The Mechanism Of α-crystallin Stimulate RGCs Axon Regeneration

As one of lentogenic factors, alpha-crystallin can promote axon regeneration after optic nerve injury. So far, the mechanism is still poorly defined. Subsequent death of retinal ganglion cells(RGCs) was one reason of the failure in axon regeneration. However, promoting RGCs survival only can ont result in long axon regeneration. Recent research on molecular inhibition in central nervous microenviroment have indicated the inhibitory microenviroment plays an important role in the failure of regeneration. There are many inhibitors of regeneration, such as Nogo, myelin associated glycoprotein,oligodendrocyte myelin glcoprotein, chondroitin sulfate proteoglycans, ephexin and so on. Rencent studies on molecular inhibition in central nervous microenviroment have indicated a common crucial signaling event for all axonal inhibitors and repellents is the activation of RhoA/Rock signaling pathway. Alpha-crystallin can not only promote RGCs survival, but also stimulate axon regeneration. Then, can alpha-crystallin stimulate axon regeneration through RhoA/Rock signaling pathway?Objective: To study the antagonism effect of alpha-crystaliin on myelin and stimulate RGCs axon regeneration . And to study whether alpha-crystallin can stimulate axon regeneration through RhoA/Rock signaling pathway or not by analysing the expression and activation of RhoA and Rock, the phosphorylation of cofilin and myosin light chain(MLC), RGCs axon regeneration and cone growth collapse in vivo and in vitro.Methods: 1. Extracted and identified the myelin and alpha-crystallin from rats. 2. Alpha-crystallin, bovine serum albumin(BSA) and the inhibitor of RhoA/Rock were injected into vitreous cavity respectively. The expression of RhoA and Rock were analysed by western blot and the activation of RhoA assayed by affinity precipitation. At the same time, the regeneration length of optic nerve were measured by anterograde tracing using cholera toxin subunit B (CTB). 3. RGCs were cultured on myelin-coated dishes with DMEM containing alpha-crystallin , BSA, and the inhibitor of RhoA/Rock. The density of RGCs with neurite and the longest neurite of the cells were measured on day 1,,3 and 5. The phosphorylation of cofilin and MLC were assayed by western blot. And the morphology of growth cone was observed by scanning electron microscope.Results: 1. Peptide mass fingerprinting analysis showed that alpha-crystallin was composed of alphaA-crystallineand alphaB-crystalline. The results of western blot showed that myelin contained NogoA and myelin associated glycoprotein.And myelin could induced RGCs growth cone retraction.2. In normal retina, RhoA and Rock were only distributed in RGCs layer. One day after optic nerve injury, the distribution of RhoA and Rock was the same as that in normal retina. After 3 days, RhoA and Rock existed in both the RGCs and inner plexiform layers. Its immunoreactivity was abundant in RGCs layers, inner plexiform layers, inner nuclear layers and outer plexiform layers 7 days after optic nerve injury.3. Compared to the normal retina, the expression of RhoA and Rock and RhoA activation were enhanced significantly 1, 3 and 7 days after optic nerve injury.4. Compared to BSA, treatment of alpha-crystallin, C3 transferase and fasudil resulted in significant decrease in GTP-RhoA after optic nerve injury. However, the expression of RhoA and Rock can not be decrease.5. Tow weeks after optic nerve injury, a little RGCs axon regenerated into crushed region. Whereas, a lot of axons regrowed across the crushed region and entered the distal optic nerve after treated with alpha-crystallin or fasudil.6. Compared to BSA, alpha-crystallin and fasudi resulted in significant decrease in phosphorylation of cofilin and MLC. And the phosphorylation of cofilin and MLC were similar between alpha-crystallin and fasudil.7. Compared to the BSA control group, alpha-crystallin and fasudil could increase the density of RGCs with neurite significantly after cultured 1, 3 and 5 days. Alpha-crystallin and fasudil could also promote neurite outgrowth in comparison with the BSA.8. In normal RGCs axon, growth cone, filopodia and lamellipodia can be seen. The growth cone disappeared after treated with BSA and myelin, and no filopodia and lamellipodia can be seen. However, alpha-crystaliin and fasudil could antagonize myelin and inhibite growth cone retraction in a certain extent. Although, the filopodia and lamellipodia were not very typical, same processes can be seen at the end of the axon after treated with alpha-crystallin and fasudil.Conclusions: 1. The expression of RhoA and Rock was enhanced significantly, and the disstribution was extended after optic nerve injury. RhoA/Rock was involved in optic nerve degeneration.2. Alpha-crystaliin could antagonize myelin and stimulate RGCs axon regeneration.3. Inhibiting RhoA activation was one of the mechanisms of alpha-crystallin stimulating RGCs axon regeration. Alpha-crystallin could stimulate axon regeneration through RhoA/Rock signaling pathway.