells. In this study, we sought to determine if AZE treatment triggered pro-inflammatory and pro-apoptotic responses in BV2 microglial cells. BV2 microglial cells exposed to AZE at increasing concentrations (0−2000 µM) at 0, 3, 6, 12 and 24 h were assayed for cell viability (MTT) and nitric oxide release (Griess assay). Annexin V-FITC/propidium iodide (PI) staining was used to assess Apoptosis. Real-time qPCR, Western blot and immunocytochemistry were used to interrogate relevant pro- and anti-inflammatory and Other molecular targets of cell survival response. AZE (at concentrations > 1000 µM) significantly reduced cell viability, increased Bax/Bcl2 ratio and caused cell death. Results were mirrored by a robust increase in nitric oxide release, percentage of activated/polarised cells and expression of pro-inflammatory markers (IL-1β, IL-6, NOS2, CD68 and MHC-2a). Additionally, we found that AZE induced the expression of the extracellular matrix degrading Enzyme matrix metalloproteinase 9 (MMP-9) and brain derived neurotrophic factor (BDNF), two critical regulators of microglial motility and structural plasticity. Collectively, these data indicate that AZE-induced toxicity is associated with increased pro-inflammatory activity and reduced survival in BV2 microglia. This evidence may prompt for an increased monitoring of AZE consumption by humans.

Keywords

L-azetidine-2-carboxylic acid; beets; environmental toxin; microglia; multiple sclerosis; neuroinflammation; non-protein amino acid.

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