Influence of Inflammatory Pain and Dopamine on Synaptic Transmission in the Mouse ACC.

Dopamine (DA) inhibits excitatory synaptic transmission in the anterior cingulate cortex (ACC), a brain region involved in the sensory and affective processing of pain. However, the DA modulation of inhibitory synaptic transmission in the ACC and its alteration of the excitatory/inhibitory (E/I) balance remains relatively understudied. Using patch-clamp recordings, we demonstrate that neither DA applied directly to the tissue slice nor complete Freund's adjuvant (CFA) injected into the hind paw significantly impacted excitatory currents (eEPSCs) in the ACC, when recorded without pharmacological isolation. However, individual neurons exhibited varied responses to DA, with some showing inhibition, potentiation, or no response. The degree of eEPSC inhibition by DA was higher in naïve slices compared to that in the CFA condition. The baseline inhibitory currents (eIPSCs) were greater in the CFA-treated slices, and DA specifically inhibited eIPSCs in the CFA-treated, but not naïve group. DA and CFA treatment did not alter the balance between excitatory and inhibitory currents. Spontaneous synaptic activity revealed that DA reduced the frequency of the excitatory currents in CFA-treated mice and decreased the amplitude of the inhibitory currents, specifically in CFA-treated mice. However, the overall synaptic drive remained similar between the naïve and CFA-treated mice. Additionally, GABAergic currents were pharmacologically isolated and found to be robustly inhibited by DA through postsynaptic D2 receptors and G-protein activity. Overall, the study suggests that CFA-induced inflammation and DA do not significantly affect the balance between excitatory and inhibitory currents in ACC neurons, but activity-dependent changes may be observed in the DA modulation of presynaptic glutamate release in the presence of inflammation.

Etomidate-Induced Myoclonus in Sprague-Dawley Rats Involves Neocortical Glutamate Accumulation and N -Methyl- d -Aspartate Receptor Activity.

BACKGROUND: Etomidate-induced myoclonus, a seizure-like movement, is of interest to anesthetists. However, its origin in the brain and its underlying mechanism remain unclear. METHODS: Adult male Sprague-Dawley rats were anesthetized with etomidate, propofol, or lidocaine plus etomidate. We assessed the incidence of myoclonus, behavioral scores, and levels of glutamate and γ-aminobutyric acid (GABA) in the neocortex and hippocampus. To determine the origin and how N -methyl- d -aspartate receptors (NMDARs) modulate etomidate-induced neuroexcitability, the local field potential and muscular tension were monitored. Calcium imaging in vitro and immunoblotting in vivo were conducted to investigate the mechanisms underlying myoclonus. RESULTS: The incidence of etomidate (1.5 mg/kg in vivo)-induced myoclonus was higher than that of propofol (90% vs 10%, P = .0010) and lidocaine plus etomidate (90% vs 20%, P = .0050). Etomidate at doses of 3.75 and 6 mg/kg decreased the mean behavioral score at 1 (mean difference [MD]: 1.80, 95% confidence interval [CI], 0.58-3.02; P = .0058 for both), 2 (MD: 1.60, 95% CI, 0.43-2.77; P = .0084 and MD: 1.70, 95% CI, 0.54-2.86; P = .0060), 3 (MD: 1.60, 95% CI, 0.35-2.85; P = .0127 and MD: 1.70, 95% CI, 0.46-2.94; P = .0091) minutes after administration compared to etomidate at a dose of 1.5 mg/kg. In addition, 0.5 and 1 µM etomidate in vitro increased neocortical intracellular calcium signaling; this signaling decreased when the concentration increased to 5 and 10 µM. Etomidate increased the glutamate level compared to propofol (mean rank difference: 18.20; P = .003), and lidocaine plus etomidate (mean rank difference: 21.70; P = .0002). Etomidate in vivo activated neocortical ripple waves and was positively correlated with muscular tension amplitude (Spearman's r = 0.785, P < .0001). Etomidate at 1.5 mg/kg decreased the K-Cl cotransporter isoform 2 (KCC2) level compared with propofol (MD: -1.15, 95% CI, -1.47 to -0.83; P < .0001) and lidocaine plus etomidate (MD: -0.64, 95% CI, -0.96 to -0.32; P = .0002), DL-2-amino-5-phosphopentanoic acid (AP5) suppressed these effects, while NMDA enhanced them. CONCLUSIONS: Etomidate-induced myoclonus or neuroexcitability is concentration dependent. Etomidate-induced myoclonus originates in the neocortex. The underlying mechanism involves neocortical glutamate accumulation and NMDAR modulation and myoclonus correlates with NMDAR-induced downregulation of KCC2 protein expression.

Cytotoxic and anti-excitotoxic effects of selected plant and algal extracts using COMET and cell viability assays.

Excess glutamate in the central nervous system may be a major cause of neurodegenerative diseases with gradual loss and dysfunction of neurons. Primary or secondary metabolites from medicinal plants and algae show potential for treatment of glutamate-induced excitotoxicity. Three plant extracts were evaluated for impact on glutamate excitotoxicity-induced in primary cultures of retinal ganglion cells (RGC). These cells were treated separately in seven groups: control; Plicosepalus. curviflorus treated; Saussurea lappa treated; Cladophora glomerate treated. Cells were treated independently with 5, 10, 50, or 100 µg/ml of extracts of plant or alga material, respectively, for 2 h. Glutamate-treated cells (48 h with 5, 10, 50, or 100 µM glutamate); and P. curviflorus/glutamate; S. lappa/glutamate; C. glomerata/glutamate [pretreatment with extract for 2 h (50 and 100 µg/ml) before glutamate treatment with 100 µM for 48 h]. Comet and MTT assays were used to assess cell damage and cell viability. The number of viable cells fell significantly after glutamate exposure. Exposure to plant extracts caused no notable effect of viability. All tested plants extracts showed a protective effect against glutamate excitotoxicity-induced RGC death. Use of these extracts for neurological conditions related to excitotoxicity and oxidative stress might prove beneficial.

Photobiomodulation Therapy Ameliorates Glutamatergic Dysfunction in Mice with Chronic Unpredictable Mild Stress-Induced Depression.

Accumulating evidence indicates that dysfunction of the glutamatergic neurotransmission has been widely involved in the pathophysiology and treatment of depression. Photobiomodulation therapy (PBMT) has been demonstrated to regulate neuronal function both in vitro and in vivo. Herein, we aim to investigate whether the antidepressant phenotype of PBMT is associated with the improvement of glutamatergic dysfunction and to explore the mechanisms involved. Results showed that PBMT decreased extracellular glutamate levels via upregulation of glutamate transporter-1 (GLT-1) and rescued astrocyte loss in the cerebral cortex and hippocampus, which also alleviated dendritic atrophy and upregulated the expression of AMPA receptors on the postsynaptic membrane, ultimately exhibiting behaviorally significant antidepressant effects in mice exposed to chronic unpredictable mild stress (CUMS). Notably, PBMT also obtained similar antidepressant effects in a depressive mouse model subcutaneously injected with corticosterone (CORT). Evidence from in vitro mechanistic experiments demonstrated that PBMT treatment significantly increased both the GLT-1 mRNA and protein levels via the Akt/NF-κB signaling pathway. NF-κB-regulated transcription was in an Akt-dependent manner, while inhibition of Akt attenuated the DNA-binding efficiency of NF-κB to the GLT-1 promoter. Importantly, in vitro, we further found that PKA activation was responsible for phosphorylation and surface levels of AMPA receptors induced by PBMT, which is likely to rescue excitatory synaptic transmission. Taken together, our research suggests that PBMT as a feasible therapeutic approach has great potential value to control the progression of depression.

TRIM27-mediated ubiquitination of PPARγ promotes glutamate-induced cell apoptosis and inflammation.

Neurotoxicity induced by glutamate (Glu) is often used to study the signaling mechanism of neurological disorders. The identification of specific genetic factors that cause Glu-induced neurotoxicity provides evidence for the common pathways of neuronal apoptosis and inflammation. TRIM27 has been found to induce apoptosis and inflammation. Nevertheless, there is little evidence that TRIM27 is associated with Glu-induced neurotoxicity. We found that TRIM27 expression was increased in epilepsy patients and in HT22 cells following Glu treatment. Glu-mediated cell apoptosis, decreased PPARγ expression, and increased levels of cleaved Caspase-3 and IL-1ß expression in HT22 cells were significantly inhibited by TRIM27 knockdown. TRIM27 overexpression significantly induced cell apoptosis and expression of cleaved Caspase-3 and IL-1ß, but inhibited PPARγ expression in HT22 cells, which were reversed by ROZ, suggesting the involvement of PPARγ in TRIM27-mediated cell apoptosis and inflammation in HT22 cells. Mechanically, TRIM27 ubiquitinates and degrades PPARγ, following induces cleaved Caspase-3 and IL-1ß expression. Clinically, increased expression of TRIM27 in epilepsy patients was associated with decreased PPARγ expression. Taken together, our study suggests that TRIM27-mediated ubiquitination of PPARγ promotes Glu-induced HT22 cell apoptosis and IL-1ß release.

Neuroprotective effects of oolong tea extracts against glutamate-induced toxicity in cultured neuronal cells and ß-amyloid-induced toxicity in Caenorhabditis elegans.

Oolong tea, a traditional Chinese tea, is especially popular in south China and has a variety of health benefits. However, studies about its neuroprotective and neuroregenerative properties are still limited. This study explored the neuroprotective and neurite outgrowth-promoting properties of oolong tea in cultured neuronal cells (Neuro-2a and HT22) and Caenorhabditis elegans models. Ultra performance liquid chromatography was applied to identify the main natural bioactive compounds in oolong tea. Using Neuro-2a and HT22 cells, we found that oolong tea extracts had a protective effect against glutamate-induced cell death. The extracts reduced intracellular reactive oxygen species accumulation and induced gene expression of cellular antioxidant enzymes such as GPx, GSTs and SODs. These extracts also increased the average neurite length, and GAP-43 and Ten-4 mRNA expression in Neuro-2a cells. Moreover, they had protective effects against Aß-induced paralysis, chemotaxis deficiency and α-synuclein aggregation in C. elegans. This is the first study showing the neuroregenerative and neuroprotective potential of the oolong tea extracts against glutamate/Aß/α-synuclein-induced toxicity in vitro and in vivo. Our study may support oolong tea extracts as potential candidates for the prevention of neurodegenerative diseases.

Distribution of Carotenoids and Protective Effects of Zeaxanthin on Retina of Ayu Sweetfish (Plecoglossus altivelis).

Ayu sweetfish (Plecoglossus altivelis) is a diurnal freshwater fish that are surface swimmers and active under broad and short wavelength-dominated light. Biochemical analyses have shown that the ayu fish have abundant carotenoids including zeaxanthin in their integuments. Although zeaxanthin plays an important role in the physiological function of the retina, the amount and location of zeaxanthin in the ayu eye have not been accurately determined. In this study, circular dichroism spectral data and chiral high-performance liquid chromatography analysis showed that zeaxanthin was the primary carotenoid in the ayu eye, and the eye had the highest carotenoid content compared to those in the integuments, subcutaneous fat, and digestive tract. Interestingly, zeaxanthin in the ayu eyeball was expressed in the photoreceptor layer and near the retinal pigmented epithelium. In vitro assays showed that zeaxanthin could protect photoreceptors and retinal pigmented epithelial cell lines against the oxidative stress induced by exposure to L-buthionine-(S,R)-sulfoximine/glutamate. These findings indicate that zeaxanthin plays protective roles against oxidative stress in the vision of wild ayu.

N-Adamantyl-4-methylthiazol-2-amine suppresses glutamate-induced autophagic cell death via PI3K/Akt/mTOR signaling pathways in cortical neurons.

We recently reported that N-adamantyl-4-methylthiazol-2-amine (KHG26693) attenuates glutamate-induced oxidative stress and inflammation in the brain. In this study, we investigated KHG 26693 as a therapeutic agent against glutamate-induced autophagic death of cortical neurons. Treatment with KHG26693 alone did not affect the viability of cultured cortical neurons but was protective against glutamate-induced cytotoxicity in a concentration-dependent manner. KHG26693 attenuated the glutamate-induced increase in protein levels of LC3, beclin-1, and p62. Whereas glutamate decreased the phosphorylation of PI3K, Akt, and mTOR, these levels were restored by treatment with KHG26693. These results suggest that KHG26693 inhibits glutamate-induced autophagy by regulating PI3K/Akt/mTOR signaling. Finally, KHG26693 treatment also attenuated glutamateinduced increases in reactive oxygen species, glutathione, glutathione peroxidase, and superoxide dismutase levels in cortical neurons, indicating that KHG26693 also protects cortical neurons against glutamate-induced autophagy by regulating the reactive oxygen species scavenging system. [BMB Reports 2020; 53(10): 527-532].

Reduced Reelin Expression in the Hippocampus after Traumatic Brain Injury.

Traumatic brain injury (TBI) is a relatively common occurrence following accidents or violence, and often results in long-term cognitive or motor disability. Despite the high health cost associated with this type of injury, presently there are no effective treatments for many neurological symptoms resulting from TBI. This is due in part to our limited understanding of the mechanisms underlying brain dysfunction after injury. In this study, we used the mouse controlled cortical impact (CCI) model to investigate the effects of TBI, and focused on Reelin, an extracellular protein that critically regulates brain development and modulates synaptic activity in the adult brain. We found that Reelin expression decreases in forebrain regions after TBI, and that the number of Reelin-expressing cells decrease specifically in the hippocampus, an area of the brain that plays an important role in learning and memory. We also conducted in vitro experiments using mouse neuronal cultures and discovered that Reelin protects hippocampal neuronal cells from glutamate-induced neurotoxicity, a well-known secondary effect of TBI. Together our findings suggest that the loss of Reelin expression may contribute to neuronal death in the hippocampus after TBI, and raise the possibility that increasing Reelin levels or signaling activity may promote functional recovery.

Dexmedetomidine Attenuates Glutamate-Induced Cytotoxicity by Inhibiting the Mitochondrial-Mediated Apoptotic Pathway.

BACKGROUND Glutamate (GLU) is the most excitatory amino acid in the central nervous system and plays an important role in maintaining the normal function of the nervous system. During cerebral ischemia, massive release of GLU leads to neuronal necrosis and apoptosis. It has been reported that dexmedetomidine (DEX) possesses anti-oxidant and anti-apoptotic properties. The objective of this study was to investigate the effects of DEX on GLU-induced neurotoxicity in PC12 cells. MATERIAL AND METHODS PC12 cells were treated with 20 mM GLU to establish an ischemia-induced injury model. Cell viability was accessed by MTT assay. MDA content and SOD activity were analyzed by assay kits. Apoptosis rate, ROS production, intracellular Ca²âº concentration, and MMP were evaluated by flow cytometry. Western blot analysis was performed to analyze expressions of caspase-3, caspase-9, cyt-c, bax, and bcl-2. RESULTS PC12 cells treated with GLU exhibited reduced cell viability and increased apoptosis rates, which were ameliorated by pretreatment with DEX. DEX significantly increased SOD activity, reduced content of MDA, and decreased production of ROS in PC12 cells. In addition, DEX clearly reduced the level of intracellular Ca²âº and attenuated the decline of MMP. Moreover, DEX notably reduced expressions of caspase-3, caspase-9, cyt-c, and bax and increased expression of bcl-2. CONCLUSIONS Our findings suggest that DEX can protect PC12 cells against GLU-induced cytotoxicity, which may be attributed to its anti-oxidative property and reduction of intracellular calcium overload, as well as its ability to inhibit the mitochondria-mediated apoptotic pathway.

Tetrastigma hemsleyanum Vine Flavone Ameliorates Glutamic Acid-Induced Neurotoxicity via MAPK Pathways.

Glutamic acid (Glu) is a worldwide flavor enhancer with various positive effects. However, Glu-induced neurotoxicity has been reported less. Tetrastigma hemsleyanum (TH), a rare herbal plant in China, possesses high medicinal value. More studies paid attention to tuber of TH whereas vine part (THV) attracts fewer focus. In this study, we extracted and purified flavones from THV (THVF), and UPLC-TOF/MS showed THVF was consisted of 3-caffeoylquinic acid, 5-caffeoylquinic acid, quercetin-3-O-rutinoside, and kaempferol-3-O-rutinoside. In vitro, Glu caused severe cytotoxicity, genotoxicity, mitochondrial dysfunction, and oxidative damage to rat phaeochromocytoma (PC12) cells. Conversely, THVF attenuated Glu-induced toxicity via MAPK pathways. In vivo, the neurotoxicity triggered by Glu restrained the athletic ability in Caenorhabditis elegans (C. elegans). The treatment of THVF reversed the situation induced by Glu. In a word, Glu could cause neurotoxicity and THVF owns potential neuroprotective effects both in vitro and in vivo via MAPK pathways.

The Effect of Protein-Rich Extract from Bombyx Batryticatus against Glutamate-Damaged PC12 Cells Via Regulating γ-Aminobutyric Acid Signaling Pathway.

Bombyx Batryticatus (BB) is a known traditional Chinese medicine (TCM) utilized to treat convulsions, epilepsy, cough, asthma, headaches, etc. in China for thousands of years. This study is aimed at investigating optimum extraction of protein-rich extracts from BB (BBPs) using response surface methodology (RSM) and exploring the protective effects of BBPs against nerve growth factor (NGF)-induced PC12 cells injured by glutamate (Glu) and their underlying mechanisms. The results indicated optimum process of extraction was as follows: extraction time 1.00 h, ratio of liquid to the raw material 3.80 mL/g and ultrasonic power 230.0 W. The cell viability of PC12 cells stimulated by Glu was determined by CCK-8 assay. The levels of γ-aminobutyric (GABA), interleukin-1ß (IL-1ß), interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α), 5-hydroxytryptamine (5-HT) and glucocorticoid receptor alpha (GR) in PC12 cells were assayed by ELISA. Furthermore, the Ca2+ levels in PC12 cells were determined by flow cytometry analysis. Protein and mRNA expressions of GABAA-Rα1, NMDAR1, GAD 65, GAD 67, GAT 1 and GAT 3 in PC12 cells were evaluated by real-time polymerase chain reaction (RT-PCR) and Western blotting assays. Results revealed that BBPs decreased toxic effects due to Glu treatment and decreased Ca2+ levels in PC12 cells. After BBPs treatments, levels of GABA and 5-HT were increased and contents of TNF-α, IL-4 and IL-1ß were decreased in NGF-induced PC12 cells injured by Glu. Moreover, BBPs up-regulated the expressions of GABAA-Rα1, GAD 65 and GAD 67, whereas down-regulated that of NMDAR1 GAT 1 and GAT 3. These findings suggested that BBPs possessed protective effects on NGF-induced PC12 cells injured by Glu via γ-Aminobutyric Acid (GABA) signaling pathways, which demonstrated that BBPs has potential anti-epileptic effect in vitro. These findings may be useful in the development of novel medicine for the treatment of epilepsy.

The RNA-binding protein FUS/TLS undergoes calcium-mediated nuclear egress during excitotoxic stress and is required for GRIA2 mRNA processing.

Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease-linked RNA-binding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function, and disease have not been explored. Here, using primary cortical and motor neurons, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity included TAR DNA-binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS). We noted that FUS is translocated through a calcium-dependent mechanism and that its translocation coincides with striking alterations in nucleocytoplasmic transport. Furthermore, glutamate-induced up-regulation of glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type subunit 2 (GRIA2) in neurons depended on FUS expression, consistent with a functional role for FUS in excitotoxic stress. These findings reveal molecular links among prominent factors in neurodegenerative diseases, namely excitotoxicity, disease-associated RBPs, and nucleocytoplasmic transport.

KHG21834 attenuates glutamate-induced mitochondrial damage, apoptosis, and NLRP3 inflammasome activation in SH-SY5Y human neuroblastoma cells.

New compounds were screened to develop effective drugs against glutamate-induced toxicity. The present study assessed the effects of the novel thiazole derivative KHG21834 against glutamate-induced toxicity in human neuroblastoma SH-SY5Y cell cultures. Treatment of SH-SY5Y cells with KHG21834 significantly protected cells against glutamate-induced toxicity in a dose-dependent manner, with an optimum concentration of 50 µM. KHG21834 protected SH-SY5Y cells against glutamate toxicity by suppressing glutamate-induced oxidative stress by 50%. KHG21834 also attenuated glutamate-induced mitochondrial membrane potential, ATP level reductions, and intracellular Ca2+ influx. Furthermore, KHG21834 efficiently reduced glutamate-induced ER stress and NLRP3 inflammasome activation (59% and 65% of glutamate group, respectively). In addition, KHG21834 effectively attenuated glutamate-induced levels of Bax, Bcl-2, cleaved caspase-3, p-p38, p-JNK proteins, and TUNEL positive cells. To our knowledge, this is the first study showing that KHG21834 can effectively protect SH-SY5Y cells against glutamate toxicity, suggesting that this compound may be a valuable therapeutic agent for the treatment of glutamate toxicity.

Screening of novel 3α5ß-neurosteroids for neuroprotective activity against glutamate- or NMDA-induced excitotoxicity.

A broad variety of central nervous system diseases have been associated with glutamate induced excitotoxicity under pathological conditions. The neuroprotective effects of neurosteroids can combat this excitotoxicity. Herein, we have demonstrated the neuroprotective effect of novel steroidal N-methyl-D-aspartate receptor inhibitors against glutamate- or NMDA- induced excitotoxicity. Pretreatment with neurosteroids significantly reduced acute L-glutamic acid or NMDA excitotoxicity mediated by Ca2+ entry and consequent ROS (reactive oxygen species) release and caspase-3 activation. Compounds 6 (IC50 = 5.8 µM), 7 (IC50 = 12.2 µM), 9 (IC50 = 7.8 µM), 13 (IC50 = 1.1 µM) and 16 (IC50 = 8.2 µM) attenuated glutamate-induced Ca2+ entry more effectively than memantine (IC50 = 18.9 µM). Moreover, compound 13 shows comparable effect with MK-801 (IC50 = 1.2 µM) and also afforded significant protection without any adverse effect upon prolonged exposure. This drop in Ca2+ level resulted in corresponding ROS suppression and prevented glutamate-induced caspase-3 activation. Therefore, compound 13 has great potential for development into a therapeutic agent for improving glutamate-related nervous system diseases.

Double-blind, randomized, placebo-controlled study of trofinetide in pediatric Rett syndrome.

OBJECTIVE: To determine safety, tolerability, and pharmacokinetics of trofinetide and evaluate its efficacy in female children/adolescents with Rett syndrome (RTT), a debilitating neurodevelopmental condition for which no pharmacotherapies directed at core features are available. METHODS: This was a phase 2, multicenter, double-blind, placebo-controlled, parallel-group study, in which safety/tolerability, pharmacokinetics, and clinical response to trofinetide were characterized in 82 children/adolescents with RTT, aged 5 to 15 years. Sixty-two participants were randomized 1:1:1:1 to receive placebo twice a day (bid) for 14 days, followed by placebo, 50, 100, or 200 mg/kg bid of trofinetide for 42 days. Following blinded safety data review, 20 additional participants were randomized 1:1 to the 200 mg/kg or placebo bid groups. Safety assessments included adverse events, clinical laboratory tests, physical examinations, and concomitant medications. Clinician- and caregiver-based efficacy measurements assessed clinically relevant, phenotypic dimensions of impairment of RTT. RESULTS: All dose levels were well tolerated and generally safe. Trofinetide at 200 mg/kg bid showed statistically significant and clinically relevant improvements relative to placebo on the Rett Syndrome Behaviour Questionnaire, RTT-Clinician Domain Specific Concerns-Visual Analog Scale, and Clinical Global Impression Scale-Improvement. Exploratory analyses suggested that observed changes correlated with trofinetide exposure. CONCLUSION: These results, together with those from a previous adolescent/adult trial, indicate trofinetide's potential for treating core RTT symptoms and support further trials. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for children/adolescents with RTT, trofinetide was safe, well-tolerated, and demonstrated improvement over placebo at 200 mg/kg bid in functionally important dimensions of RTT.

Neuroprotective Effects of Dammarane-Type Saponins from Panax notoginseng on Glutamate-Induced Cell Damage in PC12 Cells.

Dammarane-type saponins, the main active ingredients of Panax notoginseng, have substantial neuroprotective effects in different animal models of neurodegenerative diseases. However, because these compounds have different structures, the level of protection provided by individual compounds varies, and highly active compounds can be selected based on structure-activity relationships. Glutamate is a major excitatory neurotransmitter that plays an important role in synaptic response development. However, excessive extracellular glutamate levels lead to neuronal dysfunctions in the central nervous system. Herein, we investigated the neuroprotective effects of nine saponins (compounds 1:  - 9: ) on glutamate-treated PC12 cells in the concentration range of 0.1 - 10 µM. The MTT assay revealed that these compounds increased cell viability to 65.6, 69.8, 76.9, 91.7, 74.4, 63.3, 59.9, 64.7, and 59.9%, respectively, compared with the glutamate-treated cells (44.6%). Protopanaxatriol (compound 4: ) was the most neuroprotective compound, and subsequent experiments revealed that pretreatment with compound 4: significantly reverses mitochondrial membrane potential collapse, increases superoxide dismutase activity, and decreases lactate dehydrogenase leakage, malondiadehyde levels, reactive oxygen species generation, and cell apoptosis. Compound 4: also decreased the Bax/Bcl-2 ratio, cleaved caspase-3, N-methyl-D-aspartic receptor 1, and Ca2+-/calmodulin-dependent protein kinase II expression, and inhibited glutamate-induced cytochrome C release and phosphorylation of apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase, and p38. Overall, the results indicate that protopanaxatriol has significant neuroprotective effects, and might be a promising neuroprotective agent for preventing and treating neurodegenerative diseases.

Neuroprotective Effects of Tetrahydrocurcumin against Glutamate-Induced Oxidative Stress in Hippocampal HT22 Cells.

In the central nervous system, glutamate is a major excitable neurotransmitter responsible for many cellular functions. However, excessive levels of glutamate induce neuronal cell death via oxidative stress during acute brain injuries as well as chronic neurodegenerative diseases. The present study was conducted to examine the effect of tetrahydrocurcumin (THC), a major secondary metabolite of curcumin, and its possible mechanism against glutamate-induced cell death. We prepared THC using curcumin isolated from Curcuma longa (turmeric) and demonstrated the protective effect of THC against glutamate-induced oxidative stress in HT22 cells. THC abrogated glutamate-induced HT22 cell death and showed a strong antioxidant effect. THC also significantly reduced intracellular calcium ion increased by glutamate. Additionally, THC significantly reduced the accumulation of intracellular oxidative stress induced by glutamate. Furthermore, THC significantly diminished apoptotic cell death indicated by annexin V-positive in HT22 cells. Western blot analysis indicated that the phosphorylation of mitogen-activated protein kinases including c-Jun N-terminal kinase, extracellular signal-related kinases 1/2, and p38 by glutamate was significantly diminished by treatment with THC. In conclusion, THC is a potent neuroprotectant against glutamate-induced neuronal cell death by inhibiting the accumulation of oxidative stress and phosphorylation of mitogen-activated protein kinases.

Calpain2 but not calpain1 mediated by calpastatin following glutamate-induced regulated necrosis in rat retinal neurons.

The purpose of this study is to investigate whether calpastatin (CAST) plays an important role in the regulated necrosis (RN) in rat retinal neurons under an excessive glutamate condition and furthermore to investigate whether this process is regulated by calapin1 and calpain2. In the present study, glutamate triggered CAST inhibition, calpain2 activation and retinal neuronal RN after injury. The application of CAST active peptide could provide protective effects against activated calpain2 mediated RN. However, the calpain1 activity was not changed in these processes. Finally, in vivo studies further confirmed the role of the CAST-calpain2 pathway in cellular RN in the rat retinal ganglion cell layer and inner nuclear layer after glutamate excitation. In addition, flash electroretinogram results provided evidence that the impaired visual function induced by glutamate could recover after CAST peptide treatment. This research indicated that excessive glutamate may lead to CAST inhibition and activated calpain2, but not calpain1 activation, resulting in RN.