Cortical development and pathology
Using molecular and cellular biology, our group questions the normal functions during development of three cortical dysplasia proteins (Eml1, Caspr2, Dcx) associated with the cytoskeleton and the plasma membrane, and effects of their mutation.
We focus principally on three proteins mutated in brain malformations or more subtle cortical abnormalities in human. Cortical malformations are frequent causes of drug-resistant epilepsy and intellectual disability. More subtle abnormalities may be present in idiopathic and mesial temporal lobe epilepsies and in neuropsychiatric disorders. Cortical defects can arise through abnormal neuronal proliferation, migration and/or connectivity. We use the Eml1, Caspr2 and Dcx proteins as points of entry to better understand normal cortical development and physiopathology. We found that Eml1, likely to be involved in microtubule dynamics, is mutated in severe subcortical heterotopia, associated with mis-positioned neurons in the white matter. Its role in cortical development is currently unknown. We study Eml1 in mouse neuronal progenitors and post-mitotic neurons and question potentially novel mechanisms leading to heterotopia. Mutations in Caspr2, an adhesion protein, are found in a wide spectrum of disorders, including syndromic epilepsy and autism. Better known for its roles at the nodes of Ranvier, its neurodevelopmental functions have been little-studied. It may be essential for adhesive capacities of migrating neurons and during the formation of synapses at the axonal initial segment, hypotheses we are testing. We also question the consequences of patient mutations on these functions. DCX, a microtubule-associated protein is mutated in heterotopia and severe brain gyral abnormalities such as lissencephaly. Dcx knockout mice are an excellent model to study aberrant connectivity and hyperexcitability related to migration defects, epilepsy and behavioral abnormalities. Studying mutant mice and patient mutations (in collaboration with clinicians) for these three proteins will reveal novel insights into the causes and consequences of abnormal neuronal positioning and connectivity, related to cortical malformations and neuropsychiatric disorders.
Malformations of cortical development are frequent causes of drug-resistant epilepsy and intellectual disability1. At least 40% of intractable epilepsy patients are estimated to have severe cortical malformations, and more subtle abnormalities may be underestimated in idiopathic epilepsy2, in mesial temporal lobe epilepsy3 and in neuropsychiatric disorders (e.g. 4 and see below). Cortical defects can arise through abnormal neuronal proliferation, migration and/or connectivity, and many neurodevelopmental genes play roles in several of these steps. Of interest, some mutated genes can give rise to either severe malformations or more subtle abnormalities in patients, depending on the mutation. The molecular and cellular mechanisms involved in cortex development, and physiopathology related to gene mutations, are still being elucidated. We choose to focus on the roles of 3 proteins (Eml1, Caspr2, Dcx), which when mutated in patients give rise to a spectrum of ‘neuronal migration’ disorders (cortical dysplasia). We use these proteins as points of entry to better understand key steps of normal and pathological cortical development. We are also completing studies on two further proteins (Tuba1a and SCHIP-1) involved in similar processes.
We recently identified Eml1, a protein binding to microtubules, mutated in subcortical heterotopia (SH) featuring many mis-positioned neurons in the white matter (Kielar* Phan Dinh Tuy*, in press). Eml1’s role in cortical development was never previously studied and little is known about the biochemical pathways in which it acts. We study Eml1 in mouse neuronal progenitors and post-mitotic neurons and question novel mechanisms leading to SH (Project 1). Defects in the CNTNAP2 gene, coding for the adhesion protein Caspr2, have also recently been characterized in a wide and expanding spectrum of neurodevelopmental disorders, including cortical dysplasia-focal epilepsy syndrome (CDFE)5 and autism6. Caspr2 is well-known for its roles in axo-glial contacts at the Nodes of Ranvier7, but its neurodevelopmental functions are little-studied. We question molecular and cellular functions of Caspr2 during neuronal migration and synaptogenesis, and the consequences of CNTNAP2 variants identified in patients on these functions (Project 2). DCX, a microtubule-associated protein (MAP) which also interacts with membrane proteins, is mutated in SH with severe gyral abnormalities8,9. We question the connectivity of aberrantly positioned neurons, origins and rescue of hyperexcitability and behavioral deficits in the Dcx knockout (KO) mouse (Project 3).
Project 1 : Characterization of the Eml1 protein, mutated in HeCo mice, and its role in neuronal progenitors and post-mitotic neurons
Cellular causes of heterotopia have been thought to be primarily due to neuronal migration defects. Eml1 is expressed not only in post-mitotic neurons but also in dividing cells, and ectopic progenitors were identified in HeCo cortices (Fig 1). Extremely important neuronal progenitors during corticogenesis are radial glial (RG) cells, which give rise to other subtypes of progenitors and post-mitotic neurons, and serve as guides for migrating neurons (Fig 1)10. Primary abnormalities in RG cells may hence represent an overlooked cause of heterotopia in HeCo mice and patients with EML1 mutations.
General aim : To assess Eml1’s function and to explore how its perturbation leads to cell detachment, ectopic progenitors, neuronal migration abnormalities and heterotopia.
Specific aims : We plan: (a) to study Eml1’s function with respect to microtubules (MTs), and to search for other protein partners, which may give clues about its biological role; (b) to investigate the role of Eml1 during neuronal cell division and migration, and corresponding phenotypes in mouse brains lacking Eml1.
Fig 1. (a,b) Heterotopia and cell accumulation in the dorso–medial regions of developing HeCo cortex. Nissl stained brain section from a HeCo mouse showing bilateral bands of subcortical heterotopic neurons (# b) compared to a wild–type section (a). (c) Schema of developing cortical wall. RG cells (red) have their soma in the VZ and long processes extending up to the pial surface. Other progenitors are shown in blue and cell division in green. (d) Electroporation of BLBP–Eml1–IRES–EGFP in HeCo reduces the number of BLBP–EGFP+ progenitors in the SVZ/IZ). (e) Vertical and oblique cleavage planes during division are shown. HeCo mouse brains show an excess of oblique cleavage planes which is likely to lead to asymmetric inheritance of the apical cell membrane and to cell leaving the VZ.
Project 2 : Characterizing neurodevelopmental roles of Caspr2
Heterozygous copy-number variations, missense variants and homozygous defects in CNTNAP2 have repeatedly been reported as susceptibility factors in developmental language delay, autism spectrum disorders, Gilles de la Tourette syndrome, epilepsy, CDFE, schizophrenia, and severe intellectual disability5,6,12-15. Analyses of KO mice (Cntnap2-/-) revealed impaired migration of cortical projection neurons and a reduced number of GABAergic interneurons, associated with asynchronous cortical activity and epileptic seizures16. Thus Caspr2 plays a critical role in neuronal migration. Furthermore, Caspr2 is enriched at the axon initial segments (AIS) of pyramidal cells from human temporal cortex (Fig 2), which act as sites for action potential initiation and are master integrators of synaptic events regulating excitability17,18. In the mammalian cerebral cortex, AIS are contacted by the axons of Chandelier type parvalbumin-positive GABAergic interneurons (Ch). Thus Caspr2 may be involved in the development and/or function of synapses between Ch and pyramidal cell AIS, and the mutations identified in patients may lead to hyperexcitability and network disfunction.
General aim : To elucidate essential roles of Caspr2 in immature neurons taking advantage of the availability of Cntnap2 mutant mice.
Specific aims : We plan: (a) to characterize fundamental molecular and cellular functions of Caspr2 during neuronal migration and synaptogenesis using in vivo (in utero electroporation) and in vitro (primary neurons and brain slices cultures; immunochemistry and time-lapse videomicroscopy, biochemistry) approaches; and (b) to determine the pathological consequences of mutations identified in patients on these functions.
Fig. 2. (A) AIS molecular complexes composed of ion channels (Nav, KCNQ2-3, Kv1), cell adhesion molecules and cytoskeletal scaffolds (adapted from17) (B). Immunolocalization of Nav and Caspr2 (arrows) at the AIS of a pyramidal cell (PC) from the human temporal cortex. (C) Photomicrographs showing the AIS of a PC immuno stained for Nav (Na+ Ch), Kv1.2 subunits and the GABA transporter GAT-1. (B, C, adapted from19).
Project 3 : Characterizing the origins, features and consequences of hyperexcitability in Dcx knockout (KO) cells
All type I lissencephaly gene mouse models show hippocampal heterotopia and more widespread interneuron defects as common features (20-24 and unpublished). In Dcx KO mice, CA3 hippocampal pyramidal cells are abnormally positioned (Fig 3) and this is associated with altered dendritic form, mossy fiber pruning defects, hyperexcitability and spontaneous epilepsy25,26. Our hypothesis is that aberrant position, connectivity and intrinsic defects of Dcx KO cells contribute to hyperexcitability. The molecular and cellular mechanisms leading to this phenotype and more generally behavioural and neuropsychiatric deficits, still not clear in Dcx KO mice which represent a well-characterized tool to further study this.
General aim: Fine characterization of the position and connectivity of heterotopic cells, links to epilepsy and behavioral deficits.
Specific aims: We plan to study (a) the emergence of Dcx KO neuron connectivity defects; (b) intrinsic factors affecting abnormal function; and (c) the cellular bases of behavioural abnormalities; (d) strategies during development for reducing hyperexcitability of abnormal Dcx KO cells.
Fig 3. Hippocampal heterotopia in Dcx KO mice. Two CA3 pyramidal cell layers (internal and external) are observed and hyperexcitability is associated with both layers26. Fig 3. Hétérotopie hippocampique chez les souris Dcx KO. Deux couches (interne et externe) de cellules pyramidales CA3 sont observées et l’hyperexcitabilité est associée aux deux couches26.
1 Guerrini, R., Dobyns, W.B., & Barkovich, A.J. (2008) Trends Neurosci 31, 154-162.
2 Ben-Ari, Y. (2008) Trends Neurosci 31, 626-636.
3 Sloviter, R.S., Kudrimoti, H.S., Laxer, K.D. et al. (2004) Epilepsy Res 59, 123-153.
4 Kamiya, A., Kubo, K., Tomoda, T. et al. (2005) Nat Cell Biol 7, 1167-1178.
5 Strauss, K.A., Puffenberger, E.G., Huentelman, M.J. et al. (2006) N Engl J Med 354, 1370-1377.
6 Penagarikano, O. & Geschwind, D.H. (2012) Trends Mol Med 18, 156-163.
7 Susuki, K. & Rasband, M.N. (2008) Curr Opin Cell Biol 20, 616-623.
8 des Portes, V., Pinard, J.M., Billuart, P. et al. (1998) Cell 92, 51-61.
9 Francis, F., Koulakoff, A., Boucher, D. et al. (1999) Neuron 23, 247-256.
10 Noctor, S.C., Martinez-Cerdeno, V., & Kriegstein, A.R. (2007) Novartis Found Symp 288, 59-73; discussion 73-58, 96-58.
11 Hansen, D.V., Lui, J.H. Parker, P.R.L., & Kriegstein, A.R. (2010) Nature 464, 554-561.
12 Verkerk, A.J., Mathews, C.A., Joosse, M. et al. (2003) Genomics 82, 1-9.
13 Friedman, J.I., Vrijenhoek, T., Markx, S. et al. (2008) Mol Psychiatry 13, 261-266.
14 Zweier, C., de Jong, E.K., Zweier, M. et al. (2009) Am J Hum Genet 85, 655-666.
15 Gregor, A., Albrecht, B., Bader, I. et al. (2011) BMC Med Genet 12, 106.
16 Penagarikano, O., Abrahams, B.S., Herman, E.I. et al. (2011) Cell 147, 235-246.
17 Rasband, M.N. (2010) Nat Rev Neurosci 11, 552-562.
18 Kole, M.H. & Stuart, G.J. (2012) Neuron 73, 235-247.
19 Inda, M.C., DeFelipe, J., & Munoz, A. (2006) Proc Natl Acad Sci U S A 103, 2920-2925.
20 Corbo, J.C., Deuel, T.A., Long, J.M. et al. (2002) J Neurosci 22, 7548-7557.
21 Kappeler, C., Saillour, Y., Baudoin, J.P. et al. (2006) Hum Mol Genet 15, 1387-1400.
22 Fleck, M.W., Hirotsune, S., Gambello, M.J. et al. (2000) J Neurosci 20, 2439-2450.
23 Nasrallah, I.M., McManus, M.F., Pancoast, M.M. et al. (2006) J Comp Neurol 496, 847-858.
24 Keays, D.A., Tian, G., Poirier, K. et al. (2007) Cell 128, 45-57.
25 Nosten-Bertrand, M., Kappeler, C., Dinocourt, C. et al. (2008) PLoS One 3, e2473.
26 Bazelot, M., Simonnet, J., Dinocourt, C. et al. (2012) Eur J Neurosci 35, 244-256.
- Fiona Francis DR1 CNRS (PhD, HDR)
- Laurence Goutebroze CR1 CNRS (PhD, HDR)
- Marika Nosten-Bertrand CR1 CNRS (PhD)
- Marta Garcia MC UPMC (PhD)
- Richard Belvindrah MC UPMC (PhD)
- Anne Houllier AI INSERM
- Gael Grannec TR UPMC
- Sara Bizzotto PhD student
- Ana Uzquiano-Lopez PhD student
- Giorgia Canali PhD student
- Delfina Romero, post-doc
- N. Bahi-Buisson Hôpital Necker Paris France
- J Chelly Institut Cochin Paris France
- F Artiguenave CNG Evry France
- A Depaulis Institut Neurosciences (GIN) Grenoble France
- A Houdusse Institut Curie Paris France
- R Miles / J-C Poncer CR-ICM / IFM Paris France
- R Olaso CNG Evry France
- C. Legay Paris 5 Paris France
- V Borrell Institute Neuroscience Alicante Spain
- A Croquelois, E Welker
- C Lebrand DBCM Lausanne Switzerland
- D Keays IMP Vienna Austria
- C Moores Birkbeck College London UK
- R Bayliss University of Leicester Leicester UK
- C Depienne ICM Paris France
- C Faivre-Sarrailh CRN2M Marseille France
- B Dargent CRN2M Marseille France
ANR, EU-DESIRE project. Fondation Bettencourt Schueller, Partenariat Hubert Curien (al Maqdisi), Fondation Orange, Labex Bio-Psy.
Most Recent Publications
Oegema R, McGillivray G, Leventer R, Le Moing AG, Bahi-Buisson N, Barnicoat A, Mandelstam S, Francis D, Francis F, Mancini GMS, Savelberg S, van Haaften G, Mankad K, Lequin MH.
Am J Med Genet C Semin Med Genet. 2019 Dec;181(4):627-637.
Penisson M, Ladewig J, Belvindrah R, Francis F.
Front Cell Neurosci. 2019 Aug 20;13:381.
Uzquiano A, Cifuentes-Diaz C, Jabali A, Romero DM, Houllier A, Dingli F, Maillard C, Boland A, Deleuze JF, Loew D, Mancini GMS, Bahi-Buisson N, Ladewig J2, Francis F.
Cell Rep. 2019 Aug 6;28(6):1596-1611.e10.
Collins SC, Uzquiano A, Selloum M, Wendling O, Gaborit M, Osipenko M, Birling MC, Yalcin B, Francis F.
J Anat. 2019 Sep;235(3):637-650.
Bonetto G, Hivert B, Goutebroze L, Karagogeos D, Crépel V, Faivre-Sarrailh C.
Front Cell Neurosci. 2019 May 16;13:222.
Uzquiano A, Francis F.
Brain. 2019 Apr 1;142(4):834-838.
Canali G, Goutebroze L.
J Exp Neurosci. 2018 Nov 9;12:1179069518809666.
Atherton J, Stouffer M, Francis F, Moores CA.
Acta Crystallogr D Struct Biol. 2018 Jun 1;74(Pt 6):572-584.
Canali G, Garcia M, Hivert B, Pinatel D, Goullancourt A, Oguievetskaia K, Saint-Martin M, Girault JA, Faivre-Sarrailh C, Goutebroze L.
Hum Mol Genet. 2018 Jun 1;27(11):1941-1954.
Uzquiano A, Gladwyn-Ng I, Nguyen L, Reiner O, Götz M, Matsuzaki F, Francis F.
J Neurochem. 2018 Sep;146(5):500-525.