The Establishment And Phenotype Studies Of A Transgenic Mice Model Carrying A New Human Mutant SOD1Gene

BackgroundAmyotrophic lateral sclerosis(ALS)，also known as “Lou Gehrig’s disease”or“gradually paralyzing disease”, is a severe neurodegenerative disease which showingprogressive motor dysfunction and finally demise. Pathology studies showed the depositionof insoluble proteins in ALS patients. The ALS patients have a range of clinicalmanifestations including progressive motor dysfunction, difficulty swallowing and muscleatrophy. The patients usually died in3to5years after diagnose. ALS is reported from allover the world. About80%to90%of ALS cases are reported as SALS, and the rest10%to20%as FALS which has a familial history. At present, there is no effective treatment exceptthat Rilutek can be slightly slow down disease progression. Through in-depth research onALS, we can reveal the pathogenesis of ALS and look for the treatment or prevention of thetheoretical foundation.FALS can be used as a good disease model for the study of the pathogenesis of ALS,because FALS and SALS patients present the same clinical manifestations, EMG andhistopathological features. About20%of FALS is induced by the mutation of Cu/Znsuperoxide dismutase (SOD1) gene which located on21q. SOD1is a major enzymecatalyzing the disproportionation of superoxide anion in aerobic organisms. The maincatalytic chain is O2-+O2+2H+→H2O2+O2. Since the mutation of SOD1associatingwith FALS was reported by Rosen in1993, over160SOD1mutations associated with ALShave been discovered (http://alsod.iop.kcl.ac.uk/). About130kinds of cell or animal modelswere established about mutant SOD1. These cell or animal models were used in the studiesof ALS widely.Most of the SOD1mutations are the substitution of a certain amino acid in proteincaused by missence mutations. Glycine located in93was substituted by Alanine, which iscalled the mutant of G93A, is the most frequent mutations. Subsets of the mutations are non-missence mutations which result in premature termination of translation of someprotein-coding sequence deletions. The different SOD1gene mutations cause differentFALS phenotypes including the duration, age of onset and the severity of the disease. Theseresults suggested that the main factors affecting ALS phenotype may be the mutant SOD1genotype.The early studies suggested that the pathogenesis of FALS resulted from SOD1genemutation is due to loss-of-function of SOD1enzyme. The activation and the ability to resistoxidative damage of SOD1are decreased. These effects lead to neuronal damage. Theresults of clinical studies showed that the activity of SOD1in the red blood cells of FALSpatients which expressed mutant SOD1gene is decreased about50to60%. The activity ofSOD1in the red blood cells of the mutant SOD1carrier is also decreased significantly. Thestudy of the mice which knocked out the SOD1gene indicated that the knockout gene micedo not have any motor neuron disease onset. These results suggested that the cytotoxicity ofmutant SOD1maybe gain some new functions by gain-of-function. These functions lead tothe damage of neuron. It is also believed that the conformation of mutant SOD1is unstableand misfolded which causes the formation of protein aggregates. The mutant proteininteracting with other proteins causes the physiological and pathological changes.The group led by Professor Shi Shugui has discovered an autosomal dominant FALSfamily including four generations and14patients in Chongqing. The patients display avaried phenotype from what has been reported before. By Single-strand conformationpolymorphism analysis (PCR-SSCP) and genome sequencing, the group has found that basepair insertion was occurred in the coding region of exon2. This mutation leading to thechanges of the transcription and the position of translation reading frames. The numbers ofamino acid residues of exon2reduced from32to12. The mutant SOD1protein becomes ashort peptide composed of35amino acids.In previous experiments, a new mutant SOD1gene which maybe caused the familydisease has been confirmed. The activity of neurons carrying mutant SOD1(mSOD1) wassignificantly decreased. The neuronal carrying mutant SOD1gene resulted in themorphological changes of damages. The secondary and tertiary structures of the mutantSOD1gene encoding protein were gained. To establish a mice model carrying the mutantSOD1gene, the fertilized mice zygote, which was be injected into the vector carrying mSOD1gene, was transplanted into pseudo-pregnant female mice. The mice modelcarrying the mutant SOD1gene shall provided a good experimental model for subsequentstudy of the pathogenesis of ALS. The studies of phenotype of transgenic mice carrying ahuman mutant SOD1gene may reveal the role of the mutant SOD1in the Chongqing ALSfamily.PurposeTo ascertain the association between mSOD1and ALS preliminarily, a mice modelcarrying a new mSOD1was established by pronuclear microinjection technique. Thephenotypes of transgenic mice were observed by the approaches of motion functiondetection, pathology study, proteomics study, and immunohistochemics.MethodsTo construct a PCI plasmid vector carrying mSOD1gene and inject it into the micezygote, pronuclear microinjection technique was deployed. The offspring was detected byPCR. The movement function of transgenic mice was tested by tail suspension test,Rota-Rod test, Open-field test and Footprint analysis. In mice spinal cord and cortex motorneuron, the mSOD1protein was detected with Western-Blot. The expression of mSOD1intransgenic mice spinal cord and cerebral cortex motor neuron was detected withimmunohistochemics. The ultra structural pathology of mice spinal cord and cerebral cortexmotor neuron was observed by transmission electron microscopy.ResultsAmong them,2transgenic founder mice,2transgenic F1mice,2transgenic F2miceand1transgenic F2mice carrying the mSOD1gene were identified by PCR. Transgenicmice movement dysfunction was observed at about240days since the transgenic miceoffspring was burned. In the tail suspension test, transgenic mice cannot fully extended hindlimbs. In Rota-Rod test, the time of transgenic mice on the wheel is significantly shorterthan that of wild mice.(59.17±26.27sec vs.171.83±20.98sec, P <0.01). The limping anddragging movements of transgenic mice hind limbs appeared about8months after theywere born. The space of mice foot showed that the transgenic foot space is significantlysmaller than that of wild mice (34.83±9.72mm vs.52.78±7.22mm, P<0.01). The resultsof Western-Blot showed that mSOD1encoded protein were expressed both in transgenicmice spinal cord and cerebral cortex. The results of immunohistochemistry showed that mSOD1were expressed in the cytoplasm of the spinal cord and cerebral cortex motorneurons. The dense and insoluble particles were founded in the cytoplasm of neurons bytransmission electron microscopy.ConclusionsThe mSOD1transgenic mice were successfully established. The transgenic miceshowed phenotype was similar to ALS patients. The results demonstrated that mSOD1is anew pathogenesis of ALS.