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The role of transcription factors cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER) in epileptogenesis
Neuroscience 152 (2008) 829 – 836
THE ROLE OF TRANSCRIPTION FACTORS CYCLIC-AMP RESPONSIVE ELEMENT MODULATOR (CREM) AND INDUCIBLE CYCLIC-AMP EARLY REPRESSOR (ICER) IN EPILEPTOGENESIS B. E. PORTER,a* I. V. LUND,a F. P. VARODAYAN,a R. W. WALLACEa AND J. A. BLENDYb
Toth et al., 1998; Huang et al., 2002). It has been difficult to determine which of the many changes contribute to the development of epilepsy, and which are compensatory or inconsequential. The cyclic-AMP response element binding protein (CREB) family is influenced by seizure activity and is a regulator of transcription, cell survival, axonal sprouting and synaptic plasticity; all processes altered by SE (Lonze and Ginty, 2002). The central importance of the CREB family in multiple cellular signaling cascades makes it an excellent candidate for identifying pharmacologic interventions to block SE-induced changes that lead to the development of epilepsy. The CREB transcription factor family is composed of three family members (CREB, cyclic-AMP responsive element modulator (CREM) and ATF-1) sharing a high degree of similarity and all bind to CRE sites in gene promoter regions (Shaywitz and Greenberg, 1999; Mayr and Montminy, 2001; Conkright et al., 2003). While CREB primarily increases gene transcription most of the CREM splice variants and the related gene transcript, inducible cyclicAMP early repressor (ICER), inhibit CRE-mediated transcription (Molina et al., 1993; Maronde et al., 1999; Kemp et al., 2002; Morales et al., 2003). Seizures have been found to alter CREB, CREM and ICER activity. Seizures cause an immediate increase followed by a chronic decrease in CREB phosphorylation that has been implicated in memory impairment following earlylife seizures (Moore et al., 1996; Kashihara et al., 2000; Chang et al., 2003; Pi et al., 2004). Following kainic acid or electroconvulsive seizures there is an increase in the level of CREM and to a greater extent ICER in the hippocampus, frontal cortex and cerebellum accompanied by an increase in CREM/ICER CRE binding complexes (Fitzgerald et al., 1996; Konopka et al., 1998). CREB family members play a role in neuronal survival (Bonni et al., 1999; Glover et al., 2004). In CREB null mice sensory neurons exhibit excess apoptosis and degeneration, but this effect is not observed in the CNS (Lonze and Ginty, 2002). Only when both CREB and CREM/ICER are lacking is extensive apoptosis observed in the brain (Mantamadiotis et al., 2002). ICER is increased in hippocampal and cortical neurons undergoing apoptosis after excitotoxic injury and over-expression of ICER in cultured neurons results in neuronal apoptosis (Jaworski et al., 2003; Mioduszewska et al., 2003). ICER’s pro-apoptotic mechanism of action is not known but transcriptional repression of neuronal survival molecules, such as bcl-2 may be involved (Jaworski et al., 2003).
a
Departments of Pediatrics and Neurology, The Children’s Hospital of Philadelphia at the University of Pennsylvania, 502C Abramson, 3516 Civic Center Boulevard, Philadelphia, PA 19104, USA
b
Department of Pharmacology at the University of Pennsylvania, 125 South 31st Street, Translational Research Laboratory, Philadelphia, PA 19104, USA
Abstract—Alterations in the brain that contribute to the development of epilepsy, also called epileptogenesis, are not well understood, which makes it difficult to develop strategies for preventing epilepsy. Here we have studied the role of the CRE binding transcription factors, cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER), in the development of epilepsy following pilocarpine induced status epilepticus (SE) in mice. Following SE, ICER mRNA and protein are increased in neurons. The increase in ICER, however, is not necessary for neuronal injury following SE as pilocarpine treatment induces equivalent neuronal injury in pyramidal neurons of wild type and CREM/ICER null mice. Following SE, the CREM/ICER null mice develop a more severe epileptic phenotype experiencing ⬃threefold more frequent spontaneous seizures. Together these data suggest that the increase in ICER mRNA following SE may have a role in suppressing the severity of epilepsy. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: seizures, apoptosis, epileptogenesis, cyclic-AMP response element binding protein, CREB.
It is known that brain injury or status epilepticus (SE) increases the risk of developing epilepsy but anti-epileptic drug administration after head trauma or SE has failed to prevent the development of epilepsy (Temkin, 2001; Herman, 2002; Pitkanen, 2002). Our inability to identify antiepileptogenic drugs stems in part from a poor understanding of the cascade of molecular and cellular changes that contribute to epileptogenesis, the process of developing epilepsy. Many cellular and molecular changes have been identified after SE including neuronal cell death, alterations in neurotransmitter receptor expression, increased axonal sprouting and new synapse formation (Tauck and Nadler, 1985; Buckmaster and Dudek, 1997; Brooks-Kayal et al., 1998; Dudek and Buckmaster, 1998; Sankar et al., 1998; *Corresponding author. Tel: ⫹1-267-426-5210; fax: ⫹1-215-590-3779. E-mail address: [email protected] (B. E. Porter). Abbreviations: CREB, cyclic-AMP response element binding protein; CREM, cyclic-AMP responsive element modulator; ICER, inducible cyclic-AMP early repressor; PBS, phosphate-buffered saline; SE, status epilepticus; WT, wild-type.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.10.064
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Here we have studied the expression of CREM and ICER following SE and have utilized mice deficient for these transcription factors to study their role in cell death and epileptogenesis.
ter mix, and 3 l of sample cDNA. RT-PCR was performed on the SDS-7500 PCR machine (Applied Biosystems). The RT-PCR runs consisted of first one cycle of 50° for 2 min, then one cycle of 95° for 10 min, and 40 cycles of: 95° for 15 s and 60° for 1 min. All values were normalized to cyclophilin expression to control for loading variability.
EXPERIMENTAL PROCEDURES The experiments were performed with the approval of the animal care and use committee at the University of Pennsylvania and in accordance NIH guidelines for minimizing the number and suffering of animals used.
Mouse model For all experiments CREM/ICER null mutants and wild-types (WT) are F1 hybrids (129 SVEV:C57BL/6) obtained by crossing inbred mice heterozygous for the mutation from each strain CREM/ ICER⫾129SVEV N12⫻CREM/ICER⫾C57BL/6 N15 (Kaestner et al., 1996; Conti et al., 2004). In the F1 generation, each individual mouse is heterozygous for all loci that differ between the parental strains and thus are genetically identical with the exception of the CREM/ICER locus (Branbury Conference on Genetic Background in Mice, Silva et al., 1997). This allows for us to be certain that the genetic background in all the F1 mice is similar and does not contribute to the observed phenotype.
Induction of SE Male F1 hybrids between 3 months and 6 months of age underwent methyl-scopolamine i.p. injection of 1 mg/kg (Sigma, St. Louis, MO, USA) followed 30 min later by ⬃330 mg/kg of pilocarpine HCl (Sigma) or for control animals an equivalent volume of saline. A dose of 330 mg/kg of pilocarpine HCl was chosen because it produced multiple brief stage V seizures in the 3 h following injection with an ⬃70% survival rate in this genetic background.
Scoring of SE The animals for video EEG were placed in individual cages and a reviewer blinded to their genotype watched for behavioral seizures. Animals were video recorded for 6 h following pilocarpine injection. The animals developed discreet episodes of both Racine stage IV: rearing forelimb clonus; and stage V: rearing, fore- and hind-limb clonus with falling (Racine, 1972). The number, duration and Racine seizure stage were assessed for each animal.
Scoring of spontaneous seizures Two weeks following SE animals were videotaped for at least 8 h per week for 5 weeks and spontaneous seizure activity was scored. All stage IV–V Racine class seizures were analyzed and the duration and class of each seizure was recorded.
Real time-PCR Mice were anesthetized and whole hippocampi were freshly dissected and rapidly frozen at ⫺80 °C. RNA was extracted from individual hippocampi using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). To synthesize cDNA, SuperScript II reverse transcription kit was used (Invitrogen) and cDNA samples were stored in a 1:4 dilution in ddH2O. For RT-PCR reactions, each sample was run in triplicate and each 25 l reaction contained: 1.25 l ICER or CREM Taqman primer probe (Rn00569145_m1 and Rn00565271_m1; Applied Biosystems, Foster City, CA, USA) or 1.25 l Taqman cyclophilin probe (Applied Biosystems) with 1.25 l of each cyclophilin primers (cycloREV: 5= CCC AAG GGC TCG CCA 3=; cycloFWD 5= TGC AGA CAT GGT CAA CCC C 3=, IDT Technologies, Coralville, IA, USA) and 12.5 l Taqman Mas-
CREM/ICER immunohistochemistry and TUNEL staining WT and CREM/ICER null mutant adult males were injected as described above and 72 h after pilocarpine or saline injection, mice were deeply anesthetized with ketamine/xylazine (1 l/g, i.p. Sigma) and underwent intra-cardiac perfusion of cold saline followed by 4% paraformaldehyde. Dissected brains were dehydrated in 30% sucrose, frozen on dry ice, and 25 m sections were cut on a cryostat. Primary antibodies, 1–50 dilution of CREM/ ICER antibody s.c.-440 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and 1–100 dilution of the mature neuronal marker NeuN MAB377 (Chemicon, Billerica, MA, USA) were incubated in phosphate-buffered saline (PBS), 2% horse serum, 0.2% Triton X-100. Following three washes in PBS sections were incubated overnight with anti-rabbit TRITC and anti-mouse fluorescein (Jackson ImmunoResearch, West Grove, PA, USA) and mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA).
TUNEL staining was carried out as previously described Briefly, animals were processed as described above (Gavrieli et al., 1992; Porter et al., 2004), and sections corresponding to ⬃⫺3 mm from bregma were incubated with biotin-labeled deoxyuridine triphosphate (dUTP-16) (Roche Diagnostics, Indianapolis, IN, USA), one unit of TUNEL enzyme in terminal transferase buffer for 1 h at 37 °C. A Vector ABC peroxidase kit (Vector Laboratories) was used for isolated TUNEL staining (Paxinos and Watson, 1986). Sections were developed in 3= diaminobenzidine and counterstained with Cresyl Violet dehydrated in sequential ethanol and xylene and mounted with permount (Fisher Scientific, Hampton, NH, USA). An individual blinded to the animal genotype performed manual cell counts at 400⫻ magnification and freehand area measurements of CA2/CA3 were performed with ImagePro software (Media Cybernetics, Silver Spring, MD, USA).
Western blots The hippocampus and cortex were removed and frozen on dry ice. The frozen tissue was sonicated in 50 mM Tris pH 7.0, 140 mM NaCl, 10 mM EDTA, 2% SDS, 10 mm NaF, 50 mM -glycerophosphate, 30 mM Na-pyrophosphate, 0.2 mM PMSF, and 0.5 mM IBMX, then protein was assayed using a Biorad DC kit (Biorad, Hercules, CA, USA). To 75 g of total protein we added 2 l of -mercaptoethanol and 3 l 100 mM DTT and boiled the sample for 10 min. Each sample was loaded on to a 12%–14% polyacrylamide gel separated via SDS gel electrophoresis, and transferred to nitrocellulose, 90 min at 100 V. After blocking with 5% milk in 1⫻ PBS 1 h. Membranes were incubated with 1:500 CREM-1 (x-12) antibody (Santa Cruz Biotechnology) in 5% milk/ PBS with gentle agitation overnight at 4 °C. Blots were then incubated with appropriate HRP-conjugated secondary antibodies and developed with chemiluminescence, SuperSignal West Dura, extended duration substrate with agitation for 2 min at room temperature (Pierce, Rockford, IL, USA). Blots were stripped of primary and secondary antibodies and re-probed using an anti- actin antibody diluted 1–5000 (Sigma). Each blot was scanned using an Epson scanner in conjunction with Adobe Photoshop. Data were quantified using imaging densitometry and analyzed using the NIH Image program. Data from each sample were
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expressed as a ratio of -actin to insure equal loading and transfer. t-Tests were performed using Instat software (Graphpad Software Inc. San Diego, CA, USA) on Western blot data and significance was set at P⬍0.05.
RESULTS We first studied the effects of acute pilocarpine induced seizures on ICER and CREM expression in the brain of WT mice. Hippocampi were harvested 24 h after saline or pilocarpine injections in WT mice. RNA isolated from the whole hippocampus showed a 6.1-fold increase in ICER mRNA following pilocarpine induced SE (control 1⫾0.12 (SEM); SE 6.1⫾1.6; n⫽4 and 5; P⫽0.004, two-tailed t-test; Fig. 1a). In contrast there was no change in CREM mRNA levels following pilocarpine induced SE treatment (control 1⫾0.16 (SEM); SE 0.8⫾0.22; n⫽4 and 5; Fig. 1b). We carried out Western blots to quantify protein levels of ICER. The ICER␥ protein is increased in both the hippocampus and the cortex of WT mice following SE (Hippocampus-control 1.0⫾0.19 (SEM); SE 7.0⫾1.7, P⫽0.012,
Fig. 2. ICER immunoreactive bands are increased in the brain following SE. (a, b) In the hippocampus the larger ICER splice variant band is increased by visualization and the smaller ICER␥ splice variant is increased sevenfold following SE as analyzed by densitometry (n⫽4, * P⫽0.012, two-tailed t-test). The  actin loading control is shown below. (c, d) In the cortex by visualization the larger ICER splice variant band is increased and by densitometry the smaller ICER␥ splice variant is increased 2.8-fold following SE (n⫽4, * P⫽0.002, two-tailed t-test).
Fig. 1. ICER mRNA is increased in the hippocampus 24 h after SE. (a) Results of RT-PCR showing that mice subjected to SE have 6.1-fold higher expression of ICER mRNA than methyl scopolaminesaline treated control animals (n⫽4 and 5; * P⫽0.004, two-tailed t-test). All samples were normalized to cyclophilin mRNA, serving as a control for sample concentration and amplification variability. (b) CREM mRNA levels in the hippocampus are similar to control values 24 h after SE.
two-tailed t-test; Cortex-control 1.0⫾0.1; SE 2.8⫾0.33, P⫽0.002, two-tailed t-test; n⫽4; Fig. 2a– d). By visual inspection it appears that a large ICER splice form is also increased following a seizure but we were unable to separate the large ICER form from a non-specific band and therefore could not accurately quantify the change (data not shown). Using an antibody that recognizes both CREM and ICER splice forms we find that the CREM/ICER im-
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Fig. 3. CREM/ICER immunoreactivity is increased in CA3 pyramidal and dentate gyrus neurons of the hippocampus 3 days after pilocarpine induced SE. In a WT control mouse there is minimal CREM/ICER immunoreactivity in CA3 pyramidal neurons (b) and dentate granule neurons (d); a, c, NeuN staining in green and b, d, CREM/ICER staining in red. In a WT mouse following SE there is increased CREM/ICER immunoreactivity in CA3 pyramidal neurons (f) and dentate granule neurons (h); e, g, NeuN staining shown in green; f, h, CREM/ICER staining in red (200⫻ magnification).
munoreactivity is present in neurons of multiple brain regions and the immunoreactivity is increased following SE (see Fig. 3). We then went on to study the effect of pilocarpine induced SE in WT and CREM/ICER null mice. We found that the episodes of pilocarpine induced SE were similar in WT and CREM/ICER null mutant animals as assessed by number of stage IV and V seizure and mortality. After pilocarpine injection stage IV–V seizures began on average 41.3⫾5.1 min (SEM) in the WT and 32.3.3⫾7.9 min in the CREM/ICER null mutant mice (P⫽0.345, n⫽6). Equal numbers of stage IV and V seizures occurred in the WT and CREM/ICER null mutant animals during the 6 h following the pilocarpine injection (WT 2.8⫾0.4 SEM/per animal, n⫽5 versus CREM/ICER mutant 2.8⫾0.2, SEM/per animal, n⫽5; Fig. 4). Seizures lasted from 20 to 90 s. Between stage IV and V seizures, animals assumed a head nodding behavior and were not responsive and did not struggle during tail suspension. Stage IV, V behavioral seizures spontaneously resolved by 3 h after pilocarpine injection in both the WT and CREM/ICER null mutant mice.
Mortality was also similar in the WT and CREM/ICER null mutant mice with death always occurring during a prolonged tonic seizure (WT 11/33, 30% and CREM/ICER null 9/26, 30%). TUNEL staining was negligible in the hippocampus of methyl-scopolamine/saline-treated WT or CREM/ICER null mutant animals (Fig. 5a, c). In contrast, 3 days after the pilocarpine injection there was prominent TUNEL staining in hippocampal pyramidal areas CA2/CA3 in WT and CREM/ICER null mutant animals (Fig. 5b, d). Density of TUNEL positive neurons was similar in the CA2/CA3 region of the WT and CREM/ICER null mutant animals (WT 3.6⫻10⫺3 cells/m2⫾3.1⫻10⫺4 SEM, n⫽3; CREM/ICER null mutant 3.5⫻10⫺3⫾3.3⫻10⫺4, n⫽3; Fig. 6). There were few TUNEL positive cells in other regions of the hippocampus following SE. Spontaneous behavioral seizures were assessed for at least 8 h per week beginning 2 weeks after the episode of SE. Both the WT and CREM/ICER null mutant pilocarpinetreated animals developed spontaneous seizures. The seizure semiology appeared similar between the two groups (all stage V) consisting of wild running, four limb clonus, increased body tone, tail extension followed by loss of postural control. Animals had seizures both spontaneously and during handling. The frequency of spontaneous seizures was higher in the CREM/ICER null mutant animals than WT (WT 0.029⫾0.019 seizures/h⫾SEM, N⫽5 animals; CREM/ICER null mutant animals 0.11⫾0.028 seizures/h, N⫽4 animals, P⫽0.01; Fig. 7).
DISCUSSION
Fig. 4. The number of stage IV and V seizures induced by pilocarpine was similar in WT and CREM/ICER null mice (P⫽1, two-tailed t-test).
We have shown that ICER mRNA and protein increase in neurons of the hippocampus and cortex following pilocarpine induced SE. The ICER gene and its promoter are nestled within the 3= region of the CREM gene but the increase in ICER mRNA following SE appears to be specific to the ICER transcripts as CREM mRNA does not
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Fig. 5. Following pilocarpine injection there is an increase in the density of TUNEL stained CA2–CA3 neurons in both WT and CREM/ICER null mutant animals. (a) Following methyl scopolamine and saline injection, WT animals had minimal TUNEL staining in the hippocampus (50⫻ magnification). (b) Three days after pilocarpine injection there is an increase in TUNEL staining in the WT animal’s hippocampus, primarily in CA2–CA3 region (50⫻ magnification). (c) Following methyl scopolamine and saline injection, CREM/ICER null mutant animals had minimal TUNEL staining in the hippocampus (50⫻ magnification). (d) Three days after pilocarpine injection there is an increase in TUNEL staining in the CREM/ICER null mutant animal’s hippocampus, primarily in CA2–CA3 (50⫻ magnification). (e) High power view of TUNEL positive cells in CA2–CA3 pyramidal neurons (200⫻ magnification).
increase. The increase in ICER protein is likely to inhibit CRE-mediated transcription as ICER inhibition of transcription is not thought to require post-translational modifications. CRE-regulated genes that have been implicated
in epilepsy include the neurotransmitter receptor subunits GABA (A)␣1, (B)R1, and N-methyl-D-aspartic acid R1 (Mayr and Montminy, 2001; Lahteinen et al., 2002; Lau et al., 2004; Steiger et al., 2004). In addition neuropeptides
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Fig. 6. Density measurements of CA2–CA3 TUNEL positive cells are similar in the WT and CREM/ICER null mutant animals after pilocarpine injections. WT⫽N⫽3, CREM/ICER null mutant animals⫽N⫽3.
somatostatin, corticotropin releasing factor, and the neuropeptide receptor galanin R1 receptor (Gonzalez and Montminy, 1989; Zachariou et al., 2001), and neurotrophins BDNF and NGF have all been found to contain CRE sites in their promoters and regulated by CREB family members (Tao et al., 1998; McCauslin et al., 2006). Because of the large number of genes containing CRE sites and complexity of gene regulation, further studies are needed to identify specific genes regulated by ICER following SE and how disruption of this regulation might contribute to epileptogenesis. Following neuronal activity or seizures there is an increase in CREB-mediated gene transcription including the ICER transcript of the ICER/CREM gene (Fitzgerald et al., 1996; Ishige et al., 1999; Shaywitz and Greenberg, 1999; Kashihara et al., 2000; Kang et al., 2001 and data presented). We hypothesize that the increase in ICER may act to block sustained activation of CREB signaling after SE. Because the CREM/ICER null mice have more spontaneous seizures following the episode of pilocarpine-induced SE we propose that the increase in ICER is anti-epileptic. Combining these two ideas suggests that the lack of ICER repression and persistent CREB signaling caused the increase in spontaneous seizures after SE in the CREM/ ICER null mutant animals. Persistent CREB activation following SE might lead to increased aberrant synaptic plasticity and a more robust epileptic network. There are several mechanisms by which lack of CREM/ICER might contribute to increased seizure severity following SE. A sustained increase in CREB-mediated transcription might lead to abnormal gene expression that is pro-epileptic. Lack of CREM/ICER during neurodevelopment could cause abnormal neuron structure or function contributing to more frequent spontaneous seizures. Comprehensive studies on the role of CREM/ICER during neuronal development have not been reported. CREM/ICER knockout mice have normal appearing brains with no gross cell loss (Mantamadiotis et al., 2002). Only when mice are deficient in both CREM/ICER and CREB is there increased neuronal apoptosis occurring late in embryonic development and during postnatal life, suggesting that CREB and CREM/ICER are interchangeable for promoting neuronal survival.
While CREB activation has been implicated in neuronal survival, ICER overexpression leads to neuronal apoptosis in culture (Mantamadiotis et al., 2002; Jaworski et al., 2003; Lee et al., 2005). Based on these studies an increase in ICER following SE might promote excitotoxicmediated cell death and the absence of ICER following SE should be neuroprotective and anti-epileptogenic. There was an increase in TUNEL positive cells in the CA2–CA3 region of the hippocampus in both the WT and CREM/ ICER null animals suggesting that lack of ICER did not prevent DNA damage to pyramidal neurons in this region of the hippocampus. We did not carry out cell counts in the pyramidal region to determine if all the TUNEL positive cells eventually die. Though the region of increased TUNEL staining was similar to the area of pyramidal neuronal loss in CA3 found following pilocarpine induced SE in other mice strains (Turski et al., 1984; Shibley and Smith, 2002). Our data suggest that neuronal injury as suggested by TUNEL staining in the pyramidal layer of the hippocampus following SE does not require ICER. It is possible however, that the episodes of SE in the WT and CREM/ICER null mice are not the same, and it is the different characteristics of the SE that result in the observed phenotype in the CREM/ICER null mice. Using seizure semiology and mortality we did not find a difference in pilocarpine-induced seizures in the WT and mutant animals. However the lack of intra-cranial EEG analysis limits our ability to be certain at an electrophysiological level there are not differences in pilocarpine-induced SE between the WT and mutant animals. Similar TUNEL staining in the WT and mutant animals after SE suggests no difference in the amount of cellular injury between the mutant and WT animals. Prior studies of CREM/ICER null mutant mice found no obvious CNS structural abnormalities or spontaneous seizures, which suggests that the pilocarpine-induced SE is the relevant precipitant for the spontaneous seizures in the WT and CREM/ICER null mutant mice (Conti et al., 2004).
Fig. 7. There is an increase in spontaneous seizures after pilocarpine treatment in CREM/ICER null mutant animals. Animals were monitored for behavioral seizures for 8 h per week during the period 2– 6 weeks after pilocarpine treatment. All animals, WT and CREM/ICER null mutant animals developed spontaneous seizures. The frequency of spontaneous seizures was higher in the CREM/ICER null mutant animals (* P⫽0.01, two-tailed t-test).
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We have utilized CREM/ICER null mutant animals to begin to unravel the complex cascade of regulation that leads to the development of epilepsy. The lack of CREM/ ICER gene products following SE appears to promote a more severe form of epilepsy. Future studies designed to identify genes normally repressed by ICER following SE may identify novel targets for the development of antiepileptic therapies. Acknowledgments—We thank Dr. Carlos A. Molina for an antiICER antibody and the NINDS for financial support of this project (B. E. Porter: KO8 NS044869 and I.V. Lund F31 NS051943).
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(Accepted 10 January 2008) (Available online 17 January 2008)
THE ROLE OF TRANSCRIPTION FACTORS CYCLIC-AMP RESPONSIVE ELEMENT MODULATOR (CREM) AND INDUCIBLE CYCLIC-AMP EARLY REPRESSOR (ICER) IN EPILEPTOGENESIS B. E. PORTER,a* I. V. LUND,a F. P. VARODAYAN,a R. W. WALLACEa AND J. A. BLENDYb
Toth et al., 1998; Huang et al., 2002). It has been difficult to determine which of the many changes contribute to the development of epilepsy, and which are compensatory or inconsequential. The cyclic-AMP response element binding protein (CREB) family is influenced by seizure activity and is a regulator of transcription, cell survival, axonal sprouting and synaptic plasticity; all processes altered by SE (Lonze and Ginty, 2002). The central importance of the CREB family in multiple cellular signaling cascades makes it an excellent candidate for identifying pharmacologic interventions to block SE-induced changes that lead to the development of epilepsy. The CREB transcription factor family is composed of three family members (CREB, cyclic-AMP responsive element modulator (CREM) and ATF-1) sharing a high degree of similarity and all bind to CRE sites in gene promoter regions (Shaywitz and Greenberg, 1999; Mayr and Montminy, 2001; Conkright et al., 2003). While CREB primarily increases gene transcription most of the CREM splice variants and the related gene transcript, inducible cyclicAMP early repressor (ICER), inhibit CRE-mediated transcription (Molina et al., 1993; Maronde et al., 1999; Kemp et al., 2002; Morales et al., 2003). Seizures have been found to alter CREB, CREM and ICER activity. Seizures cause an immediate increase followed by a chronic decrease in CREB phosphorylation that has been implicated in memory impairment following earlylife seizures (Moore et al., 1996; Kashihara et al., 2000; Chang et al., 2003; Pi et al., 2004). Following kainic acid or electroconvulsive seizures there is an increase in the level of CREM and to a greater extent ICER in the hippocampus, frontal cortex and cerebellum accompanied by an increase in CREM/ICER CRE binding complexes (Fitzgerald et al., 1996; Konopka et al., 1998). CREB family members play a role in neuronal survival (Bonni et al., 1999; Glover et al., 2004). In CREB null mice sensory neurons exhibit excess apoptosis and degeneration, but this effect is not observed in the CNS (Lonze and Ginty, 2002). Only when both CREB and CREM/ICER are lacking is extensive apoptosis observed in the brain (Mantamadiotis et al., 2002). ICER is increased in hippocampal and cortical neurons undergoing apoptosis after excitotoxic injury and over-expression of ICER in cultured neurons results in neuronal apoptosis (Jaworski et al., 2003; Mioduszewska et al., 2003). ICER’s pro-apoptotic mechanism of action is not known but transcriptional repression of neuronal survival molecules, such as bcl-2 may be involved (Jaworski et al., 2003).
a
Departments of Pediatrics and Neurology, The Children’s Hospital of Philadelphia at the University of Pennsylvania, 502C Abramson, 3516 Civic Center Boulevard, Philadelphia, PA 19104, USA
b
Department of Pharmacology at the University of Pennsylvania, 125 South 31st Street, Translational Research Laboratory, Philadelphia, PA 19104, USA
Abstract—Alterations in the brain that contribute to the development of epilepsy, also called epileptogenesis, are not well understood, which makes it difficult to develop strategies for preventing epilepsy. Here we have studied the role of the CRE binding transcription factors, cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER), in the development of epilepsy following pilocarpine induced status epilepticus (SE) in mice. Following SE, ICER mRNA and protein are increased in neurons. The increase in ICER, however, is not necessary for neuronal injury following SE as pilocarpine treatment induces equivalent neuronal injury in pyramidal neurons of wild type and CREM/ICER null mice. Following SE, the CREM/ICER null mice develop a more severe epileptic phenotype experiencing ⬃threefold more frequent spontaneous seizures. Together these data suggest that the increase in ICER mRNA following SE may have a role in suppressing the severity of epilepsy. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: seizures, apoptosis, epileptogenesis, cyclic-AMP response element binding protein, CREB.
It is known that brain injury or status epilepticus (SE) increases the risk of developing epilepsy but anti-epileptic drug administration after head trauma or SE has failed to prevent the development of epilepsy (Temkin, 2001; Herman, 2002; Pitkanen, 2002). Our inability to identify antiepileptogenic drugs stems in part from a poor understanding of the cascade of molecular and cellular changes that contribute to epileptogenesis, the process of developing epilepsy. Many cellular and molecular changes have been identified after SE including neuronal cell death, alterations in neurotransmitter receptor expression, increased axonal sprouting and new synapse formation (Tauck and Nadler, 1985; Buckmaster and Dudek, 1997; Brooks-Kayal et al., 1998; Dudek and Buckmaster, 1998; Sankar et al., 1998; *Corresponding author. Tel: ⫹1-267-426-5210; fax: ⫹1-215-590-3779. E-mail address: [email protected] (B. E. Porter). Abbreviations: CREB, cyclic-AMP response element binding protein; CREM, cyclic-AMP responsive element modulator; ICER, inducible cyclic-AMP early repressor; PBS, phosphate-buffered saline; SE, status epilepticus; WT, wild-type.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.10.064
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Here we have studied the expression of CREM and ICER following SE and have utilized mice deficient for these transcription factors to study their role in cell death and epileptogenesis.
ter mix, and 3 l of sample cDNA. RT-PCR was performed on the SDS-7500 PCR machine (Applied Biosystems). The RT-PCR runs consisted of first one cycle of 50° for 2 min, then one cycle of 95° for 10 min, and 40 cycles of: 95° for 15 s and 60° for 1 min. All values were normalized to cyclophilin expression to control for loading variability.
EXPERIMENTAL PROCEDURES The experiments were performed with the approval of the animal care and use committee at the University of Pennsylvania and in accordance NIH guidelines for minimizing the number and suffering of animals used.
Mouse model For all experiments CREM/ICER null mutants and wild-types (WT) are F1 hybrids (129 SVEV:C57BL/6) obtained by crossing inbred mice heterozygous for the mutation from each strain CREM/ ICER⫾129SVEV N12⫻CREM/ICER⫾C57BL/6 N15 (Kaestner et al., 1996; Conti et al., 2004). In the F1 generation, each individual mouse is heterozygous for all loci that differ between the parental strains and thus are genetically identical with the exception of the CREM/ICER locus (Branbury Conference on Genetic Background in Mice, Silva et al., 1997). This allows for us to be certain that the genetic background in all the F1 mice is similar and does not contribute to the observed phenotype.
Induction of SE Male F1 hybrids between 3 months and 6 months of age underwent methyl-scopolamine i.p. injection of 1 mg/kg (Sigma, St. Louis, MO, USA) followed 30 min later by ⬃330 mg/kg of pilocarpine HCl (Sigma) or for control animals an equivalent volume of saline. A dose of 330 mg/kg of pilocarpine HCl was chosen because it produced multiple brief stage V seizures in the 3 h following injection with an ⬃70% survival rate in this genetic background.
Scoring of SE The animals for video EEG were placed in individual cages and a reviewer blinded to their genotype watched for behavioral seizures. Animals were video recorded for 6 h following pilocarpine injection. The animals developed discreet episodes of both Racine stage IV: rearing forelimb clonus; and stage V: rearing, fore- and hind-limb clonus with falling (Racine, 1972). The number, duration and Racine seizure stage were assessed for each animal.
Scoring of spontaneous seizures Two weeks following SE animals were videotaped for at least 8 h per week for 5 weeks and spontaneous seizure activity was scored. All stage IV–V Racine class seizures were analyzed and the duration and class of each seizure was recorded.
Real time-PCR Mice were anesthetized and whole hippocampi were freshly dissected and rapidly frozen at ⫺80 °C. RNA was extracted from individual hippocampi using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). To synthesize cDNA, SuperScript II reverse transcription kit was used (Invitrogen) and cDNA samples were stored in a 1:4 dilution in ddH2O. For RT-PCR reactions, each sample was run in triplicate and each 25 l reaction contained: 1.25 l ICER or CREM Taqman primer probe (Rn00569145_m1 and Rn00565271_m1; Applied Biosystems, Foster City, CA, USA) or 1.25 l Taqman cyclophilin probe (Applied Biosystems) with 1.25 l of each cyclophilin primers (cycloREV: 5= CCC AAG GGC TCG CCA 3=; cycloFWD 5= TGC AGA CAT GGT CAA CCC C 3=, IDT Technologies, Coralville, IA, USA) and 12.5 l Taqman Mas-
CREM/ICER immunohistochemistry and TUNEL staining WT and CREM/ICER null mutant adult males were injected as described above and 72 h after pilocarpine or saline injection, mice were deeply anesthetized with ketamine/xylazine (1 l/g, i.p. Sigma) and underwent intra-cardiac perfusion of cold saline followed by 4% paraformaldehyde. Dissected brains were dehydrated in 30% sucrose, frozen on dry ice, and 25 m sections were cut on a cryostat. Primary antibodies, 1–50 dilution of CREM/ ICER antibody s.c.-440 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and 1–100 dilution of the mature neuronal marker NeuN MAB377 (Chemicon, Billerica, MA, USA) were incubated in phosphate-buffered saline (PBS), 2% horse serum, 0.2% Triton X-100. Following three washes in PBS sections were incubated overnight with anti-rabbit TRITC and anti-mouse fluorescein (Jackson ImmunoResearch, West Grove, PA, USA) and mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA).
TUNEL staining was carried out as previously described Briefly, animals were processed as described above (Gavrieli et al., 1992; Porter et al., 2004), and sections corresponding to ⬃⫺3 mm from bregma were incubated with biotin-labeled deoxyuridine triphosphate (dUTP-16) (Roche Diagnostics, Indianapolis, IN, USA), one unit of TUNEL enzyme in terminal transferase buffer for 1 h at 37 °C. A Vector ABC peroxidase kit (Vector Laboratories) was used for isolated TUNEL staining (Paxinos and Watson, 1986). Sections were developed in 3= diaminobenzidine and counterstained with Cresyl Violet dehydrated in sequential ethanol and xylene and mounted with permount (Fisher Scientific, Hampton, NH, USA). An individual blinded to the animal genotype performed manual cell counts at 400⫻ magnification and freehand area measurements of CA2/CA3 were performed with ImagePro software (Media Cybernetics, Silver Spring, MD, USA).
Western blots The hippocampus and cortex were removed and frozen on dry ice. The frozen tissue was sonicated in 50 mM Tris pH 7.0, 140 mM NaCl, 10 mM EDTA, 2% SDS, 10 mm NaF, 50 mM -glycerophosphate, 30 mM Na-pyrophosphate, 0.2 mM PMSF, and 0.5 mM IBMX, then protein was assayed using a Biorad DC kit (Biorad, Hercules, CA, USA). To 75 g of total protein we added 2 l of -mercaptoethanol and 3 l 100 mM DTT and boiled the sample for 10 min. Each sample was loaded on to a 12%–14% polyacrylamide gel separated via SDS gel electrophoresis, and transferred to nitrocellulose, 90 min at 100 V. After blocking with 5% milk in 1⫻ PBS 1 h. Membranes were incubated with 1:500 CREM-1 (x-12) antibody (Santa Cruz Biotechnology) in 5% milk/ PBS with gentle agitation overnight at 4 °C. Blots were then incubated with appropriate HRP-conjugated secondary antibodies and developed with chemiluminescence, SuperSignal West Dura, extended duration substrate with agitation for 2 min at room temperature (Pierce, Rockford, IL, USA). Blots were stripped of primary and secondary antibodies and re-probed using an anti- actin antibody diluted 1–5000 (Sigma). Each blot was scanned using an Epson scanner in conjunction with Adobe Photoshop. Data were quantified using imaging densitometry and analyzed using the NIH Image program. Data from each sample were
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expressed as a ratio of -actin to insure equal loading and transfer. t-Tests were performed using Instat software (Graphpad Software Inc. San Diego, CA, USA) on Western blot data and significance was set at P⬍0.05.
RESULTS We first studied the effects of acute pilocarpine induced seizures on ICER and CREM expression in the brain of WT mice. Hippocampi were harvested 24 h after saline or pilocarpine injections in WT mice. RNA isolated from the whole hippocampus showed a 6.1-fold increase in ICER mRNA following pilocarpine induced SE (control 1⫾0.12 (SEM); SE 6.1⫾1.6; n⫽4 and 5; P⫽0.004, two-tailed t-test; Fig. 1a). In contrast there was no change in CREM mRNA levels following pilocarpine induced SE treatment (control 1⫾0.16 (SEM); SE 0.8⫾0.22; n⫽4 and 5; Fig. 1b). We carried out Western blots to quantify protein levels of ICER. The ICER␥ protein is increased in both the hippocampus and the cortex of WT mice following SE (Hippocampus-control 1.0⫾0.19 (SEM); SE 7.0⫾1.7, P⫽0.012,
Fig. 2. ICER immunoreactive bands are increased in the brain following SE. (a, b) In the hippocampus the larger ICER splice variant band is increased by visualization and the smaller ICER␥ splice variant is increased sevenfold following SE as analyzed by densitometry (n⫽4, * P⫽0.012, two-tailed t-test). The  actin loading control is shown below. (c, d) In the cortex by visualization the larger ICER splice variant band is increased and by densitometry the smaller ICER␥ splice variant is increased 2.8-fold following SE (n⫽4, * P⫽0.002, two-tailed t-test).
Fig. 1. ICER mRNA is increased in the hippocampus 24 h after SE. (a) Results of RT-PCR showing that mice subjected to SE have 6.1-fold higher expression of ICER mRNA than methyl scopolaminesaline treated control animals (n⫽4 and 5; * P⫽0.004, two-tailed t-test). All samples were normalized to cyclophilin mRNA, serving as a control for sample concentration and amplification variability. (b) CREM mRNA levels in the hippocampus are similar to control values 24 h after SE.
two-tailed t-test; Cortex-control 1.0⫾0.1; SE 2.8⫾0.33, P⫽0.002, two-tailed t-test; n⫽4; Fig. 2a– d). By visual inspection it appears that a large ICER splice form is also increased following a seizure but we were unable to separate the large ICER form from a non-specific band and therefore could not accurately quantify the change (data not shown). Using an antibody that recognizes both CREM and ICER splice forms we find that the CREM/ICER im-
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Fig. 3. CREM/ICER immunoreactivity is increased in CA3 pyramidal and dentate gyrus neurons of the hippocampus 3 days after pilocarpine induced SE. In a WT control mouse there is minimal CREM/ICER immunoreactivity in CA3 pyramidal neurons (b) and dentate granule neurons (d); a, c, NeuN staining in green and b, d, CREM/ICER staining in red. In a WT mouse following SE there is increased CREM/ICER immunoreactivity in CA3 pyramidal neurons (f) and dentate granule neurons (h); e, g, NeuN staining shown in green; f, h, CREM/ICER staining in red (200⫻ magnification).
munoreactivity is present in neurons of multiple brain regions and the immunoreactivity is increased following SE (see Fig. 3). We then went on to study the effect of pilocarpine induced SE in WT and CREM/ICER null mice. We found that the episodes of pilocarpine induced SE were similar in WT and CREM/ICER null mutant animals as assessed by number of stage IV and V seizure and mortality. After pilocarpine injection stage IV–V seizures began on average 41.3⫾5.1 min (SEM) in the WT and 32.3.3⫾7.9 min in the CREM/ICER null mutant mice (P⫽0.345, n⫽6). Equal numbers of stage IV and V seizures occurred in the WT and CREM/ICER null mutant animals during the 6 h following the pilocarpine injection (WT 2.8⫾0.4 SEM/per animal, n⫽5 versus CREM/ICER mutant 2.8⫾0.2, SEM/per animal, n⫽5; Fig. 4). Seizures lasted from 20 to 90 s. Between stage IV and V seizures, animals assumed a head nodding behavior and were not responsive and did not struggle during tail suspension. Stage IV, V behavioral seizures spontaneously resolved by 3 h after pilocarpine injection in both the WT and CREM/ICER null mutant mice.
Mortality was also similar in the WT and CREM/ICER null mutant mice with death always occurring during a prolonged tonic seizure (WT 11/33, 30% and CREM/ICER null 9/26, 30%). TUNEL staining was negligible in the hippocampus of methyl-scopolamine/saline-treated WT or CREM/ICER null mutant animals (Fig. 5a, c). In contrast, 3 days after the pilocarpine injection there was prominent TUNEL staining in hippocampal pyramidal areas CA2/CA3 in WT and CREM/ICER null mutant animals (Fig. 5b, d). Density of TUNEL positive neurons was similar in the CA2/CA3 region of the WT and CREM/ICER null mutant animals (WT 3.6⫻10⫺3 cells/m2⫾3.1⫻10⫺4 SEM, n⫽3; CREM/ICER null mutant 3.5⫻10⫺3⫾3.3⫻10⫺4, n⫽3; Fig. 6). There were few TUNEL positive cells in other regions of the hippocampus following SE. Spontaneous behavioral seizures were assessed for at least 8 h per week beginning 2 weeks after the episode of SE. Both the WT and CREM/ICER null mutant pilocarpinetreated animals developed spontaneous seizures. The seizure semiology appeared similar between the two groups (all stage V) consisting of wild running, four limb clonus, increased body tone, tail extension followed by loss of postural control. Animals had seizures both spontaneously and during handling. The frequency of spontaneous seizures was higher in the CREM/ICER null mutant animals than WT (WT 0.029⫾0.019 seizures/h⫾SEM, N⫽5 animals; CREM/ICER null mutant animals 0.11⫾0.028 seizures/h, N⫽4 animals, P⫽0.01; Fig. 7).
DISCUSSION
Fig. 4. The number of stage IV and V seizures induced by pilocarpine was similar in WT and CREM/ICER null mice (P⫽1, two-tailed t-test).
We have shown that ICER mRNA and protein increase in neurons of the hippocampus and cortex following pilocarpine induced SE. The ICER gene and its promoter are nestled within the 3= region of the CREM gene but the increase in ICER mRNA following SE appears to be specific to the ICER transcripts as CREM mRNA does not
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Fig. 5. Following pilocarpine injection there is an increase in the density of TUNEL stained CA2–CA3 neurons in both WT and CREM/ICER null mutant animals. (a) Following methyl scopolamine and saline injection, WT animals had minimal TUNEL staining in the hippocampus (50⫻ magnification). (b) Three days after pilocarpine injection there is an increase in TUNEL staining in the WT animal’s hippocampus, primarily in CA2–CA3 region (50⫻ magnification). (c) Following methyl scopolamine and saline injection, CREM/ICER null mutant animals had minimal TUNEL staining in the hippocampus (50⫻ magnification). (d) Three days after pilocarpine injection there is an increase in TUNEL staining in the CREM/ICER null mutant animal’s hippocampus, primarily in CA2–CA3 (50⫻ magnification). (e) High power view of TUNEL positive cells in CA2–CA3 pyramidal neurons (200⫻ magnification).
increase. The increase in ICER protein is likely to inhibit CRE-mediated transcription as ICER inhibition of transcription is not thought to require post-translational modifications. CRE-regulated genes that have been implicated
in epilepsy include the neurotransmitter receptor subunits GABA (A)␣1, (B)R1, and N-methyl-D-aspartic acid R1 (Mayr and Montminy, 2001; Lahteinen et al., 2002; Lau et al., 2004; Steiger et al., 2004). In addition neuropeptides
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Fig. 6. Density measurements of CA2–CA3 TUNEL positive cells are similar in the WT and CREM/ICER null mutant animals after pilocarpine injections. WT⫽N⫽3, CREM/ICER null mutant animals⫽N⫽3.
somatostatin, corticotropin releasing factor, and the neuropeptide receptor galanin R1 receptor (Gonzalez and Montminy, 1989; Zachariou et al., 2001), and neurotrophins BDNF and NGF have all been found to contain CRE sites in their promoters and regulated by CREB family members (Tao et al., 1998; McCauslin et al., 2006). Because of the large number of genes containing CRE sites and complexity of gene regulation, further studies are needed to identify specific genes regulated by ICER following SE and how disruption of this regulation might contribute to epileptogenesis. Following neuronal activity or seizures there is an increase in CREB-mediated gene transcription including the ICER transcript of the ICER/CREM gene (Fitzgerald et al., 1996; Ishige et al., 1999; Shaywitz and Greenberg, 1999; Kashihara et al., 2000; Kang et al., 2001 and data presented). We hypothesize that the increase in ICER may act to block sustained activation of CREB signaling after SE. Because the CREM/ICER null mice have more spontaneous seizures following the episode of pilocarpine-induced SE we propose that the increase in ICER is anti-epileptic. Combining these two ideas suggests that the lack of ICER repression and persistent CREB signaling caused the increase in spontaneous seizures after SE in the CREM/ ICER null mutant animals. Persistent CREB activation following SE might lead to increased aberrant synaptic plasticity and a more robust epileptic network. There are several mechanisms by which lack of CREM/ICER might contribute to increased seizure severity following SE. A sustained increase in CREB-mediated transcription might lead to abnormal gene expression that is pro-epileptic. Lack of CREM/ICER during neurodevelopment could cause abnormal neuron structure or function contributing to more frequent spontaneous seizures. Comprehensive studies on the role of CREM/ICER during neuronal development have not been reported. CREM/ICER knockout mice have normal appearing brains with no gross cell loss (Mantamadiotis et al., 2002). Only when mice are deficient in both CREM/ICER and CREB is there increased neuronal apoptosis occurring late in embryonic development and during postnatal life, suggesting that CREB and CREM/ICER are interchangeable for promoting neuronal survival.
While CREB activation has been implicated in neuronal survival, ICER overexpression leads to neuronal apoptosis in culture (Mantamadiotis et al., 2002; Jaworski et al., 2003; Lee et al., 2005). Based on these studies an increase in ICER following SE might promote excitotoxicmediated cell death and the absence of ICER following SE should be neuroprotective and anti-epileptogenic. There was an increase in TUNEL positive cells in the CA2–CA3 region of the hippocampus in both the WT and CREM/ ICER null animals suggesting that lack of ICER did not prevent DNA damage to pyramidal neurons in this region of the hippocampus. We did not carry out cell counts in the pyramidal region to determine if all the TUNEL positive cells eventually die. Though the region of increased TUNEL staining was similar to the area of pyramidal neuronal loss in CA3 found following pilocarpine induced SE in other mice strains (Turski et al., 1984; Shibley and Smith, 2002). Our data suggest that neuronal injury as suggested by TUNEL staining in the pyramidal layer of the hippocampus following SE does not require ICER. It is possible however, that the episodes of SE in the WT and CREM/ICER null mice are not the same, and it is the different characteristics of the SE that result in the observed phenotype in the CREM/ICER null mice. Using seizure semiology and mortality we did not find a difference in pilocarpine-induced seizures in the WT and mutant animals. However the lack of intra-cranial EEG analysis limits our ability to be certain at an electrophysiological level there are not differences in pilocarpine-induced SE between the WT and mutant animals. Similar TUNEL staining in the WT and mutant animals after SE suggests no difference in the amount of cellular injury between the mutant and WT animals. Prior studies of CREM/ICER null mutant mice found no obvious CNS structural abnormalities or spontaneous seizures, which suggests that the pilocarpine-induced SE is the relevant precipitant for the spontaneous seizures in the WT and CREM/ICER null mutant mice (Conti et al., 2004).
Fig. 7. There is an increase in spontaneous seizures after pilocarpine treatment in CREM/ICER null mutant animals. Animals were monitored for behavioral seizures for 8 h per week during the period 2– 6 weeks after pilocarpine treatment. All animals, WT and CREM/ICER null mutant animals developed spontaneous seizures. The frequency of spontaneous seizures was higher in the CREM/ICER null mutant animals (* P⫽0.01, two-tailed t-test).
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We have utilized CREM/ICER null mutant animals to begin to unravel the complex cascade of regulation that leads to the development of epilepsy. The lack of CREM/ ICER gene products following SE appears to promote a more severe form of epilepsy. Future studies designed to identify genes normally repressed by ICER following SE may identify novel targets for the development of antiepileptic therapies. Acknowledgments—We thank Dr. Carlos A. Molina for an antiICER antibody and the NINDS for financial support of this project (B. E. Porter: KO8 NS044869 and I.V. Lund F31 NS051943).
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(Accepted 10 January 2008) (Available online 17 January 2008)