The Pathological And Behavioral Study In An Amyotrophic Lateral Sclerosis Poly-PR Transgenic Mouse Model

Aims: Amyotrophic lateral sclerosis(ALS)is a fatal neurodegenerative disease,and the C9ORF72 genetic mutation accounts for a remarkable percentage of familial ALS and sporadic ALS.It has been reported that poly-PR,a protein translated from C9ORF72 repeat expansions,contributes to the cellular toxicity in vitro,and that the specific expression of polyPR in Drosophila eyes causes severe neurodegeneration.However,it remains unknown whether poly-PR causes neurodegeneration,or induces selective neurotoxicity in specific neurons in vivo,or produces phenotypes in mice,which can mimic human c9orf72/amyotrophic lateral sclerosis/frontotemporal dementia(c9ALS/FTD).Methods: First,the plasmid of EGFP-(CCAAGA)28 expressing poly-PR was constructed and inserted into the Rosa 26 site in mouse using CRISPR/Cas9.The F0 chimeric mice were crossed with wild type mice to produce F1 heterozygous mice,and the produced mice were further crossed with Thy1-cre transgenic mice,leading to a specific expression of poly-PR in neuron.The cellular localization and the distribution of poly-PR in major brain regions was determined using immunohistochemical staining.Moreover,the distribution of poly-PR in major brain regions and peripheral tissues was further examined using real-time quantitative PCR(q RT-PCR).A series of behavioral tests,such as suspension tail test,cage behavioral test,rotarod test,balance beam test,footprint test and open field test,were performed to determine the motor functions of control and heterozygous mice.To determine whether poly-PR causes motor dysfunction in a sex-specific manner,the behavioral tests of male and female were measured,respectively.Besides,the disease progression in heterozygous transgenic mice was determined using rotarod test at 3,6,10,and 12 months of age.The body weight,brain weight and cerebellum weight of control and heterozygous mice at 2,5 and 12 months of age were measured respectively,and the neurodegeneration of control and heterozygous mice were determined using immunohistochemical staining.To explore the pathological mechanisms that GFP-PR28 causes neuronal deficiency,the RNA-sequencing analyses were performed and the differential expressed genes were further validated using immunohistochemical staining and Western blot analysis,and the cell processes enriched in Go pathway analysis were also validated with immunostaining.Results: Immunohistochemical staining showed that GFP-PR28 displayed nucleolar localization,major expression in neurons of the hippocampus,cortex,cerebellum,and spinal cord of heterozygous mice,but not in peripheral organs.The homozygous mice expressing poly-PR showed obvious smaller morphological volume than control mice at 20 days of age with progressively decreased body weight,as well as premature death,suggesting highly toxic of poly-PR.The heterozygous mice displayed progressive motor dysfunction,including increased numbers of hind limb foot slips in balance beam test,reduced stride length in foot print test,significantly decreased time on the rotarod.As aging,the heterozygous mice showed progressively less time on the rod compared with age-matched control mice,suggesting progressive neurodegeneration.Additionally,the heterozygous mice displayed anxiety-like behavior in the open-field test.Neuropathological analysis identified atrophied motor cortex and cerebellum,accompanied with decreased numbers of Purkinje cells and lumbar motor neurons,suggesting a correlation between motor abnormality and pathological changes.The RNA-sequencing analysis identified the downregulation of synaptic transmission-related genes and the upregulation of inflammatory-related genes.Consistently,immunohistochemical staining showed agedependent gliosis in the cerebellum and spinal cord of heterozygous mice.Conclusions: We have successfully constructed poly-PR transgenic mouse model,in which the specific expression of poly-PR in neuron causes motor neuron disease.Overall,our results showed that poly-PR transgenic mice mimic neuropathological features of c9ALS/FTD,which can be used as a tool for further mechanism research and drug screen.