Astrocyte death may occur in neurodegenerative disorders and complicates the outcome of brain ischemia, a condition associated with high extracellular levels of adenosine and glutamate. We show that pharmacological activation of A 1 adenosine and mGlu3 metabotropic glutamate receptors with N 6 -chlorocyclopentyladenosine (CCPA) and (Ϫ)2-oxa-4-aminocyclo-[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), respectively, protects cultured astrocytes against apoptosis induced by a 3-h exposure to oxygen/glucose deprivation (OGD). Protection by CCPA and LY379268 was less than additive and was abrogated by receptor blockade with selective competitive antagonists or pertussis toxin. Both in control astrocytes and in astrocytes exposed to OGD, CCPA and LY379268 induced a rapid activation of the phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases 1 and 2 (ERK1/2)/mitogen-activated protein kinase (MAPK) pathways, which are known to support cell survival. In cultures exposed to OGD, CCPA and LY379268 reduced the activation of c-Jun N-terminal kinase and p38/MAPK, reduced the levels of the proapoptotic protein Bad, increased the levels of the antiapoptotic protein Bcl-X L , and were highly protective against apoptotic death, as shown by nuclear 4Ј-6-diamidino-2-phenylindole staining and measurements of caspase-3 activity. All of these effects were attenuated by treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), which inhibit the MAPK and the PI3K pathways, respectively. These data suggest that pharmacological activation of A 1 and mGlu3 receptors protects astrocytes against hypoxic/ischemic damage by stimulating the PI3K and ERK1/2 MAPK pathways.Astrocytes, the most abundant glial cell types in the brain, provide metabolic and trophic support to neurons by several mechanisms that include the clearance of ions and environmental toxins, the supply of energy substrates, and the production of trophic factors, and modulate synaptic activity (Volterra and Meldolesi, 2005). Impairments in these functions critically affect neuronal survival.Recent studies have shown that ischemic and inflammatory insults induce astrocyte apoptotic death, and this contributes to the pathophysiology of short-and long-term neurodegenerative disorders (Takuma et al., 2004). Apoptotic astrocytes are found in Alzheimer's disease (Kobayashi et al., ABBREVIATIONS: CCPA, N 6 -chlorocyclopentyladenosine; ASK1, apoptosis-signal-regulating kinase 1; DAPI, 4Ј-6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; DPCPX, 1,3-dipropyl-8-cyclopentyl xanthine; ERK1/2, extracellular signal-regulated kinases 1 and 2; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; LY341495, (2S,1ЈS,2ЈS)-2(9-xanthylmethyl)-2-(2Ј-carboxycyclo-propyl)glycine; LY379268, (Ϫ)2-oxa-4-aminocyclo-[3.1.0] hexane-4,6-dicarboxylic acid; MAPK, mitogen-activated p...

Cysteinyl-leukotrienes (cys-LTs), potent mediators in inflammatory diseases, are produced by nervous tissue, but their cellular source and role in the brain are not very well known. In this report we have demonstrated that rat cultured astrocytes express the enzymes (5'-lipoxygenase and LTC(4) synthase) required for cys-LT production, and release cys-LTs in resting condition and, to a greater extent, in response to calcium ionophore A23187, 1 h combined oxygen-glucose deprivation or 2-methyl-thioATP, a selective P2Y(1)/ATP receptor agonist. MK-886, a LT synthesis inhibitor, prevented basal and evoked cys-LT release. In addition, 2-methyl-thioATP-induced cys-LT release was abolished by suramin, a P2 receptor antagonist, or by inhibitors of ATP binding cassette proteins involved in cys-LT release. We also showed that astrocytes express cys-LT(1) and not cys-LT(2) receptors. The stimulation of these receptors by LTD(4) activated the mitogen-activated protein kinase (MAPK) pathway. This effect was: (i) insensitive to inhibitors of receptor-coupled Gi protein (pertussis toxin) or tyrosine kinase receptors (genistein); (ii) abolished by MK-571, a cys-LT(1) selective receptor antagonist, or PD98059, a MAPK inhibitor; (iii) reduced by inhibitors of calcium/calmodulin-dependent kinase II (KN-93), Ca(2+)-dependent and -independent (GF102903X) or Ca(2+)-dependent (Gö6976) protein kinase C isoforms. LTD(4) also increased astrocyte proliferation and glial fibrillary acidic protein content, which are considered hallmarks of reactive astrogliosis. Both effects were counteracted by cell pretreatment with MK-571 or PD98059. Thus, cys-LTs released from astrocytes might play an autocrine role in the induction of reactive astrogliosis that, in brain injuries, contributes to the formation of a reparative glial scar.

Axonal demyelination is a consistent pathological sequel to chronic brain and spinal cord injuries and disorders that slows or disrupts impulse conduction, causing further functional loss. Since oligodendroglial progenitors are present in the demyelinated areas, failure of remyelination may be due to lack of sufficient proliferation and differentiation of oligodendroglial progenitors. Guanosine stimulates proliferation and differentiation of many types of cells in vitro and exerts neuroprotective effects in the central nervous system (CNS). Five weeks after chronic traumatic spinal cord injury (SCI), when there is no ongoing recovery of function, intraperitoneal administration of guanosine daily for 2 weeks enhanced functional improvement correlated with the increase in myelination in the injured cord. Emphasis was placed on analysis of oligodendrocytes and NG2-positive (NG2+) cells, an endogenous cell population that may be involved in oligodendrocyte replacement. There was an increase in cell proliferation (measured by bromodeoxyuridine staining) that was attributable to an intensification in progenitor cells (NG2+ cells) associated with an increase in mature oligodendrocytes (determined by Rip+ staining). The numbers of astroglia increased at all test times after administration of guanosine whereas microglia only increased in the later stages (14 days). Injected guanosine and its breakdown product guanine accumulated in the spinal cords; there was more guanine than guanosine detected. We conclude that functional improvement and remyelination after systemic administration of guanosine is due to the effect of guanosine/ guanine on the proliferation of adult progenitor cells and their maturation into myelin-forming cells. This raises the possibility that administration of guanosine may be useful in the treatment of spinal cord injury or demyelinating diseases such as multiple sclerosis where quiescent oligodendroglial progenitors exist in demyelinated plaques.

Inflammation is widely recognized as contributing to the pathology of acute and chronic neurodegenerative conditions. Microglial cells are pathologic sensors in the brain and activated microglia have been viewed as detrimental. Leukotriene, including cysteinylleukotrienes (CysLTs) are suggested to be involved in brain inftammation and neurological diseases andATP, by its receptors is a candidate for microglia activation.A23187 (l0f1M) stimulated microglia to co-release CysLTs and [3H]adenine based purines ([3H]ABPs), mainly ATP. The biosynthetic production of CysLTs was abolished by 10f1MMK-886, an inhibitor of 5-lipoxygenase-activating protein activity. RT-PCR analysis showed that microglia expressed both CysLT I / CysLT 2 receptors, P 2Y1 ATPreceptors and several members of the ATP binding cassette (ABC) transporters including MRP1, MRP4 and Pgpo The increase in [Ca 2+]i elicited by LTD4 (0.1 f1M) and 2MeSATP (l00J.1M), agonists for CysLT-and P 2Y1 -receptors, was abolished by the respective antagonists, BAYu9773 (0.5 f1M) and suramin (50 f1M). The stimulation of both receptor subtypes, induced a concomitant increase in the release of both [3H]ABPs and CysLTs that was blocked by the antagonists and significantly reduced by a cocktail ofABC transporter inhibitors, BAPTAIAM (intracellular Ca2+ chelator) and staurosporine (0.1 f1M, PKC blocker). P2Y antagonist was unable to antagonise the effects ofLTD 4 and BAYu9773 did not reduce the effects of2MeSATP. These data suggest that: i) the efffux of purines and cysteinyl-leukotrienes is specifically and independently controlled by the two receptor types, ii) calcium, PKC and the ABC transporter system can reasonably be considered common mechanisms underlying the release ofABPs and CysLTs from microglia. The blockade ofP 2Y1 orCysLT/CysLT 2 receptors by specific antagonists that abolished the raise in [Ca"]! and drastically reduced the concomitant efffux of both compounds, as well as the effects ofBAPTA and staurosporine support this hypothesis. In conclusion, the data of the present study suggest a cross talk between the purine and leukotriene systems in a possible autocrine/paracrine control ofthe microglia-mediated initiation and progression of an inftammatory response.Proinflammatory products of 5-lipoxygenase (5-LO) pathway, including cysteinylleukotrienes (LTC4, LTD4, LTE4) are suggested to be involved in brain inflammation and neurological diseases. In aging brain and in Alzheimer's disease an increase in the activity of 5-LO has been shown (41, 57). The inhibition of the enzyme or 5-LO activating protein (FLAP) reduced the microglia-mediated toxicity towards neuronal cells, whereas the toxicity was enhanced by the cysteinyl leukotriene LTD4 (35).

The involvement of excitatory adenosine A(2A) receptors (A(2A)Rs), which probably contribute to the pathophysiology of convulsive seizures, has never been investigated in absence epilepsy. Here, we examined the distribution and function of A(2A)Rs in the brain of Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats, a model of human absence epilepsy in which disease onset occurs 2-3 months after birth. In the cerebral areas that are mostly involved in the generation of absence seizures (somatosensory cortex, reticular and ventrobasal thalamic nuclei), A(2A)R density was lower in presymptomatic WAG/Rij rats than in control rats, as evaluated by immunohistochemistry and western blotting. Accordingly, in cortical/thalamic slices prepared from the brain of these rats, A(2A)R stimulation with the agonist 2-[4-(-2-carboxyethyl)-phenylamino]-5'-N-ethylcarboxamido-adenosine failed to modulate either cAMP formation, mitogen-activated protein kinase system, or K(+)-evoked glutamate release. In contrast, A(2A)R expression, signalling and function were significantly enhanced in brain slices from epileptic WAG/Rij rats as compared with matched control animals. Additionally, the in vivo injection of the A(2A)R agonist CGS21680, or the antagonist 5-amino-7-(2-phenylethyl)-2-(2-fuyl)-pyrazolo-(4,3-c)1,2,4-triazolo(1,5-c)-pyrimidine, in the examined brain areas of epileptic rats, increased and decreased, respectively, the number/duration of recorded spontaneous spike-wave discharges in a dose-dependent manner during a 1-5 h post-treatment period. Our results support the hypothesis that alteration of excitatory A(2A)R is involved in the pathogenesis of absence seizures and might represent a new interesting target for the therapeutic management of this disease.