Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons
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The authors examine the function of Tomosyn, in dense core vesicle fusion in neuronal cultures from mice expressing conditional alleles of tomosyn and tomosyn-2. The authors show here that while loss of tomosyns did not affect dense core vesicle exocytosis, it reduced the expression of several key dense core cargos, including BDNF. However, "rescue" experiments are needed to validate the specificity of the effects.
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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 lox P 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.
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eLife assessment
The authors examine the function of Tomosyn, in dense core vesicle fusion in neuronal cultures from mice expressing conditional alleles of tomosyn and tomosyn-2. The authors show here that while loss of tomosyns did not affect dense core vesicle exocytosis, it reduced the expression of several key dense core cargos, including BDNF. However, "rescue" experiments are needed to validate the specificity of the effects.
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Reviewer #1 (Public Review):
In this study, the authors examine the function of Tomosyn, in dense core vesicle fusion using CRE-mediated deletion in neuronal cultures from mice expressing conditional alleles of tomosyn and tomosyn-2. Tomosyn is a large soluble SNARE protein, where earlier work in multiple species suggested that it functions as a competitive inhibitor of cognate SNARE interactions impairing fusion. The authors show that while loss of tomosyns did not affect dense core vesicle exocytosis, it reduced the expression of several key dense core cargos, including BDNF. Limited (if anything opposite) impact of tomosyn loss-of-function on intracellular vesicle trafficking or Golgi function.
The authors concluded that tomosyns regulate neuropeptide and neurotrophin secretion by regulating dense core vesicle cargo production but …
Reviewer #1 (Public Review):
In this study, the authors examine the function of Tomosyn, in dense core vesicle fusion using CRE-mediated deletion in neuronal cultures from mice expressing conditional alleles of tomosyn and tomosyn-2. Tomosyn is a large soluble SNARE protein, where earlier work in multiple species suggested that it functions as a competitive inhibitor of cognate SNARE interactions impairing fusion. The authors show that while loss of tomosyns did not affect dense core vesicle exocytosis, it reduced the expression of several key dense core cargos, including BDNF. Limited (if anything opposite) impact of tomosyn loss-of-function on intracellular vesicle trafficking or Golgi function.
The authors concluded that tomosyns regulate neuropeptide and neurotrophin secretion by regulating dense core vesicle cargo production but not exocytosis.
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Reviewer #2 (Public Review):
The authors provide here a very careful and thorough analysis of the effects of tomosyn elimination in neurons, in relation to dense-core vesicles. They find strong effects on vesicle generation (size, protein composition), but not on vesicle exocytosis, in spite of tomosyn's known interaction with the exocytosis SNAREs.
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Reviewer #3 (Public Review):
Based on studies over the last two decades, tomosyns participate in processes as diverse as synaptic SNARE complex stability (Yu H et al., 2014), dendritic spine density (Saldate JJ et al., 2018), mossy fiber synaptic plasticity (Ben-Simon Y et al., 2015), inhibition of mast cell degranulation (Madera-Salcedo IK et al., 2018), insulin-stimulated GLUT4 exocytosis by adipocytes (Wang S, et al., 2020), and both basal and stimulated secretion by PC12 cells (Williams et al., 2011). In yeast, which lacks storage granules, two tomosyn orthologs control the formation of post-Golgi vesicles. The actions of tomosyn are cell-type specific and subject to regulation by phosphorylation and the ubiquitin-proteasome system (Saldate JJ et al., 2018; Williams et al., 2011; Madera-Salcedo IK et al., 2018). In beta-cells, the …
Reviewer #3 (Public Review):
Based on studies over the last two decades, tomosyns participate in processes as diverse as synaptic SNARE complex stability (Yu H et al., 2014), dendritic spine density (Saldate JJ et al., 2018), mossy fiber synaptic plasticity (Ben-Simon Y et al., 2015), inhibition of mast cell degranulation (Madera-Salcedo IK et al., 2018), insulin-stimulated GLUT4 exocytosis by adipocytes (Wang S, et al., 2020), and both basal and stimulated secretion by PC12 cells (Williams et al., 2011). In yeast, which lacks storage granules, two tomosyn orthologs control the formation of post-Golgi vesicles. The actions of tomosyn are cell-type specific and subject to regulation by phosphorylation and the ubiquitin-proteasome system (Saldate JJ et al., 2018; Williams et al., 2011; Madera-Salcedo IK et al., 2018). In beta-cells, the ability of tomosyn to decrease insulin secretion by binding syntaxin1A requires its SUMOylation (Ferdaoussi M, et al., 2017). The carefully designed and validated mouse line developed by the authors will facilitate detailed, mechanistic studies of the diverse, cell-type specific actions of tomosyns.
Using cultures derived from the hippocampi of this new mouse strain, multiple differences were observed between two-week-old WT and DKO (double knockout of tomosyn-1 and -2) cultures. Analysis of dense core vesicle release by single neurons revealed no change in their exocytosis, but identified a decrease in levels of the dense core vesicle reporter, leading to the discovery of a decrease in levels of two endogenous dense core vesicle proteins, BDNF and IA-2. In contrast, levels of two lysosomal/endocytic markers were unaltered, demonstrating granule specificity.
WT and DKO cultures were compared using mass spectrometry. Significant changes in the levels of 3% of the proteins were identified. Strikingly, levels of several additional dense core vesicle proteins were decreased in DKO cultures. In contrast, levels of multiple mitochondrial proteins were greatly increased in DKO cultures. In addition, significant increases in VGLUT2 (a marker of glutamatergic neurons) and in GAD67, GAT1, and GAT3 (GABAergic markers) confirmed the presence of widespread differences in hippocampal cultures that matured in the absence of tomosyns. Focusing on BDNF and other dense core vesicle proteins, qPCR studies revealed decreases in mRNA levels for a subset of dense core vesicle proteins.
The use of multiple culture systems allowed the authors to employ different approaches, ranging from monitoring the release of single granules expressing a dense core vesicle reporter to quantifying the accelerated trafficking of a tagged cargo protein from the ER through the TGN and into DCVs in the absence of tomosyns. While no changes in synaptic complex formation were observed, both electron microscopy and analysis of single vesicles expressing a dense core vesicle reporter revealed a decrease in granule diameter.
Weaknesses of methods and results. Within 8 h of plating, hippocampal cultures prepared from a single litter were transduced with a lentivirus encoding active or inactive mCherry-tagged Cre-recombinase, generating WT and DKO cultures; expression of Cre-recombinase was limited to neurons using the synapsin promoter. Cultures were generally examined after two weeks. Culture conditions were varied to allow comparison of dense core vesicle exocytosis by single neurons (a neuron on a glial microisland) or protein and mRNA levels in dense neuronal networks plated on coated plastic without a glial feeder layer in WT vs. DKO cultures. Whether cultures allowed to develop under these vastly different conditions respond to the absence of tomosyns in a different manner is unknown. No attempt was made to rescue any of the differences observed by expressing tomosyn in DKO neurons. Successful rescue experiments would alleviate concerns about the effects of developmental differences on the phenotypes observed.
Immunocytochemical studies revealed an approximately two-fold drop in BDNF protein levels in the soma and neurites of DKO neurons. In contrast, BDNF, which was detectable in WT cultures using mass spectrometry, was not detectable using mass spectrometry to analyze DKO cultures. No explanation for this discrepancy between immunocytochemistry and mass spectrometry is offered. Despite the fact that neither BDNF secretion nor BDNF degradation was assessed, the authors state in their Abstract that "tomosyns regulate neuropeptide and neurotrophin secretion via control of DCV cargo production".
The authors do not adequately refer to the rich literature discussing the many secretory pathways used by different cell types, referring only to synaptic vesicles and dense core vesicles. Golgi by-pass pathways are known to take membrane proteins to dendrites and tomosyns are known to play a role in the trafficking of GLUT4 from endocytic compartments to the plasma membrane. Soluble cargo proteins such as BDNF are released both constitutively and in response to stimuli. Cargo proteins (proinsulin, proANP, and growth hormone, for example) can drive the appearance of dense core vesicles.
The mass spectrometry data presented in Fig. 3 are not well incorporated into the Discussion. KIF6, which plays a role in retrograde Golgi to ER traffic, is detectable in DKO cultures, but not in WT cultures and could contribute to the accelerated trafficking phenotype observed using RUSH. Coordinate control of the expression of dense core vesicle genes has been studied in a variety of systems, ranging from mammals to C. elegans to D. melanogaster. Levels of these gene products could have been assessed using existing mass spectrometric data or by additional qPCR studies. The diminished levels of dense core vesicle reporters observed in Fig.1 remain unexplained. Intracellular degradation and increased basal secretion, neither of which was assessed, could contribute to this observation.
The authors did not take advantage of the structure/function studies used to dissect the roles of the beta-propeller and SNARE-domains of tomosyns. In yeast, loss of SR07/SR077, tomosyn orthologs which lack a SNARE-like domain, causes a defect in the exocytosis of post-Golgi vesicles and the accumulation of secretory vesicles with altered composition (Forsmark et al., 2011).Are claims and conclusions justified by data: The title of the manuscript, "SNARE protein tomosyn regulates dense core vesicle composition but not exocytosis in mammalian neurons" is misleading. The authors present no evidence that the SNARE-domain of tomosyn is necessary for its effects on dense core vesicle composition. The yeast orthologs of tomosyn, which lack a SNARE domain, affect post-Golgi vesicular trafficking via their beta-propeller domains. Hippocampal neurons are not representative of all "mammalian" neurons. In rat sympathetic neurons, tomosyn depletion results in a decrease in neurotransmitter release. A key conclusion is that tomosyns regulate neuropeptide and neurotrophin secretion by controlling cargo production, not cargo release - this conclusion is not supported by the data presented.
Likely impact of work on the field: The mouse line developed for these studies will be of great use in mechanistic studies of the multiple roles of tomosyns. The authors identified a range of parameters that are altered in hippocampal neurons which develop in the absence of tomosyns. Additional mechanistic studies are needed to directly assess the manner in which the absence of tomosyns contributes to these changes.
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