Differential mechanisms of tolerance induced by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning.

We investigated the molecular events triggered by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning, that lead to neuroprotection against excitotoxic insults (AMPA or oxygen and glucose deprivation) in rat organotypic hippocampal slices, with particular attention on glutamate receptors and on cannabinoid system. We firstly evaluated the protein expression of NMDA and AMPA receptor subunits after preconditioning using western blot analysis performed in post-synaptic densities. We observed that following NMDA, but not DHPG preconditioning, the expression of GluA1 was significantly reduced and this reduction appeared to be associated with the internalization of AMPA receptors. Whole-cell voltage clamp recordings on CA1 pyramidal neurons of organotypic slices show that 24 hr after exposure to NMDA and DHPG preconditioning, AMPA-induced currents were significantly reduced. To clarify the mechanisms induced by DHPG preconditioning, we then investigated the involvement of the endocannabinoid system. Exposure of slices to the CB1 antagonist AM251 prevented the development of tolerance to AMPA toxicity induced by DHPG but not NMDA. Accordingly, the MAG-lipase inhibitor URB602, that increases arachidonoylglycerol (2-AG) content, but not the FAAH inhibitor URB597, that limits the degradation of anandamide, was also able to induce tolerance versus AMPA and OGD toxicity, suggesting that 2-AG is responsible for the DHPG-induced tolerance. In conclusion, preconditioning with NMDA or DHPG promotes differential neuroprotective mechanisms: NMDA by internalization of GluA1-AMPA receptors, DHPG by producing the endocannabinoid 2-AG.

Effects of PARP-1 Deficiency and Histamine H4 Receptor Inhibition in an Inflammatory Model of Lung Fibrosis in Mice.

Pulmonary fibrosis is the most frequent form of interstitial lung disease. Effective therapies are not yet available; novel therapeutic approaches are needed for counteracting fibrosis. Poly(ADP-ribose) polymerases are enzymes, involved in DNA repair and cell apoptosis. PARP-1 deficient mice exhibited reduced lung fibrosis in response to bleomycin treatment compared to wild-type controls. Histamine H4 receptors (H4Rs) have been recognized as a new target for inflammatory and immune diseases, and H4R ligands reduced inflammation and oxidative stress in lung tissue. The aim of the study was to evaluate the cross-talk between PARP-1 and H4R in a model of bleomycin-induced lung fibrosis in PARP-1-/- and WT mice. Animals were treated with bleomycin or saline by intra-tracheal injection. JNJ7777120, an H4R antagonist, or VUF8430, an H4R agonist, were administered i.p for 21 days. Airway resistance to inflation was evaluated, and lung tissues were processed for PARylated protein content, oxidative stress evaluation, and histology of small bronchi. The levels of pro-inflammatory (IL-1ß and TNF-α), regulatory (IL-10), and pro-fibrotic (TGF-ß) cytokines were evaluated. The deposition of αSMA was determined by immunofluorescence analysis. The results indicate that JNJ7777120 reduces PARylated protein production, decreases oxidative stress damage, and MPO, a marker for leukocyte tissue infiltration, in PARP-1-/- mice. A significant decrease in the production of both IL-1ß and TNF-α and a significant increase in IL-10 levels are observed in mice treated with H4R antagonist, suggesting a crucial anti-inflammatory activity of JNJ7777120. The smooth muscle layer thickness, the goblet cell relative number, and collagen deposition decreased following JNJ7777120 administration. The H4R antagonist treatment also reduces TGF-ß production and αSMA deposition, suggesting an important role of JNJ7777120 in airway remodeling. Our results show that PARylation is essential for the pathogenesis of pulmonary fibrosis and propose that PARP-1 and H4Rs are both involved in inflammatory and fibrotic responses. JNJ7777120 treatment, in a condition of PARP-1 inhibition, exerts anti-inflammatory and anti-fibrotic effects, reducing airway remodeling and bronchoconstriction. Therefore, selective inhibition of H4Rs together with non-toxic doses of selective PARP-1 inhibitors could have clinical relevance for the treatment of idiopathic pulmonary fibrosis.

ERRATUM.

The poly(ADP-ribose) polymerase (PARP) enzymes play a key role in the regulation of cellular processes (e.g., DNA damage repair, genomic stability). It has been shown that PARP inhibitors (PARPIs) are selectively cytotoxic against cells having dysfunctions in genes involved in DNA repair mechanisms (synthetic lethality). Drug-induced PARP inhibition potentiates the activity of anticancer drugs such as 5-fluorouracil in enhancing DNA damage, whose repair involves PARP-1 activity. The aim of this study was to evaluate the inhibitory effects of a novel PARPI, HYDAMTIQ, on growth in human tumor cell lines characterized by different features with regard to DNA damage response pathways (BRCA mutational status, microsatellite status, and ATM expression level) and degree of sensitivity/resistance to 5-fluorouracil. HYDAMTIQ showed a more potent inhibitory effect on cell growth in a BRCA2 mutant cell line (CAPAN-1) compared with wild-type cells (C2-6, C2-12, and C2-14 CAPAN-1 clones, and MCF-7). No statistically significant difference was observed after HYDAMTIQ exposure between cells having a different MS status or a different MRE11 mutational status. HYDAMTIQ induced greater antiproliferative effects in SW620 cells expressing a low level of ATM than in H630 cells expressing a high level of ATM. Finally, the combination of HYDAMTIQ and 5-fluorouracil exerted a synergistic effect on the inhibition of SW620 cell growth and an antagonistic effect on that of H630 cell growth. Our results show that the novel PARP inhibitor HYDAMTIQ potently inhibits the growth of human tumor cells with defective DNA damage response pathways and exerts synergistic cytotoxicity in combination with 5-fluorouracil. These data provide relevant examples of synthetic lethality and evidence for further development of this novel PARPI.

Methadone Dose Adjustments, Plasma R-Methadone Levels and Therapeutic Outcome of Heroin Users: A Randomized Clinical Trial.

AIMS: We aimed to improve the retention in treatment and therapeutic outcome of methadone maintenance treatment (MMT) patients by adjusting the oral methadone dose in order to reach a "target" plasma R-methadone level (80-250 ng/mL). METHODS: A multicenter randomized controlled trial was organized. RESULTS: The intention-to-treat statistical analysis showed that repeated dose adjustments performed in order to obtain therapeutic plasma R-methadone levels did not improve retention in treatment of heroin-dependent patients. However, patients having plasma methadone levels in the "target range" at the beginning of the study had a better retention in treatment than controls. Furthermore, patients succeeding in keeping plasma R-methadone target levels (per protocol analysis) remained in treatment and improved their social scores better than controls. -Conclusion: Although the primary endpoint of this study was not demonstrated, a post hoc and a per protocol analysis suggested that patients in MMT with plasma R-methadone concentrations in the target range have a better therapeutic outcome than controls.

Opportunities for the repurposing of PARP inhibitors for the therapy of non-oncological diseases.

The recent clinical availability of the PARP inhibitor olaparib (Lynparza) opens the door for potential therapeutic repurposing for non-oncological indications. Considering (a) the preclinical efficacy data with PARP inhibitors in non-oncological diseases and (b) the risk-benefit ratio of treating patients with a compound that inhibits an enzyme that has physiological roles in the regulation of DNA repair, we have selected indications, where (a) the severity of the disease is high, (b) the available therapeutic options are limited, and (c) the duration of PARP inhibitor administration could be short, to provide first-line options for therapeutic repurposing. These indications are as follows: acute ischaemic stroke; traumatic brain injury; septic shock; acute pancreatitis; and severe asthma and severe acute lung injury. In addition, chronic, devastating diseases, where alternative therapeutic options cannot halt disease development (e.g. Parkinson's disease, progressive multiple sclerosis or severe fibrotic diseases), should also be considered. We present a preclinical and clinical action plan for the repurposing of PARP inhibitors. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Glutamate Receptor-Mediated Neurotoxicity in a Model of Ethanol Dependence and Withdrawal in Rat Organotypic Hippocampal Slice Cultures.

Long-term alcohol use can lead to alterations in brain structure and functions and, in some cases, to neurodegeneration. Several mechanisms have been proposed to explain ethanol (EtOH)-related brain injury. One of the most relevant mechanisms of alcohol-induced neurodegeneration involves glutamatergic transmission, but their exact role is not yet fully understood. We investigated the neurochemical mechanisms underlying the toxicity induced by EtOH dependence and/or withdrawal by exposing rat organotypic hippocampal slices to EtOH (100-300 mM) for 7 days and then incubating the slices in EtOH-free medium for the subsequent 24 h. EtOH withdrawal led to a dose-dependent CA1 pyramidal cell injury, as detected with propidium iodide fluorescence. Electron microscopy of hippocampal slices revealed that not only EtOH withdrawal but also 7 days chronic EtOH exposure elicited signs of apoptotic cell death in CA1 pyramidal cells. These data were supported by electrophysiological recordings of spontaneus Excitatory Post Synaptic Currents (sEPSCs) from CA1 pyramidal cells. The average amplitude of sEPSCs in slices treated with EtOH for 7 days was significantly increased, and even more so during the first 30 min of EtOH withdrawal, suggesting that the initial phase of the neurodegenerative process could be due to an excitotoxic mechanism. We then analyzed the expression levels of presynaptic (vGlut1, vGlut2, CB1 receptor, synaptophysin) and postsynaptic (PSD95, GluN1, GluN2A, GluN2B, GluA1, GluA2, mGluR1 and mGluR5) proteins after 7 days EtOH incubation or after EtOH withdrawal. We found that only GluA1 and mGluR5 expression levels were significantly increased after EtOH withdrawal and, in neuroprotection experiments, we observed that AMPA and mGluR5 antagonists attenuated EtOH withdrawal-induced toxicity. These data suggest that chronic EtOH treatment promotes abnormal synaptic transmission that may lead to CA1 pyramidal cell death after EtOH withdrawal through glutamate receptors and increased excitotoxicity.

The Inhibitory Effects of HYDAMTIQ, a Novel PARP Inhibitor, on Growth in Human Tumor Cell Lines With Defective DNA Damage Response Pathways.

The poly(ADP-ribose) polymerase (PARP) enzymes play a key role in the regulation of cellular processes (e.g., DNA damage repair, genomic stability). It has been shown that PARP inhibitors (PARPIs) are selectively cytotoxic against cells having dysfunctions in genes involved in DNA repair mechanisms (synthetic lethality). Drug-induced PARP inhibition potentiates the activity of anticancer drugs such as 5-fluorouracil in enhancing DNA damage, whose repair involves PARP-1 activity. The aim of this study was to evaluate the inhibitory effects of a novel PARPI, HYDAMTIQ, on growth in human tumor cell lines characterized by different features with regard to DNA damage response pathways (BRCA mutational status, microsatellite status, and ATM expression level) and degree of sensitivity/resistance to 5-fluorouracil. HYDAMTIQ showed a more potent inhibitory effect on cell growth in a BRCA2 mutant cell line (CAPAN-1) compared with wild-type cells (C2-6, C2-12, and C2-14 CAPAN-1 clones, and MCF-7). No statistically significant difference was observed after HYDAMTIQ exposure between cells having a different MS status or a different MRE11 mutational status. HYDAMTIQ induced greater antiproliferative effects in SW620 cells expressing a low level of ATM than in H630 cells expressing a high level of ATM. Finally, the combination of HYDAMTIQ and 5-fluorouracil exerted a synergistic effect on the inhibition of SW620 cell growth and an antagonistic effect on that of H630 cell growth. Our results show that the novel PARP inhibitor HYDAMTIQ potently inhibits the growth of human tumor cells with defective DNA damage response pathways and exerts synergistic cytotoxicity in combination with 5-fluorouracil. These data provide relevant examples of synthetic lethality and evidence for further development of this novel PARPI.

HYDAMTIQ, a selective PARP-1 inhibitor, improves bleomycin-induced lung fibrosis by dampening the TGF-ß/SMAD signalling pathway.

Idiopathic pulmonary fibrosis is a severe disease characterized by excessive myofibroblast proliferation, extracellular matrix and fibrils deposition, remodelling of lung parenchyma and pulmonary insufficiency. Drugs able to reduce disease progression are available, but therapeutic results are unsatisfactory; new and safe treatments are urgently needed. Poly(ADP-ribose) polymerases-1 (PARP-1) is an abundant nuclear enzyme involved in key biological processes: DNA repair, gene expression control, and cell survival or death. In liver and heart, PARP-1 activity facilitates oxidative damage, collagen deposition and fibrosis development. In this study, we investigated the effects of HYDAMTIQ, a potent PARP-1 inhibitor, in a murine model of lung fibrosis. We evaluated the role of PARP on transforming growth factor-ß (TGF-ß) expression and TGF-ß/SMAD signalling pathway in lungs. Mice were intratracheally injected with bleomycin and then treated with either vehicle or different doses of HYDAMTIQ for 21 days. Airway resistance to inflation and lung static compliance, markers of lung stiffness, were assayed. Histochemical and biochemical parameters to evaluate TGF-ß/SMAD signalling pathway with alpha-smooth muscle actin (αSMA) deposition and the levels of a number of inflammatory markers (tumour necrosis factor-α, interleukin-1ß, iNOS and COX-2) were performed. Bleomycin administration increased lung stiffness. It also increased lung PARP activity, TGF-ß levels, pSMAD3 expression, αSMA deposition and content of inflammatory markers. HYDAMTIQ attenuated all the above-mentioned physiological, biochemical and histopathological markers. Our findings support the proposal that PARP inhibitors could have a therapeutic potential in reducing the progression of signs and symptoms of the disease by decreasing TGF-ß expression and the TGF-ß/SMAD transduction pathway.

Kynurenic acid and zaprinast induce analgesia by modulating HCN channels through GPR35 activation.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have a key role in the control of cellular excitability. HCN2, a subgroup of the HCN family channels, are heavily expressed in small dorsal root ganglia (DRG) neurons and their activation seems to be important in the determination of pain intensity. Intracellular elevation of cAMP levels activates HCN-mediated current (Ih) and small DRG neurons excitability. GPR35, a Gi/o coupled receptor, is highly expressed in small DRG neurons, and we hypothesized that its activation, mediated by endogenous or exogenous ligands, could lead to pain control trough a reduction of Ih current. Patch clamp recordings were carried out in primary cultures of rat DRG neurons and the effects of GPR35 activation on Ih current and neuronal excitability were studied in control conditions and after adenylate cyclase activation with either forskolin or prostaglandin E2 (PGE2). We found that both kynurenic acid (KYNA) and zaprinast, the endogenous and synthetic GPR35 agonist respectively, were able to antagonize the forskolin-induced depolarization of resting membrane potential by reducing Ih-mediated depolarization. Similar results were obtained when PGE2 was used to activate adenylate cyclase and to increase Ih current and the overall neuronal excitability. Finally, we tested the analgesic effect of both GPR35 agonists in an in vivo model of PGE2-induced thermal hyperalgesia. In accord with the hypothesis, both KYNA and zaprinast showed a dose dependent analgesic effect. In conclusion, GPR35 activation leads to a reduced excitability of small DRG neurons in vitro and causes a dose-dependent analgesia in vivo. GPR35 agonists, by reducing adenylate cyclase activity and inhibiting Ih in DRG neurons may represent a promising new group of analgesic drugs.

Ethanol Toxicity During Brain Development: Alterations of Excitatory Synaptic Transmission in Immature Organotypic Hippocampal Slice Cultures.

BACKGROUND: The developing brain is particularly vulnerable to alcohol: Drinking during pregnancy can lead to a number of physical, learning, and behavioral disorders in the newborn. It has been demonstrated that immature and mature brain tissues display a differential sensitivity to ethanol (EtOH) toxicity and that cerebral structure and function are diversely impaired according to the stage of synaptic maturation. METHODS: Rat organotypic hippocampal slice cultures were exposed for 7 days to EtOH (100 to 300 mM) after 2 days (immature) or 10 days (mature) of culture in vitro; EtOH was then removed from the medium, and 24 hours later, slices were analyzed by fluorescence microscopy, Western blotting, electrophysiology, and electron microscopy to explore the molecular mechanisms of EtOH toxicity in the developing hippocampus. RESULTS: EtOH withdrawal elicited a selective CA1 pyramidal cell injury in mature slices, but not in immature slices. A significant increase in the expression of pre- and postsynaptic proteins in mature slices revealed that slice maturation is presumably associated with the development of new synapses. Incubation with chronic EtOH for 7 days and its removal from the medium induced a significant decrease in GluA1 and GluA2 expression levels; a significant reduction in the expression of synaptophysin and GluN2A was observed only after EtOH withdrawal. Whole-cell patch-clamp recordings showed that incubation with EtOH for 7 days induced a significant decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency in CA1 pyramidal cells of immature slices and a trend toward a decrease in sEPSC amplitude. Electron microscopy revealed a disorganization of neurotubuli in immature slices after chronic exposure to EtOH. CONCLUSIONS: These results indicate that prolonged incubation with EtOH and its subsequent withdrawal from the medium induce an impairment of excitatory synaptic transmission and possibly an incorrect formation of neuronal circuits in developing hippocampus in vitro, which is suggestive of mechanisms that may lead to mental retardation in fetal alcohol spectrum disorders.

Poly(ADP-Ribose)Polymerase 1 (PARP-1) Activation and Ca(2+) Permeable α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid (AMPA) Channels in Post-Ischemic Brain Damage: New Therapeutic Opportunities?

A significant number of laboratories observed that poly (ADP-ribose) polymerase (PARP) inhibitors, administered a few hours after ischemic or traumatic brain injury, may drastically reduce the subsequent neurological damage. It has also been shown that PARP inhibitors, administered for 24 hours to rats with permanent middle cerebral artery occlusion (MCAO), may reduce the number of dying neurons for a long period after surgery, thus suggesting that these agents could reduce the delayed brain damage and the neurological and cognitive impairment (dementia) frequently observed a few months after a stroke. In organotypic hippocampal slices exposed to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG), an alkylating agent able to activate PARP, a selective and delayed degeneration of the CA1 pyramidal cells which was anatomically similar to that observed after a short period of oxygen and glucose deprivation (OGD) has been described. Biochemical and electrophysiological approaches showed that MNNG exposure caused an increased expression and function of the calcium permeable α-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) channels in the CA1 but not in the CA3 hippocampal region. PARP inhibitors prevented this increase and reduced CA1 cell death. The AMPA receptor antagonist 2,3-dihydroxy-6- nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione or the selective Ca(2+) permeable AMPA channel blocker 1-Naphthyl acetyl spermine (NASPM), also reduced the MNNG-induced CA1 pyramidal cell death. Since activation of PARP-1 facilitate the expression of Ca(2+) permeable channels and the subsequent delayed cell death, PARP inhibitors administered a few hours after a stroke may not only reduce the early post-ischemic brain damage but also the late neuronal death frequently occurring after severe stroke.

Interplay between histone acetylation/deacetylation and poly(ADP-ribosyl)ation in the development of ischemic tolerance in vitro.

Ischemic tolerance is an endogenous defense program in which exposure to a subtoxic preconditioning insult results in resistance to a subsequent, otherwise lethal, episode of ischemia. Herein, we evaluated the role of histone acetylation/deacetylation in an in vitro model of preconditioning, using rat organotypic hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to CA1 injury 24 h later: tolerance was induced by exposing the slices to preconditioning bouts of NMDA (3 µM for 60 min) 24 h prior to the toxic OGD challenge. Under these conditions, CA1 damage induced by OGD was reduced. The induction of tolerance was prevented by incubating the slices with HDAC inhibitors. NMDA preconditioning was associated with a mild increase in poly(ADP-ribose) polymerase (PARP) activity that was apparently followed, 3 h later, by a mild increase in histone acetylation. Use of PARP and HDAC inhibitors suggests a possible interaction between PARP and HDAC activities in the development of ischemic tolerance. Finally, both PARP and HDAC inhibitors were able to prevent the increase in pERK1/2 induced by NMDA preconditioning. We propose a model in which mild histone acetylation and PARP activity cooperate in producing a neuroprotective response in the development of ischemic tolerance.

Arrays of microLEDs and astrocytes: biological amplifiers to optogenetically modulate neuronal networks reducing light requirement.

In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (µLEDs) with an excitation peak at 470 nm. We combined Ca2+ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the µLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing µLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the µLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and µLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.

Poly(ADP-ribose) polymerase inhibition with HYDAMTIQ reduces allergen-induced asthma-like reaction, bronchial hyper-reactivity and airway remodelling.

Activation of poly(ADP-ribose) polymerases (PARPs) is considered a key event in the molecular and cellular processes leading from acute asthma attacks to bronchial hyper-reactivity, leucocyte recruitment, chronic inflammation, airway remodelling and lung damage. The present investigation has been carried out to investigate the action of hydroxyl-dimethylaminomethyl-thieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ), a new potent PARP inhibitor, in the process leading from asthma-like events to airway damage. Ovalbumin-sensitized guinea pigs exposed two times to allergen inhalation were treated for 8 days with vehicle or HYDAMTIQ. Asthma-like signs, bronchial hyper-reactivity to methacholine, cytokine production, histamine release from mast cells, airway remodelling, collagen deposition and lung damage were evaluated. Repeated HYDAMTIQ administration (1-10 mg/kg/day i.p.) reduced lung PARP activity, delayed the appearance and reduced the severity of allergen-induced cough and dyspnoea and dampened the increased bronchial responses to methacholine. HYDAMTIQ-treated animals presented reduced bronchial or alveolar abnormalities, lower number of eosinophils and other leucocytes in the lung and decreased smooth muscle or goblet cell hyperplasia. The treatment also reduced lung oxidative stress markers, such as malondialdehyde or 8-hydroxy-2'-deoxyguanosine and the lung content of pro-inflammatory cytokines (TNF-α, interleukin (IL)-1ß, IL-5, IL-6 and IL-18). Finally, mast cells isolated from the peritoneal or pleural cavities of sensitized, HYDAMTIQ-treated animals had a reduced ability to release histamine when exposed to ovalbumin in vitro. Our findings support the proposal that PARP inhibitors could have a therapeutic potential to reduce chronic lung inflammation, airway damage and remodelling in severe unresponsive asthmatic patients.

GPR35 activation reduces Ca2+ transients and contributes to the kynurenic acid-dependent reduction of synaptic activity at CA3-CA1 synapses.

Limited information is available on the brain expression and role of GPR35, a Gi/o coupled receptor activated by kynurenic acid (KYNA). In mouse cultured astrocytes, we detected GPR35 transcript using RT-PCR and we found that KYNA (0.1 to 100 µM) decreased forskolin (FRSK)-induced cAMP production (p<0.05). Both CID2745687 (3 µM, CID), a recently described GPR35 antagonist, and GPR35 gene silencing significantly prevented the action of KYNA on FRSK-induced cAMP production. In these cultures, we then evaluated whether GPR35 activation was able to modulate intracellular Ca(2+) concentration ([Ca(2+)]i ) and [Ca(2+)]i fluxes. We found that both KYNA and zaprinast, a phosphodiesterase (PDE) inhibitor and GPR35 agonist, did not modify either basal or peaks of [Ca(2+)]i induced by challenging the cells with ATP (30 µM). However, the [Ca(2+)]i plateau phase following peak was significantly attenuated by these compounds in a store-operated Ca(2+) channel (SOC)-independent manner. The activation of GPR35 by KYNA and zaprinast was also studied at the CA3-CA1 synapse in the rat hippocampus. Evoked excitatory post synaptic currents (eEPSCs) were recorded from CA1 pyramidal neurons in acute brain slices. The action of KYNA on GPR35 was pharmacologically isolated by using NMDA and α7 nicotinic receptor blockers and resulted in a significant reduction of eEPSC amplitude. This effect was prevented in the presence of CID. Moreover, zaprinast reduced eEPSC amplitude in a PDE5- and cGMP-independent mechanism, thus suggesting that glutamatergic transmission in this area is modulated by GPR35. In conclusion, GPR35 is expressed in cultured astrocytes and its activation modulates cAMP production and [Ca(2+)]i. GPR35 activation may contribute to KYNA effects on the previously reported decrease of brain extracellular glutamate levels and reduction of excitatory transmission.

Neurological basis of AMP-dependent thermoregulation and its relevance to central and peripheral hyperthermia.

Therapeutic hypothermia is of relevance to treatment of increased body temperature and brain injury, but drugs inducing selective, rapid, and safe cooling in humans are not available. Here, we show that injections of adenosine 5'-monophosphate (AMP), an endogenous nucleotide, promptly triggers hypothermia in mice by directly activating adenosine A1 receptors (A1R) within the preoptic area (POA) of the hypothalamus. Inhibition of constitutive degradation of brain extracellular AMP by targeting ecto 5'-nucleotidase, also suffices to prompt hypothermia in rodents. Accordingly, sensitivity of mice and rats to the hypothermic effect of AMP is inversely related to their hypothalamic 5'-nucleotidase activity. Single-cell electrophysiological recording indicates that AMP reduces spontaneous firing activity of temperature-insensitive neurons of the mouse POA, thereby retuning the hypothalamic thermoregulatory set point towards lower temperatures. Adenosine 5'-monophosphate also suppresses prostaglandin E2-induced fever in mice, having no effects on peripheral hyperthermia triggered by dioxymetamphetamine (ecstasy) overdose. Together, data disclose the role of AMP, 5'-nucleotidase, and A1R in hypothalamic thermoregulation, as well and their therapeutic relevance to treatment of febrile illness.

Mild activation of poly(ADP-ribose) polymerase (PARP) is neuroprotective in rat hippocampal slice models of ischemic tolerance.

Ischemic tolerance is a phenomenon in which exposure to a mild preconditioning stress results in resistance to a subsequent lethal ischemic insult. Here we investigated the role of poly(ADP-ribose) polymerase (PARP) in the development of ischemic tolerance by using organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to selective injury of the CA1 subregion 24 h later. We developed models of pharmacological preconditioning by exposing slices to subtoxic concentrations of either N-methyl-D-aspartate (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) and then, 24 h later, to 30 min OGD. Under these conditions, we observed a significant reduction in OGD-induced CA1 damage. Exposure of slices to the PARP-1 and -2 inhibitors TIQ-A, PJ-34 and UPF 1069 during preconditioning prevented the development of OGD tolerance in a concentration-dependent manner. NMDA and DHPG preconditioning increased the activity of PARP, as detected by immunoblots using antibodies against the poly(ADP-ribose) polymer product, but was not associated with consumption of cellular NAD(+) or ATP. Neuroprotection induced by preconditioning was also prevented by the caspase inhibitor Z-VAD-FMK. The modest but significant increase in caspase-3/7 induced by preconditioning, however, was not associated with PARP-1 cleavage, as occurred with staurosporine. Finally, TIQ-A prevented the activation of ERK1/2 and Akt induced by NMDA preconditioning, suggesting that the protective mechanism evoked by PARP requires activation of these prosurvival mediators. Our results suggest that preconditioning with appropriate pharmacological stimuli may promote neuroprotective mechanisms triggered by the sublethal activation of two otherwise deleterious executioners such as PARP and caspase-3/7.

Ischemic neuroprotection by TRPV1 receptor-induced hypothermia.

Although treatment of stroke patients with mild hypothermia is a promising therapeutic approach, chemicals inducing prompt and safe reduction of body temperature are an unmet need. We measured the effects of the transient receptor potential vanilloid-1 (TRPV1) agonist rinvanil on thermoregulation and ischemic brain injury in mice. Intraperitoneal or intracerebroventricular injection of rinvanil induces mild hypothermia that is prevented by the receptor antagonist capsazepine. Both intraischemic and postischemic treatments provide permanent neuroprotection in animals subjected to transient middle cerebral artery occlusion (MCAo), an effect lost in mice artificially kept normothermic. Data indicate that TRPV1 receptor agonists are promising candidates for hypothermic treatment of stroke.

Rat hippocampal slice culture models for the evaluation of neuroprotective agents.

Organotypic slices cultured for weeks in vitro represent an extremely valuable strategy for the investigation of the long-term properties of neuronal circuits under physiological and pathological conditions. Here, we describe how to prepare rat organotypic hippocampal slice cultures and how to expose them for appropriate periods of time to excitotoxic agents or to oxygen and glucose deprivation conditions, in order to mimic the pattern of pyramidal cell damage which is observed in vivo and in other in vitro models. This preparation is very useful not only to study synaptic plasticity or the pathways and mechanisms of neurodegeneration but also to evaluate the effects of neuroprotective agents.