Abstract

Controlling the balance between synaptic excitation and inhibition is crucial for the correct operation of the brain. The trans-synaptic adhesion protein Neuroligin-2 (NL2) plays a crucial role in formation and maintenance of the inhibitory synapse by inducing clustering of the scaffold protein gephyrin and binding LHFPL4, which in concert stabilises the inhibitory GABA-A receptor in the postsynaptic domain. Whereas neural activity is known to regulate trafficking of synaptic receptors, little is known about the role of NL2 dynamics in these processes. Our work is aimed at understanding how brain activity affects NL2 stability at the synapse, and uncovering the role of protein modifications and novel protein interaction partners that regulate its membrane dynamics and endocytic sorting. Ultimately, we seek to understand how NL2 contributes to synapse development and maintenance. We will use a combination of molecular, cell biological, mouse transgenic, imaging (fixed confocal, live and super-resolution) and electrophysiological approaches to determine how NL2 is stabilised at the synapse and how its trafficking (endocytosis, recycling and degradation) is regulated. We will use both dissociated neuronal culture from WT and KO mice, as well as organotypic and acute brain slices that have been genetically manipulated by biolistic (gene gun) transfection or in utero electroporation. Our work will include live cell single molecule tracking of NL2 via Quantum Dots (for which we have developed optimised probes and antibodies) to assess the effect of neural activity and various protein interactions on stabilisation at the synapse and endocytic zones, state-of-the-art super-resolution imaging (SIM, STED, STORM) to determine the positioning of endocytic trafficking compartments and cytoskeletal regulators with respect to the synapse, and patch clamp electrophysiology to study how NL2 stability and trafficking affect inhibitory synapse formation, strength and maintenance.