Tetanus insensitive VAMP2 differentially restores synaptic and dense core vesicle fusion in tetanus neurotoxin treated neurons

Rein I Hoogstraaten,Matthijs Verhage,Linda Van Keimpema,Ruud F Toonen

doi:10.1038/s41598-020-67988-2

Rein I Hoogstraaten, Matthijs Verhage + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-67988-2

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Abstract

The SNARE proteins involved in the secretion of neuromodulators from dense core vesicles (DCVs) in mammalian neurons are still poorly characterized. Here we use tetanus neurotoxin (TeNT) light chain, which cleaves VAMP1, 2 and 3, to study DCV fusion in hippocampal neurons and compare the effects on DCV fusion to those on synaptic vesicle (SV) fusion. Both DCV and SV fusion were abolished upon TeNT expression. Expression of tetanus insensitive (TI)-VAMP2 restored SV fusion in the presence of TeNT, but not DCV fusion. Expression of TI-VAMP1 or TI-VAMP3 also failed to restore DCV fusion. Co-transport assays revealed that both TI-VAMP1 and TI-VAMP2 are targeted to DCVs and travel together with DCVs in neurons. Furthermore, expression of the TeNT-cleaved VAMP2 fragment or a protease defective TeNT in wild type neurons did not affect DCV fusion and therefore cannot explain the lack of rescue of DCV fusion by TI-VAMP2. Finally, to test if two different VAMPs might both be required in the DCV secretory pathway, Vamp1 null mutants were tested. However, VAMP1 deficiency did not reduce DCV fusion. In conclusion, TeNT treatment combined with TI-VAMP2 expression differentially affects the two main regulated secretory pathways: while SV fusion is normal, DCV fusion is absent.

Highlights

The sensitive factor attachment receptor (SNARE) proteins involved in the secretion of neuromodulators from dense core vesicles (DCVs) in mammalian neurons are still poorly characterized
VAMP2 is the major isoform for synaptic vesicle (SV) and DCV exocytosis, VAMP3 can partly take over its function in chromaffin cells and VAMP1 drives at least some SV fusion in neurons as well as calcitonin gene-related peptide (CGRP) release from trigeminal ganglionic neurons
We confirmed that lentiviral infection of tetanus neurotoxin (TeNT) light chain in cortical neurons efficiently cleaves VAMP1 and VAMP2 (Fig. 1B), the only two TeNT-sensitive VAMP proteins expressed in these neurons (Fig. 1C), as shown b efore[8,17]

Summary

Introduction

The SNARE proteins involved in the secretion of neuromodulators from dense core vesicles (DCVs) in mammalian neurons are still poorly characterized. We use tetanus neurotoxin (TeNT) light chain, which cleaves VAMP1, 2 and 3, to study DCV fusion in hippocampal neurons and compare the effects on DCV fusion to those on synaptic vesicle (SV) fusion. Both DCV and SV fusion were abolished upon TeNT expression. VAMP2 is the major isoform for SV and DCV exocytosis, VAMP3 can partly take over its function in chromaffin cells and VAMP1 drives at least some SV fusion in neurons as well as CGRP release from trigeminal ganglionic neurons. TeNT treatment combined with TI-VAMP2 expression differentially affects SV and DCV fusion and may be used as a tool to selectively inhibit DCV fusion, leaving SV fusion unchanged

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