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Ariel M. Wilson, Barbara Morquette, Elena Feinstein, Adriana Di Polo; The p53 Activators ASPP1 and ASPP2 Regulate Retinal Ganglion Cell Death In Vivo Via Regulation of Fas and Puma. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2693.
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The ASPP (Ankyrin-repeats, SH3-domain and Proline-rich-region-containing Protein) family members, ASPP1 and ASPP2, are essential regulators of p53 activity, but their role in the death of central nervous system neurons is unknown. Here, we addressed their function on retinal ganglion cell (RGC) loss in a model of acute optic nerve injury (axotomy) using novel siRNAs that selectively silence ASPP1 or ASPP2 gene expression in vivo.
Rat or mice (p53 null) RGCs were retrogradely labeled by application of Fluorogold to the superior colliculus prior to intraorbital optic nerve axotomy. siRNA against ASPP1, ASPP2 or GFP were administered by intravitreal injection at the time of axotomy and a week later. RGC densities were quantified on retinal whole mounts and data analysis and statistics were performed by a one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison post-test. ASPP1/2, Fas, Puma, and Bax protein levels were examined by retinal immunohistochemistry and western blot analysis.
Remarkable RGC survival was found in axotomized p53 knockout mice retinas compared to wild-type littermate controls, demonstrating that RGCs die in a p53-dependent manner. We show that ASPP proteins are abundantly expressed in intact and injured RGCs, and that siRNAs against ASPP1 and ASPP2 lead to RGC survival. The combination of siRNA against ASPP1 and ASPP2 with brain-derived neurotrophic factor (BDNF) further increased the survival of injured RGCs. Combined therapy of ASPP1/2 siRNA with BDNF led to ~60% of surviving RGCs in treated eye, compared to ~40% in control eyes (n=4-6/group) at 2 weeks post-lesion. Our biochemical analysis of p53 downstream effectors, revealed a marked decrease in Puma and Fas levels in siASPP1- and siASPP2-treated retinas compared to controls, whereas Bax levels remained unaffected.
Our data demonstrate that p53 is an essential regulator of RGC death, and that targeted gene silencing of the p53 activators ASPP1 or ASPP2 effectively delays RGC death after acute optic nerve lesion. Importantly, we demonstrate that the neuroprotective effect of ASPP1 or ASPP2 depletion occurs via downregulation of the p53 targets Fas and PUMA.
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