• Role of neuroligin-1 protein binding motifs

To characterize the dynamics and nanoscale distribution of neuroligin-1, we developed specific labeling strategies relying on small monomeric probes combined with high-resolution fluorescence imaging (uPAINT/STORM in primary neurons, and confocal/STED in brain slices) (Chamma et al. Nat Commun 2016) (Fig. 1). This was achieved in collaboration with M. Sainlos (IINS). To study neuroligin-1 behavior at near endogenous levels, we knocked-down endogenous neuroligin-1 in dissociated or organotypic hippocampal cultures with specific shRNAs, and rescued it by tagged and/or mutated constructs, thereby achieving an approximate 1:1 replacement both at the immunocytochemical and physiological levels. Using this strategy, we were able to introduce mutations or truncations in specific protein binding motifs. For example, the C-terminal truncation of the C-terminal PDZ domain binding motif in neuroligin-1 cause defects in synaptic density and function, and increase the membrane diffusion of neuroligin-1. In a complementary approach, we developed 15 aa divalent neuroligin-1 C-terminal peptides that efficiently compete for the interaction between endogenous neuroligin-1 and PDZ domain-containing scaffolding proteins and impair excitatory synapse differentiation. Finally, we are using similar replacement and labeling strategies to track MAM-domain GPI-Anchored (MDGAs) molecules, which are proteins that bind neuroligins in cis and prevent neurexin binding, and examine their roles on neuroligin-1 function in synapse differentiation (Toledo et al. eLife 2022).

Figure 1. Single molecule localization map of biotinylated AP-neuroligin-1 labeled with Alexa647-conjugated monomeric streptavidin, showing a homogeneous distribution in the dendritic shaft and accumulation at post-synaptic densities (labeled with the Homer1c-GFP reporter in white).

 

• Role of neuroligin-1 tyrosine phosphorylation

A few years ago, we provided evidence that neuroligin-1 was a ligand-activated adhesion molecule, i.e. binding to pre-synaptic Neurexin-1β was able to switch the affinity of neuroligin-1 towards post-synaptic scaffolds, this process being regulated by the phosphorylation of a unique intracellular tyrosine (Y782). By expressing two neuroligin-1 tyrosine point mutants (Y782A/F), we showed the differential recruitment of excitatory versus inhibitory scaffolding proteins, PSD95 versus gephyrin, respectively (Giannone et al. Cell Rep 2013). We then demonstrated that these neuroligin-1 point mutants differentially recruit AMPA receptors and impair long term potentiation (LTP), and identified the tyrosine kinase receptor family responsible for neuroligin-1 phosphorylation (i.e. Trks) (Letellier et al. Nat Commun 2018). Finally, we optogenetically triggered the phosphorylation of native neuroligin-1 using a light-gated tyrosine kinase, allowing for a control of post-synaptic differentiation without affecting neuroligin-1 expression level (Letellier et al. eLife 2020) (Fig. 2). Computer simulations of AMPA receptor diffusional trapping support a model by which neuroligin-1 tyrosine phosphorylation promotes PSD assembly and attracts surface AMPARs, thereby causing a depletion of extra-synaptic AMPA receptor pools that impairs LTP.

Figure 2. (Left) Single cell electroporation of a CA1 pyramidal neuron in an organotypic hippocampal slice with AP-neuroligin-1, and corresponding confocal imaging after labeling with mSA-Atto647. (Middle) Western blot showing the tyrosine phosphorylation of neuroligin-1 upon stimulation of a photoactivatable version of the FGF receptor 1 (optoFGFR1) with 470 nm LEDs. (Right) Dual patch- clamp recordings of CA1 neurons, one being electroporated with optoFGFR1 (green) and a neighboring non-electroporated neuron. Light stimulation of the optoFGFR1 for 24 hr results in larger AMPA receptor mediated EPSCs (blue trace) compared to the control neuron (dark trace).

 

Selected references

• Toledo A, Bimbi G, Letellier M, Tessier B, Daburon S, Favereaux A, Chamma I, Vennekens, Kristel M. Vanderlinden J, Sainlos M, de Wit J, Choquet D, Thoumine O. (2022). MDGAs are fast-diffusing molecules that delay excitatory synapse development by altering neuroligin behavior. eLife 11, e75233.
• Letellier M., Lagardère M., Tessier B., Janovjak H., Thoumine, O. (2020). Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation. Elife 9, e52027.
• Letellier M., Szíber Z., Chamma I., Saphy C., Papasideri I., Tessier B., Sainlos M., Czöndör K., Thoumine O. (2018). A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation. Nat. Commun 9, 3979.
• Chamma I., Thoumine O. (2018). Dynamics, nanoscale organization, and function of synaptic adhesion molecules. Mol Cell Neurosci 91, 95–107.
• Chamma I., Letellier M., Butler C., Tessier B., Lim K.-H., Gauthereau I., Choquet D., Sibarita J.-B., Park S., Sainlos M., Thoumine O. (2016). Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin. Nat. Commun 7, 10773.
• Giannone G., Mondin M., Grillo-Bosch D., Tessier B., Saint-Michel E., Czöndör K., Sainlos M., Choquet D., Thoumine O. (2013). Neurexin-1β binding to neuroligin-1 triggers the preferential recruitment of PSD-95 versus gephyrin through tyrosine phosphorylation of neuroligin-1. Cell Rep. 3, 1996–2007.