Amyotrophic lateral sclerosis(ALS)is a progressive neurodegenerative disease affecting both upper and lower motor neurons(MNs)with large unmet medical needs.Multiple pathological mechanisms are considered to contribut...Amyotrophic lateral sclerosis(ALS)is a progressive neurodegenerative disease affecting both upper and lower motor neurons(MNs)with large unmet medical needs.Multiple pathological mechanisms are considered to contribute to the progression of ALS,including neuronal oxidative stress and mitochondrial dysfunction.Honokiol(HNK)has been reported to exert therapeutic effects in several neurologic disease models including ischemia stroke,Alzheimer’s disease and Parkinson’s disease.Here we found that honokiol also exhibited protective effects in ALS disease models both in vitro and in vivo.Honokiol improved the viability of NSC-34 motor neuron-like cells that expressed the mutant G93A SOD1 proteins(SOD1-G93A cells for short).Mechanistical studies revealed that honokiol alleviated cellular oxidative stress by enhancing glutathione(GSH)synthesis and activating the nuclear factor erythroid 2-related factor 2(NRF2)-antioxidant response element(ARE)pathway.Also,honokiol improved both mitochondrial function and morphology via fine-tuning mitochondrial dynamics in SOD1-G93A cells.Importantly,honokiol extended the lifespan of the SOD1-G93A transgenic mice and improved the motor function.The improvement of antioxidant capacity and mitochondrial function was further confirmed in the spinal cord and gastrocnemius muscle in mice.Overall,honokiol showed promising preclinical potential as a multiple target drug for ALS treatment.展开更多
Previous studies have indicated that the pathogenesis of amyotrophic lateral sclerosis(ALS) is closely linked to 5-hydroxytryptamine(5-HT).To investigate this further,we administered 5-HT receptor antagonists to SOD1*...Previous studies have indicated that the pathogenesis of amyotrophic lateral sclerosis(ALS) is closely linked to 5-hydroxytryptamine(5-HT).To investigate this further,we administered 5-HT receptor antagonists to SOD1*G93A transgenic(ALS mouse model) and wide-type mice.This involved intraperitoneal injections of either granisetron,piboserod,or ritanserin,which inhibit the 5-HT3,5-HT4,and 5-HT2 receptors,respectively.The transgenic mice were found to have fewer5-HT-positive cells in the spinal cord compared with wide-type mice.We found that the administration of granisetron reduced the body weight of the transgenic mice,while piboserod and ritanserin worsened the motor functioning,as assessed using a hanging wire test.However,none of the 5-HT receptor antagonists affected the disease progression.We analyzed the distribution and/or expression of TAR DNA binding protein 43(TDP-43) and superoxide dismutase 1 G93A(SOD1-G93A),which fo rm abnormal aggregates in ALS.We found that the expression of these proteins increased following the administration of all three 5-HT receptor antagonists.In addition,the disease-related mislocalization of TD P-43 to the cytoplasm increased markedly for all three drugs.In ce rtain anatomical regions,the 5-HT receptor antagonists also led to a marked increase in the number of astrocytes and microglia and a decrease in the number of neurons.These results indicate that 5-HT deficiency may play a role in the pathogenesis of amyotrophic lateral sclerosis by inducing the abnormal expression and/or distribution of TDP-43 and SOD1-G93A and by activating glial cells.5-HT co uld therefore be a potential therapeutic target for amyotrophic lateral sclerosis.展开更多
Heterogenous nuclear ribonucleoprotein G is down-regulated in the spinal cord of the Tg(SOD1*G93A)1Gur(TG)amyotrophic lateral sclerosis mouse model.However,most studies have only examined heterogenous nuclear ribonucl...Heterogenous nuclear ribonucleoprotein G is down-regulated in the spinal cord of the Tg(SOD1*G93A)1Gur(TG)amyotrophic lateral sclerosis mouse model.However,most studies have only examined heterogenous nuclear ribonucleoprotein G expression in the amyotrophic lateral sclerosis model and heterogenous nuclear ribonucleoprotein G effects in amyotrophic lateral sclerosis pathogenesis such as in apoptosis are unknown.In this study,we studied the potential mechanism of heterogenous nuclear ribonucleoprotein G in neuronal death in the spinal cord of TG and wild-type mice and examined the mechanism by which heterogenous nuclear ribonucleoprotein G induces apoptosis.Heterogenous nuclear ribonucleoprotein G in spinal cord was analyzed using immunohistochemistry and western blotting,and cell proliferation and proteins(TAR DNA binding protein 43,superoxide dismutase 1,and Bax)were detected by the Cell Counting Kit-8 and western blot analysis in heterogenous nuclear ribonucleoprotein G siRNA-transfected PC12 cells.We analyzed heterogenous nuclear ribonucleoprotein G distribution in spinal cord in the amyotrophic lateral sclerosis model at various time points and the expressions of apoptosis and proliferation-related proteins.Heterogenous nuclear ribonucleoprotein G was mainly localized in neurons.Amyotrophic lateral sclerosis mice were examined at three stages:preonset(60-70 days),onset(90-100 days)and progression(120-130 days).The number of heterogenous nuclear ribonucleoprotein G-positive cells was significantly higher in the anterior horn of the lumbar spinal cord segment of TG mice at the preonset stage than that of control group but lower than that of the control group at the onset stage.The number of heterogenous nuclear ribonucleoprotein G-positive cells in both central canal and surrounding gray matter of the whole spinal cord of TG mice at the onset stage was significantly lower than that in the control group,whereas that of the lumbar spinal cord segment of TG mice was significantly higher than that in the control group at preonset stage and significantly lower than that in the control group at the progression stage.The numbers of heterogenous nuclear ribonucleoprotein G-positive cells in the posterior horn of cervical and thoracic segments of TG mice at preonset and progression stages were significantly lower than those in the control group.The expression of heterogenous nuclear ribonucleoprotein G in the cervical spinal cord segment of TG mice was significantly higher than that in the control group at the preonset stage but significantly lower at the progression stage.The expression of heterogenous nuclear ribonucleoprotein G in the thoracic spinal cord segment of TG mice was significantly increased at the preonset stage,significantly decreased at the onset stage,and significantly increased at the progression stage compared with the control group.heterogenous nuclear ribonucleoprotein G expression in the lumbar spinal cord segment of TG mice was significantly lower than that of the control group at the progression stage.After heterogenous nuclear ribonucleoprotein G gene silencing,PC12 cell survival was lower than that of control cells.Both TAR DNA binding protein 43 and Bax expressions were significantly increased in heterogenous nuclear ribonucleoprotein G-silenced cells compared with control cells.Our study suggests that abnormal distribution and expression of heterogenous nuclear ribonucleoprotein G might play a protective effect in amyotrophic lateral sclerosis development via preventing neuronal death by reducing abnormal TAR DNA binding protein 43 generation in the spinal cord.展开更多
The amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder primarily involving motoneurons in the cerebral cortex, brainstem, and spinal cord. SOD1-G93A mice, which express multiple ...The amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder primarily involving motoneurons in the cerebral cortex, brainstem, and spinal cord. SOD1-G93A mice, which express multiple copies of the mutant form of the human Cu/Zn SOD, are one of the most widely used animal models for ALS pathology. However, the onset of the disease can vary between animals of 1-2 weeks while the progression is quite fast. In order to evaluate the efficacy of any treatment, it is very important to treat all animals at the early onset of the disease, instead of at a fixed age-point. To this aim, we performed behavioral analysis and measured hSOD1 mRNA expression to identify the appearance of the first motor deficits. Rotarod and PaGE tests revealed to be the most sensitive approaches to detect the beginning of the symptomatic phase of the disease, while neurological score and weight monitoring showed significant differences only at later stages in ALS pathology. Furthermore, we found a better correlation between hSOD1 mRNA expression with disease onset than with a transgene copy number. Therefore, the association of behavioral tests and molecular analysis represents a sensible and accurate tool to early detect the murine symptoms.展开更多
The development of amyotrophic lateral sclerosis(ALS)may be related to the abnormal alterations of multiple proteins.Our previous study revealed that the expression of phosphoinositide-3-kinase regulatory subunit 4(PI...The development of amyotrophic lateral sclerosis(ALS)may be related to the abnormal alterations of multiple proteins.Our previous study revealed that the expression of phosphoinositide-3-kinase regulatory subunit 4(PIK3R4)was decreased in ALS.However,the role of PIK3R4 in ALS pathogenesis remains unknown.This study was the first to find that transfection of PC12 cells with small interfering RNA against the PIK3R4 gene significantly decreased the expression levels of PIK3R4 and the autophagy-related proteins p62 and LC3.Additionally,in vivo experiments revealed that the PIK3R4 protein was extensively expressed in the anterior horn,posterior horn,central canal,and areas surrounding the central canal in cervical,thoracic,and lumbar segments of the spinal cord in adult mice.PIK3R4 protein was mainly expressed in the neurons within the spinal lumbar segments.PIK3R4 and p62 expression levels were significantly decreased at both the pre-onset and onset stages of ALS disease in Tg(SOD1*G93A)1 Gur mice compared with control mice,but these proteins were markedly increased at the progression stage.LC3 protein expression did not change during progression of ALS.These findings suggest that PIK3R4 likely participates in the prevention of ALS progression.This study was approved by the Ethics Committee for Animal Care and Use of Jiangxi Provincial People’s Hospital,Affiliated People’s Hospital of Nanchang University(approval No.2020025)on March 26,2020.展开更多
Background:Neuron-microglia communication plays a crucial role in the motor neurons(MNs)death in amyotrophic lateral sclerosis(ALS).Neurons can express chemokine(C-X3-C motif)ligand 1(CX3CL1),which mediates microglial...Background:Neuron-microglia communication plays a crucial role in the motor neurons(MNs)death in amyotrophic lateral sclerosis(ALS).Neurons can express chemokine(C-X3-C motif)ligand 1(CX3CL1),which mediates microglial activation via interacting with its sole receptor CX3CR1 in microglia.In the present study,we aimed to investigate the dynamic changes of CX3CL1/CX3CR1 axis during microglial activation and MNs loss in SOD1G93A mouse model of ALS.Methods:qPCR,western blot and immunofluorescent staining were used to examine the mRNA and protein levels and localization of CX3CL1/CX3CR1 in the anterior horn region of spinal cord in both SOD1G93A mice and their agematched wild type(WT)littermates at 40,60,90 and 120 days of age.The M1/M2 microglial activation in the spinal cord tissues of SOD1G93A mice and WT mice were evaluated by immunofluorescent staining of M1/M2 markers and further confirmed by qPCR analysis of M1/M2-related cytokines.Results:The immunofluorescent staining revealed that CX3CL1 was predominately expressed in MNs,while CX3CR1 was highly expressed in microglia in the anterior horn region of spinal cord.Compared with age-matched WT mice,CX3CL1 mRNA level was elevated at 40 days but decreased at 90 and 120 days in the anterior horn region of spinal cords in ALS mice.Consistently,CX3CR1 mRNA level was increased at 90 and 120 days.Western blot assay further confirmed the dynamic changes of CX3CL1/CX3CR1 axis in ALS mice.Additionally,the levels of M1/M2 markers of microglia and their related cytokines in the anterior horn region of spinal cord in ALS mice were increased at 90 and 120 days.Moreover,while M1-related cytokines in ALS mice were persistently increased at 120 days,the upregulated M2-related cytokines started to decline at 120 days,suggesting an altered microglial activation.Conclusions:Our data revealed the dynamic changes of CX3CL1/CX3CR1 axis and an imbalanced M1/M2 microglial activation during ALS pathological progression.These findings may help identify potential molecular targets for ALS therapy.展开更多
Background Amyotrophic lateral sclerosis （ALS） is a fatal neurodegenerative disorder characterized by progressive death of the upper and lower motor neurons. Transgenic mice over-expressing a mutant form of the huma...Background Amyotrophic lateral sclerosis （ALS） is a fatal neurodegenerative disorder characterized by progressive death of the upper and lower motor neurons. Transgenic mice over-expressing a mutant form of the human SOD1 gene develop an ALS-like phenotype. Currently, there is no effective treatment or drug for the fatal disease. Previous studies reported potent efficacy of dl-3-n-butylphthalide （DL-NBP） for several neurodegenerative disorders and cerebral ischemia. SOD1-G93A mice are a mouse model of ALS. In this study, we investigated the efficacy of DL-NBP on this ALS mouse model. Methods Sixty SOD1-G93A female mice were divided into four groups. The vehicle control group received 0 mg.kg-1.d-~ DL-NBP. The experimental groups received DL-NBP with doses of 30, 60 or 120 mg.kgl.d1, respectively. For measurement of motor activity, the hanging wire test and rotarod test were performed. Survival statistics were analyzed by Kaplan-Meier survival curves. The body weight of each mouse was recorded twice per week. The statistical motor unit number estimation （MUNE） technique was used to estimate the number of functioning motor units in gastrocnemius muscle. Muscle morphology was evaluated by hematoxylin and eosin staining. Motor neuron quantJtation was performed by Nissl staining and microglia activation was observed by immunohistochemistry. Results Oral administration of 60 mg.kg-l-d-1 DL-NBP significantly prolonged survival （（164.78±16.67） days） of SOD1-G93A mice compared with vehicle control （（140.00＋16.89） days）. Treating mice with DL-NBP （60 mg.kg-1.d-1） significantly decreased the progression rate of motor deficits and suppressed body weight reduction. Furthermore, we found that treating SOD1-G93A mice with DL-NBP （60 mg.kgl.d1） slowed the rate of MUNE reduction （P 〈0.01）. Motor neurons were remarkably preserved in the anterior horns in mice treated with DL-NBP （60 mg.kg-1d-1） at the stage of 19 weeks （P 〈0.01）. Treating mice with DL-NBP （60 mg.kg1.d1） significantly reduced CD11b immunoreactivity compared with vehicle control mice （P 〈0.05）. No significant effect was observed in mice treated with DL-NBP of 30 or 120 mg.kg-1.d-1. Conclusions The post-disease-onset administration of DL-NBP significantly prolonged survival and improved motor performance in SOD1-G93A mice. DL-NBP mav be a Dotential theraDeutic aaent for ALS.展开更多
基金supported by the grants from National Natural Science Foundation of China(Nos.82073835 and 81872855)National Key R&D Program of China(No.2019YFC1708901)+2 种基金CAMS Innovation Fund for Medical Sciences(No.2021-I2M-1028)Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study(BZ0150,China)Disciplines construction project(201920200802,China)。
文摘Amyotrophic lateral sclerosis(ALS)is a progressive neurodegenerative disease affecting both upper and lower motor neurons(MNs)with large unmet medical needs.Multiple pathological mechanisms are considered to contribute to the progression of ALS,including neuronal oxidative stress and mitochondrial dysfunction.Honokiol(HNK)has been reported to exert therapeutic effects in several neurologic disease models including ischemia stroke,Alzheimer’s disease and Parkinson’s disease.Here we found that honokiol also exhibited protective effects in ALS disease models both in vitro and in vivo.Honokiol improved the viability of NSC-34 motor neuron-like cells that expressed the mutant G93A SOD1 proteins(SOD1-G93A cells for short).Mechanistical studies revealed that honokiol alleviated cellular oxidative stress by enhancing glutathione(GSH)synthesis and activating the nuclear factor erythroid 2-related factor 2(NRF2)-antioxidant response element(ARE)pathway.Also,honokiol improved both mitochondrial function and morphology via fine-tuning mitochondrial dynamics in SOD1-G93A cells.Importantly,honokiol extended the lifespan of the SOD1-G93A transgenic mice and improved the motor function.The improvement of antioxidant capacity and mitochondrial function was further confirmed in the spinal cord and gastrocnemius muscle in mice.Overall,honokiol showed promising preclinical potential as a multiple target drug for ALS treatment.
基金supported by the National Natural Science Foundation of China,Nos.30560042,81160161,8136019882160255+2 种基金Education Department of Jiangxi Province,Nos.GJJ13198 and GJJ170021Jiangxi Provincial Department of Science and Technology,Nos.20142BBG70062,20171 BAB215022,20192BAB205043Health and Family Planning Commission of Jiangxi Province,No.20181019 (all to RSX)。
文摘Previous studies have indicated that the pathogenesis of amyotrophic lateral sclerosis(ALS) is closely linked to 5-hydroxytryptamine(5-HT).To investigate this further,we administered 5-HT receptor antagonists to SOD1*G93A transgenic(ALS mouse model) and wide-type mice.This involved intraperitoneal injections of either granisetron,piboserod,or ritanserin,which inhibit the 5-HT3,5-HT4,and 5-HT2 receptors,respectively.The transgenic mice were found to have fewer5-HT-positive cells in the spinal cord compared with wide-type mice.We found that the administration of granisetron reduced the body weight of the transgenic mice,while piboserod and ritanserin worsened the motor functioning,as assessed using a hanging wire test.However,none of the 5-HT receptor antagonists affected the disease progression.We analyzed the distribution and/or expression of TAR DNA binding protein 43(TDP-43) and superoxide dismutase 1 G93A(SOD1-G93A),which fo rm abnormal aggregates in ALS.We found that the expression of these proteins increased following the administration of all three 5-HT receptor antagonists.In addition,the disease-related mislocalization of TD P-43 to the cytoplasm increased markedly for all three drugs.In ce rtain anatomical regions,the 5-HT receptor antagonists also led to a marked increase in the number of astrocytes and microglia and a decrease in the number of neurons.These results indicate that 5-HT deficiency may play a role in the pathogenesis of amyotrophic lateral sclerosis by inducing the abnormal expression and/or distribution of TDP-43 and SOD1-G93A and by activating glial cells.5-HT co uld therefore be a potential therapeutic target for amyotrophic lateral sclerosis.
基金supported by the National Natural Science Foundation of China,Nos.30560042,81160161,81360198,82160255Education Department of Jiangxi Province,Nos.GJJ13198 and GJJ170021+1 种基金Jiangxi Provincial Department of Science and Technology,Nos.[2014]-47,20142BBG70062,20171BAB215022,20192BAB205043Health and Family Planning Commission of Jiangxi Province,No.20181019(all to RSX).
文摘Heterogenous nuclear ribonucleoprotein G is down-regulated in the spinal cord of the Tg(SOD1*G93A)1Gur(TG)amyotrophic lateral sclerosis mouse model.However,most studies have only examined heterogenous nuclear ribonucleoprotein G expression in the amyotrophic lateral sclerosis model and heterogenous nuclear ribonucleoprotein G effects in amyotrophic lateral sclerosis pathogenesis such as in apoptosis are unknown.In this study,we studied the potential mechanism of heterogenous nuclear ribonucleoprotein G in neuronal death in the spinal cord of TG and wild-type mice and examined the mechanism by which heterogenous nuclear ribonucleoprotein G induces apoptosis.Heterogenous nuclear ribonucleoprotein G in spinal cord was analyzed using immunohistochemistry and western blotting,and cell proliferation and proteins(TAR DNA binding protein 43,superoxide dismutase 1,and Bax)were detected by the Cell Counting Kit-8 and western blot analysis in heterogenous nuclear ribonucleoprotein G siRNA-transfected PC12 cells.We analyzed heterogenous nuclear ribonucleoprotein G distribution in spinal cord in the amyotrophic lateral sclerosis model at various time points and the expressions of apoptosis and proliferation-related proteins.Heterogenous nuclear ribonucleoprotein G was mainly localized in neurons.Amyotrophic lateral sclerosis mice were examined at three stages:preonset(60-70 days),onset(90-100 days)and progression(120-130 days).The number of heterogenous nuclear ribonucleoprotein G-positive cells was significantly higher in the anterior horn of the lumbar spinal cord segment of TG mice at the preonset stage than that of control group but lower than that of the control group at the onset stage.The number of heterogenous nuclear ribonucleoprotein G-positive cells in both central canal and surrounding gray matter of the whole spinal cord of TG mice at the onset stage was significantly lower than that in the control group,whereas that of the lumbar spinal cord segment of TG mice was significantly higher than that in the control group at preonset stage and significantly lower than that in the control group at the progression stage.The numbers of heterogenous nuclear ribonucleoprotein G-positive cells in the posterior horn of cervical and thoracic segments of TG mice at preonset and progression stages were significantly lower than those in the control group.The expression of heterogenous nuclear ribonucleoprotein G in the cervical spinal cord segment of TG mice was significantly higher than that in the control group at the preonset stage but significantly lower at the progression stage.The expression of heterogenous nuclear ribonucleoprotein G in the thoracic spinal cord segment of TG mice was significantly increased at the preonset stage,significantly decreased at the onset stage,and significantly increased at the progression stage compared with the control group.heterogenous nuclear ribonucleoprotein G expression in the lumbar spinal cord segment of TG mice was significantly lower than that of the control group at the progression stage.After heterogenous nuclear ribonucleoprotein G gene silencing,PC12 cell survival was lower than that of control cells.Both TAR DNA binding protein 43 and Bax expressions were significantly increased in heterogenous nuclear ribonucleoprotein G-silenced cells compared with control cells.Our study suggests that abnormal distribution and expression of heterogenous nuclear ribonucleoprotein G might play a protective effect in amyotrophic lateral sclerosis development via preventing neuronal death by reducing abnormal TAR DNA binding protein 43 generation in the spinal cord.
文摘The amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder primarily involving motoneurons in the cerebral cortex, brainstem, and spinal cord. SOD1-G93A mice, which express multiple copies of the mutant form of the human Cu/Zn SOD, are one of the most widely used animal models for ALS pathology. However, the onset of the disease can vary between animals of 1-2 weeks while the progression is quite fast. In order to evaluate the efficacy of any treatment, it is very important to treat all animals at the early onset of the disease, instead of at a fixed age-point. To this aim, we performed behavioral analysis and measured hSOD1 mRNA expression to identify the appearance of the first motor deficits. Rotarod and PaGE tests revealed to be the most sensitive approaches to detect the beginning of the symptomatic phase of the disease, while neurological score and weight monitoring showed significant differences only at later stages in ALS pathology. Furthermore, we found a better correlation between hSOD1 mRNA expression with disease onset than with a transgene copy number. Therefore, the association of behavioral tests and molecular analysis represents a sensible and accurate tool to early detect the murine symptoms.
基金supported by the National Natural Science Foundation of China(Nos.30560042,81160161 and 81360198)Education Department of Jiangxi Province(No.GJJ170021)+1 种基金Jiangxi Provincial Department of Science and Technology(Nos.[2014]-47,20142BBG70062,20171BAB215022,20192BAB205043)Health Commission of Jiangxi Province(No.20181019)(all to RSX)。
文摘The development of amyotrophic lateral sclerosis(ALS)may be related to the abnormal alterations of multiple proteins.Our previous study revealed that the expression of phosphoinositide-3-kinase regulatory subunit 4(PIK3R4)was decreased in ALS.However,the role of PIK3R4 in ALS pathogenesis remains unknown.This study was the first to find that transfection of PC12 cells with small interfering RNA against the PIK3R4 gene significantly decreased the expression levels of PIK3R4 and the autophagy-related proteins p62 and LC3.Additionally,in vivo experiments revealed that the PIK3R4 protein was extensively expressed in the anterior horn,posterior horn,central canal,and areas surrounding the central canal in cervical,thoracic,and lumbar segments of the spinal cord in adult mice.PIK3R4 protein was mainly expressed in the neurons within the spinal lumbar segments.PIK3R4 and p62 expression levels were significantly decreased at both the pre-onset and onset stages of ALS disease in Tg(SOD1*G93A)1 Gur mice compared with control mice,but these proteins were markedly increased at the progression stage.LC3 protein expression did not change during progression of ALS.These findings suggest that PIK3R4 likely participates in the prevention of ALS progression.This study was approved by the Ethics Committee for Animal Care and Use of Jiangxi Provincial People’s Hospital,Affiliated People’s Hospital of Nanchang University(approval No.2020025)on March 26,2020.
基金This work was supported by funding from the National Natural Sciences Foundation of China(NSFC 81430021 and 81370470)the Program for Liaoning Innovative Research Team in University(LT2015009)Liaoning Science and Technology Project(2015225008).
文摘Background:Neuron-microglia communication plays a crucial role in the motor neurons(MNs)death in amyotrophic lateral sclerosis(ALS).Neurons can express chemokine(C-X3-C motif)ligand 1(CX3CL1),which mediates microglial activation via interacting with its sole receptor CX3CR1 in microglia.In the present study,we aimed to investigate the dynamic changes of CX3CL1/CX3CR1 axis during microglial activation and MNs loss in SOD1G93A mouse model of ALS.Methods:qPCR,western blot and immunofluorescent staining were used to examine the mRNA and protein levels and localization of CX3CL1/CX3CR1 in the anterior horn region of spinal cord in both SOD1G93A mice and their agematched wild type(WT)littermates at 40,60,90 and 120 days of age.The M1/M2 microglial activation in the spinal cord tissues of SOD1G93A mice and WT mice were evaluated by immunofluorescent staining of M1/M2 markers and further confirmed by qPCR analysis of M1/M2-related cytokines.Results:The immunofluorescent staining revealed that CX3CL1 was predominately expressed in MNs,while CX3CR1 was highly expressed in microglia in the anterior horn region of spinal cord.Compared with age-matched WT mice,CX3CL1 mRNA level was elevated at 40 days but decreased at 90 and 120 days in the anterior horn region of spinal cords in ALS mice.Consistently,CX3CR1 mRNA level was increased at 90 and 120 days.Western blot assay further confirmed the dynamic changes of CX3CL1/CX3CR1 axis in ALS mice.Additionally,the levels of M1/M2 markers of microglia and their related cytokines in the anterior horn region of spinal cord in ALS mice were increased at 90 and 120 days.Moreover,while M1-related cytokines in ALS mice were persistently increased at 120 days,the upregulated M2-related cytokines started to decline at 120 days,suggesting an altered microglial activation.Conclusions:Our data revealed the dynamic changes of CX3CL1/CX3CR1 axis and an imbalanced M1/M2 microglial activation during ALS pathological progression.These findings may help identify potential molecular targets for ALS therapy.
基金FENG Xin-hong and YUAN Wei contributed equally to the work. The study was supported by the grants from National Natural Science Foundation of China （No. 30971002 and No. 30911120496）.
文摘Background Amyotrophic lateral sclerosis （ALS） is a fatal neurodegenerative disorder characterized by progressive death of the upper and lower motor neurons. Transgenic mice over-expressing a mutant form of the human SOD1 gene develop an ALS-like phenotype. Currently, there is no effective treatment or drug for the fatal disease. Previous studies reported potent efficacy of dl-3-n-butylphthalide （DL-NBP） for several neurodegenerative disorders and cerebral ischemia. SOD1-G93A mice are a mouse model of ALS. In this study, we investigated the efficacy of DL-NBP on this ALS mouse model. Methods Sixty SOD1-G93A female mice were divided into four groups. The vehicle control group received 0 mg.kg-1.d-~ DL-NBP. The experimental groups received DL-NBP with doses of 30, 60 or 120 mg.kgl.d1, respectively. For measurement of motor activity, the hanging wire test and rotarod test were performed. Survival statistics were analyzed by Kaplan-Meier survival curves. The body weight of each mouse was recorded twice per week. The statistical motor unit number estimation （MUNE） technique was used to estimate the number of functioning motor units in gastrocnemius muscle. Muscle morphology was evaluated by hematoxylin and eosin staining. Motor neuron quantJtation was performed by Nissl staining and microglia activation was observed by immunohistochemistry. Results Oral administration of 60 mg.kg-l-d-1 DL-NBP significantly prolonged survival （（164.78±16.67） days） of SOD1-G93A mice compared with vehicle control （（140.00＋16.89） days）. Treating mice with DL-NBP （60 mg.kg-1.d-1） significantly decreased the progression rate of motor deficits and suppressed body weight reduction. Furthermore, we found that treating SOD1-G93A mice with DL-NBP （60 mg.kgl.d1） slowed the rate of MUNE reduction （P 〈0.01）. Motor neurons were remarkably preserved in the anterior horns in mice treated with DL-NBP （60 mg.kg-1d-1） at the stage of 19 weeks （P 〈0.01）. Treating mice with DL-NBP （60 mg.kg1.d1） significantly reduced CD11b immunoreactivity compared with vehicle control mice （P 〈0.05）. No significant effect was observed in mice treated with DL-NBP of 30 or 120 mg.kg-1.d-1. Conclusions The post-disease-onset administration of DL-NBP significantly prolonged survival and improved motor performance in SOD1-G93A mice. DL-NBP mav be a Dotential theraDeutic aaent for ALS.
