Neurochem Res (2013) 38:1941–1948 DOI 10.1007/s11064-013-1100-6
ORIGINAL PAPER
Status Epilepticus Induces Long Lasting Increase in S100A6 Expression in Astrocytes Ewelina Jurewicz • Joanna Bednarczyk • Anna Bot • Katarzyna Łukasiuk • Anna Filipek
Received: 16 May 2013 / Revised: 19 June 2013 / Accepted: 20 June 2013 / Published online: 2 July 2013 Ó Springer Science+Business Media New York 2013
Abstract In the present work we examined expression and localization of the S100A6 protein in rat brain in a model of epilepsy induced by Status Epilepticus evoked by amygdala stimulation. We demonstrate, through the use of the reverse transcriptase-polymerase chain reaction technique, that mRNA level of S100A6 was increased in cortex while, as found by immunoblotting, the level of the S100A6 protein was significantly higher in the cortex and in the CA1 area of the hippocampus at day 14 after stimulation. Immunohistochemical studies performed on rat brain slices indicated that S100A6 immunoreactivity was elevated in GFAP-positive astrocytes in the hippocampus and cortex starting from day 1, and further increased at day 4 and 14 after stimulation. Interestingly, in a subpopulation of astrocytes, up-regulation of S100A6 was associated with an increased level of b-catenin, a protein involved in regulation of S100A6 expression. Altogether, our data show a widespread and prolonged up-regulation of S100A6 in the epileptic brain and indicate that an increase in S100A6 immunoreactivity is related to astrogliosis. Keywords
Status Epilepticus S100A6 Astrocytes
Abbreviations BSA Bovine serum albumin b-ME b-Mercaptoethanol DAB 3, 30 -Diaminobenzidine NMDA N-methyl-D-aspartate PAGE Polyacrylamide gel electrophoresis
E. Jurewicz J. Bednarczyk A. Bot K. Łukasiuk A. Filipek (&) Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland e-mail:
[email protected]
PBS RT RT-PCR SDS SE
Phosphate buffered saline Room temperature Reverse transcriptase-polymerase chain reaction Sodium dodecyl sulfate Status Epilepticus
Introduction S100A6 is a low molecular weight EF-hand Ca2?-binding protein belonging to the S100 family. The latter consists of more than 20 members involved in different intracellular functions such as controlling Ca2? homeostasis or cytoskeleton organization [1]. Genes encoding 17 human S100 proteins are located in the epidermal differentiation complex on chromosome 1q21 [2]. Rearrangements within this chromosome occur quite often in neoplasia causing in consequence changes in expression of S100 proteins in different types of tumors [3]. Thus, many of the S100 proteins might be used as markers of these pathologies. As to S100A6, it is known that under normal conditions its expression is cell and tissue specific and that it is up-regulated in some tumors including those with high metastatic activity [4]. In rodent tissues S100A6 is mainly expressed in fibroblasts and epithelial cells [5], although it is also present in other tissues such as brain. Immunohistochemical studies performed on rat brain show that S100A6 is localized in glial cells and in neurons in several brain areas [6, 7]. In normal human aged brain S100A6 is found to be slightly up-regulated in astrocytes of the neocortical area [8]. Interestingly, in the case of amyotrophic lateral sclerosis (ALS), S100A6 was found to be overexpressed within astrocytes surrounding the neurodegenerative lesions [9, 10]. Moreover, in other types of dementia common for the elderly population, such as
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Alzheimer’s disease (AD), it was shown that astrocytic S100A6 was homogeneously up-regulated within the white matter while in the grey matter almost all S100A6 immunoreactivity was concentrated in astrocytes surrounding the amyloid beta deposits of senile plaques [11]. A limited number of data on expression and localization of S100A6 is available for other brain pathologies such as epilepsy. Up to now, an increased level of S100A6 mRNA has been demonstrated by microarray analysis during epileptogenesis induced by blood brain barrier breakdown, albumin or TGF-beta1 [12] and following traumatic brain injury [13]. Also, an increase in S100A6 mRNA level was found in sclerotic hippocampi derived from epileptic patients [14]. However, these data have been neither validated by other methods nor examined any further. The only report published on the S100A6 protein expression in an experimental model of epilepsy claimed that Status Epilepticus (SE) induced in mice by kainic acid application resulted in increased S100A6 immunoreactivity in astrocytes localized in the CA3 field of the hippocampus [15]. In this work we examined in details the expression and localization of S100A6 in rat brain in the amygdala stimulation model of epilepsy. In this model, development of epilepsy is induced by SE evoked by electrical stimulation of the lateral nucleus of amygdala. Following SE that lasts about two hours, animals recover and, later on, start to experience spontaneous, unprovoked seizures. We demonstrate for the first time, through the use of reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunohistochemistry, that S100A6 is widely up–regulated in the brain of epileptic animals. Increase in S100A6 immunoreactivity is prolonged and is limited to the activated astrocytes, in which an increase in b-catenin level is also observed.
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butorphanol (Butomidor, Richter Pharma AG, 0.5 mg/kg, i.p.) and then subjected to isoflurane anesthesia (2–2.5 % in 100 % O2). A stimulating and recording bipolar wire electrode (Plastic One Inc., # E363-3-2WT-SPC) was implanted into the left lateral nucleus of the amygdala (3.6 mm posterior and 5.0 mm lateral to bregma, 6.5 mm ventral to the surface of the brain) (Paxinos and Watson, 2007). A stainless steel screw electrode (Plastic One Inc., #E363/20) was implanted contralaterally into the skull over the right frontal cortex (3.0 mm anterior and 2.0 mm lateral to bregma) for a surface EEG recording. Two stainless steel screw electrodes were placed bilaterally over the cerebellum (10.0 mm posterior and 2.0 mm lateral to bregma) as a ground and reference. Socket contacts of all electrodes were placed in a multi-channel electrode pedestal (Plastic One Inc., #MS363) which was attached to the skull with dental acrylate (Duracryl Plus). After 2 weeks of recovery animals were electrically stimulated via the intraamygdala electrode to evoke SE. Stimulation consisted of a 100-ms train of 1-ms biphasic square-wave pulses (400 lA peak to peak) delivered at 60 Hz, every 0.5 s for 20 min. If the animal did not reveal SE behavior following 20 min of stimulation, stimulation was continued for the next 10 min. The SE has been stopped 1.5–2 h after stimulation by intraperitoneal injection of diazepam (20 mg/kg). When the first dose of diazepam did not suppress SE, the animal did receive subsequent doses of the drug (5 mg/kg). Timematched control animals had electrodes implanted but did not receive electrical stimulation. Starting from stimulation, rats were monitored continuously with video-EEG (24 h/day) every second day to detect spontaneous epileptic seizures. Spontaneous seizures were identified from EEG recordings by browsing the EEG manually on the computer screen. Electrographic seizure was defined as a high frequency ([8 Hz), high amplitude ([29 baseline) discharge lasting for at least 5 s.
Materials and Methods Isolation of mRNA and Preparation of Protein Fraction Rat Epilepsy Model Adult male Spraque-Dawley rats (290–360 g) were obtained from Medical Research Centre (Warsaw, Poland). Animals were housed in controlled environment (temperature 24 °C, lights on 07.00–19.00 hours) with free access to food and water. Starting from the day of surgery, each animal was housed in a separate cage. All animal procedures were approved by the Ethical Committee on Animal Research of the Nencki Institute, and conducted in accordance with the guidelines set by the European Council Directives 86/609/ ECC. Status Epilepticus was triggered by electrical stimulation of the amygdala as previously described [16]. For electrode implantation animals were first injected with
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For protein and mRNA isolation, rats were anaesthetized with carbon dioxide and decapitated with a guillotine at day 14 after SE. Control rats were sacrificed at the same time as the corresponding stimulated groups. The CA1 area of the hippocampus and a block of tissue containing the piriform nucleus, amygdala, entorhinal and piriform cortices referred to as ‘‘cortex’’ were dissected and stored at -70 °C. Total RNA was isolated using RNAeasy Mini Kit (Qiagen, #74104). Genomic DNA was digested on-column with DNase I (Qiagen, #79254) according to the manufacturer’s protocol. The concentration and quality of RNA was measured by capillary electrophoresis with RNA 6000 Nano Chip (Agilent, #5067-1511) in 2100 Bioanalyzer
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(Agilent, #G2938B). Purified total RNA was stored at -70 °C. Proteins were isolated in parallel to RNA by precipitation from the flow through fraction in 4 volumes of acetone. Subsequently the proteins were washed in 70 % ethanol and dissolved in 8 M urea containing 5 % b-ME. Protein concentration was determined using the Bio-Rad Protein Assay (#500-0006, Bio-Rad) and stored at -20 °C. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) RNA (1 lg) was reverse transcribed using M-MLV Reverse Transcriptase (Sigma). The sequences of primers for S100A6 were as follows: forward 50 -ATGGCATGCCC CCTGGATCA-30 and reverse 50 -TCAGAGCTTCATTGT AGATCAAAGCC-30 . For GAPDH, sequences of primers were the following: forward 50 -ACCACAGTCCATGC CATCAC-30 and reverse 50 -TCCACCACCCTGTTGCTG TA-30 . PCR products were separated in 1 % agarose gel containing 0.1 lg/ml ethidium bromide. Pictures of agarose gels were taken using the Ingenius BioImaging system (Syngene). SDS-PAGE and Immunoblotting Proteins (100 lg) were separated by SDS-PAGE (10 %) by the method of Laemmli [17]. After transfer onto nitrocellulose they were identified by immunoblotting using appropriate primary antibodies: rabbit anti-S100A6 polyclonal antibody (1:250, made in house), rabbit anti-b-catenin polyclonal antibody (1:1,000, Santa Cruz) or mouse anti-actin monoclonal antibody (1:1,000, MP Biochemicals). Blots were washed with TBS-T buffer (50 mM Tris pH 7.5, 200 mM NaCl, 0.05 % Tween 20) and then allowed to react with secondary antibodies, either goat anti-rabbit IgG (1:10,000, MP Biomedicals) or goat anti-mouse IgG (1:15,000, Jackson Immunoresearch Laboratories). After three washes with the TBS-T buffer and two washes with the TBS buffer (50 mM Tris pH 7.5, 200 mM NaCl) blots were developed with the ECL chemiluminescence kit (Amersham Biosciences) followed by exposure against a RETINA X-ray film. Immunohistochemistry For immunohistochemical studies brains were collected at day 1 (n = 4), day 4 (n = 4) or day 14 (n = 4) after stimulation. Two time-matched control rats were sacrificed for each time point. Rats were deeply anaesthetized by intraperitoneal injection of sodium pentobarbital (Vetbutal, Biowet, 120 mg/kg) and intracardially perfused, first for 2 min with saline and then for 20 min with cold 4 % paraformaldehyde in 0.1 M sodium phosphate buffer, pH
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7.4. After perfusion brains were isolated, additionally fixed for 4 h in 4 % paraformaldehyde, and cryoprotected with 30 % sucrose in 0.02 M potassium phosphate buffer, pH 7.4 until complete saturation. Brains were frozen in dry ice and stored at -70 °C until cutting. For immunohistochemical analysis, sections were cut coronally at a 30 lm thickness using a microtome at -20 °C. Sections were then collected to TCS, a cryoprotectant tissue collecting solution containing 30 % ethylene glycol, 25 % glycerol and 0.05 M sodium phosphate buffer, pH 7.4, and stored at -20 °C. Prior to the reaction with relevant antibody, sections were washed (69 10 min) in PBS and blocked for 1 h at RT in PBS containing 1.5 % normal goat serum and 0.3 % Triton X-100. Then sections were incubated, overnight at 4 °C, with serum containing polyclonal antibodies against S100A6 (made in house) diluted 1:250 in PBS containing 1 % BSA followed by biotinylated goat anti-rabbit IgG (Millipore) diluted 1:1,000 for 1 h at RT. After washes with PBS (39 10 min), sections were incubated for 1 h at RT with the streptavidin–horseradish peroxidase complex (Vector Laboratories) diluted 1:500. The reaction was developed with 3, 30 -diaminobenzidine (Sigma) in PBS. The unspecific staining was determined by omission of primary antibody. Sections were analyzed with a Nikon Eclipse 80i epifluorescence microscope. Immunofluorescence Staining For double immunofluorescence staining sections were washed and blocked as for immunohistochemistry and incubated overnight at 4 °C with serum containing polyclonal antibodies against S100A6 (made in house) or with polyclonal antibodies against b-catenin (Santa Cruz) diluted 1:500 in PBS containing 0.3 % BSA. Sections were then washed (39 10 min) in PBS and incubated for 1 h at RT with secondary donkey anti-mouse antibodies conjugated with Alexa Fluor 555 (Molecular Probes) diluted 1:200 in PBS containing 0.3 % BSA. Next, sections were washed three times in PBS (39 10 min) and incubated for 2 h at RT with antibodies against the astroglial marker, GFAP (Sigma) diluted 1:1000 in PBS. After washing in PBS (39 10 min) sections were mounted on slides using the Vectashield mounting medium (Vector Laboratories) and analyzed using a confocal microscope (Leica Carl Zeiss LSM780 spectral confocal, lens 409). Fluorescein was excited with argon laser (488 nm), while cyanine 3 with helium–neon laser (543 nm).
Results Data obtained up to now have indicated an increase in S100A6 mRNA and protein level in human epileptic tissue
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and in the brain of animals in experimental models of epilepsy [14, 15]. However, these studies did not provide data concerning the localization and time course of S100A6 expression. Therefore, in the present work we analyzed the expression of S100A6 in more detail using a well characterized model of epilepsy in which the epileptic process is initiated by electrically evoked SE. As it could be seen in Fig. 1, the level of S100A6 mRNA in brain is increased by about 40 % in the cortex at day 14 after stimulation (Fig. 1b), while in the CA1 area of the hippocampus the difference is not statistically significant (Fig. 1a). The level of GAPDH mRNA indicates that a similar amount of total RNA was used in the reaction. Interestingly, when the protein level was examined using immunoblotting, at day 14 after stimulation, an
Fig. 1 S100A6 mRNA level in brain tissue of control (C) and SE rats at day 14. (a) CA1 area of hippocampus and (b) cortex. The level of mRNA was analyzed by RT-PCR. The level of mRNA for GAPDH indicates that a similar amount of total RNA was used in the reaction. Results of the densitometric analysis of 3 independent experiments are presented as a mean ± SD. *p B 0.05
Fig. 2 S100A6 protein level in brain tissue of control (C) and SE rats at day 14. (a) CA1 area of hippocampus and (b) cortex. In each case 100 lg of protein from the lysate were applied on the gel and then analyzed by immunoblotting with antiS100A6 polyclonal antibodies. The level of actin indicates that a similar amount of protein was loaded on the gel. Results of the densitometric analysis of 4 independent experiments are presented as a mean ± SD. **p B 0.01; ***p B 0.001
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increase in the amount of S100A6 was seen both in the CA1 area of the hippocampus (Fig. 2a) and in the cortex (Fig. 2b). Similar intensity of the bands representing actin, both in the control and the epilepsy model, indicates that the amount of protein loaded on the gel was comparable. To determine the cellular localization of the S100A6 protein, we performed immunohistochemical staining of control brains and brains of animals sacrificed 1, 4 or 14 days after stimulation. Figures 3 and 4 show representative images from the hippocampus and cortex, respectively. In control brain, weak S100A6 immunostaining is seen in cells morphologically resembling astrocytes (arrows), both in the hippocampus and in the cortex. Following stimulation, the intensity of S100A6 immunostaining in the examined brain areas gradually increases. At day 1 after SE, the reaction with anti-S100A6 antibodies is
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Fig. 3 S100A6 expression in normal hippocampus and 1, 4 and 14 days after amygdala stimulation induced SE. Panels on the right display S100A6 immunoreactivity in dentate gyrus while panels on the left—in the CA1 area of hippocampus. Note the presence of sparse and weak immunoreactivity in control hippocampus in cells morphologically resembling astrocytes and increase in the number of S100A6-positive cells as well as in staining intensity following SE (arrows). g granule cell layer, H hilus, ml molecular layer, slm stratum lacunosum moleculare, so stratum oriens, sp pyramidal cell layer, sr stratum radiatum
already stronger than in the control brain and 4 and 14 days after SE the immunostaining is even more intensive. Cells exhibiting an up-regulated level of S100A6 morphologically resemble activated astrocytes. To prove that S100A6 expressing cells are indeed astrocytes, we performed double immunostaining with antibodies against the S100A6 protein and a marker of astrocytes, GFAP. As presented in Fig. 5, there is strong
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Fig. 4 S100A6 expression in the entorhinal and piriform cortices in control animals and 1, 4 and 14 days after amygdala stimulation induced SE. Panels on the left display S100A6 immunoreactivity in the entorhinal cortex while panels on the right—in the piriform cortex. Note the presence of sparse and weak immunoreactivity in control cortex in cells morphologically resembling astrocytes and increase in the number of S100A6-positive cells as well as in staining intensity following SE (arrows). I layer I, II layer II, III layer III
co-localization of S100A6 and GFAP, both in control and stimulated brain sections in all examined areas. This indicates that S100A6 is expressed and up-regulated after stimulation in the astrocytic type of cells. It has been previously shown, that b-catenin, a protein involved in cell proliferation is engaged in regulation of S100A6 gene expression [18]. Thus, we examined whether the same mechanism operates in the epileptic brain. For that we analyzed by immunoblotting the level of b-catenin in the CA1 area of the hippocampus of control and SE rats at day 14. As it is shown in Fig. 6a, an increase in the amount of b-catenin by about 70 % was seen after
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Fig. 5 Co-localization of S100A6 with the astrocytic marker, GFAP, in hippocampus and cortex of control brain and 14 days after amygdala stimulation induced SE. S100A6 immunoreactivity (green) is seen in panels on the left while GFAP immunorectivity (red) in the middle panels. Panels on the right present the merge of images of S100A6 and GFAP immunoreactivities; colocalization (arrows) is visible in yellow. Nuclei are stained with DAPI (blue). Note that following SE virtually all GFAP-positive astrocytes contain strong S100A6 immunostaining. Scale bar is 20 lm (Color figure online)
stimulation. To check that b-catenin expressing cells are indeed astrocytes, we performed double immunostaining with antibodies against b-catenin and GFAP. Similarly to the astrocytic staining of S100A6 shown in Fig. 5, bcatenin expression was observed in some GFAP positive cells in the hippocampus (Fig. 6b) and cortex (not shown) at the 14th day after stimulation. No b-catenin expression was observed in GFAP positive cells in the hippocampus (Fig. 6b) and in the cortex (not shown) of control animals.
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Discussion S100A6 is an EF-hand Ca2?-binding protein that is expressed, among other tissues, in brain, both in neurons and in glial cells [6, 7]. Since little is known about the localization and function of S100A6 in brain pathology, in this work we examined in details the expression and localization of S100A6 in rat brain following SE using the amygdala stimulation model of epilepsy [19]. By applying
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Fig. 6 (a) b-catenin level in the CA1 area of the hippocampus of control (C) and SE rats at day 14. In each case 20 lg of protein from the lysate were applied on the gel and then analyzed by immunoblotting with anti-b-catenin polyclonal antibodies. The level of actin indicates that a similar amount of protein was loaded on the gel. Results of the densitometric analysis of 3 independent experiments are presented as a mean ± SD. *p B 0.05. (b) Co-localization of bcatenin with the astrocytic marker, GFAP, in the CA1 field of the hippocampus of control brain and 14 days after amygdala stimulation
induced SE. Left panels represent b-catenin immunoreactivity, middle panels represent GFAP immunoreactivity and right panels present merged images where b-catenin is visible in green, GFAP in red and DAPI in blue. Co-localization of b-catenin and GFAP is visible in yellow (arrows). Note that following SE some astrocytes contain strong b-catenin immunostaining, while no b-catenin is expressed in GFAP positive cells in control brain. so stratum oriens, sp pyramidal cell layer, sr stratum radiatum. Scale bar is 50 lm (Color figure online)
RT-PCR and Western blotting, we found an increased expression of S100A6 in the examined areas of stimulated brain. Results of our immunohistochemical and immunofluorescence analysis indicate that, following SE, S100A6 expression is up-regulated in astrocytes which, as judged by their morphology (enlarged size and increased thickness of processes), are activated. Our data show for the first time that up-regulation of S100A6 expression following SE in rat is widespread and long lasting. Recently, an increase in S100A6 immunoreactivity has been shown to occur 3 days following kainic acid induced SE in the CA3 area of the hippocampus in ICR (imprinting
control region) mice [15]. Our data indicate that the increase in S100A6 expression following SE is not limited to CA3 but occurs in other areas as well. This increase is also long lasting and persists for at least 14 days. The results showing astrocytic up-regulation of S100A6 are in agreement with the earlier reports indicating an increase of S100A6 expression in astrocytes in conditions involving neurodegeneration/neuroinflammation. For example, up-regulation of S100A6 mRNA or protein level was observed following epileptogenic stimuli, which induced marked neurodegeneration [12, 13, 15]. Also, it has been shown that in ALS, S100A6 was present in
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astrocytes surrounding the neurodegenerative lesions [9, 10] and in AD, S100A6 was homogeneously up-regulated in astrocytes of the white matter and in those surrounding the amyloid beta deposits of senile plaques within the grey matter [11]. Moreover, an increase in S100A6 in astrocytes has been also observed in hypoglossal nucleus after axotomy and in the hippocampus after kainic acid treatment in mice, in areas where neurodegeneration was observed [15]. Thus, the above cited data together with our results showing a widespread and prolonged up-regulation of S100A6 in the astrocytes of epileptic brain indicate that S100A6 might be a universal marker of astrogliosis associated with the process of neurodegeneration. Status Epilepticus induced by amygdala stimulation, used in this study, evokes strong neurodegeneration in well-defined locations including the CA1/CA3 fields of the hippocampus, the entorhinal and piriform cortex [19]. In our studies, S100A6 up-regulation was strongest in areas marked by intense neurodegeneration following SE. This is best manifested when an area that is strongly damaged, such as CA1, is compared to that resistant to neurodegeneration e.g., the dentate gyrus. Astrocytes with a high level of expression of S100A6 were observed in CA1, but not in the dentate gyrus. This increase is associated with an increase in b-catenin level suggesting that b-catenin might regulate S100A6 expression [18]. Interestingly, the increase in S100A6 level induced by kainic acid in ICR mice can be partially prevented by NMDA receptor antagonist, MK-801 [15]. Since MK-801 has strong neuroprotective properties in SE models [20], it might be argued that the level of neurodegeneration determines the level of S100A6 induction in astrocytes. Thus, all these data suggest that S100A6 expression is related to brain damage that induces astrogliosis and glial scar formation. Acknowledgments We thank Prof. W. Les´niak (Nencki Institute of Experimental Biology) for critical reading of this manuscript and Ms. E. Nosecka (Nencki Institute of Experimental Biology) for animal surgery. This work was supported by grants: N N303 548439, 2012/04/M/NZ3/00425 to A.F. and 888/N-ESF-EuroEPINOMICS/10/ 2011/0 to K.Ł. as well as by statutory funds from the Nencki Institute of Experimental Biology. Conflict of interest
No author has any conflict of interest to declare.
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