Myoclonin1/EFHC1 in cell division, neuroblast migration, synapse/dendrite formation in juvenile myoclonic epilepsy

This chapter presents recent works on Myocloni1/ EFHC1 a protein encoded by an epilepsy causing gene of juvenile myoclonic epilepsy (JME), one of the most frequent forms of idiopathic or genetic generalized epilepsies. Myoclonin 1/EFHC1 is a microtubule-associated protein involved in the regulation of cell division. In vitro, EFHC1 loss of function disrupted mitotic spindle organization, impaired M phase progression, induced microtubule bundling and increased apoptosis. EFHC1 impairment in the rat developing neocortex by ex vivo and in utero electroporation caused a marked disruption of radial migration. This effect was a result of cortical progenitors failing to exit the cell cycle. On the other hand, defects in the radial glia scaffold organization and in the locomotion of postmitotic neurons. Mutant analysis of Defhc1 lossand gain-of-function alleles in vivo in Drosophila revealed a number of neuronal defects, including abnormal synaptic development characterized by extensive satellite bouton formation, increased frequency of spontaneous neurotransmitter release, and aberrations in dendritic arbour morphogenesis. Thus, Myoclonin 1/ EFHC1 is a regulator of cell division and neuronal migration during cortical development synaptic bouton and dendritic morphogenesis. Disruption of these properties lead to JME, being now therefore considered as a developmental disease. Juvenile myoclonic epilepsy (JME) is the most frequent form of idiopathic/genetic generalized epilepsy. It accounts for 2–12 % of all epilepsies. JME symptoms of myoclonias and tonicclonic convulsions appear in adolescence in an otherwise normal person with normal neurological and cognitive functions (Delgado-Escueta and Bacsal, 1984)1. Five percent of these patients, five percent of their affected family members and eight percent of a 252 patient Corresponding author : Prof Thierry GRISAR, MD,PhD, University of Liege, GIGA Neuroscience Blvd de l’Hopital 1, 4000 Liege BELGIUM e-mail : tgrisar@ulg.ac.be. Japer's Basic M ecanism s of he Eppsies Japer's Basic M ecanism s of he Eppsies cohort followed for over 20 years only have seizures when triggered by external factors such as alcohol use, fatigue, menstruation and sleep deprivation. To understand the disease mechanisms underlying the stages of susceptibility and epileptogenesis in JME, many have tried to define its complex heritability and identify the genes corresponding to the fifteen chromosomal loci so far linked to the disease2 (see also chapter ...in this book). In 2004, Suzuki et al. identified several heterozygous missense mutations in a gene called EFHC1 3 in different unrelated families with JME probands. Since then, heterozygous non sense, deletion frame shifts and novel missense mutations have been identified in various populations of Italy, Austria and Chile 4–7 .Nine percent of sporadic JME cases detected in families consecutively seen in epilepsy clinics of Mexico and Honduras and 3 % of clinic patients from Japan carry mutations in EFHC1. This represents the highest number and percentage of mutations found for a juvenile myoclonic epilepsy causing gene of any population group 7. The gene encodes a 75-kDa protein with three DM10 domains of unknown function and a single EF-hand motif, a Ca2+-binding domain. The transcript is observed in many cell type of human tissue including brain (in particular in ependymal or periventricular cells) but also best expressed in dividing cells with highest levels in lung and testis 3. It was first proposed that EFHC1 was “pro-apoptotic” when overexpression in hippocampal neurons in vitro induced apoptotic cell death. This effect was significantly reduced by any of the five mutations associated with JME. Patch-clamp analysis of BHK (Baby Hamster Kidney) cells transfected with Cav2.3 VDCC (voltage-dependent calcium channel) and EFHC1 showed significantly increased R-type Ca2+ currents. So, the pro-apoptotic effect of EFHC1 was assigned to this enhancing effect on Ca2+ through Cav2.3 VDCC 3. In 2005, another research group pointed out that EFHC1 is orthologous to Rib72, an axonemal protein of Chlamydomonas reinhardtii. They demonstrated that EFHC1 is abundantly expressed in mouse tissues that have motile cilia or flagella, including the brain, and suggested that it plays a role in the intrinsic properties of these organelles 8. One of our laboratories previously reported that the subcellular distribution of EFHC1 in different cell lines varied during the cell cycle. In interphase cells, the protein is present in the cytoplasm and nucleus, except nucleoli and is particularly concentrated at the centrosome. During mitosis, EFHC1 is localized at spindle poles of the mitotic spindle and also at the midbody during cytokinesis 9. These results suggest that EFHC1 could play an important role during cell division and in particular during brain development since mRNA expression is higher at embryonic stages as compared to adult 10. More recently, we demonstrated that EFHC1 is a microtubule-associated protein (MAP) playing a key role in neuronal migration 11. In this chapter we review these putative roles of Myoclonin 1 / EFHC1 or during brain development and during adulthood. We posit an hypothesis that JME is a developmental disease involving neuronal migration and synaptic bouton and dendritic morphogenesis. EFHC1/MYOCLONIN1, A PROTEIN OF UNKNOWN FUNCTION Myoclonin 1 /EFHC1 gene is located on chromosome 6 (6p11–12) between markers D6S1960 and D6S11024, spans 72 kb and contains 11 exons. This gene encodes a protein of 640 amino acids. A domain search identified three tandemly repeated so called DM10 domains, a motif with unknown function. This protein also contains a single EF-hand, a well known Ca2+binding motif, from which it was named EFHC1 for EF-hand Containing 1 (Figure 1). This motif is located at the C-terminus between amino acid 578 and 606 and encoded by a nucleotide sequence present in exon 10. Page 2 Myoclonin1/EFHC1 in cell division, neuroblast migration, synapse/dendrite formation in juvenile myoclonic epilepsy Japer's Basic M ecanism s of he Eppsies Japer's Basic M ecanism s of he Eppsies Figure 1. Schematic representation of the EFHC1/Myoclonin1 gene, the long and short forms of the EFHC1 protein and the different mutations found co segregated with the JME phenotype The transcript undergo alternative splicing in exon 4, resulting in a C-terminally truncated protein, eliminating the EF-hand domain and two DM10 sequences. This last short form therefore only contains 278 amino acids, the first 240 being common with the entire molecule.