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

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.

An active vesicle priming machinery suppresses axon regeneration upon adult CNS injury

“…Among these components were the vesicle-associated membrane protein (VAMP) family members VAMP1 and VAMP2. Therefore, we assessed whether overexpression of tetanus toxin light chain (TeNT-LC), which cleaves VAMP1, -2, and -3 but leaves other VAMP protein family members intact ( Hoogstraaten et al., 2020 ; Humeau et al., 2000 ; McMahon et al., 1993 ; Schiavo et al., 1992 ), would promote axon growth or regeneration. We generated an AAV encoding TeNT-LC separated from mCherry by a P2A peptide, injected it into the sciatic nerves of adult wild-type mice, and plated the neurons 3 weeks later.…”

Dynamin controls neuropeptide secretion by organizing dense-core vesicle fusion sites

“…Neuromodulators such as oxytocin and dopamine are proposed to depend on volume transmission (12) and use transient fusion sites (13,14). Although fusion sites are poorly defined, DCV fusion is known to use largely the same release machinery as SVs, such as the neuronal SNARE [soluble N-ethylmaleimidesensitive factor (NSF) attachment protein (SNAP) receptor] proteins SNAP-25 and synaptobrevin/VAMP2, and key SNARE regulators such as Munc13 and RIM (6,(15)(16)(17)(18). How these components are brought together (transiently) to form DCV fusion sites remains elusive.…”

Spiking Neural Network with Linear Computational Complexity for Waveform Analysis in Amperometry

“…An example of a computational task requiring the classification of amperometric waveforms is tracking processes of cell exocytosis based on vesicle fusion taking place inside cells. This approach is used in cancer metastases’ analysis [ 27 ] and early diagnostics of diseases such as Alzheimer’s [ 28 ], cholestasis [ 29 ], hypoxia [ 30 ], thrombosis [ 31 ], and tetanus [ 32 ]. The main problem is the need to analyze signals not coming from a single cell and a single set of CNT electrodes, but from tens of thousands of cells and a huge number of electrode sets.…”