Spreading Of TDP-43 Pathology Via Pyramidal Tract Induces ALS-like Phenotypes In TDP-43 Transgenic Mice

BackgroundAmyotrophic lateral sclerosis is a progressively developing motor neuron degenerative disease.The etiology and pathogenesis of amyotrophic lateral sclerosis are still unclear.Patients with ALS have severe clinical symptoms and extremely poor prognosis.The abnormal folding and extensive accumulation of Transactive response DNA-binding protein 43 kDa(TDP-43)in the cytoplasm of motor neurons is the main pathological sign of sporadic ALS.A large number of studies have shown that TDP-43 protein has the properties of prion protein,which can be like a "seed" to induce normal TDP-43 protein to fold abnormally and aggregate to form inclusion bodies,and spread from cell to cell.Postmortem histological studies have identified stereotypical spreading patterns of phosphorylated TDP-43(pTDP-43)pathology,and these findings suggest that pTDP-43 positive inclusions may spread along axons in the central nervous system.The formation and dissemination of TDP-43 positive inclusion bodies in the central nervous system may be the underlying pathological mechanism of ALS.Studies performed in vitro have demonstrated that pathological TDP-43 could transmit from cell to cell along axon in a self-templating manner.Unfortunately,experimental evidence in vivo is still largely lacking.No animal model has been established to test whether the spreading of TDP-43 pathology could induce the pathological changes and clinical phenotypes of ALS.Objective1.To demonstrate the hTDP-43 fibrils could be made from pure recombinant hTDP43 protein and have the seeding property;2.To demonstrate that the local injection of hTDP-43 fibrils in the primary motor cortex(M1)of hTDP-43 transgenic mice could induce the pathological formation of TDP-43 in situ;3.To demonstrate that TDP-43 pathology induced by the injection of hTDP-43 fibrils in the M1 area of hTDP-43 transgenic mice could spread anteriorly along the pyramidal tract;4.To demonstrate that the spreading of pathological TDP-43 anterogradely along the pyramidal tracts in hTDP-43 transgenic mice could induce ALS-like phenotypes.Method1.Thyl-e(IRES-TARDBP)1 mice was constructed using CRISPR/Cas9 technology;2.TDP-43 fibrils was synthesized from TDP-43 monomers and identified by transmission electron microscopy,ThT staining,Western blot,and PK resistance experiments;3.TDP-43 preformed fibrils(TDP-43 PFFs)were injected into the left caudal part of M1 region(Ml-C)of Thyl-e(IRES-TARDBP)1 mice(TDP-43 PFFs M1-C mice)or the left lateral part of M1 region(M1-L)of Tyl-e(IRES-TARDBP)1 mice(TDP-43 PFFs M1-L mice),respectively;4.Detecting the TDP-43 pathology,neuron loss and brain atrophy in different brain regions and spinal cord of mice at different time points;5.Evaluating the motor function,swallowing function,gait,learning and memory function,and electrophysiological changes of mice at different time points.Result1.TDP-43 fibrils could be synthesized from solely pure recombinant TDP-43 protein and have the ability to promote fibrillization of surrounding monomeric TDP-43;2.TDP-43 PFFs injected Thy1-e(IRES-TARDBP)1 mice developed obvious pTDP43 pathology in situ as 1 month post injection(mpi);3.TDP-43 PFFs injected Thyl-e(IRES-TARDBP)1 mice developed time-depended TDP-43 pathology,neuron loss,and brain atrophy in the brain regions and spinal cord along pyramidal tract;4.TDP-43 PFFs injected Thyl-e(IRES-TARDBP)1 mice developed ALS-Iike syndromes,including motor dysfunction,electrophysiological abnormalities,learning and memory deficits in TDP-43 PFFs M1-C mice,and swallowing disorder in TDP-43 PFFs M1-L mice in a time-dependent manner.Conclusion1.TDP-43 fibrils could be synthesized from solely pure recombinant TDP-43 protein and have the seeding property.2.Injection of hTDP-43 fibrils in the M1 region of Thyl-e(IRES-TARDBP)11 mice could induce TDP-43 pathology in situ;3.TDP-43 pathology induced by the injection of hTDP-43 PFFs into the M1 region in Thyl-e(IRES-TARDBP)1 mice could spread anteriorly along the pyramidal tract;4.The spreading of TDP-43 pathology along pyramidal tract in Thy1-e(IRESTARDBP)1 mice could induce ALS-like phenotype.