Compartmentalized citrullination in Muller glial endfeet during retinal degeneration

Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell–specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate in MG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a “citrullination bunker” that initiates and sustains citrullination in retinal degeneration.

JR5558 mice B6.Cg-Crb1 rd8 Jak3 m1J /Boc (Stock No: 005558; Jax labs; also called JR5558) mice were obtained (2 females, 1 male) and retinas imaged (see also below) at 1 month and 2 months of age using the Micron III Imaging System (Phoenix, AZ). This allowed us to visualize the spontaneous development of multiple retinal lesions prior to euthanasia and subsequent analysis by immunofluorescence staining.

Mice and Laser Injury
Mice were provided anesthesia with an intraperitoneal (IP) injection of ketamine/xylazine and placed on a warming blanket. Phenylephrine hydrochloride and tropicamide were used together to dilate the pupil. An application of GenTeal Tears lubricant eye gel (Alcon Laboratories, Inc) was immediately applied to the cornea to prevent tissue drying and additionally applied prior to imaging serving as a contact lens for the imaging objective of the Micron III Imaging System (Phoenix, AZ). Once the retinal pigment epithelium (RPE) was brought into focus, the red-light guide laser was employed to focus the beam on the retinal location for delivery of the laser burn (Meridian Merilas Nd-Yag 532 alpha green laser photocoagulator) integrated with the Micron III imaging system. The laser beam of 50 m in diameter was employed to deliver 250 mW at 100 msec focal lesions. Six such laser lesions were delivered to the RPE in an equally spaced pattern surrounding the optic nerve. Care was taken to position the laser burns avoiding retinal blood vessels. Mice were subsequently recovered on warming blankets until fully ambulatory and returned to their housing units. At different time points, injured and control mice were euthanized by CO2 inhalation and cervical dislocation. Immediately after sacrifice, eyes were enucleated, frozen on dry ice, and maintained at -80°C.

Western Blot (WB) Analysis
Cornea, lens and vitreous were carefully separated from the posterior eye cups of mouse eyes. Protein from the retina/choroid tissue pooled from 2 eyes/sample was extracted in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% Sodium dodecyl sulfate) containing a cocktail of protease inhibitors (Roche, Indianapolis, IN) plus 1 mM sodium vanadate and 1 mM phenylmethyl sulfonyl fluoride. Samples were incubated on ice for 25 minutes with occasional vortexing and then centrifuged at 14,000 x g for 5 minutes at 4°C. The supernatant was removed, sheared through the tip of a p200 pipette, and centrifuged again at 14,000 x g for 5 minutes. The supernatants were normalized after adjusting to protein content (Bradford assay). Protein samples were denatured by boiling at 100°C for 10 min in presence of freshly added reducing agent ß-mercaptoethanol into Laemmeli buffer prior to loading on precast Criterion TGX-Stain-Free gels (BIO-RAD, CA). Gels were imaged (ChemiDoc XRS+ Gel Imaging System, BIO-RAD, CA) for protein loading prior to transfer to membranes using a semi-dry apparatus (Trans-Blot Turbo Transfer System, BIO-RAD, CA). Blots were blocked in 5% non-fat dry milk in Trisbuffered saline for 1-2 h at 4°C, followed by incubation overnight at 4°C in antibody solution prepared in blocking buffer.

Immunofluorescence (IF) staining
For murine samples, eyes were enucleated immediately after euthanasia, rinsed in phosphate buffered saline (1X PBS), and embedded in optimum cutting temperature compound (OCT; Tissue-Tek, Radnor, PA), and stored at -80°C. Briefly, 12 micron cryosections were air dried for 30 minutes at room temperature, fixed with 4% paraformaldehyde/PBS for 10 minutes, and then washed with PBS three times for 5 minutes each. Samples were probed with primary antibodies diluted in DAKO background reducing solution (Agilent Tech, Santa Clara, CA, Cat# S3022). After three washes with PBS slides were then incubated with secondary antibodies for 1 hour at room temperature (RT), and washed 3 times for 10 minutes before imaging.
For human samples, deidentified human donor eyes (n=3 controls and n=4 wet-AMD; range 77 to 89 years of age), procured within 8 hours of death, were dissected in buffered formalin and then the posterior eye cup imaged using Spectral Domain-Optical Coherence Tomography (Spectralis; Heidelberg Engineering) as described (Pang et al., Ophthalmology 2015). 8.2 mm, 7 line scans on macular crossing the optic disc were obtained. 30° x 30° autofluorescence fundus images were obtained with 488 nm argon blue laser and averaged from 9 images. 8 mm tissue punches of the macula were collected and cryopreserved for immunostaining. 12 micron sections of cryopreserved human maculae were collected on slides and dried overnight at 37°C. Sections were fixed in 4% PFA/PBS for 15 minutes, stained with DAPI for five minutes, and then blocked 3 hours at 37°C. Slides were incubated with primary antibodies at 4°C and then secondary antibodies for 1 hour at room temperature. Sections were imaged using confocal microscopy. Additional slides were dried overnight and fixed in 4% PFA/PBS for 15 minutes and stained with DAPI before being imaged by confocal microscopy for assessment of tissue autofluorescence. The imaging parameters employed for detection of antibodies were maintained for autofluorescence. Six different sets of sections from each donor eye were obtained and randomly chosen for analysis to ensure adequate representation of the macula tissues.

Confocal microscopy
Stained sections were examined and captured on a Ziess LSM800 confocal microscope. Mouse tissue was imaged and orthogonal projections were generated to analyze protein colocalization. Human tissue was imaged by confocal microscopy and maximum intensity projection was created from Z-stacks.

Rigor and Reproducibility
To randomize the analysis, different batches of C57Bl/6 mice were procured and subjected to experimental injury over a 2.5-year-period. To control for technical variability in the experimental injury model, three trained individuals performed the laser injuries at different times in this study, and as such, eyes from such separate experiments were collectively used for analysis. Also, different batches of PAD4cKO mice and littermates were derived and subjected to experimental injury. Mice of both sexes were employed, and the sex of mice were recorded, although the analyses was performed masked to the sex of the animal. As no significant differences were noted in the results after unmasking, these findings represent responses of male and female mice to the laser injury.