HHS Public Access Author manuscript Author Manuscript
Biochem Biophys Res Commun. Author manuscript; available in PMC 2017 June 29. Published in final edited form as:
Biochem Biophys Res Commun. 2017 May 20; 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. Wizemana and Royce Mohana,* aDepartment
of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA
Abstract Author Manuscript Author Manuscript
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) 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.
Graphical abstract
Author Manuscript
*
Corresponding author: Royce Mohan, MS., PhD., Department of Neuroscience, L4023, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030-3401, Tel: 860 679-2020, Fax: 860 679 8766, [email protected]. Authors do not have any conflicts of interest.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Wizeman and Mohan
Page 2
Author Manuscript Keywords Retinal gliosis; injury; citrullination; PAD4; GFAP
Author Manuscript
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.
Author Manuscript
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].
Author Manuscript
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
Biochem Biophys Res Commun. Author manuscript; available in PMC 2017 June 29.
Wizeman and Mohan
Page 3
Author Manuscript
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
Author Manuscript
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
Author Manuscript
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. 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
Author Manuscript
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).
Biochem Biophys Res Commun. Author manuscript; available in PMC 2017 June 29.
Wizeman and Mohan
Page 4
2.7. Protein Extraction and Western Blotting
Author Manuscript
Protein extraction, fractionation into soluble and insoluble fractions for intermediate filaments, and western blotting was performed as previously described [28]. 2.8. Intravitreal injections
Author Manuscript
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 eyecups 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
Biochem Biophys Res Commun. Author manuscript; available in PMC 2017 June 29. Published in final edited form as:
Biochem Biophys Res Commun. 2017 May 20; 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. Wizemana and Royce Mohana,* aDepartment
of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA
Abstract Author Manuscript Author Manuscript
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) 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.
Graphical abstract
Author Manuscript
*
Corresponding author: Royce Mohan, MS., PhD., Department of Neuroscience, L4023, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030-3401, Tel: 860 679-2020, Fax: 860 679 8766, [email protected]. Authors do not have any conflicts of interest.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Wizeman and Mohan
Page 2
Author Manuscript Keywords Retinal gliosis; injury; citrullination; PAD4; GFAP
Author Manuscript
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.
Author Manuscript
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].
Author Manuscript
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
Biochem Biophys Res Commun. Author manuscript; available in PMC 2017 June 29.
Wizeman and Mohan
Page 3
Author Manuscript
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
Author Manuscript
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
Author Manuscript
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. 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
Author Manuscript
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).
Biochem Biophys Res Commun. Author manuscript; available in PMC 2017 June 29.
Wizeman and Mohan
Page 4
2.7. Protein Extraction and Western Blotting
Author Manuscript
Protein extraction, fractionation into soluble and insoluble fractions for intermediate filaments, and western blotting was performed as previously described [28]. 2.8. Intravitreal injections
Author Manuscript
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 eyecups 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
