Tetanus Toxin Is Transported in a Novel Neuronal Compartment Characterized by a Specialized pH Regulation

Stephanie Bohnert,Giampietro Schiavo

doi:10.1074/jbc.m506750200

Stephanie Bohnert, Giampietro Schiavo

Open Access

https://doi.org/10.1074/jbc.m506750200

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Abstract

Tetanus toxin binds specifically to motor neurons at the neuromuscular junction. There, it is internalized into vesicular carriers undergoing fast retrograde transport to the spinal cord. Despite the importance of this axonal transport pathway in health and disease, its molecular and biophysical characterization is presently lacking. We sought to fill this gap by determining the pH regulation of this compartment in living motor neurons using a chimera of the tetanus toxin binding fragment (TeNT HC) and a pH-sensitive variant of the green fluorescent protein (ratiometric pHluorin). We have demonstrated that moving retrograde carriers display a narrow range of neutral pH values, which is kept constant during transport. Stationary TeNT HC-positive organelles instead exhibit a wide spectrum of pH values, ranging from acidic to neutral. This distinct pH regulation is due to a differential targeting of the vacuolar (H+) ATPase, which is not present on moving TeNT HC compartments. Accordingly, inhibition of the vacuolar (H+) ATPase under conditions that completely abolish the intracellular accumulation of acidotrophic dyes does not affect axonal retrograde transport of TeNT HC. However, a functional vacuolar (H+) ATPase is required for early steps of TeNT HC trafficking following endocytosis, and it is localized to axonal vesicles containing TeNT HC. Altogether, these findings indicate that the vacuolar (H+ ATPase plays a specific role in early sorting events directing TeNT HC to axonal carriers but not in their subsequent progression along the retrograde transport route, which escapes acidification and targeting to degradative organelles.

Highlights

A highly specific zinc endoprotease, which is responsible for the cleavage of synaptic SNARE proteins necessary for neurotransmitter release [2]
We have recently developed an assay to follow the retrograde transport of TeNT in motor neurons (MNs) using fluorescently tagged versions of the nontoxic TeNT HC binding fragment [5]
Restriction endonucleases and DNA-modifying enzymes were from New England Biolabs. pGEX-4TVSV-G-Kin-TeNT HC encoding the residues 855–1,314 of TeNT fused at the amino terminus with a protein kinase A phosphorylation site, the vesicular stomatitis virus protein G (VSV-G) epitope, and glutathione S-transferase (GST) [3] were linearized with NdeI

Summary

Introduction

A highly specific zinc endoprotease, which is responsible for the cleavage of synaptic SNARE proteins necessary for neurotransmitter release [2]. We have recently developed an assay to follow the retrograde transport of TeNT in MNs using fluorescently tagged versions of the nontoxic TeNT HC binding fragment [5] In these cells, TeNT and TeNT HC are internalized and transported in morphologically identical organelles with overlapping speed distributions [6]. TeNT HC shares this compartment with the nerve growth factor and the low affinity neurotrophin receptor p75NTR [5] These findings validate TeNT HC as an ideal tool for dissecting the molecular machinery controlling axonal retrograde transport and the trafficking of neurotrophin receptors and their ligands in living MNs. To date, the precise characterization of the intraluminal pH of this axonal retrograde pathway and its relevance in the regulation of transport in MNs remain unclear. The distinct pH regulation of these compartments is due to a differential targeting of the vacuolar (Hϩ) ATPase (vATPase), which activity is required for early events in the formation and/or sorting of axonal TeNT HC vesicles but not for their subsequent axonal transport

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