The Effect Of TDP-43 In Axonal Transport Of Cortical Neurons And Pathogenic Mechanism

Background and purpose:Amyotrophic lateral sclerosis(ALS)is a neurodegenerative disease characterized by progressive and irreversible degeneration of upper motor neuron(UMN)and lower motor neuron(LMN).UMN death leads to hyper-excitability and spasticity,while LMN degeneration causes weakness,fasciculation and muscular atrophy,followed by progressive paralysis.This pathology has accelerated progression when compared to other neurodegenerative diseases,leading patient to death within about 2–5 years after the onset of disease symptoms.The emergence of riluzole can delay the progression of symptoms in some patients.It has not been widely used due to its high cost and unsatisfactory efficacy.Exploring the causes of ALS has become a hot spot in neurology.Frontotemporal dementia(FTD)is also a neurodegenerative disease,which is characterized by degeneration of the frontotemporal neuron cell body or degeneration of the cell body secondary to axonal injury,leading to personality changes,speech disorders,and behavior disorders.There is still no treatment method.The clinical manifestations of ALS and FTD can occur at the same time,and have the same pathological and genetic characteristics,and are now considered the part of the same disease spectrum.Mislocalization and abnormal deposition of TDP-43 in the cytoplasm of neurons(TDP-43 proteinopathy)is a pathological sign of amyotrophic lateral sclerosis(ALS)and frontotemporal dementia(FTD).TDP-43 is an important ALS pathogenicity-related protein discovered in recent years,but the pathological mechanism related to TDP-43 is still unclear.Previous studies have shown that TDP-43 assists in the processing and transport of transcripts in axons,and by modifying axon transport to enhance axon growth,it is expected to become a therapeutic target.Neurons are higly polarized,and their axons sometimes extend more than 1,000 times the diameter of their cell bodies.The mRNA transport and protein synthesis in the axons are particularly important for the maintenance of axon function and nutritional support,attract a lof of attention from the nervous community.The transport of neuronal mRNA firstly coupled to axons through RNA-binding protein(RBP)mRNA,and then recognizes the sequence and/or structure-specific signals present in the target mRNA.The combination of mRNA and RBP stabilizes the mRNA and transports it to the end of the axon to maintain the growth and function of the axon.Incorrect expression or dysfunction of RBP is related to severe neurodevelopmental dysplasia and neurological diseases.Based on the above theory,this study hypothesized that the failure of TDP-43 to transport mRNA to axons may promote neurodegeneration in ALS and FTD.The purpose of this study is to screen the target of TDP-43 transport in neuronal axons,confirm the effectiveness of the target,and detect the association with TDP-43 through the characteristics of the 5'TOP of ribosomal protein mRNA,and to study the ribosome The protective effect of protein mRNA on TDP-43 provides a certain experimental basis for understanding the mechanism of TDP-43 in TDP-43 protein diseases such as ALS and FTD.Research method:1.Use a special culture device to isolate neurites from primary cortical neurons in mice.2.Transfect mouse cortical neurons with two different sequences of TDP-43 sh RNA and control sh RNA.3.Collect cortical neuron axons,compare the expression of the two mRNAs,and screen the mRNA whose expression decreases in neuron axons when TDP-43 is down-regulated by microarray analysis.4.Mouse cortical neurons were laid flat in microfluidic chambers,and the axons were subjected to in situ hybridization of ribosomal protein mRNA and TDP-43 immunostaining.5.TDP-43 and ribosomal protein mRNA are co-expressed in Neuro 2a cells,and the amount of ribosomal protein mRNA co-precipitated with TDP-43 is measured by quantitative RT-PCR.6.Detect the co-precipitation of Rpl41 mRNA,Rplp1 mRNA,Rpl41 mRNA lacking 5'UTR,Rpl41 mRNA lacking 3'UTR or 5'UTR and TDP-43 by immunoprecipitation method.7.3'UTR ribosomal protein mRNA with MS2 binding sequence at UTR was co-transfected with NLS-MS2-Venus plasmid into mouse cortical neurons,and defect co-localization of RP-MS2 bs and RP mRNA.8.Use the method of detecting mRNA in living cells to detect the transport distance of Rpl41 mRNA and Rplp1 mRNA in mouse cortical neuron axons and the number of RP particles,and to detect Rpl41 mRNA and Rplp1 mRNA in mouse cortex when TDP-43 expression is down-regulated The transport distance and the number of transport particles in the axon of a neuron.9.TDP-43 sh RNA was transfected into mouse cortical neurons to detect the changes in axon extension and the effect of ribosomal protein on axon extension.Result:1.Cytoplamic ribosomal protein mRNA as targets for axonal transport by TDP-43(1)The neurites of mouse cortical neurons isolated by a special culture device expressing MAP2 and Tau,but not Histone H1,Histone H3,and Lamin A.(2)Knock down the expression of TDP-43,a total of 1,050 kinds of mRNA expression in mouse cortical neuron axons are reduced,including 46 kinds of cytoplasmic ribosomal protein mRNA,1 kind of mitochondrial ribosomal protein mRNA and 4 kinds of eukaryotic extension factor.2.Ribosomal protein mRNAs are transported by TDP-43(1)Ribosomal protein mRNAs in mouse cortical neurons are distributed granularly along axons.(2)TDP-43 is co-localized with Rpl41 mRNA,Rplp1 mRNA,Rps7 mRNA and Rpl26 mRNA,and the colocalization ratio between them is significantly higher than that between TDP-43 and GAPDH mRNA(p<0.05).(3)The average percentage of RP mRNA co-localized with TDP-43 is about80%,while the percentage of GAPDH mRNA is 17.7%.(4)Knock down the expression of TDP-43,and the fluorescence intensity of Rpl41,Rpl26,Rps7,and Rplp1 mRNA co-localized with TDP-43 detected by in situ hybridization decreased significantly(p<0.05).(5)Both Rpl41 mRNA,Rpl41 mRNA lacking 5'UTR(Rpl41 mRNA ?5'UTR)or Rpl41 mRNA lacking 3'UTR(Rpl41 mRNA ?5'UTR)were co-precipitated with TDP-43.The relative expression of Rpl41 co-precipitated with TDP-43,Rpl41 without 5'UTR,and Rpl41 without 3'UTR were significantly higher than the relative expression of GAPDH mRNA co-precipitated with TDP-43(p<0.005).The relative expression of Rpl41 mRNA co-precipitated with TDP-43 was significantly higher than the relative expression of Rpl41 mRNA without 5'UTR co-precipitated with TDP-43(p<0.001),while co-precipitation with TDP-43 There was no significant difference between the relative expression of Rpl41 mRNA without 3'UTR and Rpl41 mRNA lacking 3'UTR..(6)The transport distance of Rpl41 mRNA and Rplp1 mRNA of the axon and the number of RP particles containing 5'UTR were significantly more than those of the control(p<0.05).The mRNA and Rplp1 mRNA containing 5'UTR were transported the axon.The distance and the number of particles were significantly higher than those of Rpl41 and Rplp1 without 5'UTR(p<0.05).Cortical neurons transfected with TDP-43 sh RNA contained 5'UTR in the number of Rpl41 and Rplp1 mRNA particles and the transport distance was significantly less than that of the cortical neuron transfected with negative sh RNA(p<0.05).There was no significant difference in the number of particles and transport distance of Rpl41 and Rplp1 mRNA lacking 5'UTR in cortical neurons transfected with TDP-43 sh RNA and cortical neurons transfected with negative sh RNA.3.Ribosomal protein has protective effects against TDP-43 dysfunction in axons(1)In the negative control,sh RNA and pc DNA were simultaneously transfected into cortical neurons,and TDP-43 sh RNA and pc DNA were simultaneously transfected into cortical neurons.The longest neurite length of the TDP-43 sh RNA group was significantly shorter than that of the negative control sh RNA group(P<0.05).TDP-43 sh RNA and high-concentration TDP-43 were simultaneously transfected into cortical neurons.The high-concentration TDP-43 group significantly improved the neurite length(P<0.001).(2)TDP-43 sh RNA and pc DNA were simultaneously transfected into cortical neurons.TDP-43 sh RNA and several ribosomal proteins were transfected into cortical neurons simultaneously.The Rpl41,Rpl26,and Rps7 groups containing5'UTR significantly prolonged neurite length(P<0.01),but the Rpl41,Rpl26,and Rps7 groups lacking 5'UTR had no effect on the neurite length.conclusion:1.Ribosomal protein mRNA is the target of TDP-43 axonal transport in cortical neurons.2.Ribosomal protein mRNA is located in axons in the form of particles.TDP-43 is a component of RNA particles and is necessary for the transport of RP mRNA.3.The 5'UTR of ribosomal protein mRNA plays a key role in its binding and transport with TDP-43.4.TDP-43-mediated transport of RP mRNA in axons promotes axon development,and ribosomal protein has a protective effect on axon extension,the most significant protective effect is of themRpl26.