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Elene Tsitsilashvili,1 Maia Sepashvili,1,2 Marika Chikviladze,1 Lali Shanshiashvili,1,2,* David Mikeladze1,2,* 1Institute of Chemical Biology, Ilia State University, Tbilisi, Georgia; 2Department of Biochemistry, I. Beritashvili Center of Experimental Biomedicine, Tbilisi, Georgia *These authors contributed equally to this work Purpose: During a neuronal injury, a variety of immune cells infiltrate into the local microenvironment at the demyelination site. After the destruction of the intact myelin sheath, its major constituent myelin basic protein (MBP) dissociates from the plasma membrane and acts as a free ligand on the infiltrated immune cells. MBP exhibits charge microheterogeneity as a result of post-translational modifications, but the effect of various isomers of MBP on the activity of macrophages is not known.Materials and methods: MBP was isolated and purified from bovine brain white matter. RAW 264.7 macrophages were cultured in DMEM supplemented with heat-inactivated fetal bovine serum. For evaluation of macrophage polarization following treatment of RAW 264.7 cells with MBP charge isomers, inducible nitric oxide synthase (iNOS) expression (M1 phenotype marker) and arginase-1 expression (M2 phenotype marker) were determined in cell lysates by ELISA. To assess Rac activity, G-LISA Rac Activation Assay system was used. The expression of receptor for advanced glycation end-products (RAGE) and high mobility group box 1 (HMGB1) protein were assayed by Western blot analysis.Results: Our results have shown that minimally modified C1 component of MBP increases the expression of arginase-1 in cells, decreases the expression of iNOS, does not change the secretion of HMGB1 protein, but significantly elevates surface expression of RAGE, and in parallel, increases the activity of small GTPase Rac. On the other hand, highly modified deiminated isomer C8-MBP increases the secretion of HMGB1 protein but does not change the expression of arginase-1 or the content of RAGE.Conclusion: These data indicate that deiminated C8 isomer of MBP tends to polarize RAW macrophages into M1 phenotypes, whereas C1 enhances the activity of M2 phenotype markers. Keywords: Arginase-1, iNOS, HMGB1, RAGE

A defining feature of tumor inflammation is the polarization of M1 into M2 macrophages which promotes tumor growth, angiogenesis, invasion, and metastasis. The mechanism(s) that control the M1-M2 transition remain to be determined. During inflammatory states, activated innate cells, as well as tumor cells, release amino acid glutamate that induce innate cell migration by activating class I/5 metabotropic glutamate receptors (mGluR1/5). Mutation of mGluR1/5 detected in several tumors triggers a downstream signaling cascade leading to activation of Rho family GTPase, Rac1, and Rac2. Rac overexpression in tumors may play a role in cancer progression. Targeting the Rac pathway is an approach to cancer treatment.The sigma1R (Sig1R) is an endoplasmic reticulum (ER)-resident molecular chaperone expressed at the mitochondria-associated ER membranes (MAM). Cell treatment with sigma antagonists causes rounding, detachment and growth inhibition of several cancer cells, however whether Sig1R protects cancer cells from death remains to be elucidated.To investigate the interplay between SigR1, Rac, and mGluR5, immunoprecipitation experiments were performed. Sig1R interacted with the Rac1 and Rac2. This interaction was increased in the presence of sigma-agonists, decreased by sigma-antagonists. Sig1R-Rac interaction was enhanced by GppNHp, suggesting that active Rac interacted with Sig1R. Specific association/dissociation of Sig1R with an inositol-3-phospate receptor (IP3R) and BiP was also found. p21-activated kinases (PAK) downstream effectors of Rac. PAK can phosphorylate various regulatory proteins, including pro-apoptotic Bad. Sigma-agonists increase PAK-dependent phosphorylation, causing dissociation of Bad from mitochondria, thus sigma-ligands could operate through SigR1/Rac complex by changing the PAK activity. Rac1 is be involved in the assembly and activation of ROS-generating enzyme - NADPH oxidase (NOX). ROS are increased in the presence of sigma-agonist (pentazocine), and sigma-antagonist (haloperidol) eliminated this effect, suggesting that that sigma-ligands could operate through SigR1/Rac complex by changing the NOX activity. Sigma 1-receptor induces immunosuppression through modulation of IP3R and Rac in macrophages. In activated macrophages, the binding of SigR1 to IP3R sigma ligands was shown to be decreased, and binding of SigR1 to Rac2 was increased. SigR1 was immunoprecipitated with mGluR5 suggesting that intracellular glutamate receptor may be part of a macromolecular complex in MAM. We next transfected mGluR5 cDNAs into macrophages. This decreased the binding of SigR1 to IP3R. Immunostimulants, like LPS, induced the association of SigR1 with Rac only in non-transformed cells., whereas sigma-agonists increased the binding of SigR1 to Rac in mGluR5-overexpressing macrophages that had a decreased sensitivity to the immunostimulants. Our data suggest that Sig1R dissociates from IP3R, and then associates with Rac1 and Rac2, and through this association/dissociation, sigma ligands can regulate Rac activity. The intracellular glutamate receptor may participate in induction of SigR1-Rac association. This association could lead to activation of downstream effectors (Pak and NOX) of Rac, to initiation of actin rearrangement and production of ROS. This may induce the M1→M2 transition and immunosuppression contributing to progression of tumorigenesis. [ABSTRACT FROM AUTHOR]

The deamination of arginine and its conversion to citrulline is a modification observed in positively charged proteins such as histones or myelin basic protein (MBP). This reaction is catalyzed by peptidyl arginine deiminase (PAD), whose abnormal activation is associated with autoimmune diseases like rheumatoid arthritis and multiple sclerosis. However, the mechanisms that trigger PAD activation and the pathophysiological processes involved in hypercitrullination remain unknown. In this study, we investigated the interaction between PAD and various charged isomers of MBP, each differing in the degree of post-translational modification. Immunoprecipitation experiments were conducted to examine the binding between PAD and the different charge isomers of MBP. Our findings revealed that the phosphorylated forms of MBP (C3 and C4) exhibited a higher affinity for PAD compared to the unmodified (C1) and fully citrullinated forms (C8). Additionally, we observed that only in the presence of the unmodified C1 isomer did PAD undergo autocitrullination, which was inhibited by the endogenous guanidine-containing component, creatine. In the presence of other isomers, PAD did not undergo autocitrullination. Furthermore, we found that the unmodified isomer of MBP-C1 contains methylated arginines, which were not affected by the pre-treatment with PAD. Based on our findings, we propose that the increased phosphorylation of central threonines in the original MBP may trigger PAD activation, leading to increased citrullination of the protein and subsequent disorganization of the myelin sheath. These insights contribute to a better understanding of the underlying mechanisms in autoimmune diseases associated with hypercitrullination, potentially opening new avenues for therapeutic interventions., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: During the course of demyelinating inflammatory diseases, myelin-derived proteins, including myelin basic protein(MBP), are secreted into extracellular space. MBP shows extensive post-translational modifications, including deimination/citrullination. Deiminated MBP is structurally less ordered, susceptible to proteolytic attack, and more immunogenic than unmodified MBP. This study investigated the effect of the deiminated/citrullinated isomer of MBP(C8) and the unmodified isomer of MBP(C1) on cultured primary astrocytes., Methods: MBP charge isomers were isolated/purified from bovine brain. Primary astrocyte cultures were prepared from the 2-day-old Wistar rats. For evaluation of glutamate release/uptake a Fluorimetric glutamate assay was used. Expression of peroxisome proliferator-activated receptor-gamma(PPAR-γ), excitatory amino acid transporter 2(EAAT2), the inhibitor of the nuclear factor kappa-B(ikB) and high mobility group-B1(HMGB1) protein were assayed by Western blot analysis. IL-17A expression was determined in cell medium by ELISA., Results: We found that MBP(C8) and MBP(C1) acted differently on the uptake/release of glutamate in astrocytes: C1 increased glutamate uptake and did not change its release, whereas C8 decreased glutamate release but did not change its uptake. Both isomers increased the expression of PPAR-γ and EAAT2 to the same degree. Western blots of cell lysates revealed decreased expression of ikB and increased expression of HMGB1 proteins after treatment of astrocytes by C8. Moreover, C8-treated cells released more nitric oxide and proinflammatory IL-17A than C1-treated cells., Conclusions: These data suggest that the most immunogenic deiminated isomer C8, in parallel to the decreases in glutamate release, elicits an inflammatory response and enhances the secretion of proinflammatory molecules via activation of nuclear factor kappa B(NF-kB)., Summary Statement: The most modified-citrullinated myelin basic protein charge isomer decreases glutamate release, elicits an inflammatory response and enhances the secretion of proinflammatory molecules via activation of nuclear factor kappa B in astrocytes., Competing Interests: The authors declare that they have no conflicts of interest., (© 2024 The Authors.)

Increased p-cresol levels reportedly alter brain dopamine metabolism and exacerbate neurological disorders in experimental animals. In contrast to toxic concentrations, low doses of p-cresol may have distinct effects on neuronal metabolism. However, the role of p-cresol in synapse remodeling, neurite outgrowth, and other anabolic processes in neurons remains elusive. We propose that low doses of p-cresol affect neuronal cell structural remodeling compared with the high concentration-mediated harmful effects. Thus, the effects of p-cresol on the secretion of brain-derived neurotrophic factor (BDNF) and neurofilament subunit expression were examined using rat pheochromocytoma cells (PC-12 cells). We observed that low doses of p-cresol potentiated nerve growth factor-induced differentiation via secretion of BDNF in cultured PC-12 cells. Opioidergic compounds modulated these p-cresol effects, which were reversed by oxytocin. We propose that this effect of p-cresol has an adaptive and compensatory character and can be attributed to the induction of oxidative stress. Accordingly, we hypothesize that low doses of p-cresol induce mild oxidative stress, stimulating BDNF release by activating redox-sensitive genes. Given that the intestinal microbiome is the primary source of endogenous p-cresol, the balance between gut microbiome strains (especially Clostridium species) and opioidergic compounds may directly influence neuroplasticity., Competing Interests: Conflict of interests: The authors declare that there are no conflicts of interest., (© 2022 the Author(s), licensee AIMS Press.)

Mislocalization and abnormal expression of N-methyl-D-aspartate glutamate receptor (NMDAR) subunits is observed in several brain disorders and pathological conditions. Recently, we have shown that intraperitoneal injection of the gut neurotoxin p-cresol induces autism-like behavior and accelerates seizure reactions in healthy and epilepsy-prone rats, respectively. In this study, we evaluated the expression of GLUN2B and GLUN2A NMDAR subunits, and assessed the activity of cAMP-response element binding protein (CREB) and Rac1 in the hippocampi and nucleus accumbens of healthy and epilepsy-prone rats following p-cresol administration. We have found that subchronic intraperitoneal injection of p-cresol induced differential expression of GLUN2B and GLUN2A between the two brain regions, and altered the GLUN2B/GLUN2A ratio, in rats in both groups. Moreover, p-cresol impaired CREB phosphorylation in both brain structures and stimulated Rac activity in the hippocampus. These data indicate that p-cresol differently modulates the expression of NMDAR subunits in the nucleus accumbens and hippocampi of healthy and epilepsy-prone rats. We propose that these differences are due to the specificity of interactions between dopaminergic and glutamatergic pathways in these structures., Competing Interests: Conflict of interest: The authors declare no conflict of interest., (© 2020 the Author(s), licensee AIMS Press.)

Background: Macrophages are a functionally heterogeneous cell population and depending on microenvironments they polarize in two main groups: M1 and M2. Glutamic acid and glutamate receptors may participate in the regulation of macrophage plasticity. To investigate the role of glutamatergic systems in macrophages physiology, we performed the transfection of mGluR5 cDNAs into RAW-264.7 cells., Results: Comparative analysis of modified (RAW-mGluR5 macrophages) and non-modified macrophages (RAW-macrophages) has shown that the RAW-mGluR5 macrophages absorbed more glutamate than control cells and the amount of intracellular glutamate correlated with the expression of excitatory amino acid transporters -2 (EAAT-2). Besides, our results have shown that RAW-mGluR5 macrophages expressed a higher level of peroxisome proliferator-activated receptor γ (PPAR-γ) and secreted more IL-10, high mobility group box 1 proteins (HMGB1) and Galectin-3 than control RAW-macrophages., Conclusions: We propose that elevation of intracellular glutamate and expression of mGluR5 may initiate the metabolic rearrangement in macrophages that could contribute to the formation of an immunosuppressive phenotype.

The association of myelin basic protein charge isomers with the lipid part of the myelin membrane was investigated at the microscopic (molecular) level in a model membrane system, using optical waveguide lightmode spectrometry to determine with high precision the kinetics of association and dissociation to planar phospholipid membranes under controlled hydrodynamic conditions and over a range of protein concentrations. Detailed analysis of the data revealed a rich and intricate behaviour and clearly showed that the membrane protein affinity is characterized by at least four independent parameters: (i) the association rate coefficient characterizing the protein-membrane interaction energy as the protein approaches the fluid-membrane interface; (ii) the protein-membrane adhesion, i.e., the probability that it will remain at the membrane after arrival; (iii) the protein conformation at the membrane; and (iv) the protein's tendency to cluster at the membrane. Some of these parameters varied in characteristic ways as the bulk solution concentration of the protein was varied, giving further clues to the detailed molecular comportment of the protein. The parameters and their characteristic variations with bulk concentration were markedly different for the different isomers. Implications of these results for neurological disorders involving demyelination, such as multiple sclerosis, are discussed.