Afobazole Modulates Microglial Function via Activation of Both σ-1 and σ-2 Receptors

Abstract

Microglial cells play a critical role in the neuroinflammatory response that accompanies various diseases of the central nervous system, such as ischemic stroke, and ATP is a major signaling molecule regulating the response of these cells to these pathophysiological conditions. Experiments were carried out to determine the effects of afobazole on microglial function and to identify the molecular mechanisms by which afobazole affects microglial cells. Afobazole was found to inhibit migration of microglial cells in response to ATP and UTP chemoattraction in a concentration-dependent manner. Inhibition of either -1 or -2 receptors decreased the effects of afobazole on microglia. In addition to inhibiting microglial cell migration, activation of receptors by afobazole decreased intracellular calcium elevation produced by focal application of ATP and UTP in isolated microglial cells. Furthermore, afobazole blocked membrane currents elicited by rapid application of ATP in microglial cells. Taken together, our data indicate that afobazole inhibits microglial response to P2Y and P2X purinergic receptor activation by functioning as a pan-selective -receptor agonist. In addition to modulating response to purinergic receptor activation, the effects of afobazole on microglial survival during in vitro ischemia were assessed. Application of afobazole during in vitro ischemia decreased microglial cell death during the ischemic episode and after a 24-h recovery period. Moreover, when afobazole was only applied after the ischemic episode, a significant enhancement in cell survival was still observed. Thus, afobazole acts via receptors to decrease microglial response to ATP and provides cytoprotection during and after ischemia.

Afobazole Modulates Neuronal Response to Ischemia and Acidosis via Activation of σ-1 Receptors

“…Taken together, our data suggest that afobazole acts as a -receptor agonist and that it specifically activates -1 receptors. Additional data collected in our laboratory suggests that afobazole may also act as a -2-receptor agonist (Cuevas et al, 2011). Given that neither metaphit nor the -1-receptor-selective antagonists completely eliminated the effects of afobazole, the possibility exists that this drug is also acting via mechanisms other than receptors to prevent [Ca 2ϩ ] i overload.…”

In Vitro Evaluation of Guanidine Analogs as Sigma Receptor Ligands for Potential Anti-Stroke Therapeutics

“…The current study shows that p-BrDPhG also inhibits microglial activation, and this DTG analog is 4-fold more potent than the parent compound and a second pan-selective s agonist, afobazole (Hall et al, 2009;Cuevas et al, 2011b). Unlike ischemia-and acidosis-evoked [Ca 21 ] i dysregulation, which appears to be exclusively affected by s-1 receptor activation, both s-1 and s-2 receptors can modulate microglial migration (Cuevas et al, 2011b).…”

“…Recently, our laboratory showed that activation of s receptors with pan-selective ligands, such as o-DTG or afobazole, modulates microglial activation and inhibits microglial cell migration in response to chemoattractants, such as ATP (Cuevas et al, 2011b). This effect on microglial cell ] i (6S.E.M.)…”

“…Guanidine Analogs as Sigma Receptor Ligands activation has been postulated to be a mechanism by which s receptor activation reduces ischemic stroke injury (Cuevas et al, 2011b). For this reason, experiments were performed to determine whether p-BrDPhG is also more potent than o-DTG in preventing microglial cell migration.…”

“…The migration assays were performed using a 48-well microchemotaxis chamber (Neuro Probe, Inc., Gaithersburg, MD) as described previously (Cuevas et al, 2011b). The bottom wells of the chamber were filled with DMEM containing the chemoattractant, ATP (100 mM), and 0.1% DMSO.…”