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• W1799202546 abstract "Peptidylarginine deiminases (PADs), which are a group of posttranslational modification enzymes, are involved in protein citrullination (deimination) by the conversion of peptidylarginine to peptidylcitrulline in a calcium concentration-dependent manner. Among the PADs, PAD2 is widely distributed in various tissues and is the only type that is expressed in brain. To elucidate the involvement of protein citrullination by PAD2 in the pathogenesis of brain-specific prion diseases, we examined the profiles of citrullinated proteins using the brains of scrapie-infected mice as a prion disease model. We found that, compared with controls, increased levels of citrullinated proteins of various molecular weights were detected in different brain sections of scrapie-infected mice. In support of this data, expression levels of PAD2 protein as well as its enzyme activity were significantly increased in brain sections of scrapie-infected mice, including hippocampus, brain stem, and striatum. Additionally, the expression levels of PAD2 mRNA were increased during scrapie infection. Moreover, PAD2 immunoreactivity was increased in scrapie-infected brains, with staining detected primarily in reactive astrocytes. Using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry, various citrullinated proteins were identified in the brains of scrapie-infected mice, including glial fibrillary acidic protein, myelin basic protein, enolases, and aldolases. This study suggests that accumulated citrullinated proteins and abnormal activation of PAD2 may function in the pathogenesis of prion diseases and serve as potential therapeutic targets. Peptidylarginine deiminases (PADs), which are a group of posttranslational modification enzymes, are involved in protein citrullination (deimination) by the conversion of peptidylarginine to peptidylcitrulline in a calcium concentration-dependent manner. Among the PADs, PAD2 is widely distributed in various tissues and is the only type that is expressed in brain. To elucidate the involvement of protein citrullination by PAD2 in the pathogenesis of brain-specific prion diseases, we examined the profiles of citrullinated proteins using the brains of scrapie-infected mice as a prion disease model. We found that, compared with controls, increased levels of citrullinated proteins of various molecular weights were detected in different brain sections of scrapie-infected mice. In support of this data, expression levels of PAD2 protein as well as its enzyme activity were significantly increased in brain sections of scrapie-infected mice, including hippocampus, brain stem, and striatum. Additionally, the expression levels of PAD2 mRNA were increased during scrapie infection. Moreover, PAD2 immunoreactivity was increased in scrapie-infected brains, with staining detected primarily in reactive astrocytes. Using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry, various citrullinated proteins were identified in the brains of scrapie-infected mice, including glial fibrillary acidic protein, myelin basic protein, enolases, and aldolases. This study suggests that accumulated citrullinated proteins and abnormal activation of PAD2 may function in the pathogenesis of prion diseases and serve as potential therapeutic targets. Accumulation of misfolded proteins, posttranslational modification of proteins, alteration of free ion distribution, and perturbation of cellular redox homeostasis are general features of progressive neurodegenerative disorders. These changes have been observed consistently as part of the neuropathogenesis and neuropathology of prion diseases. Prion diseases are characterized by various neurological symptoms and common histopathological features such as spongiform degeneration of the central nervous system, reactive gliosis, neuronal loss, and, in some cases, formation of amyloid plaques.1Prusiner SB Prions.Proc Natl Acad Sci USA. 1998; 95: 13363-13383Crossref PubMed Scopus (5194) Google Scholar It has been reported that all prion diseases are associated with the aberrant metabolism of prion protein (PrP). Conversion of the cellular prion protein (PrPC) into an abnormal, protease-resistant and infectious isoform (PrPSc) is believed to be a principal molecular basis of prion diseases,2Aguzzi A Heikenwalder M Polymenidou M Insights into prion strains and neurotoxicity.Nat Rev Mol Cell Biol. 2007; 8: 552-561Crossref PubMed Scopus (269) Google Scholar and the accumulation of PrPSc in the central nervous system is thought to be responsible for neuronal loss and/or astrocytosis.3Cronier S Laude H Peyrin JM Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death.Proc Natl Acad Sci USA. 2004; 101: 12271-12276Crossref PubMed Scopus (127) Google Scholar In general, the pathogenic mechanisms of neurodegenerative disorders are not fully delineated; prion diseases are no exception to this uncertainty. Alteration of intracellular calcium (Ca2+) distribution and Ca2+-related proteins have a critical role in synaptic dysfunction and neuronal cell death in neurodegenerative diseases. In cultured cells, prion infection induced abnormalities in Ca2+ homeostasis by altering receptor-mediated intracellular Ca2+ responses,4Wong K Qiu Y Hyun W Nixon R VanCleff J Sanchez-Salazar J Prusiner SB DeArmond SJ Decreased receptor-mediated calcium response in prion-infected cells correlates with decreased membrane fluidity and IP3 release.Neurology. 1996; 47: 741-750Crossref PubMed Scopus (65) Google Scholar, 5Takenouchi T Iwamaru Y Imamura M Kato N Sugama S Fujita M Hashimoto M Sato M Okada H Yokoyama T Mohri S Kitani H Prion infection correlates with hypersensitivity of P2X7 nucleotide receptor in a mouse microglial cell line.FEBS Lett. 2007; 581: 3019-3026Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar suggesting a possible role of Ca2+ in the neuronal cell death seen in prion diseases. Peptidylarginine deiminases (PADs) are known to be directly affected by Ca2+ homeostasis and convert peptidylarginine to peptidylcitrulline (protein citrullination or deimination) in a Ca2+ concentration-dependent manner.6Inagaki M Takahara H Nishi Y Sugawara K Sato C Ca2+-dependent deimination-induced disassembly of intermediate filaments involves specific modification of the amino-terminal head domain.J Biol Chem. 1989; 264: 18119-18127Abstract Full Text PDF PubMed Google Scholar, 7Vossenaar ER Zendman AJ van Venrooij WJ Pruijn GJ PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease.Bioessays. 2003; 25: 1106-1118Crossref PubMed Scopus (787) Google Scholar This modification of proteins decreases their positive charge resulting in changing the functions of native proteins.8Tarcsa E Marekov LN Mei G Melino G Lee SC Steinert PM Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin.J Biol Chem. 1996; 271: 30709-30716Crossref PubMed Scopus (310) Google Scholar PADs are found as five different isoforms in various mammalian tissues such as brain, spinal cord, spleen, and skeletal muscle.9Watanabe K Akiyama K Hikichi K Ohtsuka R Okuyama A Senshu T Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues.Biochim Biophys Acta. 1988; 966: 375-383Crossref PubMed Scopus (132) Google Scholar Among them, only PAD type II (PAD2) is expressed in adult rat brains and its cellular localization was found in glial cells.10Vincent SR Leung E Watanabe K Immunohistochemical localization of peptidylarginine deiminase in the rat brain.J Chem Neuroanat. 1992; 5: 159-168Crossref PubMed Scopus (35) Google Scholar, 11Asaga H Senshu T Combined biochemical and immunocytochemical analyses of postmortem protein deimination in the rat spinal cord.Cell Biol Int. 1993; 17: 525-532Crossref PubMed Scopus (45) Google Scholar, 12Akiyama K Sakurai Y Asou H Senshu T Localization of peptidylarginine deiminase type II in a stage-specific immature oligodendrocyte from rat cerebral hemisphere.Neurosci Lett. 1999; 274: 53-55Crossref PubMed Scopus (55) Google Scholar In a very recent report, PAD2 expression was detected in cultured Schwann cells.13Keilhoff G Prell T Langnaese K Mawrin C Simon M Fansa H Nicholas AP Expression pattern of peptidylarginine deiminase in rat and human Schwann cells.Dev Neurobiol. 2008; 68: 101-114Crossref PubMed Scopus (26) Google Scholar Previous reports indicate that PAD2 is involved in the citrullination of various cerebral proteins under neurodegenerative conditions.14Asaga H Ishigami A Protein deimination in the rat brain after kainate administration: citrulline-containing proteins as a novel marker of neurodegeneration.Neurosci Lett. 2001; 299: 5-8Crossref PubMed Scopus (61) Google Scholar Recently, it has been reported that the abnormal accumulation of citrullinated proteins including glial fibrillary acidic protein (GFAP) and vimentin were found in Alzheimer's disease (AD)-afflicted hippocampus; increased expression of PAD2 and its enzyme activity were detected during neurodegenerative changes and were accompanied by impairment of intracellular Ca2+ homeostasis.15Ishigami A Ohsawa T Hiratsuka M Taguchi H Kobayashi S Saito Y Murayama S Asaga H Toda T Kimura N Maruyama N Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer's disease.J Neurosci Res. 2005; 80: 120-128Crossref PubMed Scopus (198) Google Scholar In multiple sclerosis (MS) patients, previous studies have revealed that citrullinated myelin basic protein (MBP) was increased to 45% of total MBP compared to healthy adults16Moscarello MA Wood DD Ackerley C Boulias C Myelin in multiple sclerosis is developmentally immature.J Clin Invest. 1994; 94: 146-154Crossref PubMed Scopus (283) Google Scholar and has been implicated in the pathological mechanism of MS.17Moscarello MA Mastronardi FG Wood DD The role of citrullinated proteins suggests a novel mechanism in the pathogenesis of multiple sclerosis.Neurochem Res. 2007; 32: 251-256Crossref PubMed Scopus (209) Google Scholar Therefore, PAD and citrullinated proteins can be used as important factors for the diagnosis of various human diseases.7Vossenaar ER Zendman AJ van Venrooij WJ Pruijn GJ PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease.Bioessays. 2003; 25: 1106-1118Crossref PubMed Scopus (787) Google Scholar To our knowledge, there are no data available regarding citrullination by PAD2 in prion diseases. Here we report for the first time that increased citrullinated proteins including GFAP, MBP, and several newly identified proteins were found in the brains of scrapie-infected mice along with increased expression of PAD2 protein and its enzyme activity. These findings suggest a possible role of citrullination in the induction of pathological changes in the brains of scrapie-infected mice. C57BL/6J mice, 4 to 6 weeks of age, were obtained from the Experimental Animal Center of Hallym University. The original stock of ME7 scrapie strain was kindly provided by Dr. Alan Dickinson of Agriculture and Food Research Council and Medical Research Council Institute (Edinburgh, UK): this scrapie strain was maintained by serial intracerebral passage of brain homogenate from terminally affected mice. Mice were inoculated intracerebrally with 30 μl of 1% (w/v) brain homogenate in 0.01 mol/L phosphate-buffered saline (PBS) prepared from ME7-injected C57BL mice or from control mice that had been injected with normal brain homogenate. The mice were then sacrificed under 16.5% urethane at 150 ± 10 days after inoculation with ME7 scrapie strain, a time when clinical manifestations of disease were evident. To perform a time course study, brains were also collected at different time points (50, 100, and 150 days after inoculation). Mice inoculated with normal brain homogenate remained healthy throughout the same period. For immunohistochemistry, mice were perfused transcardially with PBS followed by 4% paraformaldehyde in PBS (pH 7.4). The brains were removed immediately, postfixed in the same fixative for 2 hours at room temperature, rinsed with PBS, dehydrated with ethanol, and embedded in paraffin. Brains from control and scrapie-infected mice were homogenized in modified RIPA buffer containing 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 2 mmol/L ethylenediaminetetraacetic acid, 1% Triton X-100, 1% Nonidet P-40, 0.25% sodium deoxycholate, and protease inhibitors.18Choi EK Zaidi NF Miller JS Crowley AC Merriam DE Lilliehook C Buxbaum JD Wasco W Calsenilin is a substrate for caspase-3 that preferentially interacts with the familial Alzheimer's disease-associated C-terminal fragment of presenilin 2.J Biol Chem. 2001; 276: 19197-19204Crossref PubMed Scopus (66) Google Scholar The homogenates were rocked at 4°C for 1 hour and centrifuged at 18,000 × g at 4°C for 10 minutes to remove cell debris. The supernatant was collected and protein concentration was determined with a BCA protein assay kit (Pierce, Rockford, IL). Citrullinated proteins were detected by Western blot analysis as described previously.19Senshu T Sato T Inoue T Akiyama K Asaga H Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane.Anal Biochem. 1992; 203: 94-100Crossref PubMed Scopus (152) Google Scholar Briefly, equal amounts of protein (50 μg/lane) were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane using an electrotransfer system (Bio-Rad, Hercules, CA). For detection of citrullinated proteins, citrulline residues on the polyvinylidene difluoride membrane were chemically modified by overnight incubation at 37°C in modification reagent [1 vol of 1% diacetyl monoxime/0.5% antipyrine/1 N acetic acid, and 2 vol of a mixture of 85% H3PO4/98% H2SO4/H2O (20/25/55) containing 0.025% FeCl3].20Rothnagel JA Rogers GE Citrulline in proteins from the enzymatic deimination of arginine residues.Methods Enzymol. 1984; 107: 624-631Crossref PubMed Scopus (51) Google Scholar The membrane was then blocked with 5% nonfat dry milk in PBST (8 mmol/L Na2HPO4, 2 mmol/L KH2PO4, 138 mmol/L NaCl, 2.7 mmol/L KCl, pH 7.4, 0.05% Tween 20) for 2 hours at room temperature, then probed with an anti-modified citrulline antibody at 1:1000 (Upstate, Lake Placid, NY) in PBST overnight at 4°C. For the detection of other target proteins, the transferred polyvinylidene difluoride membranes were directly probed with either monoclonal anti-PAD2 antibody (1:2000), mouse monoclonal anti-neuron-specific enolase (1:2000) (AbFrontier, Seoul, Republic of Korea), rabbit polyclonal anti-aldolase C (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-MBP (1:3000) (Abcam, Cambridge, MA), or mouse monoclonal anti-β actin (1:10,000) (Sigma, St. Louis, MO) in 5% nonfat dry milk in PBST. These membranes were then incubated with the appropriate secondary antibody-conjugated to horseradish peroxidase. Bound antibodies were visualized by chemiluminescent substrate as described by the manufacturer (Amersham Biosciences, Piscataway, NJ). Total RNA was extracted from brain samples of control and scrapie-infected mice using TRI-reagent (Sigma) according to the manufacturer's protocols. Complementary DNA (cDNA) was generated using the Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) according to the instructions of the manufacturer. RT-PCR was performed using primers specific for the PAD2 (665 bp; forward, 5′-CTGCGGTCTCTGGGTCCTTCCTGTA-3′ and reverse, 5′-GACCAGGCGAGAGAACAGAAATAGC-3′) and β-actin (196 bp; forward, 5′-TGTGATGGACTCCGGTGACGG-3′ and reverse, 5′- ACA GCTTCTCTTTGATGTCACGC-3′) genes. The PCR products were separated by electrophoresis on a 1% agarose gel and visualized under UV light. The PAD2 activity was determined as described previously.9Watanabe K Akiyama K Hikichi K Ohtsuka R Okuyama A Senshu T Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues.Biochim Biophys Acta. 1988; 966: 375-383Crossref PubMed Scopus (132) Google Scholar Briefly, the reaction mixture containing 100 mmol/L Tris-HCl, pH 7.5, 10 mmol/L CaCl2, 5 mmol/L dithiothreitol, with or without 10 mmol/L benzoyl-l-arginine ethyl ester (Sigma), and 0.5 mg of brain protein in a final volume of 120 μl was incubated at 50°C for 1 hour. After incubation, the reaction was stopped by adding final 1 mol/L perchloric acid. Samples were cooled down on ice for 20 minutes and then centrifuged at 18,000 × g for 5 minutes at room temperature. Aliquots of 120 μl of supernatant were mixed with 380 μl of dH2O and 500 μl of color developing reagent and incubated at 95°C for 15 minutes. The samples were cooled to room temperature and then the absorbance was measured at 540 nm by enzyme-linked immunosorbent assay reader (VersaMax; Molecular Devices, Sunnyvale, CA). Quantification of citrulline was determined by comparison with appropriate standards. One unit of the enzyme is defined as the amount of enzyme that deiminates 1 μmol/L of the substrate (benzoyl-l-arginine ethyl ester) in 1 minute at 50°C. Immunohistochemical procedures were performed using the ABC kit (Vector, Burlingame, CA) by a modification of the avidin-biotin-peroxidase method. Briefly, 6-μm sections of brain were deparaffinized with xylene and hydrated in a graded ethanol series, and then treated with 0.3% hydrogen peroxide in methyl alcohol for 20 minutes to block endogenous peroxidase. The sections were exposed to normal donkey serum (Jackson ImmunoResearch, West Grove, PA), and then incubated with mouse monoclonal antibody for PAD2 (1:500) overnight at 4°C. After washing, the sections were treated sequentially with biotinylated anti-mouse IgG and avidin-biotin-peroxidase complex, developed with diaminobenzidine-hydrogen peroxide solution (0.003% 3,3-diaminobenzidine and 0.03% hydrogen peroxide in 50 mmol/L Tris buffer), and finally counterstained with hematoxylin. For staining of citrullinated proteins, the sections were incubated in modification reagent for 2 hours at 37°C before initiation of the immunohistochemistry protocol. After three washes in PBS buffer, the sections were exposed to 10% normal donkey serum for 1 hour at room temperature and then rabbit polyclonal antibody to modified citrulline (1:4000) for 1 hour at 37°C. The subsequent procedures were performed as described above for PAD2 staining. For double-immunofluorescence staining, the sections were incubated in the following order: 10% normal donkey serum in PBS for 1 hour, rabbit polyclonal anti-PAD2 (1:100)21Ishigami A Ohsawa T Asaga H Akiyama K Kuramoto M Maruyama N Human peptidylarginine deiminase type II: molecular cloning, gene organization, and expression in human skin.Arch Biochem Biophys. 2002; 407: 25-31Crossref PubMed Scopus (92) Google Scholar overnight at 4°C, lissamine rhodamine sulfonyl chloride (LRSC)-conjugated donkey anti-rabbit IgG (1:200) (Jackson ImmunoResearch) for 1 hour at room temperature, washed, and blocked with 10% normal goat serum in PBS for 1 hour at room temperature and then incubated with various primary antibodies as follows: mouse monoclonal anti-GFAP antisera (1:400; DAKO, Copenhagen, Denmark), mouse monoclonal anti-NeuN (1:50) (Chemicon, Temecula, CA), mouse monoclonal anti-MBP (1:3000) overnight at 4°C and finally washed and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1:200) (Jackson ImmunoResearch). For microglia staining, the lectin Griffonia simplicifolia (GSA) was optimized by incubating the sections in 0.5 mg/ml of trypsin in 0.05 mol/L Tris-buffered saline containing 1 mmol/L CaCl2 (pH 7.6) for 5 minutes at 37°C. Biotinylated lectin GSA B4-isolectin (Sigma) was then added for 1 hour at room temperature, followed by fluorescein isothiocyanate-labeled streptavidin (Zymed, San Francisco, CA). The sections were examined with a LSM 510 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany). Protein extraction and 2-DE were performed as reported previously.22Toda T Satoh M Sugimoto M Goto M Furuichi Y Kimura N A comparative analysis of the proteins between the fibroblasts from Werner's syndrome patients and age-matched normal individuals using two-dimensional gel electrophoresis.Mech Ageing Dev. 1998; 100: 133-143Crossref PubMed Scopus (21) Google Scholar Briefly, 200 μg of brain protein of control and ME7 scrapie-infected mice was dissolved in a rehydration buffer containing 8 mol/L urea, 2% CHAPS, 65 mmol/L dithiothreitol, 0.5% immobilized pH gradient (IPG) buffer (Bio-Rad), 40 mmol/L Tris-HCl, and 0.002% bromophenol blue. The brain homogenates were applied to the IPG Readystrip, 7 cm, pH 3 to 10 linear gradient (Bio-Rad). The IPG strips were rehydrated for 16 hours at 20°C using the PROTEAN isoelectric focusing cell (Bio-Rad) according to the manufacturer's instructions. Briefly, isoelectric focusing was conducted at 250 V for 15 minutes, linearly increased throughout 2 hours to a maximum of 4000 V, and then run to accumulate a total of 20,000 Vhours. The gel strips were equilibrated before second dimensional electrophoresis for 15 minutes in 50 mmol/L Tris-HCl (pH 8.8) containing 6 mol/L urea, 30% glycerol, 2% sodium dodecyl sulfate, 0.002% bromophenol blue, and 80 mmol/L dithiothreitol or 0.025% iodoacetamide. The gel strips were then separated in 12% polyacrylamide gels to perform the second dimensional electrophoresis. The 2-DE gels were then exposed to Coomassie Brilliant Blue G-250 (CBBG-250) or silver staining. Duplicated 2-DE gels were also transferred on polyvinylidene difluoride membrane and then used for the detection of citrullinated proteins using an antibody to modified citrulline. The protein spots of immunoblotting-matched citrullinated proteins were subjected to in-gel trypsin digestion, and digested peptides were analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (the Proteomics Service by Genomine, Pohang, Republic of Korea). The obtained peptide mass fingerprints spectra were analyzed by searching the Genomine local database and the National Centre for Biotechnology Information, nonredundant protein database with ProFound (http://prowl.rockefeller.edu/prowl-cgi/profound.exe). Quantitative results were expressed as means ± SEM. The probability of statistical differences between control and scrapie-infected groups was determined by a one-way analysis of variance test as indicated. Differences were considered significant at P < 0.001, P < 0.01, and P < 0.05. We first used Western blot analysis as described in the Materials and Methods to examine the citrullinated proteins from different brain regions of control and ME7 scrapie-infected mice. Total proteins were extracted from whole brains as well as from various dissected brain regions including cerebral cortex, hippocampus, striatum, cerebellum, and brain stem of control and scrapie-infected mice. In general, citrullinated proteins in most brain sections of scrapie-infected mice were markedly increased compared to controls (Figure 1, asterisks). In all brain regions, most bands from 10 kDa to 100 kDa were increased in scrapie. Those bands between 100 kDa and 150 kDa in whole brain, cerebellum, and brain stem were increased during scrapie infection, whereas no difference in citrullination was seen in other regions. Next, we performed immunohistochemical staining of citrullinated proteins in different brain sections of control and scrapie-infected mice. As shown in Figure 2, citrullinated proteins (indicated by arrows) were more prominent in the scrapie-infected brains (Figure 2, F–J) than in control brains (Figure 2, A–E). These results correlated with the increased citrullinated proteins observed in scrapie-infected mice using Western blot analysis (Figure 1). We also observed similar results in the brains of 87V scrapie-infected mice (data not shown), indicating the occurrence of increased citrullination after infection is a general phenomenon. Posttranslational modification of protein arginine residues to citrulline is mediated by PAD2 enzyme in the brain. Therefore, we examined whether the expression levels of PAD2 in the brains are increased after ME7 scrapie infection. As can be seen in Figure 3A, the expression levels of PAD2 protein appeared to be increased in scrapie-infected mice in the following brain sections: cerebral cortex, hippocampus, striatum, and brain stem. However the expression level of PAD2 in cerebellum did not appear to be different between control and infected mice. Densitometric analysis of the gels showed that the relative intensity of the PAD2 bands was significantly higher in all sections of scrapie-infected brains compared to controls except in the cerebellum (Figure 3B). In the next experiment, we measured expression levels of PAD2 mRNA in whole brains of both control and ME7 scrapie-infected mice using PAD2-specific primers as described in the Materials and Methods. As can be seen in Figure 3C, RT-PCR analysis showed increased PAD2 mRNA in the whole brain of ME7 scrapie-infected mice compared to levels in controls. This result suggests that up-regulated expression of PAD2 protein seen in most brain regions is caused by an increase of gene expression in scrapie-infected mice. Next, to confirm the increased PAD2 expression and to determine the cellular localization of PAD2, immunohistochemical analysis was performed using various brain sections from control and scrapie-infected mice (Figure 4, A–E and F–J, respectively). In scrapie-infected brains, PAD2 immunoreactivity was more intense compared to control brains and these results correlated with the expression patterns of PAD2 protein in results of Western blot analysis (Figure 3A). According to previous reports, PAD2 is mainly expressed in glial cells such as astrocytes, oligodendrocytes, and microglial cells.10Vincent SR Leung E Watanabe K Immunohistochemical localization of peptidylarginine deiminase in the rat brain.J Chem Neuroanat. 1992; 5: 159-168Crossref PubMed Scopus (35) Google Scholar, 11Asaga H Senshu T Combined biochemical and immunocytochemical analyses of postmortem protein deimination in the rat spinal cord.Cell Biol Int. 1993; 17: 525-532Crossref PubMed Scopus (45) Google Scholar, 12Akiyama K Sakurai Y Asou H Senshu T Localization of peptidylarginine deiminase type II in a stage-specific immature oligodendrocyte from rat cerebral hemisphere.Neurosci Lett. 1999; 274: 53-55Crossref PubMed Scopus (55) Google Scholar To further characterize the subcellular localization of PAD2, we performed double-immunofluorescence staining using GFAP, MBP, NeuN, and B4-isolectin as a marker for astrocytes, oligodendrocytes, neurons, and microglia, respectively. Interestingly, immunoreactivity was mainly observed in activated astrocytes in the brains of scrapie-infected mice. As can be seen in Figure 5, PAD2 was mainly co-localized with GFAP-positive astrocytes and in a few B4-isolectin-positive microglia, but not with MBP or NeuN. These data suggest that up-regulation of PAD2 expression in reactive astrocytes is responsible for the increased citrullinated proteins in the brains of scrapie-infected mice.Figure 5Cellular localization of PAD2 in scrapie-infected brains. The brain sections were doubly immunostained with anti-PAD2 and one of the following antibodies: astrocyte-specific GFAP, oligodendrocyte-specific MBP, neuron-specific NeuN, or microglia-specific B4-isolectin antibodies. Slides were examined under confocal laser-scanning microscopy. Note that PAD2-positive cells were strongly co-localized in cells positive for GFAP (arrows) with very few B4-isolectin-positive microglia (arrows). Scale bars = 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate whether the increase in citrullinated proteins is attributable to higher levels of PAD2 enzyme activity and whether the increased PAD2 expression is correlated with its enzyme activity in scrapie-infected mice, we analyzed PAD2 enzyme activity in the brain homogenates of various sections from both control and ME7 scrapie-infected mice using benzoyl-l-arginine ethyl ester as a substrate as previously described.9Watanabe K Akiyama K Hikichi K Ohtsuka R Okuyama A Senshu T Combined biochemical and immunochemical comparison of peptidylarginine deiminases present in various tissues.Biochim Biophys Acta. 1988; 966: 375-383Crossref PubMed Scopus (132) Google Scholar PAD2 enzyme activity was significantly increased approximately twofold in whole brains as well as in hippocampus, striatum, and brain stem of scrapie-infected brains compared to controls (Figure 6A): whole brain [2.41-fold, control: 0.322 ± 0.052 (mean ± SEM, units); infected: 0.776 ± 0.039], hippocampus (2.19-fold, control: 0.342 ± 0.081; infected: 0.748 ± 0.120), striatum (2.48-fold, control: 0.256 ± 0.009; infected: 0.636 ± 0.082), and brain stem (1.99-fold, control: 0.589 ± 0.028; infected: 1.172 ± 0.055). The difference from controls was not significant in cerebral cortex (control: 0.193 ± 0.022; infected: 0.294 ± 0.032) or in cerebellum (control: 0.312 ± 0.041; infected: 0.419 ± 0.035). To determine whether the amount of PAD2 protein is associated with changes in enzyme activity, we compared the expression level of PAD2 protein in 50 μg of total protein from whole brain and from different brain regions of control and scrapie-infected mice. As shown in Figure 6B, the expression levels" @default.
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• W1799202546 title "Accumulation of Citrullinated Proteins by Up-Regulated Peptidylarginine Deiminase 2 in Brains of Scrapie-Infected Mice" @default.
• W1799202546 cites W147522787 @default.
• W1799202546 cites W1593204115 @default.
• W1799202546 cites W1824155914 @default.
• W1799202546 cites W1896684749 @default.
• W1799202546 cites W1976038427 @default.
• W1799202546 cites W1977225908 @default.
• W1799202546 cites W1980325059 @default.
• W1799202546 cites W1986153062 @default.
• W1799202546 cites W1987396543 @default.
• W1799202546 cites W1994244586 @default.
• W1799202546 cites W1995734706 @default.
• W1799202546 cites W2002255871 @default.
• W1799202546 cites W2004369117 @default.
• W1799202546 cites W2005699747 @default.
• W1799202546 cites W2008253975 @default.
• W1799202546 cites W2010116135 @default.
• W1799202546 cites W2016595119 @default.
• W1799202546 cites W2017212166 @default.
• W1799202546 cites W2018748658 @default.
• W1799202546 cites W2022680168 @default.
• W1799202546 cites W2023300261 @default.
• W1799202546 cites W2025322670 @default.
• W1799202546 cites W2025620957 @default.
• W1799202546 cites W2037491812 @default.
• W1799202546 cites W2038902835 @default.
• W1799202546 cites W2040068123 @default.
• W1799202546 cites W2045932700 @default.
• W1799202546 cites W2048795651 @default.
• W1799202546 cites W2051252146 @default.
• W1799202546 cites W2052517492 @default.
• W1799202546 cites W2053849276 @default.
• W1799202546 cites W2055040525 @default.
• W1799202546 cites W2058042264 @default.
• W1799202546 cites W2059796686 @default.
• W1799202546 cites W2063296711 @default.
• W1799202546 cites W2065809729 @default.
• W1799202546 cites W2067903005 @default.
• W1799202546 cites W2073560352 @default.
• W1799202546 cites W2074456202 @default.
• W1799202546 cites W2085894361 @default.
• W1799202546 cites W2089215677 @default.
• W1799202546 cites W2092225357 @default.
• W1799202546 cites W2092905357 @default.
• W1799202546 cites W2093407362 @default.
• W1799202546 cites W2104503776 @default.
• W1799202546 cites W2113191032 @default.
• W1799202546 cites W2116042754 @default.
• W1799202546 cites W2116299484 @default.
• W1799202546 cites W2119395643 @default.
• W1799202546 cites W2125110523 @default.
• W1799202546 cites W2133202761 @default.
• W1799202546 cites W2151898539 @default.
• W1799202546 cites W2152722274 @default.
• W1799202546 cites W2159254316 @default.
• W1799202546 cites W2160090073 @default.
• W1799202546 cites W2162165372 @default.
• W1799202546 cites W2324660492 @default.
• W1799202546 cites W2326526316 @default.
• W1799202546 cites W411587403 @default.
• W1799202546 cites W4247800480 @default.
• W1799202546 doi "https://doi.org/10.2353/ajpath.2008.080388" @default.
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