Reconstitution of synaptic Ion channels from rodent and human brain in Xenopus oocytes: a biochemical and electrophysiological characterization.

Disruption in the expression and function of synaptic proteins, and ion channels in particular, is critical in the pathophysiology of human neuropsychiatric and neurodegenerative diseases. However, very little is known regarding the functional and pharmacological properties of native synaptic human ion channels, and their potential changes in pathological conditions. Recently, an electrophysiological technique has been enabled for studying the functional and pharmacological properties of ion channels present in crude membrane preparation obtained from post-mortem frozen brains. We here extend these studies by showing that human synaptic ion channels also can be studied in this way. Synaptosomes purified from different regions of rodent and human brain (control and Alzheimer's) were characterized biochemically for enrichment of synaptic proteins, and expression of ion channel subunits. The same synaptosomes were also reconstituted in Xenopus oocytes, in which the functional and pharmacological properties of the native synaptic ion channels were characterized using the voltage clamp technique. We show that we can detect GABA, (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and NMDA receptors, and modulate them pharmacologically with selective agonists, antagonists, and allosteric modulators. Furthermore, changes in ion channel expression and function were detected in synaptic membranes from Alzheimer's brains. Our present results demonstrate the possibility to investigate synaptic ion channels from healthy and pathological brains. This method of synaptosomes preparation and injection into oocytes is a significant improvement over the earlier method. It opens the way to directly testing, on native ion channels, the effects of novel drugs aimed at modulating important classes of synaptic targets. Disruption in the expression and function of synaptic ion channels is critical in the pathophysiology of human neurodegenerative diseases. We here show that synaptosomes purified from rodent and human frozen brain (control and Alzheimer disease) can be studied both biochemically and functionally. This method opens the way to directly testing the effects of novel drugs on native ion channels.

Role of adenosine in oligodendrocyte precursor maturation.

Differentiation and maturation of oligodendroglial cells are postnatal processes that involve specific morphological changes correlated with the expression of stage-specific surface antigens and functional voltage-gated ion channels. A small fraction of oligodendrocyte progenitor cells (OPCs) generated during development are maintained in an immature and slowly proliferative or quiescent state in the adult central nervous system (CNS) representing an endogenous reservoir of immature cells. Adenosine receptors are expressed by OPCs and a key role of adenosine in oligodendrocyte maturation has been recently recognized. As evaluated on OPC cultures, adenosine, by stimulating A1 receptors, promotes oligodendrocyte maturation and inhibits their proliferation; on the contrary, by stimulating A2A receptors, it inhibits oligodendrocyte maturation. A1 and A2A receptor-mediated effects are related to opposite modifications of outward delayed rectifying membrane K(+) currents (IK) that are involved in the regulation of oligodendrocyte differentiation. Brain A1 and A2A receptors might represent new molecular targets for drugs useful in demyelinating pathologies, such as multiple sclerosis (MS), stroke and brain trauma.

The selective antagonism of P2X7 and P2Y1 receptors prevents synaptic failure and affects cell proliferation induced by oxygen and glucose deprivation in rat dentate gyrus.

Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X7 and P2Y1 receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y1 receptor) and BBG (selective antagonist of P2X7 receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2'-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU+ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU+ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y1 antagonist reduced the number of BrdU+ cells at this time. The data demonstrate that P2X7 and P2Y1 activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y1 receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.

Adenosine A2A receptors modulate acute injury and neuroinflammation in brain ischemia.

The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A(2A) receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A(2A) receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A(2A) receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A(2A) receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A(2A) receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A(2A) receptor agonists a wide therapeutic time-window of hours and even days after stroke.

Adenosine A2A receptors inhibit delayed rectifier potassium currents and cell differentiation in primary purified oligodendrocyte cultures.

Oligodendrocyte progenitor cells (OPCs) are a population of cycling cells which persist in the adult central nervous system (CNS) where, under opportune stimuli, they differentiate into mature myelinating oligodendrocytes. Adenosine A(2A) receptors are Gs-coupled P1 purinergic receptors which are widely distributed throughout the CNS. It has been demonstrated that OPCs express A(2A) receptors, but their functional role in these cells remains elusive. Oligodendrocytes express distinct voltage-gated ion channels depending on their maturation. Here, by electrophysiological recordings coupled with immunocytochemical labeling, we studied the effects of adenosine A(2A) receptors on membrane currents and differentiation of purified primary OPCs isolated from the rat cortex. We found that the selective A(2A) agonist, CGS21680, inhibits sustained, delayed rectifier, K(+) currents (I(K)) without modifying transient (I(A)) conductances. The effect was observed in all cells tested, independently from time in culture. CGS21680 inhibition of I(K) current was concentration-dependent (10-200 nM) and blocked in the presence of the selective A(2A) antagonist SCH58261 (100 nM). It is known that I(K) currents play an important role during OPC development since their block decreases cell proliferation and differentiation. In light of these data, our further aim was to investigate whether A(2A) receptors modulate these processes. CGS21680, applied at 100 nM in the culture medium of oligodendrocyte cultures, inhibits OPC differentiation (an effect prevented by SCH58261) without affecting cell proliferation. Data demonstrate that cultured OPCs express functional A(2A) receptors whose activation negatively modulate I(K) currents. We propose that, by this mechanism, A(2A) adenosine receptors inhibit OPC differentiation.

UDP-glucose enhances outward K(+) currents necessary for cell differentiation and stimulates cell migration by activating the GPR17 receptor in oligodendrocyte precursors.

In the developing and mature central nervous system, NG2 expressing cells comprise a population of cycling oligodendrocyte progenitor cells (OPCs) that differentiate into mature, myelinating oligodendrocytes (OLGs). OPCs are also characterized by high motility and respond to injury by migrating into the lesioned area to support remyelination. K(+) currents in OPCs are developmentally regulated during differentiation. However, the mechanisms regulating these currents at different stages of oligodendrocyte lineage are poorly understood. Here we show that, in cultured primary OPCs, the purinergic G-protein coupled receptor GPR17, that has recently emerged as a key player in oligodendrogliogenesis, crucially regulates K(+) currents. Specifically, receptor stimulation by its agonist UDP-glucose enhances delayed rectifier K(+) currents without affecting transient K(+) conductances. This effect was observed in a subpopulation of OPCs and immature pre-OLGs whereas it was absent in mature OLGs, in line with GPR17 expression, that peaks at intermediate phases of oligodendrocyte differentiation and is thereafter downregulated to allow terminal maturation. The effect of UDP-glucose on K(+) currents is concentration-dependent, blocked by the GPR17 antagonists MRS2179 and cangrelor, and sensitive to the K(+) channel blocker tetraethyl-ammonium, which also inhibits oligodendrocyte maturation. We propose that stimulation of K(+) currents is responsible for GPR17-induced oligodendrocyte differentiation. Moreover, we demonstrate, for the first time, that GPR17 activation stimulates OPC migration, suggesting an important role for this receptor after brain injury. Our data indicate that modulation of GPR17 may represent a strategy to potentiate the post-traumatic response of OPCs under demyelinating conditions, such as multiple sclerosis, stroke, and brain trauma.

Amyloid-ß oligomer synaptotoxicity is mimicked by oligomers of the model protein HypF-N.

Protein misfolded oligomers are thought to be the primary pathogenic species in many protein deposition diseases. Oligomers by the amyloid-ß peptide play a central role in Alzheimer's disease pathogenesis, being implicated in synaptic dysfunction. Here we show that the oligomers formed by a protein that has no link with human disease, namely the N-terminal domain of HypF from Escherichia coli (HypF-N), are also synaptotoxic. HypF-N oligomers were found to (i) colocalize with post-synaptic densities in primary rat hippocampal neurons; (ii) induce impairment of long-term potentiation in rat hippocampal slices; and (iii) impair spatial learning of rats in the Morris Water Maze test. By contrast, the native protein and control nontoxic oligomers had none of such effects. These results raise the importance of using HypF-N oligomers as a valid tool to investigate the pathogenesis of Alzheimer's disease, with advantages over other systems for their stability, reproducibility, and costs. The results also suggest that, in the context of a compromised protein homeostasis resulting from aggregation of the amyloid ß peptide, a number of oligomeric species sharing common synaptotoxic activity can arise and cooperate in the pathogenesis of the disease.

Effects of oxygen and glucose deprivation on synaptic transmission in rat dentate gyrus: role of A2A adenosine receptors.

The hippocampus is comprised of two distinct subfields that show different responses to hypoxic-ischemic brain injury: the CA1 region is particularly susceptible whereas the dentate gyrus (DG) is quite resistant. Our aim was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of A(2A) adenosine receptor antagonism to recovery of synaptic activity after OGD. Extracellular recordings of field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG in brain slices prepared from male Wistar rats were used. A 9-min OGD is needed in the DG to always induce the appearance of anoxic depolarization (AD) and the irreversible block of synaptic activity, as recorded up to 24 h from the end of the insult, whereas only 7-min OGD is required in the CA1 region. Selective antagonism of A(2A) adenosine receptors by ZM241385 significantly prevents or delays the appearance of AD and protects from the irreversible block of neurotransmission induced by 9-min OGD in the DG. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone of DG in slices prepared from 5-bromo-2'-deoxyuridine (BrdU) treated rats was investigated. Slices were further incubated with an immature neuronal marker, doublecortin (DCX). The number of BrdU(+) cells was significantly decreased 6 h after 9-min OGD and this effect was antagonized by ZM241385. After 24 h from the end of 9-min OGD, the number of BrdU(+) cells returned to that found before OGD and increased arborization of tertiary dendrites of DCX(+) cells was observed. The adenosine A(2A) antagonist ZM241385 protects from synaptic failure and from decreased proliferation of immature neuronal cells at a precocious time after OGD.

3-Hydroxy-1H-quinazoline-2,4-dione derivatives as new antagonists at ionotropic glutamate receptors: molecular modeling and pharmacological studies.

Based on our 3-hydroxy-7-chloroquinazoline-2,4-dione derivatives, previously reported as antagonists at ionotropic glutamate receptors, we synthesized new 3-hydroxyquinazoline-2,4-diones bearing a trifluoromethyl group at the 7-position and different groups at position 6. Glycine/NMDA, AMPA and kainate receptor binding data showed that the 7-trifluoromethyl residue increased AMPA and kainate receptor affinity and selectivity, with respect to the 7-chlorine atom. Among the probed 6-substituents, the 6-(1,2,4-triazol-4-yl) group (compound 8) was the most advantageous for AMPA receptor affinity and selectivity. Derivative 8 demonstrated to be effective in decreasing neuronal damage produced by oxygen and glucose deprivation in organotypic rat hippocampal slices and also showed anticonvulsant effects in pentylenetetrazole-induced convulsions. The previously reported kainate receptor antagonist 6-(2-carboxybenzoyl)-amino-7-chloro-3-hydroxyquinazoline-2,4-dione 3 prevented the failure of neurotransmission induced by oxygen and glucose deprivation in the CA1 region of rat hippocampal slices.