The senescence marker P16^ink4a is expressed in ocular astrocytes

Abstract

Purpose : Persistent senescent cells are believed to accelerate aging and contribute to the pathology of a number of age-related disorders. Both anterior and posterior segments of the eye are subject to age-related degenerative changes with great impact on quality of life as a result. The aim of this study was to identify cells expressing the senescence marker P16^ink4a in the normal aging mouse retina.

Methods : The expression levels of P16^ink4a mRNA and protein were determined in retinal lysates using quantitative real-time PCR (qRT-PCR) and western blotting, respectively. Cells expressing P16^ink4a were identified by immunohistochemistry on ocular cryo sections. Mice expressing enhanced green fluorescent protein (EGFP) under the control of the Nrl promoter (Nrl-EGFP, C57BL/6J:Sjl/j) at different ages from post-natal day 3 to 1 year-old were used in the present study. For immunohistochemistry investigations n=3. For qRT-PCR and western blot n=8.

Results : Immunohistochemical analysis showed P16^ink4a expression in the optic nerve, and the corneal squamous epithelium at all ages investigated but was only detected in the retinal nerve fibre layer in mice older than 3 weeks old. Ocular sections incubated with purified IgG fraction from the host species of the primary antibody gave no signal in either cell type. Co-staining for the astrocyte marker GFAP and P16^ink4a showed co-localization in the inner retina in stellate shaped branching cells indicating that P16^ink4a appears to be expressed in ocular astrocytes. The pattern was not consistent with activated Müller cells. Quantitative analyses of P16^ink4a mRNA and protein levels showed age dependent differences in retinal expression levels.

Conclusions : We identified expression of the widely used senescent marker P16^ink4a in ocular astrocytes as well as in corneal squamous epithelium cells in normal mice aged three days to one year. Expression in the corneal epithelium was expected, since these cells have a shortened lifespan. Expression of P16^ink4a in neonatal ocular astrocytes however indicates that it may be involved in normal astrocyte maturation, possibly through increased turnover in the immediate post-natal period. The persistence however of P16^ink4a expression in the adult astrocytes is unexplained and raises the possibility of continued astrocyte turnover even into adulthood.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.