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. Author manuscript; available in PMC: 2017 Jun 29.
Published in final edited form as: Biochem Biophys Res Commun. 2017 Apr 8;487(1):134–139. doi: 10.1016/j.bbrc.2017.04.031

Expression of Peptidylarginine Deiminase 4 in an Alkali Injury Model of Retinal Gliosis

John W Wizeman a, Royce Mohan a,*
PMCID: PMC5489915  NIHMSID: NIHMS868357  PMID: 28400047

Abstract

Citrullination is an important posttranslational modification that occurs during retinal gliosis. We examined the expression of peptidyl arginine deiminases (PADs) to identify the PADs that mediate citrullination in a model of alkali-induced retinal gliosis. Mouse corneas were exposed to 1.0 N NaOH and posterior eye tissue from injured and control uninjured eyes was evaluated for transcript levels of various PADs by reverse-transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qPCR). Retinas were also subjected to immunohistochemistry (IHC) for glial fibrillary acidic protein (GFAP), citrullinated species, PAD2, and PAD4 and tissue levels of GFAP, citrullinated species, and PAD4 were measured by western blots. In other experiments, the PAD4 inhibitor streptonigrin was injected intravitreally into injured eyes ex vivo to test inhibitory activity in an organ culture system. We found that uninjured retina and choroid expressed Pad2 and Pad4 transcripts. Pad4 transcript levels increased by day 7 post-injury (p <0.05), whereas Pad2 levels did not change significantly (p >0.05) by qPCR. By IHC, PAD2 was expressed in uninjured eyes along ganglion cell astrocytes, but in injured retina PAD2 was downregulated at 7 days. On the other hand, PAD4 showed increased staining in the retina upon injury revealing a pattern that overlapped with filamentous GFAP staining in Müller glial processes by 7 days. Injury-induced citrullination and soluble GFAP protein levels were reduced by PAD4 inhibition in western blot experiments of organ cultures. Together, our findings for the first time identify PAD4 as a novel injury-inducible druggable target for retinal gliosis.

Keywords: Retinal gliosis, injury, citrullination, PAD4, GFAP

Graphical abstract

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1. Introduction

Retinal gliosis is a reactive process that underlies most retinal pathologies [1],[2]. Gliosis in all areas of the central nervous system (CNS) has many similar characteristics [3],[4]. In the retina, gliosis occurs in Müller glial cells and ganglion cell layer astrocytes [2],[5]. These cells provide trophic and structural support to neuronal cells of the retina [1],[2]. The glial cell reactivity that is observed in injury [6],[7],[8],[9] and disease models [10] is characterized by the upregulation of the type III intermediate filaments (IFs) vimentin and glial fibrillary acidic protein (GFAP) [6],[7]. GFAP and vimentin are cytoskeletal proteins that are normally expressed in uninjured astrocytes and Müller cells, respectively; their chronic upregulation is considered pathological [2],[11],[12]. Thus, the precise control of these IFs, physiologically and pharmacologically [13],[6], has remained a challenge.

One mechanism by which IFs are regulated in vivo is through posttranslational modifications (PTM) [14], such as citrullination [15]. Citrullination is a calcium-dependent PTM to arginine residues that removes its positive charge [16]. This enzymatic reaction is mediated by the peptidyl arginine deiminases (PADs, EC 3.5.3.15) [17]. In mammals, there are 5 PADs, which often have disparate tissue expression profiles [16].

In cultured astrocytes, PAD2 and citrullination levels rise when cells are subjected to increases in mechanical pressure [18]. Citrullination and PAD levels are higher in autoimmune disease [19] as well as several CNS disorders, including Alzheimer’s disease [20],[21], glaucoma [22], and multiple sclerosis [23]. PAD2 is expressed in the retina and optic nerve of patients with glaucoma [22] and increased in age-related macular degeneration [24]. PAD3 is expressed in human neural stem cells [25], and PAD4 is found in the brains of Alzheimer’s [26] and multiple sclerosis [27] patients. However, it remained unknown which PAD enzyme is overactive in injury-induced gliosis.

We used an ocular injury model that induces retinal gliosis [6],[7] to identify changes in the expression of PADs in the retina. We have previously shown that GFAP and vimentin protein become elevated [6] and these proteins are deiminated after injury [28]. Total citrullinated proteins also increase after injury [28]. These changes occur within one hour of injury and can be mitigated by the pan-PAD inhibitor Cl-amidine, which reduces global citrullination and production of aberrant GFAP species [28]. In this study, we determined the contribution of PADs to citrullination in the retina and identify PAD4 as the principal PAD enzyme induced in Müller glia after injury.

2. Materials and methods

2.1. Ethics Statement

All animal experiments were conducted in accordance with procedures approved by IACUC committee of the University of Connecticut Health Center and NIH guidelines. Mice were housed in specific pathogen free cages in designated laboratory animal housing facilities.

2.2. Alkali Injury

Corneal alkali injuries were performed in equal numbers of male and female mice of the 129S6/SvEVTac strain (Taconic, Hudson, NY) [6],[7] with a minor modification as previously described [28].

2.3. RNA extraction, RT-PCR and qPCR

RNA was extracted from the retina choroid using a Qiagen RNeasy Mini Kit (Qiagen) according to manufacturers instructions. RNA was treated with DNAse (Ambion) to remove any contaminating genomic DNA. RNA was then reverse transcribed into corresponding cDNA using SuperScript III Reverse Transcriptase (Thermo Fisher) and diluted to 75 ng/ul. PCR primer pairs and conditions employed (Table 1) are outlined. Triplicate samples were analyzed by qPCR for Gapdh, Pad2 and Pad4 on a Bio-Rad CFX96 Touch real time PCR detection system. Fold change (normalized to Gapdh levels) was compared to uninjured and is expressed as fold change +/− standard deviation. Statistical differences were identified by two-tailed t-test.

Table 1.

RT-PCR and qPCR primers and amplification conditions

Gene Primers RT-PCR qPCR
Gapdh Forward - ATG ACA TCA AGA AGG TGG TG
Reverse - CAT ACC AGG AAA TGA GCT TG		 1. 94°C, 3m
2. 94°C, 30s
3. 45°C, 30s
4. 72°C, 60s
Repeat 2–4, 35X
5. 72°C, 5m		 1. 95°C, 60s
2. 95°C, 15s
3. 60°C, 30s
Repeat 2–3, 40X
Pad2 Forward - GTT ATG TTC AAG GGC CTG GGA GGC ATG
Reverse - TAG CAC GAT CAT GTT CAC CAT GTT AGG		 1. 94°C, 3m
2. 94°C, 40s
3. 57°C, 30s
4. 72°C, 40s
Repeat 2–4, 35X
5. 72°C, 7m		 1. 95°C, 3m
2. 95°C, 30s
3. 55°C, 30s
Repeat 2–3, 39X
Pad3 Forward - TTC TCC GAG ACC CCC ATC TT
Reverse - TTA TTC CTC ACC CGG CAC AC		 1. 94°C, 3m
2. 94°C, 60s
3. 58°C, 60s
4. 72°C, 3m
Repeat 2–4, 40X
5. 72°C, 10m
Pad4 Forward - CGA TTG GCT CTT TGT GGG TC
Reverse - CCG AAA ACC CTG CTT GTC C

*Forward - TCT TTG TGG GTC ACG TGG ATG AGT
*Reverse - AGC TCC TGG AAC AGC TGA TAG CAA (*second set of primers used for qPCR)		 1. 94°C, 3m
2. 94°C, 30s
3. 52°C, 30s
4. 72°C, 45s
Repeat 2–4, 34X
5. 72°C, 5m		 1. 95°C, 3m
2. 95°C, 15s
3. 51.5°C, 45s
Repeat 2–3, 40X
Gfap Forward - TCC TGG AAC AGC AAA ACA AG
Reverse - CAG CCT CAG GTT GGT TTC AT		 1. 95°C, 10m
2. 94°C, 30s
3. 55.5°C, 30s
4. 72°C, 30s
Repeat 2–4, 39X
5. 72°C, 5m		 1. 95°C, 1m
2. 95°C, 15s
3. 60°C, 30s
Repeat 2–3, 40X
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2.5. Immunohostochemistry

Immunohistochemistry (IHC) was performed as previously described [28], with modifications to secondary antibody staining. Slides were incubated with secondary antibodies at specified concentrations for 1–2 hours at room temperature in the dark, and subsequently washed 5 times for 10 minutes prior to imaging.

2.6. Antibodies

Primary antibodies used for western blotting (WB) and IHC were as follows: GFAP (IHC: EMD Millipore AB5541 1:500; WB: AB7260 1:4,000), PAD2 (Abcam ab50257 1:50), PAD4 (IHC: Abcam ab50247 1:100; WB: Biolegend clone O94H5, 1:1,000), β-III Tubulin (Abcam ab18207 1:100), F95 antibody for citrullinated proteins (EMD Millipore MABN328 1:50), GAPDH (Santa Cruz sc-25778 1:1,000), goat anti-mouse IgM 488 (Alexa 1:500), goat anti-rabbit 488 (Alexa 1:500), goat anti-chicken 594 (Alexa 1:500), goat anti-rabbit 594 (Alexa 1:500), goat anti-rabbit HRP (Santa Cruz 1:1,000), goat anti-mouse IgM HRP (Jackson Immunoresearch 1:5,000).

2.7. Protein Extraction and Western Blotting

Protein extraction, fractionation into soluble and insoluble fractions for intermediate filaments, and western blotting was performed as previously described [28].

2.8. Intravitreal injections

Mice were injured in vivo and after 10 minutes humanely sacrificed by CO2 inhalation. After sacrifice, eyes were enucleated and kept in 1X Dulbecco’s phosphate buffered saline (PBS) +Antibiotic/Antimycotic (A/A, GE Life Sciences), on ice until injection. An initial puncture of the outer layers of the globe was made using a Becton Dickinson 30-gauge x ½ in needle, keeping intact the globe structure. Streptonigrin (Cat# S1014, Sigma) was solubilized in dimethly sulfoxide (DMSO) and injected (50:50 mix with PBS) intravitreally using a Hamilton Syringe, to achieve ~25 nM final concentration (assuming a final volume of ~5μl). Vehicle controls were injected with 50:50 DMSO:PBS mix. This method allowed us to precisely deliver the drug in the enucleated eye. Vehicle and streptonigrin treated eye-cups were incubated in PBS for 5 hours in 5% CO2 at 37 °C. The posterior eye-cups were dissected from the anterior eye and posterior tissues subjected to protein [28] or RNA extractions as described.

2.9. Statistical Analysis

Each sample for western blot analysis contained pooled protein extracts from three separate mouse eyes, and entire injury experiments were repeated three times to obtain quantitative results. Data represented are the mean of three experiments (n=3) normalized to GAPDH. The data was analyzed to obtain the means and ± standard deviation (SD) using t-tests, with a difference of p <0.05 considered statistically significant.

3. Results

3.1. Pad2 and Pad4 transcripts are present in the retina

We initially measured the mRNA levels of PADs in the mouse posterior eye-cup. Pad2 mRNA was detected in uninjured and injured eye-cups (Fig. 1A), with no significant difference between transcript levels from samples of uninjured or 7 day mice, as determined by qPCR (Fig. 1B). Pad4 mRNA was observed in injured and uninjured samples (Fig. 1A). Notably, Pad4 mRNA increased by greater than 4-fold early after injury and remained high, rising by 24-fold at 7 days post-injury, as measured by qPCR (Fig. 1C). Based on their reported expression patterns, we did not screen for Pad1 [29] or Pad6 [30]. Pad3 was not detected in the posterior eye-cup.

Fig. 1. Pad2 and Pad4 mRNAs and protein expression in the retina.

Fig. 1

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PCR analysis of Pad2 and Pad4 mRNAs in the uninjured posterior eye-cup (unj) and eye-cups at 1 hour (1 h) and 7 days (7 d) post injury. (A) RT-PCR products from different time points run on a 1.2% agarose gel. Negative controls (-) contained water. (B) Quantitation of fold change (normalized to Gapdh levels) identified by qPCR of Pad2 transcripts at different time points post-injury compared to uninjured samples. (C) Quantitation of fold change (normalized to Gapdh levels) identified by qPCR of Pad4 transcripts at different time points compared to uninjured qPCR results (*p < 0.05, t-test). (D) Western blot analysis of PAD4 protein in posterior eye-cups from uninjured mice (Unj, lane 1) and eye-cups from mice 7 days post injury (7 d, lane 2). Proteins immunoreactive against PAD4 antibody were identified in uninjured and injured eye-cups at ~65–70 kDa. Immunoreactivity against a ~50 kDa species is identifiable in the injured eye (lane 2). (E) Densitometric quantitation of immunoreactive PAD4 bands normalized to GAPDH with ImageJ (*p < 0.05, t-test).

3.2. PAD4 protein is upregulated in the injured retina

The increase in Pad4 mRNA in the injured condition suggests that PAD4 mediates the citrullination of protein targets in posterior eye cup tissue extracts. Thus, we measured PAD4 in injured and uninjured eyes by western blot. In the uninjured and injured samples, we observed a significant increase in a ~65–70-kDa species that was immunoreactive for PAD4 antibody at 7 days postinjury (Fig. 1D, Unj =1.91 ±0.38; Injured =3.59 ±0.42). Basal levels of PAD4 were also seen in the uninjured posterior eye-cup. Notably, an anti-PAD4-immunoreactive cleavage product appeared at 50 kDa in the injured eye (Fig. 1D; arrow).

3.3. PAD2 is expressed in astrocytes in the uninjured retina

Having identified the PADs that are present in injured and uninjured retina, we determined the sources of PAD in the retina after injury. Citrullinated proteins are expressed along GFAP and vimentin filaments [28], indicating that the modified species accumulated in Müller glial cells or astrocytes in the injured retina. We measured the expression of PAD2 and found in the uninjured eye that PAD2 was expressed in the inner layers of the retina (Fig. 2A, C), including GFAP-positive cells in the ganglion cell layer and inner plexiform layer. However, seven days after injury, we observed minimal PAD2 expression (Fig. 2D–F). GFAP was upregulated in Müller glial cells extending throughout the layers of the inner retina after injury, as reported [28]. The ganglion cell layer was disorganized, consistent with what we have demonstrated in this model of alkali injury [28].

Fig. 2. PAD2 localization in the Uninjured and Injured Retina.

Fig. 2

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Cryosections from uninjured and injured eyes were stained using antibodies against GFAP (red) and PAD2 (green). Tissue sections were examined under fluorescent microscope at 20X magnification from uninjured eyes (A–C), and 7-day (D–F) post-injury eyes. GFAP (A) and PAD2 (B) expression overlap was mostly restricted to the ganglion cell layer (GCL) astrocytes in the uninjured retina. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bar = 100 μm

3.4. PAD4 expression in the uninjured and injured retina

Next, we examined PAD4 expression after injury. In the uninjured eye, there was no distinct cellular expression pattern of PAD4 (Fig. 3B) by IHC, despite the appearance of an immunoreactive species by western blot. Seven days after injury, PAD4 was observed extending to the inner nuclear layer of the inner retina (Fig. 3F), aligning with GFAP expression in Müller glial cells. Notably, the expression of PAD4 did not always coincide at a 1:1 ratio with GFAP filaments (Fig. 3E, F, arrowheads). PAD4 immunoreactivity assumed a filament-like pattern (Fig. 3E bracket), with PAD4-positive processes penetrating into the vitreous (Fig. 3E, asterisk).

Fig. 3. Localization of PAD4 protein in the Uninjured and Injured Retina.

Fig. 3

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Cryosections from uninjured and injured eyes were stained using antibodies against GFAP (red) and PAD4 (green). Tissue sections were examined under fluorescent microscope at 20X magnification from uninjured eyes (A–C), and 7-day (D–F) post-injury eyes. (E, bracket) PAD4 expression in the inner retina is observed in glial processes in association with filaments. GFAP filaments with variable (low) PAD4 immunoreactivity (F, arrowheads). Protrusion of PAD4-positive cells into the vitreal space (E, F, asterisk). Scale bar = 100 μm

3.5. Inhibition of PAD4 prevents GFAP expression

Identifying PAD4 as a potential source of citrullination in Müller glial cells prompted us to examine whether PAD4 inhibition alters GFAP expression. Streptonigrin is a potent and irreversible PAD4 selective inhibitor [31],[32]. To measure the effects of streptonigrin, we modified an established ocular explant system [28],[6] to allow for short-term assessments of retinal glial reactivity. Streptonigrin or vehicle was delivered by intravitreal injection to injured eyes after enucleation and incubated for 5 hours in an organ culture model [28].

As anticipated, streptonigrin inhibited the citrullination response after injury, reducing levels of the F95-reactive species by western blot (Fig. 4A). Notably, streptonigrin downregulated GFAP at a dose of 25 nM (Fig. 4B, C; Veh =2.78 ± 0.57; Streptonigrin =1.56 ± 0.12). Next, to determine whether the drug affected Gfap transcript levels, we employed qPCR to analyze vehicle- and streptonigrin-treated eyes. Gfap transcript levels were not altered by streptonigrin treatment (Fig. 4D; Vehicle 1.3667 ± 0.02663; streptonigrin =1.37057 ± 0.02288; p = 0.7431). These data show that PAD4 inhibition reduces the major citrullinated protein species, corresponding with the downregulation of GFAP protein levels.

Fig. 4.

Fig. 4

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Intravitreal Injection of Streptonigrin downregulates GFAP expression in Injury Western blot analysis of citrullinated proteins (A), and GFAP (B, C) in streptonigrin (Stn) treated eyes. Mice were injured and sacrificed 10 minutes after injury. Eyes were enucleated and injected with either DMSO (veh) or streptonigrin (Stn, 25 nM) and incubated in PBS +A/A for 5 hours at 37°C. Posterior eye-cups were dissected and soluble lysate fractionated by SDS-PAGE, blotted and probed with the F95 antibody (A, citrullinated proteins) or GFAP antibody (B). Quantitation of GFAP bands normalized to GAPDH by ImageJ (C) (*p < 0.05, t-test). In separate analysis, posterior eye-cups from vehicle and streptonigrin-treated eyes were also extracted for RNA and Gfap transcript levels analyzed by qPCR employing Gapdh transcript levels for normalization (D). No differences were found between vehicle and streptonigrin treated samples (p = 0.7431, t-test).

4. Discussion

Retinal gliosis occurs as a rapid stress response to alkali injury [28]. In this study, we found that Pad4 expression rises in the injured mouse retina and that this PAD isozyme localizes to Müller glial processes, concomitant with upregulation of GFAP. We also observed that inhibition of PAD4-driven citrullination downregulates GFAP in the injured retina. Because GFAP is overexpressed during retinal stress and disease [1] and is a major target of citrullination in injured retina [28], PAD4 could be a potential druggable target of glial reactivity.

The finding that PADs are present in the uninjured retina indicates that they are expressed at basal levels, allowing potentially rapid response to changes in calcium concentrations [33],[34]. Calcium levels increase in Müller glial cells after stretching [35], which cause alterations in cytoskeletal mechanical function [36]. PAD2 is known to citrullinate vimentin [37]. PADs 1–4 are more enzymatically active in the presence of calcium, creating an ideal system to respond to changes that are induced by enhanced mechanosensitivity from Müller glia on increases in intraocular pressure [38],[33]. The propagation of calcium waves between Müller glia and astrocytes also provides a mechanism to synchronize the retinal response across the retina [39],[40],[41].

The effects of citrullination on the IFs in vivo remain largely unknown. We have recorded citrullination of GFAP and vimentin filaments as early as 1 hour after injury and have demonstrated that the levels of two citrullinated GFAP isoforms increase in the soluble protein extracts [28]. The presence of these novel soluble GFAP isoforms in injured retinal tissues suggests that there are non-cytoskeletal functions of GFAP, perhaps relevant to its activity as a chaperone and in protein transport [42],[14]. In this context, citrullination could have a function in governing the solubility of GFAP, similar to that of phosphorylation for vimentin [14],[43].

Unlike the dynamic reversibility of phosphorylation, citrullination is an irreversible PTM. Thus, citrullination of GFAP could be a permanent function-altering mechanism that contributes to chronic changes in Müller glia. Because GFAP becomes abundant after injury, the increases in soluble GFAP on citrullination could relay the disruptive effects through its binding partners in signal transduction [44]. Alternatively, soluble GFAP might become antigenic, driving autoimmune responses [45]. Our findings that PAD4 localizes along Müller cell processes suggest that possible interactions between enzyme and substrate occur in Müller glia and lead to possible solubilization of GFAP filaments via citrullination [15],[28]. Considering that PAD4 contains a nuclear localization sequence and PAD4 can be found in the nucleus [46], another possible mechanism involves the activation of PADs in Müller cell endfeet and astrocytes through increases in calcium, which could affect the transport of PAD4 toward Müller nuclei along GFAP and vimentin filaments to trigger transcriptional responses. Considering the rapid kinetics of this acute reaction after injury [28] such responses would precede subsequent wound-healing mechanisms [9] in the alkali injury model.

Taken together, this study is the first to report PAD4 overexpression during gliosis in a model of retinal injury. Thus, it will be important to know also whether PAD4 is also upregulated in retinal diseases [10] or other models of injury [8] as well.

Highlights.

  • Retinal gliosis is associated with increased citrullination during injury

  • PAD4 expression is induced in Muller glia and localizes along GFAP filaments

  • PAD4 inhibition reduces protein citrullination and GFAP protein levels

Acknowledgments

Funding: This work was supported in part by NIH grant R01EY016782 and the John A. and Florence Mattern Solomon Endowed Chair in Vision Biology and Eye Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

Authors do not have any conflicts of interest.

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