The Role Of PRb Phosphorylation During The Inner Ear Development And Hair Cell Regeneration

ObjectivesThe hair cell damage and loss induced by aging, noise and ototoxic drugs is the primary reason for human hearing loss and the disorder of balance. The regeneration of hair cells and following reconstruction of organ of Corti is a ideal therapy for hearing recovery. Numberous functional hair cells could be generated by deleting Rb1genes at embryonic and postnatal stage using transgenic mice model, while, Rbl gene also takes great part in the hair cell survival. Therefore, the functional modification of pRb could provide a promising approach for both hair cell regeneration and survival. The function of pRb is correlated with phosphorylation, however, the roles of pRb during the early development of inner ear and hair cell regeneration are still unknown, meanwhile, the mechanism of regulating the phosphorylation of pRb in inner ear is still needed to be clarified. In our studies, the neuromasts of lateral line in zebrafish and explanted otic vesicles from chicken embryos will be employed to investigate the roles of pRb in hair cell regeneration and early inner ear development, two small molecules RRD-251and U0126will be used to explore the mechanism of pRb phosphorylation in inner ear. Furthermore, a new culture system for adult inner ear will be developed and discussed for adult hair cell regeneration.Methods1. The mechanisms of hair cell damage and regeneration in neuromasts of zebrafish induced by neomycin were investigated, furthermore, RRD-251was used to disrupt the phosphorylation of pRb, resulted in less cell proliferation and hair cell regeneration.2. The proliferation, apoptosis and differentiation of progenitor cells in explanted otic vesicles were studied after exposure to RRD-251and U0126, both of which inhibiting the phosphorylation of pRb through cell cycle independent or cell cycle dependent pathways, respectively. In order to exploit the mechanism under the phosphorylation of pRb, Western Blot was used to investigate the phosphorylation of pRb and ERK, while, qRT-PCR was used to test the expression of genes involved in proliferation and cell cycle regulation.3. In our studies, the co-culture system of adult sensory epithelium and spiral ganglion was established, the survival of both hair cells and supporting cells was investigated by live image; the maintenance and variation on the properties of supporting cells were exploited by immunostaining; mTOR pathway, an important pathway involved in regeneration, and the proliferation of supporting cells were valued during long term culture; Interestingly, we investigated the survival and innervations of neurons in spiral ganglion; Finally, adenovirus carried exogenous GFP was used to infect the cultured adult inner ear sensory epithelium。Results1. Apoptosis and necrosis were involved in the mechanism of hair cell death in neuromasts induced by neomycin in zebrafish lateral line. During early stage of regeneration, within24h after exposure to neomycin, new hair cells mainly came from supporting cells or precursor cells directly transdifferentiation, while, during a relatively late stage, from24h to48h, the proliferation of progenitor cells was the main source of new hair cells. The hair cell from proliferation was decreased by using RRD-251which inhibiting the phosphorylation of pRb by disrupting the interaction of Rb-Raf-1, while, there was no effect on hair cell transdifferentiation.2.The phosphorylation of pRb played an important role during the early development of chicken inner ear. The proliferation was decreased, apoptosis was increased significantly by inhibiting pRb phosphorylation through two independent pathway. While, the neuroblasts were more sensitive to RRD-251which inhibiting pRb phosphorylation through cell cycle independent pathway, and the cells of prosensory region were more sensitive to U0126which targeted cell cycle dependent pRb phosphorylation. There was additive effects within the two different pathways. The abolished pRb phosphorylation was followed by widespread downregulation of genes involved in cell proliferation of cell cycle regulation. Ccnb2, Ccnb3, Ccne2, Cdc2, Raf-1, Myb and Myc were downregulated by RRD-251, while, the expressions of Ccnb3, Ccne2, Cdc2and Myc was reduced by U0126. Furthermore, there was no differences on the expression of Rb1treated by RRD-251or U0126.3. During the culture, outer hair cells died within150min after dissection, while, the process was significant delayed by using Necrostatin-1, a necrosis inhibitor; The inner hair cells died according to culture period, there was a few inner hair cells after14d in culture medium, and the hair cell markers, Myo7a and Espin were detected. The connection between some remaining inner hair cells and neurons through TuJ1(+) process was also detected; Compared to hair cells, supporting cells could survive very well during the long term culture(21d), especially for the apex and mid-apex region; The specific markers for supporting cells, sox-2and Jagged-1, were detected by immunostaining; Meanwhile, the survived supporting cells could be infected by Adenovirus and expressed exogenous gene GFP at a relatively high efficiency(60-70%); Furthermore, mTOR pathway was active after culture, however, there was no proliferation among the sensory epithelium.Conclusions:1.The RRD-251could decrease the proliferation and hair cell regeneration in zebrafish neuromasts induced by neomycin through abolishing pRb phosphorylation mediated by Raf-1;2.The phosphorylation of pRb played an important role on proliferation, apoptosis and differentiation during the early development of chicken inner ear; There were two different pathway involved in regulation of pRb phosphorylation, including cell cycle dependent pathway and cell cycle independent pathway, which will be investigate during the hair cell regeneration;3. We established a new adult inner ear culture system and created an exogenous gene expression model based virus infection, which will provide a great advantage to adult hair cell regeneration.