Publications by authors named "Olivier Thoumine"

50 Publications

SlitC-PlexinA1 mediates iterative inhibition for orderly passage of spinal commissural axons through the floor plate.

Elife 2020 12 21;9. Epub 2020 Dec 21.

Institut NeuroMyoGène - CNRS UMR 5310 - INSERM U1217 de Lyon- UCBL Lyon 1, Faculté de Médecine et de Pharmacie, Lyon, France.

Spinal commissural axon navigation across the midline in the floor plate requires repulsive forces from local Slit repellents. The long-held view is that Slits push growth cones forward and prevent them from turning back once they became sensitized to these cues after midline crossing. We analyzed with fluorescent reporters Slits distribution and FP glia morphology. We observed clusters of Slit-N and Slit-C fragments decorating a complex architecture of glial basal process ramifications. We found that PC2 proprotein convertase activity contributes to this pattern of ligands. Next, we studied Slit-C acting via PlexinA1 receptor shared with another FP repellent, the Semaphorin3B, through generation of a mouse model baring PlexinA1 mutation abrogating SlitC but not Sema3B responsiveness, manipulations in the chicken embryo, and ex vivo live imaging. This revealed a guidance mechanism by which SlitC constantly limits growth cone exploration, imposing ordered and forward-directed progression through aligned corridors formed by FP basal ramifications.
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http://dx.doi.org/10.7554/eLife.63205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775108PMC
December 2020

FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins.

Sci Rep 2020 11 17;10(1):19954. Epub 2020 Nov 17.

CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Univ. Bordeaux, 33000, Bordeaux, France.

Fluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these issues, we present here a robust computer program, called FluoSim, which is an interactive simulator of membrane protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. FluoSim integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the localization and intensity of thousands of independent molecules in 2D cellular geometries, providing simulated data directly comparable to actual experiments. FluoSim was thoroughly validated against experimental data obtained on the canonical neurexin-neuroligin adhesion complex at cell-cell contacts. This unified software allows one to model and predict membrane protein dynamics and localization at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments.
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http://dx.doi.org/10.1038/s41598-020-75814-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672080PMC
November 2020

Alternative splicing at neuroligin site A regulates glycan interaction and synaptogenic activity.

Elife 2020 09 11;9. Epub 2020 Sep 11.

Djavad Mowafaghian Centre for Brain Health and Department of Psychiatry, University of British Columbia, Vancouver, Canada.

Post-transcriptional mechanisms regulating cell surface synaptic organizing complexes that control the properties of connections in brain circuits are poorly understood. Alternative splicing regulates the prototypical synaptic organizing complex, neuroligin-neurexin. In contrast to the well-studied neuroligin splice site B, little is known about splice site A. We discovered that inclusion of the positively charged A1 insert in mouse neuroligin-1 increases its binding to heparan sulphate, a modification on neurexin. The A1 insert increases neurexin recruitment, presynaptic differentiation, and synaptic transmission mediated by neuroligin-1. We propose that the A1 insert could be a target for alleviating the consequences of deleterious mutations, supported by assays with the autism-linked neuroligin-1-P89L mutant. An enrichment of neuroligin-1 A1 in GABAergic neuron types suggests a role in synchrony of cortical circuits. Altogether, these data reveal an unusual mode by which neuroligin splicing controls synapse development through protein-glycan interaction and identify it as a potential therapeutic target.
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http://dx.doi.org/10.7554/eLife.58668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486126PMC
September 2020

Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation.

Elife 2020 04 23;9. Epub 2020 Apr 23.

University of Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France.

Neuroligins (Nlgns) are adhesion proteins mediating trans-synaptic contacts in neurons. However, conflicting results around their role in synaptic differentiation arise from the various techniques used to manipulate Nlgn expression level. Orthogonally to these approaches, we triggered here the phosphorylation of endogenous Nlgn1 in CA1 mouse hippocampal neurons using a photoactivatable tyrosine kinase receptor (optoFGFR1). Light stimulation for 24 hr selectively increased dendritic spine density and AMPA-receptor-mediated EPSCs in wild-type neurons, but not in Nlgn1 knock-out neurons or when endogenous Nlgn1 was replaced by a non-phosphorylatable mutant (Y782F). Moreover, light stimulation of optoFGFR1 partially occluded LTP in a Nlgn1-dependent manner. Combined with computer simulations, our data support a model by which Nlgn1 tyrosine phosphorylation promotes the assembly of an excitatory post-synaptic scaffold that captures surface AMPA receptors. This optogenetic strategy highlights the impact of Nlgn1 intracellular signaling in synaptic differentiation and potentiation, while enabling an acute control of these mechanisms.
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http://dx.doi.org/10.7554/eLife.52027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180054PMC
April 2020

Vangl2 acts at the interface between actin and N-cadherin to modulate mammalian neuronal outgrowth.

Elife 2020 01 7;9. Epub 2020 Jan 7.

INSERM, Neurocentre Magendie, U1215, Bordeaux, France.

Dynamic mechanical interactions between adhesion complexes and the cytoskeleton are essential for axon outgrowth and guidance. Whether planar cell polarity (PCP) proteins, which regulate cytoskeleton dynamics and appear necessary for some axon guidance, also mediate interactions with membrane adhesion is still unclear. Here we show that Vangl2 controls growth cone velocity by regulating the internal retrograde actin flow in an N-cadherin-dependent fashion. Single molecule tracking experiments show that the loss of decreased fast-diffusing N-cadherin membrane molecules and increased confined N-cadherin trajectories. Using optically manipulated N-cadherin-coated microspheres, we correlated this behavior to a stronger mechanical coupling of N-cadherin with the actin cytoskeleton. Lastly, we show that the spatial distribution of Vangl2 within the growth cone is selectively affected by an N-cadherin-coated substrate. Altogether, our data show that Vangl2 acts as a negative regulator of axonal outgrowth by regulating the strength of the molecular clutch between N-cadherin and the actin cytoskeleton.
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http://dx.doi.org/10.7554/eLife.51822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946565PMC
January 2020

miRNA-Dependent Control of Homeostatic Plasticity in Neurons.

Front Cell Neurosci 2019 5;13:536. Epub 2019 Dec 5.

University of Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France.

Homeostatic plasticity is a form of plasticity in which neurons compensate for changes in neuronal activity through the control of key physiological parameters such as the number and the strength of their synaptic inputs and intrinsic excitability. Recent studies revealed that miRNAs, which are small non-coding RNAs repressing mRNA translation, participate in this process by controlling the translation of multiple effectors such as glutamate transporters, receptors, signaling molecules and voltage-gated ion channels. In this review, we present and discuss the role of miRNAs in both cell-wide and compartmentalized forms of homeostatic plasticity as well as their implication in pathological processes associated with homeostatic failure.
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http://dx.doi.org/10.3389/fncel.2019.00536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906196PMC
December 2019

TSPAN5 Enriched Microdomains Provide a Platform for Dendritic Spine Maturation through Neuroligin-1 Clustering.

Cell Rep 2019 10;29(5):1130-1146.e8

CNR, Institute of Neuroscience, Milan 20129, Italy. Electronic address:

Tetraspanins are a class of evolutionarily conserved transmembrane proteins with 33 members identified in mammals that have the ability to organize specific membrane domains, named tetraspanin-enriched microdomains (TEMs). Despite the relative abundance of different tetraspanins in the CNS, few studies have explored their role at synapses. Here, we investigate the function of TSPAN5, a member of the tetraspanin superfamily for which mRNA transcripts are found at high levels in the mouse brain. We demonstrate that TSPAN5 is localized in dendritic spines of pyramidal excitatory neurons and that TSPAN5 knockdown induces a dramatic decrease in spine number because of defects in the spine maturation process. Moreover, we show that TSPAN5 interacts with the postsynaptic adhesion molecule neuroligin-1, promoting its correct surface clustering. We propose that membrane compartmentalization by tetraspanins represents an additional mechanism for regulating excitatory synapses.
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http://dx.doi.org/10.1016/j.celrep.2019.09.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899445PMC
October 2019

A super-resolution platform for correlative live single-molecule imaging and STED microscopy.

Nat Methods 2019 12 21;16(12):1263-1268. Epub 2019 Oct 21.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.

Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative single-molecule imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.
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http://dx.doi.org/10.1038/s41592-019-0611-8DOI Listing
December 2019

A Spatiotemporal Sequence of Sensitization to Slits and Semaphorins Orchestrates Commissural Axon Navigation.

Cell Rep 2019 Oct;29(2):347-362.e5

University of Lyon, University of Lyon 1 Claude Bernard Lyon1, NeuroMyoGene Institute, CNRS UMR5310, INSERM U1217, 8 Avenue Rockefeller, 69008 Lyon, France. Electronic address:

Accurate perception of guidance cues is crucial for cell and axon migration. During initial navigation in the spinal cord, commissural axons are kept insensitive to midline repellents. Upon midline crossing in the floor plate, they switch on responsiveness to Slit and Semaphorin repulsive signals and are thus propelled away and prevented from crossing back. Whether and how the different midline repellents control specific aspects of this navigation remain to be elucidated. We set up a paradigm for live-imaging and super-resolution analysis of PlexinA1, Neuropilin-2, and Robo1/2 receptor dynamics during commissural growth cone navigation in chick and mouse embryos. We uncovered a remarkable program of sensitization to midline cues achieved by unique spatiotemporal sequences of receptor allocation at the growth-cone surface that orchestrates receptor-specific growth-cone behavior changes. This reveals post-translational mechanisms whereby coincident guidance signals are temporally resolved to allow the generation of specific guidance responses.
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http://dx.doi.org/10.1016/j.celrep.2019.08.098DOI Listing
October 2019

Differential role of pre- and postsynaptic neurons in the activity-dependent control of synaptic strengths across dendrites.

PLoS Biol 2019 06 5;17(6):e2006223. Epub 2019 Jun 5.

RIKEN Center for Brain Science, Wako, Saitama, Japan.

Neurons receive a large number of active synaptic inputs from their many presynaptic partners across their dendritic tree. However, little is known about how the strengths of individual synapses are controlled in balance with other synapses to effectively encode information while maintaining network homeostasis. This is in part due to the difficulty in assessing the activity of individual synapses with identified afferent and efferent connections for a synapse population in the brain. Here, to gain insights into the basic cellular rules that drive the activity-dependent spatial distribution of pre- and postsynaptic strengths across incoming axons and dendrites, we combine patch-clamp recordings with live-cell imaging of hippocampal pyramidal neurons in dissociated cultures and organotypic slices. Under basal conditions, both pre- and postsynaptic strengths cluster on single dendritic branches according to the identity of the presynaptic neurons, thus highlighting the ability of single dendritic branches to exhibit input specificity. Stimulating a single presynaptic neuron induces input-specific and dendritic branchwise spatial clustering of presynaptic strengths, which accompanies a widespread multiplicative scaling of postsynaptic strengths in dissociated cultures and heterosynaptic plasticity at distant synapses in organotypic slices. Our study provides evidence for a potential homeostatic mechanism by which the rapid changes in global or distant postsynaptic strengths compensate for input-specific presynaptic plasticity.
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http://dx.doi.org/10.1371/journal.pbio.2006223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6576792PMC
June 2019

Biophysical mechanisms underlying the membrane trafficking of synaptic adhesion molecules.

Neuropharmacology 2020 06 1;169:107555. Epub 2019 Mar 1.

Univ. Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000, Bordeaux, France; CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000, Bordeaux, France. Electronic address:

Adhesion proteins play crucial roles at synapses, not only by providing a physical trans-synaptic linkage between axonal and dendritic membranes, but also by connecting to functional elements including the pre-synaptic neurotransmitter release machinery and post-synaptic receptors. To mediate these functions, adhesion proteins must be organized on the neuronal surface in a precise and controlled manner. Recent studies have started to describe the mobility, nanoscale organization, and turnover rate of key synaptic adhesion molecules including cadherins, neurexins, neuroligins, SynCAMs, and LRRTMs, and show that some of these proteins are highly mobile in the plasma membrane while others are confined at sub-synaptic compartments, providing evidence for different regulatory pathways. In this review article, we provide a biophysical view of the diffusional trapping of adhesion molecules at synapses, involving both extracellular and intracellular protein interactions. We review the methodology underlying these measurements, including biomimetic systems with purified adhesion proteins, means to perturb protein expression or function, single molecule imaging in cultured neurons, and analytical models to interpret the data. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.
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http://dx.doi.org/10.1016/j.neuropharm.2019.02.037DOI Listing
June 2020

A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation.

Nat Commun 2018 09 28;9(1):3979. Epub 2018 Sep 28.

Interdisciplinary Institute for Neuroscience, UMR 5297, Univ. Bordeaux, F-33000, Bordeaux, France.

To better understand the molecular mechanisms by which early neuronal connections mature into synapses, we examined the impact of neuroligin-1 (Nlg1) phosphorylation on synapse differentiation, focusing on a unique intracellular tyrosine (Y782), which differentially regulates Nlg1 binding to PSD-95 and gephyrin. By expressing Nlg1 point mutants (Y782A/F) in hippocampal neurons, we show using imaging and electrophysiology that Y782 modulates the recruitment of functional AMPA receptors (AMPARs). Nlg1-Y782F impaired both dendritic spine formation and AMPAR diffusional trapping, but not NMDA receptor recruitment, revealing the assembly of silent synapses. Furthermore, replacing endogenous Nlg1 with either Nlg1-Y782A or -Y782F in CA1 hippocampal neurons impaired long-term potentiation (LTP), demonstrating a critical role of AMPAR synaptic retention. Screening of tyrosine kinases combined with pharmacological inhibitors point to Trk family members as major regulators of endogenous Nlg1 phosphorylation and synaptogenic function. Thus, Nlg1 tyrosine phosphorylation signaling is a critical event in excitatory synapse differentiation and LTP.
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http://dx.doi.org/10.1038/s41467-018-06220-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162332PMC
September 2018

Pre-post synaptic alignment through neuroligin-1 tunes synaptic transmission efficiency.

Elife 2018 07 25;7. Epub 2018 Jul 25.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, UMR 5297, F-33000, Bordeaux, France.

The nanoscale organization of neurotransmitter receptors regarding pre-synaptic release sites is a fundamental determinant of the synaptic transmission amplitude and reliability. How modifications in the pre- and post-synaptic machinery alignments affects synaptic currents, has only been addressed with computer modelling. Using single molecule super-resolution microscopy, we found a strong spatial correlation between AMPA receptor (AMPAR) nanodomains and the post-synaptic adhesion protein neuroligin-1 (NLG1). Expression of a truncated form of NLG1 disrupted this correlation without affecting the intrinsic AMPAR organization, shifting the pre-synaptic release machinery away from AMPAR nanodomains. Electrophysiology in dissociated and organotypic hippocampal rodent cultures shows these treatments significantly decrease AMPAR-mediated miniature and EPSC amplitudes. Computer modelling predicts that ~100 nm lateral shift between AMPAR nanoclusters and glutamate release sites induces a significant reduction in AMPAR-mediated currents. Thus, our results suggest the synapses necessity to release glutamate precisely in front of AMPAR nanodomains, to maintain a high synaptic responses efficiency.
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http://dx.doi.org/10.7554/eLife.31755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070337PMC
July 2018

Dynamics, nanoscale organization, and function of synaptic adhesion molecules.

Mol Cell Neurosci 2018 09 17;91:95-107. Epub 2018 Apr 17.

Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, 33077 Bordeaux, France; Interdisciplinary Institute for Neuroscience, University of Bordeaux, 33077 Bordeaux, France. Electronic address:

Synaptic adhesion molecules not only provide a physical link between pre- and post-synaptic membranes, but also contribute to synaptic differentiation and plasticity by organizing functional elements, in particular neurotransmitter receptors. The wealth of existing adhesive protein families including many isoforms and splice variants, calls for systematic identification of the levels and exchange rates of each of those protein members at specific synapse types. Complementary to electron microscopy to identify individual synaptic contacts and biochemistry to reveal protein-protein interactions, recent super-resolution light microscopy methods combined with appropriate fluorescent labeling provide a way to measure the dynamics and sub-micron organization of selective molecular components, and their inter-relations at the synapse. In this review, we summarize current knowledge on the dynamics, nanoscale localization, and function of key synaptic adhesion complexes.
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http://dx.doi.org/10.1016/j.mcn.2018.04.007DOI Listing
September 2018

A Triad of Crystals Sheds Light on MDGA Interference with Neuroligation.

Neuron 2017 Aug;95(4):729-732

Centre National de la Recherche Scientifique, Aix-Marseille Université, "Architecture et Fonction des Macromolécules Biologiques" laboratory, Faculté des Sciences de Luminy, 13288 Marseille, France. Electronic address:

Neurexins and neuroligins form trans-synaptic complexes that promote synapse development. In this issue of Neuron, Aricescu and colleagues (Elegheert et al., 2017) complement and strengthen two recent reports by the Kim and Rudenko teams (Kim et al., 2017; Gangwar et al., 2017) to dissect the molecular determinants by which MDGAs challenge the neurexin-neuroligin partnership.
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http://dx.doi.org/10.1016/j.neuron.2017.08.001DOI Listing
August 2017

Defective Gpsm2/Gα signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome.

Nat Commun 2017 04 7;8:14907. Epub 2017 Apr 7.

INSERM, Neurocentre Magendie, U1215, 146 rue Leo-Saignat, F-33077 Bordeaux, France.

Mutations in GPSM2 cause Chudley-McCullough syndrome (CMCS), an autosomal recessive neurological disorder characterized by early-onset sensorineural deafness and brain anomalies. Here, we show that mutation of the mouse orthologue of GPSM2 affects actin-rich stereocilia elongation in auditory and vestibular hair cells, causing deafness and balance defects. The G-protein subunit Gα, a well-documented partner of Gpsm2, participates in the elongation process, and its absence also causes hearing deficits. We show that Gpsm2 defines an ∼200 nm nanodomain at the tips of stereocilia and this localization requires the presence of Gα, myosin 15 and whirlin. Using single-molecule tracking, we report that loss of Gpsm2 leads to decreased outgrowth and a disruption of actin dynamics in neuronal growth cones. Our results elucidate the aetiology of CMCS and highlight a new molecular role for Gpsm2/Gα in the regulation of actin dynamics in epithelial and neuronal tissues.
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http://dx.doi.org/10.1038/ncomms14907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5385604PMC
April 2017

Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.

Nat Protoc 2017 04 9;12(4):748-763. Epub 2017 Mar 9.

Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, Bordeaux, France.

Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.
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http://dx.doi.org/10.1038/nprot.2017.010DOI Listing
April 2017

Synaptogenic Assays Using Neurons Cultured on Micropatterned Substrates.

Methods Mol Biol 2017 ;1538:29-44

Interdisciplinary Institute for Neuroscience, University of Bordeaux, UMR 5297, 146 rue Leo Saignat, F-33000, Bordeaux, France.

One of the difficulties for studying the mechanisms of synaptogenesis stems from the spatial unpredictability of contact formation between neurons, and the involvement of many parallel adhesive pathways mediating axon/dendrite recognition. To circumvent these limitations, we describe here a method allowing the investigation of synaptic contacts at controlled locations with high precision and statistics. Specifically, primary neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with purified synaptogenic adhesion molecules. Coating the substrates with the homophilic adhesion molecule SynCAM triggers the formation of functional presynaptic structures in axons, while neurexin elicits postsynapses in dendrites from neurons expressing the counter receptor neuroligin. This assay can be combined with various imaging techniques including immunocytochemistry to screen the accumulation of synaptic components, long-term live cell recordings to probe the kinetics of neurite growth and synapse differentiation, as well as high resolution single molecule tracking.
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http://dx.doi.org/10.1007/978-1-4939-6688-2_3DOI Listing
January 2018

Nanoscale organization of synaptic adhesion proteins revealed by single-molecule localization microscopy.

Neurophotonics 2016 Oct 3;3(4):041810. Epub 2016 Nov 3.

Centre National de la Recherche Scientifique, Interdisciplinary Institute for Neuroscience, UMR 5297, 147 rue Léo-Saignat, Bordeaux Cedex 33077, France; University of Bordeaux, Interdisciplinary Institute for Neuroscience, 147 rue Léo-Saignat, Bordeaux Cedex 33077, France.

The advent of superresolution imaging has created a strong need for both optimized labeling strategies and analysis methods to probe the nanoscale organization of complex biological structures. We present a thorough description of the distribution of synaptic adhesion proteins at the nanoscopic scale, namely presynaptic neurexin-[Formula: see text] ([Formula: see text]), and its two postsynaptic binding partners neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2). We monitored these proteins in the membrane of neurons by direct stochastic optical reconstruction microscopy, after live surface labeling with Alexa647-conjugated monomeric streptavidin. The small probe ([Formula: see text]) efficiently penetrates into crowded synaptic junctions and reduces the distance to target. We quantified the organization of the single-molecule localization data using a tesselation-based analysis technique. We show that Nlg1 exhibits a fairly disperse organization within dendritic spines, while LRRTM2 is organized in compact domains, and [Formula: see text] in presynaptic terminals displays a dual-organization pattern intermediate between that of Nlg1 and LRRTM2. These results suggest that part of [Formula: see text] interacts transsynaptically with Nlg1 and the other part with LRRTM2.
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http://dx.doi.org/10.1117/1.NPh.3.4.041810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5093229PMC
October 2016

Molecular determinants for the strictly compartmentalized expression of kainate receptors in CA3 pyramidal cells.

Nat Commun 2016 Sep 27;7:12738. Epub 2016 Sep 27.

Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, University of Bordeaux, 146 rue Léo-Saignat, F-33076 Bordeaux, France.

Distinct subtypes of ionotropic glutamate receptors can segregate to specific synaptic inputs in a given neuron. Using functional mapping by focal glutamate uncaging in CA3 pyramidal cells (PCs), we observe that kainate receptors (KARs) are strictly confined to the postsynaptic elements of mossy fibre (mf) synapses and excluded from other glutamatergic inputs and from extrasynaptic compartments. By molecular replacement in organotypic slices from GluK2 knockout mice, we show that the faithful rescue of KAR segregation at mf-CA3 synapses critically depends on the amount of GluK2a cDNA transfected and on a sequence in the GluK2a C-terminal domain responsible for interaction with N-cadherin. Targeted deletion of N-cadherin in CA3 PCs greatly reduces KAR content in thorny excrescences and KAR-EPSCs at mf-CA3 synapses. Hence, multiple mechanisms combine to confine KARs at mf-CA3 synapses, including a stringent control of the amount of GluK2 subunit in CA3 PCs and the recruitment/stabilization of KARs by N-cadherins.
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http://dx.doi.org/10.1038/ncomms12738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052629PMC
September 2016

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin.

Nat Commun 2016 Mar 16;7:10773. Epub 2016 Mar 16.

Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, 33077 Bordeaux, France.

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures.
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http://dx.doi.org/10.1038/ncomms10773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4799371PMC
March 2016

The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors.

Neuron 2015 Aug;87(4):764-80

VIB Center for the Biology of Disease, 3000 Leuven, Belgium; Center for Human Genetics, KU Leuven, 3000 Leuven, Belgium. Electronic address:

The formation, function, and plasticity of synapses require dynamic changes in synaptic receptor composition. Here, we identify the sorting receptor SorCS1 as a key regulator of synaptic receptor trafficking. Four independent proteomic analyses identify the synaptic adhesion molecule neurexin and the AMPA glutamate receptor (AMPAR) as major proteins sorted by SorCS1. SorCS1 localizes to early and recycling endosomes and regulates neurexin and AMPAR surface trafficking. Surface proteome analysis of SorCS1-deficient neurons shows decreased surface levels of these, and additional, receptors. Quantitative in vivo analysis of SorCS1-knockout synaptic proteomes identifies SorCS1 as a global trafficking regulator and reveals decreased levels of receptors regulating adhesion and neurotransmission, including neurexins and AMPARs. Consequently, glutamatergic transmission at SorCS1-deficient synapses is reduced due to impaired AMPAR surface expression. SORCS1 mutations have been associated with autism and Alzheimer disease, suggesting that perturbed receptor trafficking contributes to synaptic-composition and -function defects underlying synaptopathies.
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http://dx.doi.org/10.1016/j.neuron.2015.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692362PMC
August 2015

Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones.

Proc Natl Acad Sci U S A 2015 Jun 18;112(22):6997-7002. Epub 2015 May 18.

Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique (CNRS), University of Bordeaux, 33077 Bordeaux, France;

Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin-coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N-cadherin construct competing for the coupling between actin and endogenous N-cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N-cadherin, and further revealed a 20% actin population confined at N-cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N-cadherin and actin turnover well, validating this mechanism. Such a combination of short- and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration.
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http://dx.doi.org/10.1073/pnas.1423455112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4460488PMC
June 2015

Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines.

Mol Biol Cell 2015 Mar 7;26(5):859-73. Epub 2015 Jan 7.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Unité Mixte de Recherche 5297, F-33000 Bordeaux, France Interdisciplinary Institute for Neuroscience, Centre Nationale de la Recherche Scientifique, Unité Mixte de Recherche 5297, F-33000 Bordeaux, France

The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin-coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain.
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http://dx.doi.org/10.1091/mbc.E14-06-1086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342023PMC
March 2015

Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion.

EMBO J 2014 Dec 7;33(23):2745-64. Epub 2014 Oct 7.

Interdisciplinary Institute for Neuroscience, University Bordeaux UMR 5297, Bordeaux, France CNRS, Interdisciplinary Institute for Neuroscience UMR 5297, Bordeaux, France

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity.
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http://dx.doi.org/10.15252/embj.201488837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282554PMC
December 2014

miR-92a regulates expression of synaptic GluA1-containing AMPA receptors during homeostatic scaling.

Nat Neurosci 2014 Aug 13;17(8):1040-2. Epub 2014 Jul 13.

1] University of Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France. [2] CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, Bordeaux, France. [3].

We investigated whether microRNAs could regulate AMPA receptor expression during activity blockade. miR-92a strongly repressed the translation of GluA1 receptors by binding the 3' untranslated region of rat GluA1 (also known as Gria1) mRNA and was downregulated in rat hippocampal neurons after treatment with tetrodotoxin and AP5. Deleting the seed region in GluA1 or overexpressing miR-92a blocked homeostatic scaling, indicating that miR-92a regulates the translation and synaptic incorporation of new GluA1-containing AMPA receptors.
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http://dx.doi.org/10.1038/nn.3762DOI Listing
August 2014

Micropatterned substrates coated with neuronal adhesion molecules for high-content study of synapse formation.

Nat Commun 2013 ;4:2252

University of Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux, France.

Studying the roles of different proteins and the mechanisms involved in synaptogenesis is hindered by the complexity and heterogeneity of synapse types, and by the spatial and temporal unpredictability of spontaneous synapse formation. Here we demonstrate a robust and high-content method to induce selectively presynaptic or postsynaptic structures at controlled locations. Neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with various synaptogenic adhesion molecules. When plated on neurexin-1β-coated micropatterns, neurons expressing neuroligin-1 exhibit specific dendritic organization and selective recruitment of the postsynaptic scaffolding molecule PSD-95. Furthermore, functional AMPA receptors are trapped at neurexin-1β dots, as revealed by live-imaging experiments. In contrast, neurons plated on SynCAM1-coated substrates exhibit strongly patterned axons and selectively assemble functional presynapses. N-cadherin coating, however, is not able to elicit synapses, indicating the specificity of our system. This method opens the way to both fundamental and therapeutic studies of various synaptic systems.
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http://dx.doi.org/10.1038/ncomms3252DOI Listing
April 2014

Assembly of synapses: biomimetic assays to control neurexin/neuroligin interactions at the neuronal surface.

Curr Protoc Neurosci 2013 Jul;Chapter 2:Unit 2.19

Université Bordeaux, Bordeaux Imaging Center, Bordeaux, France.

The role of adhesion molecules in the assembly of synapses in the nervous system is an important issue. To characterize the role of neurexin/neuroligin adhesion complexes in synapse differentiation, various imaging assays can be performed in primary hippocampal cultures. First, to temporally control contact formation, biomimetic assays can be performed using microspheres coated with purified neurexin or with antibody clusters that aggregate neurexin. These models are combined with live fluorescence imaging to study the dynamics of accumulation of post-synaptic components, including scaffolding molecules and glutamate receptors. To demonstrate that AMPA receptors can be recruited to nascent neurexin/neuroligin contacts through lateral diffusion, the mobility of AMPA receptors in the neuronal membrane is monitored by tracking individual quantum dots (QDs) conjugated to antibodies against AMPA receptors. Experiments monitoring the attachment and detachment of Nrx-coated QDs to measure the rates of neurexin/neuroligin interaction can also be performed. Each of these assays is detailed in this unit.
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http://dx.doi.org/10.1002/0471142301.ns0219s64DOI Listing
July 2013