Targeting Nitric Oxide Production in Microglia with Novel Imidazodiazepines for Nonsedative Pain Treatment

Abstract

The goal of this research is the identification of new treatments for neuropathic pain. We characterized the GABAergic system of immortalized mouse and human microglia using electrophysiology and qRT-PCR. Cells from both species exhibited membrane current changes in response to γ-aminobutyric acid, with an EC 50 of 260 and 1940 nM, respectively. Human microglia expressed high levels of the γ-aminobutyric acid type A receptor (GABA A R) α 3 subunit, which can assemble with β 1 and γ 2 /δ subunits to form functional GABA A Rs. Mouse microglia contained α 2 , α 3 , and α 5 , in addition to β 1−3 , γ 1−2 , and δ, mRNA, enabling a more diverse array of GABA A Rs than human microglia. Benzodiazepines are well-established modulators of GABA A R activity, prompting a screen of a library of diverse benzodiazepines in microglia for cellular effects. Several active compounds were identified by reduction of nitric oxide (NO) in interferon gamma and lipopolysaccharide activated microglia. However, further investigation with GABA A R antagonists flumazenil, picrotoxin, and bicuculline demonstrated that GABA A Rs were not linked to the NO response. A screen of 48 receptors identified the κ-opioid receptor and to a lesser extent the μ-opioid receptor as molecular targets, with opioid receptor antagonist norbinaltorphimine reversing benzodiazepine induced reduction of microglial NO. Functional assays identified the downregulation of inducible NO synthase as the mode of action of imidazodiazepines MP-IV-010 and GL-IV-03. Like other κ-opioid receptor agonists, GL-IV-03 reduced the agitation response in both phases of the formalin nociception test. However, unlike other κ-opioid receptor agonists, MP-IV-010 and GL-IV-03 did not impair sensorimotor coordination in mice. Thus, MP-IV-010 and GL-IV-03 represent a new class of nonsedative drug candidates for inflammatory pain.

Activation of locus coeruleus-spinal cord noradrenergic neurons alleviates neuropathic pain in mice via reducing neuroinflammation from astrocytes and microglia in spinal dorsal horn

“…It has been confirmed that the NE receptors exist on both astrocytes and microglia derived from the brain [ 11 , 12 ], and the SDH [ 13 ]. For example, NE inhibited microglia activation and cytokine expression in vitro [ 14 ].…”

Dual Molecules Targeting 5-HT6 and GABA-A Receptors as a New Approach to Combat Depression Associated with Neuroinflammation

“…Interestingly it is possible to simultaneously target inflammatory responses and impaired GABA-ergic signaling with molecules modulating the activity of GABA-A receptors . Both microglia and neurons express GABA-A receptors, and modulation of microglia activity via GABA-A receptors decreases secreting proinflammatory cytokines IL-6 and TNF-α. − At the same time, modulation of neuronal GABA-A receptor activity might regulate the impaired GABA-signaling …”

Crosstalk Between GABAergic Neurotransmission and Inflammatory Cascades in the Post-ischemic Brain: Relevance for Stroke Recovery

“…In addition, astrocytes mediate neurotransmitter homeostasis through non-synaptic interactions such as uptake via GAT2 and GAT3 and metabolic conversion (Schousboe et al, 2013 ). Furthermore, with the exception of GAD, microglia also express ionotropic and metabotropic receptors as well as GABA-T (Kuhn et al, 2004 ; Lee et al, 2011 ; Nieman et al, 2020 ). However, the function of these receptors and transporters is poorly understood.…”

“…In contrast to astrocytes, microglia express the γ2-subunit (Höft et al, 2014 ; Nieman et al, 2020 ). It has been demonstrated that microglia actively survey and interact with synapses, extending their processes over the bulbous area of the synaptic buttons (Wake et al, 2009 ).…”