Human alpha 1-antitrypsin protects neurons and glial cells against oxygen and glucose deprivation through inhibition of interleukins expression.

BACKGROUND: Death due to cerebral stroke afflicts a large number of neuronal populations, including glial cells depending on the brain region affected. Drugs with a wide cellular range of protection are needed to develop effective therapies for stroke. Human alpha 1-antitrypsin (hAAT) is a serine proteinase inhibitor with potent anti-inflammatory, anti-apoptotic and immunoregulatory activities. This study aimed to test whether hAAT can protect different kind of neurons and glial cells after the oxygen and glucose deprivation (OGD). METHODS: Addition of hAAT to mouse neuronal cortical, hippocampal and striatal cultures, as well as glial cultures, was performed 30 min after OGD induction and cell viability was assessed 24 h later. The expression of different apoptotic markers and several inflammatory parameters were assessed by immunoblotting and RT-PCR. RESULTS: hAAT had a concentration-dependent survival effect in all neuronal cultures exposed to OGD, with a maximal effect at 1-2 mg/mL. The addition of hAAT at 1 mg/mL reduced the OGD-mediated necrotic and apoptotic death in all neuronal cultures. This neuroprotective activity of hAAT was associated with a decrease of cleaved caspase-3 and an increase of MAP2 levels. It was also associated with a reduction of pro-inflammatory cytokines protein levels and expression, increase of IL-10 protein levels and decrease of nuclear localization of nuclear factor-kappaB. Similar to neurons, addition of hAAT protected astrocytes and oligodendrocytes against OGD-induced cell death. CONCLUSIONS: Human AAT protects neuronal and glial cells against OGD through interaction with cytokines. GENERAL SIGNIFICANCE: Human AAT could be a good therapeutic neuroprotective candidate to treat ischemic stroke.

Loss of striatal 90-kDa ribosomal S6 kinase (Rsk) is a key factor for motor, synaptic and transcription dysfunction in Huntington's disease.

Huntington's disease (HD) is characterized by motor dysfunction due to the expression of mutant huntingtin that promotes degeneration of striatal GABAergic medium-sized spiny neurons. Here we explore the role of the 90-kDa ribosomal S6 kinase (Rsk) in the physiopathology of HD. First, we show a reduction of Rsk1 and 2 protein levels in the striatum of two HD mouse models, R6/1 and Hdh(Q7/Q111) knock-in mice, at ages when they suffer from motor disturbances. Interestingly, the analysis of post-mortem samples from HD patients revealed a significant reduction of both Rsk forms in the putamen and caudate, but not in the cortex. Rsk1 and 2 levels were also reduced in the striatum of BDNF heterozygous mice, and upon BDNF neutralization in striatal cultures, suggesting that striatal loss of BDNF could be involved in the decrease of Rsk levels. Finally, we injected recombinant adeno-associated-virus (AAV5)-Rsk in the striatum of R6/1 mice at the onset of motor symptoms. Four weeks later, we found higher Rsk levels in the striatum accompanied by improvements in motor coordination, enhanced expression of synaptic markers and increased expression of genes related to synaptic plasticity, such as cfos and egr1. Altogether, we identified Rsk as a key factor in striatal alterations associated with motor deficits in HD.

Prostaglandin E2 EP2 activation reduces memory decline in R6/1 mouse model of Huntington's disease by the induction of BDNF-dependent synaptic plasticity.

Huntington's disease (HD) patients and mouse models show learning and memory impairment even before the onset of motor symptoms. Deficits in hippocampal synaptic plasticity have been involved in the HD memory impairment. Several studies show that prostaglandin E2 (PGE2) EP2 receptor stimulates synaptic plasticity and memory formation. However, this role was not explored in neurodegenerative diseases. Here, we investigated the capacity of PGE2 EP2 receptor to promote synaptic plasticity and memory improvements in a model of HD, the R6/1 mice, by administration of the agonist misoprostol. We found that misoprostol increases dendritic branching in cultured hippocampal neurons in a brain-derived neurotrophic factor (BDNF)-dependent manner. Then, we implanted an osmotic mini-pump system to chronically administrate misoprostol to R6/1 mice from 14 to 18weeks of age. We observed that misoprostol treatment ameliorates the R6/1 long-term memory deficits as analyzed by the T-maze spontaneous alternation task and the novel object recognition test. Importantly, administration of misoprostol promoted the expression of hippocampal BDNF. Moreover, the treatment with misoprostol in R6/1 mice blocked the reduction in the number of PSD-95 and VGluT-1 positive particles observed in hippocampus of vehicle-R6/1 mice. In addition, we observed an increase of cAMP levels in the dentate ` of WT and R6/1 mice treated with misoprostol. Accordingly, we showed a reduction in the number of mutant huntingtin nuclear inclusions in the dentate gyrus of R6/1 mice. Altogether, these results suggest a putative therapeutic effect of PGE2 EP2 receptor in reducing cognitive deficits in HD.

Ras protein activation is a key event in activity-dependent survival of cerebellar granule neurons.

Neuronal activity promotes the survival of cerebellar granule neurons (CGNs) during the postnatal development of cerebellum. CGNs that fail to receive excitatory inputs will die by apoptosis. This process could be mimicked in culture by exposing CGNs to either a physiological concentration of KCl (5 mm or K5) plus N-methyl-d-aspartate (NMDA) or to 25 mm KCl (K25). We have previously described that a 24-h exposure to NMDA (100 µm) or K25 at 2 days in vitro induced long term survival of CGNs in K5 conditions. Here we have studied the molecular mechanisms activated at 2 days in vitro in these conditions. First we showed that NMDA or K25 addition promoted a rapid stimulation of PI3K and a biphasic phosphorylation on Ser-473 of Akt, a PI3K substrate. Interestingly, we demonstrated that only the first wave of Akt phosphorylation is necessary for the NMDA- and K25-mediated survival. Additionally, we detected that both NMDA and K25 increased ERK activity with a similar time-course. Moreover, our results showed that NMDA-mediated activation of the small G-protein Ras is necessary for PI3K/Akt pathway activation, whereas Rap1 was involved in NMDA phosphorylation of ERK. On the other hand, Ras, but not Rap1, mediates K25 activation of PI3K/Akt and MEK/ERK pathways. Because neuroprotection by NMDA or K25 is mediated by Ras (and not by Rap1) activation, we propose that Ras stimulation is a crucial event in NMDA- and K25-mediated survival of CGNs through the activation of PI3K/Akt and MEK/ERK pathways.

Delineating the factors and cellular mechanisms involved in the survival of cerebellar granule neurons.

Cerebellar granule neurons (CGNs) constitute the most abundant neuronal population in the mammalian brain. Their postnatal generation and the feasibility to induce their apoptotic death in vitro make them an excellent model to study the effect of several neurotransmitters and neurotrophins. Here, we first review which factors are involved in the generation and proliferation of CGNs in the external granule layer (EGL) and in the regulation of their differentiation and migration to internal granule layer (IGL). Special attention was given to the role of several neurotrophins and the NMDA subtype of glutamate receptor. Then, using the paradigm of potassium deprivation in cultured CGNs, we address several extracellular factors that promote the survival of CGNs, with particular emphasis on the cellular mechanisms. The role of specific protein kinases leading to the regulation of transcription factors and recent data involving the small G protein family is also discussed. Finally, the participation of some members of Bcl-2 family and the inhibition of mitochondria-related apoptotic pathway is also considered. Altogether, these studies evidence that CGNs are a key model to understand the development and the survival of neuronal populations.

Brain-gut photobiomodulation restores cognitive alterations in chronically stressed mice through the regulation of Sirt1 and neuroinflammation.

BACKGROUND: Chronic stress is an important risk factor for the development of major depressive disorder (MDD). Recent studies have shown microbiome dysbiosis as one of the pathogenic mechanisms associated with MDD. Thus, it is important to find novel non-pharmacological therapeutic strategies that can modulate gut microbiota and brain activity. One such strategy is photobiomodulation (PBM), which involves the non-invasive use of light. OBJECTIVE/HYPOTHESIS: Brain-gut PBM could have a synergistic beneficial effect on the alterations induced by chronic stress. METHODS: We employed the chronic unpredictable mild stress (CUMS) protocol to induce a depressive-like state in mice. Subsequently, we administered brain-gut PBM for 6 min per day over a period of 3 weeks. Following PBM treatment, we examined behavioral, structural, molecular, and cellular alterations induced by CUMS. RESULTS: We observed that the CUMS protocol induces profound behavioral alterations and an increase of sirtuin1 (Sirt1) levels in the hippocampus. We then combined the stress protocol with PBM and found that tissue-combined PBM was able to rescue cognitive alterations induced by CUMS. This rescue was accompanied by a restoration of hippocampal Sirt1 levels, prevention of spine density loss in the CA1 of the hippocampus, and the modulation of the gut microbiome. PBM was also effective in reducing neuroinflammation and modulating the morphology of Iba1-positive microglia. LIMITATIONS: The molecular mechanisms behind the beneficial effects of tissue-combined PBM are not fully understood. CONCLUSIONS: Our results suggest that non-invasive photobiomodulation of both the brain and the gut microbiome could be beneficial in the context of stress-induced MDD.

Increased PKA signaling disrupts recognition memory and spatial memory: role in Huntington's disease.

Huntington's disease (HD) patients and mouse models show learning and memory impairment even before the onset of motor symptoms. However, the molecular events involved in this cognitive decline are still poorly understood. Here, using three different paradigms, the novel object recognition test, the T-maze spontaneous alternation task and the Morris water maze, we detected severe cognitive deficits in the R6/1 mouse model of HD before the onset of motor symptoms. When we examined the putative molecular pathways involved in these alterations, we observed hippocampal cAMP-dependent protein kinase (PKA) hyper-activation in naïve R6/1 mice compared with wild-type (WT) mice, whereas extracellular signal-regulated kinase 1/2 and calcineurin activities were not modified. Increased PKA activity resulted in hyper-phosphorylation of its substrates N-methyl-D-aspartate receptor subunit 1, Ras-guanine nucleotide releasing factor-1 and striatal-enriched protein tyrosine phosphatase, but not cAMP-responsive element binding protein or the microtubule-associated protein tau. In correlation with the over-activation of the PKA pathway, we found a down-regulation of the protein levels of some phosphodiesterase (PDE) 4 family members. Similar molecular changes were found in the hippocampus of R6/2 mice and HD patients. Furthermore, chronic treatment of WT mice with the PDE4 inhibitor rolipram up-regulated PKA activity, and induced learning and memory deficits similar to those seen in R6 mice, but had no effect on R6/1 mice cognitive impairment. Importantly, hippocampal PKA inhibition by infusion of Rp-cAMPS restored long-term memory in R6/2 mice. Thus, our results suggest that occlusion of PKA-dependent processes is one of the molecular mechanisms underlying cognitive decline in R6 animals.

Striatal-enriched protein tyrosine phosphatase expression and activity in Huntington's disease: a STEP in the resistance to excitotoxicity.

Striatal-enriched protein tyrosine phosphatase (STEP) is highly expressed in striatal projection neurons, the neuronal population most affected in Huntington's disease. Here, we examined STEP expression and phosphorylation, which regulates its activity, in N-terminal exon-1 and full-length mutant huntingtin mouse models. R6/1 mice displayed reduced STEP protein levels in the striatum and cortex, whereas its phosphorylation was increased in the striatum, cortex, and hippocampus. The early increase in striatal STEP phosphorylation levels correlated with a deregulation of the protein kinase A pathway, and decreased calcineurin activity at later stages further contributes to an enhancement of STEP phosphorylation and inactivation. Accordingly, we detected an accumulation of phosphorylated ERK2 and p38, two targets of STEP, in R6/1 mice striatum at advanced stages of the disease. Activation of STEP participates in excitotoxic-induced cell death. Because Huntington's disease mouse models develop resistance to excitotoxicity, we analyzed whether decreased STEP activity was involved in this process. After intrastriatal quinolinic acid (QUIN) injection, we detected higher phosphorylated STEP levels in R6/1 than in wild-type mice, suggesting that STEP inactivation could mediate neuroprotection in R6/1 striatum. In agreement, intrastriatal injection of TAT-STEP increased QUIN-induced cell death. R6/2, Tet/HD94, and Hdh(Q7/Q111) mice striatum also displayed decreased STEP protein and increased phosphorylation levels. In Tet/HD94 mice striatum, mutant huntingtin transgene shutdown reestablished STEP expression. In conclusion, the STEP pathway is severely downregulated in the presence of mutant huntingtin and may participate in compensatory mechanisms activated by striatal neurons that lead to resistance to excitotoxicity.

Activation of Elk-1 participates as a neuroprotective compensatory mechanism in models of Huntington's disease.

The transcription factor Elk-1 has been revealed as neuroprotective against toxic stimuli. In this study, we explored the neuroprotective capacity of Elk-1 in Huntington's disease. To this aim, we used two exon-1 mutant huntingtin (mhtt) mouse models (R6/1 and R6/2), and a full-length mhtt striatal cell model (STHdh(Q111/Q111) ). Analysis of Elk-1 and pElk-1(Ser383) in the striatum of R6 mice revealed increased levels during the disease progression. Similarly, Elk-1 and pElk-1(Ser383) levels were increased in STHdh(Q111/Q111) cells when compared with wild-type cells. In addition, we observed a predominant nuclear localization of Elk-1 in STHdh(Q111/Q111) cells, and in the striatum of 30-week-old R6/1 mice. Nuclear Elk-1 did not colocalize with mhtt aggregates, suggesting a higher transcriptional activity. In agreement, the knock-down of Elk-1 decreased immediate early genes expression in STHdh(Q111/Q111) cells, but not in wild-type cells. Interestingly, reduction of Elk-1 levels by siRNAs transfection promoted cell death and caspase 3 cleavage in STHdh(Q111/Q111) cells, but not in wild-type cells. In summary, we propose that increased protein levels, phosphorylation and nuclear localization of Elk-1 observed in exon-1 and full-length Huntington's disease models could be a compensatory mechanism activated by striatal cells in response to the presence of mhtt that contributes to neuroprotection.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Protects Striatal Cells and Improves Motor Function in Huntington's Disease Models: Role of PAC1 Receptor.

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by the expression of mutant huntingtin (mHtt). One of the main features of HD is the degeneration of the striatum that leads to motor discoordination. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that acts through three receptors named PAC1R, VPAC1R, and VPAC2R. In the present study, we first investigated the effect of PACAP on STHdhQ7/Q7 and STHdhQ111/Q111 cells that express wild-type Htt with 7 and mHtt with 111 glutamines, respectively. Then we explored the capacity of PACAP to rescue motor symptoms in the R6/1, a murine model of HD. We found that PACAP treatment (10-7 M) for 24 h protects STHdhQ111/Q111 cells from mHtt-induced apoptosis. This effect is associated with an increase in PAC1R transcription, phosphorylation of ERK and Akt, and an increase of intracellular c-fos, egr1, CBP, and BDNF protein content. Moreover, the use of pharmacological inhibitors revealed that activation of ERK and Akt mediates these antiapoptotic and neurotrophic effects of PACAP. To find out PAC1R implication, we treated STHdh cells with vasoactive intestinal peptide (VIP), which exhibits equal affinity for VPAC1R and VPAC2R, but lower affinity for PAC1R, in contrast to PACAP which has same affinity for the three receptors. VIP reduced cleaved caspase-3 protein level, without promoting the expression of c-fos, egr1, CBP, and the neurotrophin BDNF. We next measured the protein level of PACAP receptors in the striatum and cortex of R6/1 mice. We observed a specific reduction of PAC1R at the onset of motor symptoms. Importantly, the intranasal administration of PACAP to R6/1 animals restored the motor function and increased the striatal levels of PAC1R, CBP, and BDNF. In conclusion, PACAP exerts antiapoptotic and neurotrophic effects in striatal neurons mainly through PAC1R. This effect in HD striatum allows the recovery of motor function and point out PAC1R as a therapeutic target for treatment of HD.

Altered cholesterol homeostasis contributes to enhanced excitotoxicity in Huntington's disease.

Recent findings suggest that altered cholesterol homeostasis may contribute to the pathophysiology of Huntington's disease (HD). To understand the underlying mechanisms, here we used a combination of two-photon microscopy, epifluorescence, and biochemical methods to visualize and quantify lipid distribution in cell cultures expressing mutant huntingtin. Such expression promotes lipid imbalance, and cholesterol accumulation in cellular and murine models and in HD-affected human brains. Interestingly, cells expressing mutant huntingtin also showed higher content of ordered domains in their plasma membranes. These findings correlated with high levels of caveolin-1 and glycosphingolipid GM1, two well-defined markers of cholesterol-enriched domains, at the cell surface. In addition, cells expressing mutant huntingtin showed increased localization of NMDA receptors with cholesterol-enriched domains, contributing to increased NMDA receptor susceptibility to excitotoxic insults. Treatment with simvastatin or ß-cyclodextrin, two cholesterol-lowering drugs, reduced the content of ordered domains at the cell surface, which in turn, protected cells against NMDA-mediated excitotoxicity. Taken together, our results indicate that mutant huntingtin produces accumulation of cholesterol and alters its cellular distribution that contributes to NMDA-mediated excitotoxicity. Administration of drugs that recover this effect, such as simvastatin could be beneficial for the treatment of HD.

Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide and Vasoactive Intestinal Peptide Against Cognitive Decline in Neurodegenerative Diseases.

Cognitive impairment is one of the major symptoms in most neurodegenerative disorders such as Alzheimer's (AD), Parkinson (PD), and Huntington diseases (HD), affecting millions of people worldwide. Unfortunately, there is no treatment to cure or prevent the progression of those diseases. Cognitive impairment has been related to neuronal cell death and/or synaptic plasticity alteration in important brain regions, such as the cerebral cortex, substantia nigra, striatum, and hippocampus. Therefore, compounds that can act to protect the neuronal loss and/or to reestablish the synaptic activity are needed to prevent cognitive decline in neurodegenerative diseases. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two highly related multifunctional neuropeptides widely distributed in the central nervous system (CNS). PACAP and VIP exert their action through two common receptors, VPAC1 and VPAC2, while PACAP has an additional specific receptor, PAC1. In this review article, we first presented evidence showing the therapeutic potential of PACAP and VIP to fight the cognitive decline observed in models of AD, PD, and HD. We also reviewed the main transduction pathways activated by PACAP and VIP receptors to reduce cognitive dysfunction. Furthermore, we identified the therapeutic targets of PACAP and VIP, and finally, we evaluated different novel synthetic PACAP and VIP analogs as promising pharmacological tools.

Decreased Myocyte Enhancer Factor 2 Levels in the Hippocampus of Huntington's Disease Mice Are Related to Cognitive Dysfunction.

People suffering from Huntington's disease (HD) present cognitive deficits. Hippocampal dysfunction has been involved in the HD learning and memory impairment, but proteins leading this dysregulation are not fully characterized. Here, we studied the contribution of the family of transcription factors myocyte enhancer factor 2 (MEF2) to the HD cognitive deficits. To this aim, we first analyzed MEF2 protein levels and found that they are reduced in the hippocampus of exon-1 (R6/1) and full-length (HdhQ7/Q111) mutant huntingtin (mHTT) mice at the onset of cognitive dysfunction. By the analysis of MEF2 mRNA levels and mHTT-MEF2 interaction, we discarded that reduced MEF2 levels are due to changes in the transcription or sequestration in mHTT aggregates. Interestingly, we showed in R6/1 primary hippocampal cultures that reduction of MEF2 is strongly related to a basal and non-apoptotic caspase activity. To decipher the involvement of hippocampal decreased MEF2 in memory impairment, we used the BML-210 molecule that activates MEF2 transcriptional activity by the disruption MEF2-histone deacetylase class IIa interaction. BML-210 treatment increased the number and length of neurites in R6/1 primary hippocampal cultures. Importantly, this effect was prevented by transduction of lentiviral particles containing shRNA against MEF2. Then, we demonstrated that intraperitoneal administration of BML-210 (150 mg/Kg/day) for 4 days in R6/1 mice improved cognitive performance. Finally, we observed that BML-210 treatment also promoted the activation of MEF2-dependent memory-related genes and the increase of synaptic markers in the hippocampus of R6/1 mice. Our findings point out that reduced hippocampal MEF2 is an important mediator of cognitive dysfunction in HD and suggest that MEF2 slight basal activation could be a good therapeutic option.

Reduced calcineurin protein levels and activity in exon-1 mouse models of Huntington's disease: role in excitotoxicity.

Calcineurin is a serine/threonine phosphatase involved in the regulation of glutamate receptors signaling. Here, we analyzed whether the regulation of calcineurin protein levels and activity modulates the susceptibility of striatal neurons to excitotoxicity in R6/1 and R6/1:BDNF+/- mouse models of Huntington's disease. We show that calcineurin inhibition in wild-type mice drastically reduced quinolinic acid-induced striatal cell death. Moreover, calcineurin A and B were differentially regulated during disease progression with a specific reduction of calcineurin A protein levels and calcineurin activity at the onset of the disease in R6/1:BDNF+/- mice. Analysis of the conditional mouse model Tet/HD94 showed that mutant huntingtin specifically controls calcineurin A protein levels. Finally, calcineurin activation induced by intrastriatal quinolinic acid injection in R6/1 mouse was lower than in wild-type mice. Therefore, reduction of calcineurin activity by alteration of calcineurin A expression participates in the pathophysiology of Huntington's disease and contributes to the excitotoxic resistance observed in exon-1 mouse models.

Estradiol facilitates neurite maintenance by a Src/Ras/ERK signalling pathway.

Different reports suggest the estrogens are involved in neuritic outgrowth, maintenance of dendritic morphology and spine formation in the CNS. However, the molecular mechanisms regulated by estrogens on neuronal integrity are not fully understood. We have addressed the relationship between 17beta-estradiol-dependent ERK pathway stimulation and the maintenance of neuritic morphology in cerebellar granule cell cultures (CGC). We report that 17beta-estradiol clearly activates ERK phosphorylation in CGC cultured in low potassium via ERalpha localized in the plasma membrane and without the activation of the insulin-like growth factor-I receptor. 17beta-estradiol activates the ERK pathway through Ras-dependent Src kinase activity. A concomitant activation of the cAMP-response element-binding protein (CREB) is observed. Moreover, we demonstrate that 17beta-estradiol-mediated ERK activation is involved in the maintenance of neuritic arborisation and neuronal morphology in proapoptotic conditions.

Calcineurin is involved in the early activation of NMDA-mediated cell death in mutant huntingtin knock-in striatal cells.

Excitotoxicity has been proposed as one of the mechanisms involved in the specific loss of striatal neurons that occurs in Huntington's disease. Here, we studied the role of calcineurin in the vulnerability of striatal neurons expressing mutant huntingtin to excitotoxicity. To this end, we induced excitotoxicity by adding NMDA to a striatal precursor cell line expressing full-length wild-type (STHdh(Q7/Q7)) or mutant (STHdh(Q111/Q111)) huntingtin. We observed that cell death appeared earlier in STHdh(Q111/Q111) cells than in STHdh(Q7/Q7) cells. Interestingly, these former cells expressed higher levels of calcineurin A that resulted in a greater increase of its activity after NMDA receptor stimulation. Moreover, transfection of full-length mutant huntingtin in different striatal-derived cells (STHdh(Q7/Q7), M213 and primary cultures) increased calcineurin A protein levels. To determine whether high levels of calcineurin A might account for the earlier activation of cell death in mutant huntingtin knock-in cells, wild-type cells were transfected with calcineurin A. Calcineurin A-transfected STHdh(Q7/Q7) cells displayed a significant increase in cell death compared with that recorded in green fluorescent protein-transfected cells after NMDA treatment. Notably, addition of the calcineurin inhibitor FK-506 produced a more robust reduction in cell death in mutant huntingtin knock-in cells than it did in wild-type cells. These results suggest that high levels of calcineurin A could account for the increased vulnerability of striatal cells expressing mutant huntingtin to excitotoxicity.

Striatal-enriched protein tyrosine phosphatase modulates nociception: evidence from genetic deletion and pharmacological inhibition.

The information from nociceptors is processed in the dorsal horn of the spinal cord by complex circuits involving excitatory and inhibitory interneurons. It is well documented that GluN2B and ERK1/2 phosphorylation contributes to central sensitization. Striatal-enriched protein tyrosine phosphatase (STEP) dephosphorylates GluN2B and ERK1/2, promoting internalization of GluN2B and inactivation of ERK1/2. The activity of STEP was modulated by genetic (STEP knockout mice) and pharmacological (recently synthesized STEP inhibitor, TC-2153) approaches. STEP(61) protein levels in the lumbar spinal cord were determined in male and female mice of different ages. Inflammatory pain was induced by complete Freund's adjuvant injection. Behavioral tests, immunoblotting, and electrophysiology were used to analyze the effect of STEP on nociception. Our results show that both genetic deletion and pharmacological inhibition of STEP induced thermal hyperalgesia and mechanical allodynia, which were accompanied by increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Striatal-enriched protein tyrosine phosphatase heterozygous and knockout mice presented a similar phenotype. Furthermore, electrophysiological experiments showed that TC-2153 increased C fiber-evoked spinal field potentials. Interestingly, we found that STEP(61) protein levels in the lumbar spinal cord inversely correlated with thermal hyperalgesia associated with age and female gender in mice. Consistently, STEP knockout mice failed to show age-related thermal hyperalgesia, although gender-related differences were preserved. Moreover, in a model of inflammatory pain, hyperalgesia was associated with increased phosphorylation-mediated STEP(61) inactivation and increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Collectively, the present results underscore an important role of spinal STEP activity in the modulation of nociception.

Novel epigallocatechin-3-gallate (EGCG) derivative as a new therapeutic strategy for reducing neuropathic pain after chronic constriction nerve injury in mice.

Neuropathic pain is common in peripheral nerve injury and often fails to respond to ordinary medication. Here, we investigated whether the two novel epigallocatechin-3-gallate (EGCG) polyphenolic derivatives, compound 23 and 30, reduce the neuropathic pain in mice chronic constriction nerve injury (CCI). First, we performed a dose-response study to evaluate nociceptive sensation after administration of EGCG and its derivatives 23 and 30, using the Hargreaves test at 7 and 21 days after injury (dpi). We daily administered EGCG, 23 and 30 (10 to 100 mg/Kg; i.p.) during the first week post-CCI. None of the doses of compound 23 caused significant pain diminution, whereas 50mg/kg was optimal for both EGCG and 30 to delay the latency of paw withdrawal. With 50 mg/Kg, we showed that EGCC prevented the thermal hyperalgesia from 7 to 21 dpi and compound 30 from 14 to 56 dpi. To evaluate the molecular mechanisms underpinning why EGCG and compound 30 differentially prevented the thermal hyperalgesia, we studied several biochemical parameters in the dorsal horn of the spinal cord at 14 and 56 dpi. We showed that the effect observed with EGCG and compound 30 was related to the inhibition of fatty acid synthase (FASN), a known target of these polyphenolic compounds. Additionally, we observed that EGCG and compound 30 reduced the expression of CCI-mediated inflammatory proteins and the nuclear localization of nuclear factor-kappa B at 14 dpi, but not at 56 dpi. We also strongly detected a decrease of synaptic plasma membrane levels of N-methyl-D-asparte receptor 2B in CCI-mice treated with compound 30 at 56 dpi. Altogether, compound 30 reduced the chronic thermal hyperalgesia induced by CCI better than the natural compound EGCG. Thus, our findings provide a rationale for the preclinical development of compound 30 as an agent to treat neuropathic pain.

Histamine H2-receptor antagonist ranitidine protects against neural death induced by oxygen-glucose deprivation.

BACKGROUND AND PURPOSE: Administration of histamine receptor antagonists has been reported to produce contradictory results, either reducing or increasing neural damage induced by ischemia. In this study, we investigated the neuroprotective effects of histamine H2-receptor antagonists in an "in vitro" model of ischemia. METHODS: Cultured rat brain cortical neurons were exposed to oxygen-glucose deprivation (OGD) in the presence or absence of different histaminergic drugs. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay. Necrosis and apoptosis were quantified by staining cells with propidium iodide and Hoechst 33258. Caspase 3 activation was determined by immunocytochemistry and Western blot. RESULTS: Pretreatment with H2 antagonists effectively reduced neuronal cell death induced by OGD. Ranitidine decreased the number of necrotic and apoptotic cells. Caspase 3 activation and alteration of the neuronal cytoskeleton were also prevented by ranitidine pretreatment. The neuroprotective effect of ranitidine was still evident when added 6 hours after OGD. CONCLUSIONS: H2-receptor antagonists protected against OGD-induced neuronal death. Ranitidine attenuated cell death even when administered after OGD. These data suggest that this drug, which is currently used for the treatment of gastric ulcers, may be useful in promoting recovery after ischemia.