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Ariel Wilson, Vince Chiodo, Sanford L Boye, Nicholas Brecha, William W Hauswirth, Adriana Di Polo; Inhibitor of Apoptosis Stimulating Protein of p53 (iASPP) is required for retinal ganglion cell survival after axonal injury. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4036.
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p53 apoptotic activity is tightly regulated by the apoptosis-stimulating proteins of p53 (ASPP) family members: ASPP1, ASPP2 and iASPP. We previously showed that pro-apoptotic members ASPP1 and ASPP2 contribute to the p53-dependent death of retinal ganglion cells (RGC), however the role of the p53 inhibitor iASPP in the central nervous system is unknown. Here, we addressed the role of iASPP on RGC survival in a model of acute optic nerve injury (axotomy) using loss-of-function and gain-of-function experiments in vivo.
iASPP knockdown was carried out by intravitreal injection of small interference RNA (si-iASPP). Overexpression of iASPP in RGCs was achieved by intraocular delivery of a tyrosine mutant serotype 2 adeno-associated virus (AAV.iASPP). Phosphoserine immunoprecipitation was performed on retinal lysates of intact, axotomized, and iASPP overexpressing retinas. iASPP, Fas/CD95, PUMA, Noxa and Bax protein levels were examined by retinal immunohistochemistry and western blot analysis. RGC immunolabeling was performed with an RBPMS antibody. RGC densities were assessed by quantification of Brn3a-positive cells on retinal whole mounts followed by statistical analysis using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison post-test.
Our data demonstrate that iASPP is expressed by intact and injured RGCs, and that iASPP phosphoserine levels, which increase iASPP affinity towards p53, are significantly reduced following axotomy. We show that iASPP downregulation by siRNA exacerbates RGC death, whereas selective AAV-mediated overexpression of iASPP promotes robust RGC survival. iASPP overexpression results in an increase of iASPP phosphoserine levels following axotomy compared to retinas treated with a control virus. Analysis of p53 downstream targets demonstrate that increasing iASPP levels in RGCs leads to downregulation of pro-apoptotic PUMA and Fas/CD95.
Our study demonstrates a novel role for iASPP in the death of RGCs, and provides further evidence of the importance of ASPP family in CNS neuronal survival after axonal injury.
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