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Philippe D'Onofrio, Mark Magharious, Adam C. Hollander, Nardos Tassew, Kinga Szydlowska, Michael Tymianski, Philippe P. Monnier, Paulo D. Koeberle; Involvement Of Caspase-6 And Caspase-8 In Neuronal Apoptosis And The Regenerative Failure Of Injured Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5448.
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Apoptosis of Retinal Ganglion Cells (RGCs) is a hallmark of Glaucoma. Optic nerve transection is a model of adult RGC apoptosis. Ninety percent of RGCs die within the first 14 days postaxotomy. Caspases constitute a family of proteases that act as apoptotic executioners, several of which have previously been implicated in RGC apoptosis after axotomy. We examined the role of caspase-6 in RGC apoptosis and the failure of axon regeneration after optic nerve injury.
Optic nerve transection or crush were performed in adult rats. Caspase inhibitors were delivered by intraocular injections. Z-VAD-FMK (pan caspase inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), Z-VEID-FMK (caspase-6 inhibitor), Z-IETD-FMK (caspase-8 inhibitor) and a Small Isatin Michael Acceptor (SIMA13a; caspase-6 inhibitor) were administered at 3 days postaxotomy. RGC survival was quantified in fixed flatmounted retinas at 14 days postaxotomy. Axon regeneration was quantified at 21 days after optic nerve crush in longitudinal frozen sections of nerve immunolabeled for GAP-43. Western blots were used to quantify caspase-6 or -8 activation after axotomy. RGC survival and regeneration were concurrently studied in vitro, using retinal explants that were cultured on chondroitin sulphate proteoglycans (CSPG) or myelin.
Treatment of retinal whole mounts with Z-VEID, a selective inhibitor of caspase-6, enhanced ganglion cell survival (p<0.01), similar to Z-LEHD or Z-VAD. Moreover, retinal explants treated with Z-VEID extended neurites on myelin but not CSPG. Intraocular delivery of Z-VEID or SIMA13a also resulted in a 2 to 3-fold increase in RGC survival in vivo (p<0.001) and increased the number of regenerating axons per nerve section at three different distances beyond the lesion site (p<0.01). In searching for downstream effectors for caspase-6, we identified caspase-8. We then showed that caspase-6 inhibition by Z-VEID reduced the activation and cleavage of caspase-8 in western blots. Furthermore, we investigated the role of caspase-8 in the injured retina both in vitro and in vivo and observed that Z-IETD promoted survival and regeneration to an extent similar to that obtained with caspase-6 inhibition by Z-VEID.
Our findings show that caspase-6 and caspase-8 are components of a cellular pathway that prevents neuronal survival and regeneration in injured adult retinal ganglion cells.
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