The Effect And Mechanism Of CX3CL1/CX3CR 1 In ALS Mice Model

Amyotrophic lateral sclerosis?ALS? is a fast-progressing neurodege-nerative fatal disease characterized by the death of motor neuron?MN?in the brainstem,spinal cord,and motor cortex.ALS is classified as two forms:sporadic?90-95%? and familial-type ALS?FALS??5-10%?.sporadic defined as having no obvious genetically inherited component.FALS defined as having family history of the disease.Despite genetic differences,these two forms of ALS are clinically indistinguishable.The most common symptoms are muscle atrophy,twitching and paralysis.In the most advanced stages,within 3-5 years of disease onset,ALS patients will succumb to the disease due to dyspnea and dysphagia.However,there are no effective cure methods for ALS.Nearly 20% of familial cases are associated with gene mutation of Cu²⁺Zn²⁺ superoxide dismutase?mSOD1?.Transgenic mSOD1 mouse are used as the gold standard in ALS research.In both human ALS and the transgenic mSOD1 mouse,neuroin-flammation and autophagy play an important role in the pathogenesis of ALS.The CX3CR1 is expressed primarily on microglia in the central nervous system?CNS?.The specific ligand for CX3CR1 is the chemokine CX3CL1,which is expressed on neuronal cells.The CX3CL1/CX3CR1 axis,have mainly been studied in mediating microglial activities.The microglia branches continuously survey neuronal surfaces in the healthy brain,when neuronal damage occurs,microglia rapidly react to protect or to eliminate neurons.CX3CL1/CX3CR1 signaling is involved in this process,modulating different aspects of microglia biology important for neuron protection,like the production of soluble factors directly involved in neuron survival,the modulation of phagocytic activity.In Alzheimer's disease?AD?,CX3CR1deficient microglia show increased phagocytic activity that markedly attenuates the extent of amyloid deposition.Knocking down CX3CR1 in these mice rescued this neuronal loss suggesting that microglia play a role in A?-induced neuronal death.In mouse experimental allergic encephalomyelitis?EAE?,the disruption in CX3CL1 signaling that occurs on Bone marrow cells in CX3CR1^-/- mice show an altered control of NK cells entry into the brain that contributes to increased demyelination,axonal damage and reduced calbindin positive neurons in the cerebellum in these animals CX3CR1 plays the opposite role in different animal models,which has aroused our great interest in CX3CR1.In amyotrophic lateral sclerosis?ALS?,in the lumbar spinal cords of CX3CR1^-/- mice show more neuronal cell loss.There is no more research about CX3CR1^-/- mice with ALS.In this study,we investigate the role of the CX3CL1/CX3CR1 in ALS pathology and may identify a new therapeutic target to modulate microglial activation and slow the progression of ALS.Part one Changes of CX3CR1,CX3CL1,inflammatory cytokines and autophagy in ALS mice modelObjective:We observed the expression of CX3CR1,CX3CL1,inflammatory cytokines and autophagy proteins of G93A-SOD1transgenic mice?male? at three periods:pre-onset,onset and end-stage.Methods:G93A-SOD1 transgenic mice carrying a mutant human SOD1 gene containing the amino acid substitution Gly93?Ala were obtained from The Jackson Laboratory?Bar Harbor,ME,USA? and bred into a B6/SJL background.The mice were genotyped using PCR with DNA extracted from tail tissues.We selected three time points of onset,onset and end-stage for observation.Three to five mice were taken at each time point.The expression of CX3CR1,CX3CL1,SOD1 and inflammatory cytokines and autophagy related proteins were studied by western blotting.Results:1.All positive G93A-SOD1 transgenic male mice included in the experiment were identified by PCR;2.CX3CR1western blotting shows greatly increased in ES SOD1^G93A/CX3CR1^+/+ mice compared to pre-onset SOD1^G93A/CX3CR1^+/+ mice.CX3CL1western blotting shows greatly decreased in ES SOD1^G93A/CX3CR1^+/+ mice compared to pre-onset SOD1^G93A/CX3CR1^+/+ mice.NEUN western blotting shows greatly decreased in ES SOD1^G93A/CX3CR1^+/+ mice compared to pre-onset SOD1^G93A/CX3CR1^+/+ mice.CD11b and SOD1 western blotting show greatly increased in ES SOD1^G93A/CX3CR1^+/+ mice compared to pre-onset SOD1^G93A/CX3CR1^+/+ mice;3.IL-1? and INOS western blotting shows greatly increased in ES SOD1^G93A/CX3CR1^+/+ mice compared to pre-onset SOD1^G93A/CX3CR1^+/+ mice.ARGINASE-1 and TGF-? western blotting shows greatly decreased in ES SOD1^G93A/CX3CR1^+/+ mice compared to pre-onset SOD1^G93A/CX3CR1^+/+ mice;4.As ALS disease progresses,the autophagy pathway is impaired and abnormal aggregation of mutant SOD1 protein is increased.Part two Knockout of CX3CR1 gene influence the survival,onset and behavior of ALS mice modelObjective:Through the above experiments,we observed different changes of CX3CR1 and CX3CL1 protein expression over time.So we considered that CX3CR1 and CX3CL1 might be involved in the disease progression of ALS.Therefore,we selected SOD1^G93A/CX3CR1^-/- mice to observe the changes of body weight,survival time,onset period and behavior.Methods:Male G93A-SOD1 mutation(SOD1^G93A) transgenic mice 20 were bred with CX3CR1^-/- females.F1 nontransgenic/CX3CR1^-/-or nontransgenic/CX3CR1^+/- females were bred with SOD1^G93A/CX3CR1^+/- males to produce F2 SOD1^G93A/CX3CR1^-/-,SOD1^G93A/CX3CR1^+/-,SOD1^G93A/CX3CR1^+/+.Throughout the study,mice were housed under a 12:12h light/dark cycle with controlled temperature and humidity.Food and water were given twice a week,to ensure mice had enough food and water at all times for free access.Results:1.Using a defined indicator of terminal state and Kaplan-Meier analysis,survival was significantly reduced in male SOD1^G93A/CX3CR1^-/- mice as compared with male SOD1^G93A/CX3CR1^+/+ mice and without significant genotype-related difference in female SOD1^G93A/CX3CR1^-/- mice as compared with female SOD1^G93A/CX3CR1^+/+ mice;2.No significant delay in disease onset was found between SOD1^G93A/CX3CR1^-/- mice and SOD1^G93A/CX3CR1^+/+ mice;3.The records showed a worse function of paw in male SOD1^G93A/CX3CR1^-/- mice than the others as the footprints appeared closer and limping.Part three Knockout of CX3CR1 gene influence microglia and inflammatory cytokines of ALS mice and research the mechanismsObjective:survival was significantly reduced in male SOD1^G93A/CX3CR1^-/- mice as compared with male SOD1^G93A/CX3CR1^+/+ mice.CX3CR1 gene is involved in the progression of ALS disease.The CX3CR1 is expressed primarily on microglia in the central nervous system?CNS?.Therefore,we further observed the changes of microglia and inflammatory factors in the CX3CR1 knockout ALS mice model and studied the related mechanisms.Methods:We selected ES and pre-onset stage of male SOD1^G93A/CX3CR1^-/- mice and SOD1^G93A/CX3CR1^+/+ mice for research.The mice were genotyped using PCR with DNA extracted from tail tissues.The mice were deeply anesthetized with a lethal dose of Chloral Hydrate and perfused transcardially with saline,then 4%paraformaldehyde.We separated the intumescentia lumbalis from the spinal cord and sectioned 18?m thick using a Leica vibratome.IBA 1,NEUN,PP65,IKB-?and IKK-?in lumbar spinal cord were observed by immunofluorescence staining.The lumbar spinal cord tissue samples were rapidly frozen with liquid nitrogen.IL-1?,INOS,ARGINASE-1,TGF-?,TNF-?,P65,PP65,IKB-?and IKK-?in lumbar spinal cord were observed by western blot.Results:1.IBA-1immunohistochemistryshowsgreatlyincreasedin SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice.NEUN immunohistochemistry shows greatly decreased in SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice.CD11b western blotting shows greatly increased in SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice.NEUN western blotting shows greatly increased in SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice;2.IL-1??INOS western blotting shows greatly increased in SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice.ARGINASE-1?TGF-?western blotting shows greatly increased in SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice.The TNF-?was highly up-regulated in the lumbar spinal cord at disease pre-onset in SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice;3.IKB-?immunohistochemistry and western blotting shows greatly decreased in ES SOD1^G93A/CX3CR1^-/- mice compared to ES SOD1^G93A/CX3CR1^+/+ mice.Phospho-p65 and IKK-?immunohistochemistry and Western blotting shows greatly increased in ES SOD1^G93A/CX3CR1^-/- mice compared to ES SOD1^G93A/CX3CR1^+/+ mice.Part four Knockout of CX3CR1 gene influence the autophagy pathway in ALS mice modelObjective:Autophagy has been shown to inhibit the formation of inflammatory signaling protein complexes.Therefore,in this part of the experiment,we observed the expression of SOD1 protein in CX3CR1knockout ALS mice model and studied the mechanism of autophagy.Methods:We selected ES of male SOD1^G93A/CX3CR1^-/- mice and SOD1^G93A/CX3CR1^+/+ mice for research.The mice were genotyped using PCR with DNA extracted from tail tissues.The mice were deeply anesthetized with a lethal dose of Chloral Hydrate and perfused transcardially with saline,then 4% paraformaldehyde.We separated the intumescentia lumbalis from the spinal cord and sectioned 18?m thick using a Leica vibratome.P62,SOD1 in lumbar spinal cord were observed by immunofluorescence staining.The lumbar spinal cord tissue samples were rapidly frozen with liquid nitrogen.SOD1,TBK1,P-TBK1,OPTN,LC3 and P62 in lumbar spinal cord were observed by western blot.Results:1.P62,SOD1 immunohistochemistry shows greatly increased in SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice.The interaction between endogenous p62 and mutant SOD1 in ES SOD1^G93A/CX3CR1^-/- mice was increased compared to ES SOD1^G93A/CX3CR1^+/+ mice.SOD1 western blotting shows greatly increased in SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice;2.OPTN,P62 western blotting shows greatly increased in SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice.P-TBK1 and LC3-II/LC3-I western blotting shows greatly decreased in SOD1^G93A/CX3CR1^-/- mice compared to SOD1^G93A/CX3CR1^+/+ mice.Conclusions:1.Microglia in SOD1^G93A/CX3CR1^-/- mice appear to switch from an M2microglial phenotype observed at the beginning of pathology to more an M1phenotype as disease advances with increasing expressions of pro-inflammatory cytokines,including TNF-?,INOS and IL-1?.Activated microglia produces these inflammatory mediators,orchestrating neuroin-flammation,and neurodegeneration.Loss of CX3CR1 in male SOD1^G93A/CX3CR1^-/- mice induces NF-kB activation.NF-kB activation induces an inflammatory?M1? microglia phenotype that causes MN death.2.Loss of CX3CR1 in male SOD1^G93A/CX3CR1^-/- mice impaired autophagy and further exacerbated SOD1 aggregation.Misfolded and aggregated SOD1 proteins may promote pro-inflammatory M1 microglia,amplifying motoneuron injury.3.The combined effect of above cause significantly reduced of survival inmaleSOD1^G93A/CX3CR1^-/- miceascomparedwithmale SOD1^G93A/CX3CR1^+/+mice and a worse function of paw in male SOD1^G93A/CX3CR1^-/- mice than the others as the footprints appeared closer and limping.