# Unraveling Botulinum Neurotoxin A Light-Chain-Induced Signaling Pathways: A Phosphoproteomic Analysis in a Controlled Cellular Model

## TL;DR

This study uses a controlled cell model to explore how botulinum toxin affects cell signaling, revealing key pathways and proteins involved in its toxicity.

## Contribution

A novel inducible cell model for studying BoNT/A toxicity and phosphoproteomic insights into its signaling effects.

## Key findings

- 75 proteins showed upregulated phosphorylation, enriched in pathways like PI3K-AKT and Ras signaling.
- 27 proteins were downregulated, linked to ERBB and thyroid hormone signaling pathways.
- Hsp90ab1 and Map2k1 emerged as central hub molecules in upregulated and downregulated networks, respectively.

## Abstract

Botulinum neurotoxin type A (BoNT/A), among the most potent known toxins, is widely used in cosmetic medicine. However, its toxicity mechanisms remain poorly understood due to a lack of suitable models. Here, we generated a doxycycline (DOX)-inducible Neuro-2a cell line stably expressing the BoNT/A light chain (ALC). ALC expression was confirmed by GFP and FLAG tag antibodies, and its activity was validated through cleavage of the substrate SNAP-25. Using this model, combined with natural toxin infection of cells, phospho-antibody microarray analysis revealed significant alterations in host phosphorylation networks in both ALC-expressing and toxin-infected cells. Among the shared phosphorylation changes, 75 proteins showed upregulation, while 27 were downregulated. Upregulated phosphorylation events were enriched in pathways such as PI3K-AKT signaling, EGFR tyrosine kinase inhibitor resistance, and Ras signaling, whereas downregulated events were associated with the ERBB and thyroid hormone signaling pathways. Key alterations were observed in AKT signaling, with protein–protein interaction analysis identifying Hsp90ab1 and Map2k1 as central hub molecules for upregulated and downregulated proteins, respectively. This study establishes a robust Neuro-2a-based model system to study BoNT/A toxicity and provides insights into toxin-induced phosphorylation network changes, offering a valuable platform for therapeutic screening and mechanistic exploration.