ED Sciences de la Vie et de la Santé
Localization, dynamics and nanoscale organization of the synaptic adhesion molecule neuroligin-1.
by Adèle DROUET (Institut Interdisciplinaire de Neurosciences)
The defense will take place at 14h00 - Amphi Centre Broca Nouvelle Aquitaine Centre Broca Nouvelle Aquitaine, 146 rue Léo Saignat 33076 Bordeaux Cedex (France)
in front of the jury composed of
- Aude PANATIER - Directrice de recherche - Université de Bordeaux - Examinateur
- Jean-Louis BESSEREAU - Professeur des universités - praticien hospitalier - Université Claude Bernard Lyon 1 - Rapporteur
- Fabrice ANGO - Directeur de recherche - Université de Montpellier - Rapporteur
- Sabine LéVI - Directrice de recherche - Université Paris Sciences & Lettres - Examinateur
- Jérôme EZAN - Chargé de recherche - Université de Bordeaux - Examinateur
The family of cell adhesion molecules called Neuroligins are essential for synapse development and function, and also the target of deleterious genetic mutations associated with autism spectrum disorders in humans. In this PhD work, I aimed to elucidate the synaptic localization, membrane dynamics, and nanoscale organization of the specific isoform neuroligin-1 (NLGN1). Due to the lack of efficient antibodies for visualizing and purifying endogenous NLGN1, alternative strategies involving gene knock-out, overexpression, or replacement with of NLGN1 with recombinant forms have been developed, leading to controversial results. First, I used such strategies to study the dynamics and nanoscale organization of tagged NLGN1 proteins, demonstrating a significant synaptic residence time that suggests stabilization of NLGN1 at synapses through multiple extracellular and intracellular interactions. Subsequently, I used a new bAP-NLGN1 knock-in mouse line developed in our team, which allows bypassing manipulation of NLGN1 expression levels. In this mouse model, endogenous NLGN1 is fused to a small biotin acceptor peptide (bAP), which can be biotinylated in a cell-specific fashion, then labeled with streptavidin conjugates, which has a very high affinity for biotin. After demonstrating by immunocytochemistry that the insertion of the bAP tag has no effect on the size or number of excitatory and inhibitory synapses formed (compared to a wild-type mouse group), I used this new tool for the purification and visualization of endogenous NLGN1 in dissociated neuron cultures. Through pull-down assays, I showed that NLGN1 can bind other isoforms NLGN2 and NLGN3, as well as PSD-95 and gephyrin, the scaffolding proteins of excitatory and inhibitory synapses, respectively. Using epifluorescence microscopy to visualize endogenous NLGN1, I demonstrated its localization at both excitatory and inhibitory synapses. Finally, using dSTORM super-resolution imaging, I showed that NLGN1 molecules are organized into nanodomains, whose number positively scales with the size of the postsynapse, and which are aligned with presynaptic protein domains (neurexin-1β, RIM1/2). This work thus provides new insights into the localization and nanoscale organization of endogenous NLGN1, challenging the historical view that NLGN1 is specific to excitatory synapses. In the future, this new knock-in mouse model will allow the study of NLGN1 localization in 3D culture models (neurospheres), including in non-neuronal cells (astrocytes). It could also serve as a standard for adjusting the expression level of mutated NLGN1 to that of the endogenous protein, in order to study the impact of these mutations on synaptic organization and function.