Kynurenic Acid and Its Analog SZR104 Exhibit Strong Antiinflammatory Effects and Alter the Intracellular Distribution and Methylation Patterns of H3 Histones in Immunochallenged Microglia-Enriched Cultures of Newborn Rat Brains.

Melinda Szabo,Karolina Dulka,Karoly Gulya,András Mihály,Ferenc Fülöp,Noémi Lajkó,László Vécsei,István Szatmári

doi:10.3390/ijms23031079

Melinda Szabo, Karolina Dulka + Show 6 more

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https://doi.org/10.3390/ijms23031079

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Abstract

Kynurenic acid (KYNA) is implicated in antiinflammatory processes in the brain through several cellular and molecular targets, among which microglia-related mechanisms are of paramount importance. In this study, we describe the effects of KYNA and one of its analogs, the brain-penetrable SZR104 (N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-hydroxyquinoline-2-carboxamide), on the intracellular distribution and methylation patterns of histone H3 in immunochallenged microglia cultures. Microglia-enriched secondary cultures made from newborn rat forebrains were immunochallenged with lipopolysaccharide (LPS). The protein levels of selected inflammatory markers C–X–C motif chemokine ligand 10 (CXCL10) and C–C motif chemokine receptor 1 (CCR1), histone H3, and posttranslational modifications of histone H3 lys methylation sites (H3K9me3 and H3K36me2, marks typically associated with opposite effects on gene expression) were analyzed using quantitative fluorescent immunocytochemistry and western blots in control or LPS-treated cultures with or without KYNA or SZR104. KYNA and SZR104 reduced levels of the inflammatory marker proteins CXCL10 and CCR1 after LPS-treatment. Moreover, KYNA and SZR104 favorably affected histone methylation patterns as H3K9me3 and H3K36me2 immunoreactivities, and histone H3 protein levels returned toward control values after LPS treatment. The cytoplasmic translocation of H3K9me3 from the nucleus indicated inflammatory distress, a process that could be inhibited by KYNA and SZR104. Thus, KYNA signaling and metabolism, and especially brain-penetrable KYNA analogs such as SZR104, could be key targets in the pathway that connects chromatin structure and epigenetic mechanisms with functional consequences that affect neuroinflammation and perhaps neurodegeneration.

Highlights

Several recent experimental and clinical studies have found that endogenous tryptophan metabolites, including kynurenic acid (KYNA), are involved in several neurophysiological and neuropathophysiological mechanisms [1,2,3]
chemokine receptor 1 (CCR1) Levels In CD11b/c-labeled microglial cells taken from unchallenged and treated microglia-enriched secondary cell cultures, there was a robust increase in the amount of immunoreactivity of the inflammation marker C–X–C motif chemokine ligand 10 (CXCL10) in LPS-treated microglia (Figure 1)
Quantitative light microscopic microdensitometric analysis of CXCL10 protein expression revealed that LPS challenge significantly elevated CXCL10 immunoreactivity in microglial cells, whereas KYNA alone, SZR104 alone, or either combined with LPS significantly depleted the CXCL10 immunoreactive signal to control levels (Figure 2)

Summary

Introduction

Several recent experimental and clinical studies have found that endogenous tryptophan metabolites, including kynurenic acid (KYNA), are involved in several neurophysiological and neuropathophysiological mechanisms [1,2,3]. Since KYNA does not pass the blood–brain barrier, researchers are attempting to synthesize KYNA analogs that can penetrate this barrier [6] and to provide possible treatments for neurodegenerative or neuroinflammatory disorders [7,8]. One such analog, N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-hydroxyquinoline2-carboxamide (SZR104; Table S1), was recently synthesized [9,10] and applied successfully against pentylenetetrazole-induced seizures, and in doing so significantly it decreased the seizure-evoked field potentials [11]. KYNA and SZR104 exhibit antiinflammatory properties both in vitro and in vivo; they markedly inhibit the lipopolysaccharide (LPS)-stimulated phagocytotic activity of cultured microglial cells and display potent immunosuppressive capabilities in an animal model of epilepsy [5]

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