Excitotoxicity effects of glutamate on human neuroblastoma SH-SY5Y cells via oxidative damage.
ABSTRACT: To investigate the mechanisms of excitotoxic effects of glutamate on human neuroblastoma SH-SY5Y cells.SH-SY5Y cell viability was measured by MTT assay. Other damaged profile was detected by lactate dehydrogenase (LDH) release and by 4', 6-diamidino-2-phenylindole (DAPI) staining. The cytosolic calcium concentration was tested by calcium influx assay. The glutamate-induced oxidative stress was analyzed by cytosolic glutathione assay, superoxide dismutase (SOD) assay and extracellular malondialdehyde (MDA) assay.Glutamate treatment caused damage in SH-SY5Y cells, including the decrease of cell viability, the increase of LDH release and the alterations of morphological structures. Furthermore, the concentration of cytoplasmic calcium in SH-SY5Y cells was not changed within 20 min following glutamate treatment, while cytosolic calcium concentration significantly increased within 24 h after glutamate treatment, which could not be inhibited by MK801, an antagonist of NMDA receptors, or by LY341495, an antagonist of metabotropic glutamate receptors. On the other hand, oxidative damage was observed in SH-SY5Y cells treated with glutamate, including decreases in glutathione content and SOD activity, and elevation of MDA level, all of which could be alleviated by an antioxidant Tanshinone IIA (Tan IIA, a major active ingredient from a Chinese plant Salvia Miltiorrhiza Bge).Glutamate exerts toxicity in human neuroblastoma SH-SY5Y cells possibly through oxidative damage, not through calcium homeostasis destruction mediated by NMDA receptors.
Project description:Glutamate excitotoxicity is associated with many neurological diseases, including cerebral ischemia and neurodegenerative diseases. Tanshinone IIA, a diterpenoid naphthoquinone from Salvia miltiorrhiza, has been shown to suppress presynaptic glutamate release, but its protective mechanism against glutamate-induced neurotoxicity is lacking. Using SH-SY5Y human neuroblastoma cells, we show here that excessive glutamate exposure decreases cell viability and proliferation and increases LDH release. Pretreatment with tanshinone IIA, however, prevents the decrease in cell viability and proliferation and the increase in LDH release induced by glutamate. Tanshinone IIA also attenuates glutamate-induced oxidative stress by reducing reactive oxygen species level and malondialdehyde and protein carbonyl contents and by enhancing activities and protein levels of superoxide dismutase and catalase. We then show that tanshinone IIA prevents glutamate-induced mitochondrial dysfunction by increasing mitochondrial membrane potential and ATP content and by reducing mitochondrial protein carbonyl content. Moreover, tanshinone IIA can inhibit glutamate-induced apoptosis through regulation of apoptosis-related protein expression and MAPK activation, including elevation of Bcl-2 protein level, decrease in Bax and cleaved caspase-3 levels, and suppression of JNK and p38 MAPK activation. Collectively, our findings demonstrate that tanshinone IIA protects SH-SY5Y cells against glutamate toxicity by reducing oxidative stress and regulating apoptosis and MAPK pathways.
Project description:AIM: We have investigated the effects of lysophosphatidylcholine (LPC), a product of lipid peroxidation, on Abeta(1-42)-induced SH-SY5Y cell apoptosis. METHODS: The viability of cultured SH-SY5Y cells was measured using a CCK-8 kit. Apoptosis was determined by Chip-based flow cytometric assay. The mRNA transcription of Bcl-2, Bax, and caspase-3 were detected by using reverse transcription and real-time quantitative PCR and the protein levels of Bax and caspase-3 were analyzed by Western blotting. The cytosolic calcium concentration of SH-SY5Y cells was tested by calcium influx assay. G2A expression in SH-SY5Y cells was silenced by small interfering RNA. RESULTS: Long-term exposure of SH-SY5Y cells to LPC augmented the neurotoxicity of Abeta(1-42). Furthermore, after LPC treatment, the Bax/Bcl-x(L) ratio and the expression levels, as well as the activity of caspase-3 were, elevated, whereas the expression level of TRAF1 was reduced. Because LPC was reported to be a specific ligand for the orphan G-protein coupled receptor, G2A, we investigated LPC-mediated changes in calcium levels in SH-SY5Y cells. Our results demonstrated that LPC can enhance the Abeta(1-42)-induced elevation of intracellular calcium. Interestingly, Abeta(1-42) significantly increased the expression of G2A in SH-SY5Y cells, whereas knockdown of G2A using siRNA reduced the effects of LPC on Abeta(1-42)-induced neurotoxicity. CONCLUSION: The effects of LPC on Abeta(1-42)-induced apoptosis may occur through the signal pathways of the orphan G-protein coupled receptor.
Project description:The present study investigates the involvement of the IL-6 family of cytokines, activation of the transcription factor Signal Transducer and Activator of Transcription-3 (STAT3), and the role of Suppressor Of Cytokine Signaling-3 (SOCS3) in regulating excitotoxic neuronal death in vitro. Biochemical evidence demonstrates that in primary cortical neurons and SH-SY5Y neuroblastoma cells, IL-6 cytokine family members, OSM and IL-6 plus the soluble IL-6R (IL-6/R), prevent NMDA and glutamate-induced neuronal toxicity. As well, OSM and IL-6/R induce tyrosine and serine phosphorylation of STAT3 in primary cortical neurons and SH-SY5Y cells. Studies using Pyridine 6 (P6), a pan-JAK inhibitor, demonstrate that the protective effect of OSM and IL-6/R on neuronal death is mediated by the JAK/STAT3 signaling pathway. In parallel to STAT3 phosphorylation, OSM and IL-6/R induce SOCS3 expression at the mRNA and protein level. P6 treatment inhibits SOCS3 expression, indicating that STAT3 is required for OSM and IL-6/R-induced SOCS3 expression. Lentiviral delivery of SOCS3, an inhibitor of STAT3 signaling, into primary neurons and SH-SY5Y cells inhibits OSM and IL-6/R-induced phosphorylation of STAT3, and also reverses the protective effect of OSM and IL-6/R on NMDA and glutamate-induced neurotoxicity in primary cortical neurons. In addition, treatment with IL-6 cytokines increases expression of the anti-apoptotic protein Bcl-xL and induces activation of the Akt signaling pathway, which are also negatively regulated by SOCS3 expression. Thus, IL-6/R and OSM-induced SOCS3 expression may be an important factor limiting the neuroprotective effects of activated STAT3 against NMDA and glutamate-induced neurotoxicity.
Project description:Whole-genome profiling of SH-SY5Y cells was done on neuroblastoma SH-SY5Y stably transfected with cDNAs coding for SOD1WT or the mutant SOD1(G93A) protein. Five wt SOD versus five mutant SOD
Project description:Whole-genome profiling of SH-SY5Y cells was done on neuroblastoma SH-SY5Y stably transfected with cDNAs coding for SOD1WT or the mutant SOD1(G93A) protein. Overall design: Five wt SOD versus five mutant SOD
Project description:The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on excitotoxic neuronal damage was investigated in vitro.The study was performed in human SH-SY5Y neuroblastoma cells exposed to N-methyl-D-aspartate (NMDA). Cell viability was measured by dye exclusion. Reactive oxygen species (ROS) and caspase-3 activity were measured fluorimetrically. Calpain I activity and the activation (phosphorylation) of Akt and glycogen synthase kinase-3beta (GSK-3beta) were assayed by Western blotting.NMDA induced concentration-dependent, receptor-mediated, death of SH-SY5Y cells, ranging from 11 to 25% (0.25-5 mM). Cell death induced by 1 mM NMDA (21%) was preceded by a significant accumulation of intracellular ROS and by a rapid activation of the calcium-activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinase and this preceded the detrimental activation of the downstream kinase, GSK-3beta. BEO (0.0005-0.01%) concentration dependently reduced death of SH-SY5Y cells caused by 1 mM NMDA. In addition to preventing ROS accumulation and activation of calpain, BEO (0.01%) counteracted the deactivation of Akt and the consequent activation of GSK-3beta, induced by NMDA. Results obtained by using specific fractions of BEO, suggested that monoterpene hydrocarbons were responsible for neuroprotection afforded by BEO against NMDA-induced cell death.Our data demonstrate that BEO reduces neuronal damage caused in vitro by excitotoxic stimuli and that this neuroprotection was associated with prevention of injury-induced engagement of critical death pathways.
Project description:Glutamate-mediated neurodegeneration resulting from excessive activation of glutamate receptors is recognized as one of the major causes of various neurological disorders such as Alzheimer's and Huntington's diseases. However, the underlying mechanisms in the neurodegenerative process remain unidentified. Here, we investigate the real-time dynamic structural and mechanical changes associated with the neurodegeneration induced by the activation of N-methyl-D-aspartate (NMDA) receptors (a subtype of glutamate receptors) at the nanoscale. Atomic force microscopy (AFM) is employed to measure the three-dimensional (3-D) topography and mechanical properties of live SH-SY5Y cells under stimulus of NMDA receptors. A significant increase in surface roughness and stiffness of the cell is observed after NMDA treatment, which indicates the time-dependent neuronal cell behavior under NMDA-mediated neurodegeneration. The present AFM based study further advance our understanding of the neurodegenerative process to elucidate the pathways and mechanisms that govern NMDA induced neurodegeneration, so as to facilitate the development of novel therapeutic strategies for neurodegenerative diseases.
Project description:Daidzein isolated from soybean (Glycine max) has been widely studied for its antioxidant and anti-inflammatory activities. However, the protective effects of 7,8,4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, on 6-hydroxydopamine (OHDA)-induced neurotoxicity are not well understood. In the current study, 7,8,4'-THIF significantly inhibited neuronal cell death and lactate dehydrogenase (LDH) release induced by 6-OHDA in SH-SY5Y cells, which were used as an in vitro model of Parkinson' disease (PD). Moreover, pretreatment with 7,8,4'-THIF significantly increased the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) and decreased malondialdehyde (MDA) activity in 6-OHDA-induced SH-SY5Y cells. In addition, 7,8,4'-THIF significantly recovered 6-OHDA-induced cleaved caspase-3, cleaved caspase-9, cleaved poly-ADP-ribose polymerase (PARP), increased Bax, and decreased Bcl-2 levels. Additionally, 7,8,4'-THIF significantly restored the expression levels of phosphorylated c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK 1/2), phosphatidylinositol 3-kinases (PI3K)/Akt, and glycogen synthase kinase-3 beta (GSK-3β) in 6-OHDA-induced SH-SY5Y cells. Further, 7,8,4'-THIF significantly increased the reduced tyrosine hydroxylase (TH) level induced by 6-OHDA in SH-SY5Y cells. Collectively, these results suggest that 7,8,4'-THIF protects against 6-OHDA-induced neuronal cell death in cellular PD models. Also, these effects are mediated partly by inhibiting activation of the MAPK and PI3K/Akt/GSK-3β pathways.
Project description:Dysfunction of D-amino acid oxidase (DAO) and DAO activator (DAOA)/G72 genes have been linked to neuropsychiatric disorders. The glutamate hypothesis of schizophrenia has proposed that increased DAO activity leads to decreased D-serine, which subsequently may lead to N-methyl-D-aspartate (NMDA) receptor hypofunction. It has been shown that DAOA binds to DAO and increases its activity. However, there are also studies showing DAOA decreases DAO activity. Thus, the effect of DAOA on DAO is controversial. We aimed to understand the effect of DAOA on DAO activity in neuron-like (SH-SY5Y), astrocyte-like (1321N1) and kidney-like (HEK293) human cell lines. DAO activity was measured based on the release of hydrogen peroxide and its interaction with Amplex Red reagent. We found that DAOA increases DAO activity only in HEK293 cells, but has no effect on DAO activity in SH-SY5Y and 1321N1 cells. This might be because of different signaling pathways, or due to lower DAO and DAOA expression in SH-SY5Y and 1321N1 cells compared to HEK293 cells, but also due to different compartmentalization of the proteins. The lower DAO and DAOA expression in neuron-like SH-SY5Y and astrocyte-like 1321N1 cells might be due to tightly regulated expression, as previously reported in the human post-mortem brain. Our simulation experiments to demonstrate the interaction between DAOA and human DAO (hDAO) showed that hDAO holoenzyme [hDAO with flavine adenine dinucleotide (FAD)] becomes more flexible and misfolded in the presence of DAOA, whereas DAOA had no effect on hDAO apoprotein (hDAO without FAD), which indicate that DAOA inactivates hDAO holoenzyme. Furthermore, patch-clamp analysis demonstrated no effect of DAOA on NMDA receptor activity in NR1/NR2A HEK293 cells. In summary, the interaction between DAO and DAOA seems to be cell type and its biochemical characteristics dependent which still needs to be elucidated.
Project description:Objective:This study investigated whether Panax notoginseng saponins (PNS) extracted from Panax notoginseng (Bruk.) F. H. Chen played a neuroprotective role by affecting the EGFR/PI3K/AKT pathway in oxygen-glucose deprived (OGD) SH-SY5Y cells. Materials and Methods:Different groups of OGD SH-SY5Y cells were treated with varying doses of PNS, PNS?+?AG1478 (a specific inhibitor of EGFR), or AG1478 for 16 hours. CCK8, Annexin V-FITC/PI apoptosis analysis, and LDH release analysis were used to determine cell viability, apoptosis rate, and amounts of LDH. Quantitative real-time PCR (q-RT-PCR) and western blotting were used to measure mRNA and proteins levels of p-EGFR/EGFR, p-PI3K/PI3K, and p-AKT/AKT in SH-SY5Y cells subjected to OGD. Results:PNS significantly enhanced cell viability, reduced apoptosis, and weakened cytotoxicity by inhibiting the release of LDH. The mRNA expression profiles of EGFR, PI3K, and AKT showed no difference between model and other groups. Additionally, ratios of p-EGFR, p-PI3K, and p-AKT to EGFR, PI3K, and AKT proteins expression, respectively, all increased significantly. Conclusions:These findings indicate that PNS enhanced neuroprotective effects by activating the EGFR/PI3K/AKT pathway and elevating phosphorylation levels in OGD SH-SY5Y cells.