Brain activity relies on the precisely regulated secretion of different chemical messengers, and most neurons co-release multiple neurotransmitters (Nusbaum et al., 2017). Classical small-molecule neurotransmitters (e.g. glutamate, γ-aminobutyric acid) are released from SVs, whereas neuropeptides and neurotrophic factors are packaged in and secreted from DCVs. SV and DCV substantially differ in their morphology and mechanisms of formation (reviewed in De Camilli and Jahn, 1990; Gondré-Lewis et al., 2012). While SVs are loaded with their content directly at synapses, where they subsequently fuse with the presynaptic membrane, DCVs are formed in neuronal somas at the trans-Golgi network (TGN), from where they travel to the release sites that are not confined to synapses. Despite these and many other differences between the two main types of neuronal secretory vesicles, Ca²⁺-triggered exocytosis of both SV and DCV in most neurons requires an identical basic machinery consisting of SNARE proteins: syntaxin-1 and SNAP25 at the plasma membrane, and VAMP2 on the vesicle (Südhof, 2013; Shimojo et al., 2015; Arora et al., 2017; Hoogstraaten et al., 2020). The SNARE domains of these proteins form a tight complex that brings the two membranes together and drives vesicle fusion. In addition to the basic SNARE machinery, many other proteins participate in the fusion process (reviewed in Südhof, 2013; Rizo and Xu, 2015), of which some are differentially required for SV and DCV exocytosis (Persoon et al., 2019; Moro et al., 2021). The differences in molecular machinery may explain why SV and DCV exocytosis are triggered upon different stimulation paradigms and at different locations within neurons.

Tomosyn (STXBP5) is a large, evolutionary conserved SNARE protein discovered as an interactor of syntaxin-1 in rat brain (Fujita et al., 1998; Masuda et al., 1998). Tomosyn is expressed in a wide range of specialized secretory cell types, such as neurons, platelets, and neuroendocrine cells, suggesting a fundamental role of tomosyn in regulated secretion. Mammals express a second tomosyn gene, STXBP5L, which encodes tomosyn-2 with a nearly 80% amino acid similarity (Groffen et al., 2005). The two genes have a partially overlapping expression pattern in the mouse brain suggesting functional redundancy (Groffen et al., 2005; Barak et al., 2010). Mutations in both genes were found in patients with neurodevelopmental disorders, suggesting a role of tomosyns in human brain function (Matsunami et al., 2013; Cukier et al., 2014; De Rubeis et al., 2014; Kumar et al., 2015).

Tomosyn is comprised of a C-terminal SNARE domain and N-terminal WD40 repeats folded into two seven-bladed β-propellers. The SNARE-domain of tomosyn is homologous to the SNARE domain of VAMP2 and mediates binding to syntaxin-1 and SNAP25. The resulting complex is remarkably similar in structure and stability to the fusogenic complex of syntaxin-1/SNAP25 with VAMP2 (Pobbati et al., 2004). However, in contrast to VAMP2 and most other SNARE proteins, tomosyn does not contain a transmembrane anchor and, therefore, cannot drive membrane fusion. This feature led to a hypothesis that tomosyn functions as an inhibitor of fusion by competing with VAMP2 for binding to syntaxin-1/SNAP25 (Hatsuzawa et al., 2003). In support of this hypothesis, overexpression of tomosyn in neuroendocrine PC12 cells and adrenal chromaffin cells inhibits the fusion of secretory vesicles (Fujita et al., 1998; Hatsuzawa et al., 2003; Yizhar et al., 2004), and, conversely, loss of tomosyn in the nematode, fruit fly and mouse brain increases SV release probability and enhances synaptic transmission (Gracheva et al., 2006; McEwen et al., 2006; Chen et al., 2011; Sauvola et al., 2021; Sakisaka et al., 2008; Ben-Simon et al., 2015). In addition, loss of tomosyn in C. elegans augments neuropeptide secretion, as evidenced by decreased DCV abundance at synapses and increased accumulation of a DCV cargo in scavenger cells (Gracheva et al., 2007; Ch’ng et al., 2008). Together, these studies suggest an evolutionary conserved role of tomosyn as an inhibitor of secretory vesicle exocytosis.

However, several lines of evidence argue against this conclusion. First, rat sympathetic neurons show a decrease in neurotransmitter release from SV upon depletion of tomosyn (Baba et al., 2005). Second, platelets from Stxbp5-null mice show severely reduced exocytosis of all types of secretory granules, which results in excessive bleeding and impaired thrombus formation in vivo (Zhu et al., 2014; Ye et al., 2014). Finally, the loss of SRO7 and SRO77, two yeast orthologs of mammalian tomosyns, causes a strong defect in the exocytosis of post-Golgi vesicles (Lehman et al., 1999). Collectively, these studies suggest a positive role of tomosyn and its yeast orthologs in exocytosis. Thus, despite the clear importance of tomosyn in regulated secretion, its role in this process remains controversial.

In the brain, the role of mammalian tomosyn has been studied with a focus on SV exocytosis, whereas its effect on DCV exocytosis is unknown. In this study, we addressed this question using a mouse model with the inducible loss of all tomosyn and tomosyn-2 isoforms and show that tomosyns do not affect neuronal DCV exocytosis but, paradoxically, regulate intracellular levels of overexpressed and endogenous secretory cargos. Thus, tomosyns differentially modulate two regulated secretory pathways in neurons (secretion from SVs and DCVs) and have an additional role in the regulation of DCV composition.

To explore the role of tomosyn in neuronal secretion, we generated a new mouse model with a conditional deletion of tomosyn (Stxbp5) and tomosyn-2 (Stxbp5l) (Figure 1—figure supplement 1A). Primary hippocampal neurons isolated from these mice showed a complete loss of tomosyns expression when transduced with a lentivirus encoding Cre-recombinase, as shown by Western blot (WB) and immunocytochemistry (ICC) (Figure 1—figure supplement 1B–C). As a control, we used neurons isolated from the same litter but expressing a defective Cre-recombinase that lacks the catalytic domain (ΔCre). Double knockout (DKO) neurons developed normally in culture and did not show any defects in neurite outgrowth and synapse formation as evidenced by normal dendrite length and density of synaptophysin puncta (Figure 1—figure supplement 1D–G).

To examine DCV fusion, we used a modification of a validated reporter consisting of NPY fused to super-ecliptic pHluorin (Figure 1A, Figure 1—figure supplement 2; Persoon et al., 2018; Nassal et al., 2022). When expressed in primary neurons, this reporter localized almost exclusively to DCVs, as indicated by its co-localization with endogenous DCV markers IA-2 and chromogranins (CHGA, CHGB) (Figure 1—figure supplement 2). Fusion of labeled DCVs with the plasma membrane was stimulated by repetitive bursts of action potentials delivered at 50 Hz to single neurons grown on glial micro-islands. Individual fusion events were detected as transient increases in fluorescence intensity along neurites, as exemplified in Figure 1B. Our stimulation paradigm resulted in a steep accumulation of fusion events that plateaued shortly before the end of the stimulation (Figure 1C). Most of these events (∼80%) were previously shown to be of axonal origin (Persoon et al., 2018). Virtually no fusion events were detected in neurons of both genotypes in the absence of electric stimulation (first 30 s of recordings or in the period after stimulation). Surprisingly, the total number of evoked fusion events was similar between control and DKO neurons, with a tendency (p=0.09) towards a decrease in DKO neurons (Figure 1D). The intracellular pool of the DCV reporter, visualized by a short application of 50 mM NH₄Cl to neurons to dequench intravesicular NPY-pHluorin, was decreased by 28% in DKO neurons (Figure 1E). The released fraction, i.e., the number of fusion events normalized to the intracellular pool of vesicles, was nearly identical between the genotypes and comprised approximately 8% of the intracellular content, consistent with the results of our previous studies (Persoon et al., 2018; Moro et al., 2020; Figure 1F). Also, the averaged fluorescence peak of individual fusion events, an estimate of NPY loading per vesicle, was not affected by the loss of tomosyns (Figure 1G). Thus, tomosyns are dispensable for the evoked fusion of neuronal DCVs in hippocampal neurons.

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Tomosyn was proposed to act as an inhibitor of vesicle fusion by competing with VAMP2 for the binding to syntaxin-1 and SNAP25. Hence, loss of tomosyns may result in the increased formation of ternary synaptic SNARE complexes consisting of syntaxin-1, SNAP25, and VAMP2. These complexes can be detected based on their resistance to SDS, as was shown for tomosyn-deficient fly brains (Sauvola et al., 2021). We examined levels of the assembled complexes in DKO neurons by SDS-PAGE on non-boiled neuronal lysates, since SDS-resistant complexes disassemble at temperatures higher than 60 °C (Hayashi et al., 1994; Otto et al., 1997; Figure 1H). As expected, a band of approximately 80 kDa that corresponds to the ternary complexes was readily detected with a syntaxin-1 antibody in non-boiled lysates and absent in lysates heated at 95 °C. This band shifted in size upon expression of tetanus toxin light chain (TeNT), which results in cleavage of VAMP2, thus confirming that the detected complexes contain VAMP2 (Figure 1—figure supplement 3C). No difference in the intensity of the 80 kDa band was observed between control and DKO cultures. The same results were obtained when non-boiled lysates were probed with antibodies against SNAP25 and VAMP2 (Figure 1—figure supplement 3A–B). These data suggest that tomosyns do not interfere with the bulk formation of syntaxin-1 containing synaptic SNARE complexes in cultured neurons. Interestingly, in addition to the 80 kDa band, non-boiled WT lysates showed a band of approximately 200 kDa, detected with antibodies against syntaxin-1 and SNAP25. This band was absent in DKO lysates and, therefore, likely represents a complex of tomosyn with syntaxin-1 and SNAP25, suggesting that this complex is resistant to SDS in vivo. This 200 kDa complex was also detected in control neurons with an antibody against tomosyn (Figure 1H).

Despite normal DCV exocytosis, intracellular levels of the DCV reporter were decreased in DKO neurons (Figure 1E). This decrease was also observed in tetrodotoxin (TTX)-silenced network cultures of DKO neurons that were not subjected to electric stimulation (Figure 2A–B). Activity of the synapsin promoter used to drive expression of the DCV reporter was not significantly altered in DKO neurons, as shown by normal expression of enhanced green fluorescent protein (EGFP) from the same promoter (Figure 2—figure supplement 1). Re-expression of the most ubiquitous tomosyn isoform (m-isoform of Stxbp5) restored levels of the overexpressed NPY-pHluorin in DKO neurons (Figure 2A–B). These data suggest that tomosyns affect the amount of the overexpressed DCV cargo and/or the number of DCVs in hippocampal neurons.

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We next examined if endogenous DCV cargos are also affected by the loss of tomosyns. Levels of BDNF, one of the most common DCV cargos in excitatory hippocampal neurons (Dieni et al., 2012), were decreased by more than half in DKO neurons as shown by ICC and WB analysis (Figure 2C–F). Levels of IA-2 (PTPRN), a transmembrane DCV-resident protein commonly used as a DCV marker, were also lower, by 25%, in DKO neurons as indicated by ICC, and by approximately 50% as evidenced by WB analysis, which allows to distinguish a proteolytically processed (mature) form of IA-2 that runs at approximately 65 kDa (Hermel et al., 1999; Figure 2—figure supplement 2). The cellular distribution of the two DCV markers was similar between the genotypes, with a comparable reduction in somas and neurites of DKO neurons and no re-distribution between the two compartments as detected by ICC. Markers of lysosomes/late endosomes, LAMP1, and LIMP2, were not affected by the loss of tomosyns, as shown by ICC and WB (Figure 2—figure supplement 3). Re-expression of tomosyn (isoform 1 m, NM_001081344) largely restored BDNF levels in DKO neurons (to approximately 80% of WT levels), as shown by ICC and WB (Figure 2C–D and G–H). The SNARE-domain of tomosyn was dispensable for the partial rescue of BDNF levels, since expression of a truncated tomosyn mutant lacking the SNARE-domain (ΔSN) rescued BDNF levels in DKO to the same extent as re-expression of the full-length (FL) tomosyn (Figure 2G–H). Taken together, these data suggest that tomosyns affect intracellular levels of some endogenous DCV cargos in a SNARE-domain independent manner.

Levels of BDNF and other secretory cargos are known to be regulated by lysosomal degradation. We, therefore, tested if an increase in BDNF degradation could account for its decreased levels in the absence of tomosyns. Inhibition of lysosomal proteases by leupeptin for 24 hr resulted in a twofold increase of BDNF levels in neurons of both genotypes but failed to raise BDNF expression in DKO to WT levels (Figure 2G and I), indicating that increased cargo degradation does not explain the decreased BDNF levels in DKO neurons.

We next asked whether levels of any other (secretory) proteins are affected by the loss of tomosyns. To address this question in an unbiased manner, we compared the proteome of cultured control and DKO neurons by mass spectrometry. We detected approximately 8500 proteins, of which around 3% were differentially expressed (log₂ fold change threshold 0.3) (Figure 3A). Gene ontology (GO) analysis of proteins decreased in DKO neurons indicating a strong enrichment of proteins related to the cytoskeleton and secretory granules (Figure 3B). As expected, both tomosyns were not detected in DKO neurons (Figure 3C). Interestingly, BDNF was also barely detected in DKO (only in one out of six biological replicates), while readily detected across all replicates in control conditions (Figure 3C), confirming a strong decrease in BDNF levels in DKO neurons. Many common DCV cargos, such as granins VGF and SCG2, cargo processing enzymes PCSK1 and CPE, and transmembrane DCV proteins IA-2 (PTPRN) and PAM were also decreased, albeit to a different extent, in DKO (Figure 3D). Most of the up-regulated proteins were components of the mitochondrial electron transport chain, suggesting that an increase in oxidative phosphorylation is a metabolic feature of DKO neurons (Figure 3B and D, Supplementary file 1). In contrast to the proteins regulating oxidative phosphorylation, most other mitochondrial proteins, for example, proteins responsible for mitochondrial DNA replication and transcription, were unchanged in DKO. A selected number of synaptic proteins were affected in DKO neurons but, in contrast to the DCV-resident proteins, SV proteins did not follow any general trend in DKO neurons: while some proteins (glutamate transporter VGLUT2, glutamate decarboxylase GAD67) were strongly increased in DKO, others (e.g. SYT12 and SV2B) were reduced to a similar extent, and many (e.g. main glutamate transporter VGLUT1, vesicular GABA transporter VGAT) were not significantly altered (Figure 3D). In conclusion, Cre-mediated loss of both tomosyns leads to selective changes in neuronal proteome with a reduction in cytoskeletal and DCV cargo proteins, an increase in mitochondrial proteins driving oxidative respiration, and a re-arrangement of synaptic proteome.

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Next, we examined the ultrastructural morphology of DCVs in DKO and control neurons by electron microscopy (Figure 4). We found DCVs with normal dense cores sparsely distributed among neurites in neurons of both genotypes. Most presynaptic sections did not contain any DCVs, in line with previous reports (Sorra et al., 2006; Persoon et al., 2018). Percent of synaptic sections containing one or several DCVs was decreased in DKO by 30% (12% of all synaptic sections in DKO versus 17% in control), indicating lowered DCV abundancy in DKO. In addition, DCVs in DKO neurons were slightly reduced in diameter (average 65.01±2.08 nm in DKO versus 71.52±1.62 nm in control), with a high proportion of DKO DCVs being only 40–50 nm in diameter (16% of all DCVs in DKO versus 4% in control) (Figure 4A–B). Such a reduction in diameter translates to an approximately 25% decrease in volume. In contrast, the diameter of SV was not affected by the loss of tomosyns (Figure 4D–E). Other parameters of synaptic ultrastructure, such as SV number per synapse, length of the active zone, and length of postsynaptic density, were also unchanged in DKO neurons (Figure 4D and F–H) and DKO somata showed no obvious abnormalities in organelle morphology (Figure 4D). Hence, loss of tomosyns leads to a reduction in DCV size and abundancy, without affecting ultrastructure and the number of SVs.

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The fact that the abundancy, composition, and size of neuronal DCVs are affected in DKO neurons suggests that tomosyns may regulate DCV biogenesis. DCVs are formed at the TGN in a sequence of membrane fusion/fission events ensuring correct packaging and processing of DCV cargos. We found that tomosyn was enriched at the TGN of cultured hippocampal neurons, as evidenced by the partial colocalization with a TGN marker, TGN38 (Figure 5—figure supplement 1A–B). As reported previously, tomosyn also colocalized with mature DCVs and showed an enrichment at the synapses (Figure 5—figure supplement 1C–F; Geerts et al., 2017), although colocalization of tomosyn with the DCV marker IA-2 was less extensive than with the synaptic marker synaptophysin, possibly due to the fact that IA-2 is also present in endosomes (Solimena et al., 1996). The TGN area, visualized by immunostaining of the TGN marker TMEM87A, was slightly decreased in DKO neurons as compared to controls, suggesting a functional importance of tomosyn at the Golgi (Figure 5A–B).

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We next tested whether tomosyn regulates the packaging and export of a neuropeptide cargo from the TGN using a modification of the Retention Using the Selective Hooks (RUSH) method (Boncompain et al., 2012). In this method, streptavidin-binding peptide (SBP)-tagged secretory cargo is retained in the endoplasmic reticulum (ER) until the addition of biotin due to the expression of the ER-localized streptavidin. Biotin disrupts SBP/streptavidin interaction and induces a synchronized transport of the cargo from the ER along the secretory pathway (Figure 5C–D). As a neuropeptide cargo, we used NPY fused to SBP and EGFP, as described previously (Emperador-Melero et al., 2018). In the absence of biotin, NPY-EGFP-SBP was diffusely distributed along cell soma and processes, in agreement with its localization to the ER. Addition of biotin induced a wave of NPY-EGFP-SBP accumulating in the Golgi first, and then leaving the Golgi as discrete puncta (Figure 5E, Figure 5—video 1). These puncta co-localized with an endogenous DCV marker, CHGA, indicating correct packaging of the NPY fusion protein into DCVs during the RUSH assay (Figure 5—figure supplement 2). Formation of such puncta and clearance of NPY-EGFP-SBP from the Golgi was observed in neurons of both genotypes. We next compared the speed of NPY-EGFP-SBP flux through the Golgi by plotting EGFP intensity within the Golgi area over time (Figure 5F). The peak of fluorescence intensity in the Golgi was reached 5.6 min faster in DKO than in control, indicating that the speed of Golgi filling is increased in DKO (Figure 5G). The speed of Golgi clearance – determined by fitting a first order exponential decay curve to the data after the peak – was also increased (by 20%) in DKO neurons (Figure 5H). Thus, loss of tomosyns does not impede export of the overexpressed neuropeptide cargo from Golgi but, conversely, accelerates it.

We also traced the fate of newly made RUSH vesicles 90 min after the addition of biotin. After the exit from Golgi, these vesicles traveled predominantly in one neurite, morphologically identified as the axon (Figure 5I). Most axonal vesicles (67%) traveled in the anterograde direction with a similar speed in neurons of both genotypes (Figure 5J–K), indicating that the transport of newly made DCVs is not affected by the loss of tomosyns.

We then asked if the loss of tomosyns affected mRNA expression of secretory cargos that were significantly decreased in DKO as evidenced by mass spectrometry and WB analysis. We detected a decrease in the expression of various transcript variants of Bdnf (Figure 6A) and other common DCV cargos, most prominently Vgf and Pcsk1, encoding a prohormone convertase (Figure 6B). Interestingly, the decrease in mRNA was less pronounced than the decrease at the protein level, as detected by WB (for BDNF) or mass spectrometry (for VGF and PCSK1) (Figure 6—figure supplement 1). The number of neuropeptides was decreased at the mRNA level in DKO as well (Figure 6C). Grp (gastrin related peptide) and Pnoc (nociceptin, nocistatin) showed the strongest reduction (by 50% and 35%, respectively), while mRNA of genes encoding cholecystokinin, NPY, and tachykinins displayed a reduction by around 30%. It is not clear whether any of these peptides were also decreased at the protein level, since most processed neuropeptides could not be detected in our mass spectrometry-based proteomics analysis due to their small size. Based on single-cell transcriptomics data (Saunders et al., 2018), expression of the affected genes was not confined to a specific neuronal population (e.g. glutamatergic or GABAergic neurons). Strikingly, re-expression of tomosyn (isoform 1 m) from DIV2 onwards did not restore altered transcription of the affected genes, suggesting that tomosyn does not mediate these changes (Figure 6D). Since no annotated genes and functional elements, other than Stxbp5 and Stxbp5l, were predicted to be affected by recombination of the Stxbp5 /Stxbp5 l loci in our mouse model, we tested whether the expression of Cre recombinase per se results in the detected transcriptional defects. Indeed, Cre expression in wild-type neurons not bearing introduced loxP sites led to a decrease in the mRNA expression of Bdnf, Vgf, Pcsk1, and Grp when compared to ΔCre-expressing wild-type neurons, with the size effect comparable to the one observed in Stxbp5/5l^lox neurons (Figure 6E). Expression of Gapdh, used as housekeeping gene control, was not affected by Cre. Thus, changes in DCV cargos transcription detected in DKO neurons are caused by direct effects of Cre expression, rather than by loss of tomosyns.

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We next asked whether this effect of Cre expression contributes to the decrease of DCV cargos in DKO at the protein level. Lentivirus-mediated expression of Cre in wild-type neurons resulted in a decrease of BDNF levels by 26% (Figure 6F–G), as compared to >50% reduction in BDNF documented in DKO (Cre-expressing Stxbp5/5l^lox) neurons (Figure 2E–F). Expression of ΔCre that lacks the catalytic domain did not affect BDNF levels when compared to wild-type neurons expressing mCherry under the same promoter (Figure 6F–G), suggesting that recombinase activity is necessary for the effect of Cre on BDNF levels. Cre expression in wild-type neurons did not affect tomosyn levels. These data indicate that unspecific Cre recombinase activity contributes to the reduction in BDNF protein levels in DKO neurons, but to a lesser extent than the loss of tomosyns. In line with this conclusion, BDNF protein levels in DKO were largely rescued by the re-expression of tomosyn (Figure 2C–D and G–H).

To test if the latter conclusion also applies to a DCV reporter overexpressed under the control of a synapsin promoter, we tested the effects of Cre on the levels of NPY-pHluorin. Cre expression in wild-type neurons did not affect levels of NPY-pHluorin as shown by ICC (Figure 6—figure supplement 1), while the same reporter showed an approximately 20% reduction upon expression of Cre in Stxbp5/5l^lox neurons that was rescued by re-expression of tomosyn (Figure 2A–B). Thus, while Cre expression impairs transcription of endogenous DCV cargos in both wild-type and Stxbp5/5l^lox neurons, it affects protein levels of the overexpressed DCV reporter only in Stxbp5/5l^lox but not in wild-type neurons.

In this study, we examined the role of tomosyns in the exocytosis of neuropeptide-containing DCVs in mammalian neurons. Using a pHluorin-based DCV fusion reporter, we found that the conditional loss of tomosyns does not alter the number of DCV exocytosis events but lowers intracellular levels of the DCV reporter. We corroborated the latter finding by showing that levels of endogenous DCV cargos, such as BDNF, are decreased in tomosyn DKO neurons by three independent approaches (ICC, WB, and mass spectrometry). The effect on the DCV pool and/or composition was accompanied by a smaller DCV size and increased speed of the DCV reporter flux through the TGN. Taken together, these data suggest that tomosyn is dispensable for control of DCV fusion, but, paradoxically, has an impact on the intracellular levels of DCV cargos.

The lack of the effect on neuronal DCV fusion is surprising, given that tomosyn regulates SV release in neurons and secretory granule exocytosis in neuroendocrine cells and platelets. However, phenotypes caused by the loss of tomosyn expression in different cell types are discrepant. While the loss of tomosyn resulted in enhanced SV fusion in the mouse brain and invertebrate nervous system (Sakisaka et al., 2008; Ben-Simon et al., 2015; Gracheva et al., 2006; McEwen et al., 2006; Chen et al., 2011; Sauvola et al., 2021), tomosyn deficiency in platelets, INS-1E cells and rat superior cervical ganglion neurons led to an opposite phenotype – a strong reduction in secretion (Ye et al., 2014; Zhu et al., 2014; Cheviet et al., 2006; Baba et al., 2005). No unifying model explains the discrepant effects of tomosyn on secretory vesicle fusion. Altogether, the discrepant effects of tomosyn on fusion in different cell types suggest that tomosyn does not act solely as an inhibitor of fusogenic SNARE complex formation.

DCVs and SVs differ in release properties, and recent studies shed light on some components of the molecular machinery contributing to these differences (Persoon et al., 2019; Moro et al., 2021). In contrast to SVs, which are released in response to a single electric pulse, neuronal DCVs require prolonged high-frequency stimulation for induction of exocytosis (Hartmann et al., 2001; Xia et al., 2009; Persoon et al., 2018). Several factors can explain the reluctance of DCVs to fuse, among them the loose coupling of DCVs to the sites of calcium entry, requiring an increase in calcium levels reaching beyond an immediate radius of a calcium channel (Mansvelder and Kits, 2000). The difference in calcium sensitivity between SVs and DCVs may facilitate frequency-dependent release of different types of chemical signals from neurons and ensure that neuropeptides are secreted predominantly at high firing rates. Interestingly, the inhibitory effect of tomosyn on DCV fusion in adrenal chromaffin cells is calcium-dependent and could be overridden by an increase in intracellular calcium (Yizhar et al., 2004). Thus, it is possible that high-frequency stimulation and a massive increase in intracellular calcium required for the induction of neuronal DCV exocytosis overrides the regulatory effect of tomosyn.

Despite the lack of effect on DCV exocytosis, loss of tomosyns resulted in the decreased intracellular levels of overexpressed (NPY-pHluorin) and endogenous (BDNF, IA-2) DCV cargos (Figure 2, Figure 2—figure supplement 1). Importantly, this phenotype was reproduced in DKO neurons grown under three different culture conditions that vary in cell density and the ratio of neurons to glia (single neurons grown on glial microislands, sparse network cultures on glial feeder layer, and dense network cultures on coated plastic). This phenotype is consistent with the effect of tomosyn’s loss in platelets, i.e. a strong decrease in many secretory cargos, despite the fact that the release of these cargos was reduced, not enhanced (Ye et al., 2014). Tomosyn-deficient platelets showed more than 50% decrease in the levels of alpha-granules cargos, such as factor V and platelet factor 4 (PF4), although alpha-granule count was not affected. Similarly, loss of tomosyn orthologs SRO7/77 in yeast resulted in the altered composition of secretory vesicles and re-routing of a secretory cargo, sodium pumping ATPase Ena1p, from secretory vesicles into the degradative pathway (vacuole) (Forsmark et al., 2011; Wadskog et al., 2006). Despite the fact that alpha-granules in platelets and secretory vesicles in yeast differ from neuronal DCVs in many aspects, taken together, our published data suggest a general role of tomosyn and its orthologs in sorting cargos into various types of secretory granules. The enrichment of tomosyn at the TGN (Figure 5—figure supplement 1) and decreased DCV and Golgi size in DKO neurons (Figures 4A–C–5A–B) are consistent with such a role.

Importantly, the loss of tomosyns in neurons did not abolish the formation of new secretory granules, as evidenced by the normal budding of NPY-vesicles in the RUSH assay (Figure 5E, Figure 5—video 1). However, NPY flux through the Golgi was slightly accelerated in DKO (Figure 5F–H), supporting the hypothesis that tomosyn regulates the sorting of the soluble cargo at the TGN. The decreased size of DCVs suggests that loading of DCVs with secretory cargo is impaired in DKO, since DCV size is regulated by the amount of loaded cargo, at least in non-neuronal cell types (Clermont et al., 1993; Boquist and Lorentzon, 1979). On the other hand, the peak amplitude of individual NPY-pHluorin fusion events, an estimate of NPY loading per fusing DCV, was not affected in DKO (Figure 1G). This discrepancy suggests that smaller DCVs in DKO may be reluctant to fuse due to the deficiency in specific transmembrane cargos necessary for DCV exocytosis. Another important observation is that interfering with lysosomal activity did not rescue cargo levels in DKO neurons (Figure 2G), indicating that cargo is not mis-sorted to lysosomes in the absence of tomosyns.

Re-expression of tomosyn rescued protein, but not mRNA expression, of DCV cargos in DKO neurons (Figure 6D). We found that the recombinase activity of Cre, irrespective of the presence of the loxP sites, affects the expression of some DCV cargos (Bdnf, Grp, Pcsk1), explaining the inability of tomosyn to rescue transcription of these genes. It has been long acknowledged that Cre expression can have deleterious side effects (Schmidt et al., 2000; Loonstra et al., 2001; Heidmann and Lehner, 2001; Forni et al., 2006; Naiche and Papaioannou, 2007), although, to our knowledge, none were reported for primary neuronal cultures. Cre can catalyze recombination between pseudo (cryptic) loxP sites (Thyagarajan et al., 2000). Our data suggest that Cre expression in postmitotic neurons affects neuronal transcriptome and warrants caution when interpreting data on the proteome DKO neurons.

Notwithstanding this effect, the current study provides robust evidence that the decrease of BDNF at the protein level is caused, to a large extent, specifically by the loss of tomosyns, and not by the effects of Cre on transcription. First, BDNF levels were decreased by more than 50% in DKO neurons (Figure 2C–D and E–F), and only by around 26% in Cre-expressing wild-type neurons (Figure 6F–G). Second, BDNF levels in DKO were restored to approximately 80% of control levels by re-expression of tomosyn (Figure 2C–D). Finally, in support of the specific effects of tomosyn on DCV cargos, levels of the overexpressed NPY-pHluorin levels were decreased in DKO, but not in Cre-expressing wild-type neurons, and this decrease was rescued by the re-expression of tomosyn (Figure 2A–B, Figure 6—figure supplement 1).

Interestingly, the SNARE domain of tomosyn was dispensable for the rescue of BDNF levels in DKO, suggesting that the N-terminal WD40-domain mediates this effect of tomosyn (Figure 2G–H). The main structural feature of the WD40-domain are two bulky beta-propellers comprised of multiple WD40 repeats. This domain constitutes the largest part of tomosyn’s molecule, and it is best conserved among all tomosyn orthologs, including the yeast SRO7/77 that, in fact, lack the canonical SNARE domain. Previous studies showed that WD40-domain alone is not sufficient to rescue synaptic transmission defects caused by the loss of tomosyn orthologs in the fly and nematode (Burdina et al., 2011; Sauvola et al., 2021). On the other hand, overexpression of the WD40 domain alone has an inhibitory effect on secretion from bovine chromaffin and rat pheochromocytoma PC12 cells (Yizhar et al., 2007; Bielopolski et al., 2014). This effect has been attributed to the binding of specific loops in the WD40 propeller region to SNAP25 (Bielopolski et al., 2014). Several other interaction partners of tomosyn were reported to bind to the WD40-propellers, such as Rab3A and synaptotagmin (Yamamoto et al., 2010; Cazares et al., 2016), suggesting that the WD40 domain may serve as a scaffold for the assembly of the dynamic protein complexes. The strength of these interactions can be regulated by posttranslational modifications of the WD40 domain, such as phosphorylation (by PKA, ROCK) and SUMOylation (Baba et al., 2005; Gladycheva et al., 2007; Ferdaoussi et al., 2017). A recent study identified tomosyn as an inhibitor of RhoA in the WD40-domain dependent manner (Shen et al., 2020). RhoA is a small GTPase that regulates, among other processes, membrane trafficking at the Golgi (Quassollo et al., 2015). Remarkably, Sro7 was originally identified in a genome-wide screen as a multicopy suppressor of RHO3, an ortholog of Rho GTPases in yeast, suggesting an evolutionary conserved link between tomosyn and Rho GTPases (Matsui and Toh-E, 1992). Further studies are required to validate the potential regulation of Rho by tomosyn and to assess a role of this hypothetical pathway at the Golgi.

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Author details

1. Aygul Subkhangulova

   Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit (VU) Amsterdam, Amsterdam, Netherlands

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Writing – review and editing

   For correspondence

   a.subkhangulova@vu.nl

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8843-0678

2. Miguel A Gonzalez-Lozano

   Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit (VU) Amsterdam, Amsterdam, Netherlands

   Present address

   Department of Cell Biology, Harvard Medical School, Boston, United States

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7837-151X

3. Alexander JA Groffen

   Department of Human Genetics, Center for Neurogenomics and Cognitive Research (CNCR), Amsterdam University Medical Center (UMC), Amsterdam, Netherlands

   Contribution

   Resources, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0046-4027

4. Jan RT van Weering

   Department of Human Genetics, Center for Neurogenomics and Cognitive Research (CNCR), Amsterdam University Medical Center (UMC), Amsterdam, Netherlands

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

5. August B Smit

   Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit (VU) Amsterdam, Amsterdam, Netherlands

   Contribution

   Conceptualization, Supervision, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

6. Ruud F Toonen

   Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit (VU) Amsterdam, Amsterdam, Netherlands

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9900-4233

7. Matthijs Verhage

   1. Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit (VU) Amsterdam, Amsterdam, Netherlands
   2. Department of Human Genetics, Center for Neurogenomics and Cognitive Research (CNCR), Amsterdam University Medical Center (UMC), Amsterdam, Netherlands

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review and editing

   For correspondence

   matthijs@cncr.vu.nl

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6085-7503

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