# Nogo Receptor Antagonist LOTUS Promotes Neurite Outgrowth through Its Interaction with Teneurin-4

## TL;DR

LOTUS promotes neurite outgrowth by interacting with Teneurin-4, offering new insights into nerve regeneration in the adult brain.

## Contribution

LOTUS promotes neurite outgrowth independently of Nogo receptor antagonism by interacting with Teneurin-4.

## Key findings

- LOTUS directly binds to Teneurin-4, a cell adhesion molecule, promoting neurite outgrowth.
- RNAi knockdown of Teneurin-4 inhibits neurite outgrowth on LOTUS-coated substrates.
- A soluble form of Teneurin-4 reduces the promoting effect of LOTUS on neurite outgrowth.

## Abstract

Neuronal growth is an essential process for establishing functional neural networks in developing brain and restoring dysfunctional networks caused by injuries in the adult brain. In the adult central nervous system, injured neurons fail to re-extend their axons due to several axon growth inhibitors mainly expressed in glial cells. We previously reported that LOTUS protein helps injured adult neurons to regenerate by blocking the inhibitors. In this study, we found that LOTUS itself promotes axon growth through an independent mechanism from the blockade of the inhibitors. LOTUS directly binds to Teneurin-4, a cell adhesion molecule expressed on the surface of neurons and the binding promotes axon growth. Thus, LOTUS was found to have two functions in axon growth. This finding may provide a new insight into neural network formation in developing brain and neural regrowth mechanism following the adult injured brain. These functions of LOTUS protein are useful for developing new therapeutic approaches to promote nerve regeneration after injury in the adult central nervous system.

Neurite outgrowth is a crucial process for organizing neuronal circuits in neuronal development and regeneration after injury. Regenerative failure in the adult mammalian central nervous system (CNS) is attributed to axonal growth inhibitors such as the Nogo protein that commonly binds to Nogo receptor-1 (NgR1). We previously reported that lateral olfactory tract usher substance (LOTUS) functions as an endogenous antagonist for NgR1 in forming neuronal circuits in the developing brain and improving axonal regeneration in the adult injured CNS. However, another molecular and cellular function of LOTUS remains unknown. In this study, we found that cultured retinal explant neurons extend their neurites on the LOTUS-coating substrate. This action was also observed in cultured retinal explant neurons derived from Ngr1-deficient mouse embryos, indicating that the promoting action of LOTUS on neurite outgrowth may be mediated by unidentified LOTUS-binding protein(s). We therefore screened the binding partner(s) of LOTUS by using a liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS analysis and pull-down assay showed that LOTUS interacts with Teneurin-4 (Ten-4), a cell adhesion molecule. RNAi knockdown of Ten-4 inhibited neurite outgrowth on the LOTUS substrate in retinoic acid (RA)-treated Neuro2A cells. Furthermore, a soluble form of Ten-4 attenuates the promoting action on neurite outgrowth in cultured retinal explant neurons on the LOTUS substrate. These results suggest that LOTUS promotes neurite outgrowth by interacting with Ten-4. Our findings may provide a new molecular mechanism of LOTUS to contribute to neuronal circuit formation in development and to enhance axonal regeneration after CNS injury.