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Neuroligation et spécificité synaptique

Contrôle de l’équilibre excitation/inhibition synaptique par phosphorylation tyrosine de la neuroligine

Le 18 juillet 2013

Neurexin-1ß Binding to Neuroligin-1 Triggers the Preferential Recruitment of PSD-95 versus Gephyrin through Tyrosine Phosphorylation of Neuroligin-1. 
Giannone G, Mondin M, Grillo-Bosch D, Tessier B, Saint-Michel E, Czöndör K, Sainlos M, Choquet D, Thoumine O.
Cell Rep. 2013 Jun 112013 
Equipe d'Olivier Thoumine, IINS
Equipe de Daniel Choquet

Olivier Thoumine what is the biological paradigm underlying this study?
This study just published in Cell Reports started several years ago, when Grégory Giannone joined the team of Daniel Choquet after a very productive post-doctoral stay in the laboratory of Mike Sheetz, a specialist of mechanotransduction. Gregory had worked on the role of integrin adhesion molecules in cell locomotion using fibroblast cell models. In these cells, integrins display a very specific response to ligands from the extracellular matrix such as fibronectin. Upon ligand binding, these heterodimeric adhesion molecules trigger phosphotyrosine signaling cascades that allow a strengthening of their connection to the underlying actin cytoskeleton (Giannone and Sheetz Trends Cell Biol 2006). The sensing of the extracellular matrix rigidity, generally referred to as mechanotransduction, may play important roles in developmental and pathological situations such as tumor progression.

Receptor multimerization versus ligand activation 

When Gregory joined the team of Daniel Choquet, he wanted to understand whether neuronal adhesion molecules could also display such a a ligand-activated behavior. He focused on the trans-synaptic neurexin/neuroligin adhesion complex. Neurexin is mostly pre-synaptic and binds through its extracellular domain to neuroligin situated in the post-synaptic membrane. Neurexins and neuroligins exist in a variety of isoforms and splice variants, and specific combinations between these molecules are thought to guide the formation of either excitatory or inhibitory synapses. One striking property of these molecules is their ability to trigger synapse formation. For example, when expressed in co-cultured heterologous cells, or presented on microspheres as purified proteins, Nlg1 or Nrx1β induce pre- and post-synapses in contacting neurites, respectively (Scheiffele et al., Cell 2000; Graf et al., Cell 2004; Nam and Chen, PNAS 2005). These reports suggest that the recruitment of scaffolding molecules including gephyrin and PSD-95 at inhibitory and excitatory synapses, respectively, depended mainly on Nlg1 aggregation. However, other studies supported more complex activity-dependent signaling mechanisms associated with Nrx1β/Nlg1 functions. We tested here the hypothesis that Nrx1β binding to Nlg1 could selectively regulate Nlg1 association to scaffolding molecules.

A ligand-binding activation of neuroligin-1
Gregory started to use Quantum dots (Qdots) coated with neurexin to follow the mobility of individual neuroligin molecules in the neuronal membrane. He found that neuroligin molecules often stopped at synapses, similarly to what was described for AMPA receptors in the group of Daniel Choquet (Bats et al., Neuron 2007). Specifically, neuroligin immobilization at synapses relied on the interaction between the C-terminal PDZ domain binding motif of Nlg1 and the scaffolding protein at excitatory synapses, PSD-95. He then compared the behavior of Qdots coated with ‘non activating’ antibodies directed to an N-terminal HA tag on neuroligin 1. These Qdots were much more diffusive, and stopped more seldom at synapses than Qdots coated with neurexin. This suggested that neurexin binding to neuroligin-1 could somehow trigger the anchoring of neuroligin-1 to PSD-95. Strikingly, addition of purified soluble neurexin immobilized anti-HA coated Qdots within minutes, suggesting that the ligand activation of neuroligin was a fairly rapid mechanism.

 Identification of the neuroligin-1 tyrosine phosphorylation
We then designed another assay based on the aggregation of neuroligin-1 using either purified neurexin or anti-HA antibody cross-linked with secondary antibodies Similarly to the Qdot results, we showed that PSD-95 was recruited to neurexin clusters, within 30 min, but not to clusters made with anti-HA antibodies. The large cluster statistics obtained allowed a broad screening of the underlying activation mechanism. We found that pharmacological inhibitors of tyrosine kinases and phosphatases modified the recruitment of PSD-95 by neurexin clusters. We thus decided to focus on a single tyrosine residue (Y782) located in the intracellular tail of neuroligin-1, and to test if its phosphorylation state could regulate neuroligin function. This tyrosine belongs to a consensus region conserved among all Nlgs, and critical for binding to gephyrin, a major scaffold at inhibitory synapses. After considerable efforts in biochemistry, Béatrice Tessier, our team engineer, showed that Nlg1 could be phosphorylated at residue Y782.


Neuroligin-1 tyrosine phosphorylation controls the balance between excitatory and inhibitory synapses
Furthermore, Matthieu Sainlos and Dolors Grillo, bio-organic chemists in the team of Daniel Choquet, showed using pull-down of purified gephyrin by Nlg1 peptides in vitro that phosphorylation of Y782 prevented gephyrin binding. They further designed two Nlg1 tyrosine point mutants (Y782F and Y782A) that either bound gephyrin or not, thus mimicking the effect of non-phosphorylated and phosphorylated Nlg1, respectively. Magali Mondin, post-doc in the team, then expressed those Nlg1 point mutants in neurons and measured the recruitment of endogenous PSD-95 and gephyrin at neurexin clusters and actual synapses. Her results showed that Y782 phosphorylation controls the preferential binding of Nlg1 to PSD-95 versus gephyrin, and the formation of inhibitory and excitatory synapses accordingly. We thus propose that ligand-induced changes in the Nlg1 phosphotyrosine level control the balance between excitatory and inhibitory scaffold assembly during synapse formation and stabilization.

Consequences of this new concept
This strongly collaborative study shows that Nrx1β adhesion to Nlg1 promotes a fast and direct interaction between Nlg1 and PSD-95, not only through receptor clustering, but also involving a specific ligand-binding effect linked to phosphotyrosine signaling. Thus, Nlg1 can be considered, like integrins (Miyamoto et al., Science 1995), or ephrins (Palmer et al., Mol Cell 2002) as a ligand-activated adhesion molecule. This is the first study demonstrating that neuroligin-1 function is regulated by phosphorylation. The next step is to identify the kinases and phosphatases regulating neuroligin-1 tyrosine phosphorylation, and the mechanisms of activation at synapses by neurexin binding. Our data support a competitive model by which unoccupied tyrosine-unphosphorylated Nlg1 binds more to gephyrin, preventing PSD-95 accessibility, whereas ligand-occupied phosphorylated Nlg1 interacts less with gephyrin, and is accessible to PSD-95 binding. This process may play an important role in regulating the balance between excitatory and inhibitory synapse formation. Finally, since neuroligin dysfuntions are associated with autism and mental retardation (Südhof, Nature 2008), pharmacological compounds modifying neuroligins phosphotyrosine level could be envisioned as novel therapeutic strategies against these disorders.

Grégory Giannone (gregory.giannone @
Dernière mise à jour le 01.09.2013