Regulation of Postsynaptic Function by the Dementia-Related ESCRT-III Subunit CHMP2B

The charged multivesicular body proteins (Chmp1–7) are an evolutionarily conserved family of cytosolic proteins that transiently assembles into helical polymers that change the curvature of cellular membrane domains. Mutations in human CHMP2B cause frontotemporal dementia, suggesting that this protein may normally control some neuron-specific process. Here, we examined the function, localization, and interactions of neuronal Chmp2b. The protein was highly expressed in mouse brain and could be readily detected in neuronal dendrites and spines. Depletion of endogenous Chmp2b reduced dendritic branching of cultured hippocampal neurons, decreased excitatory synapse density in vitro and in vivo, and abolished activity-induced spine enlargement and synaptic potentiation. To understand the synaptic effects of Chmp2b, we determined its ultrastructural distribution by quantitative immuno-electron microscopy and its biochemical interactions by coimmunoprecipitation and mass spectrometry. In the hippocampus in situ, a subset of neuronal Chmp2b was shown to concentrate beneath the perisynaptic membrane of dendritic spines. In synaptoneurosome lysates, Chmp2b was stably bound to a large complex containing other members of the Chmp family, as well as postsynaptic scaffolds. The supramolecular Chmp assembly detected here corresponds to a stable form of the endosomal sorting complex required for transport-III (ESCRT-III), a ubiquitous cytoplasmic protein complex known to play a central role in remodeling of lipid membranes. We conclude that Chmp2b-containing ESCRT-III complexes are also present at dendritic spines, where they regulate synaptic plasticity. We propose that synaptic ESCRT-III filaments may function as a novel element of the submembrane cytoskeleton of spines

AIMTOR, a BRET Biosensor for Live Recording of mTOR Activity in Cell Populations and Single Cells

International audienceMammalian target of rapamycin (mTOR) controls many crucial cellular functions, including protein synthesis, cell size, energy metabolism, lysosome and mitochondria biogenesis, and autophagy. Consequently, deregulation of mTOR signaling plays a role in numerous pathological conditions such as cancer, metabolic disorders and neurological diseases. Developing new tools to monitor mTOR spatiotemporal activation is crucial to better understand its roles in physiological and pathological conditions. However, the most widely used method to report mTOR activity relies on the quantification of specific mTOR-phosphorylated substrates by western blot. This approach requires cellular lysate preparation, which restricts the quantification to a single time point. Here, we present a simple protocol to study mTOR activity in living cells in real time using AIMTOR, an intramolecular BRET-based (bioluminescence resonance energy transfer) biosensor that we recently designed (Bouquier et al., 2020). We describe transfection of AIMTOR in the C2C12 cell line and procedures to monitor BRET in a cell population using a plate reader and in single cells by microscopy. Importantly, this protocol is transposable to any cell line and primary cells. In addition, several subcellular compartment-specific versions of AIMTOR have been developed, enabling compartmentalized assessment of mTOR activity. This protocol describes how to use the sensitive AIMTOR biosensor to investigate mTOR signaling dynamics in living cells

Fast and high resolution single-cell BRET imaging

Resonance Energy Transfer (RET)-based technologies are used to report protein-protein interactions in living cells. Among them, Bioluminescence-initiated RET (BRET) provides excellent sensitivity but the low light intensity intrinsic to the bioluminescent process hampers its use for the localization of protein complexes at the sub-cellular level. Herein we have characterized the methodological conditions required to reliably perform single-cell BRET imaging using an extremely bright luciferase, Nanoluciferase (Nluc). With this, we achieved an unprecedented performance in the field of protein-protein interaction imaging in terms of temporal and spatial resolution, duration of signal stability, signal sensitivity and dynamic range. As proof-of-principle, an Nluc-containing BRET-based sensor of ERK activity enabled the detection of subtle, transient and localized variations in ERK activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors. This development will improve our comprehension of both the spatio-temporal dynamics of protein-protein interactions and the activation patterns of specific signaling pathways

Gelatinase Biosensor Reports Cellular Remodeling During Epileptogenesis

International audienceEpileptogenesis is the gradual process responsible for converting a healthy brain into an epileptic brain. This process can be triggered by a wide range of factors, including brain injury or tumors, infections, and status epilepticus. Epileptogenesis results in aberrant synaptic plasticity, neuroinflammation and seizure-induced cell death. As Matrix Metalloproteinases (MMPs) play a crucial role in cellular plasticity by remodeling the extracellular matrix (ECM), gelatinases (MMP-2 and MMP-9) were recently highlighted as key players in epileptogenesis. In this work, we engineered a biosensor to report in situ gelatinase activity in a model of epileptogenesis. This biosensor encompasses a gelatinase-sensitive activatable cell penetrating peptide (ACPP) coupled to a TAMRA fluorophore, allowing fluorescence uptake in cells displaying endogenous gelatinase activities. In a preclinical mouse model of temporal lobe epilepsy (TLE), the intrahippocampal kainate injection, ACPPs revealed a localized distribution of gelatinase activities, refining temporal cellular changes during epileptogenesis. The activity was found particularly but not only in the ipsilateral hippocampus, starting from the CA1 area and spreading to dentate gyrus from the early stages throughout chronic epilepsy, notably in neurons and microglial cells. Thus, our work shows that ACPPs are suitable molecular imaging probes for detecting the spatiotemporal pattern of gelatinase activity during epileptogenesis, suggesting their possible use as vectors to target cellular reactive changes with treatment for epileptogenesis

mGlu1 receptor canonical signaling pathway contributes to the opening of the orphan GluD2 receptor

International audienc

Paroles d’expert.e.s autour de l’affaire Renvois relatifs à la Loi sur la tarification de la pollution causée par les gaz à effet de serre

Ce texte revient sur une affaire portée devant la Cour suprême du Canada au cours de l’année 2021 dans laquelle plusieurs provinces canadiennes ont contesté la Loi sur la tarification de la pollution par les gaz à effet de serre, adoptée par le fédéral. Cette loi établit à l’échelle fédérale une norme minimale sur la tarification des gaz à effet de serre dans le but de réduire les émissions du pays et contribuer à l’atteinte des objectifs de l’Accord de Paris. Les provinces ont argué devant la Cour que la tarification des gaz à effet de serre ne relève pas de la compétence fédérale en ce que la réglementation des ressources naturelles est une compétence provinciale. Le 25 mai 2021, la Cour suprême du Canada a débouté les trois provinces en validant la compétence fédérale en la matière.À l’aide d’entrevues réalisées avec des intervenant.e.s dans ce litige, le présent texte donne la parole à Amir Attaran, Nathalie Chalifour, Stewart Elgie et David Robitaille, quatre professeurs qui sont membres du Centre du droit de l’environnement et de la durabilité mondiale (CDEDM) de l’Université d’Ottawa, ayant participé à la défense dans cette affaire historique. Ceux-ci transmettent de manière vulgarisée leur expérience devant la Cour suprême et les arguments qu’ils ont fait valoir pour mener à une décision de la Cour en faveur de la tarification fédérale des gaz à effet de serre. Cette démarche apporte un éclairage intéressant et des informations de première main sur une décision qui constitue un pas de plus vers l’aboutissement de contentieux climatiques au Canada. L’article donne également une perspective très instructive sur le fonctionnement de la Cour suprême et l’organisation des plaidoiries des experts interrogés.This text looks back at a case brought before the Supreme Court of Canada in 2021 in which several Canadian provinces challenged the federal Greenhouse Gas Pollution Pricing Act. This law establishes a minimum standard for greenhouse gas pricing at the federal level, with the aim of reducing the country’s emissions and contributing to achieving the objectives of the Paris Agreement. The provinces argued before the Court that greenhouse gas pricing did not fall under federal jurisdiction, as the regulation of natural resources is a provincial area of competence. On May 25, 2021, the Supreme Court of Canada ruled against the three provinces, validating federal jurisdiction in the matter.Through interviews with stakeholders in the dispute, this text gives the floor to Amir Attaran, Nathalie Chalifour, Stewart Elgie and David Robitaille, four Professors who are members of the Centre for Environmental Law and Global Sustainability (CELGS) at the University of Ottawa, who participated in the defence of this historic case. They convey in simple words their experience before the Supreme Court and the arguments they put forward to lead to the Court’s decision in favour of federal greenhouse gas pricing. This approach provides interesting insights and first-hand information on a decision that represents another step towards the successful outcome of climate litigation in Canada. This article also provides a highly instructive perspective on the workings of the Supreme Court and the organization of the oral arguments of the experts interviewed

Identification of TRIO-GEFD1 chemical inhibitors using the yeast exchange assay.

BACKGROUND INFORMATION: Rho GTPases are involved in many biological processes and participate in cancer development. Their activation is catalysed by exchange factors [RhoGEFs (Rho GTPase guanine nucleotide-exchange factor)] of the Dbl family. RhoGEFs display proto-oncogenic features, thus appearing as candidate targets for anticancer drugs. Dominant-negative Rho GTPase mutants have been widely used to block RhoGEF signalling. However, these tools suffer from limitations, due to the high number of RhoGEFs and the complex mechanisms that control Rho GTPase activation. RESULTS: RhoG-T17N is a poor inhibitor of its exchange factor TRIO-GEFD1 (first exchange domain of the exchange factor TRIO) in vivo: although it binds to TRIO-GEFD1, RhoG-T17N does not block the downstream signalling. Using the yeast exchange assay, we show that in the presence of TRIO-GEFD1, RhoG-T17N can bind to its effectors, which illustrates how negative mutants may produce misleading interpretations and emphasizes the need for new types of RhoGEF inhibitors. In that prospect, we adapted the yeast exchange assay method to identify RhoGEF inhibitors. Using this novel approach, we screened a 3500-chemical-compound library and identified a potential inhibitor of TRIO-GEFD1. This molecule inhibited TRIO-GEFD1 in vitro. Among the chemical analogues of this compound, we identified two molecules with better inhibitory activity. The three TRIO-GEFD1 inhibitors had no effect on ARHGEF17 and ARNO [ARF (ADP-ribosylation factor) nucleotide-binding-site opener], two exchange factors for RhoA and Arf1 respectively. CONCLUSIONS: The development of RhoGEF inhibitors appears as a valuable tool for the study of Rho GTPase signalling pathways. The yeast exchange assay adaptation we present here is suitable to screen for chemical or peptide libraries and identify candidate inhibitors

Whole-brain characterization of apoptosis after sevoflurane anesthesia reveals neuronal cell death patterns in the mouse neonatal neocortex

Abstract In the last two decades, safety concerns about general anesthesia (GA) arose from studies documenting brain cell death in various pharmacological conditions and animal models. Nowadays, a thorough characterization of sevoflurane-induced apoptosis in the entire neonatal mouse brain would help identify and further focus on underlying mechanisms. We performed whole-brain mapping of sevoflurane-induced apoptosis in post-natal day (P) 7 mice using tissue clearing and immunohistochemistry. We found an anatomically heterogenous increase in cleaved-caspase-3 staining. The use of a novel P7 brain atlas showed that the neocortex was the most affected area, followed by the striatum and the metencephalon. Histological characterization in cortical slices determined that post-mitotic neurons were the most affected cell type and followed inter- and intracortical gradients with maximal apoptosis in the superficial layers of the posterodorsal cortex. The unbiased anatomical mapping used here allowed us to confirm sevoflurane-induced apoptosis in the perinatal period, neocortical involvement, and indicated striatal and metencephalic damage while suggesting moderate hippocampal one. The identification of neocortical gradients is consistent with a maturity-dependent mechanism. Further research could then focus on the interference of sevoflurane with neuronal migration and survival during development

Cell Type-Specific mRNA Dysregulation in Hippocampal CA1 Pyramidal Neurons of the Fragile X Syndrome Mouse Model

Fragile X syndrome (FXS) is a genetic disorder due to the silencing of the Fmr1 gene, causing intellectual disability, seizures, hyperactivity, and social anxiety. All these symptoms result from the loss of expression of the RNA binding protein fragile X mental retardation protein (FMRP), which alters the neurodevelopmental program to abnormal wiring of specific circuits. Aberrant mRNAs translation associated with the loss of Fmr1 product is widely suspected to be in part the cause of FXS. However, precise gene expression changes involved in this disorder have yet to be defined. The objective of this study was to identify the set of mistranslated mRNAs that could contribute to neurological deficits in FXS. We used the RiboTag approach and RNA sequencing to provide an exhaustive listing of genes whose mRNAs are differentially translated in hippocampal CA1 pyramidal neurons as the integrative result of FMRP loss and subsequent neurodevelopmental adaptations. Among genes differentially regulated between adult WT and Fmr1−/y mice, we found enrichment in FMRP-binders but also a majority of non-FMRP-binders. Interestingly, both up- and down-regulation of specific gene expression is relevant to fully understand the molecular deficiencies triggering FXS. More importantly, functional genomic analysis highlighted the importance of genes involved in neuronal connectivity. Among them, we show that Klk8 altered expression participates in the abnormal hippocampal dendritic spine maturation observed in a mouse model of FXS