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Brian Choi, Philippe M D'Onofrio, Paulo D Koeberle; Inhibition of Primary Cilia Resorption Increases Retinal Ganglion Cell Survival After Axotomy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1756.
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© ARVO (1962-2015); The Authors (2016-present)
Primary cilia inhibit cell division but it is unknown whether they can hault the abortive cell cycle re-entry exhibited by RGCs (retinal ganglion cells) during apoptosis. Aurora Kinase A, HDAC6 (histone deacetylase 6), Plk1 (Polo-like kinase 1), HEF1 (Human enhancer of filmanentation 1), and Nde1 (neurodevelopment protein 1) are proteins involved in the dissolution of the primary cilia and are elevated at various stages of the cell cycle. This study investigates the hypothesis that inhibiting the pathways responsible for ciliary resorption can prevent apoptotic cell cycle re-entry in RGCs after optic nerve transection.
Adult Sprague-Dawley rats received intraorbital optic nerve transections. At 3 and 8 days postaxotomy, animals received 4uL intraocular injections of an Aurora A kinase inhibitor I (AAi; N=6) or Tubastatin A (TBA; N=4), an inhibitor of HDAC6, delivered at 10mM or 20 mM concentrations respectively. To silence Plk1, HEF1, and Nde1 expression, Adeno-associated virus serotype 2 (AAV2) vectors were intraocularly injected 1 week before axotomy to express shRNAs against these proteins (N=4 each). 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. Data was analyzed using a one-way ANOVA followed by Tukey’s post-hoc test in order to identify statistically significant differences between control and experimental groups. To examine the effects of cilia disruption in RGCs, Ift88 (intraflagellar transport) shRNAs were used to interfere the trafficking system responsible for cilia maintenance. Survival after cilia knockdown was quantified 7 days after axotomy.
RGC cilia were significantly shorter and in lesser amounts in axotomized retinas compared to normal. At 14 days postaxotomy retinas treated with AAi, TBA, or shRNAs had significantly higher RGC density compared to controls. Ift88 shRNA treated retina’s had significantly reduced RGC survival compared to those treated with scrambled control shRNA.
Our findings suggest that the primary cilia may play a role in keeping RGCs in a post mitotic state. Interfering with the pathways involved in cilia resorption appears to halt apoptotic cell cycle re-entry. These findings support a novel role of the primary cilium in regulating the survival of RGCs after injury.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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