Role Of GSK3Signaling In Neuronal Axon Regeneration

With the rapid development of modern transportation, sports,construction work, the incidence of spinal cord injury(SCI) increasedyear by year, its high morbidity and medical expense brought thepatient’s family and the society heavy burden. Spinal cord injury destroythe neuron in the central nervous system at first, and then broken axonswhich lead to nerve tracts interrupted, in addition, interrupted nerveconduction circuits are unable to launch self-regenerate and repair,which makes the occurrence of spinal cord injury serious and difficultrecovery of neurological dysfunction. Consequently, after spinal cordinjury, whether the axons could regenerated and establish synapticconnections or not is crucial to the neurological function recovery.Glycogen synthase kinase-3(GSK3) The serine/threonine kinase, akey enzyme in the process of glycogen synthesis was discovered in1980s. With the deepening of research, it was found that GSK3, whichexists in rest cells in its activated state, is widely expressed in humantissues, and it can phosphorylate a variety of downstream substrates,such as cytoskeletal proteins, transcription factors and trans-membranesignaling molecules. In addition to the regulation function of metabolism,GSK3is also play a key role in various physiological and pathological processes, for instance, cell proliferation, immune response,inflammation and tumorigenesis. Recent studies have shown that, GSK3regulation effect throughout the nervous system, which controls thedevelopment, degeneration, regeneration process of the whole nervoussystem, however, its mechanism is still remain elusive.GSK3contains two kind of isoforms, GSK3a and GSK3b, due to itshigh expression level in the nervous system, conventional experimentalmethods, such as inhibitory reagents or simply SiRNA gene silencing,are not only inadequate to completely modify its activity and expressionlevel, but also incompetent to detect the function of each isoforms ofGSK3. In addition, a series of complicated pathophysiology changeshappened in the surrounding environment, such as nerve degenerationand glial scar formation, which are impossible for in vitro cultureexperiment to simulate.Thus, it is incompetent for previous experimental methods to studythe molecular regulation mechanism of GSK3of neurons, and this isprecisely one of the reasons which cause the vastly differentexperimental results.Based on the background, we utilize the GSK3transgenic mice,take the advantages of the in vivo electroperation, we establish GSK3and its isoforms knock-out and overexpression animal models.Together the DRG culture experiment and the in vivo nerve crush procedure, we examined the influence of nerve axon regeneration whichcaused by total and isoforms GSK3expression level modification.Finally, we studied the PI3K-GSK3signal transduction of axonalregeneration system. Through this study, we not only clarified the role ofGSK3in axonal regeneration process, but also elaborated PI3K-GSK3pathway regulating mechanism of nerve regeneration.Experimental ResearchSection1. GSK3a affects the axon regeneration process of mouseObjectives: To determine GSK3a knockout or overexpressioneffects on axonal regenerationMethods: In vitro experiments, taking adult GSK3a KO/KItransgenic mice and WT mice separate them into the experimental groupand the control group. After the in vitro cell culture, neuron was fixedand stained with Tuj-1antibody, measure the length of the axonregeneration and statistical analysis. In vivo experiments, mice weregrouped as above, each group of mice were performed the left L4/L5DRG GFP electroperation process, sciatic nerve crush2days later. Fixand measure the nerve axon regeneration length of left sciatic andstatistical analysis.Results: Axon regeneration length of GSK3a KO/KI and WTmouse has no significantly differenceConclusion: GSK3a knockout or overexpression has no effect on axon regeneration.Section2. GSK3b affects the axon regeneration process of mouseObjectives: To determine GSK3b knockout or overexpressioneffects on axonal regenerationMethods: We use the GSK3a WT/b flox/flox genotype mice,together with in vitro or in vivo electroporation method to transfectCMV-Cre in to the mouse to obtained GSK3b KO mouseIn vitro experiments, taking adult GSK3b KO/KI transgenicmice(Knock in mouse and GSK3S9A plasmid) and WT mice,separatethem into the experimental group and the control group.After the invitro cell culture, neuron was fixed and stained with Tuj-1antibody,measure the length of the axon regeneration and statistical analysis. Invivo experiments, mice were grouped as above, each group of mice wereperformed the left L4/L5DRG GFP electroperation process, sciaticnerve crush3days later. Fix and measure the nerve axon regenerationlength of left sciatic and statistical analysis.Results: Axon length of GSK3b KO group was significantly longerthan the control group (P <0.05), whereas overexpression group lengthwas significantly shorter than the control group (P <0.05).Conclusion: GSK3b knockout promotes axon regeneration,overexpression inhibits axon regeneration.Section3. Total GSK3affects the axon regeneration process of mouseObjectives: To determine GSK3double knockout or double knockin effects on axonal regenerationMethods: We use the GSK3a KO/b flox/flox genotype mice toobtained GSK3DKO mouse. In vitro experiments, taking adult GSK3DKO/KI transgenic mice and WT mice, separate them into theexperimental group and the control group.After the in vitro cell culture,neuron was fixed and stained with Tuj-1antibody, measure the length ofthe axon regeneration and statistical analysis. In vivo experiments, micewere grouped as above, each group of mice were performed the leftL4/L5DRG GFP electroperation process, sciatic nerve cruch3days later.Fix and measure the nerve axon regeneration length of left sciatic andstatistical analysis.Results: Axon length of GSK3DKO group was significantlyshorter than the control group (P <0.05), whereas axon length betweenoverexpression and WT group has no significant difference.Conclusion: GSK3express level is necessary for axon regeneration.Section4. Study of PI3K-GSK3signaling pathway regulatingmechanismObjectives: To determine the regulation relationship betweenPI3K-GSK3and GSK3regulatory mechanismMethods: In vitro experiments, taking GSK3DKI and WT mouse as the experimental and the control group, performed post sciatic nervetransection and primary cultures. Measure the axon length and thenperform WB to detect the phosphorylation level of GSK3a/b andsubstrate CRMP2. In vivo experiments, same as section3in vivoExperiment. Drug Intervention experiment: take adult GSK3DKI micefor DMSO/LY/BIO/LY+BIO groups, culture the DRG neurons withreagents, then observe the phosphorylation level of GSK3a/b andsubstrate of each group by WB.Results: No significant differences between GSK3DKI group andWT group axon length and substrate phosphorylation levels (P>0.05).Conclusion: PI3K is upstream of GSK3, negatively regulates GSK3in a Akt independent manner.ConclusionIn this study, we systematically analyze the axon regenerationregulatory function of GSK3molecules, we learned that a certain levelof expression of GSK3is necessary for axon regeneration, absence ofGSK3b could promote axon regeneration. PI3K kinase negativelyregulates GSK3in a Akt independent manner.