SNX5 targets a monoamine transporter to the TGN for assembly into dense core vesicles by AP-3.

The time course of signaling by peptide hormones, neural peptides, and other neuromodulators depends on their storage inside dense core vesicles (DCVs). Adaptor protein 3 (AP-3) assembles the membrane proteins that confer regulated release of DCVs and is thought to promote their trafficking from endosomes directly to maturing DCVs. We now find that regulated monoamine release from DCVs requires sorting nexin 5 (SNX5). Loss of SNX5 disrupts trafficking of the vesicular monoamine transporter (VMAT) to DCVs. The mechanism involves a role for SNX5 in retrograde transport of VMAT from endosomes to the TGN. However, this role for SNX5 conflicts with the proposed function of AP-3 in trafficking from endosomes directly to DCVs. We now identify a transient role for AP-3 at the TGN, where it associates with DCV cargo. Thus, retrograde transport from endosomes by SNX5 enables DCV assembly at the TGN by AP-3, resolving the apparent antagonism. A novel role for AP-3 at the TGN has implications for other organelles that also depend on this adaptor.

Dynamin deficiency causes insulin secretion failure and hyperglycemia.

Pancreatic ß cells operate with a high rate of membrane recycling for insulin secretion, yet endocytosis in these cells is not fully understood. We investigate this process in mature mouse ß cells by genetically deleting dynamin GTPase, the membrane fission machinery essential for clathrin-mediated endocytosis. Unexpectedly, the mice lacking all three dynamin genes (DNM1, DNM2, DNM3) in their ß cells are viable, and their ß cells still contain numerous insulin granules. Endocytosis in these ß cells is severely impaired, resulting in abnormal endocytic intermediates on the plasma membrane. Although insulin granules are abundant, their release upon glucose stimulation is blunted in both the first and second phases, leading to hyperglycemia and glucose intolerance in mice. Dynamin triple deletion impairs insulin granule exocytosis and decreases intracellular Ca2+ responses and granule docking. The docking defect is correlated with reduced expression of Munc13-1 and RIM1 and reorganization of cortical F-actin in ß cells. Collectively, these findings uncover the role of dynamin in dense-core vesicle endocytosis and secretory capacity. Insulin secretion deficiency in the absence of dynamin-mediated endocytosis highlights the risk of impaired membrane trafficking in endocrine failure and diabetes pathogenesis.

Rab2 drives axonal transport of dense core vesicles and lysosomal organelles.

Fast axonal transport of neuropeptide-containing dense core vesicles (DCVs), endolysosomal organelles, and presynaptic components is critical for maintaining neuronal functionality. How the transport of DCVs is orchestrated remains an important unresolved question. The small GTPase Rab2 mediates DCV biogenesis and endosome-lysosome fusion. Here, we use Drosophila to demonstrate that Rab2 also plays a critical role in bidirectional axonal transport of DCVs, endosomes, and lysosomal organelles, most likely by controlling molecular motors. We further show that the lysosomal motility factor Arl8 is required as well for axonal transport of DCVs, but unlike Rab2, it is also critical for DCV exit from cell bodies into axons. We also provide evidence that the upstream regulators of Rab2 and Arl8, Ema and BORC, activate these GTPases during DCV transport. Our results uncover the mechanisms underlying axonal transport of DCVs and reveal surprising parallels between the regulation of DCV and lysosomal motility.

Neuromodulator release in neurons requires two functionally redundant calcium sensors.

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.

Quantitative analysis of dense-core vesicle fusion in rodent CNS neurons.

Neuropeptides are essential signaling molecules secreted by dense-core vesicles (DCVs). They contribute to information processing in the brain, controlling a variety of physiological conditions. Defective neuropeptide signaling is implicated in several psychiatric disorders. Here, we provide a protocol for the quantitative analysis of DCV fusion events in rodent neurons using pH-sensitive DCV fusion probes and custom-written analysis algorithms. This method can be used to study DCV fusion mechanisms and is easily adapted to investigate fusion principles of other secretory organelles. For complete details on the use and execution of this protocol, please refer to Persoon et al. (2019).

Down-regulation of habenular calcium-dependent secretion activator 2 induces despair-like behavior.

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.

Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging.

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.