Background And Purpose: Voltage sensitivity is a common feature of many membrane proteins, including some G-protein coupled receptors (GPCRs). However, the functional consequences of voltage sensitivity in GPCRs are not well understood.
Experimental Approach: In this study, we investigated the voltage sensitivity of the post-synaptic metabotropic glutamate receptor mGlu and its impact on synaptic transmission.
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.
View Article and Find Full Text PDFBackground: Shank3 is a scaffolding protein essential for the organization and function of the glutamatergic postsynapse. Monogenic mutations in gene are among the leading genetic causes of Autism Spectrum Disorders (ASD). The multiplicity of Shank3 isoforms seems to generate as much functional diversity and yet, there are no tools to study endogenous Shank3 proteins in an isoform-specific manner.
View Article and Find Full Text PDFMetabotropic glutamate receptor Type 3 (mGlu3) controls the sleep/wake architecture, which plays a role in the glutamatergic pathophysiology of schizophrenia. Interestingly, mGlu3 receptor expression is decreased in the brain of schizophrenic patients. However, little is known about the molecular mechanisms regulating mGlu3 receptors at the cell membrane.
View Article and Find Full Text PDFShank3 monogenic mutations lead to autism spectrum disorders (ASD). Shank3 is part of the glutamate receptosome that physically links ionotropic NMDA receptors to metabotropic mGlu5 receptors through interactions with scaffolding proteins PSD95-GKAP-Shank3-Homer. A main physiological function of the glutamate receptosome is to control NMDA synaptic function that is required for plasticity induction.
View Article and Find Full Text PDFMammalian 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.
View Article and Find Full Text PDFBackground: mTOR signaling is an essential nutrient and energetic sensing pathway. Here we describe AIMTOR, a sensitive genetically encoded BRET (Bioluminescent Resonance Energy Transfer) biosensor to study mTOR activity in living cells.
Results: As a proof of principle, we show in both cell lines and primary cell cultures that AIMTOR BRET intensities are modified by mTOR activity changes induced by specific inhibitors and activators of mTORC1 including amino acids and insulin.
Front Synaptic Neurosci
April 2020
Epileptogenesis 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.
View Article and Find Full Text PDFDopamine receptor D1 modulates glutamatergic transmission in cortico-basal ganglia circuits and represents a major target of L-DOPA therapy in Parkinson's disease. Here we show that D1 and metabotropic glutamate type 5 (mGlu5) receptors can form previously unknown heteromeric entities with distinctive functional properties. Interacting with Gq proteins, cell-surface D1-mGlu5 heteromers exacerbated PLC signaling and intracellular calcium release in response to either glutamate or dopamine.
View Article and Find Full Text PDFFragile X syndrome (FXS) is a genetic disorder due to the silencing of the 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 product is widely suspected to be in part the cause of FXS.
View Article and Find Full Text PDFResonance 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).
View Article and Find Full Text PDFThe orphan Glutamate receptor Delta2 (GluD2) intrinsic ion channel activity is indirectly triggered by glutamate through stimulation of the metabotropic glutamate receptor 1 (mGlu1), in cerebellar Purkinje cells. However, the mechanisms of GluD2 ion channel opening are entirely unknown. In this work, we investigated the signaling pathways underlying the mGlu1-induced GluD2 current, performing whole-cell voltage-clamp recordings from mGlu1 and GluD2 transfected HEK293 cells.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFQuantitative spatio-temporal characterization of protein interactions in living cells remains a major challenge facing modern biology. We have investigated in living neurons the spatial dependence of the stoichiometry of interactions between two core proteins of the N-methyl-D-aspartate (NMDA)-receptor-associated scaffolding complex, GKAP (also known as DLGAP1) and DLC2 (also known as DYNLL2), using a novel variation of fluorescence fluctuation microscopy called two-photon scanning number and brightness (sN&B). We found that dimerization of DLC2 was required for its interaction with GKAP, which, in turn, potentiated GKAP self-association.
View Article and Find Full Text PDFThe powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue.
View Article and Find Full Text PDFNetworks of signaling molecules are activated in response to environmental changes. How are these signaling networks dynamically integrated in space and time to process particular information? To tackle this issue, biosensors of single signaling pathways have been engineered. Bioluminescence resonance energy transfer (BRET)-based biosensors have proven to be particularly efficient in that matter due to the high sensitivity of this technology to monitor protein-protein interactions or conformational changes in living cells.
View Article and Find Full Text PDFAccumulating work over the past decade has shown that peptide aptamer screening represents a valid strategy for inhibitor identification that can be applied to a variety of different targets. Because of the screening method in cells and the highly combinatorial libraries available, this approach yields rapidly highly specific candidate inhibitors. Once a hit peptide has been identified, its interaction strength and affinity towards its target protein can be optimized even more, in order to increase its inhibition efficiency when subsequently applied in vivo.
View Article and Find Full Text PDFCyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2-depleted cells display a cortical distribution of actin filaments and increased migration.
View Article and Find Full Text PDFRhoGEFs (guanine nucleotide exchange factors of the Rho GTPase family) are upstream regulators of cell adhesion and migration pathways, thus representing attractive yet relatively unexplored targets for the development of anti-invasive drugs. We screened for chemical inhibitors of TrioN, the N-terminal GEF domain of the multidomain Trio protein, and identified ITX3 as a nontoxic inhibitor. In transfected mammalian cells, ITX3 blocked TrioN-mediated dorsal membrane ruffling and Rac1 activation while having no effect on GEF337-, Tiam1-, or Vav2-mediated RhoA or Rac1 activation.
View Article and Find Full Text PDFGuanine nucleotide exchange factors (GEFs) activate the Rho GTPases by accelerating their GDP/GTP exchange rate. Some RhoGEFs have been isolated based on their oncogenic potency, and strategies to inhibit their activity are therefore actively being sought. In this study we devise a peptide inhibitor screening strategy to target the GEF activity of Tgat, an oncogenic isoform of the RhoGEF Trio, based on random mutations of the Trio inhibitor TRIP alpha, which we previously isolated using a peptide aptamer screen.
View Article and Find Full Text PDFBackground 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.
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