Calpain-1 C2L domain peptide protects mouse hippocampus-derived neuronal HT22 cells against glutamate-induced oxytosis.
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ABSTRACT:
SUBMITTER: Sugawara M
PROVIDER: S-EPMC8374356 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Sulfation is a common modification of extracellular glycans, tyrosine residues on proteins, and steroid hormones, and is important in a wide variety of signaling pathways. We investigated the role of sulfation on endogenous oxidative stress, such as glutamate-induced oxytosis and erastin-induced ferroptosis, using mouse hippocampal HT22 cells. Sodium chlorate competitively inhibits the formation of 3'-phosphoadenosine 5'-phosphosulfate, the high energy sulfate donor in cellular sulfation reactions. The treatment of HT22 cells with sodium chlorate decreased sulfation of heparan sulfate proteoglycans and chondroitin sulfate proteoglycans. Sodium chlorate and β-d-xyloside, which prevents proteoglycan glycosaminoglycan chain attachment, exacerbated both glutamate- and erastin-induced cell death, suggesting that extracellular matrix influenced oxytosis and ferroptosis. Moreover, sodium chlorate enhanced the generation of reactive oxygen species and influx of extracellular Ca2+ in the process of oxytosis and ferroptosis. Interestingly, sodium chlorate did not affect antioxidant glutathione levels. Western blot analysis revealed that sodium chlorate enhanced erastin-induced c-Jun N-terminal kinase phosphorylation, which is preferentially activated by cell stress-inducing signals. Collectively, our findings indicate that sulfation is an important modification for neuroprotection against oxytosis and ferroptosis in neuronal hippocampal cells.
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Project description:Neuron-glia interactions are essential for the central nervous system's homeostasis. Microglial cells are one of the key support cells in the brain that respond to disruptions in such homeostasis. Although their participation in neuroinflammation is well known, studies investigating their role in ferroptosis, an iron-dependent form of nonapoptotic cell death, are lacking. To address this issue, we explored whether microglial (BV-2 cells) activation products can intensify, mitigate or block oxidative and/or ferroptotic damage in neuronal cells (HT22 cell line). Cultured BV-2 microglial cells were stimulated with 5-100 ng/mL lipopolysaccharide (LPS) for 24 h and, after confirmation of microglial activation, their culture medium (conditioned media; CM) was transferred to neuronal cells, which was subsequently (6 h later) exposed to glutamate or tert-butyl hydroperoxide (t-BuOOH). As a major finding, HT22 cells pretreated for 6 h with CM exhibited a significant ferroptosis-resistant phenotype characterized by decreased sensitivity to glutamate (15 mM)-induced cytotoxicity. However, no significant protective effects of LPS-activated microglial cell-derived CM were observed in t-BuOOH (30 µM)-challenged cells. In summary, activated microglia-derived molecules may protect neuronal cells against ferroptosis. The phenomenon observed in this work highlights the beneficial relationship between microglia and neurons, highlighting new possibilities for the control of ferroptosis.
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Project description:Protein kinase A (PKA) is a ser/thr kinase that is critical for maintaining essential neuronal functions including mitochondrial homeostasis, bioenergetics, neuronal development, and neurotransmission. The endogenous pool of PKA is targeted to the mitochondrion by forming a complex with the mitochondrial scaffold A-kinase anchoring protein 121 (AKAP121). Enhanced PKA signaling via AKAP121 leads to PKA-mediated phosphorylation of the fission modulator Drp1, leading to enhanced mitochondrial networks and thereby blocking apoptosis against different toxic insults. In this study, we show for the first time that AKAP121/PKA confers neuroprotection in an in vitro model of oxidative stress induced by exposure to excess glutamate. Unexpectedly, treating mouse hippocampal progenitor neuronal HT22 cells with an acute dose or chronic exposure of glutamate robustly elevates PKA signaling, a beneficial compensatory response that is phenocopied in HT22 cells conditioned to thrive in the presence of excess glutamate but not in parental HT22 cells. Secondly, redirecting the endogenous pool of PKA by transiently transfecting AKAP121 or transfecting a constitutively active mutant of PKA targeted to the mitochondrion (OMM-PKA) or of an isoform of AKAP121 that lacks the KH and Tudor domains (S-AKAP84) are sufficient to significantly block cell death induced by glutamate toxicity but not in an oxygen deprivation/reperfusion model. Conversely, transient transfection of HT22 neuronal cells with a PKA-binding-deficient mutant of AKAP121 is unable to protect against oxidative stress induced by glutamate toxicity suggesting that the catalytic activity of PKA is required for AKAP121's protective effects. Mechanistically, AKAP121 promotes neuroprotection by enhancing PKA-mediated phosphorylation of Drp1 to increase mitochondrial fusion, elevates ATP levels, and elicits an increase in the levels of antioxidants GSH and superoxide dismutase 2 leading to a reduction in the level of mitochondrial superoxide. Overall, our data supports AKAP121/PKA as a new molecular target that confers neuroprotection against glutamate toxicity by phosphorylating Drp1, to stabilize mitochondrial networks and mitochondrial function and to elicit antioxidant responses.
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Project description:BackgroundThe objective of our study was to analyze the neuroprotective effects of ginsenoside derivatives Rb1, Rb2, Rc, Rd, Rg1, and Rg3 against glutamate-mediated neurotoxicity in HT22 hippocampal mouse neuron cells.MethodsThe neuroprotective effect of ginsenosides were evaluated by measuring cell viability. Protein expressions of mitogen-activated protein kinase (MAPK), Bcl2, Bax, and apoptosis-inducing factor (AIF) were determined by Western blot analysis. The occurrence of apoptotic and death cells was determined by flow cytometry. Cellular level of Ca2+ and reactive oxygen species (ROS) levels were evaluated by image analysis using the fluorescent probes Fluor-3 and 2',7'-dichlorodihydrofluorescein diacetate, respectively. In vivo efficacy of neuroprotection was evaluated using the Mongolian gerbil of ischemic brain injury model.ResultReduction of cell viability by glutamate (5 mM) was significantly suppressed by treatment with ginsenoside Rb2. Phosphorylation of MAPKs, Bax, and nuclear AIF was gradually increased by treatment with 5 mM of glutamate and decreased by co-treatment with Rb2. The occurrence of apoptotic cells was decreased by treatment with Rb2 (25.7 μM). Cellular Ca2+ and ROS levels were decreased in the presence of Rb2, and in vivo data indicated that Rb2 treatment (10 mg/kg) significantly diminished the number of degenerated neurons.ConclusionOur results suggest that Rb2 possesses neuroprotective properties that suppress glutamate-induced neurotoxicity. The molecular mechanism of Rb2 is by suppressing the MAPKs activity and AIF translocation.
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Project description:Epilepsy is the most common childhood neurologic disorder. Status epilepticus (SE), which refers to continuous epileptic seizures, occurs more frequently in children than in adults, and approximately 40-50% of all cases occur in children under 2 years of age. Conventional antiepileptic drugs currently used in clinical practice have a number of adverse side effects. Drug-resistant epilepsy (DRE) can progressively develop in children with persistent SE, necessitating the development of novel therapeutic drugs. During SE, the persistent activation of neurons leads to decreased glutamate clearance with corresponding glutamate accumulation in the synaptic extracellular space, increasing the chance of neuronal excitotoxicity. Our previous study demonstrated that after developmental seizures in rats, E-64d exerts a neuroprotective effect on the seizure-induced brain damage by modulating lipid metabolism enzymes, especially ApoE and ApoJ/clusterin. In this study, we investigated the impact and mechanisms of E-64d administration on neuronal excitotoxicity. To test our hypothesis that E-64d confers neuroprotective effects by regulating autophagy and mitochondrial pathway activity, we simulated neuronal excitotoxicity in vitro using an immortalized hippocampal neuron cell line (HT22). We found that E-64d improved cell viability while reducing oxidative stress and neuronal apoptosis. In addition, E-64d treatment regulated mitochondrial pathway activity and inhibited chaperone-mediated autophagy in HT22 cells. Our findings indicate that E-64d may alleviate glutamate-induced damage via regulation of mitochondrial fission and apoptosis, as well as inhibition of chaperone-mediated autophagy. Thus, E-64d may be a promising therapeutic treatment for hippocampal injury associated with SE.
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Project description:Previous studies suggest that reducing the numbers of adult-born neurons in the dentate gyrus (DG) of the mouse increases susceptibility to severe continuous seizures (status epilepticus; SE) evoked by systemic injection of the convulsant kainic acid (KA). However, it was not clear if the results would be the same for other ways to induce seizures, or if SE-induced damage would be affected. Therefore, we used pilocarpine, which induces seizures by a different mechanism than KA. Also, we quantified hippocampal damage after SE. In addition, we used both loss-of-function and gain-of-function methods in adult mice. We hypothesized that after loss-of-function, mice would be more susceptible to pilocarpine-induced SE and SE-associated hippocampal damage, and after gain-of-function, mice would be more protected from SE and hippocampal damage after SE. For loss-of-function, adult neurogenesis was suppressed by pharmacogenetic deletion of dividing radial glial precursors. For gain-of-function, adult neurogenesis was increased by conditional deletion of pro-apoptotic gene Bax in Nestin-expressing progenitors. Fluoro-Jade C (FJ-C) was used to quantify neuronal injury and video-electroencephalography (video-EEG) was used to quantify SE. Pilocarpine-induced SE was longer in mice with reduced adult neurogenesis, SE had more power and neuronal damage was greater. Conversely, mice with increased adult-born neurons had shorter SE, SE had less power, and there was less neuronal damage. The results suggest that adult-born neurons exert protective effects against SE and SE-induced neuronal injury.
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Project description:Vitis vinifea has been used for traditional medicines, food, beverages, and dietary antioxidant supplements. The chemical compositions and biological activities of the fruits and seeds have been extensively investigated. However, the biological effects of the leaves are limited, and its anti-neurodegeneration or antiaging activities are little known. The current work aims to study the beneficial effects of V. vinifera leaf extract on neuroprotective effects in HT22 cells, antiaging, and oxidative stress resistance properties in the Caenorhabditis elegans model. The ethanol extract was characterized by phytochemical composition using gas/liquid chromatography-mass spectrometry and reversed-phase high-performance liquid chromatography. The beneficial effects of V. vinifera ethanol (VVE) extract on antioxidant properties, neuroprotective effects, and the underlying mechanisms were studied by in vitro and in vivo studies. In HT22 cells, we found that VVE has a protective effect against glutamate-mediated oxidative stress-induced cell death. The gene expression of cellular antioxidant enzymes such as CAT, SODs, GSTs, and GPx was upregulated by VVE treatment. Moreover, VVE was also shown to alleviate oxidative stress and attenuate reactive oxygen species accumulation in C. elegans. We demonstrated that VVE could upregulate the expression of stress-response genes gst-4 and sod-3 and downregulate the expression of hsp-16.2. Our results suggest that the oxidative stress resistance properties of VVE are possibly involved in DAF-16/FoxO transcription factors. VVE reduced age-related markers (lipofuscin) while did not extend the life span of C. elegans under normal conditions. This study reports the neuroprotective effect and antioxidant activity of V. vinifera leaf extract and suggests its potential as a dietary or alternative supplement to defend against oxidative stress and age-related diseases.
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Project description:In traditional herbal medicine, the Polyscias fruticosa has been frequently used for the treatment of ischemia and inflammation. Oxidative stress mediated by elevated glutamate levels cause neuronal cell death in ischemia and various neurodegenerative diseases. However, so far, the neuroprotective effects of this plant extract against glutamate-mediated cell death have not been investigated in cell models. The current study investigates the neuroprotective effects of ethanol extracts of Polyscias fruticosa (EEPF) and elucidates the underlying molecular mechanisms of EEPFs relevant to neuroprotection against glutamate-mediated cell death. The oxidative stress-mediated cell death was induced by 5 mM glutamate treatment in HT22 cells. The cell viability was measured by a tetrazolium-based EZ-Cytox reagent and Calcein-AM fluorescent dye. Intracellular Ca2+ and ROS levels were measured by fluorescent dyes, fluo-3 AM and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), respectively. Protein expressions of p-AKT, BDNF, p-CREB, Bax, Bcl-2, and apoptosis-inducing factor (AIF) were determined by western blot analysis. The apoptotic cell death was measured by flow cytometry. The in vivo efficacy of EEPF was evaluated using the Mongolian gerbil mouse by surgery-induced brain ischemia. EEPF treatment showed a neuroprotective effect against glutamate-induced cell death. The EEPF co-treatment reduced the intracellular Ca2+ and ROS and apoptotic cell death. Furthermore, it recovered the p-AKT, p-CREB, BDNF, and Bcl-2 levels decreased by glutamate. The EEPF co-treatment suppressed the activation of apoptotic Bax, the nuclear translocation of AIF, and mitogen-activated protein kinase (MAPK) pathway proteins (ERK1/2, p38, JNK). Further, EEPF treatment significantly rescued the degenerative neurons in the ischemia-induced Mongolian gerbil in vivo model. EEPF exhibited neuroprotective properties that suppress glutamate-mediated neurotoxicity. The underlying mechanism of EEPF is increasing the level of p-AKT, p-CREB, BDNF, and Bcl-2 associated with cell survival. It has therapeutic potential for the treatment of glutamate-mediated neuropathology.
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