Modulation of Hippocampal Network Oscillation by PICK1-Dependent Cell Surface Expression of mGlu3 Receptors.

Metabotropic 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. Subcellular receptor localization is strongly dependent on protein-protein interactions. Here we show that mGlu3 interacts with PICK1 and that this scaffolding protein is important for mGlu3 surface expression and function in hippocampal primary cultures. Disruption of their interaction via an mGlu3 C-terminal mimicking peptide or an inhibitor of the PDZ domain of PICK1 altered the functional expression of mGlu3 receptors in neurons. We next investigated the impact of disrupting the mGlu3-PICK1 interaction on hippocampal theta oscillations in vitro and in vivo in WT male mice. We found a decreased frequency of theta oscillations in organotypic hippocampal slices, similar to what was previously observed in mGlu3 KO mice. In addition, hippocampal theta power was reduced during rapid eye movement sleep, non-rapid eye movement (NREM) sleep, and wake states after intraventricular administration of the mGlu3 C-terminal mimicking peptide. Targeting the mGlu3-PICK1 complex could thus be relevant to the pathophysiology of schizophrenia.SIGNIFICANCE STATEMENT Dysregulation of the glutamatergic system might play a role in the pathophysiology of schizophrenia. Metabotropic glutamate receptors Type 3 (mGlu3) have been proposed as potential targets for schizophrenia. Understanding the molecular mechanisms regulating mGlu3 receptor at the cell membrane is critical toward comprehending how their dysfunction contributes to the pathogenesis of schizophrenia. Here we describe that the binding of the signaling and scaffolding protein PICK1 to mGlu3 receptors is important for their localization and physiological functions. The identification of new proteins that associate specifically to mGlu3 receptors will advance our understanding of the regulatory mechanisms associated with their targeting and function and ultimately might provide new therapeutic strategies to counter these psychiatric conditions.

Restoring glutamate receptosome dynamics at synapses rescues autism-like deficits in Shank3-deficient mice.

Shank3 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. Intact glutamate receptosome supports glutamate receptors activation and plasticity induction, while glutamate receptosome disruption blocks receptors activity, preventing the induction of subsequent plasticity. Despite possible impact on metaplasticity and cognitive behaviors, scaffold interaction dynamics and their consequences are poorly defined. Here, we used mGlu5-Homer interaction as a biosensor of glutamate receptosome integrity to report changes in synapse availability for plasticity induction. Combining BRET imaging and electrophysiology, we show that a transient neuronal depolarization inducing NMDA-dependent plasticity disrupts glutamate receptosome in a long-lasting manner at synapses and activates signaling pathways required for the expression of the initiated neuronal plasticity, such as ERK and mTOR pathways. Glutamate receptosome disruption also decreases the NMDA/AMPA ratio, freezing the sensitivity of the synapse to subsequent changes of neuronal activity. These data show the importance of a fine-tuning of protein-protein interactions within glutamate receptosome, driven by changes of neuronal activity, to control plasticity. In a mouse model of ASD, a truncated mutant form of Shank3 prevents the integrity of the glutamate receptosome. These mice display altered plasticity, anxiety-like, and stereotyped behaviors. Interestingly, repairing the integrity of glutamate receptosome and its sensitivity to the neuronal activity rescued synaptic transmission, plasticity, and some behavioral traits of Shank3∆C mice. Altogether, our findings characterize mechanisms by which Shank3 mutations cause ASD and highlight scaffold dynamics as new therapeutic target.

AIMTOR, a BRET biosensor for live imaging, reveals subcellular mTOR signaling and dysfunctions.

BACKGROUND: 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. We further engineered several versions of AIMTOR enabling subcellular-specific assessment of mTOR activities. We then used AIMTOR to decipher mTOR signaling in physio-pathological conditions. First, we show that mTORC1 activity increases during muscle cell differentiation and in response to leucine stimulation in different subcellular compartments such as the cytosol and at the surface of the lysosome, the nucleus, and near the mitochondria. Second, in hippocampal neurons, we found that the enhancement of neuronal activity increases mTOR signaling. AIMTOR further reveals mTOR-signaling dysfunctions in neurons from mouse models of autism spectrum disorder. CONCLUSIONS: Altogether, our results demonstrate that AIMTOR is a sensitive and specific tool to investigate mTOR-signaling dynamics in living cells and phenotype mTORopathies.

Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons.

Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine.

The stoichiometry of scaffold complexes in living neurons - DLC2 functions as a dimerization engine for GKAP.

Quantitative 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. In the dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of two DLC2 and two GKAP monomers, whereas, in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP monomers. Disruption of the GKAP-DLC2 interaction strongly destabilized the oligomers, decreasing the spine-preferential localization of GKAP and inhibiting NMDA receptor activity. Hence, DLC2 serves a hub function in the control of glutamatergic transmission by ordering GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2-GKAP interactions in glutamatergic signaling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes.

Rho-GTPase-activating protein interacting with Cdc-42-interacting protein 4 homolog 2 (Rich2): a new Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase-activating protein that controls dendritic spine morphogenesis.

Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.

GKAP-DLC2 interaction organizes the postsynaptic scaffold complex to enhance synaptic NMDA receptor activity.

At glutamatergic brain synapses, scaffolding proteins regulate receptor location and function. The targeting and organization of scaffolding proteins in the postsynaptic density (PSD) is poorly understood, but it is known that a core protein of the glutamatergic receptor postsynaptic scaffold complex, guanylate-kinase-associated protein (GKAP) interacts with dynein light chain 2 (DLC2, also known as DYNLL2), a protein associated with molecular motors. In the present study, we combined BRET imaging, immunostaining and electrophysiological recording to assess the role of the GKAP-DLC2 interaction in the functional organization of the glutamatergic synapse. We found that GKAP-DLC2 interaction in dendritic spine stabilizes scaffolding protein expression at the PSD and enhances synaptic NMDA receptor activity. Moreover, the GKAP-DLC2 functional interaction is favored by sustained synaptic activity. These data identify a regulatory pathway of synaptic transmission that depends on activity-induced remodelling of the postsynaptic scaffold protein complex.

Voltage tunes mGlu5 receptor function, impacting synaptic transmission.

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 mGlu5 and its impact on synaptic transmission. Using biosensors and electrophysiological recordings in non-excitable HEK293T cells or neurons. KEY RESULTS: We found that mGlu5 receptor function is optimal at resting membrane potentials. We observed that membrane depolarization significantly reduced mGlu5 receptor activation, Gq-PLC/PKC stimulation, Ca2+ release and mGlu5 receptor-gated currents through transient receptor potential canonical, TRPC6, channels or glutamate ionotropic NMDA receptors. Notably, we report a previously unknown activity of the NMDA receptor at the resting potential of neurons, enabled by mGlu5. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that mGlu5 receptor activity is directly regulated by membrane voltage which may have a significant impact on synaptic processes and pathophysiological functions.

The Shank3Venus/Venus knock in mouse enables isoform-specific functional studies of Shank3a.

Background: Shank3 is a scaffolding protein essential for the organization and function of the glutamatergic postsynapse. Monogenic mutations in SHANK3 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. Methods: In this study, we created a novel transgenic mouse line, the Shank3Venus/Venus knock in mouse, which allows to monitor the endogenous expression of the major Shank3 isoform in the brain, the full-length Shank3a isoform. Results: We show that the endogenous Venus-Shank3a protein is localized in spines and is mainly expressed in the striatum, hippocampus and cortex of the developing and adult brain. We show that Shank3Venus/+ and Shank3Venus/Venus mice have no behavioral deficiency. We further crossed Shank3Venus/Venus mice with Shank3ΔC/ΔC mice, a model of ASD, to track the Venus-tagged wild-type copy of Shank3a in physiological (Shank3Venus/+) and pathological (Shank3Venus/ΔC) conditions. We report a developmental delay in brain expression of the Venus-Shank3a isoform in Shank3Venus/ΔC mice, compared to Shank3Venus/+ control mice. Conclusion: Altogether, our results show that the Shank3Venus/Venus mouse line is a powerful tool to study endogenous Shank3a expression, in physiological conditions and in ASD.

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

Mammalian 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. Graphic abstract: AIMTOR protocol overview from seeding cells to live BRET recording.

Procedures for Culturing and Genetically Manipulating Murine Hippocampal Postnatal Neurons.

Neuronal hippocampal cultures are simple and valuable models for studying neuronal function. While embryonic cultures are widely used for different applications, mouse postnatal cultures are still challenging, lack reproducibility and/or exhibit inappropriate neuronal activity. Yet, postnatal cultures have major advantages such as allowing genotyping of pups before culture and reducing the number of experimental animals. Herein we describe a simple and fast protocol for culturing and genetically manipulating hippocampal neurons from P0 to P3 mice. This protocol provides reproducible cultures exhibiting a consistent neuronal development, normal excitatory over inhibitory neuronal ratio and a physiological neuronal activity. We also describe simple and efficient procedures for genetic manipulation of neurons using transfection reagent or lentiviral particles. Overall, this method provides a detailed and validated protocol allowing to explore cellular mechanisms and neuronal activity in postnatal hippocampal neurons in culture.

The atypical chemokine receptor 3 interacts with Connexin 43 inhibiting astrocytic gap junctional intercellular communication.

The atypical chemokine receptor 3 (ACKR3) plays a pivotal role in directing the migration of various cellular populations and its over-expression in tumors promotes cell proliferation and invasiveness. The intracellular signaling pathways transducing ACKR3-dependent effects remain poorly characterized, an issue we addressed by identifying the interactome of ACKR3. Here, we report that recombinant ACKR3 expressed in HEK293T cells recruits the gap junction protein Connexin 43 (Cx43). Cx43 and ACKR3 are co-expressed in mouse brain astrocytes and human glioblastoma cells and form a complex in embryonic mouse brain. Functional in vitro studies show enhanced ACKR3 interaction with Cx43 upon ACKR3 agonist stimulation. Furthermore, ACKR3 activation promotes ß-arrestin2- and dynamin-dependent Cx43 internalization to inhibit gap junctional intercellular communication in primary astrocytes. These results demonstrate a functional link between ACKR3 and gap junctions that might be of pathophysiological relevance.

D1-mGlu5 heteromers mediate noncanonical dopamine signaling in Parkinson's disease.

Dopamine 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. In rodent models of Parkinson's disease, D1-mGlu5 nanocomplexes were strongly upregulated in the dopamine-denervated striatum, resulting in a synergistic activation of PLC signaling by D1 and mGlu5 receptor agonists. In turn, D1-mGlu5-dependent PLC signaling was causally linked with excessive activation of extracellular signal-regulated kinases in striatal neurons, leading to dyskinesia in animals treated with L-DOPA or D1 receptor agonists. The discovery of D1-mGlu5 functional heteromers mediating maladaptive molecular and motor responses in the dopamine-denervated striatum may prompt the development of new therapeutic principles for Parkinson's disease.

SNAP23-Kif5 complex controls mGlu1 receptor trafficking.

Metabotropic glutamate receptors are expressed at excitatory synapses and control synaptic transmission in mammalian brain. These receptors are involved in numerous patho-physiological functions. However, little is known about the molecular determinants responsible for their intracellular transport and membrane targeting. Here we investigated the nature of the molecular motor and adaptor protein responsible for trafficking and membrane localization of the group I metabotropic glutamate mGlu1 postsynaptic receptor in cultured hippocampal neurons. In proteomic studies, we identified the synaptosome-associated protein 23 (SNAP23) and the molecular motor Kif5 kinesin as proteins interacting with mGlu1 receptor. We showed that SNAP23, but not Kif5, directly interacts with mGlu1 receptor carboxyl terminus. Using a recombination approach to impair or enhance the interaction between SNAP23 and Kif5, we found that the SNAP23-Kif5 complex controls the trafficking of mGlu1 receptor along microtubules. Additional fluorescence recovery after cleavage experiments allowed us to identify a role of the complex in the receptor cell surface targeting. In conclusion, our study indicates that along dendritic processes Kif5-SNAP23 complex contributes to proper mGlu1 receptor trafficking and cell surface expression.

Image Processing for Bioluminescence Resonance Energy Transfer Measurement-BRET-Analyzer.

A growing number of tools now allow live recordings of various signaling pathways and protein-protein interaction dynamics in time and space by ratiometric measurements, such as Bioluminescence Resonance Energy Transfer (BRET) Imaging. Accurate and reproducible analysis of ratiometric measurements has thus become mandatory to interpret quantitative imaging. In order to fulfill this necessity, we have developed an open source toolset for Fiji-BRET-Analyzer-allowing a systematic analysis, from image processing to ratio quantification. We share this open source solution and a step-by-step tutorial at https://github.com/ychastagnier/BRET-Analyzer. This toolset proposes (1) image background subtraction, (2) image alignment over time, (3) a composite thresholding method of the image used as the denominator of the ratio to refine the precise limits of the sample, (4) pixel by pixel division of the images and efficient distribution of the ratio intensity on a pseudocolor scale, and (5) quantification of the ratio mean intensity and standard variation among pixels in chosen areas. In addition to systematize the analysis process, we show that the BRET-Analyzer allows proper reconstitution and quantification of the ratiometric image in time and space, even from heterogeneous subcellular volumes. Indeed, analyzing twice the same images, we demonstrate that compared to standard analysis BRET-Analyzer precisely define the luminescent specimen limits, enlightening proficient strengths from small and big ensembles over time. For example, we followed and quantified, in live, scaffold proteins interaction dynamics in neuronal sub-cellular compartments including dendritic spines, for half an hour. In conclusion, BRET-Analyzer provides a complete, versatile and efficient toolset for automated reproducible and meaningful image ratio analysis.

Scribble1/AP2 complex coordinates NMDA receptor endocytic recycling.

The appropriate trafficking of glutamate receptors to synapses is crucial for basic synaptic function and synaptic plasticity. It is now accepted that NMDA receptors (NMDARs) internalize and are recycled at the plasma membrane but also exchange between synaptic and extrasynaptic pools; these NMDAR properties are also key to governing synaptic plasticity. Scribble1 is a large PDZ protein required for synaptogenesis and synaptic plasticity. Herein, we show that the level of Scribble1 is regulated in an activity-dependent manner and that Scribble1 controls the number of NMDARs at the plasma membrane. Notably, Scribble1 prevents GluN2A subunits from undergoing lysosomal trafficking and degradation by increasing their recycling to the plasma membrane following NMDAR activation. Finally, we show that a specific YxxR motif on Scribble1 controls these mechanisms through a direct interaction with AP2. Altogether, our findings define a molecular mechanism to control the levels of synaptic NMDARs via Scribble1 complex signaling.

Scaffold remodeling in space and time controls synaptic transmission.

Scaffolding proteins that are associated with glutamate receptors in dendritic spines govern the location and function of receptors to control synaptic transmission. Unraveling the spatio-temporal dynamics of protein-protein interactions within components of the scaffolding complex will bring to light the function of these interactions. Combining bioluminescence resonance energy transfer (BRET) imaging to electrophysiological recordings, we have recently shown that GKAP, a core protein of the scaffolding complex, interacts with DLC2, a protein associated with molecular motors. Synaptic activity-induced GKAP-DLC2 interaction in spines stabilizes the scaffolding complex and enhances the NMDA currents. Interestingly, this work placed emphasis on the bioarchitectural dependence of protein-protein interaction dynamics. Depending on physiological conditions, the modulation in space and time of protein-protein interaction is acutely regulated, engendering a subtle control of synaptic transmission in the state of the individual synapse.

Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability.

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor-mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.