Dependent Activator Protein Research Articles

GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

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.

Ca2+ -dependent activator protein for secretion 1 promotes spontaneous recovery in ischemic stroke by regulating BDNF secretion.

Ischemic stroke triggers a cascade of events that facilitates neural protection and spontaneous recovery, which accounts for a major part of functional recovery. Despite the cellular and molecular facilitations on neural protection, the molecular mechanisms of spontaneous recovery have not been fully understood. Ca2+ -dependent activator protein for secretion 1 (CAPS1), a member of CAPS family, plays a major role in synaptic transmission and synaptic effectiveness by regulating vesicle exocytosis. Here, the molecular mechanism of CAPS1 in spontaneous recovery after ischemic stroke was studied. In this study, transient left middle cerebral artery occlusion (MCAO) was used as the ischemic stroke model. The whole brain magnetic resonance imaging (MRI) and neurological score analysis showed decreased infarct volume and neurological scores at 7 days as compared with 1day after MCAO, suggesting the spontaneous recovery. Elisa and Western blot analysis showed elevated BDNF and CAPS1 expression levels in bilateral hippocampus at both 1day and 3 days after MCAO. Then, inhibition of CAPS1 by adeno-associated virus (AAV) microinjection in the hippocampus attenuated the spontaneous recovery of both motor and memory impairment induced by MCAO. In addition, elevated p-TrkB levels were detected after MCAO, which were reduced by CAPS1-AAV microinjection, indicating that CAPS1 could induce BDNF secretion after ischemic stroke. Moreover, we found elevated combination of CAPS1 with dense core vesicles (DCV) in the hippocampus at both 1day and 3 days after MCAO, which could also be inhibited by CAPS1-AAV microinjection, indicating the potential mechanism of CAPS1 in regulating BDNF release after MCAO. Finally, we found that CAPS1/BDNF signaling could influence the neurogenesis in the hippocampus after MCAO. In conclusion, CAPS1 regulates neurogenesis by up-regulating BDNF release in the hippocampus, which finally facilitate spontaneous recovery after ischemic stroke.

Loss of CAPS2/Cadps2 leads to exocrine pancreatic cell injury and intracellular accumulation of secretory granules in mice.

The type 2 Ca2+-dependent activator protein for secretion (CAPS2/CADPS2) regulates dense-core vesicle trafficking and exocytosis and is involved in the regulated release of catecholamines, peptidergic hormones, and neuromodulators. CAPS2 is expressed in the pancreatic exocrine acinar cells that produce and secrete digestive enzymes. However, the functional role of CAPS2 in vesicular trafficking and/or exocytosis of non-regulatory proteins in the exocrine pancreas remains to be determined. Here, we analyzed the morpho-pathological indicators of the pancreatic exocrine pathway in Cadps2-deficient mouse models using histochemistry, biochemistry, and electron microscopy. We used whole exosome sequencing to identify CADPS2 variants in patients with chronic pancreatitis (CP). Caps2/Cadps2-knockout (KO) mice exhibited morphophysiological abnormalities in the exocrine pancreas, including excessive accumulation of secretory granules (zymogen granules) and their amylase content in the cytoplasm, deterioration of the fine intracellular membrane structures (disorganized rough endoplasmic reticulum, dilated Golgi cisternae, and the appearance of empty vesicles and autophagic-like vacuoles), as well as exocrine pancreatic cell injury, including acinar cell atrophy, increased fibrosis, and inflammatory cell infiltration. Pancreas-specific Cadps2 conditional KO mice exhibited pathological abnormalities in the exocrine pancreas similar to the global Cadps2 KO mice, indicating that these phenotypes were caused either directly or indirectly by CAPS2 deficiency in the pancreas. Furthermore, we identified a rare variant in the exon3 coding region of CADPS2 in a non-alcoholic patient with CP and showed that Cadps2-dex3 mice lacking CAPS2 exon3 exhibited symptoms similar to those exhibited by the Cadps2 KO and cKO mice. These results suggest that CAPS2 is critical for the proper functioning of the pancreatic exocrine pathway, and its deficiency is associated with a risk of pancreatic acinar cell pathology.

Task-Related c-Fos Expression in the Posterior Parietal Cortex During the "Rubber Tail Task" Is Diminished in Ca2+-Dependent Activator Protein for Secretion 2 (Caps2)-Knockout Mice.

Rubber hand illusion (RHI), a kind of body ownership illusion, is sometimes atypical in individuals with autism spectrum disorder; however, the brain regions associated with the illusion are still unclear. We previously reported that mice responded as if their own tails were being touched when rubber tails were grasped following synchronous stroking to rubber tails and their tails (a “rubber tail illusion”, RTI), which is a task based on the human RHI; furthermore, we reported that the RTI response was diminished in Ca2+-dependent activator protein for secretion 2-knockout (Caps2-KO) mice that exhibit autistic-like phenotypes. Importance of the posterior parietal cortex in the formation of illusory perception has previously been reported in human imaging studies. However, the local neural circuits and cell properties associated with this process are not clear. Therefore, we aimed to elucidate the neural basis of the RTI response and its impairment by investigating the c-Fos expression in both wild-type (WT) and Caps2-KO mice during the task since the c-Fos expression occurred soon after the neural activation. Immediately following the delivery of the synchronous stroking to both rubber tails and actual tails, the mice were perfused. Subsequently, whole brains were cryo-sectioned, and each section was immunostained with anti-c-Fos antibody; finally, c-Fos positive cell densities among the groups were compared. The c-Fos expression in the posterior parietal cortex was significantly lower in the Caps2-KO mice than in the WT mice. Additionally, we compared the c-Fos expression in the WT mice between synchronous and asynchronous conditions and found that the c-Fos-positive cell densities were significantly higher in the claustrum and primary somatosensory cortex of the WT mice exposed to the synchronous condition than those exposed to the asynchronous condition. Hence, the results suggest that decreased c-Fos expression in the posterior parietal cortex may be related to impaired multisensory integrations in Caps2-KO mice.

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.

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.

Neuronal Ca2+ -dependent activator protein 1 (NCDAP1) induces neuronal cell death by activating p53 pathway following traumatic brain injury.

Overactivation of N-methyl-d-aspartate glutamate receptors (NMDARs) after traumatic brain injury (TBI) contributes to excitotoxic cell death. The hyperactivation of NMDARs results in toxic levels of intracellular Ca2+ and in the activation of p53-mediated apoptosis pathway. Neuronal Ca2+ -dependent activator protein 1 (NCDAP1) was identified as an epileptogenic gene of unknown function in our laboratory. In this study, we investigated the expression and cellular localization of NCDAP1 in rat models of fluid percussion-induced TBI. NCDAP1 expression increased in the ipsilateral cortex and hippocampus adjacent to the lesion of the TBI rats compared with that in the sham-operated controls. In addition, NCDAP1 was co-expressed with neuronal marker (NeuN), and the results of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining suggest that NCDAP1 is involved in neuronal apoptosis that occurs after brain injury. In addition, the expression levels of p53, Bax, and active caspase-3 correlated with those of NCDAP1. To further investigate the function of NCDAP1, primary cultured neurons were employed to establish an apoptosis model. The expression of NCDAP1 was induced by NMDA-induced Ca2+ influx, and the knockdown of NCDAP1 by siRNA decreased apoptosis caused by treatment with NMDA. Silencing of NCDAP1 also reduced p53 expression, whereas the over-expression of NCDAP1 induced cell death and up-regulated the expression of p53. The inhibition of p53 with pifithrin alpha or siRNA counteracted the effects of NCDAP1. Based on our data, we suggest that NCDAP1 plays an important role in p53-mediated neuronal apoptosis following TBI.

Rubber tail illusion is weakened in Ca2+-dependent activator protein for secretion 2 (Caps2)-knockout mice

Body ownership is a fundamental aspect of self-consciousness. Illusion of body ownership is caused by updating body representation through multisensory integration. Synchronous visuotactile stimulation of a hand and rubber hand leads to illusory changes in body ownership in humans, but this is impaired in individuals with autism spectrum disorder (ASD). We previously reported that mice also exhibit body ownership illusion. With synchronous stroking of a tail and rubber tail, mice responded as if their own tails were being touched when the rubber tails were grasped (‘rubber tail illusion’). However, it remains unknown whether deficits in illusion of body ownership occur in mouse models of autism. Here, we examined whether the ‘rubber tail illusion’ occurred in Ca2+-dependent activator protein for secretion 2-knockout (Caps2-KO) mice, which exhibit autistic-like phenotypes. During the synchronous stroking, response rates were significantly lower in Caps2-KO mice than in wild-type mice. There were no significant differences between the response rates of wild-type and Caps2-KO mice during the asynchronous stroking. The ‘rubber tail illusion’ was weak in Caps2-KO mice, suggesting that Caps2-KO mice experienced weaker visuotactile integration during the task. The rubber tail task will be a useful tool in mouse models of autism to evaluate atypical sensory processing.

CAPS1 promotes colorectal cancer metastasis via Snail mediated epithelial mesenchymal transformation.

Colorectal cancer (CRC) is a common gastrointestinal cancer with high mortality rate mostly due to metastasis. Ca2+-dependent activator protein for secretion 1 (CAPS1) was originally identified as a soluble factor that reconstitutes Ca2+-dependent secretion. In this study, we discovered a novel role of CAPS1 in CRC metastasis. CAPS1 is frequently up-regulated in CRC tissues. Increased CAPS1 expression is associated with frequent metastasis and poor prognosis of CRC patients. Overexpression of CAPS1 promotes CRC cell migration and invasion in vitro, as well as liver metastasis in vivo, without affecting cell proliferation. CAPS1 induces epithelial-mesenchymal transition (EMT), including decreased E-cadherin and ZO-1, epithelial marker expression, and increased N-cadherin and Snail, mesenchymal marker expression. Snail knockdown reversed CAPS1-induced EMT, cell migration and invasion. This result indicates that Snail is required for CAPS1-mediated EMT process and metastasis in CRC. Furthermore, CAPS1 can bind with Septin2 and p85 (subunit of PI3K). LY294002 and wortmanin, PI3K/Akt inhibitors, can abolish CAPS1-induced increase of Akt/GSK3β activity, as well as increase of Snail protein level. Taken together, CAPS1 promotes colorectal cancer metastasis through PI3K/Akt/GSK3β/Snail signal pathway-mediated EMT process.

CADPS2 gene expression is oppositely regulated by LRRK2 and alpha-synuclein

The Ca2+-dependent activator protein for secretion 2 (CADPS2) is a member of the CAPS/CADPS protein family that plays crucial roles in synaptic vesicle dynamics. Genomic variability in the CADPS2 gene has been associated to autism spectrum disorders and Alzheimer's disease, both characterized by altered neurotransmission. Biological evidence also linked CADPS2 to Parkinson's disease (PD), as a disease-causing mutation in leucine-rich repeat kinase 2 (LRRK2) was reported to increase CADPS2 gene and protein expression. Furthermore, restoration of CADPS2 physiologic levels was able to provide neuroprotection in patient-derived neurons, consistent with the synaptic dysfunction postulated to underlie PD. However, little is known about the influence of PD-related proteins on transcriptional regulation of critical synaptic genes such as CADPS2. Here we aimed at investigating the transcriptional effects of LRRK2 and alpha-synuclein (aSyn) on CADPS2 gene expression, using a combination of in silico analyses and cell biology techniques. First, we identified a predicted promoter in the human CADPS2 genomic sequence, which we then utilized in a luciferase-based gene reporter assay. This approach enabled us to disclose a differential effect of high levels of LRRK2 and aSyn on CADPS2 promoter activity. Specifically, CADPS2 transcriptional activity was enhanced by high cellular levels of LRRK2 and reduced by overexpression of aSyn. Consistently, CADPS2 mRNA levels were diminished in aSyn overexpressing cells.Our results indicate that LRRK2 and aSyn participate in the dysregulation of CADPS2 by altering transcription and support the hypothesis that synaptic dysfunctions, through different mechanisms, might contribute to the neuronal defects of diseases such as PD.

Fast cAMP Modulation of Neurotransmission via Neuropeptide Signals and Vesicle Loading

Cyclic AMP (cAMP) signaling augments synaptic transmission, but because many targets of cAMP and protein kinase A (PKA) may be involved, mechanisms underlying this pathway remain unclear. To probe this mechanism, we used optogenetic stimulation of cAMP signaling by Beggiatoa-photoactivated adenylyl cyclase (bPAC) in Caenorhabditis elegans motor neurons. Behavioral, electron microscopy (EM), and electrophysiology analyses revealed cAMP effects on both the rate and on quantal size of transmitter release and led to the identification of a neuropeptidergic pathway affecting quantal size. cAMP enhanced synaptic vesicle (SV) fusion by increasing mobilization and docking/priming. cAMP further evoked dense core vesicle (DCV) release of neuropeptides, in contrast to channelrhodopsin (ChR2) stimulation. cAMP-evoked DCV release required UNC-31/Ca2+-dependent activator protein for secretion (CAPS). Thus, DCVs accumulated in unc-31 mutant synapses. bPAC-induced neuropeptide signaling acts presynaptically to enhance vAChT-dependent SV loading with acetylcholine, thus causing increased miniature postsynaptic current amplitudes (mPSCs) and significantly enlarged SVs.

Analysis of gene expression in Ca2+-dependent activator protein for secretion 2 (Cadps2) knockout cerebellum using GeneChip and KEGG pathways

In the mouse cerebellum, Ca2+-dependent activator protein for secretion 2 (CADPS2, CAPS2) is involved in regulated secretion from dense-core vesicles (DCVs), which contain neuropeptides including brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). Capds2 knockout (KO) mice show impaired cerebellar development in addition to autistic-like behavioral phenotypes. To understand the molecular impact caused by loss of Capds2, we analyzed gene expression profiles in the Capds2 KO cerebellum using a GeneChip microarray and the KEGG Pathway database. Significant differential expression was observed in 1211 of 22,690 (5.34%) genes represented on the chip. The expression levels of exocytosis-related genes (Stx5a, Syt6), genes encoding secretory (Fgf2, Fgf4, Edn2) and synaptic proteins (Grin2b, Gabbr1), neurotrophin signaling-associated genes (Sos1, Shc1, Traf6, Psen2), and a gene for Rett syndrome (Mecp2) were significantly changed. Taken together, these results suggest that deregulated gene expression caused by loss of Capds2 may cause developmental deficits and/or pathological symptoms, resulting in autistic-like phenotypes.

Interaction of Ca2+-dependent activator protein for secretion 1 (CAPS1) with septin family proteins in mouse brain

The Ca2+-dependent activator protein for secretion 1 (CAPS1) protein plays a regulatory role in the dense-core vesicle exocytosis pathway. To clarify the functions of this protein in the brain, we searched for novel interaction partners of CAPS1 by mass spectrometry. We identified a specific interaction of CAPS1 with septin family proteins. We also demonstrated that the C-terminal region of the CAPS1 protein binds to part of the deduced GTP-binding domain of septin proteins. It is possible that a tertiary complex of septin, CAPS, and syntaxin contributes to dense-core vesicle trafficking and exocytosis in neurons.

Lack of stress responses to long-term effects of corticosterone in Caps2 knockout mice.

Chronic stress is associated with anxiety and depressive disorders, and can cause weight gain. Ca2+-dependent activator protein for secretion 2 (CAPS2) is involved in insulin release. Caps2 knockout (KO) mice exhibit decreased body weight, reduced glucose-induced insulin release, and abnormal psychiatric behaviors. We chronically administered the stress hormone corticosterone (CORT), which induces anxiety/depressive-like behavior and normally increases plasma insulin levels, via the drinking water for 10 weeks, and we examined the stress response in KO mice. Chronic CORT exposure inhibited stress-induced serum CORT elevation in wild-type (WT) mice, but not in KO mice. Poor weight gain in CORT-treated animals was observed until week 6 in WT mice, but persisted for the entire duration of the experiment in KO mice, although there is no difference in drug*genotype interaction. Among KO mice, food consumption was unchanged, while water consumption was higher, over the duration of the experiment in CORT-treated animals, compared with untreated animals. Moreover, serum insulin and leptin levels were increased in CORT-treated WT mice, but not in KO mice. Lastly, both WT and KO mice displayed anxiety/depressive-like behavior after CORT administration. These results suggest that Caps2 KO mice have altered endocrine responses to CORT administration, while maintaining CORT-induced anxiety/depressive-like behavior.

Secretory Vesicle Priming by CAPS Is Independent of Its SNARE-Binding MUN Domain

Priming of secretory vesicles is a prerequisite for their Ca(2+)-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca(2+)-dependent activator protein for secretion (CAPS) also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin byMUN domains of CAPSs, and is therefore not redundant with Munc13 action.

Axonal localization of Ca2+-dependent activator protein for secretion 2 is critical for subcellular locality of brain-derived neurotrophic factor and neurotrophin-3 release affecting proper development of postnatal mouse cerebellum.

Ca2+-dependent activator protein for secretion 2 (CAPS2) is a protein that is essential for enhanced release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from cerebellar granule cells. We previously identified dex3, a rare alternative splice variant of CAPS2, which is overrepresented in patients with autism and is missing an exon 3 critical for axonal localization. We recently reported that a mouse model CAPS2Δex3/Δex3 expressing dex3 showed autistic-like behavioral phenotypes including impaired social interaction and cognition and increased anxiety in an unfamiliar environment. Here, we verified impairment in axonal, but not somato-dendritic, localization of dex3 protein in cerebellar granule cells and demonstrated cellular and physiological phenotypes in postnatal cerebellum of CAPS2Δex3/Δex3 mice. Interestingly, both BDNF and NT-3 were markedly reduced in axons of cerebellar granule cells, resulting in a significant decrease in their release. As a result, dex3 mice showed developmental deficits in dendritic arborization of Purkinje cells, vermian lobulation and fissurization, and granule cell precursor proliferation. Paired-pulse facilitation at parallel fiber-Purkinje cell synapses was also impaired. Together, our results indicate that CAPS2 plays an important role in subcellular locality (axonal vs. somato-dendritic) of enhanced BDNF and NT-3 release, which is indispensable for proper development of postnatal cerebellum.

CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis

Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca(2+)-triggered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca(2+)-triggered fusion were unknown. Ca(2+)-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca(2+)-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP2, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP2-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP2 for its activity. Munc13 is cytoplasmic, but Ca(2+)-dependent translocation to PIP2-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP2-rich membrane domains is facilitated by sequential PIP2-dependent activation of CAPS and PIP2-dependent recruitment of Munc13. PIP2 hydrolysis only occurs under strong Ca(2+) influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca(2+)-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.

Mouse Models of Mutations and Variations in Autism Spectrum Disorder-Associated Genes: Mice Expressing Caps2/Cadps2 Copy Number and Alternative Splicing Variants

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by disturbances in interpersonal relationships and behavior. Although the prevalence of autism is high, effective treatments have not yet been identified. Recently, genome-wide association studies have identified many mutations or variations associated with ASD risk on many chromosome loci and genes. Identification of the biological roles of these mutations or variations is necessary to identify the mechanisms underlying ASD pathogenesis and to develop clinical treatments. At present, mice harboring genetic modifications of ASD-associated gene candidates are the best animal models to analyze hereditary factors involved in autism. In this report, the biological significance of ASD-associated genes is discussed by examining the phenotypes of mouse models with ASD-associated mutations or variations in mouse homologs, with a focus on mice harboring genetic modifications of the Caps2/Cadps2 (Ca2+-dependent activator protein for secretion 2) gene.

New insights concerning insulin synthesis and its secretion in rat hippocampus and cerebral cortex: Amyloid-β1–42-induced reduction of proinsulin level via glycogen synthase kinase-3β

The reduction of insulin levels in hippocampal areas is associated with Alzheimer's disease. The present study using rat brain explores the mechanisms of insulin synthesis and secretion, as well as amyloid-β1–42 (Aβ1–42)-induced reduction of proinsulin expression. After confirming the expression of insulin mRNA and proinsulin in rat brain, we visualized and analyzed the motion of insulin secretion in rat hippocampal neurons using pH-sensitive green fluorescent protein (pHluorin) fused to the insulin. In the rat hippocampal neurons expressing insulin–pHluorin, time-lapse confocal laser scanning microscopy revealed the appearance of fluorescent spots induced by depolarization after stimulation with 50mM KCl. In these fluorescent spots, Ca2+-dependent activator protein for secretion 2 (CAPS2), which is the regulator of the dense-core vesicle involving neuronal peptides, was co-localized with insulin–pHluorin. However, Aβ1–42-induced reduction of proinsulin in rat hippocampal neurons was inhibited by treatment with lithium and transfection with glycogen synthase kinase-3β (GSK-3β) siRNA. These results demonstrate that synthesized insulin is secreted from rat hippocampal and cortical neuron's dense-core vesicles, and that activation of GSK-3β in Aβ1–42-induced Alzheimer's model hippocampal neurons decreases the insulin synthesis.