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.

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.

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.

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 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.

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.

Different biochemical correlates for different neuropsychiatric abnormalities in patients with cirrhosis.

UNLABELLED: The diagnosis of hepatic encephalopathy (HE) relies on clinical, neurophysiological, psychometric and laboratory variables. The relationships between such tests remain debated. The aim of this study was to determine the laboratory correlates/prognostic value of neurophysiological/psychometric abnormalities in patients with cirrhosis. Seventy-two patients and 14 healthy volunteers underwent EEG and paper-and-pencil psychometry (PHES). Blood was obtained for C reactive protein (CRP), interleukin 6 (IL6), tumor necrosis factor (TNF)α, ammonia and indole/oxindole. Patients were followed prospectively for a median of 22 months in relation to the occurrence of death, transplantation and HE-related hospitalizations. Thirty-three patients had normal PHES and EEG, 6 had abnormal PHES, 18 abnormal EEG and 13 abnormal PHES and EEG. Patients with abnormal PHES had higher CRP (17 ± 22 vs 7 ± 6, P < 0.01), IL6 (32 ± 54 vs 12 ± 13, P < 0.05) and TNFα (17 ± 8 vs 11 ± 7, P < 0.001) levels than those with normal PHES. Patients with abnormal EEG had higher indole (430 ± 270 vs 258 ± 255, P < 0.01) and ammonia (66 ± 35 vs 45 ± 27, P < 0.05) levels than those with normal EEG. Psychometric test scores showed significant correlations with CRP, TNFα and IL6; EEG indices with ammonia and IL6. CRP and TNFα concentrations were independent predictors of abnormal PHES, ammonia and indole of abnormal EEG on multivariate analysis. Seven patients were lost to follow-up; of the remaining 65, 20 died and 14 underwent transplantation; 15 developed HE requiring hospitalization. PHES and EEG performance were independent predictors of HE and death (P < 0.05). CONCLUSION: PHES and EEG abnormalities in patients with cirrhosis have partially different biochemical correlates and independently predict outcome.

Kynurenic acid: a metabolite with multiple actions and multiple targets in brain and periphery.

It is usually assumed that kynurenic acid (KYNA) modifies neuronal function because it antagonizes the glycine site of the NMDA receptors and/or the neuronal cholinergic α7 nicotine receptors. It is not clear, however, whether the basal levels of KYNA found in brain extracellular spaces are sufficient to interact with these targets. Another reported target for KYNA is GPR35, an orphan receptor negatively coupled to G(i) proteins. GPR35 is expressed both in neurons and other cells (including glia, macrophages and monocytes). KYNA affinity for GPR35 in native systems has not been clarified and the low-affinity data widely reported in the literature for the interaction between KYNA and human or rat GPR35 have been obtained in modified expression systems. Possibly by interacting with GPR35, KYNA may also reduce glutamate release in brain and pro-inflammatory cytokines release in cell lines. The inhibition of inflammatory mediator release from both glia and macrophages may explain why KYNA has analgesic effects in inflammatory models. Furthermore, it may also explain why, KYNA administration (200 mg/kg ip × 3 times) to mice treated with lethal doses of LPS, significantly reduces the number of deaths. Finally, KYNA has been reported as an agonist of aryl hydrocarbon receptor (AHR), a nuclear protein involved in the regulation of gene transcription and able to cause immunosuppression after binding with dioxin. Thus, KYNA has receptors in the nervous and the immune systems and may play interesting regulatory roles in cell function.

Poly(ADP-ribose) polymerase-1 is a nuclear epigenetic regulator of mitochondrial DNA repair and transcription.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a NAD-consuming enzyme with an emerging key role in epigenetic regulation of gene transcription. Although PARP-1 expression is characteristically restricted to the nucleus, a few studies report the mitochondrial localization of the enzyme and its ability to regulate organelle functioning. Here, we show that, despite exclusive nuclear localization of PARP-1, mitochondrial homeostasis is compromised in cell lines exposed to PARP-1 pharmacological inhibitors or small interfering RNA. PARP-1 suppression reduces integrity of mitochondrial DNA (mtDNA), as well as expression of mitochondria-encoded respiratory complex subunits COX-1, COX-2, and ND-2. Accordingly, PARP-1 localizes at promoters of nuclear genes encoding both the mtDNA repair proteins UNG1, MYH1, and APE1 and the mtDNA transcription factors TFB1M and TFB2M. It is noteworthy that poly(ADP-ribosyl)ation is required for nuclear gene expression of these mitochondrial proteins. Consistent with these findings, PARP-1 suppression impairs mitochondrial ATP production. Our results indicate that PARP-1 plays a central role in mitochondrial homeostasis by epigenetically regulating nuclear genes involved in mtDNA repair and transcription. These data might have important implications in pharmacology of PARP-1 inhibitors as well as clinical oncology and aging.

Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis.

During the last several years, evidence that various enzymes hydrolyze NAD into bioactive products prompted scientists to revisit or design strategies able to increase intracellular availability of the dinucleotide. However, plasma membrane permeability to NAD and the mitochondrial origin of the dinucleotide still wait to be clearly defined. Here, we report that intracellular NAD contents increased upon exposure of cell lines or primary cultures to exogenous NAD (eNAD). NAD precursors could not reproduce the effects of eNAD, and they were not found in the incubating medium containing eNAD, thereby suggesting direct cellular eNAD uptake. We found that in mitochondria of cells exposed to eNAD, NAD and NADH as well as oxygen consumption and ATP production were increased. Conversely, DNA repair, a well known NAD-dependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-N'-nitro-N-nitrosoguanidine. In particular, eNAD reduced staurosporine-induced loss of mitochondrial membrane potential and ensuing caspase activation. Of importance, pharmacological inhibition or silencing of the NAD-dependent enzyme SIRT1 abrogated the ability of eNAD to provide protection from staurosporine, having no effect on eNAD-dependent protection from C2-ceramide or N-methyl-N'-nitro-N-nitrosoguanidine. Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.

Inhibition of nicotinamide phosphoribosyltransferase: cellular bioenergetics reveals a mitochondrial insensitive NAD pool.

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.

PARP-1 inhibition prevents CNS migration of dendritic cells during EAE, suppressing the encephalitogenic response and relapse severity.

BACKGROUND: Pharmacological inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) are currently evaluated in clinical trials for various malignancies but, interestingly, also proved of remarkable efficacy in preclinical models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE). OBJECTIVES: The objectives of the study were to determine molecular mechanisms underlying suppression of the encephalitogenic response by these drugs; likewise, whether clinically-relevant post-treatment paradigms with PARP-1 inhibitors could prevent EAE relapses. METHODS: Adopted both in vitro techniques (bone marrow-derived cultured DC) as well as in vivo models of chronic or relapsing-remitting (RR) EAE. RESULTS: We report that two structurally unrelated PARP-1 inhibitors negatively regulated NFκB activation, as well as maturation, cytokine production and APC function of cultured mouse bone marrow-derived dendritic cells (DCs). PARP-1 inhibitors also reduced the number and APC function of DCs migrating in the draining lymph nodes of ovalbumin-immunized mice. In C57Bl mice with chronic EAE or SJL mice with RR EAE, pharmacological inhibition of PARP-1 reduced CNS DC migration and demyelination as well as neurological impairment to an extent similar to that achieved with the potent immunosuppressant cyclosporine A. Remarkably, PARP-1 inhibitors injected after the first phase of disease reduced relapse incidence and severity, as well as the spinal cord number of autoreactive Th17 cells. Under this clinically-relevant treatment paradigm, PARP inhibitors also suppressed epitope spreading of the encephalitogenic response. CONCLUSIONS: Overall, data underscore the potential relevance of PARP-1 inhibitors to MS therapy and suppression of autoimmunity.

Brain ischemic preconditioning does not require PARP-1.

BACKGROUND AND PURPOSE: Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in ischemic preconditioning of the heart and cultured neurons, but its role in brain ischemic preconditioning is unknown. Summary of Report- We report that 5-minute bilateral common carotid artery occlusion (BCCAO) in the mouse prompted reduction of infarct volumes triggered 24 hours later by 20-minute middle cerebral artery occlusion (MCAO). Pharmacological PARP-1 inhibition between BCCAO and MCAO did not impair preconditioning. The contents of the PARP-1 substrate NAD, those of its product poly(ADP-ribose), caspase-3 activation, and PARP-1 expression did not change after BCCAO within the preconditioned tissue. PARP-1 KO mice were similarly protected by the 5-minute BCCAO. CONCLUSIONS: Data demonstrate that, at variance with the heart, PARP-1 is dispensable for brain ischemic preconditioning.

Peripheral and splanchnic indole and oxindole levels in cirrhotic patients: a study on the pathophysiology of hepatic encephalopathy.

OBJECTIVES: Intestinal bacteria metabolize tryptophan into indole, which is then further metabolized into oxindole, a sedative compound putatively involved in the pathophysiology of hepatic encephalopathy (HE). The aim of this study was to measure indole and oxindole levels in patients with cirrhosis with or without HE and to establish whether an intestinal production and a hepatic metabolism of these substances exist. METHODS: We studied 10 healthy subjects (controls) and 51 cirrhotic patients: 17 without HE, 14 with a minimal HE, 8 with overt HE, and 12 who had undergone a transjugular intrahepatic portosystemic shunt (TIPS) procedure. In the last group, blood was collected from the artery, and the portal and hepatic veins during TIPS construction and from the peripheral veins before, immediately after, and at weekly intervals during the first month after TIPS. RESULTS: Plasma indole levels were significantly higher in patients with overt HE. Oxindole levels were higher in cirrhotics than in controls. Indole and ammonia were significantly correlated (r=0.66). Peripheral and splanchnic determinations showed that indole was produced in the intestine and cleared by the liver, similar to ammonia. TIPS implantation increased both indole and ammonia levels. After TIPS, the psychometric performance worsened in 4 of the 12 patients. The increase in indole plasma concentrations in these four patients was higher than in those who remained stable after undergoing TIPS. CONCLUSIONS: Indole correlates with HE and has a significant intestinal production and hepatic extraction; its level increases after TIPS and is related to psychometric performance. These data suggest that indole may be involved in the pathophysiology of HE.

Histone deacetylase (HDAC) inhibitors reduce the glial inflammatory response in vitro and in vivo.

Histone deacetylase inhibitors (HDACi) are emerging tools for epigenetic modulation of gene expression and suppress the inflammatory response in models of systemic immune activation. Yet, their effects within the brain are still controversial. Also, whether HDACs are expressed in astrocytes or microglia is unclear. Here, we report the identification of transcripts for HDAC 1-11 in cultured mouse glial cells. Two HDACi such as SAHA and ITF2357 induce dramatic increase of histone acetylation without causing cytotoxicity of cultured cells. Of note, the two compounds inhibit expression of pro-inflammatory mediators by LPS-challenged glial cultures, and potentiate immunosuppression triggered by dexamethasone in vitro. The anti-inflammatory effect is not due to HDACi-induced transcription of immunosuppressant proteins, (including SOCS-1/3) or microRNA-146. Rather, it is accompanied by direct alteration of transcription factor DNA binding and ensuing transcriptional activation. Indeed, both HDACi impair NFkappaB-dependent IkappaBalpha resynthesis in glial cells exposed to LPS, and, among various AP1 subunits and NFkappaB p65, affect the DNA binding activity of c-FOS, c-JUN and FRA2. Importantly, ITF2357 reduces the expression of pro-inflammatory mediators in the striatum of mice iontophoretically injected with LPS. Data demonstrate that mouse glial cells have ongoing HDAC activity, and its inhibition suppresses the neuroinflammatory response because of a direct impairment of the transcriptional machinery.

Poly(ADP-ribose)polymerase 1 (PARP-1) and postischemic brain damage.

Poly(ADP-ribose)polymerases (PARPs) are enzymes that are able to catalyze the transfer of ADP-ribose units from NAD to substrate proteins and are particularly abundant in cell nuclei where they play key roles in the maintenance of genomic integrity, control of cell cycle and gene expression. Brain ischemia overactivates PARPs and PARP-deficient mice or animal treated with PARP inhibitors have a drastically reduced brain damage in various stroke models. PARP 'overactivation' occurs not only in neurons but also in astrocytes, microglial cells, endothelia, and infiltrating leukocytes. The ensuing cell death occurs through various molecular mechanisms: a) excessive ATP use for NAD synthesis and inhibition of mitochondrial function with subsequent energy failure (particularly important in neurons); b) apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus (present in neurons, endothelial, and other cells); c) excessive expression of inflammatory mediators (well demonstrated in glial cells) or d) reduced expression of prosurvival factors. Thus PARPs seem to play key roles in postischemic brain damage and are now considered interesting targets for therapies aimed at reducing stroke pathology.

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.

mGlu1alpha receptors are co-expressed with CB1 receptors in a subset of interneurons in the CA1 region of organotypic hippocampal slice cultures and adult rat brain.

Recent studies have demonstrated a functional interaction between group I metabotropic glutamate (mGlu) receptors and the cannabinoid system in the modulation of synaptic transmission. By using antisera directed against mGlu1alpha and CB1 cannabinoid receptors, we examined their distribution in the CA1 region of rat organotypic hippocampal slice cultures. Immunoreactive mGlu1alpha and CB1 elements were localized in non-principal cells, with a labeling distribution that was very similar to the pattern previously observed in the adult rat brain. Double-immunofluorescence staining and confocal microscopy showed that a subset of interneurons, mainly located in the stratum radiatum, was double-labeled for both mGlu1alpha and CB1 receptors. Co-localization of the two receptor subtypes was confirmed in hippocampal sections from adult rat brain. By using the "mirror technique" in adjacent sections, we observed that the double-labeled cells for mGlu1alpha and CB1 receptors were also immunopositive for the cholecystokinin peptide. Quantitative analysis revealed that in the stratum radiatum the majority (92%) of the CB1-positive cells and 19% of the mGlu1alpha-positive cells expressed both receptors. Triple immunofluorescence staining showed partial co-labeling of mGlu1alpha- and CB1-immunopositive cells with the vesicular glutamate transporter 3 or calbindin. Our results demonstrate that mGlu1alpha and CB1 receptors co-exist in a subpopulation of inhibitory neurons in the stratum radiatum of the hippocampus that is suggestive of the Schaffer collateral-associated interneurons. Hence, additional functional mechanisms underlying the cooperation between these two receptor subtypes may exist.

Neuroprotection by group I mGlu receptors in a rat hippocampal slice model of cerebral ischemia is associated with the PI3K-Akt signaling pathway: a novel postconditioning strategy?

Ischemic postconditioning is defined as a repetitive series of brief interruptions of reperfusion applied immediately after ischemia. In this study, postconditioning was investigated by first exposing rat organotypic hippocampal slices to 30min oxygen-glucose deprivation (OGD), which promotes selective CA1 pyramidal cell death, and 5min later to either a brief period (3min) of OGD or to a low dose (10microM) of 3,5-dihydroxyphenylglycine (DHPG) for 30min. Both protocols attenuated CA1 neuronal injury, as revealed 24h later by measuring the intensity of propidium iodide fluorescence in this region. The beneficial effects were observed when DHPG postconditioning was applied up to 15min after OGD, but not at later time points, and was not additive with the neuroprotective effects of a preconditioning DHPG treatment. The attenuation of the OGD-induced CA1 injury evoked by postconditioning was prevented when mGlu1 and mGlu5 receptor antagonists and inhibitors of phosphatidylinositol 3-kinase and Akt activity were present in the incubation medium during the 5min recovery period after OGD and the 30min exposure to DHPG. The PI3K inhibitor was also able to prevent the reduction of NMDA toxicity induced by the DHPG treatment. Finally, DHPG increased the phosphorylation of Akt in a transient and mGlu1/mGlu5-dependent manner. Our results show that activation of the mGlu1/mGlu5-PI3K-Akt signaling pathway plays a crucial role in the mechanisms of postconditioning evoked by DHPG and point to this strategy as a possible novel therapeutic tool for stroke and cerebral ischemia.