A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase (GAD) activity. An effective isolation and purification procedure of GAD from a cell-f...A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase (GAD) activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps: 30%—90% saturation (NH4)2SO4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel fil-tration, and resource Q anion-exchange chromatography. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37℃, respectively. The purified GAD had a half-life of 50min at 45℃ and the Km value of the enzyme from Lineweaver-Burk plot was found to be 8.22. 5′-pyridoxal phosphate (PLP) had little effect on the regulation of its activity.展开更多
Backgrounds:Treatment with levodopa enhances recovery of lost neurological functions in preclinical stroke models and patients.Here,we studied whether dopamine signaling modulates GABAergic neurotransmission in parval...Backgrounds:Treatment with levodopa enhances recovery of lost neurological functions in preclinical stroke models and patients.Here,we studied whether dopamine signaling modulates GABAergic neurotransmission in parvalbumin-positive interneurons after experimental stroke.Methods:Following block randomization,mice were subjected to experimental stroke induced by photothrombosis(PT).Two days after the insult,mice were treated either with the D1 receptor antagonist by R(+)-SCH-23390(0.1 mg/kg),the selective D1 receptor agonist(R)-(+)-SKF-38393 hydrochloride(1 mg/kg),the D2 receptor agonist R(−)-2,10,11-trihydroxy-Npropyl-noraporphine hydrobromide hydrate(TNPA)(1 mg/kg),the D2 receptor antagonist S-(−)-eticlopride hydrochloride(0.3 mg/kg),or vehicle(saline)by daily intraperitoneal injection for five consecutive days,respectively.Recovery of function was assessed by paw placement and foot fault test before and on Days 2 and 7 after surgery.Results:Mice treated with TNPA showed a statistically significant improvement of recovery compared to all other treatment conditions.Synthesis of gamma-aminobutyric acid(GABA)was quantified by levels of full-length and cleaved glutamate acid decarboxylase 67 and 65(GAD65 and GAD67)in the peri-infarct area and homotypic regions of the contralateral cortex.Compared to the other treatments,TNPA significantly reduced the level of the GAD67 isoform both in the ischemic and contralateral hemispheres.Levels of GAD65 were found significantly higher in the contralateral hemisphere in TNPA-treated mice after PT accompanied by an increase in the 58 kDa-truncated form.Conclusion:Our results point toward reduced GABA synthesis in a D2 receptor-mediated mechanism possibly contributing to counteract functional inhibition after stroke.展开更多
Biosynthesis of the functional factor𝛾γ-aminobutyric acid(GABA)in bacteria involves two key proteins an intra-cellular glutamate decarboxylase(GadB)and a membrane-bound antiporter(GadC).Efficient co-expressio...Biosynthesis of the functional factor𝛾γ-aminobutyric acid(GABA)in bacteria involves two key proteins an intra-cellular glutamate decarboxylase(GadB)and a membrane-bound antiporter(GadC).Efficient co-expression of suitable GadB and GadC candidates is crucial for improving GABA productivity.In this study,gadBΔC11 of Lacti-plantibacillus plantarum and gadCΔC41 of Escherichia coli were inserted into the designed double promoter(P T7lac and P BAD)expression system.Then,E.coli Lemo21(DE3)was chosen as the host to minimize the toxic effects of GadCΔC41 overexpression.Furthermore,a green and high-efficiency GABA synthesis system using dormant engineered Lemo21(DE3)cells as biocatalysts was developed.The total GABA yield reached 829.08 g/L with a 98.7%conversion ratio within 13 h,when engineered E.coli Lemo21(DE3)cells were concentrated to an OD 600 of 20 and reused for three cycles in a 3 M L-glutamate solution at 37℃,which represented the highest GABA productivity ever reported.Overall,expanding the active pH ranges of GadB and GadC toward physiological pH and employing a tunable expression host for membrane-bound GadC production is a promising strategy for high-level GABA biosynthesis in E.coli.展开更多
Epilepsy is a neurological disorder characterized by high morbidity,high recurrence,and drug resistance.Enhanced signaling through the excitatory neurotransmitter glutamate is intricately associated with epilepsy.Meta...Epilepsy is a neurological disorder characterized by high morbidity,high recurrence,and drug resistance.Enhanced signaling through the excitatory neurotransmitter glutamate is intricately associated with epilepsy.Metabotropic glutamate receptors(mGluRs)are G protein-coupled receptors activated by glutamate and are key regulators of neuronal and synaptic plasticity.Dysregulated mGluR signaling has been associated with various neurological disorders,and numerous studies have shown a close relationship between mGluRs expression/activity and the development of epilepsy.In this review,we first introduce the three groups of mGluRs and their associated signaling pathways.Then,we detail how these receptors influence epilepsy by describing the signaling cascades triggered by their activation and their neuroprotective or detrimental roles in epileptogenesis.In addition,strategies for pharmacological manipulation of these receptors during the treatment of epilepsy in experimental studies is also summarized.We hope that this review will provide a foundation for future studies on the development of mGluR-targeted antiepileptic drugs.展开更多
Glutamate excitotoxicity has been shown to play an important role in glaucoma, and glutamate can induce ferroptosis. The p38 mitogenactivated protein kinase(MAPK) pathway inhibitor SB202190 has a potential ability to ...Glutamate excitotoxicity has been shown to play an important role in glaucoma, and glutamate can induce ferroptosis. The p38 mitogenactivated protein kinase(MAPK) pathway inhibitor SB202190 has a potential ability to suppress ferroptosis, and its downstream targets, such as p53, have been shown to be associated with ferroptosis. However, whether ferroptosis also occurs in retinal ganglion cells in response to glutamate excitotoxicity and whether inhibition of ferroptosis reduces the loss of retinal ganglion cells induced by glutamate excitotoxicity remain unclear. This study investigated ferroptosis in a glutamate-induced glaucoma rat model and explored the effects and molecular mechanisms of SB202190 on retinal ganglion cells. A glutamate-induced excitotoxicity model in R28 cells and an N-methyl-D-aspartate-induced glaucoma model in rats were used. In vitro experiments showed that glutamate induced the accumulation of iron and lipid peroxide and morphological changes of mitochondria in R28 cells, and SB202190 inhibited these changes. Glutamate induced the levels of p-p38 MAPK/p38 MAPK and SAT1 and decreased the expression levels of ferritin light chain, SLC7A11, and GPX4. SB202190 inhibited the expression of iron death-related proteins induced by glutamate. In vivo experiments showed that SB202190 attenuated N-methyl-D-aspartate-induced damage to rat retinal ganglion cells and improved visual function. These results suggest that SB202190 can inhibit ferroptosis and protect retinal ganglion cells by regulating ferritin light chain, SAT1, and SLC7A11/Gpx4 pathways and may represent a potential retina protectant.展开更多
The inflammatory microenvironment and neurotoxicity can hinder neuronal regeneration and functional recovery after spinal cord injury.Ruxolitinib,a JAK-STAT inhibitor,exhibits effectiveness in autoimmune diseases,arth...The inflammatory microenvironment and neurotoxicity can hinder neuronal regeneration and functional recovery after spinal cord injury.Ruxolitinib,a JAK-STAT inhibitor,exhibits effectiveness in autoimmune diseases,arthritis,and managing inflammatory cytokine storms.Although studies have shown the neuroprotective potential of ruxolitinib in neurological trauma,the exact mechanism by which it enhances functional recovery after spinal cord injury,particularly its effect on astrocytes,remains unclear.To address this gap,we established a mouse model of T10 spinal cord contusion and found that ruxolitinib effectively improved hindlimb motor function and reduced the area of spinal cord injury.Transcriptome sequencing analysis showed that ruxolitinib alleviated inflammation and immune response after spinal cord injury,restored EAAT2 expression,reduced glutamate levels,and alleviated excitatory toxicity.Furthermore,ruxolitinib inhibited the phosphorylation of JAK2 and STAT3 in the injured spinal cord and decreased the phosphorylation level of nuclear factor kappa-B and the expression of inflammatory factors interleukin-1β,interleukin-6,and tumor necrosis factor-α.Additionally,in glutamate-induced excitotoxicity astrocytes,ruxolitinib restored EAAT2 expression and increased glutamate uptake by inhibiting the activation of STAT3,thereby reducing glutamate-induced neurotoxicity,calcium influx,oxidative stress,and cell apoptosis,and increasing the complexity of dendritic branching.Collectively,these results indicate that ruxolitinib restores glutamate homeostasis by rescuing the expression of EAAT2 in astrocytes,reduces neurotoxicity,and effectively alleviates inflammatory and immune responses after spinal cord injury,thereby promoting functional recovery after spinal cord injury.展开更多
Objective To study the developmental changes of glutamic acid decarboxylase-67 (GAD-67, a GABA synthetic enzyme) in normal and hypoxic ischemic (HI) brain. Methods C57/BL6 mice on postnatal day (P) 5, 9, 21 and 60, co...Objective To study the developmental changes of glutamic acid decarboxylase-67 (GAD-67, a GABA synthetic enzyme) in normal and hypoxic ischemic (HI) brain. Methods C57/BL6 mice on postnatal day (P) 5, 9, 21 and 60, corresponding developmentally to premature, term, juvenile and adult human brain were investigated by using both Western blot and immunohistochemistry methods either in normal condition or after hypoxic ischemic insult. Results The immunoreactivity of GAD67 was up regulated with brain development and significant difference was seen between mature (P21, P60) and immature (P5, P9) brain. GAD67 immunoreactivity decreased in the ipsilateral hemisphere in all the ages after hypoxia ischemia (HI) insult, but, significant decrease was only seen in the immature brain. Double labeling of GAD67 and cell death marker, TUNEL, in the cortex at 8h post-HI in the P9 mice showed that (15.6±7.0)% TUNEL positive cells were GAD67 positive which was higher than that of P60 mice. Conclusion These data suggest that GABAergic neurons in immature brain were more vulnerable to HI insult than that of mature brain.展开更多
Maintaining glutamate homeostasis after hypoxic ischemia is important for synaptic function and neural cell activity,and regulation of glutamate transport between astrocyte and neuron is one of the important modalitie...Maintaining glutamate homeostasis after hypoxic ischemia is important for synaptic function and neural cell activity,and regulation of glutamate transport between astrocyte and neuron is one of the important modalities for reducing glutamate accumulation.However,further research is needed to investigate the dynamic changes in and molecular mechanisms of glutamate transport and the effects of glutamate transport on synapses.The aim of this study was to investigate the regulatory mechanisms underlying Notch pathway mediation of glutamate transport and synaptic plasticity.In this study,Yorkshire neonatal pigs(male,age 3 days,weight 1.0–1.5 kg,n=48)were randomly divided into control(sham surgery group)and five hypoxic ischemia subgroups,according to different recovery time,which were then further subdivided into subgroups treated with dimethyl sulfoxide or a Notch pathway inhibitor(N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester).Once the model was established,immunohistochemistry,immunofluorescence staining,and western blot analyses of Notch pathway-related proteins,synaptophysin,and glutamate transporter were performed.Moreover,synapse microstructure was observed by transmission electron microscopy.At the early stage(6–12 hours after hypoxic ischemia)of hypoxic ischemic injury,expression of glutamate transporter excitatory amino acid transporter-2 and synaptophysin was downregulated,the number of synaptic vesicles was reduced,and synaptic swelling was observed;at 12–24 hours after hypoxic ischemia,the Notch pathway was activated,excitatory amino acid transporter-2 and synaptophysin expression was increased,and the number of synaptic vesicles was slightly increased.Excitatory amino acid transporter-2 and synaptophysin expression decreased after treatment with the Notch pathway inhibitor.This suggests that glutamate transport in astrocytes-neurons after hypoxic ischemic injury is regulated by the Notch pathway and affects vesicle release and synaptic plasticity through the expression of synaptophysin.展开更多
Adipose mesenchymal stem cells(ADSCs)have protective effects against glutamate-induced excitotoxicity,but ADSCs are limited in use for treatment of optic nerve injury.Studies have shown that the extracellular vesicles...Adipose mesenchymal stem cells(ADSCs)have protective effects against glutamate-induced excitotoxicity,but ADSCs are limited in use for treatment of optic nerve injury.Studies have shown that the extracellular vesicles(EVs)secreted by ADSCs(ADSC-EVs)not only have the function of ADSCs,but also have unique advantages including non-immunogenicity,low probability of abnormal growth,and easy access to target cells.In the present study,we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography.In addition,R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium,downregulation ofα-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor(AMPAR)subunit GluA2,and phosphorylation of GluA2 and protein kinase C alpha in vitro.A protein kinase C alpha agonist,12-O-tetradecanoylphorbol 13-acetate,inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells.These findings suggest that ADSCEVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.展开更多
基金Supported by the National Natural Science Foundation of China (No.30570411)the Research Plan of Zhejiang Province, China.
文摘A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase (GAD) activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps: 30%—90% saturation (NH4)2SO4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel fil-tration, and resource Q anion-exchange chromatography. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37℃, respectively. The purified GAD had a half-life of 50min at 45℃ and the Km value of the enzyme from Lineweaver-Burk plot was found to be 8.22. 5′-pyridoxal phosphate (PLP) had little effect on the regulation of its activity.
基金Swedish Brain Fund,Grant/Award Numbers:FO2020-0138,FO2022-0154Hans-Gabriel and Alice Trolle Wachtmeister Foundation+1 种基金Skåne(ALF)Crafoord Foundation。
文摘Backgrounds:Treatment with levodopa enhances recovery of lost neurological functions in preclinical stroke models and patients.Here,we studied whether dopamine signaling modulates GABAergic neurotransmission in parvalbumin-positive interneurons after experimental stroke.Methods:Following block randomization,mice were subjected to experimental stroke induced by photothrombosis(PT).Two days after the insult,mice were treated either with the D1 receptor antagonist by R(+)-SCH-23390(0.1 mg/kg),the selective D1 receptor agonist(R)-(+)-SKF-38393 hydrochloride(1 mg/kg),the D2 receptor agonist R(−)-2,10,11-trihydroxy-Npropyl-noraporphine hydrobromide hydrate(TNPA)(1 mg/kg),the D2 receptor antagonist S-(−)-eticlopride hydrochloride(0.3 mg/kg),or vehicle(saline)by daily intraperitoneal injection for five consecutive days,respectively.Recovery of function was assessed by paw placement and foot fault test before and on Days 2 and 7 after surgery.Results:Mice treated with TNPA showed a statistically significant improvement of recovery compared to all other treatment conditions.Synthesis of gamma-aminobutyric acid(GABA)was quantified by levels of full-length and cleaved glutamate acid decarboxylase 67 and 65(GAD65 and GAD67)in the peri-infarct area and homotypic regions of the contralateral cortex.Compared to the other treatments,TNPA significantly reduced the level of the GAD67 isoform both in the ischemic and contralateral hemispheres.Levels of GAD65 were found significantly higher in the contralateral hemisphere in TNPA-treated mice after PT accompanied by an increase in the 58 kDa-truncated form.Conclusion:Our results point toward reduced GABA synthesis in a D2 receptor-mediated mechanism possibly contributing to counteract functional inhibition after stroke.
基金This work was supported by Natural Science Foundation of Zhe-jiang Province(LY23B060001)Zhejiang Provincial Key R&D Pro-gram of China(2021C02049)+2 种基金China Postdoctoral Science Founda-tion(2020M671337)National Natural Science Foundation of China(31670804,31971372)Ningbo"Scientific and Technological In-novation 2025″Key Project(2020Z080,2020Z088).
文摘Biosynthesis of the functional factor𝛾γ-aminobutyric acid(GABA)in bacteria involves two key proteins an intra-cellular glutamate decarboxylase(GadB)and a membrane-bound antiporter(GadC).Efficient co-expression of suitable GadB and GadC candidates is crucial for improving GABA productivity.In this study,gadBΔC11 of Lacti-plantibacillus plantarum and gadCΔC41 of Escherichia coli were inserted into the designed double promoter(P T7lac and P BAD)expression system.Then,E.coli Lemo21(DE3)was chosen as the host to minimize the toxic effects of GadCΔC41 overexpression.Furthermore,a green and high-efficiency GABA synthesis system using dormant engineered Lemo21(DE3)cells as biocatalysts was developed.The total GABA yield reached 829.08 g/L with a 98.7%conversion ratio within 13 h,when engineered E.coli Lemo21(DE3)cells were concentrated to an OD 600 of 20 and reused for three cycles in a 3 M L-glutamate solution at 37℃,which represented the highest GABA productivity ever reported.Overall,expanding the active pH ranges of GadB and GadC toward physiological pH and employing a tunable expression host for membrane-bound GadC production is a promising strategy for high-level GABA biosynthesis in E.coli.
基金supported by the Natural Science Foundation of Hunan Province,No.2021JJ30389(to JG)the Key Research and Development Program of Hunan Province of China,Nos.2022SK2042(to LL)and 2020SK2122(to ET)。
文摘Epilepsy is a neurological disorder characterized by high morbidity,high recurrence,and drug resistance.Enhanced signaling through the excitatory neurotransmitter glutamate is intricately associated with epilepsy.Metabotropic glutamate receptors(mGluRs)are G protein-coupled receptors activated by glutamate and are key regulators of neuronal and synaptic plasticity.Dysregulated mGluR signaling has been associated with various neurological disorders,and numerous studies have shown a close relationship between mGluRs expression/activity and the development of epilepsy.In this review,we first introduce the three groups of mGluRs and their associated signaling pathways.Then,we detail how these receptors influence epilepsy by describing the signaling cascades triggered by their activation and their neuroprotective or detrimental roles in epileptogenesis.In addition,strategies for pharmacological manipulation of these receptors during the treatment of epilepsy in experimental studies is also summarized.We hope that this review will provide a foundation for future studies on the development of mGluR-targeted antiepileptic drugs.
基金supported by the National Natural Science Foundation of China,Nos.81974132,81770927Hunan Provincial Health Commission,No.20220702839+1 种基金the Natural Science Foundation of Hunan Province of China,No.2022JJ30076National Key R&D Program of China,No.2021YFA1101202(all to WS)。
文摘Glutamate excitotoxicity has been shown to play an important role in glaucoma, and glutamate can induce ferroptosis. The p38 mitogenactivated protein kinase(MAPK) pathway inhibitor SB202190 has a potential ability to suppress ferroptosis, and its downstream targets, such as p53, have been shown to be associated with ferroptosis. However, whether ferroptosis also occurs in retinal ganglion cells in response to glutamate excitotoxicity and whether inhibition of ferroptosis reduces the loss of retinal ganglion cells induced by glutamate excitotoxicity remain unclear. This study investigated ferroptosis in a glutamate-induced glaucoma rat model and explored the effects and molecular mechanisms of SB202190 on retinal ganglion cells. A glutamate-induced excitotoxicity model in R28 cells and an N-methyl-D-aspartate-induced glaucoma model in rats were used. In vitro experiments showed that glutamate induced the accumulation of iron and lipid peroxide and morphological changes of mitochondria in R28 cells, and SB202190 inhibited these changes. Glutamate induced the levels of p-p38 MAPK/p38 MAPK and SAT1 and decreased the expression levels of ferritin light chain, SLC7A11, and GPX4. SB202190 inhibited the expression of iron death-related proteins induced by glutamate. In vivo experiments showed that SB202190 attenuated N-methyl-D-aspartate-induced damage to rat retinal ganglion cells and improved visual function. These results suggest that SB202190 can inhibit ferroptosis and protect retinal ganglion cells by regulating ferritin light chain, SAT1, and SLC7A11/Gpx4 pathways and may represent a potential retina protectant.
基金supported by the National Natural Science Foundation of China,No.82272484(to XC).
文摘The inflammatory microenvironment and neurotoxicity can hinder neuronal regeneration and functional recovery after spinal cord injury.Ruxolitinib,a JAK-STAT inhibitor,exhibits effectiveness in autoimmune diseases,arthritis,and managing inflammatory cytokine storms.Although studies have shown the neuroprotective potential of ruxolitinib in neurological trauma,the exact mechanism by which it enhances functional recovery after spinal cord injury,particularly its effect on astrocytes,remains unclear.To address this gap,we established a mouse model of T10 spinal cord contusion and found that ruxolitinib effectively improved hindlimb motor function and reduced the area of spinal cord injury.Transcriptome sequencing analysis showed that ruxolitinib alleviated inflammation and immune response after spinal cord injury,restored EAAT2 expression,reduced glutamate levels,and alleviated excitatory toxicity.Furthermore,ruxolitinib inhibited the phosphorylation of JAK2 and STAT3 in the injured spinal cord and decreased the phosphorylation level of nuclear factor kappa-B and the expression of inflammatory factors interleukin-1β,interleukin-6,and tumor necrosis factor-α.Additionally,in glutamate-induced excitotoxicity astrocytes,ruxolitinib restored EAAT2 expression and increased glutamate uptake by inhibiting the activation of STAT3,thereby reducing glutamate-induced neurotoxicity,calcium influx,oxidative stress,and cell apoptosis,and increasing the complexity of dendritic branching.Collectively,these results indicate that ruxolitinib restores glutamate homeostasis by rescuing the expression of EAAT2 in astrocytes,reduces neurotoxicity,and effectively alleviates inflammatory and immune responses after spinal cord injury,thereby promoting functional recovery after spinal cord injury.
基金This work was supported by the Natural Science Foundation of China （30470598）.
文摘Objective To study the developmental changes of glutamic acid decarboxylase-67 (GAD-67, a GABA synthetic enzyme) in normal and hypoxic ischemic (HI) brain. Methods C57/BL6 mice on postnatal day (P) 5, 9, 21 and 60, corresponding developmentally to premature, term, juvenile and adult human brain were investigated by using both Western blot and immunohistochemistry methods either in normal condition or after hypoxic ischemic insult. Results The immunoreactivity of GAD67 was up regulated with brain development and significant difference was seen between mature (P21, P60) and immature (P5, P9) brain. GAD67 immunoreactivity decreased in the ipsilateral hemisphere in all the ages after hypoxia ischemia (HI) insult, but, significant decrease was only seen in the immature brain. Double labeling of GAD67 and cell death marker, TUNEL, in the cortex at 8h post-HI in the P9 mice showed that (15.6±7.0)% TUNEL positive cells were GAD67 positive which was higher than that of P60 mice. Conclusion These data suggest that GABAergic neurons in immature brain were more vulnerable to HI insult than that of mature brain.
基金supported by the National Natural Science Foundation of China,Nos.81871408 and 81271631(to XMW)National Science Foundation for Young Scientists of China,No.81801658(to YZ)+1 种基金Outstanding Scientific Fund of Shengjing Hospital,No.201402(to XMW)345 Talent Support Project of Shengjing Hospital,No.30B(to YZ)。
文摘Maintaining glutamate homeostasis after hypoxic ischemia is important for synaptic function and neural cell activity,and regulation of glutamate transport between astrocyte and neuron is one of the important modalities for reducing glutamate accumulation.However,further research is needed to investigate the dynamic changes in and molecular mechanisms of glutamate transport and the effects of glutamate transport on synapses.The aim of this study was to investigate the regulatory mechanisms underlying Notch pathway mediation of glutamate transport and synaptic plasticity.In this study,Yorkshire neonatal pigs(male,age 3 days,weight 1.0–1.5 kg,n=48)were randomly divided into control(sham surgery group)and five hypoxic ischemia subgroups,according to different recovery time,which were then further subdivided into subgroups treated with dimethyl sulfoxide or a Notch pathway inhibitor(N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester).Once the model was established,immunohistochemistry,immunofluorescence staining,and western blot analyses of Notch pathway-related proteins,synaptophysin,and glutamate transporter were performed.Moreover,synapse microstructure was observed by transmission electron microscopy.At the early stage(6–12 hours after hypoxic ischemia)of hypoxic ischemic injury,expression of glutamate transporter excitatory amino acid transporter-2 and synaptophysin was downregulated,the number of synaptic vesicles was reduced,and synaptic swelling was observed;at 12–24 hours after hypoxic ischemia,the Notch pathway was activated,excitatory amino acid transporter-2 and synaptophysin expression was increased,and the number of synaptic vesicles was slightly increased.Excitatory amino acid transporter-2 and synaptophysin expression decreased after treatment with the Notch pathway inhibitor.This suggests that glutamate transport in astrocytes-neurons after hypoxic ischemic injury is regulated by the Notch pathway and affects vesicle release and synaptic plasticity through the expression of synaptophysin.
基金supported by the National Key R&D Program of China,No.2016YFC1201800(to JFH)the Key Research and Development Program of Hunan Province,Nos.2018SK2090(to JFH),2022SK2079(to JFH)+2 种基金the Natural Science Foundation of Hu nan Province,No.2021JJ30891(to DC)the Human Resource Bank Program of Hunan Province,No.2020TP3003(to JFH)the School-Enterprise Joint Program of Central South University,No.2021XQLH092(to TQD)。
文摘Adipose mesenchymal stem cells(ADSCs)have protective effects against glutamate-induced excitotoxicity,but ADSCs are limited in use for treatment of optic nerve injury.Studies have shown that the extracellular vesicles(EVs)secreted by ADSCs(ADSC-EVs)not only have the function of ADSCs,but also have unique advantages including non-immunogenicity,low probability of abnormal growth,and easy access to target cells.In the present study,we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography.In addition,R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium,downregulation ofα-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor(AMPAR)subunit GluA2,and phosphorylation of GluA2 and protein kinase C alpha in vitro.A protein kinase C alpha agonist,12-O-tetradecanoylphorbol 13-acetate,inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells.These findings suggest that ADSCEVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.
