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Brian Choi, Philippe M D'Onofrio, Paulo D Koeberle; Primary Cilia Enhances Retinal Ganglion Cell Survival after Axotomy by Abortive Cell Cycle Inhibition. Invest. Ophthalmol. Vis. Sci. 2018;59(9):6149. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
A number of neuronal pathologies cause abortive cell cycle re-entry in post-mitotic neurons, including the optic nerve transection model of glaucoma. Inhibition of cell cycle progression is neuroprotective. Primary cilia are ubiqitious, solitary, finger-like appendages of the cell body that act as gatekeepers of the cell cycle by sequestering the centriole. The resorption of the cilia by disassembly proteins HEF1, NDE1, PLK1, Trichoplein, and Kif2a promotes cell cycle entry in dividing cells. This study aims to protect RGCs (Retinal Ganglion Cells) from axtomy induced apopotsis by inhibiting abortive cell cycling through the stabilization of the primary cilia.
Adult Sprague-Dawley rats received intraorbital optic nerve transections. At 1,4, and 7 days postaxotomy, the retinas were harvested and cryosectioned to examine for cell cycle entry by immunofluorescence directed against pRb (phosphorylated Retinoblastoma). In another set of rats, AAV2 vectors encoding shRNA against HEF1, NDE1, PLK1, Trichoplein and kif2a were injected into the vitreous chamber 3 weeks prior to optic nerve transection. At 14 days postaxotomy RGCs were imaged by immunofluorescence directed against RBPMS (RNA Binding Protein with Multiple Splicing) and survival was quantified from fixed, flat-mounted retinas. Reduction of pRB by the shRNAs was also quantified in infected cells by densitometry of the immunofluorescent signal. Statistically significant differences between control and experimental groups were calculated using an unpaired Student's t-test.
At 1,4, and 7 days post axotomy, RGCs demonstrated nuclear expression of pRb, which was absent in naive retinas, demonstrating abortive cell cycling after injury. Knockdown HEF1, NDE1, PLK1, Trichoplein and kif2a signifincantly increased RGC survival compared to control (scrambled) shRNA treatment (p<0.05, n=3) at 14 days postaxotomy. Furthermore, there was significant reduction of pRb expression in RGCs at 1 day postaxotomy in HEF1 and PLK1 shRNA treated retinas compared to controls (p<0.05, n=3).
Our findings suggest that the primary cilia may play a role in keeping RGCs in a post mitotic state. Knockdown of cilia resorption proteins appears to halt the signalling cascades that lead to apoptotic cell cycle re-entry.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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