Actl6a regulates autophagy via Sox2-dependent Atg5 and Atg7 expression to inhibit apoptosis in spinal cord injury

Abstract

Introduction

Spinal cord injury (SCI) is a severe central nervous system disorder with limited treatment options. While autophagy plays a protective role in neural repair, its regulatory mechanisms in SCI remain unclear. Actin-like protein 6A (Actl6a) influences cell fate and neural development, yet its specific role in SCI repair is not well understood. This study investigates Actl6a’s function in regulating autophagy and apoptosis via the transcription factor Sox2 in SCI.

Objectives

This study aims to determine if Actl6a promotes neural survival post-SCI by regulating autophagy-related genes Atg5 and Atg7 through Sox2. It also examines how the demethylase Fto modulates Actl6a mRNA stability via m6A methylation.

Methods

In vitro experiments were conducted using primary neurons and HT-22 hippocampal cells exposed to hydrogen peroxide (H₂O₂)-induced oxidative stress. Actl6a expression was manipulated by knockdown or overexpression. For in vivo studies, a rat SCI model was established with AAV-Actl6a injected at the injury site to induce Actl6a overexpression. Autophagy and apoptosis markers were analyzed using immunofluorescence, Western blotting, and qPCR. Additionally, m6A dot blot and RNA immunoprecipitation (RIP) assays were performed to assess Fto’s role in regulating Actl6a mRNA methylation and stability.

Results

Actl6a expression significantly decreased after SCI, resulting in increased apoptosis. Overexpressing Actl6a enhanced autophagy, reduced apoptosis, and improved neurological function in SCI models. Mechanistically, Actl6a and Sox2 collaboratively upregulated Atg5 and Atg7 expression, promoting autophagy. Fto’s modulation of Actl6a mRNA stability via m6A demethylation further influenced autophagy and apoptosis.

Conclusion

Actl6a, through interaction with Sox2, plays a critical role in modulating autophagy and reducing apoptosis in SCI, with Fto’s m6A modification affecting Actl6a stability. This Fto/Actl6a/Sox2 axis is a promising therapeutic target for SCI repair.