Antitumor effect of the antimicrobial peptide GLI13-8 derived from domain of the avian β-defensin-4

We previously reported that GLII3-8, one of cationic anti- microbial peptides from linear avian β-defensin-4 （RL38） analogs, exhibited high antimicrobial activities against both Gram-negative and Gram-positive bacteria. In the present study, we reported the in vitro cytotoxicity of GLI13-8 using the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphe- nyltetrazolium bromide assay. Results showed that the cytotoxicity of GLI13-8 in three human carcinoma cells （HepG2, SGC7901, and A375） was in a dose-dependent manner. When the concentration of GLI13-8 is 〈128/xM, it had no toxicity towards the normal human fibroblasts （MRC-5）. The Annexin-V-FITC/PI staining assay, the Hoechst 33258/PI staining assay, the permeability of fluor- escein macromolecules and scanning electron microscope assays, mitochondrial membrane potential assay, caspases- 3 and poly ADP-ribose polymerase （PARP） assays have been carried out. Results indicated that apoptosis was induced by GLI13-8 in HepG2 cells, and demonstrated that GLI13-8 induced loss of mitochondriai membrane poten- tial, disruption of HepG2 cell membranes, and activation of caspase-3 and PARP. These findings suggested that GLI13-8 may be an effective agent for HepG2 cells.     

Flavonoids from tartary buckwheat induce G2/M cell cycle arrest and apoptosis in human hepatoma HepG2 cells

The cytotoxicity and antioxidant activity on human hepa toma cell line HepG2 of three flavonoids homogenous com pounds from tartary buckwheat seeds and bran, namely quercetin, isoquercetin, and rutin, were investigated. The total antioxidant competency detection results indicated that the antioxidant capacity of quercetin was the strongest in a biological response system. A [3-（4,5-dimethylthiazol-2-yl）-2,5diphenyltetrazolium bromide] assay showed that quercetin exhibited the strongest cytotoxic effects against the HepG2 cell line. Flow cytometric analysis indicated that quercetin significantly increased the production of reactive oxygen species, and led to the G2/M phase arrest accom panied by an increase of apoptotic cell death after 48 h of incubation. Quercetininduced cell apoptosis was shown to involve p53 and p21 upregulation, Cyclin D1, Cdk2, and Cdk7 downregulation. These results suggested that the in duction of G2/M arrest, apoptosis, and cell death by quer cetin may associate with increased expression of p53 and p21, decrease of Cyclin D1, Cdk2, and Cdk7 levels, and generation of reactive oxygen species in cells. This study will help to better understand and fully utilize medicinal resources of plant flavonoids.     

Binding activity of H-Ras is necessary for in vivo inhibition of ASK1 activity

H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASKl-induced apoptosis in vivo, which was reversed only partially by addition of RafS621 A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASKI activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.     

Nerve growth factor prevents the apoptosis-associated increase in acetylcholinesterase activity after hydrogen peroxide treatment by activating Akt

Acetylcholinesterase (ACHE) is thought to play an important role during apoptosis.Our resultsshowed that H_2O_2 induced AChE activity,a functional marker in apoptosis,increases in neuronal-like PC 12cells.Glutathione, which is involved in cellular redox homeostasis,inhibited the increase of AChE activity,suggesting that reactive oxygen species (ROS) play a key role in this process.Further investigation showedthat the elevation of AChE was observed after the degradation of Akt, release of cytochrome c from mitochondriainto the cytosol,and activation of caspase family members.When nerve growth factor (NGF) was present,with the maintenance of Akt level,the elevation of AChE,the cytochrome c diffusion,as well as apoptosiswere markedly attenuated in H202-treated PC 12 cells. However,wortmannin,an inhibitor of the PI3K/Aktpathway,accelerated the apoptosis and increased the AChE activity.The overexpression of constitutivelyactivated Akt,which is a downstream signalling element of the NGF receptor TrkA,delayed mitochondrialcollapse and inhibited elevation of AChE activity.Thus, NGF prevented apoptosis and elevation of AChEactivity by activating the Akt pathway and stabilizing the function of mitochondria.     

Hydrogen peroxide induces the activation of the phospholipase C-γ1 survival pathway in PC12 cells： protective role in apoptosis

It has been reported that phospholipase C-γ1 （PLC-γ1） plays an important protective role in hydrogen peroxide （H2O2）-induced pheochromocytoma （PC） 12 cells death. However, most studies have used high doses of H2O2 and the downstream targets of PLC-γ1 activation remain to be identified. The present study was designed to examine the roles of PLC-γ1 signaling pathway in the apoptosis of PC12 cells induced by low dose of H2O2, as well as the downstream factors involved in this pathway. Low-dose treatment of H2O2 resulted in PLC-γ1 tyrosine phosphorylation in a time-dependent manner and H2O2 killed the PC12 cells by inducing necrosis. In contrast, pretreatment of PCI2 cells with U73122, a specific inhibitor of PLC, markedly increased the percentage of dead cells. The mode of cell death was converted to apoptosis as determined by Hoechst/PI nuclear staining and fluorescence microscopy. Western blot analysis demonstrated that the expression of Bcl-2 protein and the activation of pro-caspase-3 were not significantly affected by low dose of H2O2 alone. However, after pretreatment with U73122, Bcl-2 protein expression was dramatically decreased and the activation of pro-caspase-3 was significantly increased. We concluded that PLC-γ1 plays an important protective role in H2O2-induced PC12 cells death. Bcl-2 and caspase-3 probably participate in the signaling pathway as downstream factors.     

Protective Effects of Vitamin E against Oxidative Damage Induced by Aβ1-40Cu（Ⅱ） Complexes

β-amyloid peptide (Aβ) is considered to be responsible for the formation of senile plaques,which is the hallmark of Alzheimer's disease (AD).Oxidative stress,manifested by protein oxidation andlipid peroxidation,among other alterations,is a characteristic of AD brain.A growing body of evidence hasbeen presented in support of Aβ_(1-40) forming an oligomeric complex that binds copper at a CuZn superoxidedismutase-like binding site. Aβ_(1-40)Cu(Ⅱ) complexes generate neurotoxic hydrogen peroxide (H_2O_2) from O_2via Cue reduction,though the precise reaction mechanism is unclear.The toxicity of Aβ_(1-40) or the Aβ_(1-40)Cu(Ⅱ)complexes to cultured primary cortical neurons was partially attenuated when ( )-α-tocopherol (vitamin E)as free radical antioxidant was added at a concentration of 100 μM.The data derived from lactate dehydro-genase (LDH) release and the formation of H_2O_2 confirmed the results from the MTT assay.These findingsindicate that copper binding to Aβ_(1-40) can give rise to greater production of H_2O_2, which leads to a break-down in the integrity of the plasma membrane and subsequent neuronal death.Groups treated with vitaminE exhibited much slighter damage,suggesting that vitamin E plays a key role in protecting neuronal cellsfrom dysfunction or death.     

Mechanisms of arsenic trioxide induced apoptosis of human cervical cancer HeLa cells and protection by Bcl-2

It was recently reported that arsenic trioxide (As_2O_3) can induce complete remission in patients with acute promyelocytic leukemia (APL). In this present article, the biological effect of As_2O_3 on human cervical cancer HeLa cells and HeLa cells overexpressing Bcl-2 is studied. By MTT and colony forming ability assays, morphology alteration, flow cytometric analysis, DNA gel electrephoresis and in situ cell death detection (TUNEL), it was found that As_2O_3 inhibited the growth of HeLa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR, Northern blot, Western blot analysis revealed that As_2O_3 induced HeLa cell apoptosis possibly via decreasing the expression of c-myc and viral genes. HeLa cells overexpressing Bcl-2 partly resist As_2O_3 induced apoptosis, which might be relative to preventing the cells from As_2O_3 caused G2/M block, downregulation of c-myc gene expression and inhibition of viral gene expression was also noted, However, it was found that As_2O_3 at a high concentratio     

A Lectin from Chinese Mistletoe Increases γδ T Cell-mediated Cytotoxicity through Induction of Caspase-dependent Apoptosis

In this study, a mistletoe lectin (ML) was purified from Chinese mistletoe and the effect of this 60 kDa Chinese ML on human γδ T cell cytotoxicity, apoptosis and modulation of the cytokine network was studied. The cytotoxic properties of δ T cells was evaluated by using a ~(51)Cr release test and employed fluorescence-activated cell sorting and enzyme-linked immunosorbent assay analysis to quantify translocation of the cell membrane phospholipid, phosphatidylserine and nuclear DNA fragmentation during apoptosis. It was found that: (ⅰ) ML effectively stimulated γδ T cell proliferation in a dose- and time-dependent manner; (ⅱ) ML increased γδ T cell cytotoxicity; (ⅲ) ML could modulate lipopolysaccharide-induced cytokine release in a pro-inflammatory manner by increasing tumor necrosis factor (TNF)-α release and inhibiting the release of anti-inflammatory interleukin (IL)-10; (ⅳ) ML induced apoptosis in caspase-dependent and CD95-independent manner. The results indicated that ML is a potent immunomodulator to human γδ T cell cytotoxicity, apoptosis and cytokine production.     

The herbal compound geniposide rescues formaldehyde-induced apoptosis in N2a neuroblastoma cells

The herbal medicine Tong Luo Jiu Nao(TLJN)contains geniposide(GP)and ginsenoside Rg1 at a molar ratio of 10:1.Rg1 is the major component of another herbal medicine,panax notoginseng saponin(PNS).TLJN has been shown to strengthen brain function in humans,and in animals it improves learning and memory.We have previously shown that TLJN reduces amyloidogenic processing in Alzheimer’s disease(AD)mouse models.Together this suggests TLJN may be a potential treatment for patients with dementia.Because chronic damage of the central nervous system by formaldehyde(FA)has been presented as a risk factor for age-associated cognitive dysfunction,in the present study we investigated the protective effect of both TLJN and GP in neuron-like cells exposed to FA.FA-exposed murine N2a neuroblastoma cells were incubated with TLJN,its main ingredient GP,as well as PNS,to measure cell viability and morphology,the rate of apoptosis and expression of genes encoding Akt,FOXO3,Bcl2 and p53.The CCK-8 assay,cytoskeletal staining and flow cytometry were used to test cell viability,morphology and apoptosis,respectively.Fluorescent quantitative real-time PCR(qRT-PCR)was used to monitor changes in gene expression,and HPLC to determine the rate of FA clearance.Treatment of N2a cells with 0.09 mmol L?1 FA for 24 h significantly reduced cell viability,changed cell morphology and promoted apoptosis.Both TLJN and GP conferred neuroprotection to FA-treated N2a cells,whereas PNS,which had to be used at lower concentrations because of its toxicity,did not.Our data demonstrate that TLJN can rescue neuronal damage caused by FA and that its main ingredient,GP,has a major role in this efficacy.This presents purified GP as a drug or lead compound for the treatment of AD.     

Selective protection by phosphatidic acid against staurosporine-induced neuronal apoptosis

Phosphatidic acid, the main product of lipid breakdown through phospholipase D activation, has been implicated in important signal transduction pathways able to influence cell fate in many ways. The purpose of this work was to determine possible effects of phosphatidic acid on neuronal cell death pathways. Here we used cerebellar granular cell cultures and cell death was triggered with either staurosporine or H₂O₂. Cell viability was quantified by spectrophotometry, using the 3- (4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl-tetrazolium bromide (MTT) test. Staurosporine (1-3 μM) or H₂O₂ (50-800 μM) induced cell death in a dose-dependent manner. Using fluorescent staining (propidium iodide or annexin V-Cy3/6-carboxyfluorescein) we showed that cell death was mostly apoptotic in staurosporine treated cells and mostly non-apoptotic (necrotic) in H2O2 treated cells. Phosphatidic acid was able to increase cell viability in staurosporine-, but not in H₂O₂ - treated cells. We therefore conclude that phosphatidic acid has neuroprotective potential in neurons exposed to stimuli that trigger apoptosis.     

Cytoprotective effect of polypeptide from Chlamys farreri on neuroblastoma (SH-SY5Y) cells following H2O2 exposure involves scavenging ROS and inhibition JNK phosphorylation

Oxidative stress has long been linked to cell death in many neurodegenerative conditions. Treatment with antioxidants is a promising approach for slowing disease progression. In this study, we used the neuroblastoma SH-SY5Y cells as an in vitro model to first assess the effect of polypeptide from Chlamys farreri (PCF), a natural marine antioxidant, on H₂O₂-induced neuronal cell death. Pre-treatment of SH-SY5Y cells with PCF inhibited H₂O₂-induced cell death in a concentration-dependent manner. In parallel, intracellular reactive oxygen species generation and lipid peroxidation were inhibited by PCF. Under severe H₂O₂ insult, PCF promoted endogenous antioxidant defense components including glutathione peroxidase, catalase, superoxide dismutase, and glutathione. PCF also protected DNA from oxidative damage and enhanced the removal of 8-oxo-7,8-dihydro-2'-deoxyguanosine from DNA. Further, we found that PCF potentially prevented H₂O₂–induced cell apoptosis. When investigated mitogen-activated protein kinase signaling pathway, we found that pre-treatment of cells with PCF significantly blocked H₂O₂–induced phosphorylation of c- Jun N-terminal kinase of the mitogen-activated protein kinase family. However, PCF had little inhibitory effect on the H₂O₂–induced activation of extracellular signal-regulated kinase. Taken together, these data demonstrate that PCF prevents oxidative stress-induced reactive oxygen species production and c- Jun N-terminal kinase activation and may be useful in the treatment of neurodegenerative diseases.     

Amyloid β-Mediated Oxidative and Metabolic Stress in Rat Cortical Neurons: No Direct Evidence for a Role for H2O2 Generation

Abstract: H₂O₂ and free radical-mediated oxidative stresses have been implicated in mediating amyloid β(1–40) [Aβ(1–40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H₂O₂-scavenging enzyme catalase protects neurons in culture against Aβ-mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H₂O₂. Aβ-mediated elevation in intracellular H₂O₂ production is suppressed by addition of a potent H₂O₂ scavenger without any significant neuroprotection. Three intracellular biochemical markers of H₂O₂-mediated oxidative stress were unchanged by Aβ treatment: (a) glyceraldehyde-3-phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. Ionspray mass spectra of Aβ in the incubation medium indicated that Aβ itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24-h exposure of neurons to Aβ. Our results challenge a pivotal role for H₂O₂ generation in mediating Aβ toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.     

BCL-2-Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells

Abstract: Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H₂O₂ treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H₂O₂-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H₂O₂ but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H₂O₂ exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.     

Inhibition of Dopamine Release by Prostaglandin EP3 Receptor via Pertussis Toxin-Sensitive and -Insensitive Pathways in PC12 Cells

Abstract: Hydrogen peroxide (H₂O₂) is produced from several sources in brain and may be involved in neurodegeneration and second messenger signaling. Little is known about the effects of H₂O₂ on transmitter storage in brain synaptic vesicles. Neurotransmitter uptake into synaptic vesicles is driven by an electrochemical proton gradient generated by the vacuolar H⁺-ATPase (V-ATPase) in the vesicle membrane. We report here that the V-ATPase in bovine brain synaptic vesicles is highly sensitive to inhibition by micromolar concentrations of H₂O₂. Glutamate uptake by the vesicles is also inhibited, very likely as a secondary consequence of ATPase inactivation. Dithiothreitol or reduced glutathione reverse H₂O₂-induced inhibition of the V-ATPase, and ATP or GTP partially protect the ATPase from inhibition by H₂O₂. These and other results suggest that the mechanism of inhibition of the V-ATPase by H₂O₂ involves oxidation of a reactive cysteine sulfhydryl group in the ATP binding site. Inhibition of V-ATPase activity would decrease the amount of transmitter stored in synaptic vesicles and thus down-regulate transmitter release during episodes of oxidative stress or in response to second messenger signaling.     

Stress-induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide

Reactive oxygen species (ROS) have traditionally been viewed as a toxic group of molecules; however, recent publications have shown that these molecules, including H₂O₂, can also strongly promote cell survival. Even though the retina has a large capacity to produce ROS, little is known about its non-mitochondrial sources of these molecules, in particular the expression and function of NADPH oxidase (Nox) proteins which are involved in the direct generation of superoxide and indirectly H₂O₂. This study demonstrated that 661W cells, a retina-derived cell line, and mouse retinal explants express Nox2, Nox4 and certain of their well-established regulators. The roles of Nox2 and Nox4 in producing pro-survival H₂O₂ were determined using 661W cells and some of the controlling factors were identified. To ascertain if this phenomenon could have physiological relevance, the novel technique of time-lapse imaging of dichlorofluorescein fluorescence (generated upon H₂O₂ production) in retinal explants was established and it showed that explants also produce a burst of H₂O₂. The increase in H₂O₂ production was partly blocked by an inhibitor of Nox proteins. Overall, this study demonstrates a pro-survival role of Nox2 and Nox4 in retina-derived cells, elucidates some of the regulatory mechanisms and reveals that a similar phenomenon exists in retinal tissue as a whole.     

Induction of proteins during phage reactivation induced by UV irradiation, oxygen and peroxide in Bacteroides fragilis

Abstract Phage reactivation systems in Bacteroides fragilis were induced by far-UV irradiation, O₂ and H₂O₂. These three treatments also induced the synthesis of 3, 6, and 4 protein bands, respectively, which were easily detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Two proteins with apparent M _r s of approx. 90 000 and 70 000 were induced by all three treatments. Caffeine completely inhibited UV- and O₂-induced phage reactivation and prevented the synthesis of the M _r 90 000 and M _r 70 000 proteins. The results suggest that these two proteins may be involved in phage reactivation processes induced by UV, O₂ and H₂O₂ in B. fragilis .     

Insulin/PI3K signaling protects dentate neurons from oxygen–glucose deprivation in organotypic slice cultures

It is known that ischemia/reperfusion induces neurodegeneration in the hippocampus in a subregion‐dependent manner. This study investigated the mechanism of selective resistance/vulnerability to oxygen–glucose deprivation (OGD) using mouse organotypic hippocampal cultures. Analysis of propidium iodide uptake showed that OGD‐induced duration‐ and subregion‐dependent neuronal injury. When compared with the CA1–3 subregions, dentate neuronal survival was more sensitive to inhibition of phosphatidylinositol 3‐kinase (PI3K)/Akt signaling under basal conditions. Dentate neuronal sensitivity to PI3K/Akt signaling activation was inversely related to its vulnerability to OGD‐induced injury; insulin/insulin‐like growth factor 1 pre‐treatment conferred neuroprotection to dentate neurons via activation of PI3K/Akt signaling. In contrast, CA1 and CA3 neurons were less sensitive to disruptions of endogenous PI3K/Akt signaling and protective effects of insulin/insulin‐like growth factor 1, but more vulnerable to OGD. OGD‐induced injury in CA1 was reduced by inhibition of NMDA receptor or mitogen‐activated protein kinase signaling, and was prevented by blocking NMDA receptor in the presence of insulin. The CA2 subregion was distinctive in its response to glutamate, OGD, and insulin, compared with other CA subregions. CA2 neurons were sensitive to the protective effects of insulin against OGD‐induced injury, but more resistant to glutamate. Distinctive distribution of insulin receptor β and basal phospho‐Akt was detected in our slice cultures. Our results suggest a role for insulin signaling in subregional resistance/vulnerability to cerebral ischemia.     

The ketogenic diet increases mitochondrial glutathione levels

The ketogenic diet (KD) is a high-fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague–Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of β-hydroxybutyrate (BHB). KD-fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet-fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate-limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD-fed rats, as well as up-regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD-fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H₂O₂ production and H₂O₂-induced mtDNA damage. Isolated hippocampal mitochondria from KD-fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H₂O₂ production and mtDNA damage. Together, the results demonstrate that the KD up-regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant-induced damage.     

Activation of the histaminergic H3 receptor induces phosphorylation of the Akt/GSK-3β pathway in cultured cortical neurons and protects against neurotoxic insults

Stimulation of histamine H₃ receptors (H₃R) activates G_i/o-proteins that inhibit adenylyl cyclase and triggers MAPK and phospholipase A₂. In a previous study, we showed that H₃R-mediated phosphorylation of Akt at Ser473 occurs in primary cultures of rat cortical neurons, but neither the downstream targets nor the function of such activation were explored. In this report we address these questions. Western blotting experiments showed that H₃R-mediated activation of Akt in cultured rat cortical neurons was inhibited by LY 294004 and U0126, suggesting that it depends on phosphoinositide-3-kinase and mitogen-activated protein kinase kinase. H₃R activation phosphorylated, hence inactivated, the Akt downstream effector glycogen synthase kinase-3β, increased the expression of the antiapoptotic protein Bcl-2 and protected cultured rat and mouse cortical neurons from neurotoxic insults in a dose-dependent manner. All these effects were inhibited by the H₃R antagonist inverse/agonist thioperamide. Mouse cortical cells expressed H₃R as revealed by immunostaining experiments, and stimulation of H₃R phoshorylated Akt and decreased caspase 3 activity. Hence, we uncovered a yet unexplored action of the H₃R that may help understand the impact of H₃R signaling in the CNS.