Region-Specific Phosphorylation Determines Neuroligin-3 Localization to Excitatory Versus Inhibitory Synapses.

in Biological psychiatry by Bekir Altas, Liam P Tuffy, Annarita Patrizi, Kalina Dimova, Tolga Soykan, Cheryl Brandenburg, Andrea J Romanowski, Julia R Whitten, Colin D Robertson, Saovleak N Khim, Garrett W Crutcher, Mateusz C Ambrozkiewicz, Oleksandr Yagensky, Dilja Krueger-Burg, Matthieu Hammer, He-Hsuan Hsiao, Pawel R Laskowski, Lydia Dyck, Adam C Puche, Marco Sassoè-Pognetto, John J E Chua, Henning Urlaub, Olaf Jahn, Nils Brose, Alexandros Poulopoulos

TLDR

  • The study investigates how a protein called neuroligin-3 is involved in the brain. Neuroligin-3 is important for the development and function of synapses, which are the connections between neurons. The study found that neuroligin-3 is located differently in different parts of the brain and that it is involved in the balance of excitation and inhibition in the brain. The study also found a way to control how neuroligin-3 is located in the brain, which could be useful for treating neurological disorders.

Abstract

Neuroligin-3 is a postsynaptic adhesion molecule involved in synapse development and function. It is implicated in rare, monogenic forms of autism, and its shedding is critical to the tumor microenvironment of gliomas. While other members of the neuroligin family exhibit synapse-type specificity in localization and function through distinct interactions with postsynaptic scaffold proteins, the specificity of neuroligin-3 synaptic localization remains largely unknown. We investigated the synaptic localization of neuroligin-3 across regions in mouse and human brain samples after validating antibody specificity in knockout animals. We raised a phospho-specific neuroligin antibody and used phosphoproteomics, cell-based assays, and in utero CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) knockout and gene replacement to identify mechanisms that regulate neuroligin-3 localization to distinct synapse types. Neuroligin-3 exhibits region-dependent synapse specificity, largely localizing to excitatory synapses in cortical regions and inhibitory synapses in subcortical regions of the brain in both mice and humans. We identified specific phosphorylation of cortical neuroligin-3 at a key binding site for recruitment to inhibitory synapses, while subcortical neuroligin-3 remained unphosphorylated. In vitro, phosphomimetic mutation of that site disrupted neuroligin-3 association with the inhibitory postsynaptic scaffolding protein gephyrin. In vivo, phosphomimetic mutants of neuroligin-3 localized to excitatory postsynapses, while phospho-null mutants localized to inhibitory postsynapses. These data reveal an unexpected region-specific pattern of neuroligin-3 synapse specificity, as well as a phosphorylation-dependent mechanism that regulates its recruitment to either excitatory or inhibitory synapses. These findings add to our understanding of how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition.

Overview

  • The study investigates the synaptic localization of neuroligin-3 across regions in mouse and human brain samples. The authors raise a phospho-specific neuroligin antibody and use phosphoproteomics, cell-based assays, and in utero CRISPR/Cas9 knockout and gene replacement to identify mechanisms that regulate neuroligin-3 localization to distinct synapse types. The study aims to understand how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition.

Comparative Analysis & Findings

  • Neuroligin-3 exhibits region-dependent synapse specificity, largely localizing to excitatory synapses in cortical regions and inhibitory synapses in subcortical regions of the brain in both mice and humans. The study identifies specific phosphorylation of cortical neuroligin-3 at a key binding site for recruitment to inhibitory synapses, while subcortical neuroligin-3 remained unphosphorylated. In vitro, phosphomimetic mutation of that site disrupted neuroligin-3 association with the inhibitory postsynaptic scaffolding protein gephyrin. In vivo, phosphomimetic mutants of neuroligin-3 localized to excitatory postsynapses, while phospho-null mutants localized to inhibitory postsynapses. These data reveal an unexpected region-specific pattern of neuroligin-3 synapse specificity, as well as a phosphorylation-dependent mechanism that regulates its recruitment to either excitatory or inhibitory synapses.

Implications and Future Directions

  • The study's findings add to our understanding of how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition. The study identifies a phosphorylation-dependent mechanism that regulates neuroligin-3 recruitment to either excitatory or inhibitory synapses, which could have implications for the development of therapies for neurological disorders. Future research could explore the role of neuroligin-3 in other neurological disorders, such as schizophrenia or depression, and investigate the potential therapeutic targets for modulating neuroligin-3 function.
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