The Roles Of NeuroD1 In Peripheral Axonal Regeneration And Its Potential Mechanisms

During the development of the nervous system,the axons of immature neurons stretch to develop the synaptic connections with target cells or organs.After the axon of the adult neuron is damaged,the axon of the distal segment of the injury will be disintegrated,and axon regeneration will occur at the proximal axon under appropriate conditions.The regeneration capacity of neurons decreased as the neurons mature.The pro-regenerative cellular changes after axon injury are thought to recapitulate some of the developmental processes.As most important functional regulating molecules,transcription factors are regarded as essential for initiating the axonal growth or re-growth.Recently some studies have shown that manipulation of transcription factors that act as master controlling factors of axon growth during development might represent another avenue for promoting axon regeneration in adults.NeuroDl,a member of bHLH transcription factor family,has been shown play a critical role in development of the nervous system.It is widely expressed in the embryonic neurons,and then is downregulated during the development.Plenty of studies demonstrated that lack of NeuroDl results in failure of neuron maturation and process elongation.However,it remains unclear whether NeuroD1 expression in mature neurons play roles to promote axon regrowth and functional recovery after peripheral nerve injury.Present study was mainly designed to figure out this issue.Since the previous studies had demonstrated that NeuroD1 can regulate the expression of neurotrophin receptorsand cytoskeletal rearrangement,some factors involved in these two aspectswere also detected to explore the potential mechanism of NeuroD1 on the axonal regeneration.Methods1.pAAV-Flex-CAG-NeuroD1-P2A-mCherry and rAAV-hSYN1-Cre-WPE-pA were used as vectors to infect the neurons in the lumber spinal cord of adult mice.pAAV-Flex-CA-P2A-mCherry and rAAV-hSYN1-Cre-WPE-pA were microinject with the same protocol for the control group.Seven days or twenty-one days after virus injection,the expression of NeuroD1 was detected by Western blot and immunofluorescence.2.Sciatic nerve crush was performed in the AAV virus infected mice.Three after the nerve injury surgry,the axon regeneration was detected by immunofluorescence and western blot with the axon regeneration marker GAP43.Forthween days after nerve injury,the functional recovery of the nerve conduction and moto-function were assessed by Cat Walk test and CMAP neurophysiological test.The wet weight ratio of the gastrocnemius muscle was measured after the behavioral and neurophysiological test.The cross-sectional area of the myofibers and the rate of the innervated motor endplate were detected with histology and immunochemistry.3.pCAG-NeuroD1-IRES-GFP retroviral vector was used to treat PC 12 cells to induce the cells overexpress NeuroD1.Then the cells were neuronal inducedfor 7 days.Finally,the cells were trypsized and????to minic axon injury.RT-PCR analysis and western blot were used to screen the possible target genes of NeuroD1.4.Sciatic nerve crush was performed as above described.The expressions of BDNF,TrkB and Spastin in motoneurons of lumbar spinal cord were detected 3d after nerve injury.Results1.NeuroD1 overexpression promotes sciatic nerve regeneration in mice:1.1 AAV-NeuroD1 virus under the control of hSYN1-Cre vector transfectded spinal cord neurons with high Specificity.Immunofluorescence and Western blot showed that adult spinal cord neurons with AAV-con didn't express NeuroD1 protein.While the infected neurons in NeuroD1 group were stably overexpress NeuroD1 protein 7 days or 21 days after virus injection.1.2 Both of the length of GAP43-positive regenerative axons and the number of GAP43-positive axons in the distal trunk of the injured nerve of NeuroD1 group were significantly greater than that of the control group.Western blot results also showed that the total protein expression level of GAP43 in the distal nerve was significantly greater in the NeuroD1 group than that in the control group.1.3 The wet weight ratio and cross-section hematoxylin staining of gastrocnemius showed that NeuroD1 overexpression improved target muscle atrophy.Double staining of a-BTX and NF showed that NeuroD1 overexpression promoted neural re-innervation of the target muscle tissue.1.4 The SFI values obtained from GatWalk analysis were significantly greater in the NeuroD1 group than that in the control group1.5 Neuroelectrophysiological results showed that the amplitude of NeuroD1 group was larger,and the difference was statistically significant,suggesting that neurodegeneration in NeuroD1 group was better.2.The mechanism of NeuroD1 promoting axonal regeneration2.1 Immunofluorescence staining showed that PC12 cells were DCX and TuJ1 positive after induction,while uninduced PC 12 cells were negative.2.2 PC 12 cells were transfected retrovirus.Immunofluorescence and western blot results showed that the expression of NeuroD1 in RV-NeuroD1 group was significantly increased.2.3 The length of regenerative neurites of cells with RV-NeuroD1 transfetion is significantly longer than that of untransfected cells and cells with RV-con transfection in the same dish 24h after replating.2.4 Q-PCR anlysis showed that the mRNA expression of TrkB,Spastin and P80-katanin were significantly increased,while no differences were seen in the mRNA expression of TrkA,TrkC,P75 and P60-katanin.Western blot assay confirmed that NeuroD1 overexpression increased the protein expression of TrkB and Spastin.2.5 Immunofluorescence staining of spinal cord tissue showed that the expression of BDNF and its receptors TrkB and Spastin in anterior horn motor neurons of AAV-NeuroD1 group were significantly greater than that in adjacent untransfected neurons and AAV-con groups.Western blot showed that total protein levels of TrkB,BDNF and Spastin in lumbar spinal cord of AAV-NeuroD1 group were also significantly greater than that in the control group.Conclusion:In this study,the spinal cord neurons were transfected with viral vectors with high specificity and overexpressed NeuroD1.The sciatic nerve crush model was used for the model of peripheral nerve injury.By which,the effects of NeuroD1 on peripheral nerve regeneration were investigated from the aspects of morphology,behavior and electrophysiology.Present results confirmed that overexpression of NeuroD1 in spinal cord neurons significantly promote the regeneration of axons after injury.And the mechanism may be related to that NeuroD1 could upregulate the expression of neurotrophic factor BDNF and its receptor TrkB as well as the microtubule-severing protein Spastin.