Diffusion Of Pathological TDP-43 Along Corticospinal Tract Axons Induces ALS Like Phenotype In Atg5^+/- Mice

BackgroundAmyotrophy amyotrophic lateral sclerosis(ALS)is a kind of control voluntary muscle motor neuron death due to the progressive neurodegenerative disease.ALS affects both the upper motor neuron(UMN)in the cerebral cortex and the lower motor neuron(LMN)in the brainstem and spinal cord.The main clinical manifestations are progressive muscle weakness,spasms,stiffness,convulsions,and atrophy,which in severe cases lead to speech problems,dysphagia,and eventually dyspnea.Some people with ALS may also show mild cognitive or behavioral dysfunction in the middle and late stages.Behavioral dysfunction includes repeated phrases or gestures,apathy,and loss of inhibition.Cognitive impairments include language impairments,executive impairments,social cognition and verbal memory impairments.However,the sensory and autonomic nervous systems are generally unaffected,and most people with ALS see no significant decrease in hearing,vision,touch,smell,or taste.Currently there is no treatment that can cure or prevent the progression of ALS.Amyotrophic lateral sclerosis is mainly characterized by phosphorylated TDP-43(pTDP-43)positive inclusion bodies in neurons and glial cells.TDP-43 is a highly conserved and widely expressed RNA/DNA-binding protein belonging to the heterogeneous cell nuclear ribonucleoprotein(HnRNP)family.TDP-43 plays a key role in cell function,including the regulation of RNA metabolism,mRNA transport and microRNA formation.TDP-43 plays an important role in the formation of stress particles and protects nerve cells from oxidative stress and other cellular damage.According to the data,TDP-43 protein is present in the nucleus under normal conditions.When the gene mutation leads to the dysfunction of TDP-43,the abnormal aggregation of TDP-43 in the cytoplasm results in the decreased content of TDP-43 protein in the nucleus and the loss of its function.It eventually leads to RNA metabolism,mitochondrial dysfunction,abnormal cytoskeleton and muscle linkage,and autophagy dysfunction,etc.Thus,mutations and dysfunction of TDP-43 lead to severe neurological disorders.The research results show that the TDP-43 mainly through the ubiquitin-proteasome system and lysosomal degradation pathway is cleared,this phenomenon was observed in many neurodegenerative diseases,including muscle atrophy degeneration amyotrophic lateral sclerosis,frontal temporal lobe(FTLD),Alzheimer’s disease(AD)and Huntington’s disease(HD),etc.Therefore,autophagy is essential to maintain the stability of central nervous system(CNS)function.Autopsy results of amyotrophic lateral sclerosis showed that under pathological conditions,TDP-43 in the nucleus migrated into the cytoplasm and aggregated,and hyperphosphorylation and ubiquitination formed inclusion bodies to induce cellular neurotoxicity.However,the pathogenesis of ALS remains largely unknown.In view of the basis of the above studies,the present study intended to induce pathological TDP-43 aggregation by injecting TDP-43PFFS into the motor cortex of ATG5+/-mice,and further investigate the mechanism of pathological TDP-43 aggregation spread along the axons of the corticospinal tract.ObjectiveIn this study,by injecting TDP-43PFFs into the motor cortex of Atg5+/-mice,it was proved that the autophagy-deficient mice were more likely to induce the formation of pathological TDP-43 aggregates,and the mechanism of the spread of pathological TDP-43 aggregates along the axons of the corticospinal tract was explored.It was further demonstrated that TDP-43PFFs could induce the motor phenotype similar to ALS in ATG5+/-mice.Methods1.The TDP-43 monomer with a concentration of 0.5mg/ml was stirred by a vortex mixer at a speed of 600rpm for 2h at 37℃ to obtain TDP-43PFFs.The physical properties of the TDP-43 fiber were examined by transmission electron microscopy.2.Male Atg5+/-mice aged 8 weeks were used as the experimental group and C57 mice of the same age as the control group.Under the stereoscopic locator,TDP-43PFFs,Phosphate Buffer Saline(PBS)and PBS buffer were injected into the left primary motor cortex(M1)of ATG5+/-mice on the 5th layer or the 7th cervical spinal cord segment(C7)on the left anterior horn of ATG5+/-mice,5μl each,at a rate of 0.2μ/min.3.In every 2 weeks before and after injection of TDP-43 PFFs needle electromyography,turn bar test,drawing experiment,footprint experiment and behavioral science,movement function index to evaluate two groups of mice,measurement of two groups of mice weight every week,and each month after the injection of execution groups of mice,the anatomy of the biceps and H&E staining to observe the fiber morphology.After perfusion,the brain and spinal cord were collected for immunohistochemistry and western blot to quantitatively detect the level of pathological TDP-43 inclusion bodies in the tissues of mice.The date and cause of death of each mouse were recorded.4.SPSS24.0(IBM,Armonk,New York,USA)was used for statistical analysis.The behavioral and electrophysiological data of mice were expressed as mean ± standard deviation(SD).Two-way analysis of variance was used for comparison between groups.Kaplan-Meier survival curve was used to analyze the incidence and survival.Log-rank sum test and χ2 value were used for significance test.The significance of all statistical tests were p<0.05.Results1.pTDP-43 immunoreactive(pTDP-43-ir)was detected 2 months after TDP-43 PFFs prepared in vitro was injected into the 5th layer of the left M1 or the left anterior horn of C7 in ATG5+/-mice.pTDP-43 immunoreactive(pTDP-43-ir)was detected in neurons and glial cells of the 2nd,3rd,and 5th layers in the area surrounding the injection of M1.In ATG5+/-mice injected with C7-TDP-43 PFFs,pTDP-43-ir pathological changes were observed in neurons adjacent to the anterior horn of the left spinal cord after injection.2.Compared with the control group,the pathological manifestations of pTDP-43ir in the cerebral cortex,neurons and glial cells were mainly distributed in the 5th layer of the left M1 in the experimental group when the mice were injected with TDP-43PFFs month post-injection(MPI)for about 6 months;In hippocampal CA1 region,pTDP-43-IR staining of left neurons and glial cells was deeper than that of contralateral cells.pTDP-43-ir was detected in the medulla oblongata,in the nerve fibers and glial cytoplasm of the hypoglossal nucleus on both sides,and in the left corticospinal tract.Deposits of pTDP-43 neurons and glial cells are seen on the right side of the enlarged anterior horn.In ATG5+/-mice injected with C7-TDP-43PFFs,pTDP-43 staining was more pronounced in neurons and glial cells in the right hypoglossal nucleus,hippocampal CA1 region,and layer 5 of M1.pTDP-43-ir was detected in the nerve fibers of the left corticospinal tract and in the glial cytoplasm at about 5 MPI injection.In addition,we injected TDP-43PFFs into C7 in C57BL/6 mice.It took about 18 MPI to detect the formation of TDP-43-ir pathology in the neurons and glial cytoplasm of the fifth layer of M1.3.At about 2 MPI in mice injected with M1-TDP-43PFFs,spontaneous activity,including fibrillation potential,flutter potential and positive apex wave,was detected in the right biceps brachii,T10 paraspinous muscle,tibialis anterior muscle and gastrocnemius muscle.At 5 MPI,the biceps brachii,T10 parspinous muscle,tibialis anterior muscle and gastrocnemius muscle all showed abnormal spontaneous activity,and there was no significant difference between the two sides.Compared with the control group,the M1-TDP-43 PFFs ATG5+/-injection mice in 2,5,and 7 MPI moto unit action potentials(MUAP)average amplitude and duration increased significantly.Biopsies were performed on bilateral biceps in ATG5+/-mice injected with M1-TDP-43PFFS at 7 MPI.H&E staining of the right biceps showed muscular atrophy,round muscle fibers,and central nuclear muscle fibers.4.The life span of ATG5+/-mice injected with TDP-43pFFs was significantly shortened compared with the control group injected with PBS.ATi5+/-mice injected with C7-TDP-43pFFs gradually lost weight approximately 8 weeks after injection,performed poorly on the rod-turning and wire-hanging tests,and showed significant deficits in muscle strength,motor coordination,and balance.In the footprint test,the step spacing was significantly shortened and the base width was significantly increased.About 10 weeks after injection,there was a significant decrease in motor ability during the rod rotation test and the wire hanging test.ConclusionBy injecting TDP-43PFFs into the 5th layer of left M1 or the left anterior horn of C7,phosphorylated TDP-43-positive inclusion bodies were induced to form in ATG5+/-mice and spread anterograde and retrograde along the axons of the corticospinal tract.ATG5+/-mice showed ALS like neuropathology and motor phenotype,demonstrating that autophagy deficiency promotes the formation and diffusion of pathological TDP-43 in vivo.