Study On The Molecular Mechanism Of Retrodifferentiation And Transdifferentiation Of Rat Retinal Müller Cell During Retinal Degeneration

Retinitis pigmentosa (RP) results in retinal pigment epithelial (RPE) dysfunction and the apoptosis of photoreceptor which lead to vision loss. RP is one of the most common hereditary diseases caused visual loss and blindness.Etiopathogenisis of the RP has not been clarified yet. After the onset of RP, the missed photoceptors can hardly be repaired, and there are currently no effective measures to cure RP. Unlike mammalian, Muller glia in the nonmammalian and amphibian retina have been shown to act as stem/progenitor cells to regenerate retinal neurons after injury throughout lifetime. However, some studies have demonstrated that the limited potentiality of endogenous stem cells (transient) was only observed in mammalian Muller cells during acute retinal injury.It has become a bottleneck problem to seek a safe and effective way to activate stem cells potentiality of retinal Muller cells for realizing the retinal nerve regeneration of higher vertebrates. Study on the expression profiles of Muller cells during retinal degeneration and regeneration, and the regulatory mechanism may reveal the mystery of the adult mammalian retinal regeneration.Acute retinal degeneration induced by NaIO3 is a classic retinitis pigmentosa model, meanwhile, Royal College of Surgeons (RCS) rat is an animal model for inherited retinal pigmentosa, the pathogenesis and turnover of which are similar with that of human. Researches both in the domestic and foreign have confirmed that BMSCs transplantation has significantly curative effect on retinal degenerative diseases, which are good for studying retinal regeneration.It has been widely accepted that both damaged retinal cells and transplanted BMSCs can release one or more NTs(Neurotrophic factors),which influence the survival, migration, adhesion, selfrenewal, and differentiation of stem cells, stimulate stem cell entering the cell cycle by suppressing p27kipl, promote stem cell differentiation towards to neural fate by suppressing Hesl,and inhibit glia fate]. Unfortunately, it is not clear how these signaling pathways change and whether play a role on Muller cell during retinal degeneration and regeneration.Based on the above research status, we hypothesize that acute retinal degeneration may produce limited NTs such as NGF and so on, transiently activating retina endogenous stem cells, Muller cells, and promoting them retrodifferentiation via NGF/TrkA signaling pathway. With the disappearance of the NTs, Miiller cells eventually walked towards gliosis. Similarly, in RCS rats suffered by inherited retinal pigmentosa, the grafted BMSCs may activate retrodifferentiation of Miiller cell by sustained releasing NTs, inhibit glial hyperplasia, and then promote Muller cells neural regeneration, which may restore visual function in the processes of RP. This study would explore the regulatory mechanism of retrodifferentiation and transdifferentiation of retinal endogenous stem cells during acute retinal degeneration and retinal regeneration, which would provide more theory basis for neural regenerate regulation of endogenous stem cells.This study consists of two parts:Part One:Study on the molecular mechanism of Muller cells retrodifferentiation and transdifferentiation during the rat acute pigmentary degeneration of retinaAcute retinal degeneration model is established by intraperitoneal injection of NaIO3 in normal LE rats, and PBS-treated rats are used as control. The retinal frozen sections were made. Double-label immunofluorescence staining and qRT-PCR were performed. We found that the retinal Miiller cells re-entered the cell cycle and dedifferentiated 3 to 7 days after injury. In detail, increased expressing of cell proliferation marker BrdU, PH3 and PCNA were observed in Muller cells, and the expression of retinal stem cells marker such as SOX2, PAX6, increased in Muller cells. Imoportantly, the expression of retinal neurons marker such as crx and rhodopsin presented in Muller cells, which suggested Muller cells transdifferentiated towards to neural fate. At the same time, using the RayBiotech array assay, ELISA, immunofluorescence staining and qRT-PCR, we found increased NGF expression in NaI03 treated retinas only on the 3rd days post-injury, increased NGF/TrkA signal in Muller cells, and reduced expression of p27klpl,the cyclin-dependent kinase inhibitor, which is also a downstream of NGF/TrkA signal, resulting upregulating nucleoprotein Cyclin Dl expression, promoting transient Muller cell re-enter the cell cycle and proliferataion. In addition, decreased inhibitory transcription factor Hesl expression, the downstream of NGF/TrkA signaling pathways, promote neural differentiation of Miiller cells.28 days after the degeneration, NGF/TrkA signaling pathways is closed, the levels of Muller cells retrodifferentiation and neural differentiation drop. Meanwhile, GFAP expression increased leading Muller cells to glia fate. In order to conform the regulating pattern of such molecular mechanism observed in retinal damage, we performed rBMSCs subretinal space transplantation in RCS rat, which is an stable and classic model of chronic and inherited retinal pigmentosa.Part Two:Study on the molecular mechanism of Muller cells retrodifferentiation and transdifferentiation after grafted rBMSCs in RCS ratRetinal neural regeneration model is established by rBMSCs transplantation on RCS rats. RCS rats were divided into three groups, they were rBMSCs treated (RCS/BMSCs), PBS treated (RCS/PBS) and untreated RCS rats (RCS/Unt.) respectively. ERG wave forms were preserved and the ERG b-wave amplitudes were significantly increased after 2,4,8 weeks of rBMSCs transplantation compared with control rats treated with PBS or untreated. ONL thicknesses statistically increased after transplantation. By double-label immunofluorescence staining and qRT-PCR, we found that expression of cell proliferation marker BrdU and PCNA, retinal stem cells marker SOX2 and PAX6, as well as retinal neurons marker crx and opsin increased in Miiller cells 2 to 4 weeks after transplantation, which suggest the retinal Miiller cells re-enter the cell cycle and dedifferentiate and transdifferentiate towards to retinal neurons. Cocultivated with rBMSCs in vitro, Miiller cells was activated to dedifferentiate, expressing of proliferation markers BrdU, retinal stem cell marker SOX2, transdifferentiating and expressing retinal photoreceptor precursor cells marker crx by using double-label cell immunofluorescence staining.Using the ELISA technology, we found rBMSCs can produce and secrete large amounts of NGF, which is the 3-5 times of control. At the same time, NGF activeted Muller cells retrodifferentiation and neuralization, in vivo and vitro. Some of the effect of rBMSCs on Muller cells retrodifferentiation was transiently depressed by co-transplantation or co-culture of rBMSC with the NGF neutralizing antibody.Double-labled immunofluorescence staining and Western-blot confirmed that NGF receptor, TrkA, expression in Muller cells increased, the same as downstream molecule of NGF/TrkA signal, p-PI3K, p-Akt and p-CREB. Eight weeks after transplantation, NGF/TrkA signal drop, the levels of Miiller cells retrodifferentiation go down to baseline. The increased expressions of downstream molecular of NGF/TrkA signaling pathways, p-PI3K, p-Akt and p-CREB were confirmed in co-culture system in vitro, at protein levels using western-blot assay. After adding 252a, the TrkA blockers, in the co-culture system, NGF/TrkA signaling pathways expression is reduced, and Muller cells retrodifferentiation is also suppressed.Different from transient releasing of NGF and Muller cells activation during acute retinal degeneration, grafted rBMSCs showed stable and continued potential in secreting of NGF and activating of Miiller cells retrodifferentiation and neurogenesis, as well as restoring of visual function. NGF/TrkA signaling played a great role for Miiller cells activation either in retinal degeneration or in retinal regeneration.Conclusion:1. During the process of rat acute retinal degeneration, Muller cells transiently expressed retinal precursor cell and retinal neuron markers. It suggests that Muller cells presented an endogenous stem cell potential, namely, they can transiently dedifferentiat and transdifferentiate. ERG recording showed that the visual function was restored in different level in the short term. On the molecular level, we found that the level of secreted NGF in retina transiently only increased at early stage of retinal degeneration, and its receptor, TrkA, was activated too. These results that transiently activating endogenous stem cells by acute retinal degeneration, provide theory basis for role of endogenous stem cells during retinal degeneration.2. The first time, in vivo and vitro, rBMSCs activated Muller cells re-enter the cell cycle, dedifferentiate and transdifferentiate, and play a role in nerve regeneration. These results provide theory basis for exogenous stem cell via activating endogenous stem cells.3. Study in vitro showed that rBMSCs can steadily and continuously produce and secrete NGF. In vivo and vitro, NGF actived Muller cells re-enter cell cycle and transdifferentiate towards neurons, which revealed one of the mechanisms for therapy of retinal degenerative diseases by rBMSCs transplantation.4. In our study, NGF/TrkA signaling pathway plays an important role in deciding Muller cells differentiation fate, stimulating retrodifferentiation by suppressed p27kipl, promoting neural differentiation by suppressed Hesl, and inhibiting glia fate. These results clarify the possible molecular mechanisms regulating Muller cell retrodifferentiation by rBMSCs.