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.

Neuritic plaques in Alzheimer’s disease (AD) brains refer to β-amyloid (Aβ) plaques surrounded by dystrophic neurites (DNs), activated microglia and reactive astrocytes. Most recently, we showed that DNs form sequentially in three layers during plaque growth. Although lysosomal proteins such as LAMP1 are found in DNs, it is not clear how many and how early lysosomal proteins are involved in forming neuritic plaques. To answer this unmet question, we examined APP knock-in (APPNL-G-F), 5xFAD and APP/PS1ΔE9 mouse brains and found that the lysosomal activator proteins saposins (SAPs) and LAMP1 were accumulated to surround Aβ plaques at the earliest stage, namely the 1st layer of DNs. Noticeably, lysosomal hydrolases were not detectable in these early DNs, suggesting that DNs at this early stage likely enrich dysfunctional lysosomes. In old AD mouse brains and in the later stage of human AD brains, SAP-C+-DNs and LAMP1+-DNs were gradually reduced in concomitant with the growth of amyloid plaques. Remarkably, the observed LAMP1 immunoreactivity near plaques in aged AD mouse and human brains were actually associated with disease-associated microglia rather than neuronal sources, likely reflecting more severely impaired lysosomal functions in neurons. Western blot analyses showed increased levels of SAP-C in AD mouse brains, and Aβ oligomers induced elevated levels of SAP-C in cellular assays. The elevated protein levels of SAP-C in AD mouse brains during plaque growth potentially contributed lysosomal membrane leakage and loss of hydrolases. Together, our study indicates that lysosomal functions are impaired by being entrapped in DNs early during plaque growth, and this may viciously facilitate growth of amyloid plaques.

Retinal scarring with vision loss continues to be an enigma in individuals with advanced age‐related macular degeneration (AMD). Müller glial cells are believed to initiate and perpetuate scarring in retinal degeneration as these glial cells participate in reactive gliosis and undergo hypertrophy. We previously showed in the murine laser‐induced model of choroidal neovascularization that models wet‐AMD that glial fibrillary acidic protein (GFAP) expression, an early marker of reactive gliosis, increases along with its posttranslational modification citrullination. This was related to increased co‐expression of the citrullination enzyme peptidyl arginine deiminase‐4 (PAD4), which also colocalizes to GFAP filaments. However, whether such hypercitrullination in Müller glial drives fibrotic pathology has remained understudied. Here, using male and female C57Bl6 mice subjected to laser injury, we investigated in a temporal study how citrullination impacts GFAP and PAD4 dynamics. We found that high molecular weight citrullinated species that accumulate in Müller glia corresponded with dynamic changes in GFAP and PAD4 showing their temporal redistribution from polymeric cytoskeletal to soluble protein fractions using immunostaining and western blot analysis. In conditional glial‐specific PAD4 knockout (PAD4cKO) mice subjected to laser injury, there was a stark reduction of citrullination and of polymerized GFAP filaments. These injured PAD4cKO retinas showed improved lesion healing, as well as reduced fibronectin deposition in the subretinal space at 30 days. Taken together, these findings reveal that pathologically overexpressed PAD4 in reactive Müller glia governs GFAP filament dynamics and alters their stability, suggesting chronic PAD4‐driven hypercitrullination may be a target for retinal fibrosis.

Citrullination is a protein posttranslational modification (PTM) involving the irreversible modification of arginine residues on proteins to citrulline. Citrullinated proteins have recently been recognized as biomarkers in several major autoimmune and inflammatory diseases, such as rheumatoid arthritis and the central nervous system disease multiple sclerosis. Moreover, targeting the peptidyl arginine deiminases (PADs), the enzymes responsible for protein citrullination, with small molecule inhibitors has also been shown to be effective in reducing disease burden in several preclinical models. We recently reported that mice subjected to corneal chemical injury elicits retinal gliosis, wherein activated Muller glia displayed elevated levels of citrullinated glial fibrillary acidic protein (GFAP) (Mol Vision 2016; 22:1137–1155). Inhibition of PAD activity using a small molecule inhibitor potently decreased expression of citrullinated targets, identifying PADs as potential druggable targets for retinal pathology. Chemical injury to the cornea causes inner retinal pathology, and hence, initiates GFAP polymerization at Muller glial end feet. We found that the isozyme PAD4 was induced and also localized along glial processes (BBRC, 2017 487;134–139). As retinal gliosis, including altered citrullinated proteins, have been reported in tissue from age‐related macular degeneration (AMD) patients, we explored the idea that PAD4 may be responsible for the altered citrullinated proteome in AMD. Here we have exploited a laser injury model of AMD in mice, where laser ablation of 50 micrometer regions in the retinal pigment epithelium (RPE) causes focal lesions. We employed this model to determine if PAD4 expression is co‐regulated with retinal gliosis, and to also determine the localization of PAD4. Our findings show GFAP expression is induced as early as 1‐day post injury, as evidenced by western blot analysis. Time course studies using immunohistochemical analysis to examine polymerization of GFAP filaments show that this process initiates at the end feet of Muller glia and progresses into longer filaments that stain in the outer layers of the retina. Surprisingly, PAD4 distribution overlapping with GFAP reveals this polarized staining pattern despite the focal injury activating gliosis occurs in the RPE. Our findings reveal for the first time a previously unrecognized feature about the polarized distribution of PAD4 in retinal pathology, and enlists the hypothesis that a possible signal relay from the outer retina to the inner retina drives these events. Towards future studies testing this hypothesis, three questions can be asked. One, how are increased calcium levels required for PAD4 activity transmitted proximally to the Muller end feet? Two, because the retina is accessible for non‐invasive optical imaging, can this localization of PAD4 help to inform about early retinal pathology using PAD4‐targeted biomarker probes? Three, because PAD4 inhibitors are currently available, can this newly discovered localization within the inner retina be exploited for delivery of precision medicine for AMD?Support or Funding InformationJohn A. and Florence Mattern Solomon Endowed Chair in Vision Biology and Eye Diseases; Connecticut Bioscience Innovation Fund; NIH R01 EY016782This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.