Elucidating molecular and cellular Causes of heteroTOPIA – ECTOPIA

Task 1: Biochemistry and cell biology
• Microtubule function by biochemistry, cell transfections, and videomicroscopy
• In utero electroporation, immunohistochemistry and confocal microscopy of mouse brain slices (embryonic day E13.5 and 15.5)
• Protein purification, mass spectrometry and STRING database analysis
• Regarding the identification of Eml1’s partners, pull down and immunoprecipitation followed by protein gels and Western blot.
Task 2: Clinical characterizations, molecular analyses and functional studies
• Patient classification. Whole exome sequencing.
• Design and clonage of shRNA for candidate genes and scramble controls into a pCAGMIR30 vector.
• Cell transfections, immunocytochemistry and confocal microscopy
• Sub-cellular studies using fractionation and microscopical experiments, protein gels, Western blot, quantitative PCR
• Immunohistochemistry of brain slices (vibratome sections, E14.5, 15.5, 17.5, P0…)
Task 3: Bioinformatics
• Gene expression
• Data analyses
• Descriptive statistics (Student t-test)
• Data integration

We recently found that Eml1, likely to be involved in microtubule and membrane dynamics, is mutated in subcortical heterotopia in mouse and human and is associated with mis-positioned neurons in the white matter and by abnormal neuronal progenitors during cortical development (published by our groups in Nature Neuroscience 2014). To characterize the EML1 protein we are analyzing its microtubule function as well as characterizing defects in dividing mutant cells in brain slices. We have identified a list of protein partners of Eml1 which we are now further studying. To characterize patients suffering from subcortical heterotopia, we performed trio based Whole exome sequencing in a small cohort of individuals showing subcortical heterotopia similar to EML1 mutation (i.e. an EML-like phenotype). From this analysis so far, DNA from several patients presented candidate gene variations which were selected for further analysis. We have hence begun studying one of the mutated genes in the mouse brain during development. We started to study the effects of downregulation of the protein and mRNA expression, its overexpression and its possible interacting partners using a neuroblastoma cell line, which resembles neuronal progenitor cells. We also performed the analysis of gene expression data in order to identify possible biochemical pathways affected by the inactivation of the Eml1 gene, responsible for heterotopia in the murine model. From this, we identified a set of differentially expressed genes. Potential interactions between these genes allowed us to build a connected interaction network. A first integration of gene expression with exome sequencing data allowed us to infer a small network of highly connected genes.

Having recently identified EML1 as a new heterotopia gene, we propose a natural follow-up study, important to further elucidate its function and the disrupted pathways in which it acts. The protein shows a vesicular localization associated with microtubules, both in mitotic cells and post-mitotic neurons. Using biochemistry and cell biology, we will further question its roles particularly in dividing cells. We will continue to identify interacting partners. We will further study the causes of the heterotopia phenotype in mutant mice.
We are continuing to study the rare disorder of giant sub-cortical heterotopia. Several other patients with sub-cortical heterotopia have been identified within France. We are now extending our search for such patients in discussion with other European laboratories (in the framework of European consortiums) who have cortical malformation patient cohorts. We plan to enlarge our cohort to similar forms of sub-cortical heterotopia in the context of Aicardi syndrome.
We also plan to make further links between interacting partners of Eml1, the perturbed gene networks identified in HeCo brain tissue and the genes found mutated in giant heterotopia patients, to reveal subcellular mechanisms important in radial glial cells and to help direct future functional and genetic studies. Our preliminary analyses provide interesting snapshots of de-regulated pathways and are likely, with further work, to lead to further pertinent functional experiments, querying specific aspects of radial glial cell and neuronal function. Further, more global analyses are still required to have an overall perspective of the entire dataset. A database (‘Ectopia-DB’) allowing cross querying between the different datasets will be constructed by a dedicated bioinformatician and made available to the community.

1. Kielar M*., Phan Dinh Tuy F*., Bizzotto S*., Lebrand C*., de Juan C, Poirier K., Oegema R., Mancini GM, Bahi-Buisson N., Olaso R., Le Moing A. G., Boutourlinsky K., Boucher D., Carpentier W., Berquin P., Deleuze JF., Belvindrah R., Borrell V, Welker E., Chelly J., Croquelois A#., Francis F#. (2014). Mutations in Eml1 lead to ectopic progenitors and neuronal heterotopia in mouse and human. Nat Neurosci. 17, 923–933.
2. Bizzotto S and Francis F (2015) Morphological and functional aspects of progenitors perturbed in cortical malformations. Front Cell Neurosci 9:30. doi: 10.3389/fncel.2015.00030
3. Kielar M., Phan Dinh Tuy F., Bizzotto S., Belvindrah R., Croquelois A., Francis F. (2014). [Mutations in Eml1/EML1 lead to ectopic progenitors and neuronal heterotopia in mouse and human]. Mutations du gène EML1/Eml1, progéniteurs neuronaux et hétérotopies chez l’homme et la souris Med Sci (Paris) 30 : 1087–1090.

Cortical malformations are severe neurodevelopmental disorders associated with drug-resistant epilepsy and intellectual disability. During brain development, cytoskeletal and associated proteins play critical roles in cortical neuron generation, migration and differentiation. Abnormal functioning of these proteins, is one of the major underlying causes of so-called ‘neuronal migration disorders’ which result in aberrantly positioned cortical neurons present in the white matter (‘heterotopia’) and severe cortical malformations. Heterotopic cells and perturbed neuronal networks are likely to be the underlying defects giving rise to severe epilepsy and cognitive deficits, although this still remains little elucidated.
The DCX, TUBA1A and LIS1 genes are mutated in type 1 lissencephaly and band heterotopia. Further patients exist, with typical or atypical forms of these disorders, for which no mutations have been identified so far. We recently identified the Echinoderm microtubule associated protein like 1 gene, Eml1, mutated in a spontaneous mouse model (HeCo), which, unlike the type I lissencephaly mouse mutants, shows severe band heterotopia. Importantly, we also showed that EML1 is mutated in a human family with a rare, severe and atypical form of giant heterotopia (Kielar et al., submitted). This protein has never previously been studied during cortical development and is likely to interact in different biochemical pathways than DCX and LIS1. Studying the function of this protein hence provides an entry point to identify novel aspects of cortical development. We find that EML1 associates with microtubules (MTs) in a vesicle-like pattern, in both proliferating cells and differentiating neurons. HeCo mouse mutants show abnormalities of neuronal progenitor cells and perturbed proliferation. Indeed, our data suggest that ectopic progenitors are the primary defect in this model, and that abnormal neuronal positioning, resulting in severe heterotopia, is a secondary effect. This represents a little-studied concept for these ‘neuronal migration disorders’.
A major objective of this project, combining molecular and cellular biologists, clinicians and bioinformaticians, is to elucidate the role of Eml1 and biochemical pathways in which it acts, during normal cortical development and to identify how the mutation of this protein perturbs neuronal progenitor, in particular radial glial stem cell, polarity and division. The mechanisms maintaining such cells in the proliferative zones of the developing cortex are little understood and our project will identify key mechanisms controlling these critical processes. Our objectives are as follows: 1) To identify Eml1-interacting proteins, to provide clues to the protein complexes in which it acts, which may be involved in MT dynamics and the end stages of radial glial cell division. Using fluorescent proteins, we will image the nucleus, MT cytoskeleton and apical processes of these cells during the cell cycle using videomicroscopy; 2) To identify mutations in other genes coding for proteins in the same biochemical pathway as EML1, by sequencing the exomes of ten unrelated patients with severe forms of giant heterotopia, who show no mutations in EML1; 3) To understand how the perturbation of gene and protein networks leads to the abnormal phenotypes observed in HeCo neuronal progenitors, by analyzing in detail previously obtained transcriptome data, and integrating it with Eml1-proteome and patient exome datasets. Thus, ectopic and perturbed proliferation, identified in HeCo mice, may represent the primary mechanism leading to heterotopia formation in this model, and giant heterotopia in human, for which we will identify the underlying bases in this project, revealing novel and fundamental aspects of brain development.