Periodic Reporting for period 4 - VERTICAL CITY (Versatility of scaffold complexes in vivo to control synaptic plasticity) Reporting period: 2019-12-01 to 2021-05-31 Summary of the context and overall objectives of the project Memory is a dynamic process. At all time we have to encode new information and recall old ones. Brain plasticity is thought to be the basis of memory encoding. Each of our sensorial or emotional experience will indeed transform our brain. There is no specific area where memories are stored. Memory is spread all over the brain. This spreading comes from the biological processes that sustain the encoding and storage of new information. When stimulated by new information, a neuron will make contact (synapse) with other nervous cells. The more it is solicited, the more new synapses it will form with other neurons, as numerous mnesic traces speared in brain areas defined by the nature of stimuli. Thus, specific connections between neurons and neuronal communication efficiency control the encoding of information and its storage. The efficiency of synaptic transmission is controlled at synapses by the release of a chemical messenger (neurotransmitter) by the activated neuron, and the activation of a receptor’s neurotransmitter on the connected neuron. Receptors and associated scaffolds, together called receptosome, are relatively stable structures, but indeed exchange of individual adaptor proteins can occur on a short time scale and in a highly regulated manner, which provides fine-tuning, speed, and specificity to the receptor signaling. Therefore, understanding how receptor function is affected by the composition and dynamics of complexes is an essential biological concern that will offer the opportunity to target exclusively the therapeutically relevant signaling pathway of a given receptor. We showed that in the brain, receptosome dynamics is involved in fine-tuning synaptic transmission and plasticity, which is crucial for cognitive functions.We have established links between molecular events, neuronal signaling and memory performance. More than correlations, this project enabled live recording of molecular events and cellular signaling during memory encoding. The herein developed technologies enabled to monitor the versatility of protein-protein interactions in space and time ranging from in cellulo to in vivo BRET imaging in freely behaving animals. To conclude, our work enables a better understanding of the functional significance of oligomer remodeling in the physiological synaptic plasticity and highlights the need to restore it in neurological disorders, like we did in parkinson dease or austism spectrum disorder mouse models. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The overall goal of this proposal was to understand the molecular mechanisms that allow the learning and memory processes. The neuronal communication (and its efficiency) is driven by plastic and specific connections between neurons, called synapses, which are finely refined by our cognitive experiences. At synapses, a neurotransmitter released from the pre-synaptic terminal activates post-synaptic receptors which will transduce their activation by initiating specific signaling pathways in the post-synaptic neuron. Receptors functions at synapses rely on their ability to engage in specific protein-protein interactions and to form complexes that are dynamically regulated by stimuli. Intracellular proteins (scaffolds) interact with receptors to control their specific sub-cellular targeting, allow receptor cell-surface expression, specify their cellular effectors and modify specific connections between neurons, enabling concerted activities of neuronal ensembles to trigger physiological functions. Thus understanding how proteins are activated as free molecules or part of complexes was an essential biological issue to offer new possibilities to target exclusively the therapeutically relevant signaling pathway of a given receptor. 1) We first developped technological approaches to follow protein-protein interaction dynamics and receptor signaling in cellulo and and in vivo. These developments encompase new imaging technologies (1, 2, 5, 6, 7), biosensors engineering to report signaling pathways involved in plasiticty, like mTOR, ERK, or MMP9 activation (1, 9, 10, 13), protocols to follow signalings from neuronal ensembles in vitro and in vivo (6, 7, 11), and molecular tools to constrain protein-protein interaction dynamics and neuronal signalings (3, 8, 12, 14).2) We used these state-of-the-art technologies to identify functional consequences of protein-protein interactions dynamics in physiological conditions and deficiencies in mouse models of neurological disorders. Briefly, we found the importance of protein-protein interaction dynamics in the receptor trafficking to a specific subcellular compartment of the neuron (12), specific cellular signaling (1, 3, 9, 10), structural (4) and functional (3, 14) neuronal plasticty. Besides, we identified and repaired protein-protein interaction deficiencies in mouse models of L-Dopa-induced dyskinesia (8) and autisum spectrum disorders (14), which improved related cognitive behaviors. 3) Finally, we identified new receptors associated proteins involved in neuronal plasticity and memory deficiencies (4), a screening that defines new therapeutic target opportunities for future studies.1- Fast and high resolution single-cell BRET imaging2- Agonist-Specific Recruitment of Arrestin Isoforms Differentially Modify Delta Opioid Receptor Function3- Elevated CaMKIIα and Hyperphosphorylation of Homer Mediate Circuit Dysfunction in a Fragile X Syndrome Mouse Model4- Cell Type-Specific mRNA Dysregulation in Hippocampal CA1 Pyramidal Neurons of the Fragile X Syndrome Mouse Model5- Fluorescent-Based Strategies to Investigate G Protein-Coupled Receptors: Evolution of the Techniques to a Better Understanding6- Image Processing for Bioluminescence Resonance Energy Transfer Measurement—BRET-Analyzer7- Fast confocal fluorescence imaging in freely behaving mice8- D1-mGlu5 heteromers mediate noncanonical dopamine signaling in Parkinson’s disease9- AIMTOR, a BRET biosensor for live imaging, reveals subcellular mTOR signaling and dysfunctions10- Gelatinase Biosensor Reports Cellular Remodeling During Epileptogenesis11- Procedures for Culturing and Genetically Manipulating Murine Hippocampal Postnatal Neurons12- SNAP23–Kif5 complex controls mGlu1 receptor trafficking13- AIMTOR, a BRET Biosensor for Live Recording of mTOR Activity in Cell Populations and Single Cells14- Restoring glutamate receptosome dynamics at synapses rescues autism-like deficits in Shank3-deficient mice Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) First, this proposal is groundbreaking because it makes the link between molecular events, neuronal signaling and memory performance. More then correlations, this project enabled live recording of molecular events and cellular signaling during memory encoding. Second, we proposed new specific therapeutic targets to treat mental deficiencies: By opposition to pharmaceutical compound interfering with the ligand-biding pocket of the receptor, we here proposed to play on scaffold interactions. This strategy only modifies a specific altered function of glutamate receptor without modifying other functions of the receptor (thus, avoiding undesired side effects). Third, within the scope of this proposal innovative powerful tools / technics have been developed, of high interest for the broad community of researchers in life sciences to highlight the versatility of protein-protein interactions in space and time ranging from in cellulo to in vivo BRET imaging in freely behaving animals. illustration-for-summary2.jpg
