Molecular and cellular mechanisms underlying neural synapse formation and specialization based on the cell adhesion molecules neurexins, neuroligins, and LRRTMs – SynAdh

The team of O. Thoumine (IINS, Bordeaux) contributes expertise in the biology of synaptic adhesion molecules, culture of neurons, high-resolution imaging, informatics simulation, and electrophysiology. The team of M. Sainlos (IINS, Bordeaux) contributes expertise in the design, production and functional characterization of peptides able to modulate interaction of the cell adhesion molecules with their extra and intracellular partners. The team of P. Marchot and Y. Bourne (Marseille) contribute expertise in the biochemical, pharmacological and structural characterization of adhesion proteins and the design and functional characterization of peptides aimed at competing for their extracellular interactions.

• The affinity of LRRTM2 for the neurexins increases proportionally with their length, a feature that assigns a primary, ‘catcher’ role to their LNS6 domain and a secondary, ‘stabilizer’ role to the LNS1-5/EGF1-3 domains for promoting trans-synaptic partnerships.

• Monomeric streptavidin permits to label synaptic adhesion molecules for super-resolution imaging, by combining specificity, accessibility, absence of reticulation, and short distance relative to the target protein (Chamma et al, Nat Comm 2016; Neurophotonics in revision 2016; Nature Protocols, submitted). This new tool has allowed us to initiate several collaborations aimed to study the membrane distribution of several synaptic proteins (TSPN5 with Maria Passafaro, Milan; SynCAM with T. Biederer, Boston; kainate receptor with C. Mulle, Bordeaux).

• Being inspired by these molecular dynamics approaches we developed a software based on real time calculation of the positions of thousands of individual molecules, thus permitting modeling of the fluorescence imaging experiments, particularly in super-resolution conditions (Lagardère et al, in preparation). We have been in contact with several private companies (ExploraNova, Nikon, Leica) in order to launch this software in the market, to help experimenters in the design and interpretation of imaging experiments. The software will also be useful for educational purposes, with students in bioimaging techniques as well as the general public.

• A combination of immunocytochemistry and electrophysiology experiments led us show that a single tyrosine residue within the intracellular domain of NLGN1 is determinant for regulation of excitatory versus inhibitory synapse formation (Letellier et al., in preparation).

• Dimeric peptides that compete with interaction of NLGN1 with its scaffolding proteins (PSD-95, S-SCAM) appear to selectively perturb excitatory synapse assembly.

We will continue to develop new orthogonal probes based on monomeric streptavidin to label synaptic membrane proteins, with the aim to establish a super-resolution multi-color map of the synaptic cleft (project of Ingrid Chamma, CR2 candidate at the CNRS in 2017). A new collaboration has been initiated between M. Sainlos and U. Rothbauer (Tuebingen) in order to use their new VhH BC2T for multi-spectral labeling.

We will also exploit this approach to study the membrane trafficking of adhesion molecules bearing autism-linked mutations, in particular the MDGA proteins, described as new regulators of the neurexin-neuroligin interaction (consortium ERA-NET Neuron 2015).

Being inspired by the single molecule pull-down approaches learned at UC Berkeley, with Vincent Studer (IINS) and as a partnership with the Alveole et Nikon companies, we are developing a new technical facility permitting detection of individual molecules tethered on a substrate, their stoichiometries and their interactions (funding: Labex BRAIN Transfert).

Finally, we will extend our physiology approaches to study the influence of the optogenetically generated synaptic activity onto the synaptic adhesion dynamics in brain slices (recruitment for a CR1 position at the CNRS in 2016 of M. Letellier, initially a laureate of the “ANR Retour-Post-doctorant “SynSpe””). This approach will be coupled to in-depth imaging by the SoSPIM technique, in collaboration with the team of JB Sibarita (Bordeaux).

. CHAMMA I, LETELLIER M, Butler, TESSIER B, Lim KH, GAUTHEREAU I, Choquet D, Sibarita JB, Park S, SAINLOS M*, THOUMINE O* (2016). Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin. Nature Commun, 7:10773. (*Co-last authors).
Highlight in Nature Methods 13, 290 (2016).

2. CHAMMA I, Levet F, Sibarita JB, SAINLOS M, THOUMINE O. Nanoscale organization of synaptic adhesion proteins revealed by single molecule localization microscopy. Neurophotonics, in revision.

3. CHAMMA I, Rossier O., Giannone G., THOUMINE O*, M. SAINLOS*. (*Co-last authors). Optimized labeling of membrane proteins in confined cellular environments using monomeric streptavidin: applications to super-resolution imaging. Nature Protocols, submitted.

We have set up a new technique for labeling of synaptic adhesion molecules, based on the replacement of endogenous proteins by new molecules bearing a N-terminal peptidic tag of 15 amino acid residues, permitting the covalent addition of a biotine moiety via an enzymatic reaction catalyzed by biotine ligase. The biotine moiety is then labeled with a small probe, monomeric streptavidin, conjugated with an organic fluorophore. This label is compatible with most super-resolution techniques and permits unprecedented visualization of proteins in the synaptic cleft.

4. LETELLIER M, CHAMMA I, SAPHY C, PAPASIDERI I, TESSIER B, SAINLOS M, CZÖNDÖR K, THOUMINE O. A unique tyrosine residue in the intracellular domain of neuroligin-1 regulates excitatory versus inhibitory synapse differentiation. In preparation.

By using NLGN1 point mutants expressed in cultured neurons along with a combination of biochemistry, immunocytochemistry and electrophysiology approaches, we show that a single tyrosine residue within the intracellular domain of NLGN1 is determinant for regulation of excitatory versus inhibitory synapse formation.

Assembly of the myriad of synapses enabling communication between neurons is a crucial process of the CNS development and dictates the generation, maintenance and functioning of neural circuitries. Moreover, the function and specificity of synapses make them the locus of expression of most neurological disorders. This project aims at better characterizing the molecular and cellular mechanisms underlying synapse formation, maturation and specification.

At the level of individual axon/dendrite contacts, synaptogenesis is a complex process initiated by recognition of specific adhesion proteins and followed by the recruitment of scaffolding molecules and functional receptor-channels. Among cell-adhesion molecules, the neurexins (NRXN), neuroligins (NLGN) and leucine-rich repeat transmembrane proteins (LRRTM) are implicated in synapse formation, differentiation and functional validation. In the mammalian brain, specific recognition between isoforms and splice variants of these molecules dictate the formation of, mainly : i) excitatory synapses relying on presynaptic glutamate release in front of AMPARs and NMDARs, which are stabilized at the postsynapse by NLGN1, LRRTM2, and PDZ domain-containing scaffolding molecules such as PSD-95; ii) inhibitory synapses relying on presynaptic GABA release, which activates GABARs stabilized by NLGN2 and scaffolding molecules such as gephyrin. Pathological mutations in the NRXN and NLGN genes are related to autism, X-linked mental retardation and schizophrenia, supporting the need of studying how adhesion molecules modulate synapse formation and functioning. However, the mechanisms by which these molecules, besides maintaining together axonal and dendritic membranes, dynamically assemble functional pre- and postsynaptic elements are still unclear.

The study of synaptogenesis is hindered by the limited spatial resolution of conventional microscopy and the lack of selective molecules able to promote or perturb synapse formation. This project is aimed at overcoming these limitations by developing i) new strategies for protein labeling with small fluorescent probes combined with super-resolution imaging, ii) new peptidic ligands designed after the 3D structures of the extra- and intracellular domains of the adhesion proteins and to be used as interaction modulators. This project should lead to a better understanding of the role of adhesion molecules in the assembly of synapses and to the rational design of new therapeutic agents alleviating neurodevelopmental disorders.

To address this multi-disciplinary and ambitious project, we built a consortium of three teams with complementary expertise. The first two teams, of O. Thoumine and M. Sainlos from the same institute (IINS, Bordeaux), have been collaborating for several years and interact on a day-to-day basis. They will bring their respective expertise in the biology of synaptic adhesion molecules, cell culture systems, high-resolution imaging and electrophysiology, and in the design and production of peptides mimicking the interaction of adhesion molecules to their extra- and intracellular partners. The third team associates P. Marchot, a biochemist and pharmacologist, and Yves Bourne, a structural biologist, who have strong collaborative records and now work in the same lab (AFMB, Marseille). They will add their complementary expertise in documenting the structure-function relationships of various ligand-receptor and protein-protein complexes and their functional implications.

Our program involves three main collaborative tasks:
Task 1. Structure-function analysis of the extracellular NRXN/LRRTM complexes and design of peptidic effectors of the NRXN interactions with NLGN and LRRTM
Task 2. Intracellular interactions of the NRXNs, NLGNs and LRTTMs with scaffolding molecules, characterization of novel peptidic effectors, and high-resolution microscopy of the complexes
Task 3: Signaling and other functions mediated by the NRXNs, NLGNs and LRRTMs