Botulinum protease‐cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells

Jason Arsenault,Enrico Ferrari,Dhevahi Niranjan,Charlotte Leese,Sabine A G Cuijpers,Aleksander Rust,John A O'Brien,Bazbek Davletov,Thomas Binz

doi:10.1111/jnc.12645

Abstract

Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed toward their partner target SNAREs: synaptosomal-associated protein of 25 kDa and syntaxin. SNARE proteins are normally membrane bound, but can be cleaved and released by botulinum neurotoxins. We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA-transfection reagents. Following administration of the C and D proteases into normally refractory Neuro2A neuroblastoma cells, the SNARE proteins were cleaved with high efficiency within hours. Remarkably, botulinum protease exposures led to cytotoxicity evidenced by spectrophotometric assays and propidium iodide penetration into the nuclei. Direct delivery of SNARE fragments into the neuroblastoma cells reduced viability similar to botulinum proteases' application. We observed synergistic cytotoxic effects of the botulinum proteases, which may be explained by the release and interaction of soluble SNARE fragments. We show for the first time that previously observed cytotoxicity of botulinum neurotoxins/C in neurons could be achieved in cells of neuroendocrine origin with implications for medical uses of botulinum preparations.

Highlights

Direct delivery of botulinum proteases into neuroblastoma cells First, we ascertained the presence of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins syntaxin, synaptosomal-associated protein of 25 kDa (SNAP25), and synaptobrevin in mouse neuroblastoma mouse neuroblastoma 2A (N2A) cells by confocal microscopy
We explored a possibility of delivering botulinum proteases, which cleave membrane-embedded SNAREs, into normally resistant N2A cells
As it has been observed that transfection reagents are capable of causing the internalization of the Botulinum A and E proteases targeting SNAP25 (Kuo et al 2010), we tried an array of transfection reagents to deliver the type C and D proteases, which, respectively, target t-SNAREs and the v-SNARE

Summary

Introduction

Immunoreactivity is not necessarily a measure of protein presence and other studies have been able to identify syntaxin fragments in cell extracts (Tsukamoto et al 2012) If these fragments are free to diffuse and to engage SNAP25 and synaptobrevin or other non-neuronal SNAREs (Fasshauer et al 1999), they may potentially interfere with the scrupulous fusion events. The soluble SNARE fragments were able to form ternary complexes and when transduced, triggered loss of cell viability mimicking the type C and D protease effects

Methods

Results

Conclusion