Calcium-dependent Activator Protein For Secretion Research Articles

Calcium-dependent activator protein for secretion 2 is involved in dopamine release in mouse midbrain neurons.

The Ca2+-dependent activator protein for secretion (CAPS/CADPS) family protein facilitates catecholamine release through the dense-core vesicle exocytosis in model neuroendocrine cell lines. However, it remains unclear if it induces dopamine release in the central neurons. This study aimed to examine the expression and function of CADPS2, one of the two CADPS paralogs, in dopamine neurons of the mouse midbrain. This study shows that CADPS2 was expressed in tyrosine hydroxylase and the vesicular monoamine transporter 2 (VMAT2)-positive dopaminergic neurons of the midbrain samples and primary mesencephalic cell cultures. Subcellular fractions rich in dopamine were collected using immunoaffinity for CADPS2 from midbrain protein extracts. Cell imaging using fluorescent false neurotransmitter FFN511 as a substrate for VMAT2 showed decreased activity-dependent dopamine release in Cadps2-deficient cultures, compared to that in wild-type cultures. These results suggest that CADPS2 is involved in dopamine release from the central neurons, indicating its involvement in the central dopamine pathway.

Conditional Degradation of UNC-31/CAPS Enables Spatiotemporal Analysis of Neuropeptide Function.

Neuropeptide release from dense-core vesicles in Caenorhabditis elegans is promoted by UNC-31, ortholog of the calcium-dependent activator protein for secretion (CAPS). Loss of UNC-31 causes multiple phenotypes in C. elegans including reduced motility, retention of late-stage eggs, and reduction in evoked synaptic release. However, the ability to analyze UNC-31 function over discrete timescales and in specific neurons is lacking. Here, we generated and validated a tool to enable UNC-31 expression and spatiotemporal functional analysis. We show that endogenously tagged UNC-31 is expressed in major ganglia and nerve cords from late embryonic stages through to adult. Using the auxin-inducible degradation system, we depleted UNC-31 postembryonically from the hermaphrodite nervous system and revealed defects in egg laying, locomotion, and vesicle release that were comparable to those in unc-31 null mutant animals. In addition, we found that depleting UNC-31 specifically from the BAG sensory neurons causes increased intestinal fat storage, highlighting the spatial sensitivity of this system. Together, this protein degradation tool may be used to facilitate studies of neuropeptide function at precise cellular and temporal scales.SIGNIFICANCE STATEMENT Animal behavior and physiology is controlled by neuropeptides that are released from specific neuronal sources. The ability to dissect discrete neuropeptide functions requires precise manipulation of neuropeptide release. We have developed and validated a tool that enables precise spatiotemporal regulation of neuropeptide release that will enable researchers to examine neuropeptide function at exceptional resolution.

Curcumin-Attenuated TREM-1/DAP12/NLRP3/Caspase-1/IL1B, TLR4/NF-κB Pathways, and Tau Hyperphosphorylation Induced by 1,2-Diacetyl Benzene: An in Vitro and in Silico Study.

We aimed to evaluate the effects of 1,2-diacetylbenzene (DAB) and curcumin on neuroinflammation induced by DAB via triggering receptor expressed on myeloid cells 1 (TREM-1), Toll-like receptor 4 (TLR4), and NLR family pyrin domain containing 3 (NLP3)/calcium-dependent activator protein for secretion 1 (CAPS1)/interleukin 1 beta (IL1B) pathways; tau hyperphosphorylation; reactive oxygen species (ROS); and advanced glycation end-product (AGE) in microglia cells; and explore the molecular mechanisms involved in the key genes induced by DAB and targeted by curcumin in silico analysis. In this study, Western blot, quantitative polymerase chain reaction, and immunocytochemistry were used as the key methods in vitro. In silico analysis, GeneMANIA, ToppFun feature, Metascape, CHEA3, Cytoscape, Autodock, and MIENTURNET were the core approaches used. Curcumin inhibited both the DAB-induced TREM-1/DAP12/NLRP3/caspase-1/IL1B pathway and the TLR4/NF-κB pathway. In BV2 cells, curcumin inhibited ROS, AGE, hyperphosphorylation, glycogen synthase kinase-3β (GSK-3β), and β-amyloid while activating nuclear factor erythroid 2-related factor 2 (Nrf2) expression. In silico studies showed that tumor necrosis factor (TNF), IL6, NFKB1, IL10, and IL1B, as well as MTF1 and ZNF267, were shown to be important genes and transcription factors in the pathogenesis of cognitive impairment produced by DAB and curcumin. Three significant miRNAs (hsa-miR-26a-5p, hsa-miR-203a-3p, and hsa-miR-155-5p) implicated in the etiology of DAB-induced cognitive impairment and targeted by curcumin were also identified. Inflammation and cytokine-associated pathways, Alzheimer's disease, and cognitive impairment were characterized as the most significant biological processes implicated in genes, miRNAs, and transcription factors induced by DAB and targeted by curcumin. Our findings provide new insight into fundamental molecular mechanisms implicated in the pathogenesis of cognitive impairment caused by DAB, particularly the effects of neuroinflammation. Furthermore, this study suggests that curcumin might be a promising therapeutic molecule for cognitive impairment treatment through modulating neuroinflammatory responses.

Localization of the Priming Factors CAPS1 and CAPS2 in Mouse Sensory Neurons Is Determined by Their N-Termini.

Both paralogs of the calcium-dependent activator protein for secretion (CAPS) are required for exocytosis of synaptic vesicles (SVs) and large dense core vesicles (LDCVs). Despite approximately 80% sequence identity, CAPS1 and CAPS2 have distinct functions in promoting exocytosis of SVs and LDCVs in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms underlying these differences remain enigmatic. In this study, we applied high- and super-resolution imaging techniques to systematically assess the subcellular localization of CAPS paralogs in DRG neurons deficient in both CAPS1 and CAPS2. CAPS1 was found to be more enriched at the synapses. Using – in-depth sequence analysis, we identified a unique CAPS1 N-terminal sequence, which we introduced into CAPS2. This CAPS1/2 chimera reproduced the pre-synaptic localization of CAPS1 and partially rescued synaptic transmission in neurons devoid of CAPS1 and CAPS2. Using immunoprecipitation combined with mass spectrometry, we identified CAPS1-specific interaction partners that could be responsible for its pre-synaptic enrichment. Taken together, these data suggest an important role of the CAPS1-N terminus in the localization of the protein at pre-synapses.

CADPS functional mutations in patients with bipolar disorder increase the sensitivity to stress.

Bipolar disorder is a severe and chronic psychiatric disease resulting from a combination of genetic and environmental risk factors. Here, we identified a significant higher mutation rate in a gene encoding the calcium-dependent activator protein for secretion (CADPS) in 132 individuals with bipolar disorder, when compared to 184 unaffected controls or to 21,070 non-psychiatric and non-Finnish European subjects from the Exome Aggregation Consortium. We found that most of these variants resulted either in a lower abundance or a partial impairment in one of the basic functions of CADPS in regulating neuronal exocytosis, synaptic plasticity and vesicular transporter-dependent uptake of catecholamines. Heterozygous mutant mice for Cadps+/- revealed that a decreased level of CADPS leads to manic-like behaviours, changes in BDNF level and a hypersensitivity to stress. This was consistent with more childhood trauma reported in families with mutation in CADPS, and more specifically in mutated individuals. Furthermore, hyperactivity observed in mutant animals was rescued by the mood-stabilizing drug lithium. Overall, our results suggest that dysfunction in calcium-dependent vesicular exocytosis may increase the sensitivity to environmental stressors enhancing the risk of developing bipolar disorder.

RNA editing-mediated regulation of calcium-dependent activator protein for secretion (CAPS1) localization and its impact on synaptic transmission.

Calcium-dependent activator protein for secretion 1 (CAPS1) is a SNARE accessory protein that facilitates formation of the SNARE complex to enable neurotransmitter release. Messenger RNAs encoding CAPS1 are subject to a site-specific adenosine-to-inosine (A-to-I) editing event resulting in a glutamate-to-glycine (E-to-G) substitution in the C-terminal domain of the encoded protein product. The C-terminal domain of CAPS1 is necessary for its synaptic enrichment and Cadps RNA editing has been shown previously to enhance the release of neuromodulatory transmitters. Using mutant mouse lines engineered to solely express CAPS1 protein isoforms encoded by either the non-edited or edited Cadps transcript, primary neuronal cultures from mouse hippocampus were used to explore the effect of Cadps editing on neurotransmission and CAPS1 synaptic localization at both glutamatergic and GABAergic synapses. While the editing of Cadps does not alter baseline evoked neurotransmission, it enhances short-term synaptic plasticity, specifically short-term depression, at inhibitory synapses. Cadps editing also alters spontaneous inhibitory neurotransmission. Neurons that solely express edited Cadps have a greater proportion of synapses that contain CAPS1 than neurons that solely express non-edited Cadps for both glutamatergic and GABAergic synapses. Editing of Cadps transcripts is regulated by neuronal activity, as global network stimulation increases the extent of transcripts edited in wild-type hippocampal neurons, whereas chronic network silencing decreases the level of Cadps editing. Taken together, these results provide key insights into the importance of Cadps editing in modulating its own synaptic localization, as well as the modulation of neurotransmission at inhibitory synapses in hippocampal neurons.

CAPS1 is involved in hippocampal synaptic plasticity and hippocampus-associated learning

Calcium-dependent activator protein for secretion 1 (CAPS1) is a key molecule in vesicular exocytosis, probably in the priming step. However, CAPS1’s role in synaptic plasticity and brain function is elusive. Herein, we showed that synaptic plasticity and learning behavior were impaired in forebrain and/or hippocampus-specific Caps1 conditional knockout (cKO) mice by means of molecular, physiological, and behavioral analyses. Neonatal Caps1 cKO mice showed a decrease in the number of docked vesicles in the hippocampal CA3 region, with no detectable changes in the distribution of other major exocytosis-related molecules. Additionally, long-term potentiation (LTP) was partially and severely impaired in the CA1 and CA3 regions, respectively. CA1 LTP was reinforced by repeated high-frequency stimuli, whereas CA3 LTP was completely abolished. Accordingly, hippocampus-associated learning was severely impaired in adeno-associated virus (AAV) infection-mediated postnatal Caps1 cKO mice. Collectively, our findings suggest that CAPS1 is a key protein involved in the cellular mechanisms underlying hippocampal synaptic release and plasticity, which is crucial for hippocampus-associated learning.

CAPS1 Suppresses Tumorigenesis in Cholangiocarcinoma.

CAPS1 (calcium-dependent activator protein for secretion) is a multi-domain protein involved in regulating exocytosis of synaptic vesicles and dense-core vesicles. However, the expression and function of CAPS1 in cholangiocarcinoma (CCA) remains unclear. In the present study, we explored the role of CAPS1 in CCA carcinogenesis. CAPS1 expression was explored using western blotting and immunohistochemistry in four CCA cell lines and clinical samples from 90 cases of CCA. The clinical significance of CAPS1 was analyzed. The biological function of CAPS1 in CCA cells was detected in vitro and in vivo. The underlying mechanism of CAPS1 function was explored by detecting the expression of critical molecules in its associated signaling pathways. The mechanism of CAPS1 downregulation in tumor tissues was explored using in silico prediction and luciferase reporter assays. CAPS1 expression was reduced in CCA cell lines and human tumor tissues. Loss of CAPS1 in tumor tissues was closely associated with poor prognosis of patients with CCA. Moreover, CAPS1 expression correlated significantly with tumor-node-metastasis stage, lymph node metastasis, and vascular invasion. Lentivirus-mediated CAPS1 overexpression substantially prevented clone formation, cell proliferation, and cell cycle progression. CAPS1 overexpression also suppressed carcinogenesis in nude mice. Mechanistically, CAPS1 overexpression greatly accelerated the ERK and p38 MAPK signal pathways. In addition, microRNA miR-30e-5p negatively regulated CAPS1 expression. These data showed that CAPS1 functions as a tumor suppressor in CCA. Reduced CAPS1 expression could indicate poor prognosis of patients with CCA.

The priming factor CAPS1 regulates dense-core vesicle acidification by interacting with rabconnectin3β/WDR7 in neuroendocrine cells

Vacuolar-type H+-ATPases (V-ATPases) contribute to pH regulation and play key roles in secretory and endocytic pathways. Dense-core vesicles (DCVs) in neuroendocrine cells are maintained at an acidic pH, which is part of the electrochemical driving force for neurotransmitter loading and is required for hormonal propeptide processing. Genetic loss of CAPS1 (aka calcium-dependent activator protein for secretion, CADPS), a vesicle-bound priming factor required for DCV exocytosis, dissipates the pH gradient across DCV membranes and reduces neurotransmitter loading. However, the basis for CAPS1 binding to DCVs and for its regulation of vesicle pH has not been determined. Here, MS analysis of CAPS1 immunoprecipitates from brain membrane fractions revealed that CAPS1 associates with a rabconnectin3 (Rbcn3) complex comprising Dmx-like 2 (DMXL2) and WD repeat domain 7 (WDR7) proteins. Using immunofluorescence microscopy, we found that Rbcn3α/DMXL2 and Rbcn3β/WDR7 colocalize with CAPS1 on DCVs in human neuroendocrine (BON) cells. The shRNA-mediated knockdown of Rbcn3β/WDR7 redistributed CAPS1 from DCVs to the cytosol, indicating that Rbcn3β/WDR7 is essential for optimal DCV localization of CAPS1. Moreover, cell-free experiments revealed direct binding of CAPS1 to Rbcn3β/WDR7, and cell assays indicated that Rbcn3β/WDR7 recruits soluble CAPS1 to membranes. As anticipated by the reported association of Rbcn3 with V-ATPase, we found that knocking down CAPS1, Rbcn3α, or Rbcn3β in neuroendocrine cells impaired rates of DCV reacidification. These findings reveal a basis for CAPS1 binding to DCVs and for CAPS1 regulation of V-ATPase activity via Rbcn3β/WDR7 interactions.

Aspects of excitatory/inhibitory synapses in multiple brain regions are correlated with levels of brain-derived neurotrophic factor/neurotrophin-3

Appropriate synapse formation during development is necessary for normal brain function, and synapse impairment is often associated with brain dysfunction. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are key factors in regulating synaptic development. We previously reported that BDNF/NT-3 secretion was enhanced by calcium-dependent activator protein for secretion 2 (CADPS2). Although BDNF/NT-3 and CADPS2 are co-expressed in various brain regions, the effect of Cadps2-deficiency on brain region-specific BDNF/NT-3 levels and synaptic development remains elusive. Here, we show developmental changes of BDNF/NT-3 levels and we assess disruption of excitatory/inhibitory synapses in multiple brain regions (cerebellum, hypothalamus, striatum, hippocampus, parietal cortex and prefrontal cortex) of Cadps2 knockout (KO) mice compared with wild-type (WT) mice. Compared with WT, BDNF levels in KO mice were reduced in young/adult hippocampus, but increased in young hypothalamus, while NT-3 levels were reduced in adult cerebellum and young hippocampus, but increased in adult parietal cortex. Immunofluorescence of vGluT1, an excitatory synapse marker, and vGAT, an inhibitory synapse marker, in adult KO showed that vGluT1 was higher in the cerebellum and parietal cortex but lower in the hippocampus, whereas vGAT was lower in the hippocampus and parietal cortex compared with WT. Immunolabeling for both vGluT1 and vGAT was increased in the parietal cortex but vGAT was decreased in the cerebellum in adult KO compared with WT. These data suggest that CADPS2-mediated secretion of BDNF/NT-3 may be involved in development and maturation of synapses and in the balance between inhibitory and excitatory synapses.

Paralogs of the Calcium-Dependent Activator Protein for Secretion Differentially Regulate Synaptic Transmission and Peptide Secretion in Sensory Neurons.

The two paralogs of the calcium-dependent activator protein for secretion (CAPS) are priming factors for synaptic vesicles (SVs) and neuropeptide containing large dense-core vesicles (LDCVs). Yet, it is unclear whether CAPS1 and CAPS2 regulate exocytosis of these two vesicle types differentially in dorsal root ganglion (DRG) neurons, wherein synaptic transmission and neuropeptide release are of equal importance. These sensory neurons transfer information from the periphery to the spinal cord (SC), releasing glutamate as the primary neurotransmitter, with co-transmission via neuropeptides in a subset of so called peptidergic neurons. Neuropeptides are key components of the information-processing machinery of pain perception and neuropathic pain generation. Here, we compared the ability of CAPS1 and CAPS2 to support priming of both vesicle types in single and double knock-out mouse (DRG) neurons using a variety of high-resolution live cell imaging methods. While CAPS1 was localized to synapses of all DRG neurons and promoted synaptic transmission, CAPS2 was found exclusively in peptidergic neurons and mediated LDCV exocytosis. Intriguingly, ectopic expression of CAPS2 empowered non-peptidergic neurons to drive LDCV fusion, thereby identifying CAPS2 as an essential molecular determinant for peptidergic signaling. Our results reveal that these distinct functions of both CAPS paralogs are based on their differential subcellular localization in DRG neurons. Our data suggest a major role for CAPS2 in neuropathic pain via control of neuropeptide release.

Calcium-dependent activator protein for secretion 2 (CADPS2) deficiency causes abnormal synapse development in hippocampal mossy fiber terminals

Hippocampal mossy fibers (MFs) project from dentate gyrus granule cells onto the CA2–CA3 region. MF-mediated synaptic transmission plays an important role in hippocampal learning and memory. However, the molecular mechanisms underlying MF synaptic development and subsequent functional organization are not fully understood. We previously reported that calcium-dependent activator protein for secretion 2 (CADPS2, also known as CAPS2) regulates the secretion of dense-core vesicles (DCVs). Because CADPS2 is strongly expressed in MF terminals, we hypothesized that CADPS2 regulates the development and functional organization of MF synapses by controlling the secretion of DCVs and their contents. To test this, we compared the synaptic microstructures of hippocampal MF terminals in Cadps2 knockout (KO) mice and wild-type (WT) mice by electron microscopy (EM). On postnatal day 15 (P15), KO mice exhibited morphological abnormalities in MF boutons, including smaller bouton size, a larger number of DCVs and a smaller number of post-synaptic densities (PSDs), compared with WT mice. In adults (P56), MF boutons were larger in KO mice. Synaptic vesicles (SVs) were increased but with a lower density compared with the WT. Furthermore, the number of SVs was decreased near the active zone. Moreover, MF-innervated CA3 postsynapses in KO mice displayed aberrant structures at the postsynaptic density (PSD), with an increased number of PSDs (likely because of a larger number of perforated PSDs), compared with WT mice. Taken together, our findings suggest that CADPS2 plays a critical role in MF synaptic development and functional organization.

CAPS2 deficiency affects environmental enrichment-induced adult neurogenesis and differentiation/survival of newborn neurons in the hippocampal dentate gyrus.

Hippocampal adult neurogenesis is observed in the subgranular zone of the dentate gyrus (DG), and is associated with hippocampal memory formation and several psychiatric disorders including autism spectrum disorder (ASD). Calcium-dependent activator protein for secretion 2 (CAPS2) is a candidate gene related to ASD, and is highly expressed in the hippocampal DG region, with Caps2 knockout (KO) mice exhibiting ASD-like behavior. Accordingly, CAPS2 is potentially associated with hippocampal adult neurogenesis, the relationship between CAPS2 and adult neurogenesis has not yet been investigated. Here, we determined whether deficit of the Caps2 gene affects hippocampal adult neurogenesis and maturation of newborn neurons. To induce adult neurogenesis, we used the environmental enrichment (EE) condition. Both wild-type (WT) and Caps2 KO mice were housed in control or EE conditions for 3 or 14days. Hippocampal levels of brain-derived neurotrophic factor (BDNF) can be used as a physiological EE conditioned marker, and were increased at 14days in the EE condition in both WT and KO mice. Newborn cells during control and EE conditions were labeled by BrdU, and the labeled cells co-immunostained with the immature and mature neuron markers, calretinin (CR) and NeuN. The ratio of CR/BrdU and NeuN/BrdU double positive cells to all of BrdU positive cells were significantly increased in WT mice housed in the EE condition for 14days compared with the control condition. Whereas KO mice in the EE condition showed no significant increase of newborn neurons. These findings suggest that CAPS2 deficiency strongly impairs hippocampal adult neurogenesis and maturation of newborn neurons.

CAPS-1 requires its C2, PH, MHD1 and DCV domains for dense core vesicle exocytosis in mammalian CNS neurons

CAPS (calcium-dependent activator protein for secretion) are multi-domain proteins involved in regulated exocytosis of synaptic vesicles (SVs) and dense core vesicles (DCVs). Here, we assessed the contribution of different CAPS-1 domains to its subcellular localization and DCV exocytosis by expressing CAPS-1 mutations in four functional domains in CAPS-1/-2 null mutant (CAPS DKO) mouse hippocampal neurons, which are severely impaired in DCV exocytosis. CAPS DKO neurons showed normal development and no defects in DCV biogenesis and their subcellular distribution. Truncation of the CAPS-1 C-terminus (CAPS Δ654-1355) impaired CAPS-1 synaptic enrichment. Mutations in the C2 (K428E or G476E) or pleckstrin homology (PH; R558D/K560E/K561E) domain did not. However, all mutants rescued DCV exocytosis in CAPS DKO neurons to only 20% of wild type CAPS-1 exocytosis capacity. To assess the relative importance of CAPS for both secretory pathways, we compared effect sizes of CAPS-1/-2 deficiency on SV and DCV exocytosis. Using the same (intense) stimulation, DCV exocytosis was impaired relatively strong (96% inhibition) compared to SV exocytosis (39%). Together, these data show that the CAPS-1 C-terminus regulates synaptic enrichment of CAPS-1. All CAPS-1 functional domains are required, and the C2 and PH domain together are not sufficient, for DCV exocytosis in mammalian CNS neurons.

BAIAP3, a C2 domain-containing Munc13 protein, controls the fate of dense-core vesicles in neuroendocrine cells.

Dense-core vesicle (DCV) exocytosis is a SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptor)-dependent anterograde trafficking pathway that requires multiple proteins for regulation. Several C2 domain-containing proteins are known to regulate Ca2+-dependent DCV exocytosis in neuroendocrine cells. In this study, we identified others by screening all (∼139) human C2 domain-containing proteins by RNA interference in neuroendocrine cells. 40 genes were identified, including several encoding proteins with known roles (CAPS [calcium-dependent activator protein for secretion 1], Munc13-2, RIM1, and SYT10) and many with unknown roles. One of the latter, BAIAP3, is a secretory cell-specific Munc13-4 paralog of unknown function. BAIAP3 knockdown caused accumulation of fusion-incompetent DCVs in BON neuroendocrine cells and lysosomal degradation (crinophagy) of insulin-containing DCVs in INS-1 β cells. BAIAP3 localized to endosomes was required for Golgi trans-Golgi network 46 (TGN46) recycling, exhibited Ca2+-stimulated interactions with TGN SNAREs, and underwent Ca2+-stimulated TGN recruitment. Thus, unlike other Munc13 proteins, BAIAP3 functions indirectly in DCV exocytosis by affecting DCV maturation through its role in DCV protein recycling. Ca2+ rises that stimulate DCV exocytosis may stimulate BAIAP3-dependent retrograde trafficking to maintain DCV protein homeostasis and DCV function.

Caenorhabditis elegansMale Copulation Circuitry Incorporates Sex-Shared Defecation Components To Promote Intromission and Sperm Transfer

Sexual dimorphism can be achieved using a variety of mechanisms, including sex-specific circuits and sex-specific function of shared circuits, though how these work together to produce sexually dimorphic behaviors requires further investigation. Here, we explore how components of the sex-shared defecation circuitry are incorporated into the sex-specific male mating circuitry in Caenorhabditis elegans to produce successful copulation. Using behavioral studies, calcium imaging, and genetic manipulation, we show that aspects of the defecation system are coopted by the male copulatory circuitry to facilitate intromission and ejaculation. Similar to hermaphrodites, male defecation is initiated by an intestinal calcium wave, but circuit activity is coordinated differently during mating. In hermaphrodites, the tail neuron DVB promotes expulsion of gut contents through the release of the neurotransmitter GABA onto the anal depressor muscle. However, in the male, both neuron and muscle take on modified functions to promote successful copulation. Males require calcium-dependent activator protein for secretion (CAPS)/unc-31, a dense core vesicle exocytosis activator protein, in the DVB to regulate copulatory spicule insertion, while the anal depressor is remodeled to promote release of sperm into the hermaphrodite. This work shows how sex-shared circuitry is modified in multiple ways to contribute to sex-specific mating.

CAPS1 RNA Editing Promotes Dense Core Vesicle Exocytosis

Calcium-dependent activator protein for secretion 1 (CAPS1) plays a distinct role in the priming step of dense core vesicle (DCV) exocytosis. CAPS1 pre-mRNA is known to undergo adenosine-to-inosine RNA editing in its coding region, which results in a glutamate-to-glycine conversion at a site in its C-terminal region. However, the physiological significance of CAPS1 RNA editing remains elusive. Here, we created mutant mice in which edited CAPS1 was solely expressed. These mice were lean due to increased energy expenditure caused by physical hyperactivity. Electrophysiological and biochemical analyses demonstrated that the exocytosis of DCVs was upregulated in the chromaffin cells and neurons of these mice. Furthermore, we showed that edited CAPS1 bound preferentially to the activated form of syntaxin-1A, a component of the exocytotic fusion complex. These findings suggest that RNA editing regulates DCV exocytosis invivo, affecting physical activity.

CAPS1 Negatively Regulates Hepatocellular Carcinoma Development through Alteration of Exocytosis-Associated Tumor Microenvironment.

The calcium-dependent activator protein for secretion 1 (CAPS1) regulates exocytosis of dense-core vesicles (DCVs) in neurons and neuroendocrine cells. The role of CAPS1 in cancer biology remains unknown. The purpose of this study was to investigate the role of CAPS1 in hepatocellular carcinoma (HCC). We determined the levels of CAPS1 in eight hepatoma cell lines and 141 HCC specimens. We evaluated the prognostic value of CAPS1 expression and its association with clinical parameters. We investigated the biological consequences of CAPS1 overexpression in two hepatoma cell lines in vitro and in vivo. The results showed that loss of CAPS1 expression in HCC tissues was markedly correlated with aggressive tumor phenotypes, such as high-grade tumor node metastasis (TNM) stage (p = 0.003) and absence of tumor encapsulation (p = 0.016), and was associated with poor overall survival (p = 0.008) and high recurrence (p = 0.015). CAPS1 overexpression inhibited cell proliferation and migration by changing the exocytosis-associated tumor microenvironment in hepatoma cells in vitro. The in vivo study showed that CAPS1 overexpression inhibited xenograft tumor growth. Together, these results identified a previously unrecognized tumor suppressor role for CAPS1 in HCC development.

Molecular Network of Tinnitus Focusing on BDNF Signal and Autophagy in Auditory Cells

Objectives: Recent studies show that the expression of brain-derived neurotrophic factor (BDNF) is decreasing in patients with depression and severe tinnitus. However, the relationship between the incidence of tinnitus and the role of BDNF remains unclear in auditory cells. The aim of this study is to consider the molecular network of tinnitus focusing on BDNF signal and autophagy in auditory cells. Methods: We used auditory cell line (HEI-OC1) in this study. Tunicamycin was used as endoplasmic reticulum (ER) stress. The viability of HEI-OC1 was determined by cell viability assays. Morphological observation was performed under transmission electron microscope (TEM). Western blot analysis was performed. Results: The cell viability after treatment of tunicamycin was decreased in dose- and time-dependent matter. Autophagosomes and autolysososmes were confirmed into the cytoplasm of HEI-OC1 under TEM. The expression of LC3-II was increased in HEI-OC1 after treatment of tunicamycin. These results mean that autophagy was consistently induced in tunicamycin-treated cells. The expression of CHOP was decreased after peaking at 12 hours after the treatment of tunicamycin. The expression of BDNF, which has the potential to become a biomarker of tinnitus, and calcium-dependent activator protein for secretion 2 (CAPS2) that promotes BDNF section, were decreased in tunicamycin-treated cells. The expression of Bcl-2 that control apoptosis and Beclin1 that regulate autophagy were decreased in tunicamycin-treated cells. Conclusions: Our results lead to the suggestion that the autophagy-mediated regulation of cell death and molecular network focusing on BDNF signal play a major part of the incidence of tinnitus.

Molecular Mechanism of Tinnitus from the Standpoint of Autophagy

Objectives: Recent studies described that the expression of BDNF was decreasing in patients with depression and severe tinnitus. However, the relationship between the incidence of tinnitus and the role of BDNF still remain unclear in auditory cells. The aim of this study is to consider the molecular mechanism of tinnitus from the standpoint of the autophagy-based regulation of cell death and BDNF signaling pathway in auditory cells. Methods: We used auditory cell line HEI-OC1 in this study. Tunicamycin was used as endoplasmic reticulum (ER) stress. The viability of HEI-OC1 was determined by cell viability assays. Immunofluorescent confocal laser microscopy was used. Western blot was performed. Results: It was confirmed that autophagy was consistently induced in HEI-OC1 after treatment of tunicamycin. The cell viability after treatment of tunicamycin was decreased in dose- and time-dependent matter. The expression of BDNF which has the potential to become a biomarker of tinnitus and calcium-dependent activator protein for secretion 2 (CAPS2), which promotes BDNF section, were decreased in tunicamycin treated HEI-OC1 cells. The expression of Bcl-2 which controls apoptosis and Beclin1 that regulates autophagy were decreased in tunicamycin treated HEI-OC1 cells. Conclusions: We consider that Bcl-2 and Beclin 1 serve as adapter proteins between BDNF signaling pathway and autophagy in auditory cells under ER stress. Our results led to the suggestion that the autophagy-mediated regulation of cell death and BDNF signaling pathway play a major part of the incidence of tinnitus.