• Journal of Microbiology and Biotechnology
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• v.28 no.1
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• pp.40-49
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• 2018

We evaluated the antioxidant activity and neuronal cell-protective effect of fucoidan extract from Ecklonia cava (FEC) on hydrogen peroxide ($H_2O_2$)-induced cytotoxicity in PC-12 and MC-IXC cells to assess its protective effect against oxidative stress. Antioxidant activities were examined using the ABTS radical scavenging activity and malondialdehyde-inhibitory effect, and the results showed that FEC had significant antioxidant activity. Intracellular ROS contents and neuronal cell viability were investigated using the DCF-DA assay and MTT reduction assay. FEC also showed remarkable neuronal cell-protective effect compared with vitamin C as a positive control for both $H_2O_2$-treated PC-12 and MC-IXC cells. Based on the neuronal cell-protective effects, mitochondrial function was analyzed in PC-12 cells, and FEC significantly restored mitochondrial damage by increasing the mitochondrial membrane potential (${\Delta}{\Psi}m$) and ATP levels and regulating mitochondrial-mediated proteins (p-AMPK and BAX). Finally, the inhibitory effects against acetylcholinesterase (AChE), which is a critical hydrolyzing enzyme of the neurotransmitter acetylcholine in the cholinergic system, were investigated ($IC_{50}$ value = 1.3 mg/ml) and showed a mixed (competitive and noncompetitive) pattern of inhibition. Our findings suggest that FEC may be used as a potential material for alleviating oxidative stress-induced neuronal damage by regulating mitochondrial function and AChE inhibition.

• Journal of Nutrition and Health
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• v.48 no.5
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• pp.451-456
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• 2015

Purpose: Neuronal apoptotic events induced by aging and hypoxic/ischemic conditions is an important risk factor in neurodegenerative diseases such as ischemia stroke and Alzheimer's disease. The peel of Citrus sunki Hort. ex Tanaka has long been used as a traditional medicine, based on multiple biological activities including anti-oxidant, anti-inflammation, and anti-obesity. In the current study, we examined the actions of fermented C. sunki peel extract against cobalt chloride ($CoCl_2$)-mediated hypoxic death in human neuroblastoma SH-SY5Y cells. Methods: Cell viability was measured by trypan blue exclusion. Expression of apoptosis related proteins and release of cytochrome c were detected by western blot. Production of intracellular reactive oxygen species (ROS) and apoptotic morphology were examined using 2',7'-dichlorofluorescin diacetate (DCF-DA) and 4',6-diamidino-2-phenylindole (DAPI) staining. Results: Exposure to $CoCl_2$, a well-known mimetic agent of hypoxic/ischemic condition, resulted in neuronal cell death via caspase-3 dependent pathway. Extract of fermented C. sunki peel significantly rescued the $CoCl_2$-induced neuronal toxicity with the cell viability and appearance of apoptotic morphology. Cytoprotection with fermented C. sunki peel extract was associated with a decrease in activities of caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). In addition, increase in the intracellular ROS and release of cytochrome c from mitochondria to the cytosol were inhibited by treatment with extract of fermented C. sunki peel. Conclusion: Based on these data, fermented C. sunki peel extract might have a protective effect against $CoCl_2$-induced neuronal injury partly through generation of ROS and effectors involved in mitochondrial mediated apoptosis.

• BMB Reports
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• v.43 no.5
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• pp.369-374
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• 2010

The PC12 is the widely used cell line to study neuronal differentiation. We had extensively investigated the details of protein expression in differentiated PC12 cells by proteomic analysis. The cells were incubated at the presence of nerve growth factor. We had analyzed the expression changes in the differentiating PC12 cells by 2-dimensional electrophoresis and the identification of the proteins using MALDI-TOF MS. By comparing expression pattern in the time course, we identified the candidate genes which are associated with neuronal differentiation. Among these genes, we performed real-time PCR analysis to validate $Idh3{\alpha}$ expression by the time course. To identify the function of $Idh3{\alpha}$ in neuronal differentiation stage, the transfection of $Idh3{\alpha}$ to PC12 cells was performed. As a result, we proved that up-regulation of $Idh3{\alpha}$ causes reduction in neural differentiation of PC12 cells. Based on these data, we suggest that $Idh3{\alpha}$ plays a role to the neuronal differentiation.

• Molecules and Cells
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• v.43 no.10
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• pp.848-855
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• 2020

Cells assemble stress granules (SGs) to protect their RNAs from exposure to harmful chemical reactions induced by environmental stress. These SGs release RNAs, which resume translation once the stress is relieved. During stem cell differentiation, gene expression is altered to allow cells to adopt various functional and morphological features necessary to differentiate. This process induces stress within a cell, and cells that cannot overcome this stress die. Here, we investigated the role of SGs in the progression of stem cell differentiation. SGs aggregated during the neuronal differentiation of human bone marrow-mesenchymal stem cells, and not in cell lines that could not undergo differentiation. SGs were observed between one and three hours post-induction; RNA translation was restrained at the same time. Immediately after disassembly of SGs, the expression of the neuronal marker neurofilament-M (NF-M) gradually increased. Assembled SGs that persisted in cells were exposed to salubrinal, which inhibited the dephosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), and in eIF2α/S51D mutant cells. When eIF2α/S51A mutant cells differentiated, SGs were not assembled. In all experiments, the disruption of SGs was accompanied by delayed NF-M expression and the number of neuronally differentiated cells was decreased. Decreased differentiation was accompanied by decreased cell viability, indicating the necessity of SGs for preventing cell death during neuronal differentiation. Collectively, these results demonstrate the essential role of SGs during the neuronal differentiation of stem cells.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.6
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• pp.689-696
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• 2018

Increasing evidence implicates changes in $[Ca^{2+}]_i$ and oxidative stress as causative factors in amyloid beta ($A{\beta}$)-induced neuronal cell death. Cyanidin-3-glucoside (C3G), a component of anthocyanin, has been reported to protect against glutamate-induced neuronal cell death by inhibiting $Ca^{2+}$ and $Zn^{2+}$ signaling. The present study aimed to determine whether C3G exerts a protective effect against $A{\beta}_{25-35}$-induced neuronal cell death in cultured rat hippocampal neurons from embryonic day 17 fetal Sprague-Dawley rats using MTT assay for cell survival, and caspase-3 assay and digital imaging methods for $Ca^{2+}$, $Zn^{2+}$, MMP and ROS. Treatment with $A{\beta}_{25-35}$ ($20{\mu}M$) for 48 h induced neuronal cell death in cultured rat pure hippocampal neurons. Treatment with C3G for 48 h significantly increased cell survival. Pretreatment with C3G for 30 min significantly inhibited $A{\beta}_{25-35}$-induced $[Zn^{2+}]_i$ increases as well as $[Ca^{2+}]_i$ increases in the cultured rat hippocampal neurons. C3G also significantly inhibited $A{\beta}_{25-35}$-induced mitochondrial depolarization. C3G also blocked the $A{\beta}_{25-35}$-induced formation of ROS. In addition, C3G significantly inhibited the $A{\beta}_{25-35}$-induced activation of caspase-3. These results suggest that cyanidin-3-glucoside protects against amyloid ${\beta}$-induced neuronal cell death by reducing multiple apoptotic signals.

• Nutrition Research and Practice
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• v.14 no.3
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• pp.188-202
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• 2020

BACKGROUND/OBJECTIVES: Brain aging is a major risk factor for severe neurodegenerative diseases. Conversely, L-histidine and L-carnosine are known to exhibit neuroprotective effects. The aim of this study was to examine the potential for L-histidine, L-carnosine, and their combination to mediate anti-brain aging effects in neuronal cells subjected to D-galactose-induced aging. MATERIALS/METHODS: The neuroprotective potential of L-histidine, L-carnosine, and their combination was examined in a retinoic acid-induced neuronal differentiated SH-SY5Y cell line exposed to D-galactose (200 mM) for 48 h. Neuronal cell proliferation, differentiation, and expression of anti-oxidant enzymes and apoptosis markers were subsequently evaluated. RESULTS: Treatment with L-histidine (1 mM), L-carnosine (10 mM), or both for 48 h efficiently improved the proliferation, neurogenesis, and senescence of D-galactose-treated SH-SY5Y cells. In addition, protein expression levels of both neuronal markers (β tubulin-III and neurofilament heavy protein) and anti-oxidant enzymes, glutathione peroxidase-1 and superoxide dismutase-1 were up-regulated. Conversely, protein expression levels of amyloid β (1-42) and cleaved caspase-3 were down-regulated. Levels of mRNA for the pro-inflammatory cytokines, interleukin (IL)-8, IL-1β, and tumor necrosis factor-α were also down-regulated. CONCLUSIONS: To the best of our knowledge, we provide the first evidence that L-histidine, L-carnosine, and their combination mediate anti-aging effects in a neuronal cell line subjected to D-galactose-induced aging. These results suggest the potential benefits of L-histidine and L-carnosine as anti-brain aging agents and they support further research of these amino acid molecules.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.3
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• pp.177-183
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• 2000

We examined the neurotoxic effects of 3 glutathione (GSH) depletors, buthionine sulfoximine (BSO), diethyl maleate (DEM) and phorone, under the presence of trolox, cycloheximide (CHX), pyrrolidine dithiocarbamate (PDTC) or MK-801 in primary mouse cortical cell cultures. All three depletors induced neuronal death in dose and exposure time dependent manner, and decreased total cellular GSH contents. The patterns of the neuronal death and the GSH decrements were dependent on the individual agents. DEM $(200\;{\mu}M)$ induced rapid and irreversible decrement of the GSH. BSO (1 mM) also decreased the GSH irreversibly but the rate of decrement was more progressive than that of DEM. Phorone (1 mM) reduced the GSH content to 40% by 4 hr exposure, that is comparable to the decrement of BSO, but the GSH recovered and reached over the control value by 36 hr exposure. BSO showed a minimal neurotoxicity $(0{\sim}10%)$ at the end of 24 hr exposure, but marked neuronal cell death at the end of 48 hr exposure. The BSO (1 mM)-induced neurotoxicity was markedly inhibited by trolox or CHX and partially attenuated by MK-801. DEM induced dose-dependent cytotoxicity at the end of 24 hr exposure. Over the doses of $400\;{\mu}M,$ glial toxicity also appeared. DEM $(200\;{\mu}M)-induced$ neurotoxicity was markedly inhibited by trolox or PDTC. Phorone (1 mM) induced moderate neurotoxicity (40%) at the end of 48 hr exposure. Only CHX showed significant inhibitory effect on the phorone-induced neurotoxicity. These results suggest that the GSH depletors induce neuronal injury via different mechanisms and that GSH depletors should be carefully employed in the researches of neuronal oxidative injuries.

• Natural Product Sciences
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• v.22 no.3
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• pp.187-192
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• 2016

The goal of this study was to determine whether gypenosides (GPS) exert protective effects against dopaminergic neuronal cell death in a 6-hydroxydopamine (OHDA)-lesioned rat model of Parkinson's disease (PD) with or without long-term 3,4-dihydroxyphenylalanine (L-DOPA) treatment. Rats were injected with 6-OHDA in the substantia nigra to induce PD-like symptoms; 14 days after injection, groups of 6-OHDA-lesioned animals were treated for 21 days with GPS (25 or 50 mg/kg) and/or L-DOPA (20 mg/kg). Dopaminergic neuronal cell death was assessed by counting tyrosine hydroxylase (TH)-immunopositive cells in the substantia nigra and measuring levels of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the striatum. Dopaminergic neuronal cell death induced by 6-OHDA lesions was ameliorated by GPS treatment (50 mg/kg). L-DOPA treatment exacerbated 6-OHDA-induced dopaminergic neuronal cell death; however, these effects were partially reversed by GPS treatment (25 and 50 mg/kg). These results suggest that GPS treatment is protective against dopaminergic neuronal cell death in a 6-OHDA-lesioned rat model of PD with long-term L-DOPA treatment. Therefore, GPS may be useful as a phytotherapeutic agent for the treatment of PD.

• Journal of Medicine and Life Science
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• v.15 no.2
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• pp.67-71
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• 2018

Neuronal excitotoxicity induces mitochondrial dysfunction and the release of proapoptotic proteins. Excitotoxicity, the process by which the overactivation of excitatory neurotransmitter receptors leads to neuronal cell death. Neuronal death by excitotoxicity was related to neuronal degenerative disorders and hypoxia, results from excessive exposure to excitatory neurotransmitters, such as glutamate. Glutamate acts at NMDA receptors in cultured neurons to increase the intracellular free calcium concentration. Therefore endogenous glutamate may be a key factor to regulate neuronal cell death via activating $Ca^{2+}$ signaling. For this issue, we tested some conditions to alter intracellular $Ca^{2+}$ level in dissociated hippocampal neurons of rats. Cultured hippocampal neuron were treated by KCl (20 mM), $CaCl_2$ (3.8 mM) and glutamate ($5{\mu}M$) for 24 hrs. Interestingly, The Optical Density of hippocampal neurons was increased by high KCl application in MTT assay data. This enhanced response by high KCl was dependent on synaptic $Ca^{2+}$ influx but not on intracellular $Ca^{2+}$ level. However, the number of neurons seemed to be not changed in Hoechst 33342 staining data. These results suggest that enhancement of synaptic activity plays a key role to increase mitochondrial signaling in hippocampal neurons.

• BMB Reports
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• v.54 no.6
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• pp.317-322
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• 2021

CCCTC-binding factor (CTCF), a zinc finger protein, is a transcription factor and regulator of chromatin structure. Forebrain excitatory neuron-specific CTCF deficiency contributes to inflammation via enhanced transcription of inflammation-related genes in the cortex and hippocampus. However, little is known about the long-term effect of CTCF deficiency on postnatal neurons, astrocytes, or microglia in the hippocampus of adult mice. To address this, we knocked out the Ctcf gene in forebrain glutamatergic neurons (Ctcf cKO) by crossing Ctcf-floxed mice with Camk2a-Cre mice and examined the hippocampi of 7.5-10-month-old male mice using immunofluorescence microscopy. We found obvious neuronal cell death and reactive gliosis in the hippocampal cornu ammonis (CA)1 in 7.5-10-month-old cKO mice. Prominent rod-shaped microglia that participate in immune surveillance were observed in the stratum pyramidale and radiatum layer, indicating a potential increase in inflammatory mediators released by hippocampal neurons. Although neuronal loss was not observed in CA3, and dentate gyrus (DG) CTCF depletion induced a significant increase in the number of microglia in the stratum oriens of CA3 and reactive microgliosis and astrogliosis in the molecular layer and hilus of the DG in 7.5-10-month-old cKO mice. These results suggest that long-term Ctcf deletion from forebrain excitatory neurons may contribute to reactive gliosis induced by neuronal damage and consequent neuronal loss in the hippocampal CA1, DG, and CA3 in sequence over 7 months of age.