Dissecting the neurodevelopmental role of the microtubule-associated protein EML1, mutated in subcortical heterotopias

BIZZOTTO, S; PHAN DINH TUY, F; KIELAR, M; ROMERO, D.M; HOULLIER, A; CANALI, G; WELKER, E; HOUDUSSE, A; CROQUELOIS, A; FRANCIS, F

Rio de Janeiro

Congreso; 9th Word Congress of the International Brain Research Organization; 2015

IBRO

Cortical malformations, such as lissencephaly and subcortical heterotopia, are associated with intellectual disability and epilepsy. Subcortical band heterotopia (SBH) is a malformation characterized by the presence of a band of heterotopic neurons located in the white matter below a grossly normal cortex. Mutations were identified in the microtubule-associated protein Eml1/EML1 in the spontaneous HeCo (?Heterotopic cortex?) mouse mutant, a model of SBH, and in patients exhibiting giant ribbon-like heterotopia. HeCo mice have misplaced progenitors and ectopic proliferation, which are likely to be the primary causes of their heterotopia.The role of Eml1 in neurodevelopment has not been previously studied. We found that during the neurogenic period in the mouse cortex it is expressed in both the VZ, where progenitors are located, and in the cortical plate (CP), made up of post-mitotic neurons. We aimed to explore Eml1?s function in progenitors. Our objectives were to characterize the subcellular localization of Eml1 in dividing cells in vitro and in vivo, and to study neuronal progenitor behaviour in vivo in the presence or absence of Eml1. Further objectives were to study Eml1 association with microtubules (MTs) and to search for protein partners in mouse embryonic brain extracts.We found that Eml1 shows a cell cycle-dependent localization in vitro, enriched at the spindle poles and along spindle MTs during metaphase, and in the spindle midzone and midbody region during anaphase/telophase, suggesting a role in spindle function and/or in cytokinesis. Recombinant Eml1 partially co-localizes with MTs in epithelial cells and the purified protein binds directly to MTs in vitro, although its function with respect to these polymers still remains to be explored. Proteomics studies from embryonic mouse brain extracts, using pull-down experiments and mass spectrometry, identified a list of potential protein partners, and we are focusing our analysis on networks of proteins known to be involved in cell division.To further understand its function in progenitors, we knocked down the expression of Eml1 in wild-type E14.5 mouse embryos using in utero electroporation, specifically in radial glial cell progenitors, which are predominant in the mouse embryonic VZ. Reduced expression of Eml1 leads to loss of their radial orientation and a shift of somata to more superficial positions. Eml1 re-expression in knockdown and HeCo embryos rescues the position of progenitors, restoring them to the proliferation zones. Using these assays, we are further exploring the consequences of Eml1 loss on MT dynamics and cell division in vitro and in vivo. Protein partners will be assessed for their roles in these processes. These data will contribute to understanding mechanisms of progenitor constraint in the VZ during corticogenesis, and add knowledge concerning the causes of heterotopia formation.