The role of Kinesin-1 in neuronal dense core vesicle transport, locomotion and lifespan regulation in C. elegans.

Fast axonal transport is crucial for neuronal function and is driven by kinesins and cytoplasmic dynein. We investigated the role of kinesin-1 in dense core vesicle (DCV) transport in C. elegans, using mutants in kinesin light chains (klc-1 and klc-2) and motor subunit (unc-116) expressing an ida-1::gfp transgene that labels DCVs. DCV transport in both directions was greatly impaired in an unc-116 mutant and had reduced velocities in a klc-2 mutant. In contrast, the speed of retrograde DCV transport was increased in a klc-1 mutant whilst anterograde transport was unaffected. We identified striking differences between the klc mutants in their effects on worm locomotion and responses to drugs affecting neuromuscular junction activity. We also determined lifespan, finding that unc-116 mutant was short-lived whilst the klc single mutant life-span was wild type. The ida-1::gfp transgenic strain was also short-lived, but surprisingly, klc-1 and klc-2 extended the ida-1::gfp lifespan beyond wild type. Our findings suggest that kinesin-1 not only influences anterograde and retrograde DCV transport but is also involved in regulating lifespan and locomotion, with the two KLCs playing distinct roles.

Activator of KAT3 histone acetyltransferase family ameliorates a neurodevelopmental disorder phenotype in the syntaxin 1A ablated mouse model.

Syntaxin-1A (stx1a) repression causes a neurodevelopmental disorder phenotype, low latent inhibition (LI) behavior, by disrupting 5-hydroxytryptaminergic (5-HTergic) systems. Herein, we discovered that lysine acetyltransferase (KAT) 3B increases stx1a neuronal transcription and TTK21, a KAT3 activator, induces stx1a transcription and 5-HT release in vitro. Furthermore, glucose-derived CSP-TTK21 could restore decreased stx1a expression, 5-HTergic systems in the brain, and low LI in stx1a (+/-) mice by crossing the blood-brain barrier, whereas the KAT3 inhibitor suppresses stx1a expression, 5-HTergic systems, and LI behaviors in wild-type mice. Finally, in wild-type and stx1a (-/-) mice treated with IKK inhibitors and CSP-TTK21, respectively, we show that KAT3 activator-induced LI improvement is a direct consequence of KAT3B-stx1a pathway, not a side effect. In conclusion, KAT3B can positively regulate stx1a transcription in neurons, and increasing neuronal stx1a expression and 5-HTergic systems by a KAT3 activator consequently improves the low LI behavior in the stx1a ablation mouse model.

Pre-fusion motion state determines the heterogeneity of membrane fusion dynamics for large dense-core vesicles.

AIM: In neuroendocrine cells, large dense-core vesicles (LDCVs) undergo highly regulated pre-fusion processes before releasing hormones via membrane fusion. Significant heterogeneity has been found for LDCV population based on the dynamics of membrane fusion. However, how the pre-fusion status impacts the heterogeneity of LDCVs still remains unclear. Hence, we explored pre-fusion determinants of heterogeneous membrane fusion procedure of LDCV subpopulations. METHODS: We assessed the pre-fusion motion of two LDCV subpopulations with distinct membrane fusion dynamics individually, using total internal reflection fluorescence microscopy. These two subpopulations were isolated by blocking Rho GTPase-dependent actin reorganization using Clostridium difficile toxin B (ToxB), which selectively targets the fast fusion vesicle pool. RESULTS: We found that the fast fusion subpopulation was in an active motion mode prior to release, termed "active" LDCV pool, while vesicles from the slow fusion subpopulation were also moving but in a significantly more confined status, forming an "inert" pool. The depletion of the active pool by ToxB also eliminated fast fusion vesicles and was not rescued by pre-treatment with phorbol ester. A mild actin reorganization blocker, latrunculin A, that partially disrupted the active pool, only slightly attenuated the fast fusion subpopulation. CONCLUSION: The pre-fusion motion state of LDCVs also exhibits heterogeneity and dictates the heterogeneous fusion pore dynamics. Rearrangement of F-actin network mediates vesicle pre-fusion motion and subsequently determines the membrane fusion kinetics.

Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons.

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.

Exploiting Microelectrode Geometry for Comprehensive Detection of Individual Exocytosis Events at Single Cells.

Carbon fiber microelectrodes are commonly used for real-time monitoring of individual exocytosis events at single cells. Since the nature of an electrochemical signal is fundamentally governed by mass transport to the electrode surface, microelectrode geometry can be exploited to achieve precise and accurate measurements. Researchers traditionally pair amperometric measurements of exocytosis with a ∼10-µm diameter, disk microelectrode in an "artificial synapse" configuration to directly monitor individual release events from single cells. Exocytosis is triggered, and released molecules diffuse to the "post-synaptic" electrode for oxidation. This results in a series of distinct current spikes corresponding to individual exocytosis events. However, it remains unclear how much of the material escapes detection. In this work, the performance of 10- and 34-µm diameter carbon fiber disk microelectrodes was directly compared in monitoring exocytosis at single chromaffin cells. The 34-µm diameter electrode was more sensitive to catecholamines and enkephalins than its traditional, 10-µm diameter counterpart, and it more effectively covered the entire cell. As such, the larger sensor detected more exocytosis events overall, as well as a larger quantal size, suggesting that the traditional tools underestimate the above measurements. Both sensors reliably measured l-DOPA-evoked changes in quantal size, and both exhibited diffusional loss upon adjustment of cell-electrode spacing. Finite element simulations using COMSOL support the improved collection efficiency observed using the larger sensor. Overall, this work demonstrates how electrode geometry can be exploited for improved detection of exocytosis events by addressing diffusional loss─an often-overlooked source of inaccuracy in single-cell measurements.

Damaged mitochondria coincide with presynaptic vesicle loss and abnormalities in alzheimer's disease brain.

Loss of synapses is the most robust pathological correlate of Alzheimer's disease (AD)-associated cognitive deficits, although the underlying mechanism remains incompletely understood. Synaptic terminals have abundant mitochondria which play an indispensable role in synaptic function through ATP provision and calcium buffering. Mitochondrial dysfunction is an early and prominent feature in AD which could contribute to synaptic deficits. Here, using electron microscopy, we examined synapses with a focus on mitochondrial deficits in presynaptic axonal terminals and dendritic spines in cortical biopsy samples from clinically diagnosed AD and age-matched non-AD control patients. Synaptic vesicle density within the presynaptic axon terminals was significantly decreased in AD cases which appeared largely due to significantly decreased reserve pool, but there were significantly more presynaptic axons containing enlarged synaptic vesicles or dense core vesicles in AD. Importantly, there was reduced number of mitochondria along with significantly increased damaged mitochondria in the presynapse of AD which correlated with changes in SV density. Mitochondria in the post-synaptic dendritic spines were also enlarged and damaged in the AD biopsy samples. This study provided evidence of presynaptic vesicle loss as synaptic deficits in AD and suggested that mitochondrial dysfunction in both pre- and post-synaptic compartments contribute to synaptic deficits in AD.

High-speed imaging reveals the bimodal nature of dense core vesicle exocytosis.

During exocytosis, the fusion of secretory vesicle with plasma membrane forms a pore that regulates release of neurotransmitter and peptide. Heterogeneity of fusion pore behavior has been attributed to stochastic variation in a common exocytic mechanism, implying a lack of biological control. Using a fluorescent false neurotransmitter (FFN), we imaged dense core vesicle (DCV) exocytosis in primary mouse adrenal chromaffin cells by total internal reflection fluorescence microscopy at millisecond resolution and observed strikingly divergent modes of release, with fast events lasting <30 ms and slow events persisting for seconds. Dual imaging of slow events shows a delay in the entry of external dye relative to FFN release, suggesting exclusion by an extremely narrow pore <1 nm in diameter. Unbiased comprehensive analysis shows that the observed variation cannot be explained by stochasticity alone, but rather involves distinct mechanisms, revealing the bimodal nature of DCV exocytosis. Further, loss of calcium sensor synaptotagmin 7 increases the proportion of slow events without changing the intrinsic properties of either class, indicating the potential for independent regulation. The identification of two distinct mechanisms for release capable of independent regulation suggests a biological basis for the diversity of fusion pore behavior.

Rat Pheochromocytoma PC12 Cells in Culture.

PC12 cells serve as a secretory cell model, especially suitable for studying the molecular mechanisms underlying fusion pore kinetics in regulated exocytosis of dense-core vesicles (DCVs). In this chapter, we describe a series of PC12 cell culture procedures optimized for real-time functional assays such as single-vesicle amperometry. In addition, these conditions have been widely used for single-cell biochemical assays such as the proximity ligation assay with immunostaining.

Estrogen differentially regulates transcriptional landscapes of preoptic and arcuate kisspeptin neuron populations.

Kisspeptin neurons residing in the rostral periventricular area of the third ventricle (KPRP3V) and the arcuate nucleus (KPARC) mediate positive and negative estrogen feedback, respectively. Here, we aim to compare transcriptional responses of KPRP3V and KPARC neurons to estrogen. Transgenic mice were ovariectomized and supplemented with either 17ß-estradiol (E2) or vehicle. Fluorescently tagged KPRP3V neurons collected by laser-capture microdissection were subjected to RNA-seq. Bioinformatics identified 222 E2-dependent genes. Four genes encoding neuropeptide precursors (Nmb, Kiss1, Nts, Penk) were robustly, and Cartpt was subsignificantly upregulated, suggesting putative contribution of multiple neuropeptides to estrogen feedback mechanisms. Using overrepresentation analysis, the most affected KEGG pathways were neuroactive ligand-receptor interaction and dopaminergic synapse. Next, we re-analyzed our previously obtained KPARC neuron RNA-seq data from the same animals using identical bioinformatic criteria. The identified 1583 E2-induced changes included suppression of many neuropeptide precursors, granins, protein processing enzymes, and other genes related to the secretory pathway. In addition to distinct regulatory responses, KPRP3V and KPARC neurons exhibited sixty-two common changes in genes encoding three hormone receptors (Ghsr, Pgr, Npr2), GAD-65 (Gad2), calmodulin and its regulator (Calm1, Pcp4), among others. Thirty-four oppositely regulated genes (Kiss1, Vgf, Chrna7, Tmem35a) were also identified. The strikingly different transcriptional responses in the two neuron populations prompted us to explore the transcriptional mechanism further. We identified ten E2-dependent transcription factors in KPRP3V and seventy in KPARC neurons. While none of the ten transcription factors interacted with estrogen receptor-α, eight of the seventy did. We propose that an intricate, multi-layered transcriptional mechanism exists in KPARC neurons and a less complex one in KPRP3V neurons. These results shed new light on the complexity of estrogen-dependent regulatory mechanisms acting in the two functionally distinct kisspeptin neuron populations and implicate additional neuropeptides and mechanisms in estrogen feedback.

Morphology and synaptic connections of pigment-dispersing factor-immunoreactive neurons projecting to the lateral protocerebrum in the large black chafer, Holotrichia parallela.

Pigment-dispersing factor (PDF) is a well-known output neuropeptide modulator of circadian pacemakers in insects. Here, we investigated PDF-immunoreactive (ir) neurons in the brain of the large black chafer Holotrichia parallela to search for circadian neural components, which are potentially involved in its circabidian rhythm. PDF-ir cells were exclusively detected near the accessory medulla (AME) as a cluster of ∼ 100 cells with almost homogeneous size. No other cells exhibited immunoreactivity. The PDF-ir cells send beaded fibers into the proximal half of the AME and ventral elongation in an anterior region between the medulla (ME) and lobula (LO). Neither the lamina, ME, LO, nor lobula plate receives PDF-ir fibers. Primary axons derived from the PDF-ir cells extend toward the contralateral hemisphere through the dorsolateral protocerebrum anterior to the calyx to connect the bilateral AME. The axons form varicose outgrowths exclusively in the lateral protocerebrum. Double labeling with antisynapsin revealed partial overlaps between PDF-ir varicosities and synapsin-ir puncta. Thus, it was assumed that the PDF-ir fibers form output synapses there. To verify this, we investigated the ultrastructure of the PDF-ir varicosities in the lateral protocerebrum by preembedding immunoelectron microscopy. The PDF-ir profiles contain small clear synaptic vesicles as well as both PDF-positive and PDF-negative dense-core vesicles, and the profiles form output synapses upon unknown profiles and receive synapses from other PDF-ir profiles. PDF neurons near the AME are considered to be prominent circadian pacemakers in the cockroach and flies. Their possible function in the circabidian rhythm was discussed based on these anatomical insights.

Defective axonal transport of endo-lysosomes and dense core vesicles in a Drosophila model of C9-ALS/FTD.

A GGGGCC (G4 C2 ) repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although disruptions in axonal transport are implicated in the pathogenesis of multiple neurodegenerative diseases, the underlying mechanisms causing these defects remain unclear. Here, we performed live imaging of Drosophila motor neurons expressing expanded G4 C2 repeats in third-instar larvae and investigated the axonal transport of multiple organelles in vivo. Expression of expanded G4 C2 repeats causes an increase in static axonal lysosomes, while it impairs trafficking of late endosomes (LEs) and dense core vesicles (DCVs). Surprisingly, however, axonal transport of mitochondria is unaffected in motor axons expressing expanded G4 C2 repeats. Thus, our data indicate that expanded G4 C2 repeat expression differentially impacts axonal transport of vesicular organelles and mitochondria in Drosophila models of C9orf72-associated ALS/FTD.

Full-fusion and kiss-and-run in chromaffin cells controlled by irreversible vesicle size-dependent fusion pore transitions.

Exocytosis operates through two distinct modes. Full-fusion leads to rapid expulsion of the entire content of a vesicle; kiss-and-run leads to slow and partial expulsion. These two modes have important biological consequences for endocrine regulation and synaptic transmission. Amperometry recordings of catecholamine release from chromaffin cells reveal single-vesicle fusion events corresponding to both of these modes, but classification is often difficult. This study introduces a new method of analyzing amperometry data to improve this classification. The ratio of the average amplitude to the peak amplitude differs between full-fusion and kiss-and-run, and the probability distribution of this ratio is well fitted by a double-Gaussian. Kiss-and-run events identified by this method have fusion pores with kinetic properties different from pores associated with full-fusion. They have slower transition rates and lifetime distributions indicative of irreversible transitions. The total-charge of an amperometric spike is expected to scale with vesicle volume during a full-fusion event. The cube root of this quantity should therefore scale with diameter, but the distribution of this quantity differs from the distribution of vesicle diameter seen in the electron microscope. Fusion pore lifetimes associated with full-fusion depend on vesicle size, and this makes the choice of mode size dependent. The fusion pore thus bifurcates after opening, and vesicle size influences this choice. The secretory vesicle protein synaptophysin influences the size dependence of fusion pore lifetime and the choice of release mode. Incorporating vesicle size into an analysis of release mode reconciled the kinetics of fusion pores, as well as the distributions of vesicle diameter and catecholamine content. Thus, the initial fusion pore emerges as a critical focus in endocrine regulation. By modulating the size-dependence of the mode of exocytosis, changes in the molecular makeup of the exocytotic apparatus can impact the shape and size of an amperometric event, and the speed and composition of secretion.

Imaging Neuropeptide Release at Drosophila Neuromuscular Junction with a Genetically Engineered Neuropeptide Release Reporter.

Despite the important roles of neuropeptides in a variety of physiological processes, there still lacks a method to probe neuropeptide release events in vivo with satisfying temporal and spatial resolution. Neuropeptide Release Reporter (NPRR) was recently introduced as a novel genetically encoded indicator of neuropeptide release with a high temporal resolution and peptide specificity based on GCaMP molecule. Here we describe a method for using NPRR to image selective neuropeptide release at Drosophila neuromuscular junction in semi-dissected larvae. This method provides a quantitative analysis of activity-dependent neuropeptide release as real-time changes in fluorescence intensity of GCaMP reporter with sub-second temporal resolution and single bouton specificity.

CAPS2 Deficiency Impairs the Release of the Social Peptide Oxytocin, as Well as Oxytocin-Associated Social Behavior.

Ca2+-dependent activator protein for secretion 2 (CAPS2) regulates dense-core vesicle (DCV) exocytosis to facilitate peptidergic and catecholaminergic transmitter release. CAPS2 deficiency in mice has mild neuronal effects but markedly impairs social behavior. Rare de novo Caps2 alterations also occur in autism spectrum disorder, although whether CAPS2-mediated release influences social behavior remains unclear. Here, we demonstrate that CAPS2 is associated with DCV exocytosis-mediated release of the social interaction modulatory peptide oxytocin (OXT). CAPS2 is expressed in hypothalamic OXT neurons and localizes to OXT nerve projection and OXT release sites, such as the pituitary. Caps2 KO mice exhibited reduced plasma albeit increased hypothalamic and pituitary OXT levels, indicating insufficient release. OXT neuron-specific Caps2 conditional KO supported CAPS2 function in pituitary OXT release, also affording impaired social interaction and recognition behavior that could be ameliorated by exogenous OXT administered intranasally. Thus, CAPS2 appears critical for OXT release, thereby being associated with social behavior.SIGNIFICANCE STATEMENT The role of the neuropeptide oxytocin in enhancing social interaction and social bonding behavior has attracted considerable public and neuroscientific attention. A central issue in oxytocin biology concerns how oxytocin release is regulated. Our study provides an important insight into the understanding of oxytocin-dependent social behavior from the perspective of the CAPS2-regulated release mechanism.

Synaptophysin Regulates Fusion Pores and Exocytosis Mode in Chromaffin Cells.

Synaptophysin (syp) is a major integral membrane protein of secretory vesicles. Previous work has demonstrated functions for syp in synaptic vesicle cycling, endocytosis, and synaptic plasticity, but the role of syp in the process of membrane fusion during Ca2+-triggered exocytosis remains poorly understood. Furthermore, although syp resides on both large dense-core and small synaptic vesicles, its role in dense-core vesicle function has received less attention compared with synaptic vesicle function. To explore the role of syp in membrane fusion and dense-core vesicle function, we used amperometry to measure catecholamine release from single vesicles in male and female mouse chromaffin cells with altered levels of syp and the related tetraspanner protein synaptogyrin (syg). Knocking out syp slightly reduced the frequency of vesicle fusion events below wild-type (WT) levels, but knocking out both syp and syg reduced the frequency 2-fold. Knocking out both proteins stabilized initial fusion pores, promoted fusion pore closure (kiss-and-run), and reduced late-stage fusion pore expansion. Introduction of a syp construct lacking its C-terminal dynamin-binding domain in syp knock-outs (KOs) increased the duration and fraction of kiss-and-run events, increased total catecholamine release per event, and reduced late-stage fusion pore expansion. These results demonstrated that syp and syg regulate dense-core vesicle function at multiple stages to initiate fusion, control the choice of mode between full-fusion and kiss-and-run, and influence the dynamics of both initial and late-stage fusion pores. The transmembrane domain (TMD) influences small initial fusion pores, and the C-terminal domain influences large late-stage fusion pores, possibly through an interaction with dynamin.SIGNIFICANCE STATEMENT The secretory vesicle protein synaptophysin (syp) is known to function in synaptic vesicle cycling, but its roles in dense-core vesicle functions, and in controlling membrane fusion during Ca2+-triggered exocytosis remain unclear. The present study used amperometry recording of catecholamine release from endocrine cells to assess the impact of syp and related proteins on membrane fusion. A detailed analysis of amperometric spikes arising from the exocytosis of single vesicles showed that these proteins influence fusion pores at multiple stages and control the choice between kiss-and-run and full-fusion. Experiments with a syp construct lacking its C terminus indicated that the transmembrane domain (TMD) influences the initial fusion pore, while the C-terminal domain influences later stages after fusion pore expansion.

Release of insulin granules by simultaneous, high-speed correlative SICM-FCM.

Exocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label-free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM-FCM) to study the exocytosis of individual granules of insulin from the top, nonadherent, surface of pancreatic ß-cells. Using SICM-FCM, we were first to directly follow the topographical changes associated with physiologically induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal.

Measurements of Compensatory Endocytosis by Antibody Internalization and Quantification of Endocytic Vesicle Distribution in Adrenal Chromaffin Cells.

Plasma membrane proteins are amenable to endocytosis assays since they are easily labeled by reagents applied in the extracellular medium. This has been widely exploited to study constitutive endocytosis or ligand-induced receptor endocytosis. Compensatory endocytosis is the mechanism by which components of secretory vesicles are retrieved after vesicle fusion with the plasma membrane in response to cell stimulation and a rise in intracellular calcium. Luminal membrane proteins from secretory vesicles are therefore transiently exposed at the plasma membrane. Here, we described an antibody-based method to monitor compensatory endocytosis in chromaffin cells and present an image-based analysis to quantify endocytic vesicles distribution.

HPS1 Regulates the Maturation of Large Dense Core Vesicles and Lysozyme Secretion in Paneth Cells.

HPS1, a BLOC-3 subunit that acts as a guanine nucleotide exchange factor of Rab32/38, may play a role in the removal of VAMP7 during the maturation of large dense core vesicles of Paneth cells. Loss of HPS1 impairs lysozyme secretion and alters the composition of intestinal microbiota, which may explain the susceptibility of HPS-associated inflammatory bowel disease. Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, bleeding tendency, and other chronic organ lesions due to defects in tissue-specific lysosome-related organelles (LROs). For some HPS subtypes, such as HPS-1, it is common to have symptoms of HPS-associated inflammatory bowel disease (IBD). However, its underlying mechanism is largely unknown. HPS1 is a subunit of the BLOC-3 complex which functions in the biogenesis of LROs. Large dense core vesicles (LDCVs) in Paneth cells of the intestine are a type of LROs. We here first report the abnormal LDCV morphology (increased number and enlarged size) in HPS1-deficient pale ear (ep) mice. Similar to its role in melanosome maturation, HPS1 plays an important function in the removal of VAMP7 from LDCVs to promote the maturation of LDCVs. The immature LDCVs in ep mice are defective in regulated secretion of lysozyme, a key anti-microbial peptide in the intestine. We observed changes in the composition of intestinal microbiota in both HPS-1 patients and ep mice. These findings provide insights into the underlying mechanism of HPS-associated IBD development, which may be implicated in possible therapeutic intervention of this devastating condition.

Regulating quantal size of neurotransmitter release through a GPCR voltage sensor.

Current models emphasize that membrane voltage (Vm) depolarization-induced Ca2+ influx triggers the fusion of vesicles to the plasma membrane. In sympathetic adrenal chromaffin cells, activation of a variety of G protein coupled receptors (GPCRs) can inhibit quantal size (QS) through the direct interaction of G protein Gißγ subunits with exocytosis fusion proteins. Here we report that, independently from Ca2+, Vm (action potential) per se regulates the amount of catecholamine released from each vesicle, the QS. The Vm regulation of QS was through ATP-activated GPCR-P2Y12 receptors. D76 and D127 in P2Y12 were the voltage-sensing sites. Finally, we revealed the relevance of the Vm dependence of QS for tuning autoinhibition and target cell functions. Together, membrane voltage per se increases the quantal size of dense-core vesicle release of catecholamine via Vm → P2Y12(D76/D127) → Gißγ → QS → myocyte contractility, offering a universal Vm-GPCR signaling pathway for its functions in the nervous system and other systems containing GPCRs.

CAPS2 deficiency induces proopiomelanocortin accumulation in pituitary and affects food intake behavior in mice.

Proopiomelanocortin (POMC) is a neuropeptide precursor produced in the anterior and intermediate pituitary lobes, the hypothalamic arcuate nucleus (ARC), and solitary tract nucleus. Alpha-melanocyte-stimulating hormone (α-MSH) is a cell type specific POMC derivative that is essential for regulating feeding, and energy homeostasis. However, the molecular mechanism underlying POMC/α-MSH secretion remains unclear. Ca2+-dependent activator protein for secretion 2 (CAPS2) is a regulatory protein involved in the exocytosis of dense-core vesicles containing neuropeptides. We previously reported CAPS2 localization in the intermediate pituitary lobe and reduced body weights in Caps2-knockout (Caps2-KO) mice, compared to control mice. Here, we aimed to investigate CAPS2 expression in POMC-expressing neurons and the effects of CAPS2 deficiency on the secretion of POMC-related peptides and feeding behavior phenotype. CAPS2 was localized in the POMC-expressing neurons of the intermediate pituitary lobe, hypothalamic ARC, and the paraventricular nucleus, which is innervated by hypothalamic neurons. POMC protein levels in the intermediate pituitary lobe of Caps2-KO mice were significantly higher than that in the control mice, suggesting a possible accumulation of POMC-derived peptides in the intermediate pituitary lobe of Caps2-KO mice. Moreover, administration of low-dose melanotan-2, an α-MSH receptor (MC4R) agonist, decreased food intake per body weight in Caps2-KO mice; no such effect was observed in the wildtype mice. Collectively, these results suggest that CAPS2 is involved in regulating the secretion of POMC-derived peptides, including α-MSH, is partially associated with feeding, and affects energy metabolism.