Age-related reduction in retinal deimination levels in the F344BN rat

Abstract

Increased deimination and peptidyl arginine deiminase type 2 (PAD2) expression has been observed in age-related neurodegenerative diseases without discrimination between their aging and disease component. Here, we describe reduced levels of deimination commensurate with reduced protein, mRNA and activity of peptidylarginine deiminase type 2 in the retina, optic nerve and plasma of aged rats when compared to young rats. The decrease was significant in the ganglion cell layer, inner plexiform layer and inner nuclear layer. Because our observations suggest reduced deimination is a consequence of aging, we conclude that increased deimination must be a consequence of disease. Our findings are important to understand late-onset and progressive diseases such as glaucoma, pseudoexfoliation syndrome, age-related macular degeneration and Oguchi's disease.

Fig. 1

Decreased deiminated proteins in aged rats. (A–C) Immunohistochemical analyses. The retina (A–A’ and B–B’) and the optic nerve (C’–C’) of F344BN rats probed with anti-citrulline and a secondary Alexa 488 antibody. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; PR, photoreceptor; RPE, retinal pigment epithelium. Upper and lower panels correspond to 3 months and 24 months rat tissue, respectively. (B–C) Merged images of fluorescence and differential interference contrast microscopy. (D) Representative Western analysis for protein-bound citrulline. (E) The densitometric immunoblot quantification of deiminated proteins (relative units) from F344BN retina; age in months are as indicated. Standard deviation from three independent experiments has been shown. (F) Relative quantification of deiminated proteins in blood serum by enzyme-linked immunosorbent assay.

Fig. 2

Aged retina and peptidyl arginine deiminase type 2 (PAD2). Representative Western, Northern and enzymatic activity analysis for PAD2 in the retina and the optic nerve. (A) Representative Western for PAD2 (upper panel) and Northern analysis for PAD2 and GAPDH message. (B) Densitometric quantification of PAD2 Western (hollow bars) and enzymatic activity (solid bars) of 5 µg protein from young and aged retinas. (C, C’, D, D’, E, E’) Merged DAPI and antibody stained images. The retinal cryosections were probed with monoclonal anti-PAD2, anti-Thy1 and anti-MAP2 as indicated and detected with secondary antibodies coupled to Alexa 488. Upper and lower panels correspond to 3-month-old and 24-month-old F344 BN retina, respectively.