# Differential neuronal functions of LNX1 and LNX2 revealed by behavioural analysis in single and double knockout mice

## TL;DR

This study reveals distinct roles of LNX1 and LNX2 proteins in mouse behavior, including anxiety, risk-taking, and communication.

## Contribution

The study identifies novel behavioral roles for LNX1 and LNX2 proteins and an unexpected role for LNX1 in body weight regulation.

## Key findings

- LNX2 knockout mice show more pronounced effects on anxiety and risk-taking behaviors compared to LNX1 knockout mice.
- LNX1 knockout mice exhibit altered ultrasonic vocalizations and contribute to reduced body weight in double knockouts.
- LNX1 and LNX2 differentially modulate specific behavioral tests like dark-light emergence and wire beam bridge paradigms.

## Abstract

Ligand of NUMB protein-X 1 (LNX1) and LNX2 proteins are closely related PDZ domain-containing E3 ubiquitin ligases that interact with and potentially modulate numerous synaptic and neurodevelopmentally important proteins. While both LNX1 and LNX2 are expressed in neurons, it is noteworthy that neuronal LNX1 isoforms lack the catalytic domain responsible for ubiquitination of substrates. Thus, the shared interaction partners of LNX1 and LNX2 might be differentially regulated by these proteins, with LNX1 acting as a stabilizing scaffold while LNX2 may promote their ubiquitination and degradation. Despite the identification of many LNX interacting proteins and substrates, our understanding of the distinct in vivo functions of LNX1 and LNX2 remains very incomplete.

We previously reported that mice lacking both LNX1 in the central nervous system and LNX2 globally exhibit decreased anxiety-related behaviour. Here we significantly extend this work by examining anxiety-related and risk-taking behaviours in Lnx1-/- and Lnx2-/- single knockout animals for the first time and by analysing previously unexplored aspects of behaviour in both single and double knockout animals. While the absence of both LNX1 and LNX2 contributes to the decreased anxiety-related behaviour of double knockout animals in the open field and elevated plus maze tests, the elimination of LNX2 plays a more prominent role in altered behaviour in the dark-light emergence test and wire beam bridge risk-taking paradigms. By contrast, Lnx knockout mice of all genotypes were indistinguishable from wildtype animals in the marble burying, stress-induced hyperthermia and novel object recognition tests. Analysis of the ultrasonic vocalizations of pups following maternal separation revealed significant differences in call properties and vocal repertoire for Lnx1-/- and Lnx1-/-;Lnx2-/- double knockout animals. Finally, decreased body weight previously noted in double knockout animals could be attributed largely to Lnx1 gene knockout.

These results identify specific roles of LNX1 and LNX2 proteins in modulating distinct aspects of anxiety and risk-taking behaviour and social communication in mice. They also reveal an unexpected role for neuronally expressed LNX1 isoforms in determining body weight. These novel insights into the differential neuronal functions of LNX1 and LNX2 proteins provide a foundation for mechanistic studies of these phenomena.

The online version contains supplementary material available at 10.1186/s12993-025-00276-z.