Abstracts

Myocyte-specific enhancer binding factor 2C (MEF2C) expression in the dentate gyrus during development and after pilocarpine-induced status epilepticus: a preliminary report

Realce miócito específico ligado a expressão do fator 2C (MEF2C) no giro denteado durante o desenvolvimento e após uso de pilocarpina induzindo status epilepticus: estudo preliminar

Carla Alessandra Scorza^I; Esper Abrão Cavalheiro^I; Fulvio Alexandre Scorza^{I, II}

^IDisciplina de Neurologia Experimental, Universidade Federal de São Paulo/Escola Paulista de Medicina, São Paulo SP, Brasil

^IIProgram in Brain Plasticity and Epilepsy, Harvard Medical School and Department of Neurology, Beth Israel Deaconess Medical Center, Boston, MA, USA

ABSTRACT

OBJECTIVE: As axon outgrowth and dentate granule cell neurogenesis are hallmarks of hippocampal development and are also the two morphologic changes in the structure of the dentate gyrus after status epilepticus (SE), we hypothesized that molecules involved in normal development may also play a role during epileptogenesis.

METHOD: Using in situ hybridization, we have characterized mRNA expression of myocyte-specific enhancer binding factor 2C (MEF2C) in the dentate gyrus during development (P0, P3, P7, P14 and P28) and at multiple time points following pilocarpine-induced SE (3, 7, 14, 28 days after SE).

RESULTS: It was demonstrated that MEF2C is up-regulated during development (P0, P3, P7, P14 and P28) and in the adult rat dentate gyrus following SE (3, 7, 14, 28 days after SE).

CONCLUSIONS: The molecules controlling cell-fate decisions in the developing dentate gyrus are also operative during epileptogenesis.

Key words: rat, hippocampus, epilepsy, status epilepticus, MEF2C.

RESUMO

OBJETIVO: Como o crescimento axonal e a neurogênese do giro denteado são características intrínsecas do hipocampo durante o processo de desenvolvimento, e também são duas alterações morfológicas na estrutura do giro denteado após o status epilepticus (SE), nós hipotetizamos que as moléculas envolvidas no processo normal do desenvolvimento hipocampal também podem participar do processo de epileptogênese.

MÉTODO: Utilizando hibridização in situ, caracterizamos a expressão do RNAm do fator de transcrição myocyte-specific enhancer binding factor 2C (MEF2C) no giro denteado durante o desenvolvimento (P0, P3, P7, P14 e P28) e em diferentes períodos após o SE (3, 7, 14, 28 dias após SE).

RESULTADOS: Foi demonstrado um aumento da expressão de MEF2C no giro denteado durante o desenvolvimento e no giro denteado de animais adultos após o SE.

CONCLUSÃO: As moléculas que controlam o destino celular durante o processo de desenvolvimento também estão operativas durante o processo de epileptogênese.

Palavras-chave: rato, hipocampo, epilepsia, status epilepticus, MEF2C.

Epilepsy is the commonest serious neurological condition. Temporal lobe epilepsy (TLE), the most common epilepsy in adults, is generally intractable and is suspected to be the result of recurrent excitation or inhibition circuitry¹. TLE is defined by recurring partial complex seizures arising from limbic structures of the temporal lobes, including the hippocampus, dentate gyrus, amygdala and associated temporal neocortex¹. The study of mechanisms of the epilepsies requires employment of animal models. In these lines, the pilocarpine model is the most useful TLE model that reflects the human condition². In brief, the systemic administration of the cholinergic agonist pilocarpine to rats promotes sequential behavioural and electrographic changes that can be divided into three distinct periods: a) an acute period that builds progressively into limbic status epilepticus (SE) and that lasts 24h; b) a silent period with progressive normalization of EEG and behaviour which varies from 4 to 44 days; and c) a chronic period with spontaneous recurrent seizures². From a morphological point of view, seizure-induced injury leads to various forms of plasticity in the adult rodent dentate gyrus³. While these forms of neuroplasticity during epileptogenesis have been well documented, the molecular mechanisms underlying these alterations remain poorly understood.

During the last decade, a number of transcription factors have been shown to be involved in the cellular and molecular organization of the brain. The myocyte-specific enhancer binding factor 2C (MEF2C), a member of the MEF2 sub-family of the MADS gene family, is a transcription factor expressed at high levels in the brain and has been implicated in the development of neurons⁴. MEF2C binds with basic helix-loop-helix (bHLH) proteins⁵, which in the brain are responsible for neural determination or differentiation⁶. There is more definitive evidence for interactions among MEF2 with bHLH factors. For example, multiple members of bHLH family of transcription factors have been shown to be expressed in dentate granule cells at specific developmental stages⁷, supporting their involvement in regulating dentate gyrus development. Interestingly, Elliott and colleagues⁸ demonstrated that the expression of bHLH molecules is altered during seizure-associated network reorganization. Specifically, they showed varied profiles of expression of bHLH family members following SE, with some increasing (Mash1, Id2), some decreasing (Hes5, Prox1), and others remaining unchanged NeuroD/BETA2, NeuroD2/NDRF, Id3, Rath2/Nex1, supporting the idea that molecules controlling cell-fate decisions in the developing dentate gyrus are also operative during seizure-induced plasticity.

As MEF2C has been implicated to play an important role in the process of neuronal differentiation, we pursued the hypothesis that MEF2C is a potential upstream regulator of bHLH expression that is modified following SE. To do so, using in situ hybridization, we have characterized mRNA expression of MEF2C in the dentate gyrus during development and at multiple time points following pilocarpine-induced SE.

METHOD

Animals

For adult animals (n=40), two groups of Sprague-Dawley rats (180-200g) were studied: (A) Adult male rats at different time points following pilocarpine-induced status epilepticus (SE). It is important to note that for each time point (3, 7, 14, 28 days after SE) we used n=8 animals, (B) Control group, adult male rats (n=8), that received saline instead pilocarpine. For postnatal ages, subjects were male Sprague-Dawley rats (n=25) with ages P0, P3, P7, P14 and P28 days and for each group we used n=5 animals. The rats were bred in our laboratories and the day of birth was considered as day 0. The pups were housed with their mother in individual cages until weaning at day 21. All the animals used in our study were housed under standard controlled conditions (7:00 AM/7:00 P.M. light/dark cycle; 20-22ºC; 45–55% humidity) with food and water ad libitum.

Induction of SE

All animals were treated according to protocols for animal care established by the Harvard Medical School, Boston and the National Institutes of Health, and all efforts were made to minimize animal suffering. Adult male Sprague-Dawley rats (180–200g) were given an intraperitoneal (i.p.) injection of atropine methylbromide (5 mg/kg; Sigma, St. Louis, MO, USA) followed 20 minutes later by an i.p. injection of pilocarpine hydrochloride (340 mg/kg; Sigma) to induce SE. Seizure activity was monitored behaviourally and terminated with an i.p. injection of diazepam (10 mg/kg; Elkins-Sinn, Cherry Hill, NJ, USA) after 2 hr of convulsive SE. Only rats that displayed continuous, convulsive seizure activity after pilocarpine treatment were included in these studies. Control animals received the same injections of atropine and diazepam, but received saline instead of pilocarpine.

Tissue preparation

At designated time points following SE, all animals received an anesthetic overdose of pentobarbital and transcardially perfused with 300 mL of a 4% paraformaldehyde solution in phosphate-buffered saline at pH 7.4. Brains were post-fixed in situ overnight at 4ºC and then removed and cryoprotected in 30% sucrose in PBS prior to freezing. To generate anatomically comparable sections for the in situ hybridization analysis, 20 µm frozen coronal sections were cut through the middle third of the hippocampus with a cryostat, melted onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA, USA), and stored at –20ºC for subsequent in situ analysis of mRNA. Potential variations in mRNA expression along the septo/temporal axis of the hippocampus were not assessed in this study.

Non-radioactive in situ hybridization

Non-radioactive in situ hybridization was performed essentially as described previously⁷ using a protocol obtained from Dr. David J. Anderson (California Institute of Technology). Briefly, sections were pre-treated with Proteinase K for 5 minutes prior to prehybridization for 3 hr in a solution containing yeast tRNA, Denhardt's solution, and 50% formamide. Following prehybridization, sections were incubated at their respective optimal temperatures overnight with digoxigenin-labeled probes at a final concentration of 1 µg/µl. Slides were washed the next day at high stringency and incubated with sheep anti-digoxigenin Fab fragments conjugated to alkaline phosphatase (diluted 1:2000; Roche Molecular Biochemicals, Indianapolis, IN, USA) for 2-3 hr at room temperature. Following washes, the slides were incubated in buffer containing nitrobluetetrazolium (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (BCIP) (Roche Molecular Biochemicals). In situ data were evaluated qualitatively, based on the comparison of mRNA expression patterns in comparable hippocampal sections. These sections were taken from multiple sets of animals processed for in situ hybridization on different days.

Non-radioactive in situ probe preparation

The MEF2C probe used for this study, along with the optimal in situ hybridization temperatures and template sources, were described previously⁹. Probe templates generated by PCR were derived from cDNA adult rat. Antisense and sense digoxigenin-labeled RNA probes were transcribed from each template using the Genius RNA labeling kit (Roche Molecular Biochemicals) and purified with Chromaspin-100 columns packed in DEPC water (Clontech Laboratories, Palo Alto, CA, USA).

RESULTS

Pilocarpine treatment induced the following sequence of behavioral changes: akinesia, facial automatisms, and limbic seizures consisting of forelimb clonus with rearing, salivation, and masticatory jaw movements and falling. This type of behaviour built up progressively into motor limbic seizures that recurred repeatedly and rapidly and developed into status epilepticus. After SE, animals were unresponsive to their environment and akinetic; behavior returned toward normal over a 3 to 5-day period².

In situ analysis of MEF2C mRNAs at various times following SE course indicates that this molecule is expressed during epileptogenesis. In situ hybridization of control tissue with MEF2C antisense riboprobe demonstrates expression of MEF2C throughout the dentate granule cell layer (Fig 1B). The specificity of the probe was documented using an MEF2C sense probe, with which no signal and minimal background staining were observed (Fig 1A). In addition, at 3 days following SE the MEF2C expression in adult dentate gyrus appears to be slightly elevated as compared to the control group (Fig 1C). The expression of MEF2C mRNA throughout the dentate granule cell layer increased acutely within 7 days (Figure 1D) and 14 days (Fig 1E) following SE, and remained elevated until 28 days post-SE (Fig 1F).

In situ analysis of MEF2C during development shows that MEF2C is expressed in a specific pattern in the dentate gyrus (Fig 2A). In situ hybridization of P0 tissue with MEF2C antisense riboprobe demonstrates expression of MEF2C in immature granule cells populations (Fig 2B). At P3 and P7 (Figs 2C and 2D respectively), MEF2C is distributed throughout the developing dentate granule cell layer. MEF2C is expressed at highest levels in the granule cell layer at P14 (Fig 2E) and P28 (Fig 2F), when the entire inner half of the granule cell layer has only recently been generated.

DISCUSSION

It has been known that MEF2C is expressed in a dynamic pattern during development and in the mature mouse CNS, suggesting that the molecule may play an important role at different stages of neuronal differentiation and/or maturation^4,10. Here it was demonstrated that MEF2C is up-regulated during development and in the adult rat dentate gyrus following SE, suggesting that MEF2C may also be involved in seizure-induced plasticity. Interestingly, the results in this report are not in agreement with the recent paper of Yoon and colleagues¹¹. In their study, the authors have shown that MEF2C phosphorylation was reduced immediately after electroconvulsive shock. The discrepancies in these MEF2C activation patterns may reflect different epilepsy models and time points analyzed.

During development, four MEF2 genes (A, B, C, D) are expressed in spatially and temporally specific patterns during brain development, suggesting that, similar to its expression pattern in striated muscle, MEF2 gene expression in the CNS occurs in neurons exiting the cell cycle and entering differentiation¹⁰. In addition, human studies using immunocytochemical localization of MEF2C in hippocampal surgical specimens revealed immunoreactivity in nuclei of dentate granule cells and the hilus of dentate gyrus, supporting the concept of a role for MEF2C in post-mitotic neuronal differentiation⁴. Given that, the results of this study also showed that MEF2C is expressed in dentate granule cells during specific developmental stages, it seems quite possible that MEF2C is involved is in regulating dentate gyrus development.

The persistent cell birth in the rodent dentate gyrus during postnatal and adult life is well established¹². Interestingly, neurogenesis and mossy fiber growth are important processes that occur during both development and epileptogenesis of dentate gyrus, supporting the idea that molecules guiding neurogenesis and axon outgrowth during development overlap with those expressed during epileptogenesis⁸. Numerous examples support this hypothesis, but one of the more important of them is the differential expression of bHLH molecules during development⁷ and following SE⁸. Interestingly, MEF2C binds as homodimers and heterodimers with bHLH proteins⁵, supporting the idea that interactions between MEF2 factors and bHLH proteins (e.g., Mash1) are required for activation of tissue-specific transcription in neurogenic cell lineages¹³. Based on this, we believe that the increase of MEF2C expression after 3, 7, 14 and 28 days after pilocarpine-induced-SE may also be involved with dentate gyrus plasticity.

Human temporal lobe epilepsy and rodent models of limbic epilepsy are frequently associated with a marked loss of hippocampal and dentate gyrus neurons. Seizure-induced cell loss has been attributed to a mechanism of excitotoxicity-induced necrosis¹⁴. However, it has been shown that apoptosis contributes to the degeneration following status epilepticus¹⁵ and alterations in the expression of caspases suggest an involvement of these factors in mediating apoptosis following seizures. Moreover, Ekdahl and co-workers¹⁶ have shown that caspases modulate seizure-induced neurogenesis, possibly through the regulation of apoptosis of new neurons, because this action can be suppressed by caspase inhibitors. Along these lines, MEF2C has also been implicated in mediating or modulating apoptosis responses. Okamoto and colleagues¹⁷ recently described antiapoptotic functions for MEF2C. However, in mature neurons exposed to excitotoxic injury or others forms of stress, an apoptotic effect¹⁸ mediated by caspases were demonstrated¹⁹. With these results, it is possible to support the hypothesis that MEF2C is involved with the mechanism of apoptotic degeneration following pilocarpine induced-SE.

Finally, the present results support previous evidence that MEF2C is expressed in areas of ongoing neural plasticity and raise, for the first time, the possibility of a potential role for MEF2C molecules during epileptogenesis. Nevertheless, there are some limitations to this study. Firstly, the present data is only from in situ hybridization. A description of quantification of MEF2C mRNA expression in the dentate gyrus should be carried out. Secondly, to confirm the roles of MEF2C in epileptogenesis, an imunohistochemistry study should be realized to investigate the possible changes of MEF2C protein expression in the dentate gyrus. These future studies, which are in evaluation in our laboratory, are needed to gain a better understanding of these and other possible mechanisms of MEF2C transcription factor during epileptogenesis.

ACKNOWLEDGEMENTS – The authors would like to thank Dr. Daniel H. Lowenstein and Dr. Robert C. Elliott for their suggestions and helpful review of this manuscript and to Mr. Brian Kruegel for his help with technical procedures.

Received 28 March 2008, received in final form 6 June 2008. Accepted 12 July 2008.

Dr. Fulvio Alexandre Scorza – Disciplina de Neurologia Experimental / UNIFESP/EPM - Rua Botucatu 862 - 04023-900 São Paulo SP - Brasil. E-mail: scorza.nexp@epm.br

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