Abstract
Tomosyn is a large, non-canonical SNARE protein proposed to act as an inhibitor of SNARE complex formation in the exocytosis of secretory vesicles. In the brain, tomosyn inhibits the fusion of synaptic vesicles (SVs), whereas its role in the fusion of neuropeptide-containing dense core vesicles (DCVs) is unknown. Here, we addressed this question using a new mouse model with a conditional deletion of tomosyn (Stxbp5) and its paralogue tomosyn-2 (Stxbp5l). We monitored DCV exocytosis at single vesicle resolution in tomosyn-deficient primary neurons using a validated pHluorin-based assay. Surprisingly, loss of tomosyns did not affect the number of DCV fusion events but resulted in a strong reduction of intracellular levels of DCV cargos, such as neuropeptide Y (NPY) and brain-derived neurotrophic factor (BDNF). BDNF levels were largely restored by re-expression of tomosyn but not by inhibition of lysosomal proteolysis. Tomosyn’s SNARE domain was dispensable for the rescue. The size of the trans-Golgi network and DCVs was decreased, and the speed of DCV cargo flux through Golgi was increased in tomosyn-deficient neurons, suggesting a role for tomosyns in DCV biogenesis. Additionally, tomosyn-deficient neurons showed impaired mRNA expression of some DCV cargos, which was not restored by re-expression of tomosyn and was also observed in Cre-expressing wild-type neurons not carrying loxP sites, suggesting a direct effect of Cre recombinase on neuronal transcription. Taken together, our findings argue against an inhibitory role of tomosyns in neuronal DCV exocytosis and suggests an evolutionary conserved function of tomosyns in the packaging of secretory cargo at the Golgi.
Original language | English |
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Article number | e85561 |
Pages (from-to) | 1-30 |
Number of pages | 30 |
Journal | eLife |
Volume | 12 |
Early online date | 11 Sept 2023 |
DOIs | |
Publication status | Published - 2023 |
Bibliographical note
Funding Information:We are indebted to D Schut, L Laan, I Saarlos, R Zalm, J Wortel, J Hoetjes, R Dekker, and I Paliukhovich for their excellent technical assistance. We also thank F Koopmans for his expert advice in proteomics dataset analysis. This work was supported by the DFG (German Research Foundation) postdoctoral fellowship to A Subkhangulova (DFG project number SU 1131/1–1).
Publisher Copyright:
© Subkhangulova et al.
Funding
We are indebted to D Schut, L Laan, I Saarlos, R Zalm, J Wortel, J Hoetjes, R Dekker, and I Paliukhovich for their excellent technical assistance. We also thank F Koopmans for his expert advice in proteomics dataset analysis. This work was supported by the DFG (German Research Foundation) postdoctoral fellowship to A Subkhangulova (DFG project number SU 1131/1–1).