Purchase this article with an account.
Meghan D. Lysko, Philippe M. D'Onofrio, Mark M. Magharious, Paulo D. Koeberle; The role of Receptor Interacting Proteins in Retinal Ganglion Cell apoptosis following axotomy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3483.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We have recently shown that caspase-8 (CASP8) promotes Retinal Ganglion Cell (RGC) degeneration and contributes to regenerative failure after optic nerve axotomy or crush. Nevertheless, the activators and targets of CASP8 remain unclear. CASP8 activation is associated with Tumor Necrosis Factor-Receptor 1 (TNF-R1) and TRAIL receptor signaling. We examined the role Receptor Interacting Proteins (RIPs: molecular switches in the TNF-R1 cell death pathway) in RGC apoptosis after axotomy. We also evaluated whether TRAIL plays a role in RGC death.
Optic nerve transection was performed in adult rats. Animals received intraocular injections (3 days postaxotomy; 4 microliters) of either Necrostatin-1 (Nec-1: a RIP1 phosphorylation inhibitor), Pre-084 (a Sigma-1 receptor agonist), or recombinant TRAIL protein. RIP3 and RIP5 were identified and quantified in an iTRAQ proteomic screen of axotomized retinas. In order to examine the role of RIPs in RGC apoptosis following axotomy, short interfering RNAs (siRNAs) directed against RIP1, RIP3, or RIP5 were injected into the transected optic nerve to knockdown these proteins in RGCs. RGC survival was quantified in fixed flat-mounted retinas at 7 or 14 days postaxotomy.
Intraocular delivery of Nec-1 at 3 days postaxotomy increased RGC survival by threefold (p<0.001) at 14 days, showing that RIP1 activation promotes RGC death. Furthermore, in vivo transfection of axotomized RGCs with RIP1 siRNAs enhanced RGC survival by threefold (p<0.001). The levels of RIP3 increased between 3 and 7 days postaxotomy and then steadily declined. In contrast, the expression of RIP5 remained normal until 3 days postaxotomy and then decreased continuously until 14 days. By 21 days postaxotomy, the levels of both RIP3 and RIP5 were 50% or less relative to normal retinas. siRNAs directed against RIP3 or RIP5 increased RGC survival by threefold (p<0.001) at 14 days postaxotomy. Intraocular injection of recombinant TRAIL protein did not induce RGC apoptosis in uninjured or axotomized retinas. Similarly, Pre-084, which has been shown to antagonize TRAIL activation, had no effect on RGC survival.
Our results show that RIP1 activation plays an important role in RGC apoptosis following axotomy. Furthermore, RIP3 and RIP5 also promote RGC degeneration. However, TRAIL does not affect RGC survival.
This PDF is available to Subscribers Only