Activation of Sigma Receptors With Afobazole Modulates Microglial, but Not Neuronal, Apoptotic Gene Expression in Response to Long-Term Ischemia Exposure.

Stroke continues to be a leading cause of death and serious long-term disability. The lack of therapeutic options for treating stroke at delayed time points (≥6 h post-stroke) remains a challenge. The sigma receptor agonist, afobazole, an anxiolytic used clinically in Russia, has been shown to reduce neuronal and glial cell injury following ischemia and acidosis; both of which have been shown to play important roles following an ischemic stroke. However, the mechanism(s) responsible for this cytoprotection remain unknown. Experiments were carried out on isolated microglia from neonatal rats and cortical neurons from embryonic rats to gain further insight into these mechanisms. Prolonged exposure to in vitro ischemia resulted in microglial cell death, which was associated with increased expression of the pro-apoptotic protein, Bax, the death protease, caspase-3, and reduced expression in the anti-apoptotic protein Bcl-2. Incubation of cells with afobazole during ischemia decreased the number of microglia expressing both Bax and caspase-3, and increased cells expressing Bcl-2, which resulted in a concomitant enhancement in cell survival. In similar experiments, incubation of neurons under in vitro ischemic conditions resulted in higher expression of Bax and caspase-3, while at the same time expression of Bcl-2 was decreased. However, unlike observations made in microglial cells, afobazole was unable to modulate the expression of these apoptotic proteins, but a reduction in neuronal death was still noted. The functional state of surviving neurons was assessed by measuring metabolic activity, resting membrane potential, and responses to membrane depolarizations. Results showed that these neurons maintained membrane potential but had low metabolic activity and were unresponsive to membrane depolarizations. However, while these neurons were not fully functional, there was significant protection by afobazole against long-term ischemia-induced cell death. Thus, the effects of sigma receptor activation on microglial and neuronal responses to ischemia differ significantly.

Thorase variants are associated with defects in glutamatergic neurotransmission that can be rescued by Perampanel.

The AAA+ adenosine triphosphatase (ATPase) Thorase plays a critical role in controlling synaptic plasticity by regulating the expression of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Bidirectional sequencing of exons of ATAD1, the gene encoding Thorase, in a cohort of patients with schizophrenia and healthy controls revealed rare Thorase variants. These variants caused defects in glutamatergic signaling by impairing AMPAR internalization and recycling in mouse primary cortical neurons. This contributed to increased surface expression of the AMPAR subunit GluA2 and enhanced synaptic transmission. Heterozygous Thorase-deficient mice engineered to express these Thorase variants showed altered synaptic transmission and several behavioral deficits compared to heterozygous Thorase-deficient mice expressing wild-type Thorase. These behavioral impairments were rescued by the competitive AMPAR antagonist Perampanel, a U.S. Food and Drug Administration-approved drug. These findings suggest that Perampanel may be useful for treating disorders involving compromised AMPAR-mediated glutamatergic neurotransmission.

Activation of σ1 and σ2 receptors by afobazole increases glial cell survival and prevents glial cell activation and nitrosative stress after ischemic stroke.

Activation of sigma receptors at delayed time points has been shown to decrease injury following ischemic stroke. The mixed σ1/σ2 receptor agonist, 5-ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole (afobazole), provides superior long-term outcomes compared to other σ ligands in the rat middle cerebral artery occlusion (MCAO) stroke model. Experiments using the MCAO model were carried out to determine the molecular mechanism involved in the beneficial effects of afobazole. Administration of afobazole (3 mg/kg) at delayed time points post-stroke significantly increased the number of microglia and astrocytes detected in the ipsilateral hemisphere at 96 h post-surgery. Morphological analysis of the microglia indicated that a greater number of these cells were found in the ramified resting state in MCAO animals treated with afobazole relative to MCAO vehicle controls. Similarly, fewer reactive astrocytes were detected in the injured hemisphere of afobazole-treated animals. Both the enhanced survival and reduced activation of glial cells were abolished by co-application of either a σ1 (BD-1063) or a σ2 (SM-21) receptor antagonist with afobazole. To gain further insight into the mechanisms by which afobazole lessens stroke injury, we probed the brain sections for markers of neuroinflammation (tumor necrosis factor α) and nitrosative stress (S-nitrosocysteine). Data show that afobazole significantly reduces S-nitrosocysteine levels, but does not alter tumor necrosis factor α expression 96 h after an ischemic stroke. Taken together our data indicate that afobazole acting via both σ1 and σ2 receptors decreases stroke injury by enhancing glial cell survival, blocking ischemia-induced glial cell activation, and decreasing nitrosative stress.

Stimulation of sigma receptors with afobazole blocks activation of microglia and reduces toxicity caused by amyloid-ß25-35.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the leading cause of senile dementia in the United States. Accumulation of amyloid-ß (Aß) and the effects of this peptide on microglial cells contribute greatly to the etiology of AD. Experiments were carried out to determine whether the pan-selective σ-receptor agonist afobazole can modulate microglial response to the cytotoxic Aß fragment, Aß25-35. Treatment with afobazole decreased microglial activation in response to Aß, as indicated by reduced membrane ruffling and cell migration. The effects of afobazole on Aß25-35-evoked migration were concentration dependent and consistent with σ-receptor activation. When afobazole was coapplied with either BD-1047 [N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide] or rimcazole, which are σ-1- and σ-2-selective antagonists, respectively, the inhibition of Aß25-35-induced migration by afobazole was reduced. Prolonged exposure of microglia to Aß25-35 resulted in glial cell death that was associated with increased expression of the proapoptotic protein Bax and the death protease caspase-3. Coapplication of afobazole with Aß25-35 decreased the number of cells expressing both Bax and caspase-3 and resulted in a concomitant enhancement in cell survival. Although afobazole inhibited activation of microglia cells by Aß25-35, it preserved normal functional responses in these cells after exposure to the amyloid peptide. Intracellular calcium increases induced by ATP were depressed in microglia after 24-hour exposure to Aß25-35. However, coincubation in afobazole returned these responses to near control levels. Therefore, stimulation of σ-1 and σ-2 receptors by afobazole prevents Aß25-35 activation of microglia and inhibits Aß25-35-associated cytotoxicity, suggesting that afobazole may be useful for AD therapeutics.

Afobazole activation of σ-1 receptors modulates neuronal responses to amyloid-ß25-35.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a continual decline of cognitive function. No therapy has been identified that can effectively halt or reverse its progression. One hallmark of AD is accumulation of the amyloid-ß peptide (Aß), which alone induces neuronal injury via various mechanisms. Data presented here demonstrate that prolonged exposure (1-24 hours) of rat cortical neurons to Aß25-35 results in an increase in basal intracellular Ca(2+) concentration ([Ca(2+)]i), and that coincubation with the compound afobazole inhibits these [Ca(2+)]i increases. The effect of afobazole on [Ca(2+)]i is due to activation of σ-1 receptors but could not be mimicked by a second pan-selective σ receptor agonist, 1,3-di-o-tolylguanidine (DTG). Afobazole was also found to lessen nitric oxide (NO) production in response to Aß25-35 application but did not affect elevations in reactive oxygen species elicited by the Aß fragment. The reductions in [Ca(2+)]i and NO perturbation produced by afobazole were associated with a decrease in neuronal cell death, whereas DTG failed to enhance cell survival. Examining the molecular mechanisms involved in the increased neuronal survival demonstrates that afobazole incubation results in lower expression of the proapoptotic protein Bax and the death protease caspase-3, while at the same time increasing expression of the antiapoptotic protein, Bcl-2. Given the importance of Aß neurotoxicity in AD etiology, the findings reported here suggest that afobazole may be an effective AD therapeutic agent. Furthermore, σ-1 receptors may represent a useful target for AD treatment, although not all σ ligands appear to be equally beneficial.

In vitro evaluation of guanidine analogs as sigma receptor ligands for potential anti-stroke therapeutics.

Currently, the only Food and Drug Administration-approved treatment of acute stroke is recombinant tissue plasminogen activator, which must be administered within 6 hours after stroke onset. The pan-selective σ-receptor agonist N,N'-di-o-tolyl-guanidine (o-DTG) has been shown to reduce infarct volume in rats after middle cerebral artery occlusion, even when administered 24 hours after stroke. DTG derivatives were synthesized to develop novel compounds with greater potency than o-DTG. Fluorometric Ca(2+) imaging was used in cultured cortical neurons to screen compounds for their capacity to reduce ischemia- and acidosis-evoked cytosolic Ca(2+) overload, which has been linked to stroke-induced neurodegeneration. In both assays, migration of the methyl moiety produced no significant differences, but removal of the group increased potency of the compound for inhibiting acidosis-induced [Ca(2+)](i) elevations. Chloro and bromo substitution of the methyl moiety in the meta and para positions increased potency by ≤160%, but fluoro substitutions had no effect. The most potent DTG derivative tested was N,N'-di-p-bromo-phenyl-guanidine (p-BrDPhG), which had an IC(50) of 2.2 µM in the ischemia assay, compared with 74.7 µM for o-DTG. Microglial migration assays also showed that p-BrDPhG is more potent than o-DTG in this marker for microglial activation, which is also linked to neuronal injury after stroke. Radioligand binding studies showed that p-BrDPhG is a pan-selective σ ligand. Experiments using the σ-1 receptor-selective antagonist 1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride (BD-1063) demonstrated that p-BrDPhG blocks Ca(2+) overload via σ-1 receptor activation. The study identified four compounds that may be more effective than o-DTG for the treatment of ischemic stroke at delayed time points.

A modular approach to create a neurovascular unit-on-a-chip.

In this work, we describe the fabrication and working of a modular microsystem that recapitulates the functions of the "Neurovascular Unit". The microdevice comprised a vertical stack of a poly(dimethylsiloxane) (PDMS) neural parenchymal chamber separated by a vascular channel via a microporous polycarbonate (PC) membrane. The neural chamber housed a mixture of neurons (~4%), astrocytes (~95%), and microglia (~1%). The vascular channel was lined with a layer of rat brain microvascular endothelial cell line (RBE4). Cellular components in the neural chamber and vascular channel showed viability (>90%). The neural cells fired inhibitory as well as excitatory potentials following 10 days of culture. The endothelial cells showed diluted-acetylated low density lipoprotein (dil-a-LDL) uptake, expressed von Willebrand factor (vWF) and zonula occludens (ZO-1) tight junctions, and showed decreased Alexafluor™-conjugated dextran leakage across their barriers significantly compared with controls (p < 0.05). When the vascular layer was stimulated with TNF-α for 6 h, about 75% of resident microglia and astrocytes on the neural side were activated significantly (p < 0.05 compared to controls) recapitulating tissue-mimetic responses resembling neuroinflammation. The impact of this microsystem lies in the fact that this biomimetic neurovascular platform might not only be harnessed for obtaining mechanistic insights for neurodegenerative disorders, but could also serve as a potential screening tool for central nervous system (CNS) therapeutics in toxicology and neuroinfectious diseases.