Analysis of the expression profile and distribution of EFHC1 gene transcript during rodent brain development and in the adult animal / Analise da expressão e localização do transcrito do gene EFHC1 no cerebro de roedores durante o desenvolvimento e no animal adulto

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

Epilepsy is a frequent condition that affects around 1.5% of general population and is considered worldwide as a public health problem. The term epilepsy refers to a group of chronic neurological disorders with different etiologies and prognostics. However, a common characteristic of all epilepsies is the recurrence of seizures. Juvenile myoclonic epilepsy (JME), one of the most common forms of epilepsy, is characterized by myoclonic seizures mainly at awakening. JME has been intensively studied at the molecular level. Recently, one of the putative causative genes for JME was cloned. This gene, called EFHC1, encodes a protein with 640 amino acids that has three DM10 domains, with unknown function, and a calcium binding motif, called Ef-Hand. EFHC1 protein associates with microtubules and, therefore, it actively participates in the cellular division process. In addition, it has been demonstrated that this protein induces apoptosis in cultured neurons through the association with an R-type voltage-dependent calcium channel (Cav2.3).To date, five different EFHC1 missense mutations identified in patients with JME were shown to decrease this proapoptotic function in cell models. The ortholog genes in mouse and rat, both named Efhc1, were also isolated. The murine gene encodes a protein with around 75KDa, homolog to Chlamydomonas reinhardtii Rib72 protein. Efhc1 protein presents its highest expression levels in tissues and organs that have mobile cilia and flagella, like testis, oviduct and, in the central nervous system, the ependymal cells. The aim of this study was to determine the distribution and expression profile of Efhc1 genes and evaluate the viability of functional studies by the modulation of the its expression during the development of the brain of mice and rats. Real time polymerase chain reaction (Real Time PCR) revealed that there is no difference in the expression of Efhc1 transcript between right and left hemispheres in both species. Western blot experiments corroborated this finding. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-days-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME. The technique of RNA interference (RNAi) promotes a potent and specific gene silence and it was employed in this project to study the function of Efhc1 gene in cultured mammal cells and in the brain of mice in different developmental stages. This approach has been widely used to achieve this aim. Both for cultured cells assays as for in vivo experiments, the percentage of gene silence was evaluated with Real Time PCR and Western blot. In addition, negative controls (injection only with buffer and the use of an irrelevant siRNA) were used in the experiments. Initially, we used two siRNAs, the effectors of RNAi. The siRNA molecules promote a temporary gene silence, whereas the short hairpin RNA (shRNA), another type of molecule, promotes long-term gene silence. In the experiments with mammal cells in culture, we used different cellular types, from rat cardiomyocytes to mouse neuroblastoma cells. For the in vivo experiments, different methodologies were used, such as intra-uterine surgery, hydrodynamic transfection, and association of the siRNA with a peptide derived from a rabies virus glycoprotein. A long-term Efhc1 gene silencing was obtained in Neuro2A cells. The greatest silencing percentage (60%) was observed after 48h of incubation with the siRNA and the gene remained silenced for up to 6 days. Besides, Efhc1 gene silencing in rodent Central Nervous System (decrease in gene expression of 45%) was achieved by the inoculation of the siRNA-RVG-9R complex. We believe that our results point to an association between putative EFHC1 gene function during brain development and the physiopathology of JME

ASSUNTO(S)

epilepsia mioclonica juvenil polymerase chain reaction rna interference in situ hybridization hibridização in situ gene expression expressão genica interferencia de rna reação em cadeia de polimerase juvenile myoclonic epilepsy

ACESSO AO ARTIGO

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