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.

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.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the leading cause of senile dementia in the United States. Accumulation of amyloid-b (Ab) and the effects of this peptide on microglial cells contribute greatly to the etiology of AD. Experiments were carried out to determine whether the panselective s-receptor agonist afobazole can modulate microglial response to the cytotoxic Ab fragment, Ab [25][26][27][28][29][30][31][32][33][34][35] . Treatment with afobazole decreased microglial activation in response to Ab, as indicated by reduced membrane ruffling and cell migration. The effects of afobazole on Ab 25-35 -evoked migration were concentration dependent and consistent with s-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 s-1-and s-2-selective antagonists, respectively, the inhibition of Ab 25-35 -induced migration by afobazole was reduced. Prolonged exposure of microglia to Ab 25-35 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 Ab 25-35 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 Ab [25][26][27][28][29][30][31][32][33][34][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 Ab [25][26][27][28][29][30][31][32][33][34][35] . However, coincubation in afobazole returned these responses to near control levels. Therefore, stimulation of s-1 and s-2 receptors by afobazole prevents Ab [25][26][27][28][29][30][31][32][33][34][35] activation of microglia and inhibits Ab 25-35 -associated cytotoxicity, suggesting that afobazole may be useful for AD therapeutics.

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Afobazole is an anxiolytic medication that has been previously shown to be neuroprotective both in vitro and in vivo. However, the mechanism(s) by which afobazole can enhance neuronal survival remain poorly understood. Experiments were carried out to determine whether afobazole can decrease intracellular calcium overload associated with ischemia and acidosis and whether the effects of afobazole are mediated via interaction of the compound with receptors. Fluorometric Ca 2ϩ imaging was used to resolve how application of afobazole affects intracellular Ca 2ϩ handling in cortical neurons. Application of afobazole significantly depressed, in a concentration-dependent and reversible manner, the intracellular Ca 2ϩ overload resulting from in vitro ischemia and acidosis. The IC 50 for afobazole inhibition of ischemia-evoked intracellular Ca 2ϩ overload was considerably less than that for the inhibition of [Ca 2ϩ ] i increases induced by acidosis. However, afobazole maximally inhibited only 70% of the ischemia-evoked intracellular Ca 2ϩ overload but effectively abolished intracellular Ca 2ϩ increases produced by acidosis. The effects of afobazole on ischemia-and acidosis-induced intracellular Ca 2ϩ dysregulation were inhibited by preincubating the neurons in the irreversible, pan-selective -receptor antagonist, metaphit. Moreover, the effects of afobazole on intracellular Ca 2ϩ increases triggered by acidosis and ischemia were blocked by the selective -1-receptor antagonists, BD 1063 and BD 1047, respectively. Experiments examining the effects of afobazole on neuronal survival in response to ischemia showed that afobazole was neuroprotective. Taken together, these data suggest that afobazole regulates intracellular Ca 2ϩ overload during ischemia and acidosis via activation of -1 receptors. This mechanism is probably responsible for afobazole-mediated neuroprotection.

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 , 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.

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