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author:("perros, Julie")
1.  Ring finger protein 10 is a novel synaptonuclear messenger encoding activation of NMDA receptors in hippocampus 
eLife  null;5:e12430.
Synapses and nuclei are connected by bidirectional communication mechanisms that enable information transfer encoded by macromolecules. Here, we identified RNF10 as a novel synaptonuclear protein messenger. RNF10 is activated by calcium signals at the postsynaptic compartment and elicits discrete changes at the transcriptional level. RNF10 is enriched at the excitatory synapse where it associates with the GluN2A subunit of NMDA receptors (NMDARs). Activation of synaptic GluN2A-containing NMDARs and induction of long term potentiation (LTP) lead to the translocation of RNF10 from dendritic segments and dendritic spines to the nucleus. In particular, we provide evidence for importin-dependent long-distance transport from synapto-dendritic compartments to the nucleus. Notably, RNF10 silencing prevents the maintenance of LTP as well as LTP-dependent structural modifications of dendritic spines.
eLife digest
Brain activity depends on the communication between neurons. This process takes place at the junctions between neurons, which are known as synapses, and typically involves one of the cells releasing a chemical messenger that binds to receptors on the other cell. The binding triggers a cascade of events inside the recipient cell, including the production of new receptors and their insertion into the cell membrane. These changes strengthen the synapse and are thought to be one of the ways in which the brain establishes and maintains memories.
However, in order to induce these changes at the synapse, neurons must be able to activate the genes that encode their component parts. These genes are present inside the cell nucleus, which is located some distance away from the synapse. Studies have shown that signals can be sent from the nucleus to the synapse and vice versa, enabling the two parts of the cell to exchange information. Synapses that communicate using a chemical called glutamate have been particularly well studied; but it still remains unclear how the activation of receptors at these “glutamatergic synapses” is linked to activation of genes inside the nucleus at the molecular level.
Dinamarca, Guzzetti et al. have now discovered that this process at glutamatergic synapses involves the movement of a protein messenger to the nucleus. Specifically, activation at synapses of a particularly common subtype of receptor, called NMDA, causes a protein called Ring Finger protein 10 (or RNF10 for short) to move from the synapse to the nucleus. To leave the synapse, RNF10 first has to bind to proteins called importins, which transport RNF10 into the nucleus. Once inside the nucleus, RNF10 binds to another protein that interacts with the DNA to start the production of new synaptic proteins.
Further work is required to identify the molecular mechanisms that trigger RNF10 to leave the synapse. In addition, future studies should evaluate the levels and activity of RNF10 in brain disorders in which synapses are known to function abnormally.
PMCID: PMC4805553  PMID: 26977767
NMDA receptors; dendritic spines; hippocampus; rattus; importins; synaptonuclear messengers; Rat
2.  Elevated CaMKIIα and hyperphosphorylation of Homer mediate circuit dysfunction in a Fragile X Syndrome mouse model 
Cell reports  2015;13(10):2297-2311.
Abnormal metabotropic glutamate receptor 5 (mGluR5) function, as a result of disrupted scaffolding with its binding partner Homer, contributes to the pathophysiology of Fragile X Syndrome, a common inherited from of intellectual disability and autism caused by mutations in Fmr1. How loss of Fmr1 disrupts mGluR5-Homer scaffolds is unknown, and little is known about the dynamic regulation of mGluR5-Homer scaffolds in wildtype neurons. Here we demonstrate that brief (minutes) elevations in neural activity cause CaMKIIα-mediated phosphorylation of long Homer proteins and dissociation from mGluR5 at synapses. In Fmr1 knockout cortex, Homers are hyperphosphorylated as a result of elevated CaMKIIα protein. Genetic or pharmacological inhibition of CaMKIIα or replacement of Homers with dephosphomimetics restores mGluR5-Homer scaffolds and multiple Fmr1 KO phenotypes, including circuit hyperexcitability and/or seizures. This work links translational control of an FMRP target mRNA, CaMKIIα, to the molecular, cellular and circuit level brain dysfunction in a complex neurodevelopmental disorder.
PMCID: PMC4685008  PMID: 26670047
3.  Analysis of RXR/THR and RXR/PPARG2 Heterodimerization by Bioluminescence Resonance Energy Transfer (BRET) 
PLoS ONE  2013;8(12):e84569.
Nuclear receptors (NR) regulate transcription of genes involved in many biological processes such as development, cell proliferation, differentiation and cell death. Amongst them, PPARG2 and THR control tissue glucose and lipid homeostasis which are deregulated in severe pathophysiological conditions such as metabolic syndromes.
Methodology/Principal Findings
Here, we describe a real time BRET approach to monitor heterodimerization between RXR and PPARG2 or THR in vitro or in living cells. The presence of a specific DNA target was required to induce in vitro a BRET shift reflecting heterodimerization of RXR/PPARG2 or RXR/THR. As in electrophoretic mobility shift assay (EMSA), the stringency and specificity of the BRET shift assay depended upon assay condition optimization including MgCl2 concentration. For the nuclear receptors, we found by mutagenesis analysis that each heterodimer partner must harbor an intact DNA binding domain to induce BRET and heterodimerization on a DNA target. Moreover the interaction between the PPARG2 ligand binding domain and the RXR DNA binding domain stabilized the heterodimer on its DNA target. BRET microscopy in living cells highlighted the heterodimerization of RXR/PPARG2 within the nucleus clustered in discrete foci that may represent active target gene transcription regulation regions. BRET imaging also suggested that heterodimerization between RXR and PPARG2 required the DNA binding of PPARG2.
The BRET approach described here allowed us to study the dynamic interactions which exist between NR in vitro or in living cells and can provide important information on heterodimerization modes, affinity with a given RE and subcellular localization of the heterodimers. This method could be used to study real time changes of NR heterodimers occurring on DNA depending upon cell activation, chromatin state and help to define the mechanisms of ligands or drug action designed to target NRs.
PMCID: PMC3877338  PMID: 24391967
4.  A genetically encoded IL-1β BRET sensor to monitor inflammasome activity 
Inflammation is fundamental for protecting the organism against infection and injury. However, a failure to control immune response results in chronic inflammation and several associated disorders such as pain and loss of function. Initiation of inflammation is orchestrated by cytokines, among which interleukin (IL)-1β is particularly important. IL-1β is synthesized as an inactive protein that has to be processed by the inflammasome to generate the mature bioactive form. Conventional techniques cannot monitor IL-1β activation with high spatial and temporal resolution. Here, we present a ratiometric biosensor that allows monitoring IL-1β processing in real-time, with a temporal resolution of seconds and with a single cell spatial resolution. Using this sensor, we describe for the first time the kinetic of the inflammasome activity in living macrophages. With this new probe we also demonstrated that the pro-IL-1β processing occurs all over the cytoplasm.
PMCID: PMC3437522  PMID: 22815289
5.  Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability 
The Journal of Cell Biology  2012;198(2):251-263.
Synaptic activity–dependent remodeling of the glutamate receptor scaffold complex generates a negative feedback loop that limits further NMDA receptor activation.
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.
PMCID: PMC3410417  PMID: 22801779
6.  REV, A BRET-Based Sensor of ERK Activity 
Networks 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. Extracellular signal-regulated kinases (ERK) are ubiquitously expressed and involved in many diverse cellular functions that might be encoded by the strength and spatio-temporal pattern of ERK activation. We developed a BRET-based sensor of ERK activity, called Rluc8-ERKsubstrate-Venus (REV). As expected, BRET changes of REV were correlated with ERK phosphorylation, which is required for its kinase activity. In neurons, the nature of the stimuli determines the strength, the location, or the moment of ERK activation, thus highlighting how acute modulation of ERK may encode the nature of initial stimulus to specify the consequences of this activation. This study provides evidence for suitability of REV as a new biosensor to address biological questions.
PMCID: PMC3727045  PMID: 23908646
biosensor; bioluminescence resonance energy transfer; BRET imaging; fluorescence lifetime imaging microscopy; extracellular signal-regulated kinases; spatio-temporal signaling; Rluc8-ERKsubstrate-Venus
7.  A Genetically Encoded IL-1β Bioluminescence Resonance Energy Transfer Sensor To Monitor Inflammasome Activity 
Inflammation is fundamental for protecting the organism against infection and injury. However, a failure to control immune response results in chronic inflammation and several associated disorders such as pain and loss of function. Initiation of inflammation is orchestrated by cytokines, among which IL-1β is particularly important. IL-1β is synthesized as an inactive protein that has to be processed by the inflammasome to generate the mature bioactive form. Conventional techniques cannot monitor IL-1β activation with high spatial and temporal resolution. In this study, we present a ratiometric biosensor that allows monitoring IL-1β processing in real time, with a temporal resolution of seconds and with a single-cell spatial resolution. Using this sensor, to our knowledge, we describe for the first time the kinetic of the inflammasome activity in living macrophages. With this new probe, we also demonstrated that the pro–IL-1β processing occurs all over the cytoplasm.
PMCID: PMC3437522  PMID: 22815289
8.  Scaffold remodeling in space and time controls synaptic transmission 
Bioarchitecture  2012;2(2):29-32.
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.
PMCID: PMC3383718  PMID: 22754626
bioluminescence resonance energy transfer (BRET); dendritic spine; dynein light chain 2 (DLC2); glutamate receptors; guanylate kinase-associated protein (GKAP); protein-protein interaction; scaffolding proteins; synaptic transmission
9.  A single subunit (GB2) is required for G-protein activation by the heterodimeric GABAb receptor 
The Journal of Biological Chemistry  2001;277(5):3236-3241.
Although G-protein coupled receptors (GPCRs) have been shown to assemble into functional homo or heteromers, the role of each protomer in G-protein activation is not known. Among the GPCRs, the γ-aminobutyric acid (GABA) type B receptor (GABABR) is the only one known so far that needs two subunits, GB1 and GB2, to function. The GB1 subunit contains the GABA binding site but is unable to activate G-proteins alone. In contrast the GB2 subunit which does not bind GABA, has an heptahelical domain able to activate G-proteins when assembled into dimers (Galvez et al., EMBO J. 20, 2001, 2152–2159). In the present study, we examined the role of each subunit within the GB1-GB2 heteromer, in G-protein coupling. To that aim, point mutations in the highly conserved third intracellular loop known to prevent G-protein activation of the related Ca-sensing or metabotropic glutamate receptors were introduced into GB1 and GB2. One mutation, L686P introduced in GB2 prevents the formation of a functional receptor, even though the heteromer reaches the cell surface, and even though the mutated subunit still associates with GB1 and increases GABA affinity on GB1. This was observed either in HEK293 cells where the activation of the G-protein was assessed by measurement of IP accumulation, or in cultured neurons where the inhibition of the Ca-channel current was measured. In contrast, the same mutation when introduced into GB1 does not modify the G-protein coupling properties of the heteromeric GABAB receptor either in HEK293 cells or in neurons. These data show that a single subunit in a dimeric GPCR is critical for coupling to G-proteins.
PMCID: PMC2566549  PMID: 11711539
Animals; Benzoates; pharmacokinetics; Binding; Competitive; Cell Line; Cells; Cultured; Cerebellum; cytology; GABA Antagonists; pharmacokinetics; GTP-Binding Proteins; chemistry; metabolism; Humans; Inositol Phosphates; metabolism; Kidney; Kinetics; Ligands; Mice; Mutagenesis; Site-Directed; Neurons; cytology; Organophosphorus Compounds; pharmacokinetics; Protein Binding; Protein Subunits; Receptors; GABA-B; drug effects; genetics; physiology; Recombinant Proteins; chemistry; metabolism; Transfection; gamma-Aminobutyric Acid; pharmacology

Results 1-9 (9)