Tristan Bouschet

An intrinsic mechanism of corticogenesis from embryonic stem cells

The cerebral cortex develops through the coordinated generation of dozens of neuronal subtypes, but the mechanisms involved remain unclear. Here we show that mouse embryonic stem cells, cultured without any morphogen but in the presence of a sonic hedgehog inhibitor, recapitulate in vitro the major milestones of cortical development, leading to the sequential generation of a diverse repertoire of neurons that display most salient features of genuine cortical pyramidal neurons. When grafted into the cerebral cortex, these neurons develop patterns of axonal projections corresponding to a wide range of cortical layers, but also to highly specific cortical areas, in particular visual and limbic areas, thereby demonstrating that the identity of a cortical area can be specified without any influence from the brain. The discovery of intrinsic corticogenesis sheds new light on the mechanisms of neuronal specification, and opens new avenues for the modelling and treatment of brain diseases.

Receptor-activity-modifying proteins are required for forward trafficking of the calcium-sensing receptor to the plasma membrane

The calcium-sensing receptor (CaSR) is a class III G-protein-coupled receptor (GPCR) that responds to changes in extracellular calcium concentration and plays a crucial role in calcium homeostasis. The mechanisms controlling CaSR trafficking and surface expression are largely unknown. Using a CaSR tagged with the pH-sensitive GFP super-ecliptic pHluorin (SEP-CaSR), we show that delivery of the GPCR to the cell surface is dependent on receptor-activity-modifying proteins (RAMPs). We demonstrate that SEP-CaSRs are retained in the endoplasmic reticulum (ER) in COS7 cells that do not contain endogenous RAMPs whereas they are delivered to the plasma membrane in HEK 293 cells that do express RAMP1. Coexpression of RAMP1 or RAMP3, but not RAMP2, in COS7 cells was sufficient to target the CaSR to the cell surface. RAMP1 and RAMP3 colocalised and coimmunoprecipitated with the CaSR suggesting that these proteins associate within the cell. Our results indicate that RAMP expression promotes the forward trafficking of the GPCR from the ER to the Golgi apparatus and results in mature CaSR glycosylation, which is not observed in RAMP-deficient cells. Finally, silencing of RAMP1 in the endogenously expressing HEK293 cells using siRNA resulted in altered CaSR traffic. Taken together, our results show that the association with RAMPs is necessary and sufficient to transfer the immature CaSR retained in the ER towards the Golgi where it becomes fully glycosylated prior to delivery to the plasma membrane and demonstrate a role for RAMPs in the trafficking of a class III GPCR.

Generation of cortical neurons from mouse embryonic stem cells

Embryonic stem cells (ESCs) constitute a tool of great potential in neurobiology, enabling the directed differentiation of specific neural cell types. We have shown recently that neurons of the cerebral cortex can be generated from mouse ESCs cultured in a chemically defined medium that contains no morphogen, but in the presence of the sonic hedgehog inhibitor cyclopamine. Corticogenesis from ESCs recapitulates the most important steps of cortical development, leading to the generation of multipotent cortical progenitors that sequentially produce cortical pyramidal neurons displaying distinct layer-specific identities. The protocol provides a most reductionist cellular model to tackle the complex mechanisms of cortical development and function, thereby opening new perspectives for the modeling of cortical diseases and the design of novel neurological treatments, while offering an alternative to animal use. In this protocol, we describe a method by which millions of cortical neurons can be generated in 2–3 weeks, starting from a single frozen vial of ESCs.

BCL6 controls neurogenesis through Sirt1-dependent epigenetic repression of selective Notch targets

During neurogenesis, neural stem/progenitor cells (NPCs) undergo an irreversible fate transition to become neurons. The Notch pathway is important for this process, and repression of Notch-dependent Hes genes is essential for triggering differentiation. However, Notch signaling often remains active throughout neuronal differentiation, implying a change in the transcriptional responsiveness to Notch during the neurogenic transition. We identified Bcl6, an oncogene, as encoding a proneurogenic factor that is required for proper neurogenesis of the mouse cerebral cortex. BCL6 promoted the neurogenic conversion by switching the composition of Notch-dependent transcriptional complexes at the Hes5 promoter. BCL6 triggered exclusion of the co-activator Mastermind-like 1 and recruitment of the NAD+-dependent deacetylase Sirt1, which was required for BCL6-dependent neurogenesis. The resulting epigenetic silencing of Hes5 led to neuronal differentiation despite active Notch signaling. Our findings suggest a role for BCL6 in neurogenesis and uncover Notch-BCL6-Sirt1 interactions that may affect other aspects of physiology and disease.

The calcium-sensing receptor changes cell shape via a β-arrestin-1–ARNO–ARF6–ELMO protein network

G-protein-coupled receptors (GPCRs) transduce the binding of extracellular stimuli into intracellular signalling cascades that can lead to morphological changes. Here, we demonstrate that stimulation of the calcium-sensing receptor (CaSR), a GPCR that promotes chemotaxis by detecting increases in extracellular calcium, triggers plasma membrane (PM) ruffling via a pathway that involves β-arrestin 1, Arf nucleotide binding site opener (ARNO), ADP-ribosylating factor 6 (ARF6) and engulfment and cell motility protein (ELMO). Expression of dominant negative β-arrestin 1 or its knockdown with siRNA impaired the CaSR-induced PM ruffling response. Expression of a catalytically inactive ARNO also reduced CaSR-induced PM ruffling. Furthermore, β-arrestin 1 co-immunoprecipitated with the CaSR and ARNO under resting conditions. Agonist treatment did not markedly alter β-arrestin 1 binding to the CaSR or to ARNO but it did elicit the translocation and colocalisation of the CaSR, β-arrestin 1 and ARNO to membrane protrusions. Furthermore, ARF6 and ELMO, two proteins known to couple ARNO to the cytoskeleton, were required for CaSR-dependent morphological changes and translocated to the PM ruffles. These data suggest that cells ruffle upon CaSR stimulation via a mechanism that involves translocation of β-arrestin 1 pre-assembled with the CaSR or ARNO, and that ELMO plays an essential role in this CaSR-signalling-induced cytoskeletal reorganisation.

Progressive Myoclonic Epilepsy-Associated Gene KCTD7 is a Regulator of Potassium Conductance in Neurons

The potassium channel tetramerization domain-containing protein 7 (KCTD7) was named after the structural homology of its predicted N-terminal broad complex, tramtrack and bric à brac/poxvirus and zinc finger domain with the T1 domain of the Kv potassium channel, but its expression profile and cellular function are still largely unknown. We have recently reported a homozygous nonsense mutation of KCTD7 in patients with a novel form of autosomal recessive progressive myoclonic epilepsy. Here, we show that KCTD7 expression hyperpolarizes the cell membrane and reduces the excitability of transfected neurons in patch clamp experiments. We found the expression of KCTD7 in the hippocampal and Purkinje cells of the murine brain, an expression profile consistent with our patients’ phenotype. The effect on the plasma membrane resting potential is possibly mediated by Cullin-3, as we demonstrated direct molecular interaction of KCTD7 with Cullin-3 in co-immunoprecipitation assays. Our data link progressive myoclonic epilepsy to an inherited defect of the neuron plasma membrane’s resting potential in the brain.

Bidirectional regulation of kainate receptor surface expression in hippocampal neurons

Kainate receptors (KARs) are crucial for the regulation of both excitatory and inhibitory neurotransmission, but little is known regarding the mechanisms controlling KAR surface expression. We used super ecliptic pHluorin (SEP)-tagged KAR subunit GluR6a to investigate real-time changes in KAR surface expression in hippocampal neurons. Sindbis virus-expressed SEP-GluR6 subunits efficiently co-assembled with native KAR subunits to form heteromeric receptors. Diffuse surface-expressed dendritic SEP-GluR6 is rapidly internalized following either N-methyl-d-aspartate or kainate application. Sustained kainate or transient N-methyl-d-aspartate application resulted in a slow decrease of base-line surface KAR levels. Surprisingly, however, following the initial loss of surface receptors, a short kainate application caused a long lasting increase in surface-expressed KARs to levels significantly greater than those prior to the agonist challenge. These data suggest that after initial endocytosis, transient agonist activation evokes increased KAR exocytosis and reveal that KAR surface expression is bidirectionally regulated. This process may provide a mechanism for hippocampal neurons to differentially adapt their physiological responses to changes in synaptic activation and extrasynaptic glutamate concentration.

A systems-level approach to parental genomic imprinting: the imprinted gene network includes extracellular matrix genes and regulates cell cycle exit and differentiation

Genomic imprinting is an epigenetic mechanism that restrains the expression of ∼ 100 eutherian genes in a parent-of-origin-specific manner. The reason for this selective targeting of genes with seemingly disparate molecular functions is unclear. In the present work, we show that imprinted genes are coexpressed in a network that is regulated at the transition from proliferation to quiescence and differentiation during fibroblast cell cycle withdrawal, adipogenesis in vitro, and muscle regeneration in vivo. Imprinted gene regulation is not linked to alteration of DNA methylation or to perturbation of monoallelic, parent-of-origin-dependent expression. Overexpression and knockdown of imprinted gene expression alters the sensitivity of preadipocytes to contact inhibition and adipogenic differentiation. In silico and in cellulo experiments showed that the imprinted gene network includes biallelically expressed, nonimprinted genes. These control the extracellular matrix composition, cell adhesion, cell junction, and extracellular matrix-activated and growth factor-activated signaling. These observations show that imprinted genes share a common biological process that may account for their seemingly diverse roles in embryonic development, obesity, diabetes, muscle physiology, and neoplasm.

Regulation of calcium-sensing-receptor trafficking and cell-surface expression by GPCRs and RAMPs

The calcium-sensing (CaS) receptor is a G-protein-coupled receptor (GPCR) that is of fundamental importance for extracellular calcium signalling and calcium homeostasis. The CaS receptor detects changes in free, ionized extracellular calcium concentration and initiates pathways that constantly re-adjust levels of circulating calcium. In addition, the CaS receptor is involved in processes such as stem-cell homing and regulation of neuronal-process outgrowth. To perform these functions, the CaS receptor must be appropriately targeted to the plasma membrane so that its large N-terminal calcium-sensing domain is positioned in the extracellular environment to detect dynamic changes in ionic calcium concentration. Here, we provide an overview of the molecular determinants controlling CaS receptor forward traffic and highlight the roles of CaS receptor interactors such as receptor-activity-modifying proteins and subunits of other class C GPCRs in this process.

ICR Noncoding RNA Expression Controls Imprinting and DNA Replication at the Dlk1-Dio3 Domain

Imprinted genes play essential roles in development, and their allelic expression is mediated by imprinting control regions (ICRs). The Dlk1-Dio3 locus is among the few imprinted domains controlled by a paternally methylated ICR. The unmethylated maternal copy activates imprinted expression early in development through an unknown mechanism. We find that in mouse embryonic stem cells (ESCs) and in blastocysts, this function is linked to maternal, bidirectional expression of noncoding RNAs (ncRNAs) from the ICR. Disruption of ICR ncRNA expression in ESCs affected gene expression in cis, led to acquisition of aberrant histone and DNA methylation, delayed replication timing along the domain on the maternal chromosome, and changed its subnuclear localization. The epigenetic alterations persisted during differentiation and affected the neurogenic potential of the stem cells. Our data indicate that monoallelic expression at an ICR of enhancer RNA-like ncRNAs controls imprinted gene expression, epigenetic maintenance processes, and DNA replication in embryonic cells.