Protective Effect And Mechanism Of Intrathecal Injection Of SsAAV9-DAO On ALS Transgenic Mouse Model

Amyotrophic lateral sclerosis(ALS)is an adult onset,progressive,fatal neurodegenerative disease,which is characterized by the progressive degeneration of motor neurons in the cortex,brainstem and spinal cord,resulting in systemic muscle atrophy and weakness.The patients would eventually die of respiratory muscle atrophy within 3 to 5 years after the onset.There is no effective treatment due to the complexity of the pathogenesis so far.The Prevalence of ALS is about 5~7 / 100,000,of which about 90% of the cases is sporadic amyotrophic lateral sclerosis(SALS),and the remaining 10% is familial amyotrophic lateral sclerosis(FALS),about 20% of the FALS cases is due to Cu / Zn superoxide dismutase 1 gene(SOD1)mutation.SOD1-G93 A transgenic mice have a mutation in 93 loci of SOD1 gene,resulting in a substitution of glycine by arginine,which highly simulate the pathological and clinical characteristics of ALS patients,and has been widely used in the study of the pathological mechanisms of ALS.The underlying pathogenesis of ALS is not entirely clear yet,the known mechanisms including protein misfolding,excitotoxicity,mitochondrial damage,oxidative stress and so on,in recent years,some new evidence demonstrate that D-serine(D-Ser),a kind of endogenous co-agonist of Nmethyl-D-aspartate receptor(NMDAR),has been found to increased in ALS patients,and can aggravate the death of motor neurons.The main reason for the increase of D-Ser is the decrease of D-amino acid oxidase(DAO)activity.DAO is a kind of flavin protease with flavin adenine dinucleotide(FAD)as a cofactor.It can oxidize D-amino acids such as D-alanine,D-serine,D-proline and generate the corresponding keto acid.DAO is widely distributed in mammalian central nervous system(CNS),liver and kidney,and mainly exist in the brainstem and spinal cord in CNS.Arg199 residue of the DAO protein in mammals and lower organisms such as yeast,fungi and bacteria is close to the FAD binding site,which is located between Tyr228 and His307 residues,and plays an important role in the enzyme activity.Mutations in this site(R199W)can cause adverse effects in cells,leading to the occurrence and development of ALS.It is suggested that the high level of DAO in the spinal cord is mainly responsible for the degradation of D-Ser,which modulate the survival of motor neurons by regulating NMDAR mediated neurotoxicity.However,the level of DAO in lumbar spinal cord was decreased in both FALS patients and in SOD1-G93 A model mice.We assume that increasing the level of DAO in the spinal cord of the ALS mouse model may enhance the degradation of D-Ser,thereby reduce the neurotoxicity induced by NMDAR overe activation and protect motor neurons.Since DNA was discovered as a genetic material in 1952,the use of vectors(such as viruses)carrying DNA to treat inherited diseases has been emerged.Vectors commonly used in the transduction of the central nervous system include nanoparticles containing plasmid,retroviral recombinant vectors,adeno-associated virus(AAV),adenovirus(AV)and herpes simplex virus(HSV).AAV has become one of the safest and most commonly used viral vectors for the delivery of therapeutic genes.We apply single stranded adeno-associated virus serum type 9(ssAAV9)under the control of the CMV promoter,carrying the mice DAO gene(ssAAV9-DAO),to treat SOD1-G93 A transgenic mice in symptomatic stage by intrathecal injection,for observing the effects on behavior performance and pathologicalrepresentation after increasing DAO level in spinal cord of mice.And we explored the underlying mechanisms further.We have not seen similar reports before.Part one Expression and distribution of DAO in ALS mouse modelObjective: To provide the basis for viral vector system selection,DAO levels in spinal cord of SOD1-G93 A transgenic mouse of different periods,presymptomatic stage(60 days),symptomatic stage(90days)and end stage(120~140 days),were detected to observe the changes of DAO expression with disease progression.And we further observed the cell type that DAO exist in.Methods: We selected SOD1-G93 A transgenic mice provided by the United States Jackson laboratory as an animal model,and the corresponding primer sequences and identification methods were used for DNA identification of the mice.SOD1-G93 A positive mice of 60 days,90 days and end-stage were chose as the research object,and on the SOD1-G93 A negative mice of the same age were used as negative control.There are 6 groups in total(60d positive and 60 d negative,90 d positive and 90 d negative,positive and negative of end stage)and with 4 mice in each group.The expression of DAO in lumbar spinal cord of mice was detected by Western blot analysis.Another 3 90 d positive mice were perfused and fixed for vibrate cutting into slices of 25μm,and then the main cell types including DAO were observed by immunofluorescence confocal microscopy.Results:1 compared with the negative control,the DAO expression in the lumbar spinal cord of SOD1-G93 A mice was significantly reduced at end stage(P < 0.05),and there was no significant change in other periods.2 immunofluorescence staining showed that DAO was mainly expressed in motor neurons in the spinal cord of SOD1-G93 A mice,and there was a little expression in astrocytes and microglia.Conclusion: expression of DAO in spinal cord tissue of SOD1-G93 A mice decreased significantly in end stage,demonstrates that supplement of DAO in lumbar spinal may have protective effects on SOD1-G93 A mice.DAO primarily exist in motor neurons in lumbar spinal cord of SOD1-G93 A mice,which provides a basis for selection of neuron targeting vector system.Part two Study on the gene transduce efficiency of intrathecal injection of ssAAV9 in the spinal cord of SOD1-G93 A transgenic miceObjective: We administrated ssAAV9-GFP and ssAAV9-DAO into SOD1-G93 A transgenic mice via intrathecal injection,and observed the distribution and the transduction efficiency of the green fluorescent protein(GFP)in lumbar spinal cord of SOD1-G93 A mice.And the expression of DAO and D-Ser in lumbar spinal cord tissue was detected to determine the feasibility of this administration method.Methods: We selected SOD1-G93 A transgenic mice as the research object and gave them a single intrathecal injection of ssAAV9-GFP(1×1012 vg/kg)or ssAAV9-DAO(1×1012vg/kg).After 3 weeks of treatment,some mice were perfused and fixed with 4% paraformaldehyde,the spinal cord tissue was vibrate cut into slices of 25μm,and the others were sacrificed and the lumbar spinal cords were desected freshly.The expression and distribution of GFP was observed with confocal immunofluorescence technique,the expression of DAO in lumbar spinal cord of mice was detected using Western blotting method,and the expression of D-Ser in lumbar spinal cord of mice was observed using immunohistochemical staining.Results:1 immunofluorescence staining showed that there was more GFP expression in the lumbar spinal cord.In addition,there was a little GFP expression in sacral and thoracic spinal cord.But GFP expression was rarely seen in cervical spinal cord.2 Double immunofluorescence staining showed that GFP was co-localized with neurons,but not in astrocytes and microglia,and motor neuron transduction efficiency in lumbar spinal cord of SOD1-G93 A transgenic mice was about 12%.3 Western blotting results showed that DAO expression in lumbar spinal cord of ssAAV9-DAO treated mice increased significantly,while the immunohistochemical staining showed that compared with ssAAV9-GFP treated mice,D-Ser positive cells in lumbar spinal cord of ssAAV9-DAO treated mice were significantly decreased.Conclusion: GFP is mainly expressed in the spinal cord tissue near the injection point.GFP is mainly expressed in neurons and motor neuron transduction efficiency in lumbar spinal cord of SOD1-G93 A transgenic mice is about 12%.A single intrathecal injection of ssAAV9-DAO can increase the expression of DAO in lumbar spinal cord of SOD1-G93 A,and reduce the expression of D-Ser at the same time.The results demonstrated that this method could increase the level of DAO in the spinal cord of mice and the DAO has enzyme activity.Part three Study on the effects and the underlying mechanisms of intra-thecal injection of ssAAV9-DAO on the behavior of SOD1-G93 A transgenic miceObjective: We administrated ss AAV9-DAO into SOD1-G93 A transgenic mice by intrathecal injection,and observed the effects of over expression of DAO on the survival,body weight,rotation test and histopathological changes of SOD1-G93 A mice.And explored the underlying mechanisms further.Methods: We selected SOD1-G93 A transgenic mice offered by the Jackson laboratory as ALS animal model,and made DNA identification use the primer sequences and identification methods provided by the Jackson laboratory.Littermates matched SOD1-G93 A positive mice were selected as subjects.In their 90 days of age,we selected bodyweight matched littermates(the difference of mean starting weights is within 0.3g between groups),15 pairs of male and 15 pairs of female mice were randomly assigned to treatment group and control group.They were treated with ssAAV9-DAO and ssAAV9-GFP respectively by intrathecal injection,and bodyweight,rotation changes and survival were observed.Besides,several mice given the same intervention were remained for mechanism study.3 weeks after administration,3 pairs of them were perfused and fixed with 0.05% glutaraldehyde paraformaldehyde,the spinal cord tissue was vibrate cut into slices of 25μm.Expression of SMI-32,Iba-1 and GFAP were observed by immunohistochemical staining.Axonal damage of L5 anterior root sections was observed by toluidine blue staining.4 pairs of mice were sampled for fresh material,muscle fiber of gastrocnemius was observed by H E staining.Expression of TNF-α,IKBα,IKKβ,p-P65,p-Akt,IKKγ,Bcl-2,Bax,Caspase3,Caspase9,Cd11 b and GFAP in spinal cord was detected through Western blot analysis.Results:1 Compared with ssAAV9-GFP treated mice,lifespan in ssAAV9-DAO treated group prolonged 6 days,and the difference was statistically significant(n=30,P < 0.05).Among them,lifespan of female mice was extended by 8 days,and the difference was statistically significant(n=15,P < 0.05),lifespan of male mice was also extended by 8 days,but the difference was not statistically significant(n=15,P > 0.05).There was no significant difference in bodyweight and the rotation performance of the mice between the two female groups.The rotation performance in ssAAV9-DAO treated female mice was transitorily improved at the age of 110 days(P < 0.05).2 Immunohistochemical staining results showed that,compared with ssAAV9-GFP treated group,SMI32 expression in spinal cord tissue in ssAAV9-DAO treated mice was increased,and expression of GFAP and Iba-1 was decreased.Western blotting results showed that compared to the control group,expression of Cd11 b and GFAP in lumbar spinal cord tissue in the ssAAV9-DAO treated mice decreased at the same time.In conclusion,number of motor neurons in spinal cord of the treatment group was significantly increased compared with the control group,while the proliferation of astrocytes and microglia was significantly alleviated than that of the control group.3 Toluidine blue staining of L5 ventral roots indicated that the number of remaining axons was significantly higher in the DAO-treated mice than in GFP controls.Furthermore,HE staining of the gastrocnemius demonstrated that there was more severe muscle degeneration,including atrophic fibers and central nuclei,in the GFP-treated mice.And these pathological changes were improved in ssAAV9-DAO treated group.4 Western blotting results of lumbar spinal cord showed that compared with the ssAAV9-GFP group,expression of IKKβ and IKKγ,the positive regulator of NF-κB signaling pathway,in the ssAAV9-DAO group was decreased significantly.While expression of IKBα,the negative regulatory factor,was significantly increased.In addition,expression of p-P65,TNF-α,which reflects activation of the NF-κB signaling pathway,was significantly decreased(P < 0.05).At the same time,the expression of pro-apoptotic factors such as Bax,Caspase3 and Caspase9 was significantly reduced in the ssAAV9-DAO group,and the anti-apoptotic factor Bcl-2 was significantly increased,and the expression of p-Akt was increased(P < 0.05).Conclusions: A single intrathecal injection of ssAAV9-DAO can prolong the lifespan of symptomatic SOD1-G93 A mice,but there is gender difference,and the cause of this difference is not completely clear.However,there was no significant improvement in body weight and rotation performance after ssAAV9-DAO treatment.ssAAV9-DAO treatment can reduce the loss of motor neurons and the proliferation and activation of microglia and astrocytes in lumbar spinal cord of SOD1-G93 A mice.Besides,ssAAV9-DAO treatment has some protective effects on the axon and muscle fiber.The protective effects of DAO may be related to its inhibition of the activation of NF-κB,then inhibit the inflammatory response,and inhibition of the phosphorylation of Akt and decrease the apoptotic response.The exact mechanism remains to be further studied.