| BackgroundAmyotrophic lateral sclerosis(ALS)is a neurodegenerative disease caused by the loss of upper and lower motor neurons.One of the pivotal pathological markers in diseased neurons is the mislocalization of disease-associated proteins and the formation of cytoplasmic aggregates of these proteins and their interactants due to defects in protein quality control system.This apparent imbalance in cellular protein homeostasis may be a key factor in the death of motor neurons in patients with advanced disease.Abnormal folding and aggregation of TAR DNA-binding protein43(TDP-43)in the brain and spinal cord is the main pathological manifestation of ALS.TDP-43 is a widely expressed and highly conserved nuclear protein with a variety of important functions in regulating RNA metabolism,including translation,alternative splicing,and transport.The autopsy of ALS patients found that under pathological conditions,TDP-43 can migrate from the nucleus to the cytoplasm and form hyperphosphorylated and ubiquitinated cytoplasmic inclusions.Autophagy is the major protein degradation pathway involved in the clearance of protein aggregates and damaged organelles,avoiding misfolding and abnormal aggregation of proteins,and is essential for maintaining the normal function of the central nervous system(CNS).Abnormal autophagy was observed in many neurodegenerative diseases including ALS,and the regulation of autophagy in ALS patients may affect the deposition of pathological TDP-43,which may be an effective treatment of ALS.Objective1.To demonstrate the injection of adeno-associated virus(AAV)vector,AAV9-NSE-hTDP-43,can induce the deposition of pathological TDP-43 in the motor cortex of model mice.2.To demonstrate that in the background of autophagy defects,pathological aggregates of TDP-43 are more likely to form.3.To demonstrate that the deposition of pathological TDP-43 is able to induce ALS-like dyskinesia in Atg5 KO transgenic mice.Methods1.Construct the AAV9-NSE-hTDP-43 and AAV9-Null vectors.2.Male C57BL6/J and Atg5 KO transgenic mice with similar age and body weight were selected,and the mice of each strain were divided into 2 groups,15 in each group.The mice of each strain were stereotactically injected with 5 μl AAV9-NSE-hTDP-43 or AAV9-Null AAV vectors in the left motor cortex,respectively.3.Immunohistochemistry was used to detect the deposition of pathological TDP-43 in the motor cortex of mice after 1,2,3 months post-injection.4.Regularly check the body weight of each group of mice,and use the behavioral test methods such as rotating rod,footprint test,and hanging line experiment to regularly test the motor function of each group of mice.5.Detect the cortical motor evoked potential(cMEP)to demonstrate the injury of upper motor neurons.Results1.The mice in the motor cortex injected with AAV9-NSE-hTDP-43 vectors showed pathological TDP-43 deposition.2.Atg5 KO mice injected with the AAV9-NSE-hTDP-43 vector in the motor cortex observed a more abundant accumulation of pathological TDP-43 than C57BL6/J mice.3.When pathological TDP-43 deposition occurs in the motor cortex of mice,weight loss and motor dysfunction occur gradually.Conclusions1.Injected AAV9-NSE-hTDP-43 vectors into the motor cortex of mice can induce the pathological deposition of TDP-43.2.In the background of autophagy defects,TDP-43 pathological aggregates are more likely to form.3.The deposition of pathological TDP-43 was able to induce ALS-like dyskinesia in Atg5 KO transgenic mice. |
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