Angelo O. Rosa

Antidepressant-like effect of the novel thiadiazolidinone NP031115 in mice.

Glycogen synthase kinase-3beta (GSK-3beta) is an enzyme that phosphorylates glycogen synthase, thereby inhibiting glycogen synthesis. Besides this role, it is now believed that this enzyme plays an important role in the pathophysiology of many brain diseases including depression. Some inhibitors of this enzyme have shown antidepressant effects in animal models. This study investigated the effects of a novel thiadiazolidinone NP031115, a putative GSK-3beta inhibitor, and the well-established GSK-3beta inhibitor AR-A014418 in the mouse forced swimming test (FST), a model widely used to evaluate antidepressant activity. We found that NP031115 had an IC50 of 1.23 and 6.5 microM for GSK-3beta and GSK-3alpha, respectively. NP031115 (0.5 and 5 mg/kg, i.p.), in a way similar to imipramine (15 mg/kg, i.p), fluoxetine (32 mg/kg, i.p), AR-A014418 (9 mg/kg, i.p.), and rosiglitazone (5 microg/site, i.c.v.), significantly reduced immobility time in the FST. NP031115 at the higher dose and AR-A014418 (9 mg/kg, i.p.) reduced locomotion in the open-field test. Rosiglitazone (30 microM), AR-A014418 (1 microM), PG(J2) (10 microM), and NP031115 (1, 10 and 25 microM) activate PPARgamma in CHO transfected cells. GW-9662 (10 microg/site, i.c.v, a PPARgamma antagonist) administered 15 min before NP03115 (5 mg/kg, i.p.) or co-administered with rosiglitazone (5 microg/site, i.c.v.) prevented the antidepressant-like effect of these drugs in the FST. The results of this study show that NP031115 can exhibit an antidepressant effect, likely by inhibiting GSK-3beta and enhancing PPARgamma activity.

Functional interference between glycogen synthase kinase-3 beta and the transcription factor Nrf2 in protection against kainate-induced hippocampal celldeath

Excitotoxicity mediated by glutamate receptors may underlay the pathology of several neurologic diseases. Considering that oxidative stress is central to excitotoxic damage, in this study we sought to analyze if the transcription factor Nrf2, guardian of redox homeostasis, might be targeted to prevent kainate-induced neuron death. Hippocampal slices from Nrf2 knockout mice exhibited increased oxidative stress and cell death compared to those of control mice in response to kainate, as determined with the redox sensitive probes 2,7-dichlorodihydrofluorescein diacetate (H(2)DCFAC) and propidium iodide and lactate dehydrogenase release, respectively, therefore demonstrating a role of Nrf2 in antioxidant protection against excitotoxicity. In the hippocampus of mice intraperitoneally injected with kainate we observed a rapid activation of Akt, inhibition of GSK-3beta and translocation of Nrf2 to the nucleus, but after 4 h Akt was inactive, GSK-3beta was active and Nrf2 was mostly cytosolic, therefore extending our previous studies which indicate that GSK-3beta excludes Nrf2 from the nucleus. Lithium, a GSK-3beta inhibitor, promoted Nrf2 transcriptional activity towards an Antioxidant-Response-Element (ARE) luciferase reporter and cooperated with sulforaphane (SFN) to induce this reporter and to increase the protein levels of heme oxygenase-1 (HO-1), coded by a representative ARE-containing gene. Conversely, ARE activation by SFN was attenuated by over-expression of active GSK-3beta. Finally, combined treatment with SFN and lithium attenuated oxidative stress and cell death in kainate-treated hippocampal slices of wild type mice but not Nrf2 null littermates. Our findings identify the axis GSK-3beta/Nrf2 as a pharmacological target in prevention of excitotoxic neuronal death.

Neuroprotection afforded by nicotine against oxygen and glucose deprivation in hippocampal slices is lost in α7 nicotinic receptor knockout mice

Although alpha7-receptors are considered the main target for neuroprotection, other receptor subtypes (alpha4beta2 or alpha3beta4) have also been implicated. Hence, we have used alpha7-transgenic mice, to study the hypothesis that alpha7-receptors play a dominant role in mediating neuroprotection in an in vitro model of ischemia. We have used rat and mouse hippocampal slices to establish the model of nicotinic neuroprotection against oxygen and glucose deprivation (OGD). Neuronal damage caused by OGD during 1 h plus 3 h re-oxygenation, was quantified by measuring lactate dehydrogenase (LDH) release from hippocampal slices. In rat hippocampal slices, OGD increased over twofold basal LDH release. Such increase was reduced when treated with 10-100 microM nicotine; maximal protection afforded by nicotine amounted to 46%. This neuroprotection was antagonized by the non-selective nicotinic receptor for acetylcholine (nAChR) blocker mecamylamine (10 microM). In hippocampal slices from wild-type control mice, nicotine (100 microM) decreased by 54.4% LDH release evoked by OGD plus re-oxygenation. In contrast, nicotine failed to exert neuroprotection in alpha7 knockout mice. This finding reinforces the view that the hippocampal neuroprotective effects of nicotine are predominantly linked to alpha7 receptors.

Nicotinic receptor activation by epibatidine induces heme oxygenase-1 and protects chromaffin cells against oxidative stress.

Activation of neuronal nicotinic acetylcholine receptors (nAChR) provides neuroprotection against different toxic stimuli that often lead to overproduction of reactive oxygen species (ROS) and cell death. ROS production has been related with disease progression in several neurodegenerative pathologies such as Alzheimer’s or Parkinson’s diseases. In this context, we investigated here if the exposure of bovine chromaffin cells to the potent nAChR agonist epibatidine protected against rotenone (30 μmol/L) plus oligomycin (10 μmol/L) (rot/oligo) toxicity, an in vitro model of mitochondrial ROS production. Epibatidine induced a concentration‐ and time‐dependent protection, which was maximal at 3 μmol/L after 24 h. Pre‐incubation with dantrolene (100 μmol/L) (a blocker of the ryanodine receptor channel), chelerythrine (1 μmol/L) (a protein kinase C inhibitor), or PD98059 (50 μmol/L) (a MEK inhibitor), aborted epibatidine‐elicited cytoprotection. Mitochondrial depolarization, ROS, and caspase 3 active produced by rot/oligo were also prevented by epibatidine. Epibatidine doubled the amount of heme oxygenase‐1 (HO‐1), a critical cell defence enzyme against oxidative stress. Furthermore, the HO‐1 inhibitor Sn(IV) protoporphyrin IX dichloride reversed the epibatidine protecting effects and HO‐1 inducer Co (III) protoporphyrin IX dichloride exhibited neuroprotective effects by itself. The results of this study point to HO‐1 as the cytoprotective target of nAChR activation through the following pathway: endoplasmic reticulum Ca2+‐induced Ca2+‐release activates the protein kinase C/extracellular regulated kinase/HO‐1 axis to mitigate mitochondrial depolarization and ROS production. This study provides a mechanistic insight on how nAChR activation translates into an antioxidant and antiapoptotic signal through up‐regulation of HO‐1.

Nrf2-mediated haeme oxygenase-1 up-regulation induced by cobalt protoporphyrin has antinociceptive effects against inflammatory pain in the formalin test in mice.

&NA; This study investigated the effect of haeme oxygenase‐1 (HO‐1) in nociception induced by formalin injection in the mice hind paw. Intraperitoneal (i.p.) administration of cobalt protoporphyrin (CoPP, an HO‐1 inducer, 5 mg/kg) 24 h before the test, inhibited the nociceptive response during the second phase, but not during the first phase of the formalin test. The effect of CoPP was prevented by treatment with tin protoporphyrin (SnPP, an inhibitor of HO‐1 activity) administered either by i.p. (25 mg/kg, 30 min before the test) or intraplantar (400 nmol/paw, 5 min before the test) routes. Human embryonic kidney (HEK) 293T cells treated with 10 μM CoPP expressed 20‐fold higher HO‐1 levels when compared to controls; this effect was suppressed by transfection with the dominant negative for the nuclear factor‐erythroid 2‐related factor 2 (Nrf2). Western blot analysis also revealed that CoPP treatment induced a similar 20‐fold increase in HO‐1 expression in the paw; this effect was attenuated in knockout mice for Nrf2. CoPP treatment of wild‐type, but not in Nrf2 knockout mice, resulted in a striking increase of HO‐1 stained cells surrounding the muscular tissues of the hind limbs. HO‐1 positive cells were scarce in wild‐type and in Nrf2 knockout untreated mice. CoPP‐induced HO‐1 expression in Nrf2 knockout mice was lost and correlated with the loss of antinociceptive effects. In conclusion, Nrf2‐mediated HO‐1 expression induced an antinociceptive effect at peripheral sites. These results suggest that HO‐1 modulates the inflammatory pain pathways. Hence, the development of drugs that could raise peripheral HO‐1 could be relevant in inflammatory pain treatment.

The Antinociceptive Effects of AR-A014418, a Selective Inhibitor of Glycogen Synthase Kinase-3 Beta, in Mice

UNLABELLED We investigated the antinociceptive effects of AR-A014418, a selective inhibitor of glycogen synthase kinase-3β (GSK-3β) in mice. A 30-minute pretreatment with AR-A014418 (.1 and 1 mg/kg, intraperitoneal [ip]) inhibited nociception induced by an ip injection of acetic acid. AR-A014418 pretreatment (.1 and .3 mg/kg, ip) also decreased the late (inflammatory) phase of formalin-induced licking, without affecting responses of the first (neurogenic) phase. In a different set of experiments, AR-A014418 (.1-10 μg/site) coinjected intraplantarly (ipl) with formalin inhibited the late phase of formalin-induced nociception. Furthermore, AR-A014418 administration (1 and 10 ng/site, intrathecal [it]) inhibited both phases of formalin-induced licking. In addition, AR-A014418 coinjection (10 ng/site, it) inhibited nociception induced by glutamate, N-methyl-D-aspartate (NMDA), (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1β) by 47 ± 12%, 48 ± 11%, 31 ± 8%, 46 ± 13%, and 44 ± 11%, respectively. In addition, a 30-minute pretreatment with NP031115 (3 and 10 mg/kg, ip), a different GSK-3 β inhibitor, also attenuated the late phase of formalin-induced nociception. Collectively, these results provide convincing evidence that AR-A014418, given by local, systemic, and central routes, produces antinociception in several mouse models of nociception. The AR-A014418-dependent antinociceptive effects were induced by modulation of the glutamatergic system through metabotropic and ionotropic (NMDA) receptors and the inhibition of the cytokine (TNF-α and IL-1β) signaling. PERSPECTIVE These results suggest that GSK-3β may be a novel pharmacological target for the treatment of pain.

Neuroprotection by nicotine in hippocampal slices subjected to oxygen-glucose deprivation: involvement of the alpha7 nAChR subtype.

Nicotine (NIC) is neuroprotective against glutamate and hypoxia-induced neurotoxicity, preventing neuronal death and apoptosis in primary neuronal cultures. This effect is mediated by activation of both alpha7 and alpha4beta2 subtypes of nicotinic receptors for acetylcholine (nAChR) (Kaneko et al., 1997; Hejmadi et al., 2003). Furthermore, it seems that activation of alpha7 nAChR is the mechanism by which galantamine protects against thapsigargin and beta-amyloid-induced cell death (Arias et al., 2004), as well as in neuroprotection exerted by NIC against tumor necrosis factor alpha (Gahring et al., 2003). In this context we studied possible protection produced by NIC in an oxygen-glucose deprivation (OGD) model of rat and mouse hippocampal slices. The involvement of alpha7 nAChR in neuroprotection was proved by using wild-type and alpha7 knockout (KO) mice.

Haeme oxygenase-1 overexpression via nAChRs and the transcription factor Nrf2 has antinociceptive effects in the formalin test

ABSTRACT Epibatidine has shown antinociceptive effects in various pain models, being 200‐fold more potent than morphine. Previous results from our laboratory demonstrated that HO‐1 overexpression has an antinociceptive effect in the formalin test. Furthermore, epibatidine was able to induce haeme oxygenase‐1 (HO‐1). So, the aim of this study was to investigate the effect of HO‐1 overexpression induced by epibatidine in nociception elicited by formalin injection in the mice hindpaw. Administration of epibatidine (4 μg/kg) 24 h before the test reduced the nociceptive response during the first phase and second phase of the formalin test. This effect was prevented by treatment with tin protoporphyrin (SnPP, an inhibitor of HO‐1 activity) administered via intraplantar 5 min before the test, suggesting a main role of HO‐1. Western blot analysis revealed that epibatidine treatment increased by 2‐fold HO‐1 expression in the paw; this effect was lost in knockout mice for nuclear factor‐erythroid 2‐related factor 2 (Nrf2) and was accompanied by the loss of its antinociceptive effect. Furthermore, the antinociceptive effect of epibatidine was related to the activation of alpha7 and/or alpha9 nAChRs since methyllycaconitine (MLA) and mecamylamine but not dihydro‐&bgr;‐erythroidine (DH&bgr;E) reverted this effect. Finally, we showed by flow cytometry and by immunofluorescence that white blood cells of the animals injected with epibatidine expressed more HO‐1 than control animals, and this expression was also reverted by MLA pre‐treatment. These findings demonstrate that HO‐1 induction by epibatidine has antinociceptive and anti‐inflammatory effects by the activation of MLA‐sensitive nAChRs.