# Neuronal Subtype-Specific Expression of γ-Enolase: Its Role in Neuronal Differentiation

## TL;DR

This study explores how γ-enolase, a glycolytic enzyme, supports the development of specific types of neurons, particularly cholinergic neurons, and how its activity is regulated by cathepsin X.

## Contribution

The study reveals that γ-enolase promotes neuronal differentiation, especially in cholinergic neurons, and is regulated by cathepsin X, offering new insights into neuroregeneration.

## Key findings

- γ-enolase expression is significantly upregulated in differentiated neurons, especially cholinergic-like neurons.
- Cathepsin X cleaves γ-enolase, reducing its neurotrophic effects, and inhibiting cathepsin X preserves active γ-enolase and promotes neuronal differentiation.
- A γ-enolase-derived peptide enhances neurite outgrowth and β-tubulin expression in cholinergic-like neurons.

## Abstract

Neuronal differentiation into specific subtypes is crucial for nervous system development and function, guided by neurotrophic factors. γ-Enolase, a neuron-specific glycolytic enzyme, exhibits neurotrophic-like properties and supports neuronal differentiation; however, its role in specific neuronal subtypes remains unknown. Here, we investigate the role of γ-enolase in differentiation dopaminergic-, cholinergic-, and adrenergic-like neuronal cells. Our results demonstrate that γ-enolase expression is significantly upregulated in differentiated cells, with the highest expression observed in cholinergic-like neurons. Full-length γ-enolase, compared to its truncated form, promoted enhanced neurite outgrowth and increased β-tubulin, a cytoskeletal marker. Conversely, silencing endogenous γ-enolase significantly reduced neurite length, confirming its essential role in driving neuronal morphological maturation. Furthermore, a γ-enolase-derived peptide corresponding to the active C-terminus of γ-enolase significantly promoted neurite outgrowth and increased β-tubulin expression, particularly in cholinergic-like neuronal cells. Notably, γ-enolase activity is regulated by cathepsin X, a lysosomal peptidase that cleaves γ-enolase at its C-terminus, reducing its neurotrophic effects. Confocal microscopy revealed increased co-localization of γ-enolase and cathepsin X in differentiated neuronal cells, emphasizing their interaction in cholinergic-like neurons. Inhibiting cathepsin X preserved active γ-enolase, promoted neuronal differentiation, and altered cytoskeletal marker expression. These findings suggest an important role for γ-enolase in cholinergic-like neuronal cells and propose cathepsin X as a regulatory modulator of γ-enolase activity, suggesting novel therapeutic strategies for neuroregeneration.

The online version contains supplementary material available at 10.1007/s12017-025-08902-9.