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Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

The absolute concentration and the compartmentalization of analytes in cells and organelles are crucial parameters in the development of drugs and drug delivery systems, as well as in the fundamental understanding of many cellular processes. Nanoscale secondary ion mass spectrometry (NanoSIMS) imaging is a powerful technique which allows subcellular localization of chemical species with high spatial and mass resolution, and high sensitivity. In this study, we combined NanoSIMS imaging with spatial oversampling with transmission electron microscopy (TEM) imaging to discern the compartments (dense core and halo) of large dense core vesicles in a model cell line used to study exocytosis, and to localize 13C dopamine enrichment following 4–6 h of 150 μM 13C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation. In addition, the absolute concentrations of 13C dopamine in distinct vesicle domains as well as in entire single vesicles were quantified and validated by comparison to electrochemical data. We found concentrations of 87.5 mM, 16.0 mM and 39.5 mM for the dense core, halo and the whole vesicle, respectively. This approach adds to the potential of using combined TEM and NanoSIMS imaging to perform absolute quantification and directly measure the individual contents of nanometer-scale organelles.

Transport of dense core vesicles (DCVs) in neurons is crucial for distributing molecules like neuropeptides and growth factors. We studied the experimental trajectories of dynein-driven directed movement of DCVs in the ALA neuron in  C. elegans over a duration of up to 6 seconds. We analysed the DCV movement in three strains of C. elegans : (1) with normal kinesin-1 function, (2) with reduced function in kinesin light chain 2 (KLC-2), and (3) a null mutation in kinesin light chain 1 (KLC-1). We find that DCVs move superdiffusively with displacement variance in all three strains with low reversal rates and frequent immobilization of DCVs. The distribution of DCV displacements fits a beta-binomial distribution with the mean and the variance following linear and quadratic growth patterns, respectively. We propose a simple heterogeneous random walk model to explain the observed superdiffusive retrograde transport behaviour of DCV movement. This model involves a random probability with the beta density for a DCV to resume its movement or remain in the same position. To validate our model further, we measure the first passage time for a DCV to reach a certain threshold for the first time. According to the model, the first passage time distribution should follow a beta-negative binomial distribution with the same parameters as the DCV displacement distributions. Our experimental data confirm this prediction.

This report demonstrates insoluble alpha-synuclein (aSYN)+ aggregates in human sporadic Parkinson’s disease (PD) midbrain that are linearly correlated with loss of glucocerebrosidase (GCase) activity. To identify early protein-lipid interactions that coincide with loss of lipid homeostasis, an aging study was carried out in mice with age-dependent reductions in GCase function. The analysis identified aberrant lipid-association by aSYN and hyperphosphorylated Tau (pTau) in a specific subset of neurotransmitter-containing, Secretogranin II (SgII)+ large, dense-core vesicles (LDCVs) responsible for neurotransmission of dopamine and other monoamines. The lipid vesicle-accumulation was concurrent with loss of PSD-95 suggesting synaptic destabilization. aSYN overexpression in the absence of lipid deregulation did not recapitulate the abnormal association with SgII+ vesicles. These results show lipid-dependent changes occur with age in neuronal vesicular membrane compartments that accumulate lipid-stabilized aSYN and pTau.

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.

Calcium-dependent secretion activator 2 (CAPS2) regulates the trafficking and exocytosis of neuropeptide-containing dense-core vesicles (DCVs). CAPS2 is prominently expressed in the medial habenula (MHb), which is related to depressive behavior; however, how MHb neurons cause depressive symptoms and the role of CAPS2 remains unclear. We hypothesized that dysfunction of MHb CAPS neurons might cause defects in neuropeptide secretion and the activity of monoaminergic centers, resulting in depressive-like behaviors. In this study, we examined (1) CAPS2 expression in the habenula of depression animal models and major depressive disorder patients and (2) the effects of down-regulation of MHb CAPS2 on the animal behaviors, synaptic transmission in the interpeduncular nucleus (IPN), and neuronal activity of monoamine centers. Habenular CAPS2 expression was decreased in the rat chronic restraint stress model, mouse learned helplessness model, and showed tendency to decrease in depression patients who died by suicide. Knockdown of CAPS2 in the mouse habenula evoked despair-like behavior and a reduction of the release of DCVs in the IPN. Neuronal activity of IPN and monoaminergic centers was also reduced. These results implicate MHb CAPS2 as playing a pivotal role in depressive behavior through the regulation of neuropeptide secretion of the MHb-IPN pathway and the activity of monoaminergic centers.

Pancreatic β‐cells secrete insulin stored in large dense core vesicles (LDCV) by fusion of vesicle and plasma membrane during a process called insulin exocytosis. Insulin secretion is biphasic with a fast first phase and a sustained second phase. Previous studies have pointed out that exocytosis of insulin can occur via (1) single LDCVs fusing with the plasma membrane to release their content or (2) multiple vesicles are involved during a process called compound exocytosis. Compound exocytosis represents a specialized form of secretion in which vesicles undergo homotypic fusion either before (multi‐vesicular exocytosis) or continuous fusion in a sequential manner from (sequential exocytosis) within the same site at the plasma membrane. We see that the number of multi‐vesicles is few and not localized in the vicinity of the plasma membrane. Studying the kinetics of this process and correlating it with biphasic insulin secretion is not possible since there are no specific probes to detect them. It is challenging to identify compound exocytosis with probes that exist for simple exocytosis. To advance our understanding, we need a fluorescent probe that could detect secretory vesicles undergoing compound exocytosis and allow us to distinguish it from other modes of exocytosis. Here, we used two cargo proteins (NPY and tPA) labeled with different fluorescent proteins (mCherry GFP and eGFP) and employed total internal reflection fluorescence microscopy (TIRF‐M) to capture distinct single‐granule and multi‐granular fusion events. We identified tPA‐GFP as a better probe for studying compound exocytosis, as it can detect both simple and sequential exocytosis reliably. Using these probes, we have studied the kinetics of compound exocytosis in human β‐cells. These observations, with additional experiments, may open a whole new field to study the impact of compound exocytosis on biphasic secretion of insulin. Identifying targets to increase the compound exocytosis process can help potentiate insulin secretion in diabetics. Tissue plasminogen activator tagged to GFP (tPA‐GFP) specifically enables the labelling of insulin granules, a type of large dense‐core vesicle of the pancreatic β cells. When compared to other markers widely used to understand the behavior of insulin granules, tPA enabled the detection of the most number of simple (blue arrow) and compound exocytotic (gray arrows) events usingtotal internal fluorescence reflection microscopy (TIRF microscopy). This makes it a valuable tool in the understanding of the depths of compound exocytosis, including its mechanics and the machinery involved.

Large dense‐core vesicles (LDCVs) are larger in volume than synaptic vesicles, and are filled with multiple neuropeptides, hormones, and neurotransmitters that participate in various physiological processes. However, little is known about the mechanism determining the size of LDCVs. Here, it is reported that secretogranin II (SgII), a vesicle matrix protein, contributes to LDCV size regulation through its liquid–liquid phase separation in neuroendocrine cells. First, SgII undergoes pH‐dependent polymerization and the polymerized SgII forms phase droplets with Ca2+ in vitro and in vivo. Further, the Ca2+‐induced SgII droplets recruit reconstituted bio‐lipids, mimicking the LDCVs biogenesis. In addition, SgII knockdown leads to significant decrease of the quantal neurotransmitter release by affecting LDCV size, which is differently rescued by SgII truncations with different degrees of phase separation. In conclusion, it is shown that SgII is a unique intravesicular matrix protein undergoing liquid–liquid phase separation, and present novel insights into how SgII determines LDCV size and the quantal neurotransmitter release. The mystery of what fundamentally determines the size of large dense‐core vesicles (LDCVs) in neuroendocrine cells is uncovered. Secretogranin II, an intravesicular matrix protein, undergoes liquid–liquid phase separation in vitro and in vivo to determine the size/volume of LDCVs, thus tuning the quantal neurotransmitters release.

Dense core vesicles (DCVs) can transmit signals by releasing neuropeptides from specialized synaptic regions called active zones. DCVs reach the active zone by motorized transport through a long axon. A reverse motor frequently interrupts progress by taking DCVs in the opposite direction. “Guided transport” refers to the mechanism by which outward movements ultimately dominate to bring DCVs to the synaptic region. After guided transport, DCVs alter their interactions with motors and enter a “captured” state. The mechanisms of guided transport and capture of DCVs are unknown. Here, we discovered two proteins that contribute to both processes in Caenorhabditis elegans. SAD kinase and a novel conserved protein we named Sentryn are the first proteins found to promote DCV capture. By imaging DCVs moving in various regions of single identified neurons in living animals, we found that DCV guided transport and capture are linked through SAD kinase, Sentryn, and Liprin-α. These proteins act together to regulate DCV motorized transport in a region-specific manner. Between the cell body and the synaptic region, they promote forward transport. In the synaptic region, where all three proteins are highly enriched at active zones, they promote DCV pausing by inhibiting transport in both directions. These three proteins appear to be part of a special subset of active zone-enriched proteins because other active zone proteins do not share their unique functions.

Communication between neurons through synapses includes the release of neurotransmitter-containing synaptic vesicles and of neuromodulator-containing dense-core vesicles (DCVs). Neurexins, a polymorphic family of cell surface molecules encoded by three genes in vertebrates (Nrxn1-3), have been proposed as essential presynaptic organizers and as candidates for cell type-specific or even synapse-specific regulation of synaptic vesicle exocytosis. However, it remains unknown whether Nrxn also regulate DCVs. Here we report that at least β-neurexins (βNrxn), an extracellularly smaller Nrxn variant, are involved in the distribution of presynaptic DCVs. We found that conditional deletion of all three βNrxn isoforms in mice by lentivirus-mediated Cre recombinase expression in primary hippocampal neurons reduces the number of ultrastructurally identified DCVs in presynaptic boutons. Consistently, colabeling against marker proteins revealed a diminished population of chromogranin A-positive DCVs in synapses and axons of βNrxn-deficient neurons. Moreover, we validated the impaired DCV distribution in cerebellar brain tissue from constitutive βNrxn knockout (KO) mice, where DCVs are normally abundant and βNrxn isoforms prominently expressed. Finally, we observed that the ultrastructure and marker proteins of the Golgi apparatus, responsible for packaging neuropeptides into DCVs, appear unchanged. In conclusion, based on validation from two deletion strategies in conditional and constitutive KO mice, two neuronal populations from hippocampus and cerebellum, and two experimental protocols in cultured neurons and in brain tissue, this study presented morphological evidence that the number of DCV at synapses is altered in absence of βNrxn. Our results therefore point to an unexpected contribution of βNrxn to the organization of neuropeptide and neuromodulator function, in addition to their more established role in synaptic vesicle release.