Altered Hippocampal and Striatal Expression of Endothelial Markers and VIP/PACAP Neuropeptides in a Mouse Model of Systemic Lupus Erythematosus.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the most common and severe manifestations of lupus; however, its pathogenesis is still poorly understood. While there is sparse evidence suggesting that the ongoing autoimmunity may trigger pathogenic changes to the central nervous system (CNS) microvasculature, culminating in inflammatory/ischemic damage, further evidence is still needed. In this study, we used the spontaneous mouse model of SLE (NZBWF1 mice) to investigate the expression of genes and proteins associated with endothelial (dys)function: tissue and urokinase plasminogen activators (tPA and uPA), intercellular and vascular adhesion molecules 1 (ICAM-1 and VCAM-1), brain derived neurotrophic factor (BDNF), endothelial nitric oxide synthase (eNOS) and Krüppel-like factor 4 (KLF4) and neuroprotection/immune modulation: pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), PACAP receptor (PAC1), VIP receptors 1 and 2 (VPAC1 and VPAC2). Analyses were carried out both in the hippocampus and striatum of SLE mice of two different age groups (2 and 7 months old), since age correlates with disease severity. In the hippocampus, we identified a gene/protein expression profile indicative of mild endothelial dysfunction, which increased in severity in aged SLE mice. These alterations were paralleled by moderate alterations in the expression of VIP, PACAP and related receptors. In contrast, we report a robust upregulation of endothelial activation markers in the striatum of both young and aged mice, concurrent with significant induction of the VIP/PACAP system. These data identify molecular signatures of endothelial alterations in the hippocampus and striatum of NZBWF1 mice, which are accompanied by a heightened expression of endogenous protective/immune-modulatory neuropeptides. Collectively, our results support the idea that NPSLE may cause alterations of the CNS micro-vascular compartment that cannot be effectively counteracted by the endogenous activity of the neuropeptides PACAP and VIP.

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 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.

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

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-1ß, 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.