Single-cell genotyping and transcriptomic proling in focal cortical dysplasia

Focal cortical dysplasia type II (FCDII) is a cortical malformation causing refractory epilepsy. FCDII arises from developmental somatic mutations in mTOR pathway genes, leading to focal cortical dyslamination and abnormal cytomegalic cells. Which cell types carry pathogenic mutations and how they affect cell-type-specific transcriptional programs remains unknown. To address this question, here we combined single-nucleus genotyping and transcriptomics in morphologically-identified cells using surgical cortical samples from genetically-characterized FCDII patients. Mutations were predominantly detected in glutamatergic neurons and astrocytes and only a small fraction of mutated cells exhibited cytomegalic features, revealing incomplete penetrance of FCDII-causing mutations. Moreover, we identified cell-type-specific transcriptional dysregulations in both mutated and non-mutated FCDII cells, including synapse and neurodevelopment-related pathways, that may account for epilepsy, and dysregulation of mitochondrial metabolism pathways in cytomegalic cells. Together, these findings reveal cell-autonomous and non-cell-autonomous mechanisms at play in FCDII, towards the development of precision therapies for this disorder

Mécanismes moléculaires orchestrant le guidage des axones commissuraux

Commissural neurons ensure the coordination of motor and somatosensory information between halves of the central nervous system. In the caudal part of the CNS, commissural axons, first grow toward the ventral midline, the floor plate, to cross it and reach their final target. The cellular and molecular mechanisms controlling midline crossing have been extensively studied. Ram—n y Cajal, in his neurotropic theory, suggested that floor plate cells could release diffusible factors chemo-attracting commissural axons to the ventral midline. Netrin-1, a protein discovered more than 2 decades ago, is a secreted protein expressed both by floor plate cells and ventricular zone progenitors and with long-range chemoattractive activity in vitro. Today, Netrin-1 is widely accepted as the textbook example of long-range chemoattractive guidance cue. However, our results, challenge this model by proposing a short-range mechanism of action for Netrin-1 during commissural axon guidance. Indeed, we determined that floor plate-derived netrin-1 is dispensable for commissural axon guidance. Instead, ventricular zone-derived netrin-1 is necessary and sufficient to promote the dorso-ventral extension of hindbrain commissural axons and midline crossing. We also confirmed that ventricular zone progenitors are the main Netrin-1 source for ventrally migrating precerebellar neurons. In addition, we observe that in absence of ventricular zone-derived netrin-1, commissural axons and precerebellar neurons cell bodies invade several cranial nerves. This appears to be a cell- autonomous and Dcc-dependent process. This mechanism is not conserved in the spinal cord, where both netrin-1 sources act synergistically to ensure commissural axon guidance and midline crossing. Commissural neurons are diverse and found throughout the nervous system. To analyse the molecular diversity of hindbrain and spinal cord commissural neurons, we used approaches combining mouse genetics and transcriptomics. We are currently working on some novel transcription factors that might play a role in the development of hindbrain and spinal cord commissural neurons.Les neurones commissuraux assurent la coordination des informations motrices et somatosensorielles entre les deux moitiés du système nerveux central. Dans la partie caudale du SNC, les axones commissuraux migrent d'abord vers la ligne médiane ventrale, la plaque du plancher, pour la traverser et puis atteindre leur cible finale. Les mécanismes cellulaires et moléculaires qui contrôlent le croisement de la ligne médiane ont fait l'objet d'Etudes approfondies. Ramón y Cajal, dans sa théorie neurotrophique, a suggéré que les cellules de la plaque de plancher pourraient libérer des facteurs diffusibles qui attirent les axones commissuraux vers la ligne médiane ventrale. La nétrine-1, une protéine découverte il y a plus de deux décennies, est une protéine secrétée exprimée à la fois par les cellules de la plaque de plancher et les progéniteurs de la zone ventriculaire et possède une activité chimioattractive à longue distance in vitro. Aujourd'hui, la nétrine-1 est largement acceptée comme l'exemple parfait de la chimioattraction à longue distance. Cependant, nos résultats remettent en question ce modèle, proposant un mécanisme d'action à courte portée de nétrine-1 pendant le guidage des axones commissuraux. En effet, nous avons déterminé que la nÉtrine-1 dérivée de la plaque du plancher n'est pas nécessaire pour le guidage des axones commissuraux. C’est la nétrine-1 dérivée de la zone ventriculaire qui est nécessaire et suffisante pour induire la migration dorso-ventrale des axones commissuraux du cerveau postérieur et leur croisement de la ligne médiane. Nous avons Également confirmé que les progéniteurs de la zone ventriculaire sont la principale source de Netrin-1 pour la migration ventrale des neurones précérébelleux. De plus, nous observons qu'en l'absence de nétrine-1 dérivée de la zone ventriculaire, les axones commissuraux et les neurones précérébelleux envahissent plusieurs nerfs crâniens. Il s’agit d'un procès autonome et dépendant de Dcc. A différence du tronc cérébral, dans la moelle épinière, les deux sources de nétrine-1 agissent en synergie pour assurer le guidage des axones commissuraux et le croisement des lignes médianes. Les neurones commissuraux sont une population diverse distribuée tout au long du système nerveux. Pour analyser la diversité moléculaire des neurones commissuraux du cerveau postérieur et de la moelle épinière, nous avons utilisé des approches combinant la génétique de la souris et la transcriptomique. Nous travaillons actuellement sur de nouveaux facteurs de transcription qui pourraient jouer un rôle dans le développement des neurones commissuraux du tronc et de la moelle épinière

Synergistic Activity of Floor-Plate- and Ventricular-Zone-Derived Netrin-1 in Spinal Cord Commissural Axon Guidance

International audienceIn vertebrates, commissural axons extend ventrally toward the floor plate in the spinal cord and hindbrain. Netrin-1, secreted by floor plate cells, was proposed to attract commissural axons at a distance. However, recent genetic studies in mice have shown that netrin-1 is also produced by ventricular zone (VZ) progenitors and that in the hindbrain, it represents the main source of netrin-1 for commissural axons. Here, we show that genetically deleting netrin-1 either from the VZ or the floor plate does not prevent midline crossing in the spinal cord, although axon pathfinding and fasciculation are perturbed. Strikingly, the VZ and floor plate act synergistically, as the simultaneous ablation of netrin-1 from these two sources severely impedes crossing. These results suggest that floor-plate-derived netrin-1 has a distinct impact on commissural axons in the spinal cord and hindbrain

Uncoupling axon guidance and neuronal migration in Robo3-deficient inferior olivary neurons

Inferior olivary (IO) neurons are born in the dorsal hindbrain and migrate tangentially toward the ventral midline. During their dorsoventral migration, IO neurons extend long leading processes that cross the midline, transform into axons, and project into the contralateral cerebellum. In absence of the axon guidance receptor Robo3, IO axons fail to cross the midline and project to the ipsilateral cerebellum. Remarkably, the IO cell bodies still reach the midline where they form a nucleus of abnormal cytoarchitecture. The mechanisms underlying the migration of Robo3-deficient IO neurons are unknown. Here, we used three-dimensional imaging and transgenic mice to label subsets of IO neurons and study their migratory behavior in Robo3 knockout. We show that IO migration is delayed in absence of Robo3. Strikingly, Robo3-deficient IO neurons progress toward the midline in a direction opposite to their axons. This occurs through a change of polarity and the generation of a second leading process at the rear of the cell. These results suggest that Robo3 receptor controls the establishment of neuronal polarity and the coupling of axonogenesis and cell body migration in IO neurons.Agence Nationale de la Recherche 10.13039/501100001665 Agence Nationale de la Recherche ANR-18-IAHU-01 Generalitat Valenciana 10.13039/501100003359 Generalitat Valenciana SEJIGENT 2021/026 Agencia Estatal de Investigación 10.13039/501100011033 Agencia Estatal de Investigación RYC2018-023868-I Severo Ochoa SEV-2017-0723 European Research Council ERC-2020-StG-950013.Peer reviewe

Commissural neurons transgress the cns/pns boundary in absence of ventricular zone-derived netrin 1

During the development of the central nervous system (CNS), only motor axons project into peripheral nerves. Little is known about the cellular and molecular mechanisms that control the development of a boundary at the CNS surface and prevent CNS neuron emigration from the neural tube. It has previously been shown that a subset of spinal cord commissural axons abnormally invades sensory nerves in Ntn1 hypomorphic embryos and Dcc knockouts. However, whether netrin 1 also plays a similar role in the brain is unknown. In the hindbrain, precerebellar neurons migrate tangentially under the pial surface, and their ventral migration is guided by netrin 1. Here, we show that pontine neurons and inferior olivary neurons, two types of precerebellar neurons, are not confined to the CNS in Ntn1 and Dcc mutant mice, but that they invade the trigeminal, auditory and vagus nerves. Using a Ntn1 conditional knockout, we show that netrin 1, which is released at the pial surface by ventricular zone progenitors is responsible for the CNS confinement of precerebellar neurons. We propose, that netrin 1 distribution sculpts the CNS boundary by keeping CNS neurons in netrin 1-rich domains.This work was supported by grants from the Agence Nationale de la Recherche (ANR-14-CE13-0004-01) (to A.C.). It was performed in the frame of the Labex Lifesenses (ANR-10-LABX-65) supported by French state funds managed by the Agence Nationale de la Recherche within the Investissements d'Avenir programme under ANR-11-IDEX-0004-02 (to A.C.).Peer reviewe

Loss of floor plate Netrin-1 impairs midline crossing of corticospinal axons and leads to mirror movements

International audienceIn humans, execution of unimanual movements requires lateralized activation of the primary motor cortex,which then transmits the motor command to the contralateral hand through the crossed corticospinal tract(CST). Mutations inNTN1alter motor control lateralization, leading to congenital mirror movements. Toaddress the role of midline Netrin-1 on CST development and subsequent motor control, we analyze themorphological and functional consequences of floor plate Netrin-1 depletion in conditional knockout mice.We show that depletion of floor plate Netrin-1 in the brainstem critically disrupts CST midline crossing,whereas the other commissural systems are preserved. The only associated defect is an abnormal entryof CST axons within the inferior olive. Alteration of CST midline crossing results in functional ipsilateral pro-jections and is associated with abnormal symmetric movements. Our study reveals the role of Netrin-1 in CSTdevelopment and describes a mouse model recapitulating the characteristics of human congenital mirrormovements

Construction and reconstruction of brain circuits: normal and pathological axon guidance

Perception of our environment entirely depends on the close interaction between the central and peripheral nervous system. In order to communicate each other, both systems must develop in parallel and in coordination. During development, axonal projections from the CNS as well as the PNS must extend over large distances to reach their appropriate target cells. To do so, they read and follow a series of axon guidance molecules. Interestingly, while these molecules play critical roles in guiding developing axons, they have also been shown to be critical in other major neurodevelopmental processes, such as the migration of cortical progenitors. Currently, a major hurdle for brain repair after injury or neurodegeneration is the absence of axonal regeneration in the mammalian CNS. By contrasts, PNS axons can regenerate. Many hypotheses have been put forward to explain this paradox but recent studies suggest that hacking neurodevelopmental mechanisms may be the key to promote CNS regeneration. Here we provide a seminar report written by trainees attending the second Flagship school held in Alpbach, Austria in September 2018 organized by the International Society for Neurochemistry (ISN) together with the Journal of Neurochemistry (JCN). This advanced school has brought together leaders in the fields of neurodevelopment and regeneration in order to discuss major keystones and future challenges in these respective fields

Floor-plate-derived netrin-1 is dispensable for commissural axon guidance

International audienceNetrin-1 is an evolutionarily conserved, secreted extracellular matrix protein involved in axon guidance at the central nervous system midline. Netrin-1 is expressed by cells localized at the central nervous system midline, such as those of the floor plate in vertebrate embryos. Growth cone turning assays and three-dimensional gel diffusion assays have shown that netrin-1 can attract commissural axons. Loss-of-function experiments further demonstrated that commissural axon extension to the midline is severely impaired in the absence of netrin-1 (refs 3, 7, 8, 9). Together, these data have long supported a model in which commissural axons are attracted by a netrin-1 gradient diffusing from the midline. Here we selectively ablate netrin-1 expression in floor-plate cells using a Ntn1 conditional knockout mouse line. We find that hindbrain and spinal cord commissural axons develop normally in the absence of floor-plate-derived netrin-1. Furthermore, we show that netrin-1 is highly expressed by cells in the ventricular zone, which can release netrin-1 at the pial surface where it binds to commissural axons. Notably, Ntn1 deletion from the ventricular zone phenocopies commissural axon guidance defects previously described in Ntn1-knockout mice. These results show that the classical view that attraction of commissural axons is mediated by a gradient of floor-plate-derived netrin-1 is inaccurate and that netrin-1 primarily acts locally by promoting growth cone adhesio