The Associated Mechanism Research Of Apoe With Neuronal Injury And Axonal Regeneration

BackgroundIn the mammal CNS, neurons play the role of building the neural network andtransmitting nerve signals. As the enlargement part of neuron axon terminals, growthcone guide axon extension, form the contact with target and transmit signals. Afterneurons death, the undead neurons cannot reproduce themselves to replace the losedneurons. But the injured axons can regenerate to replace the losed axons and promotethe some recovery of injured nerve functions. As the main lipoprotein, apoE may repairinjured neurons and promote axons extension after CNS injury. However, the apoEsource of injured neurons is not understood, and the different human apoE subtypes onthe growth cone and axonal regeneration remain to be elucidated.ObjectiveThis research focused on the role of apoE in injured neurons and axonalregeneration. On the side of neurons, we explored the apoE changing in injured neurons,and injured neurons whether uptook apoE from exogenous environment and the injuredneurons whether triggered astrocytes to produce and secrete apoE by some signalpathway. On the side of neuron axons and growth cone, we investigated apoE-mRNAand protein whether existed in axons and regenerated axons, observed the human apoEsubtypes whether affected on axonal regeneration and growth cone, as well as thepossible mechanism. For further discovering the possible role of apoE in the TBIpathological process and exploring the potential interfering strategy for apoE subtypesinfluence on CNS disease. Methods1. Culturing wild type/apoE KO mice primary cortical neurons and astrocytes invitro, building neuron mechanical injured model. Western blot measured the apoE levelin wild type neurons and medium. Making the conditional medium of wild typeastrocyte and apoE KO neurons, incubated with injured apoE KO neurons and wild typeastrocytes, the apoE levels in cell and medium also were measured by Western blot. TheERK inhibitor (U0126) was added in wild type astrocytes with injured apoE KOneurons conditional medium, the apoE level in above astrocytes and medium weremeasured.2. Culturing wild type/apoE KO mice primary cortical explants in vitro, buildingthe improved transected axonal model and getting the pure axons. The RT-PCR and immunofluorescence were used to test the apoE-mRNA and protein. The recombinedhuman apoE2,3and4was added in medium of explants respectively. The growth conewas dyed by the phalloidin to observe the affecting of apoE on growth cone. At thesame time, the ERK inbitor was used to explore possible pathway in above process. Inaddition, the recombined human apoE2,3and4also was added in medium oftransected axons, and to observe the affecting of apoE subtypes on axonal regeneration.Results1. Comparing to uninjured wild type neurons, the injured wild type neuronsincreased the apoE expression about2.5fold, but no significant different in medium.The injured apoE KO neurons uptook more apoE (about4fold) form the wild typeastrocytes conditional medium than the uninjured neurons. The conditional medium ofinjured apoE KO neurons can triggered astrocytes produce (about3fold) and secrete(about2fold) apoE, the ERK inhibitor can abolish above trigged effectiveness.2. The RT-PCR and immunofluorescence showed apoE-mRNA and protein waspositive in the axons and the regenerated axons. The fluorescent intensity of growthcones which respectively added recombined human apoE2,3and4was50.7±19.4, 58.5±15.4,23.4±13.5. The fluorescent intensity of growth cones which added apoE4+U0126was32.8±13.2. The growth cone fluorescent intensity incubated with apoE4was significant decreased than apoE2or apoE3. The growth cone fluorescent intensityincubated with apoE4+U0126was significant increased than incubated with apoE4. Theproportion of regenerated axons which respectively added recombined human apoE2,3and4was0.414±0.078,0.347±0.091,0.186±0.080, which demonstrated theeffectiveness of apoE4promted axonal regeneration was the weakest.Conclusion1. After mechanical injury, neurons can produce more apoE and uptake moreexogenous apoE and induce astrocytes produce and secrete apoE through ERK pathway.2. ApoE4negatively influences the growth cone; inhibiting the ERK signalpathway can weaken this negative influencing. The apoE4also negatively influencedaxonal regenerated.