The Study On Promoting Mice Retinal Ganglion Cells Axon Regeneration And The Relationship With TGFβ Signaling Pathway

Retinal ganglion cells (RGCs) are the prejection neurons of the eye, which are very important in the pathway from which the photochemical signals could be transferred to visual cortex. .RGC axons can be injured in a number of diseases, such as glaucoma,optic nerve trauma and other optic neuropathies. How to promote RGCs axon regeneration has attracted extensive investigation at both the preclinical and clinical levels. Similar to the neuronal cells in central nervous system (CNS), RGCs have rather limited potential in regenerating damaged axons.In addition; RGCs axon injury has been an important and widely used model system to study the cellular and molecular mechanism for CNS axon regeneration, which is potentially important in the development of more effective therapy to many neurological diseases. But most of the reseachers focus on how the non-permissive environment for RGCs axon regeneration, there is little articles focus on how to stimulate the intrinsic potential of RGCs to regenerate axon.In most optic neuropathies, the signaling of Transforming growth factorβis upregulated, which suggests that there is some relationship between TGFβsignaling pathway and RGC survival and axon regeneration. TGFβsuperfamily comprises numerous secreted signaling molecules and regulates a wide variety of biological processes including cell growth, apoptosis, differentiation, migration, extracellular matrix production, angiogenesis, and development.TGFβsignals are conveyed across the membrane by type ? and type II receptors. The mechanism whereby TGFβsuperfamily members initiate signal transduction is via the phosphorylation and activation of the Smad proteins by the type ? receptor. Phosphorylated Smad proteins accumulate in the nucleus where they collaborate with other transcription factors to regulate gene expression. But there is very little reference mentioned about the relationship of TGFβand RGC axon regeneration.To identify the essential signaling pathway(s) and gene(s) that are important in the regulation of RGCs survival/axon regeneration,we established an in vitro screening system combining purification and culture of primary mouse RGCs and an automatic imaging biorobot, and screened libraries containing small compounds and proteins.In a focused screen of kinase inhibitors,we indentified several kinase inhibitors that could strongly stimulate RGCs neurite growth.Among them,one TGFβreceptor inhibitor showed remarkable and dosage dependent effect to promote RGCs neurite growth.We proved that this blocker could inhibit TGFβsignaling pathway in both mRNA and protein levels. And we also proved that inhibition of TGFβsignaling pathway could promote mouse RGCs axon regeneration in cell culture, retinal explants and mice optic nerve crushed models.Hopefully we could understand the instrnsic mechanism of RGCs axon regeneration and prove some experimental support to study the mechanism of CNS axon regeneration through this experiment. The experiment totally can be divided into two parts:1 Mouse RGCs cultured in vitro and the establishment of the screening systemOur culture system, based on the work of Barres et al employed papain digestion, immunomagnetic purification of the mixed cell population of cells from the postnatal mouse retina to yield a highly enriched population of RGCs for culture. We used 0.05%DMSO as negative control. Because Forskolin could increase the level of cAMP, it was exsited in all the inhibitorsMorphology change: Mouse RGCs started to adhere the membrane after seeding 1.5h.All the RGCs adhered the membrane after 24h incubation, some of the cells got clusterd, and some of the cells started to regeneration neurites, usually 1-3/cell. After 48h incubation, the number of the cells which had neurites started getting more, and the cell bodies became bigger.Some of the cells start to die. After 72h incubation, the neurites became longer and the number of the neurites became more and more. Some of the neurites became short and some of them disrupted.The establishment of screening system: We used Hoechst to stained nuclei, Calcein AM for live cells, and Ethidium homodimer for the dead cells. Imaged were prossed using the Cellomics, and the results were quantified using Extended Neurite Outgrowth program.In a focused screen of kinase inhibitors, we indentified four kinase inhibitors that could strongly stimulate RGC neurite growth: three p38MAPK inhibitors and one TGFβReceptor inhibitor. Among them, the TGFβReceptor inhibitor showed remarkable effect to promote RGCs neurite growth.This finding strongly encourages the notion that inhibition of TGFβpathway could promote RGC neurite regeneration in vitro.The identification of mice RGCs: We used the Brn3b to idenfied mice RGCs. The percentage of RGCs which expressed Brn3b was 85%-90%. It suggested we had very good yield of RGCs.2 Blocking the TGFβsignaling pathway could promote mouse RGCs axon regeneration in vitro and in vivo.The effect of TGFβRI (AL 47070) on TGFβsignaling pathwayIn order to see whether the TGFβRI blocked the TGFβsignaling pathway,we used Western blotting to detect the level TGFβdownstream protein :Smad2, P-smad, Smad3, P-smad3.The expression level of P-smad2 and P-smad3 in mouse RGCs after treated with TGFβRI dramatically dreased than that of negative control DMSO. And there were almost no change of Smad2 and Smad3 between TGFβRI and DMSO. It meaned that phosphoralation of Smads were blocked by TGFβRI. This result suggested that TGFβRI could block TGFβRI signaling transduction.We also utilized the Realtime PCR to detect the downstream genes of TGFβsignaling pathway: IGF1, NRP2, and IL6.Compared with control Forskolin, the expression of these genes in TGFβRI group were extremely downregulated.These results showed TGFβRI could block the TGFβsignaling pathway in protein and mRNA levels.The identification of regenerated neuritesWe used the immunochemical staining to idenfy the regenerated neurites, and we found that TGFβRI(AL 47070)(4μM)could promote both mice axon and denrites regeneration.Compared the TGFβRI with well-known axon regenerating growth factor in vitro and in retinal explants culture.In cell culture and retinal explants culture, we found that blocking of TGFβsignaling could promote mouse RGCs axon regeneration, and it had more potent to promote neurite growth than that of BDNF, CNTF, GDNF which were well studied neurite promoting factor. In conclusion, we improved that TGFβRI could block TGFβsingaling pathway in both protein and gene levels. Furthermore we found that blocking of TGFβsingaling pathway in cell culture and retinal explant culture could promote axon rengeration, and the effect of TGFβRI was more potent than that of BDNF, CNTF, and GDNF.In order to see whether blocking of TGFβsingaling pathway could promote mouse RGCs axon regeneration in vivo, we established a mouse optic nerve crush model.We used nissle staining to make sure the establishment of optic nerve crush model and used the immunohistochemical staining method to see whether there were axon regeneration.After surgery, we found that the number of retinal cells in optic nerve crushed group was less than that of fellow eys, which suggested that the surgery worked. After optic nerve crush, the optic nerve was stained with GAP-43, which was well accepted axon regeneration marker. There was no GAP-43 staining in the non-surgical eye.Compared with DMSO group, the optic nerve in TGFβRI group regenerated more axons and there were axon reneration even in the distal part of optic nerve. It suggested that blocking TGFβRI signaling pathway could promote RGC axon regeneration.During this experiment, we proved that blocking of TGFβsignaling pathway could promote axon regeneration in cell culture, retinal explants culture and mouse optic nerve crush model.In conclusions, this finding strongly encourages the notion that inhibition of TGFβsignaling pathway could be utilized as a promising and novel therapeutic strategy in promting RGCs axon regeneration.