Mouse Hippocampal Cell Line HT22 Research Articles

Protective Effects of Gintonin on Reactive Oxygen Species-Induced HT22 Cell Damages: Involvement of LPA1 Receptor-BDNF-AKT Signaling Pathway.

Gintonin is a kind of ginseng-derived glycolipoprotein that acts as an exogenous LPA receptor ligand. Gintonin has in vitro and in vivo neuroprotective effects; however, little is known about the cellular mechanisms underlying the neuroprotection. In the present study, we aimed to clarify how gintonin attenuates iodoacetic acid (IAA)-induced oxidative stress. The mouse hippocampal cell line HT22 was used. Gintonin treatment significantly attenuated IAA-induced reactive oxygen species (ROS) overproduction, ATP depletion, and cell death. However, treatment with Ki16425, an LPA1/3 receptor antagonist, suppressed the neuroprotective effects of gintonin. Gintonin elicited [Ca2⁺]i transients in HT22 cells. Gintonin-mediated [Ca2⁺]i transients through the LPA1 receptor-PLC-IP3 signaling pathway were coupled to increase both the expression and release of BDNF. The released BDNF activated the TrkB receptor. Induction of TrkB phosphorylation was further linked to Akt activation. Phosphorylated Akt reduced IAA-induced oxidative stress and increased cell survival. Our results indicate that gintonin attenuated IAA-induced oxidative stress in neuronal cells by activating the LPA1 receptor-BDNF-TrkB-Akt signaling pathway. One of the gintonin-mediated neuroprotective effects may be achieved via anti-oxidative stress in nervous systems.

Protective Effect of Salidroside on Mitochondrial Disturbances via Reducing Mitophagy and Preserving Mitochondrial Morphology in OGD-induced Neuronal Injury.

Salidroside is the active ingredient extracted from Rhodiola rosea, and has been reported to show protective effects in cerebral ischemia, but the exact mechanisms of neuronal protective effects are still unrevealed. In this study, the protective effects of salidroside (1 µmol/L) in ameliorating neuronal injuries induced by oxygen-glucose deprivation (OGD), which is a classical model of cerebral ischemia, were clarified. The results showed that after 8 h of OGD, the mouse hippocampal neuronal cell line HT22 cells showed increased cell death, accompanied with mitochondrial fragmentation and augmented mitophagy. However, the cell viability of HT22 cells showed significant restoration after salidroside treatment. Mitochondrial morphology and mitochondrial function were effectively preserved by salidroside treatment. The protective effects of salidroside were further related to the prevention of mitochondrial over-fission. The results showed that mTOR could be recruited to the mitochondria after salidroside treatment, which might be responsible for inhibiting excessive mitophagy caused by OGD. Thus, salidroside was shown to play a protective role in reducing neuronal death under OGD by safeguarding mitochondrial function, which may provide evidence for further translational studies of salidroside in ischemic diseases.

Kuwanon G protects HT22 cells from advanced glycation end product-induced damage.

The incidence of diabetic encephalopathy is increasing as the population ages. Evidence suggests that formation and accumulation of advanced glycation end products (AGEs) plays a pivotal role in disease progression, but limited research has been carried out in this area. A previous study demonstrated that Kuwanon G (KWG) had significant anti-oxidative stress and anti-inflammatory properties. As AGEs are oxidative products and inflammation is involved in their generation it is hypothesized that KWG may have effects against AGE-induced neuronal damage. In the present study, mouse hippocampal neuronal cell line HT22 was used. KWG was shown to significantly inhibit AGE-induced cell apoptosis in comparison with a control treatment, as determined by both MTT and flow cytometry. Compared with the AGEs group, expression of pro-apoptotic protein Bax was reduced and expression of anti-apoptotic protein Bcl-2 was increased in the AGEs + KWG group. Both intracellular and extracellular levels of acetylcholine and choline acetyltransferase were significantly elevated after KWG administration in comparison with controls whilethe level of acetylcholinesterase decreased. These changes in protein expression were accompanied by increased levels of superoxide dismutase and glutathione peroxidase synthesis and reduced production of malondialdehyde and reactive oxygen species. Intracellular signaling pathway protein levels were determined by western blot and immunocytochemistry. KWG administration was found to prevent AGE-induced changes to the phosphorylation levels of Akt, IκB-α, glycogen synthase kinase 3 (GSK3)-α and β, p38 MAPK and NF-κB p65 suggesting a potential neuroprotective effect of KWG against AGE-induced damage was via the PI3K/Akt/GSK3αβ signaling pathway. The findings of the present study suggest that KWG may be a potential treatment for diabetic encephalopathy.

MiR-9-5p inhibits mitochondrial damage and oxidative stress in AD cell models by targeting GSK-3β.

This study aims to investigate the effects and underlying mechanisms of overexpression microRNA-9-5p (miR-9-5p) on the Aβ-induced mouse hippocampal neuron cell line HT22. Different concentrations of Aβ25-35 (10, 20, 40, 80, and 160μM) treatment were used to establish AD model in HT22 cells. The CCK-8 assay was used to measure the cell viability. The mRNA expression levels of miR-9-5p and glycogen synthase kinase-3β (GSK-3β) were determined by RT-qPCR. HT22 cell apoptosis was analyzed flow cytometry. MiR-9-5p was down-regulated in Aβ25-35-induced HT22 cells. GSK-3β is a functional target for miR-9-5p. MiR-9-5p overexpression inhibited Aβ25-35-induced mitochondrial dysfunction, cell apoptosis, and oxidative stress by regulating GSK-3β expression in HT22 cells. Furthermore, through targeting GSK-3β, overexpression of miR-9-5p partly activated nuclear factor Nrf2/Keap1 signaling, including part increases of Nrf2, HO-1, SOD-1, GCLC expression and slight decrease of Keap1 expression. Our results showed miR-9-5p may play a powerful role in the pathogenesis of AD.

Synthesis and biological evaluation of chalcone derivatives as neuroprotective agents against glutamate-induced HT22 mouse hippocampal neuronal cell death

Seventeen chalcone analogues were synthesized from 7-methoxy-3,4-dihydronaphthalen1(2H)-one and various aromatic aldehydes under basic conditions and their therapeutic properties were studied in mouse hippocampal cell line HT-22 against neuronal cell death induced by glutamate. From this study, we selected an analogue C01 as a active compound which showed significantly high neuroprotection. This compound inhibited Ca2+ influx and reactive oxygen species (ROS) accumulation inside cells. The glutamate-induced cell death was analyzed by flow cytometry and it showed that C01 significantly reduced apoptotic or dead cell induced by 5 mM glutamate. Western blot analysis indicates that glutamate-mediated activation of MAPKs were inhibited by compound C01 treatment. In addition, the C01enhanced Bcl-2 and decreased Bax, the anti and pro apoptotic proteins respectively. Further analysis showed that, C01 prevented the nuclear translocation of AIF (apoptosis inducing factor) and inhibited neuronal cell death. Taken together, compound C01 treatment resulted in decreased neurotoxicity induced by 5 mM of glutamate. Our finding confirmed that compound C01 has neuro-therapeutic potential against glutamate-mediated neurotoxicity.

Astrocyte-derived exosomes transfer miR-190b to inhibit oxygen and glucose deprivation-induced autophagy and neuronal apoptosis

ABSTRACT Our previous work has verified that astrocytes (AS)-derived exosomes (AS-Exo) inhibited autophagy and ameliorated neuronal damage in experimental ischemic stroke. However, the mechanism of AS-Exo regulation of autophagy remains unclear. The aim of this study was to investigate the regulatory mechanism of AS-Exo on neuronal autophagy. The mouse hippocampal neuronal cell line HT-22 was cultured in oxygen and glucose deprivation (OGD) condition to mimic ischemic injury. The primary astrocytes were used to isolate exosomes. Exosome labeling and uptake by HT-22 cells were observed by confocal laser microscopy. miR-190b expression was determined by qRT-PCR. HT-22 cell vitality and apoptosis were determined by CCK-8 assay and TUNEL staining, respectively. Levels of TNF-α, IL-6 and IL-1β were analyzed by ELISA. Protein levels of apoptosis-related cleaved caspase-3, Bax, Bcl-2 and autophagy-related Beclin-1, LC3-I/II, Atg7, P62 were determined by western blot. A dual-luciferase reporter assay was performed to confirm the direct interaction between miR-190b and Atg7. miR-190b expression in AS-Exo was found to be significantly higher than that in AS. AS-Exo-mediated transfer of miR-190b attenuated OGD-induced neuronal apoptosis via suppressing autophagy. Moreover, Atg7 was identified as a target of miR-190b. AS-Exo-mediated transfer of miR-190b regulated autophagy by targeting Atg7. Collectively, our data indicated that AS-Exo transferred miR-190b to inhibit OGD-induced autophagy and neuronal apoptosis.

Neuroprotective effects of Brazilian green propolis on oxytosis/ferroptosis in mouse hippocampal HT22 cells

Propolis is a sticky dark-colored substance produced by honey bees and comprises resin, balsam, wax, essential and aromatic oils, pollen, and several other substances; it is used in food and beverages to improve health and prevent diseases. We studied the neuroprotective effects of extracts of Brazilian green propolis in the mouse hippocampal cell line HT22. Ethanol extracts of Brazilian green propolis had a more potent preventive effect on oxidative stress-induced cell death, oxytosis/ferroptosis, in HT22 cells than water extracts of Brazilian green propolis, whereas it did not protect against anticancer drug-induced apoptotic cell death. Among the primary constituents of ethanol extracts of Brazilian green propolis, only artepillin C, kaempferide, and kaempferol demonstrated neuroprotective effects against oxytosis/ferroptosis. The flavonoid derivatives kaempferide and kaempferol are antioxidants with radical-scavenging abilities that additionally induce antioxidant response element-mediated transcriptional activity, suggesting that upregulation of endogenous antioxidant defense protects against oxidative stress. In contrast, artepillin C attenuated reactive oxygen species production; however, it did not induce antioxidant response element activation. These findings indicate that the ethanol extracts of Brazilian green propolis help to prevent oxidative stress-related neuronal cell death that is involved in the pathogenesis of several neurodegenerative diseases.

Effects of 5-Aza on p-Y1472 NR2B related to learning and memory in the mouse hippocampus

BackgroundWe have previously reported that 5-Aza-2-deoxycytidine (5-Aza-cdR) can repress protein serine/threonine phosphatase-1γ (PP1γ) expression and activity in the mouse hippocampus and affect the behaviour of mice in a water maze. It is well known that the phosphorylation of N-methyl-d-aspartate receptor 2B subunit (NR2B) plays a role in behaviour. In this study, we examined whether 5-Aza-cdR affects NR2B phosphorylation at tyrosine 1472 (p-Y1472 NR2B) and whether it affected the responses of the mice in a passive avoidance test. Methods5-Aza-cdR (10 μM) was administered to mice via intracerebroventricular injection (i.c.v). The learning and memory behaviour of the mice were evaluated by measuring their response in a step-down type passive avoidance test 24 h after the injection. The mRNA level of NR2B was measured by real-time PCR. NR2B and p-Y1472 NR2B protein expression in the mouse hippocampus was detected by western blot and immunofluorescence. CDK5 activity was detected by the ADP-Glo™ + CDK5/p35 Kinase Enzyme System. To further clarify whether the 5-Aza-cdR effects on behaviour were dependent on cellular proliferation or not, the effect of 5-Aza-cdR on the expression level of NR2B, the phosphorylation level of p-Y1472 NR2B, cell viability and the cell cycle were analysed using the immortalized mouse hippocampal neuronal cells neural cell line HT22 treated with 10 μM 5-Aza-cdR compared with an untreated control group. ResultsAfter injection with 5-Aza-cdR, the behaviour of the mice in the step-down test was improved, while their phosphorylation level of p-Y1472 NR2B was increased and their CDK5 activity was decreased in the hippocampus. In vitro experiments showed 10 μM 5-Aza-cdR increased the p-Y1472 NR2B phosphorylation level with inhibition of cell viability and cell cycle arrest. ConclusionsOur results suggested that the effect of 5-Aza-cdR on behaviour may be related to the increase in phosphorylation of p-Y1472 NR2B in the hippocampus.

Trehalose Activates CRE-Dependent Transcriptional Signaling in HT22 Mouse Hippocampal Neuronal Cells: A Central Role for PKA Without cAMP Elevation.

Cyclic adenosine 3′,5′monophosphate (cAMP) regulated element binding protein (CREB) is a transcription factor involved in many different signaling processes including memory storage and retrieval. The mouse hippocampal neuronal cell line HT22 is widely used as a model system for neuronal cell death and cellular signal pathway investigations. For the present work a variant of HT22 with a stably expressed CRE-luciferase (CRE-luc) reporter (HT22CRE) is introduced, characterized and used to investigate cAMP-dependent and independent CRE-dependent signal processes. Trehalose (Mykose or 1-α-Glucopyranosyl-1-α-glucopyranosid) is a naturally occurring disaccharide consisting of two α,α′,1,1-glycosidic connected glucose molecules in a wide range of organisms but usually not found in mammals. Trehalose has been shown to activate autophagy, a process which regulates the degradation and recycling of proteins and organelles. The exact processes how trehalose application works on mammalian neuronal cells is not yet understood. The present work shows that trehalose application dose-dependently elevates CRE-luc activity in HT22 cells and acts synergistically with cAMP-elevating agents. In this pathway cAMP-dependent protein kinase (PKA) appears to be the most important factor and the stress kinase p38 and protein tyrosine kinases (PTKs) act as modulators.

Artemisinin Prevents Glutamate-Induced Neuronal Cell Death Via Akt Pathway Activation.

Artemisinin is an anti-malarial drug that has been in use for almost half century. Recently, novel biological effects of artemisinin on cancer, inflammation-related disorders and cardiovascular disease were reported. However, neuroprotective actions of artemisinin against glutamate-induced oxidative stress have not been investigated. In the current study, we determined the effect of artemisinin against oxidative insult in HT-22 mouse hippocampal cell line. We found that pretreatment of artemisinin declined reactive oxygen species (ROS) production, attenuated the collapse of mitochondrial membrane potential induced by glutamate and rescued HT-22 cells from glutamate-induced cell death. Furthermore, our study demonstrated that artemisinin activated Akt/Bcl-2 signaling and that neuroprotective effect of artemisinin was blocked by Akt-specific inhibitor, MK2206. Taken together, our study indicated that artemisinin prevented neuronal HT-22 cell from glutamate-induced oxidative injury by activation of Akt signaling pathway.

The Potential Protective Effect of Curcumin on Amyloid-β-42 Induced Cytotoxicity in HT-22 Cells.

Background We aimed to investigate the effect and mechanism of curcumin (CUR) in Alzheimer's disease (AD). Methods Mouse hippocampal neuronal cell line HT-22 was treated with Aβ1–42 and/or CUR, and then cell viability was evaluated by cell counting kit 8, Beclin-l level was detected using western blotting, and the formation of autophagosomes was observed by transmission electron microscopy (TEM). Furthermore, transcriptome sequencing and analysis were performed in cells with Aβ1–42 alone or Aβ1–42 + CUR. Results Aβ1–42 treatment significantly inhibited cell viability compared with untreated cells (P < 0.01). After treatment for 48 h, CUR remarkably promoted cell viability compared with cell treated with Aβ1–42 alone (P < 0.01). Compared with cells treated with Aβ1–42 alone, the expression of Beclin-1 was slightly reduced in cells with combined treatment of Aβ1–42 with CUR (P < 0.05). Consistently, TEM results showed that CUR inhibited the formation of autophagosomes in cells treated with Aβ1–42. Furthermore, the protein-protein interaction network showed five key genes, including MYC, Cdh1, Acaca, Egr1, and CCnd1, likely involved in CUR effects. Conclusions CUR might have a potential neuroprotective effect by promoting cell viability in AD, which might be associated with cell autophagy. Furthermore, MYC, Cdh1, and Acaca might be involved in the progression of AD.

Comparison of the Pharmacological Profiles of Selective PDE4B and PDE4D Inhibitors in the Central Nervous System

Inhibition of cyclic AMP (cAMP)-specific phosphodiesterase 4 (PDE4) has been proposed as a potential treatment for a series of neuropsychological conditions such as depression, anxiety and memory loss. However, the specific involvement of each of the PDE4 subtypes (PDE4A, 4B and 4C) in different categories of behavior has yet to be elucidated. In the present study, we compared the possible pharmacological effects of PDE4B and PDE4D selective inhibitors, A-33 and D159687, in mediating neurological function in mice. Both compounds were equally potent in stimulating cAMP signaling in the mouse hippocampal cell line HT-22 leading to an increase in CREB phosphorylation. In contrast, A-33 and D159687 displayed distinct neuropharmacological effects in mouse behavioral tests. A-33 has an antidepressant-like profile as indicated by reduced immobility time in the forced swim and tail suspension tasks, as well as reduced latency to feed in the novelty suppressed feeding test. D159687, on the other hand, had a procognitive profile as it improved memory in the novel object recognition test but had no antidepressant or anxiolytic benefit. The present data suggests that inhibitors targeting specific subtypes of PDE4 may exhibit differential pharmacological effects and aid a more efficient pharmacotherapy towards neuropsychological conditions.

Twist1 Is a TNF-Inducible Inhibitor of Clock Mediated Activation of Period Genes.

BackgroundActivation of the immune system affects the circadian clock. Tumor necrosis factor (TNF) and Interleukin (IL)-1β inhibit the expression of clock genes including Period (Per) genes and the PAR-bZip clock-controlled gene D-site albumin promoter-binding protein (Dbp). These effects are due to cytokine-induced interference of E-box mediated transcription of clock genes. In the present study we have assessed the two E-box binding transcriptional regulators Twist1 and Twist2 for their role in cytokine induced inhibition of clock genes.MethodsThe expression of the clock genes Per1, Per2, Per3 and of Dbp was assessed in NIH-3T3 mouse fibroblasts and the mouse hippocampal neuronal cell line HT22. Cells were treated for 4h with TNF and IL-1β. The functional role of Twist1 and Twist2 was assessed by siRNAs against the Twist genes and by overexpression of TWIST proteins. In luciferase (luc) assays NIH-3T3 cells were transfected with reporter gene constructs, which contain a 3xPer1 E-box or a Dbp E-box. Quantitative chromatin immunoprecipitation (ChIP) was performed using antibodies to TWIST1 and CLOCK, and the E-box consensus sequences of Dbp (CATGTG) and Per1 E-box (CACGTG).ResultsWe report here that siRNA against Twist1 protects NIH-3T3 cells and HT22 cells from down-regulation of Period and Dbp by TNF and IL-1β. Overexpression of Twist1, but not of Twist2, mimics the effect of the cytokines. TNF down-regulates the activation of Per1-3xE-box-luc, the effect being prevented by siRNA against Twist1. Overexpression of Twist1, but not of Twist2, inhibits Per1-3xE-box-luc or Dbp-E-Box-luc activity. ChIP experiments show TWIST1 induction by TNF to compete with CLOCK binding to the E-box of Period genes and Dbp.Conclusion Twist1 plays a pivotal role in the TNF mediated suppression of E-box dependent transactivation of Period genes and Dbp. Thereby Twist1 may provide a link between the immune system and the circadian timing system.

Neuroprotective and Antioxidant Effects of Neuroglutam in Cerebral Ischemia.

We studied in vitro and in vivo neuroprotective and antioxidant properties of neuroglutam, a new glutamic acid derivative. In experiments on immortalized mouse hippocampal cell line HT22, neuroglutam exhibited a neuroprotective effect in the model of oxidative stress after its introduction, both before and after H2O2. In vivo study on animals treated with neuroglutam against the background of cerebral ischemia modeled by irreversible occlusion of the common carotid arteries showed that plasma level of TBA-active products was significantly lower and activities of cell antioxidant enzymes (superoxide dismutase and catalase) were higher than in control animals receiving saline under the same conditions.

CNS effects of a novel Phosphodiesterase 4B selective inhibitor A‐33

Phosphodiesterase 4 (PDE4) inhibitors such as Rolipram, have been studied extensively for their roles in alleviating mood and cognitive abnormalities, such as depression, anxiety and learning and memory deficits in rodents. However, one major hurdle in this field is the dose‐limiting side effects, i.e. nausea and vomiting. Targeting specific subtypes of PDE4 might produce similar therapeutic effects while reducing the unwanted side effects. This study aims to characterize the CNS effects of the first PDE4B selective inhibitor A‐33. In vitro studies using the mouse hippocampal cell line HT‐22 indicate that A‐33 dose‐dependently increased cAMP levels and phosphorylation of CREB (cAMP response element binding protein), which peaked at 12 h after treatment. In mice, A‐33 significantly reduced the immobility time of in the forced swim and tail suspension tests, 2 classical rodent tests for depressive behaviors. A‐33 also significantly increased expression of phosphorylated CREB in the mouse hippocampus 2h after i.p. administration of the compound, to a level that is comparable to those induced by rolipram. However, A‐33 did not exhibit any anxiolytic‐like or memory enhancing effects in the study. Moreover, A‐33 was also tested for its potential to induce emesis in a rodent surrogate test, ketamine/xylazine induced anesthesia duration. It did not cause a significant reduction of duration of anesthesia, which is consistent with a previous report stating its non‐emetogenic property in ferrets. In summary, the current study shows that A‐33 exhibits antidepressant‐like effects that is comparable to rolipram without causing any significant adverse effects.

The Neuroprotective Effect of Klotho is Mediated via Regulation of Members of the Redox System

Generation of reactive oxygen species (ROS), leading to oxidative damage and neuronal cell death, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer disease. The present study aimed to examine the mechanism by which the anti-aging protein Klotho exerts neuroprotective effects against neuronal damage associated with neurodegeneration and oxidative stress. Pretreatment of rat primary hippocampal neurons and mouse hippocampal neuronal cell line HT22 with recombinant Klotho protected these cells from glutamate and oligomeric amyloid β (oAβ)-induced cytotoxicity. In addition, primary hippocampal neurons obtained from Klotho-overexpressing mouse embryos were more resistant to both cytotoxic insults, glutamate and oAβ, compared with neurons from wild-type littermates. An antioxidative stress array analysis of neurons treated with Klotho revealed that Klotho significantly enhances the expression of the thioredoxin/peroxiredoxin (Trx/Prx) system with the greatest effect on the induction of Prx-2, an antioxidant enzyme, whose increase was confirmed at the mRNA and protein levels. Klotho-induced phosphorylation of the PI3K/Akt pathway, a pathway important in apoptosis and longevity, was associated with sustained inhibitory phosphorylation of the transcription factor forkhead box O3a (FoxO3a) and was essential for the induction of Prx-2. Down-regulation of Prx-2 expression using a lentivirus harboring shRNA almost completely abolished the ability of Klotho to rescue neurons from glutamate-induced death and significantly, but not completely, inhibited cell death mediated by oAβ, suggesting that Prx-2 is a key modulator of neuroprotection. Thus, our results demonstrate, for the first time, the neuroprotective role of Klotho and reveal a novel mechanism underlying this effect.

Anandamide protects HT22 cells exposed to hydrogen peroxide by inhibiting CB1 receptor-mediated type 2 NADPH oxidase.

Background. Endogenous cannabinoid anandamide (AEA) protects neurons from oxidative injury in rodent models; however the mechanism of AEA-induced neuroprotection remains to be determined. Activation of neuronal NADPH oxidase 2 (Nox2) contributes to oxidative damage of the brain, and inhibition of Nox2 can attenuate cerebral oxidative stress. We aimed to determine whether the neuronal Nox2 was involved in protection mediated by AEA. Methods. The mouse hippocampal neuron cell line HT22 was exposed to hydrogen peroxide (H2O2) to mimic oxidative injury of neurons. The protective effect of AEA was assessed by measuring cell metabolic activity, apoptosis, lactate dehydrogenase (LDH) release, cellular morphology, intracellular reactive oxygen species (ROS), and antioxidant and oxidant levels and Nox2 expression. Results. HT22 cells exposed to H2O2 demonstrated morphological changes, decreased LDH release, reduced metabolic activity, increased levels of intracellular ROS and oxidized glutathione (GSSG), reduced levels of superoxide dismutase (SOD), and reduced glutathione (GSH) and increased expression of Nox2. AEA prevented these effects, a property abolished by simultaneous administration of CB1 antagonist AM251 or CB1-siRNA. Conclusion. Nox2 inhibition is involved in AEA-induced cytoprotection against oxidative stress through CB1 activation in HT22 cells.

B-Raf and CRHR1 Internalization Mediate Biphasic ERK1/2 Activation by CRH in Hippocampal HT22 Cells

CRH is a key regulator of neuroendocrine, autonomic, and behavioral response to stress. CRH-stimulated CRH receptor 1 (CRHR1) activates ERK1/2 depending on intracellular context. In a previous work, we demonstrated that CRH activates ERK1/2 in limbic areas of the mouse brain (hippocampus and basolateral amygdala). ERK1/2 is an essential mediator of hippocampal physiological processes including emotional behavior, synaptic plasticity, learning, and memory. To elucidate the molecular mechanisms by which CRH activates ERK1/2 in hippocampal neurons, we used the mouse hippocampal cell line HT22. We document for the first time that ERK1/2 activation in response to CRH is biphasic, involving a first cAMP- and B-Raf-dependent early phase and a second phase that critically depends on CRHR1 internalization and β-arrestin2. By means of mass-spectrometry-based screening, we identified B-Raf-associated proteins that coimmunoprecipitate with endogenous B-Raf after CRHR1 activation. Using molecular and pharmacological tools, the functional impact of selected B-Raf partners in CRH-dependent ERK1/2 activation was dissected. These results indicate that 14-3-3 proteins, protein kinase A, and Rap1, are essential for early CRH-induced ERK1/2 activation, whereas dynamin and vimentin are required for the CRHR1 internalization-dependent phase. Both phases of ERK1/2 activation depend on calcium influx and are affected by calcium/calmodulin-dependent protein kinase II inactivation. Thus, this report describes the dynamics and biphasic nature of ERK1/2 activation downstream neuronal CRHR1 and identifies several new critical components of the CRHR1 signaling machinery that selectively controls the early and late phases of ERK1/2 activation, thus providing new potential therapeutic targets for stress-related disorders.

The plasma membrane channel ORAI1 mediates detrimental calcium influx caused by endogenous oxidative stress

The mouse hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Addition of extracellular glutamate depletes the cells of glutathione (GSH) by blocking the glutamate−cystine antiporter system xc−. GSH is the main antioxidant in neurons and its depletion induces a well-defined program of cell death called oxytosis, which is probably synonymous with the iron-dependent form of non-apoptotic cell death termed ferroptosis. Oxytosis is characterized by an increase of reactive oxygen species and a strong calcium influx preceding cell death. We found a significant reduction in store-operated calcium entry (SOCE) in glutamate-resistant HT22 cells caused by downregulation of the Ca2+ channel ORAI1, but not the Ca2+ sensors STIM1 or STIM2. Pharmacological inhibition of SOCE mimicked this protection similarly to knockdown of ORAI1 by small interfering RNAs. Long-term calcium live-cell imaging after induction of the cell death program showed a specific reduction in Ca2+-positive cells by ORAI1 knockdown. These results suggest that dysregulated Ca2+ entry through ORAI1 mediates the detrimental Ca2+ entry in programmed cell death induced by GSH depletion. As this detrimental Ca2+ influx occurs late in the course of the cell death program, it might be amenable to therapeutic intervention in diseases caused by oxidative stress.

Molecular basis for glucocorticoid induction of the Kruppel-like factor 9 gene in hippocampal neurons.

Stress has complex effects on hippocampal structure and function, which consequently affects learning and memory. These effects are mediated in part by circulating glucocorticoids (GC) acting via the intracellular GC receptor (GR) and mineralocorticoid receptor (MR). Here, we investigated GC regulation of Krüppel-like factor 9 (KLF9), a transcription factor implicated in neuronal development and plasticity. Injection of corticosterone (CORT) in postnatal d 6 and 30 mice increased Klf9 mRNA and heteronuclear RNA by 1 h in the hippocampal region. Treatment of the mouse hippocampal cell line HT-22 with CORT caused a time- and dose-dependent increase in Klf9 mRNA. The CORT induction of Klf9 was resistant to protein synthesis inhibition, suggesting that Klf9 is a direct CORT-response gene. In support of this hypothesis, we identified two GR/MR response elements (GRE/MRE) located -6.1 and -5.3 kb relative to the transcription start site, and we verified their functionality by enhancer-reporter, gel shift, and chromatin immunoprecipitation assays. The -5.3-kb GRE/MRE is largely conserved across tetrapods, but conserved orthologs of the -6.1-kb GRE/MRE were only detected in therian mammals. GC treatment caused recruitment of the GR, histone hyperacetylation, and nucleosome removal at Klf9 upstream regions. Our findings support a predominant role for GR, with a minor contribution of MR, in the direct regulation of Klf9 acting via two GRE/MRE located in the 5'-flanking region of the gene. KLF9 may play a key role in GC actions on hippocampal development and plasticity.