USP9X Inhibition Promotes Autophagy-associated Clearance of Intracellular Botulinum Neurotoxin A and Accelerates Functional Recovery.

Authors

Xuerui Xiang, Ying Liu, Lixi Li, Lizhen Pan, Kunshan Zhang, Yuyang Yan, Baogang Sun, Yunping Song, Lingjing Jin

Published in

Neurotoxicology. Pages 103586. Oct 08, 2026. Epub Oct 08, 2026.

Abstract

The prolonged neuroparalytic effect of botulinum neurotoxin type A (BoNT/A) is largely attributable to the high intracellular stability of its light chain (LC), highlighting the need for strategies to promote LC clearance. Although deubiquitinating enzymes (DUBs) regulate LC turnover through both proteasomal and non-proteasomal pathways, the latter remains poorly understood. Here, we investigated the role of the DUB USP9X in regulating LC stability. Using a stable LC-overexpressing cell model, we found that pharmacological inhibition of USP9X with Degrasyn markedly promoted LC clearance. Transcriptomic analysis following lentivirus-mediated USP9X knockdown revealed enrichment of autophagy-related pathways, accompanied by suppression of PI3K-AKT signaling. Mechanistically, USP9X knockdown did not affect total mTOR protein levels but suppressed AKT-mTOR signaling, as shown by reduced phosphorylation of AKT and mTOR. This was associated with decreased ULK1 phosphorylation at the inhibitory site Ser757, increased phosphorylation at the active site Ser555, elevated p-AMPK levels, increased LC3-II/LC3-I ratio, and reduced p62 levels. All of those pointed to enhanced autophagic activity in response to USP9X inhibition. In BoNT/A-intoxicated rats, chloroquine-mediated autophagy inhibition delayed recovery from paralysis, whereas rapamycin-induced autophagy activation accelerated muscle strength recovery. Consistently, systemic administration of Degrasyn promoted functional recovery. Collectively, these findings indicate that USP9X inhibition promotes intracellular LC clearance, likely through an autophagy-lysosome-dependent mechanism involving suppression of AKT-mTOR signaling and activation of AMPK-ULK1-related pathways. Our study identifies USP9X as a host negative regulator of LC clearance and a potential therapeutic target for accelerating recovery following botulinum toxin intoxication.

PMID:
42849818
Bibliographic data and abstract were imported from PubMed on 09 Oct 2026.

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