New Pathogenesis And Treatment Research Of Amyotrophic Lateral Sclerosis Basing On SOD1^G93A Mice

Amyotrophic lateral sclerosis(ALS)is a fatal neurodegenerative disease characterized by progressive loss of both lower and upper motor neurons(MNs)in the brain,brainstem and spinal cord,resulting in progressive weakness,atrophy of voluntary skeletal muscles and bulbar paralysis,etc.The majority of patients die within 3-5 years from respiratory failure.There is no cure for ALS,except that riluzole and edaravone can slightly delay disease progression.The exact etiology and pathogenesis of ALS have not been fully understood.About 10%of ALS is familial with dominant inheritance.Mutation in superoxide dismutase 1(SOD1)gene is the first discovered genetic cause for ALS.SOD1G93A mice carrying a mutant human SOD1 gene are able to develop similar symptoms and pathological features as ALS patient.This mouse model has been widely used in the field of ALS research.Here,we did the work from three aspect as following:(1)we investigated new pathogenesis of SOD1G93A transgenic mice by RNA sequencing(RNA-seq).(2)We investigated the dynamic changes of chemokine(C-X3-C motif)ligand 1(CX3CL 1)/CX3CR1 axis during microglial activation in SOD1G93A mice.(3)The therapeutic effect and molecular mechanism of carbamazepine(CBZ)were also studied for SOD1393A mice treatment.we had some new findings on the pathogenesis and treatment of ALS,which can provide reference for the clinical diagnosis and treatment of ALS.Part 1:Transcriptomics study of amyotrophic lateral sclerosis mouse model by RNA-sequencingIn this part,we compared the transcriptome of the forebrain(FB)and lumbar spinal anterior horn(AH)regions between male SOD1G93A mice and age-matched wild type(WT)mice at the asymptomatic stage(40 days)and symptomatic stage(90 days)of disease by RNA sequencing(RNA-seq),aiming to identify the differentially expressed genes(DEGs)and differentially alternative splicing genes(DASGs).Then all of the DEGs and DASGs were used for GO function clustering and KEGG pathway analysis to find the main pathological pathway of ALS.Several DEGs were further identified by real-time quantitative PCR(qPCR)and immunofluorescent staining.The results showed that:(1)Most DEGs were up-regulated in AH region from the symptomatic SOD1G93A mice,which had 663 DEGs(469 up regulated,194 down-regulated)when compared to asymptomatic SOD1G93A mice,and had 524 DEGs(404 up-regulated,120 down-regulated)when compared to age-matched WT mice.GO function clustering and KEGG pathway analysis showed that up-regulated DEGs were significantly enriched in inflammatory,immune and defense response pathway.(2)The symptomatic SOD1G93A mice had a large number of DASGs in AH region which were predominately involved in cytoskeleton,synaptic,and GTPase activity pathway.(3)The level of some inflammatory genes(BST2,Clqa,C3,CLEC7A,LGALS3,LY86)were verified by qPCR and immunofluorescent staining.(4)Immunofluorescent staining showed that TDP-43 protein aggregated in cytoplasm of MNs in AH region of SOD1G93A mice.(5)A few of overlapping genes were found between DEGs and DASGs.Therefore,inflammation,RNA alternative splicing,and cytoskeletal abnormalities may be involved in the pathogenesis of SOD1G93A mice.These results suggested a multi-factor mechanism in ALS progression.Part 2:Dynamic changes of CX3CL1/CX3CR1 axis during microglial activation and motor neuron loss in the spinal cord of ALS mouse modelNeurons can express chemokine(C-X3-C motif)ligand 1(CX3CL1),which serves as a signaling molecule mediating microglial activation via its sole receptor CX3CR1 in microglia.Activated microglia can protect or damage MNs.Here,qPCR,western blot and immunofluorescent staining were used to examine the dynamics of CX3CL1/CX3CR1 axis and microglia activation in the spinal cord of SOD1G93A mice at 40,60,90 and 120 days of age.All the results revealed that CX3CL1 was elevated at 40 days but decreased in the anterior horn region of spinal cords in ALS mice at 90 and 120 days.Its receptor CX3CR1 was increased at 90 and 120 days.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,the M1-related cytokines were persistently increased at 120 days,and the up-regulated M2-related cytokines started to decline in ALS mice at 120 days.Our data revealed dynamic changes in CX3CL1/CX3CR1 axis and microglial activation are involved in the pathogenesis of SOD1G931 mice.The neuroinflammatory mechanism is one of the important pathogenesis of ALS,and regulation of neuroinflammation may improve ALS.Part 3:Therapeutic effect and mechanism of carbamazepine on SOD1G93A mouse modelCarbamazepine(CBZ)could activate autophagy and promote the clearance of mutant protein aggregates.In this part,SOD1G93A mice were orally administered with CBZ from 64 days of age.We found CBZ treatment could delay the disease onset and extend lifespan of SOD1G93A mice by about 14.5%and 13.9%respectively.Furthermore,CBZ treatment reduced MNs loss by about 46.6%and ameliorated the altered muscle morphology and neuromuscular junction.Much more interestingly,mechanism study revealed that CBZ treatment activated autophagy via AMPK-ULK1 pathway and promoted the clearance of mutant SOD1 aggregation.All the results indicated that CBZ has the potential for ALS treatment and is worthy of further research.