Glaucoma is a main blinding disease in the world. It causes permanent damage of the optic nerve since its’ impossible for mammalian nerves regenerated. Glaucoma is characterized by progressive loss of retinal ganglion cells along with their optic nerve axons. Reduction of intraocular pressure (IOP), nutrition of optic nerve, or the antioxidation treatment are the therapies of glaucoma. These treatments postpone further death of the ganglion cell. However, these methods can not make the apoptosis RGCs regenerate. Therefore, for the patient with massive death of ganglion cells, we must find an effective therapy to activate the regeneration of ganglion cells to reestablish visual pathway and help these patients to regain their vision.
Atoh7 is a member of the bHLH family, and in our previous research, it had been verified that Atoh7 regulates Müller cell-derived stem cells differentiating into RGCs both in vitro and in vivo. This makes it possible for us to replace glaucoma-induced apoptosis of RGCs cells by transforming Müller cell-derived stem cells differentiating into RGCs. However, the axonal growth of the regenerated RGCs determines the reconstruction of the visual pathway. How to promote the growth of regenerated RGCs axons becomes our research focus. Studies have shown that STAT3 plays an important role in the axon growth of retinal ganglion cells. However, the specific mechanism of STAT3 promoting RGCs axon regeneration is still unknown.
STAT3 is a kind of bifunctional cytoplasmic protein coupling with tyrosine phosphorylation signal pathway in the cytoplasm (24). Normally STAT3 protein is in a non-phosphorylated form in cells, when cytokines or growth factors bind to the receptor, tyrosine kinases (JAK) that bind to receptors are activated. Activated JAK phosphorylates the 705th position of tyrosine residue (Tyr705) in the STAT3 cytoplasm. They combined to form the JAK/STAT3 homodimers. Then the homodimers entered into the nucleus, recognized specific DNA sequences and regulated the transcription of target genes. Therefore, the amount of nuclear-activated STAT3 represents the activation of the JAK/STAT3 pathway in the cell. Activated STAT3 will be dephosphorylated in the nucleus after transmitting signals and be restored to the monomeric form. And then, it will deactivate and return to the cytoplasm to participate in the next round of signal transduction. The activation degree of JAK/STAT3 in normal peripheral nerves is low. Only phosphorylated activated STAT3 may translocate into the nucleus to regulate the expression of certain genes to regenerate peripheral never. Our experimental results also confirmed that the axons of RGCs in the STAT3 overexpressed group were significantly longer than those in the STAT3 inhibited group. We hypothesized that STAT3 regulates the growth of RGCs axons through the above mechanisms.
Research showed that when adeno-associated virus vector containing STAT3 gene was injected into vitreous cavity of the optic nerve contusion model rats, they found that the number of STAT3 positive cells increased significantly as well as the expression of STAT3 protein and STAT3 mRNA increased by 4 to 6 fold after 3 weeks. And the expression of GAP-43 (growth-associated protein-43), a marker of retinal ganglion cell axon regeneration, significantly increased. The optic nerve 3D image analysis which used nerve fiber anterograde tracer CTb-594 showed that the axons of ganglion cells after STAT3 gene infected increased about 200 microns than the control group. But the axon density did not increase and formed many U-turn and right-angle turn. These results indicated that STAT3 signal transduction can increase the length of retinal ganglion cell axons and promote the extension of axonal growth. Another research showed that the use of the JAK2 inhibitor AG490 in vitro in RGCs inhibited CNTF-mediated axonal growth and significantly reduced the regenerative effect of RGCs in response to inflammatory stimuli in vivo. These results confirmed that the activation of JAK and STAT3 played an important role on the axis initial stage of outgrowth (25, 26). Studies have demonstrated that STAT3 signaling pathway plays an important role in the axon growth of RGCs(27, 28). The same results were obtained in our experiment. Deletion Pten, a negative regulator of the mammalian target of rapamycin (mTOR) pathway in adult RGCs, promotes robust axon regeneration after optic nerve injury(29). A high-throughput gene profiling study revealed that the deletion of Klf9 gene substantially promotes optic nerve regeneration in adults RGCs(30). Researches confirmed that a genetic deletion of PTEN, SOCS3, or PTEN/SOCS3 allows partial axon regeneration in the optic nerve after optic nerve crush(31, 32). Inhibition of Mdm4 in the eye and spinal cord promotes axonal regeneration and sprouting of the optic nerve after crush and of supraspinal tracts after spinal cord injury(33). Our research showed that the mRNA level of PTEN, SOCS3, Klf9, and Mdm4 decreased significantly in AAV-STAT3 group. Overexpression of Dclk2, Armcx1, c-myc, and Nrn1 are closely related to the regeneration of RGCs axon(34–37). The mRNA level of Dclk2, Armcx1, c-myc, and Nrn1 significantly increased in the AAV-STAT3 group. The inhibition of STAT3 expression inhibited the growth of the RGCs axon. The mRNA expression levels of RGCs axon-regenerated proteins were significantly opposite between the shSTAT3 group and the AAV-STAT3 group. All the above results confirmed that STAT3 overexpression could promote axonal regeneration of Müller differentiated RGCs.
Recently years, studies have shown that Rho-associated coiled-coil-containing protein kinase (Rho-ROCK) is negatively correlated with cell mitosis and nerve regeneration. Rho is a small molecule homopolymer of the GTPases superfamily and is a mammalian gene homologue of the Ras superfamily. Its biological function is mainly through its downstream effector molecule ROCK. ROCK is a serine/threonine-protein kinase whose molecular structure includes an amino-terminal catalytic domain, an intermediate domain which combined with Rho’s α-coiled-coil, a carboxy-terminal catalytic domain, and a Cys/His region. Activated Rho-GTP activates ROCK by binding to the alpha coiled-coil domain of ROCK and exposing the catalytic center of ROCK(38). Our results have shown that STAT3 could be phosphorylated by Y27632, which is an inhibitor of the Rho-ROCK signaling pathway. The STAT3 + Y27632 combination can effectively promote axon regeneration of Müller cell differentiated RGCs. The axon length of RGCs in the AAV-STAT3 + Y27632 group was the longest, and the expression of GAP-43, phosphorylated STAT3 and its downstream genes p21, Irf1 and Sprr1a was significantly increased. Inhibition of the Rho/ROCK signaling pathway not only reduced the U-turn of the axon and avoid navigation errors but also promote axon regeneration in RGCs. Our research is the first to confirmed that the combined treatment of STAT3 + Y27632 on Müller-derived RGCs can improve the differentiation rate of RGC cells and significantly increase the length of axons, which is a significant improvement for RGCs regeneration. The mRNA levels of RGCs axon-growth-related proteins Pten, Socs3, Klf9, and Mdm4 in the STAT3 + Y27632 group were significantly lower than those in the STAT3 group, while the mRNA levels of Dclk2, Armcx1, C-myc, and Nrn1 were significantly higher than those in the STAT3 intervention group. In order to analyze the mechanism underlying the effect of STAT3 + Y27632 in promoting axon growth of Müller cells differentiated RGCs, we tested the mRNA level of pluripotency genes (Esrrb, Prdm14, Sox2, and Rex1) and differentiation genes (Nestin, Eomes, Milx1, and Gata4). In the STAT3 + Y27632 group, mRNA level of pluripotency genes (Esrrb, Prdm14, Sox2, and Rex1) compared with STAT3 group increased significantly, the mRNA level of differentiation genes (Nestin, Eomes Milx1, and Gata4) decreased obviously. We concluded that the reason for overexpression of STAT3 promote axon growth could be launched Müller cells differentiated RGCs pluripotency. The combination of STAT3 and Y27632 can promote the axon growth of Müller cells differentiated RGCs better than STAT3 alone, which is the same mechanism.
Furthermore, rat chronic ocular hypertension glaucoma model were made to access the mechanism in vivo. in this study. To verify whether STAT3 + Y27632 improve growth of RGCs differentiated from stem cells, the stem cells were transfected with lentivirus PGC-FU-Atoh7-GFP. The stem cells were transplanted into the vitreous cavity of the glaucoma rat model. After 14 days, retina sections of glaucoma models were used to examine immunoreactivity for RGCs-specific marker GFP and ZO-1. The result showed that length of RGCs axons was significantly longer in the STAT3 + Y27632 group, which verified that combination STAT3 with Y27632 can promote the growth of RGCs axons. OCT results showed that the RGCs cell layer in the STAT3 + Y27632 group was significantly thicker than that in the other intervention groups. We observed a delay in peak latencies of P1 waves both in the Glaucoma group and Y27632 group. Additionally, N1-P1 amplitudes in STAT3 + Y27632 group are significantly higher compared with other groups excepted Con group. No significance was observed in the P1 latency and N1-P1 amplitude between Glaucoma group and Y27632 group. These results suggests that Müller cell differentiated RGCs transplants can integrate into preexisting retinal circui and function physiologically.
In conclusion, STAT3 combined with Y27632 can significantly promote the axon growth of ganglion cells which was dedifferentiated from retina Müller cells either in vitro or in vivo.