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A. M. Wilson, E. Feinstein, A. Di Polo; Targeted siRNA-Mediated Knockdown of p53 Family Activators ASPP1 and ASPP2 Delays Adult Retinal Ganglion Cell Death in vivo. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3720.
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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 retinal ganglion cell (RGC) death is unknown. Here, we addressed their function in a rat model of acute optic nerve injury (axotomy) using novel siRNAs that selectively silence ASPP1 and ASPP2 gene expression.
RGCs were retrogradely labeled by application of Fluorogold to the rat superior colliculus, and intraorbital optic nerve axotomy was performed one week later. siRNA against ASPP1, ASPP2 or GFP (control) were administered by intravitreal injection at the time of axotomy and one week after injury. The density of Fluorogold-labeled RGCs was quantified in 12 standard retinal areas at 1 and 2 weeks post-axotomy. ASPP protein expression was examined by retinal immunohistochemistry and western blot analysis.
Endogenous, retinal ASPP1 and ASPP2 proteins were found to be primarily expressed by RGCs, and were effectively knocked down as early as 24 hours after intravitreal injection of targeted siRNAs. ASPP1 and ASPP2 gene silencing led to robust protection of axotomized RGCs: ASPP2 and ASPP1 siRNA-injected eyes displayed 79% RGC survival (1,636 RGCs/mm2 ± 62, mean ± S.E.M., n=5) and 69% survival (1,423 RGCs/mm2 ± 19, n=4), respectively, compared to 54% that survived in GFP siRNA injected eyes (1,132±41 RGCs/mm2, n=5). Significant neuroprotection was also observed at two weeks post-axotomy: ~26% RGC survival was achieved with either ASPP1 or ASPP2, with respect to 6% in eyes treated with control siRNA.
Our data demonstrate that targeted gene silencing of ASPP1 or ASPP2 effectively delays RGC death after acute optic nerve lesion, and suggest that ASPP proteins play an important role in the demise of these neurons following axonal injury.
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