The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies

Commissural axon guidance : mechanism underlying the gain of sensitivity the midline signal Semaphorin 3B

Les mouvements locomoteurs rythmiques nécessitent l’intervention de circuits neuronaux qui coordonnent l’activité motrice des deux parties du corps. Ces circuits sont formés majoritairement par les projections des interneurones commissuraux de la moelle épinière. Des facteurs de guidage comme la Nétrine, les Slits jouent un rôle fondamental dans la mise en place de ces projections. Une étude a également montré qu’une signalisation impliquant le récepteur Neuropiline2 (Nrp2) des signaux Sémaphorines de la classe 3 (Sema3), participe au guidage de ces projections et cela uniquement après la traversée de la ligne médiane (Zou et al. 2000). Ma thèse porte sur l’étude fonctionnelle d’un ligand de la Nrp2, la Sema3B dans le développement de ce système de projections. J’ai analysé une souris invalidée pour Sema3B et observé de nombreuses erreurs de trajectoires après la traversée de la ligne médiane. Je me suis ensuite intéressée aux mécanismes sous-jacents au gain de réponse : par une approche pharmacologique et biochimique j’ai pu montrer que le signal de la plaque du plancher inhibe une activité de dégradation dépendante de la calpaine1. L’inhibition de cette voie conduit à la stabilisation d’un co-récepteur de la Nrp2, la Plexine A1 dont l’expression est très faible dans les axones n’ayant pas encore traversé la ligne médiane. Cette régulation permet alors l’assemblage d’un complexe récepteur fonctionnel de Sema3B, comprenant cette Plexine associée à la Nrp2 au niveau des cônes de croissance. J’ai identifié la molécule d’adhérence NrCAM, et le facteur neurotrophique GDNF comme étant les facteurs de la plaque du plancher déclencheurs de la réponseRhythmic locomotor movements require neuronal circuits ensuring left-right coordination. Spinal commissural projections participate to left-right coordination of limb movements by mediating reciprocal inhibition in synchrony. Extensive research of the mechanisms governing the formation of commissural pathways focused on dorsally-located spinal commissural neurons, establishing a fundamental role for multiple guidance cues derived for the midline and surrounding tissues, including Netrins, Slits and various morphogens. Semaphorin (Sema2)/Neuropilin-2 (Nrp2) signaling has been proposed to contribute to the guidance of commissural projections in the spinal cord at the post- but not pre-crossing stage (Zou et al, 2000). My PhD project aimed at analyzing the role of a Nrp2 ligand, Sema3B, in the guidance of spinal commissural projections, whose expression is dynamic and restricted to some territories, including the floor plate in which axons cross the midline. Analysis of Sema3B null mice showed that the loss of Sema3B induces a range of guidance defects of post-crossing commissural pathways. I investigated the underlying mechanisms and found that the floor plate signal induces through blockade of a calpain 1-dependant pathway the stabilization of the Nrp2 co-receptor Plexin-A1, and enable the assembly of Nrp2/Plexin-A1 sub-units into functional complexes for Sema3B in post-crossing commissural growth cones. I identified the cell adhesion molecule NrCAM and the neurotrophic factor GDNF as being the floor-platederived signals triggering the gain of respons

The RSK2-RPS6 axis promotes axonal regeneration in the peripheral and central nervous systems.

Unlike immature neurons and the ones from the peripheral nervous system (PNS), mature neurons from the central nervous system (CNS) cannot regenerate after injury. In the past 15 years, tremendous progress has been made to identify molecules and pathways necessary for neuroprotection and/or axon regeneration after CNS injury. In most regenerative models, phosphorylated ribosomal protein S6 (p-RPS6) is up-regulated in neurons, which is often associated with an activation of the mTOR (mammalian target of rapamycin) pathway. However, the exact contribution of posttranslational modifications of this ribosomal protein in CNS regeneration remains elusive. In this study, we demonstrate that RPS6 phosphorylation is essential for PNS and CNS regeneration in mice. We show that this phosphorylation is induced during the preconditioning effect in dorsal root ganglion (DRG) neurons and that it is controlled by the p90S6 kinase RSK2. Our results reveal that RSK2 controls the preconditioning effect and that the RSK2-RPS6 axis is key for this process, as well as for PNS regeneration. Finally, we demonstrate that RSK2 promotes CNS regeneration in the dorsal column, spinal cord synaptic plasticity, and target innervation leading to functional recovery. Our data establish the critical role of RPS6 phosphorylation controlled by RSK2 in CNS regeneration and give new insights into the mechanisms related to axon growth and circuit formation after traumatic lesion

Guidance landscapes unveiled by quantitative proteomics to control reinnervation in adult visual system

Abstract In the injured adult central nervous system (CNS), activation of pro-growth molecular pathways in neurons leads to long-distance regeneration. However, most regenerative fibers display guidance defects, which prevent reinnervation and functional recovery. Therefore, the molecular characterization of the proper target regions of regenerative axons is essential to uncover the modalities of adult reinnervation. In this study, we use mass spectrometry (MS)-based quantitative proteomics to address the proteomes of major nuclei of the adult visual system. These analyses reveal that guidance-associated molecules are expressed in adult visual targets. Moreover, we show that bilateral optic nerve injury modulates the expression of specific proteins. In contrast, the expression of guidance molecules remains steady. Finally, we show that regenerative axons are able to respond to guidance cues ex vivo, suggesting that these molecules possibly interfere with brain target reinnervation in adult. Using a long-distance regeneration model, we further demonstrate that the silencing of specific guidance signaling leads to rerouting of regenerative axons in vivo. Altogether, our results suggest ways to modulate axon guidance of regenerative neurons to achieve circuit repair in adult

Overexpression of phosphomimic RPS6^235D-236D induces the preconditioning effect in naive DRG and has a modest effect on sciatic nerve regeneration in WT mice.

(A, B) Western blot of ribosome purification showing a good integration of phosphomimetics RPS6 constructs (A) RPS6 240D-244D-247D or (B) RPS6235D-236D in ribosome of N2A cells. (C) Representative microphotographs of naive cultures of mature DRG neurons from WT mice 21 days after intrathecal injection of AAV8-Ctrl; AAV8-RPS6240D-244D-247D or AAV8-RPS6235D-236D showing that only overexpression of AAV8-RPS6235D-236D induces the preconditioning effect. Scale bar: 250 μm. (D, E) Graphs showing the quantification of C. (D) Longest neurite length per neuron 16 h after plating (mean ± SEM, one-way ANOVA, 3 independent DRG cultures, approximately 50 cells counted per condition per culture). (E) Distance between 2 ramifications in longest neurite (mean ± SEM; one-way ANOVA, 3 independent DRG cultures, approximately 50 cells analyzed per condition per culture). (F) Percentage of neurons growing a neurite 16 h after plating (mean ± SEM, two-way ANOVA, 10 random microscopy fields were quantified per condition). (G) Representative confocal images of sciatic nerve sections 3 days post-injury from WT mice injected intrathecally with AAV8-PLAP (control), AAV8-RPS6240D-244D-247D, or AAV8-RPS6235D-236D. Regenerating axons are labeled with anti-SCG10 antibody (white). The red dashed line indicates the injury site. Scale bar: 500 μm. (H) Quantification of regenerative axons from G (mean ± SEM, two-way ANOVA, at least 5 animals per group). (I) Regeneration index at 3 dpi (mean ± SEM, one-way ANOVA, at least 5 animals per group). (J) Representative microphotographs of mature naive DRG neurons cultures from RPS6p-/p- mice, 21 days after intrathecal injection of AAV8-Ctrl; AAV8- RPS6240D-244D-247D or AAV8-RPS6235D-236D showing that only overexpression of phosphomimic AAV8-RPS6235D-236D induces the preconditioning effect. Scale bar: 250 μm. (K–M) Graphs showing the quantification of J. (K) Longest neurite length per neuron 16 h after plating (mean ± SEM, one-way ANOVA, 3 independent DRG cultures, approximately 50 cells counted per condition per culture). (L) Distance between 2 ramifications in longest neurite (mean ± SEM, one-way ANOVA, 3 independent DRG cultures, approximately 50 cells analyzed per condition per culture). (M) Percentage of neurons growing a neurite 16 h after plating (mean ± SEM, two-way ANOVA, 10 random microscopy fields quantified per condition). ⁎⁎⁎p p p S1 Data and S1 Raw Images). (TIF)</p

The underlying data for Figs 1D, 1E, 1F, 1H, 2C, 2D, 2E, 2G, 2H, 2J, 2K, 2L, 2N, 2O, 3B, 3C, 3D, 3E, 3G, 3H, 4C, 4E, 4H, 4J, 4L, 5B, 5C, 5D, 5F, 5G, 5I, 5J, 5K, 5M, 5N, 5P, 5Q, 5R, 5T, 5U, 6B, 6C, 6D, 6F, 6G, 7D, 7F, 7G, 7H, 7J, 7L, 7N, 7Q, 7R and S1C, S1D, S2D, S1E, S2G, S2H, S2I, S3D, S3E, S3F, S3H, S3I, S3K, S3L, S3M, S4C, S4D, S6B, S6C, S6D, S7C, S7D, S7E, S7G, S7H, S7L, S7M, S8D, S8E, S8F, S8F and S8G.

The underlying data for Figs 1D, 1E, 1F, 1H, 2C, 2D, 2E, 2G, 2H, 2J, 2K, 2L, 2N, 2O, 3B, 3C, 3D, 3E, 3G, 3H, 4C, 4E, 4H, 4J, 4L, 5B, 5C, 5D, 5F, 5G, 5I, 5J, 5K, 5M, 5N, 5P, 5Q, 5R, 5T, 5U, 6B, 6C, 6D, 6F, 6G, 7D, 7F, 7G, 7H, 7J, 7L, 7N, 7Q, 7R and S1C, S1D, S2D, S1E, S2G, S2H, S2I, S3D, S3E, S3F, S3H, S3I, S3K, S3L, S3M, S4C, S4D, S6B, S6C, S6D, S7C, S7D, S7E, S7G, S7H, S7L, S7M, S8D, S8E, S8F, S8F and S8G.</p

Characterization of ShRNA-RSK2.

(A) Schematic of the plasmid constructs used to overexpress RSK1-VSVG, RSK2-Flag, RSK3-V5, RSK4-His, PLAP, or shRNA (sh-Scrambled or sh-RSK2). (B–E) Western blot showing shRNA-RSK2 specificity in N2A cells 96 h after co-transfection (mean ± SEM, one-sample t test, N = 3 transfections per group). (F) Representative microphotographs of DRG sections stained with anti-RFP (in magenta) and anti-Tuj 1 (in gray) antibodies, 21 days after intrathecal injection of AAV8-shCtrl (that co expressed the RFP). Scale bar: 25 μm. (G) Quantification of H (mean ± SEM, 3 animals, 5 DRG sections counted per animal). ⁎⁎⁎p p p S1 Data and S1 Raw Images). (TIF)</p

List of cDNAs used for in situ hybridization.

Unlike immature neurons and the ones from the peripheral nervous system (PNS), mature neurons from the central nervous system (CNS) cannot regenerate after injury. In the past 15 years, tremendous progress has been made to identify molecules and pathways necessary for neuroprotection and/or axon regeneration after CNS injury. In most regenerative models, phosphorylated ribosomal protein S6 (p-RPS6) is up-regulated in neurons, which is often associated with an activation of the mTOR (mammalian target of rapamycin) pathway. However, the exact contribution of posttranslational modifications of this ribosomal protein in CNS regeneration remains elusive. In this study, we demonstrate that RPS6 phosphorylation is essential for PNS and CNS regeneration in mice. We show that this phosphorylation is induced during the preconditioning effect in dorsal root ganglion (DRG) neurons and that it is controlled by the p90S6 kinase RSK2. Our results reveal that RSK2 controls the preconditioning effect and that the RSK2-RPS6 axis is key for this process, as well as for PNS regeneration. Finally, we demonstrate that RSK2 promotes CNS regeneration in the dorsal column, spinal cord synaptic plasticity, and target innervation leading to functional recovery. Our data establish the critical role of RPS6 phosphorylation controlled by RSK2 in CNS regeneration and give new insights into the mechanisms related to axon growth and circuit formation after traumatic lesion.</div