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TJ Hollingsworth, Marko Z. Radic, Francesco Giorgianni, Sarka Beranova-Giorgianni, Diwa Koirala, Yanming Wang, Alessandro Iannaccone; Peptidylarginine deiminase 4 (PAD4) is the primary mediator of retinal citrullination in mice. Invest. Ophthalmol. Vis. Sci. 2017;58(8):616.
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© ARVO (1962-2015); The Authors (2016-present)
Citrullination is a post-translational modification known to serve many functions in both epigenetics and inflammation. Previously, we showed PAD4 expression in retina (ARVO2015 Abstract 4636-D0011). Herein, we report on the expression of PAD2, PAD4 and retinal citrullination in murine retina with age, and show that PAD4 is essential for normal citrullination in post-natal developing retina.
Eyes from wild-type (WT) and PAD4-/- (PAD4KO) mice at 0.5, 3, and 6 months of age were fixed and cryosectioned. Sections were probed using an anti-PAD2/PAD4 antibody (Ab), (Proteintech), a specific anti-PAD4 ab (Proteintech), and F95 anti-citrullinated peptide Ab (Millipore) and fluorescent intensities quantified.
WT mice displayed PAD labeling in all retinal layers varying with age. The ganglion cell, inner nuclear (INL), and outer plexiform layers labeled most intensely, but labeling spanned the retinal thickness. In WT, both PAD2 and PAD4 levels decreased with age; however, PAD4KO animals maintained near constant PAD2 levels. Citrullination paralleled the changes in PAD4 expression, with younger animals having more citrullination. Citrullination patterning shifted from primarily nuclear to more INL cell-specific with age. PAD4 labeling was near background levels in PAD4KO mice. Using a non-specific anti-PAD Ab, residual PAD reactivity was modest, and was attributed to the other known retinal PAD, PAD2. In PAD4KO mice, minimal residual retinal citrullination was observed.
PAD expression and citrullination levels vary with age. Younger retinas exhibited the most PAD2/PAD4 expression and citrullination, while older retinas exhibited the least; however, in the absence of PAD4, PAD2 expression appears to retain a near constant level, possibly to compensate for the loss of PAD4. The primarily nuclear citrullination in WT mice suggests a role for citrullination in expression of genes involved in either retinal development, homoeostasis, or both. The physiological age-related decline in PAD4 expression and citrullination is similar to the decrease in PAD2 expression and citrullination seen in rat eyes (Bhattacharya, et al 2008). The near complete elimination of retinal citrullination seen in PAD4KO mice supports the notion that, while both PAD2 and PAD4 are expressed in the retina, PAD4 is the main mediator of retinal citrullination, at least under physiological conditions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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