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

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.

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

“…Whilst L-proline co-administration was unable to prevent AZE-driven IL6 induction at 1 h (p > 0.05), it remarkably diminished IL6 mRNAs at 6 h (*** p < 0.001 vs. AZE) and at 24 h (**** p < 0.0001 vs. AZE; Figure 2B). IBA1 gene expression was not increased in response to AZE after 1 h (p > 0.05); however, gene expression was significantly up-regulated at 6 h (** p < 0.01 vs. control; Figure 2C), but not at 24 h (p > 0.05 vs. control) corroborating our previous data [7]. Accordingly, L-proline completely prevented IBA1 up-regulation caused by AZE treatment at 6 h (* p < 0.05 vs. AZE; Figure 2D), with minor and non-significant effects at 1 and 24 h (p > 0.05 vs. AZE).…”

“…Viability and Nitric Oxide (NO) Release in BV2 Microglial Cells MTT assay was used to assess whether L-proline co-supplementation elicited any beneficial effects on cell viability and NO release in cells undergoing the same toxicity regime used in our previously established model of AZE toxicity [7]. As depicted in Figure 1A, AZE (1000 µM) caused a progressive and significant reduction in BV2 microglia viability (** p < 0.01 at 6 h and **** p < 0.0001 at 24 h vs. control, respectively).…”

“…Upon its identification, Rubenstein developed a novel unifying hypothesis linking AZE consumption to multiple sclerosis (MS) pathogenesis, in which geographical and historical aspects of the disease were also taken into account [6]. Later studies revealed that the identified NPAA triggers specific pathological alterations in at least two resident cell populations of the central nervous system (CNS), oligodendrocytes and microglia [7,8], corroborating the initial pathogenic link with MS. AZE exhibits high structural similarity with L-proline [9]. For this reason, upon its entry into cells, AZE can evade recognition by aminoacyl-tRNA synthetases (aaRS) and be misincorporated into the sequence of proline-rich proteins upon consumption [6,10].…”

Mechanical Properties and Temperature and Pressure Effects on the Crystal Structure of (2S,3S,4R)-2,3,4-(Trinitratomethyl)-1-nitroazetidine (TMNA) by Single-Crystal X-ray Diffractometry and Density Functional Theory

“…Temperature and pressure are two key thermodynamic variables that govern the phase stability of materials and impact their unit cell parameters and molecular structures. Table 1 lists the experimental unit cell parameters at 297 K, together with those at 100 K. The experimental results reveal a volume increase of 3.6% and anisotropic expansion with unit lengths c (2.1%) > a (0.91%) > b (0.57%) Figure 5 presents the results of the DFT-MD temperature simulations for the TMNA unit cell parameters at 300 K. The simulations yield mean unit cell lengths of a (Å) = 7.92 (7), b (Å) = 8.17 (7), and c (Å) = 21.27(2), which we list in Table 1 along with the experimental values at 297(1) K. Overall, the calculated values are in good agreement with the experimental values at ambient temperature. The model overpredicts the experimental lengths a, b, and c by approximately 3% and the cell volume by approximately 7%.…”

“…Azetidine [(CH 2 ) 3 NH], a saturated four-membered, heterocyclic molecule, and its derivatives have sparked significant interest in recent years because of their potential use as pharmacological products, agriculture compounds, and energetic ingredients. − The versatile azetidine scaffold presents great opportunities for building a variety of azetidine derivatives. Its versatility stems from its structural rigidity as well as from its electronegative nitrogen atom, which provides the ring nucleophilic activity, and its three carbon atoms, which offer the addition of a variety of functional groups.…”