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Several genetic models that recapitulate neurodegenerative features of Parkinson's disease (PD) exist, which have been largely based on genes discovered in monogenic PD families. However, spontaneous genetic mutations have not been linked to the pathological hallmarks of PD in non-human vertebrates.To describe the genetic and pathological findings of three Yellow-crowned parrot (Amazona ochrocepahala) siblings with a severe and rapidly progressive neurological phenotype.The phenotype of the three parrots included severe ataxia, rigidity, and tremor, while their parents were phenotypically normal. Tests to identify avian viral infections and brain imaging studies were all negative. Due to their severe impairment, they were all euthanized at age 3 months and their brains underwent neuropathological examination and proteasome activity assays. Whole genome sequencing (WGS) was performed on the three affected parrots and their parents.The brains of affected parrots exhibited neuronal loss, spongiosis, and widespread Lewy body-like inclusions in many regions including the midbrain, basal ganglia, and neocortex. Proteasome activity was significantly reduced in these animals compared to a control (P 0.05). WGS identified a single homozygous missense mutation (p.V559L) in a highly conserved amino acid within the pleckstrin homology (PH) domain of the calcium-dependent secretion activator 2 (CADPS2) gene.Our data suggest that a homozygous mutation in the CADPS2 gene causes a severe neurodegenerative phenotype with Lewy body-like pathology in parrots. Although CADPS2 variants have not been reported to cause PD, further investigation of the gene might provide important insights into the pathophysiology of Lewy body disorders. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Calcium-dependent activator protein for secretion 2 (CAPS2; also referred to as CADPS2) is a dense core vesicle-associated protein that promotes the activity-dependent release of neuropeptides including neurotrophins. Addictive drugs appear to prime neurotrophin release in multiple brain regions, but mechanistic factors are still being elucidated. Here, experimenters administered cocaine to adolescent mice at doses that potentiated later cocaine self-administration. Experimenter-administered cocaine elevated the CAPS2 protein content in the orbitofrontal cortex (OFC; but not striatum) multiple weeks after drug exposure. Meanwhile, proteins that are sensitive to brain-derived neurotrophic factor (BDNF) release and binding (phosphorylated protein kinase B and phosphoinositide 3-kinase, and GABAAα1 levels) did not differ between cocaine-exposed and naive mice in the OFC. This pattern is consistent with evidence that CAPS2 primes stimulated release of neurotrophins like BDNF, rather than basal levels. Thus, cocaine administered at behaviorally relevant doses elevates CAPS2 protein content in the OFC, and the effects are detected long after cocaine exposure.

Most psychiatric disorders are highly heritable and associated with altered brain structural and functional patterns. Data fusion analyses on brain imaging and genetics, one of which is parallel independent component analysis (pICA), enable the link of genomic factors to brain patterns. Due to the small to modest effect sizes of common genetic variants in psychiatric disorders, it is usually challenging to reliably separate disorder-related genetic factors from the rest of the genome with the typical size of clinical samples. To alleviate this problem, we propose sparse parallel independent component analysis (spICA) to leverage the sparsity of individual genomic sources. The sparsity is enforced by performing Hoyer projection on the estimated independent sources. Simulation results demonstrate that the proposed spICA yields improved detection of independent sources and imaging-genomic associations compared to pICA. We applied spICA to gray matter volume (GMV) and single nucleotide polymorphism (SNP) data of 341 unrelated adults, including 127 controls, 167 attention-deficit/hyperactivity disorder (ADHD) cases, and 47 unaffected siblings. We identified one SNP source significantly and positively associated with a GMV source in superior/middle frontal regions. This association was replicated with a smaller effect size in 317 adolescents from ADHD families, including 188 individuals with ADHD and 129 unaffected siblings. The association was found to be more significant in ADHD families than controls, and stronger in adults and older adolescents than younger ones. The identified GMV source in superior/middle frontal regions was not correlated with head motion parameters and its loadings (expression levels) were reduced in adolescent (but not adult) individuals with ADHD. This GMV source was associated with working memory deficits in both adult and adolescent individuals with ADHD. The identified SNP component highlights SNPs in genes encoding long non-coding RNAs and SNPs in genes MEF2C, CADM2, and CADPS2, which have known functions relevant for modulating neuronal substrates underlying high-level cognition in ADHD.

Parkinson’s disease (PD) etiology is associated with genetic and environmental factors that lead to a loss of dopaminergic neurons. However, the functional interpretation of PD-associated risk variants and how other midbrain cells contribute to this neurodegenerative process are poorly understood. Here, we profiled >41,000 single-nuclei transcriptomes of postmortem midbrain tissue from 6 idiopathic PD (IPD) patients and 5 matched controls. We show that PD-risk variants are associated with glia- and neuron-specific gene expression patterns. Furthermore, Microglia and astrocytes presented IPD-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signalling. IPD-microglia revealed a specific pro-inflammatory trajectory. Finally, we discovered a neuronal cell cluster exclusively present in IPD midbrains characterized by CADPS2 overexpression and a high proportion of cycling cells. We conclude that elevated CADPS2 expression is specific to dysfunctional dopaminergic neurons, which have lost their dopaminergic identity and unsuccessful attempt to re-enter the cell cycle.

The disruptor of telomeric silencing 1-like (DOT1L) mediates methylation of histone H3 at position lysine 79 (H3K79). Conditional knockout of Dot1l in mouse cerebellar granule cells (Dot1l-cKOAtoh1) led to a smaller external granular layer with fewer precursors of granule neurons. Dot1l-cKOAtoh1 mice had impaired proliferation and differentiation of granular progenitors, which resulted in a smaller cerebellum. Mutant mice showed mild ataxia in motor behavior tests. In contrast, Purkinje cell-specific conditional knockout mice showed no obvious phenotype. Genome-wide transcription analysis of Dot1l-cKOAtoh1 cerebella using microarrays revealed changes in genes that function in cell cycle, cell migration, axon guidance, and metabolism. To identify direct DOT1L target genes, we used genome-wide profiling of H3K79me2 and transcriptional analysis. Analysis of differentially methylated regions (DR) and differentially expressed genes (DE) revealed in total 12 putative DOT1L target genes in Dot1l-cKOAtoh1 affecting signaling (Tnfaip8l3, B3galt5), transcription (Otx1), cell migration and axon guidance (Sema4a, Sema5a, Robo1), cholesterol and lipid metabolism (Lss, Cyp51), cell cycle (Cdkn1a), calcium-dependent cell-adhesion or exocytosis (Pcdh17, Cadps2), and unknown function (Fam174b). Dysregulated expression of these target genes might be implicated in the ataxia phenotype observed in Dot1l-cKOAtoh1. Electronic supplementary material The online version of this article (10.1007/s12035-018-1377-1) contains supplementary material, which is available to authorized users.

Autism spectrum disorder (ASD) is a group of the neurodevelopment disorders presenting as an isolated ASD or more complex forms, where a broader clinical phenotype comprised of developmental delay and intellectual disability is present. Both the isolated and complex forms have a significant causal genetic component and submicroscopic genomic copy number variations (CNV) are the most common identifiable genetic factor in these patients. The data on microarray testing in ASD cohorts are still accumulating and novel loci are often identified; therefore, we aimed to evaluate the diagnostic efficacy of the method and the relevance of implementing it into routine genetic testing in ASD patients. A genome-wide CNV analysis using the Agilent microarrays was performed in a group of 150 individuals with an isolated or complex ASD. Altogether, 11 (7.3%) pathogenic CNVs and 15 (10.0%) variants of unknown significance (VOUS) were identified, with the highest proportion of pathogenic CNVs in the subgroup of the complex ASD patients (14.3%). An interesting case of previously unreported partial UPF3B gene deletion was identified among the pathogenic CNVs. Among the CNVs with unknown significance, four VOUS involved genes with possible correlation to ASD, namely genes SNTG2, PARK2, CADPS2 and NLGN4X. The diagnostic efficacy of aCGH in our cohort was comparable with those of the previously reported and identified an important proportion of genetic ASD cases. Despite the continuum of published studies on the CNV testing in ASD cohorts, a considerable number of VOUS CNVs is still being identified, namely 10.0% in our study.

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.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder marked by impairments in social functioning, language, communication, and behavior. Recent genome-wide association studies show some microdeletions on the 7q31-32 region, including the CADPS2 locus in autistic patients. This paper reports the case of a patient with ASD and recurrent psychotic syndrome, in which a deletion on the 7q31-32 band at the CADPS2 gene locus was evidenced, as well as a brief review of the literature on the CADPS2 gene and its association with ASD.

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.

Background Attention deficit hyperactivity disorder (ADHD) is a common psychiatric disorder with high prevalence. Candidate gene association studies have identified several genes contributing to the development of ADHD. However, these genes only interpreted 4.3% of the average heritability. Several genome-wide association studies (GWAS) of ADHD have been performed, but none of them got genome-wide significant result. One reason for the lack of significant genetic result is that ADHD is a heterogeneous disorder. Endophenotypes as the intermediate phenotypes between genes and phenotypes have been widely used in psychiatric disorders. Many neuropsychological features have been suggested to be endophenotypes of ADHD. Exploration of the genetic basis of the disease related neuropsychological traits would provide clues for the understanding of the genetic basis of the disease and further facilitate the differentiation of disease and precision prevention, diagnosis and treatment. Methods We had identified one significant locus near MICALL2 gene for inhibitory control in ADHD by using a two-stage Genome-Wide Association Study for the Word Interference Time (WIT) in the Stroop task (PMID: 28416812). The down expression of the homologous gene of MICALL2 led to hyperactive-impulsive-like behavior in zebrafish. In our new experimental study, we further tested the numbers/ activity of neurons in zebrafish with MICALL2b gene knockdown by using real-time quantitative PCR, in situ hybridization, as well as neuroD transgenic zebrafish and th transgenic zebrafish. The result showed that the catecholaminergic activity were increased at 24 hours, 48 hours and 3 days of the embryonic development and the noradrenergic activity was increased at 3 days of the embryonic development after the reduction of MICALL2b gene expression. At 3 days post fertilization, the activity of catecholaminergic neuron was tend to increase in the transgenic zebrafish. The neurobiological changes of the zebrafish with decreased MICALL2b gene function further validated the contribution of MICALL2 to ADHD. Results In addition, we tested the cognitive flexibility of ADHD patients using Trail-Make Test, and performed genome-wide association study for the shifting time in 785 ADHD patients. The top SNP is located in gene DLGAP1 (P = 5.08E-07), which was validated in several other cognitive flexibility related traits, ADHD symptoms and several other psychiatric disorders. Gene-level analysis showed the most significant gene is CADPS2, which also interacted with several well-known psychiatric disorder related genes, including BDNF, DRD2, NRG3. Pathway enrichment analysis for the potential SNPs with P Discussion Our result showed using the neuropsychological traits could advance the genetic discovery of ADHD. The identified genes and pathways would help understand the relationship between ADHD and the neuropsychological function, and the underlying genetic basis.

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.

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.

SummaryThe LRRK2 mutation G2019S is the most common genetic cause of Parkinson’s disease (PD). To better understand the link between mutant LRRK2 and PD pathology, we derived induced pluripotent stem cells from PD patients harboring LRRK2 G2019S and then specifically corrected the mutant LRRK2 allele. We demonstrate that gene correction resulted in phenotypic rescue in differentiated neurons and uncovered expression changes associated with LRRK2 G2019S. We found that LRRK2 G2019S induced dysregulation of CPNE8, MAP7, UHRF2, ANXA1, and CADPS2. Knockdown experiments demonstrated that four of these genes contribute to dopaminergic neurodegeneration. LRRK2 G2019S induced increased extracellular-signal-regulated kinase 1/2 (ERK) phosphorylation. Transcriptional dysregulation of CADPS2, CPNE8, and UHRF2 was dependent on ERK activity. We show that multiple PD-associated phenotypes were ameliorated by inhibition of ERK. Therefore, our results provide mechanistic insight into the pathogenesis induced by mutant LRRK2 and pointers for the development of potential new therapeutics.

The effect ofγ-tocotrienol, a vitamin E isomer, in modulating gene expression in cellular aging of human diploid fibroblasts was studied. Senescent cells at passage 30 were incubated with 70 μM ofγ-tocotrienol for 24 h. Gene expression patterns were evaluated using Sentrix HumanRef-8 Expression BeadChip from Illumina, analysed using GeneSpring GX10 software, and validated using quantitative RT-PCR. A total of 100 genes were differentially expressed (P<0.001) by at least 1.5 fold in response toγ-tocotrienol treatment. Amongst the genes wereIRAK3, SelS, HSPA5, HERPUD1, DNAJB9, SEPR1, C18orf55, ARF4, RINT1, NXT1, CADPS2, COG6, andGLRX5. Significant gene list was further analysed by Gene Set Enrichment Analysis (GSEA), and the Normalized Enrichment Score (NES) showed that biological processes such as inflammation, protein transport, apoptosis, and cell redox homeostasis were modulated in senescent fibroblasts treated withγ-tocotrienol. These findings revealed thatγ-tocotrienol may prevent cellular aging of human diploid fibroblasts by modulating gene expression.

Ca2+-dependent activator protein for secretion 2 (CADPS2), a secretory granule associate protein, mediates monoamine transmission and neurotrophin release. Both monoamines and neurotrophins play a crucial role in cognition, learning and memory. An aberrant splice variant of CADPS2, CADPS2ΔExon3, was reported to be associated with autism. Therefore, we examined the possible association between the expression of CADPS2/CADPS2ΔExon3 in peripheral blood and brain functions such as intelligence and memory. Quantitative polymerase chain reaction analysis was performed in 271 healthy adults (age range 20–74 years, mean ± SD 43.3 ± 15.3). Data on intelligence quotient (IQ) and memory were obtained by using full versions of the Wechsler Adult Intelligence Scale-Revised (WAIS-R), and the Wechsler Memory Scale-Revised (WMS-R), respectively. CADPS2 expression levels were not significantly associated with any scores/sub-scores of these scales. However, CADPS2ΔExon3 expression was significantly associated with lower IQ (p = 0.022; effect size: ηp2 = 0.031), particularly verbal IQ of WAIS-R (p = 0.019; ηp2 = 0.032), lower verbal memory (p = 0.026; ηp2 = 0.026) and delayed recall (p = 0.042; ηp2 = 0.021) of WMS-R. Our results suggest that CADPS2ΔExon3 affects intelligence and memory in the non-clinical population.

Ca2+-dependent activator protein for secretion 2 (CADPS2), a secretory granule associate protein, mediates monoamine transmission and the release of neurotrophins including brain-derived neurotrophic factor (BDNF) which have been implicated in psychiatric disorders. Furthermore, the expression of CADPS2deltaExon3, a defective splice variant of CADPS2, has been reported to be associated with autism. Based on these observations, we examined whether expression levels of CADPS2 and CADPS2deltaExon3 are altered in psychiatric disorders. Quantitative polymerase chain reaction analysis was performed for postmortem frontal cortex tissues (BA6) from 15 individuals with schizophrenia, 15 with bipolar disorder, 15 with major depression, and 15 controls (Stanley neuropathology consortium). The mean CADPS2 expression levels normalized to human glyceraldehyde-3phosphate dehydrogenase (GAPDH) or TATA-box binding protein levels was found to be significantly increased in the brains of the schizophrenia group, compared to the control group. On the other hand, the ratio of CADPS2deltaExon3 to total CADPS2 was similar in the 4 diagnostic groups. We then analyzed CADPS2 expression in blood samples from 121 patients with schizophrenia and 318 healthy controls; however, there was no significant difference between the two groups. Chronic risperidone treatment did not alter the expression of CADPS2 in frontal cortex of mice. The observed increase in the expression of CADPS2 may be related to the impaired synaptic function in schizophrenia.

We performed array comparative genomic hybridization utilizing a whole genome oligonucleotide microarray in a patient with the autism spectrum disorders (ASDs) and persistent hyperplastic primary vitreous (PHPV). Submicroscopic deletions in 7q31 encompassing CADPS2 (Ca(2+) -dependent activator protein for secretion 2) and TSPAN12 (one of the members of the tetraspanin superfamily) were confirmed. The CADPS2 plays important roles in the release of neurotrophin-3 and brain-derived neurotrophic factor. Mutations in TSPAN12 are a relatively frequent cause of familial exudative vitreoretinopathy. We speculate that haploinsufficiency of CADPS2 and TSPAN12 contributes to ASDs and PHPV, respectively.

Ca(2+)-dependent activator protein for secretion 2 (CAPS2 or CADPS2) regulates dense-core vesicle (DCV) exocytosis. We found that CAPS2 is involved in the secretion of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and that CAPS2 KO mice not only have deficits in neuronal development and survival but also exhibit abnormal behaviors, including impaired social interaction, hyperactivity, an abnormal sleep-wake rhythm and increased anxiety in unfamiliar environments. Moreover, we identified increased expression of a rare CAPS2 splice variant in autism patients that specifically lacks exon 3 and that is not transported to axons when exogenously expressed in mouse cortical neurons. Moreover, non-synonymous SNPs have been identified in some autistic patients. These results implicate CAPS2 in autism susceptibility. Therefore, CAPS2 KO mice will be a useful animal model to study the aspects of the neuropathology and behavior characteristics of neurodevelopmental disorders.

Recent studies have elucidated mechanisms of brain-derived neurotrophic factor (BDNF) secretion, and impaired secretion of BDNF may be involved in the pathogenesis of several neuropsychiatric diseases. The huntingtin gene, for example, has been shown to regulate vesicular transport of BDNF, which may play a role in the neurodegeneration present in Huntington's disease. In animal studies, mice lacking calcium-dependent activator protein for secretion 2 (CADPS2), which is involved in the activity-dependent release of BDNF, showed several phenotypes including autistic behavior. A single nucleotide polymorphism that results in an amino-acid change (Val66Met) in the BDNF gene has been shown to cause a decline in the function of BDNF vesicular sorting and has been reported to be associated with behavioral and intermediate phenotypes (e.g., episodic memory) in humans. In this review, we introduce recent progress in the molecular mechanisms of BDNF secretion and discuss its possible role in the pathophysiology and treatment of neuropsy

Neurotrophins are key modulators of various neuronal functions, including differentiation, survival, and synaptic plasticity, but the molecules that regulate their secretion are poorly understood. We isolated a clone that is predominantly expressed in granule cells of postnatally developing mouse cerebellum, which turned out to be a paralog of CAPS (Ca2+-dependent activator protein for secretion), and named CAPS2. CAPS2 is enriched on vesicular structures of presynaptic parallel fiber terminals of granule cells connecting postsynaptic spines of Purkinje cell dendrites. Vesicle factions affinity-purified by the CAPS2 antibody from mouse cerebella contained significant amounts of neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and chromogranin B but not marker proteins for synaptic vesicle synaptophysin and synaptotagmin. In cerebellar primary cultures, punctate CAPS2 immunoreactivities are primarily colocalized with those of NT-3 and BDNF and near those of a postsynaptic marker, postsynaptic density-95, around dendritic arborization of Purkinje cells. Exogenously expressed CAPS2 enhanced release of exogenous NT-3 and BDNF from PC12 cells and endogenous NT-3 from cultured granule cells in a depolarization-dependent manner. Moreover, the overexpression of CAPS2 in granule cells promotes the survival of Purkinje cells in cerebellar cultures. Thus, we suggest that CAPS2 mediates the depolarization-dependent release of NT-3 and BDNF from granule cells, leading to regulation in cell differentiation and survival during cerebellar development.

Ca2+-dependent activator protein for secretion (CAPS) 1 is an essential cytosolic component of the protein machinery involved in large dense-core vesicle (LDCV) exocytosis and in the secretion of a subset of neurotransmitters. In the present study, we report the identification, cloning, and comparative characterization of a second mammalian CAPS isoform, CAPS2. The structure of CAPS2 and its function in LDCV exocytosis from PC12 cells are very similar to those of CAPS1. Both isoforms are strongly expressed in neuroendocrine cells and in the brain. In subcellular fractions of the brain, both CAPS isoforms are enriched in synaptic cytosol fractions and also present on vesicular fractions. In contrast to CAPS1, which is expressed almost exclusively in brain and neuroendocrine tissues, CAPS2 is also expressed in lung, liver, and testis. Within the brain, CAPS2 expression seems to be restricted to certain brain regions and cell populations, whereas CAPS1 expression is strong in all neurons. During development, CAPS2 expression is constant between embryonic day 10 and postnatal day 60, whereas CAPS1 expression is very low before birth and increases after postnatal day 0 to reach a plateau at postnatal day 21. Light microscopic data indicate that both CAPS isoforms are specifically enriched in synaptic terminals. Ultrastructural analyses show that CAPS1 is specifically localized to glutamatergic nerve terminals. We conclude that at the functional level, CAPS2 is largely redundant with CAPS1. Differences in the spatial and temporal expression patterns of the two CAPS isoforms most likely reflect as yet unidentified subtle functional differences required in particular cell types or during a particular developmental period. The abundance of CAPS proteins in synaptic terminals indicates that they may also be important for neuronal functions that are not exclusively related to LDCV exocytosis.

Intellectual disability (ID) and autism spectrum disorders (ASDs) are complex neuropsychiatric conditions, with overlapping clinical boundaries in many patients. We identified a novel intragenic deletion of maternal origin in two siblings with mild ID and epilepsy in the CADPS2 gene, encoding for a synaptic protein involved in neurotrophin release and interaction with dopamine receptor type 2 (D2DR). Mutation screening of 223 additional patients (187 with ASD and 36 with ID) identified a missense change of maternal origin disrupting CADPS2/D2DR interaction. CADPS2 allelic expression was tested in blood and different adult human brain regions, revealing that the gene was monoallelically expressed in blood and amygdala, and the expressed allele was the one of maternal origin. Cadps2 gene expression performed in mice at different developmental stages was biallelic in the postnatal and adult stages; however, a monoallelic (maternal) expression was detected in the embryonal stage, suggesting that CADPS2 is subjected to tissue- and temporal-specific regulation in human and mice. We suggest that CADPS2 variants may contribute to ID/ASD development, possibly through a parent-of-origin effect.

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.

Sadakata et al. (1) reported that a CADPS2 isoform lacking exon 3 is aberrantly spliced in the peripheral blood of autistic patients. However, we found this splice isoform in the blood of normal subjects at a similar frequency to that of individuals with autism spectrum disorder (ASD) (95% CI of the difference, –0.06 to 0.1). Moreover, this splice variant exists as a minor isoform in cerebellar RNA of both normal individuals and individuals with ASD. Thus, exon 3 skipping likely represents a minor isoform rather than aberrant splicing and is probably not an underlying mechanism of autism. Defects of CADPS2 function might contribute to autism susceptibility, but likely not through aberrant splicing. Sadakata et al. (1) reported that 4 of 16 patients with autism expressed an exon 3–skipped variant of CADPS2 mRNA in the blood, while the CADPS2 mRNA of all 24 normal subjects included exon 3. They thus concluded that CADPS2 is aberrantly spliced in autism, and they performed further experiments showing that the subcellular localization of exogenously expressed exon 3–skipped CADPS2 is disturbed in primary cultured neocortical and cerebellar neurons. We aimed to replicate the CADPS2 findings in an independent set of peripheral blood samples from 41 children with ASD and 39 control children, following the Sadakata et al. protocols (Figure ​(Figure1A).1A). Furthermore, we performed sequencing (Figure ​(Figure1B)1B) and nested priming (Figure ​(Figure1C)1C) to validate the presence or absence of exon 3. Our results showed that, of 39 control samples, 1 was apparently homozygous for the exon 3–skipped allele in peripheral blood, 5 were heterozygous, and 33 were wild type. Of the 41 ASD samples, 5 were heterozygous for the exon 3–skipped isoform, while the rest were wild type. Analysis of these results showed no significant difference in the frequency of the exon 3–skipped allele in ASD versus control samples (P = 0.6, two-proportion z test). Although the samples tested here might differ from those tested by Sadakata et al. in their ethnicity, gender, or age distributions (Supplemental Figure 1 and Supplemental Tables 1 and 2; supplemental materials available online with this article; doi: 10.1172/JCI38620DS1), the finding of exon 3 skipping in healthy controls at a high frequency suggests that this isoform does not represent aberrant splicing and likely is not a mechanism underlying autism. Figure 1 Exon 3 skipping in CADPS2 mRNA from 41 children with ASD and 39 control children. Since Sadakata et al. extrapolate function of the exon 3–skipped isoform within the cerebellum, we additionally tested the presence of exon 3 in mRNA extracted from the cerebella of 9 control children and 5 children with ASD. All ASD and control samples were found to contain the exon 3–skipped splice variant as a minor isoform (Figure ​(Figure1D).1D). Thus, our experiments suggest that exon 3 skipping represents a normal, minor isoform of CADPS2 in the cerebellum. As we observed no difference in prevalence of this allele between ASD and control samples, we conclude that exon 3 skipping is likely not a mechanism underlying autism susceptibility or pathogenesis.

Using a novel approach to measure exocytosis in vitro from semi-intact synaptosomes, we establish that the Ca2+-dependent release of glutamate requires cytosolic factors for mobilization from the reserve pool. The cytosolic activity for glutamate release was not satisfied by CAPS, a soluble component required for norepinephrine (NE) release. Moreover, the CAPS-independent glutamate release from synaptic vesicles (SVs) was 200-fold less sensitive to Ca2+ than that required for dense core vesicles (DCVs). The differential regulation of exocytosis by CAPS, Ca2+, and potential novel cytosolic factor(s) suggests that the docking and fusion machinery controlling DCVs has diverged from that regulating glutamate-containing SVs.

Autism spectrum disorder (ASD) is one of the most common neurodevelopmental disorders and is thought to be closely associated with genetic factors. It is noteworthy that many ASD-associated genes reported by genome-wide association studies encode proteins related to synaptic formation, transmission, and plasticity. Therefore, it is essential to elucidate the relationship between deficiencies in these genes and the relevant ASD-related phenotypes using synaptic and behavioral phenotypic analysis of mice that are genetically modified for genes related to ASD (e.g., knockout or mutant mice). In this review, we focus on the behavioral-, cellular-, and circuit-level phenotypes, including synaptic formation and function, of several knockout mouse models with genetic mutations related to ASD. Moreover, we introduce our recent findings on the possible association of the dense-core vesicle secretion-related gene CAPS2/CADPS2 with ASD by using knockout mice. Finally, we discuss the usefulness and limitations of various mouse models with single gene mutations for understanding ASD.

Ca 2 + -dependent activator protein for secretion 2 (CAPS2 or CADPS2) potently promotes the release of brain-derived neurotrophic factor (BDNF). A rare splicing form of CAPS2 with deletion of exon3 (dex3) was identified to be overrepresented in some patients with autism. Here, we generated Caps2 -dex3 mice and verified a severe impairment in axonal Caps2-dex3 localization, contributing to a reduction in BDNF release from axons. In addition, circuit connectivity, measured by spine and interneuron density, was diminished globally. The collective effect of reduced axonal BDNF release during development was a striking and selective repertoire of deficits in social- and anxiety-related behaviors. Together, these findings represent a unique mouse model of a molecular mechanism linking BDNF-mediated coordination of brain development to autism-related behaviors and patient genotype.

Ca2+-dependent activator protein for secretion 2 (CAPS2 or CADPS2) facilitates secretion and trafficking of dense-core vesicles. Recent genome-wide association studies of autism have identified several microdeletions due to copy number variation (CNV) in one of the chromosome 7q31.32 alleles on which the locus for CAPS2 is located in autistic patients. To evaluate the biological significance of reducing CAPS2 copy number, we analyzed CAPS2 heterozygous mice. Our present findings suggest that adequate levels of CAPS2 protein are critical for normal brain development and behavior, and that allelic changes due to CNV may contribute to autistic symptoms in combination with deficits in other autism-associated genes.

We read with great interest the comment by Eran et al. (1) regarding our recently published CADPS2 article in the JCI (2). We appreciate their comment that “exon 3 skipping likely represents a minor isoform rather than aberrant splicing” in the blood and postmortem cerebella of both healthy and autistic individuals. However, we are concerned about the sensitivity of detection of exon 3 skipping in their experiments and have a few replies to their letter. First, the signal intensity of the exon 3–skipped CADPS2 band was considerably weaker than that of the normal band in some ASD and control blood samples (Figure 1A in ref. 1), in contrast to our results using samples from autism, but not pervasive development disorder — not otherwise specified (PDD-NOS), samples. Moreover, only a trace amount of skipped band was detected in all postmortem cerebella they analyzed (Figure 1D in ref. 1). Second, they utilized RT-PCR with a nested amplification (at 70 cycles) to detect a control sample C14 with a skipped, but no normal, band (which was called “homozygous” in their comment; Figure 1C in ref. 1) and claimed that only one sample (control sample C14) was homozygous in their study. Little quantitative gain is generally noticed when increasing the number of cycles to such an extraordinary number. We are left wondering if only the skipped band would also be detected in case C14 using an ordinary RT-PCR method (similar to our method with 48 cycles), such as that used to generate the data shown in Figure 1A (1). Third, their argument regarding one sample “homozygous for the exon 3–skipped allele” and “heterozygous” samples may not be appropriate, since the terms are usually used for genomic DNA, not mRNA. Also, it is yet unknown whether exon 3 skipping is of a cis- or trans-acting genetic origin or some other origin such as epigenetic. Fourth, considering the results of Eran et al., we assume that a balance between exon 3–skipped and normal CADPS2 is important for the local secretion property (somato-dendritic, axonal, and synaptic secretion) of CADPS2. Our JCI article indicated that exon 3–skipped CADPS2 is not transported into the axons of cultured neurons and suggested that disturbance of this balance may cause a defect in local secretion. Impaired synaptic secretion should be more serious in neurons that dominantly express exon 3–skipped CADPS2 than in those that weakly express it. Thus, excessive exon 3 skipping, together with a combination of other genetic mutations, might contribute to susceptibility to autism. Finally, we have recently succeeded in generating a mouse line expressing exon 3–skipped Cadps2 and have confirmed that exon 3 is critical for the subcellular localization of Cadps2 in neurons (our unpublished observations). Further studies will shed light on the association of exon 3 skipping with disturbed brain development and behavioral traits.

Background Ca2+-dependent activator protein 2 (CAPS2/CADPS2) is a secretory vesicle-associated protein involved in the release of neurotrophin. We recently reported that an aberrant, alternatively spliced CAPS2 mRNA that lacks exon 3 (CAPS2Δexon3) is detected in some patients with autism. Splicing variations in mouse CAPS2 and their expression and functions remain unclear. Results In this study, we defined 31 exons in the mouse CAPS2 gene and identified six alternative splicing variants, CAPS2a-f. CAPS2a is an isoform lacking exons 22 and 25, which encode part of the Munc13-1-homologous domain (MHD). CAPS2b lacks exon 25. CAPS2c lacks exons 11 and 22. CAPS2d, 2e, and 2f have C-terminal deletions from exon 14, exon 12, and exon 5, respectively. On the other hand, a mouse counterpart of CAPS2Δexon3 was not detected in the mouse tissues tested. CAPS2b was expressed exclusively in the brain, and the other isoforms were highly expressed in the brain, but also in some non-neural tissues. In the brain, all isoforms showed predominant expression patterns in the cerebellum. In the developing cerebellum, CAPS2b showed an up-regulated expression pattern, whereas the other isoforms exhibited transiently peaked expression patterns. CAPS2 proteins were mostly recovered in soluble fractions, but some were present in membrane fractions, except for CAPS2c and 2f, both of which lack the PH domain, suggesting that the PH domain is important for membrane association. In contrast to CAPS2a and 2b, CAPS2c showed slightly decreased BDNF-releasing activity, which is likely due to the C-terminal truncation of the PH domain in CAPS2c. Conclusion This study indicates that, in mouse, there are six splicing variants of CAPS2 (CAPS2a-f), and that these are subdivided into two groups: a long form containing the C-terminal MHD and a short form lacking the C-terminal MHD. These results demonstrate that the splicing variations correlate with their expression patterns and intracellular distribution, and affect BDNF release; however, whether or not the short forms possess activities other than BDNF release, for example as natural dominant-negative isoforms, remains to be determined.

Ca2+-dependent activator protein for secretion 2 (CAPS2/CADPS2) is a secretory granule-associated protein that is abundant at the parallel fiber terminals of granule cells in the mouse cerebellum and is involved in the release of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), both of which are required for cerebellar development. The human homolog gene on chromosome 7 is located within susceptibility locus 1 of autism, a disease characterized by several cerebellar morphological abnormalities. Here we report thatCAPS2knock-out mice are deficient in the release of NT-3 and BDNF, and they consequently exhibit suppressed phosphorylation of Trk receptors in the cerebellum; these mice exhibit pronounced impairments in cerebellar development and functions, including neuronal survival, differentiation and migration of postmitotic granule cells, dendritogenesis of Purkinje cells, lobulation between lobules VI and VII, structure and vesicular distribution of parallel fiber–Purkinje cell synapses, paired-pulse facilitation at parallel fiber–Purkinje cell synapses, rotarod motor coordination, and eye movement plasticity in optokinetic training. Increased granule cell death of the external granular layer was noted in lobules VI–VII and IX, in which high BDNF and NT-3 levels are specifically localized during cerebellar development. Therefore, the deficiency of CAPS2 indicates that CAPS2-mediated neurotrophin release is indispensable for normal cerebellar development and functions, including neuronal differentiation and survival, morphogenesis, synaptic function, and motor leaning/control. The possible involvement of theCAPS2gene in the cerebellar deficits of autistic patients is discussed.

The family of Ca2+-dependent activator proteins for secretion (CAPS) is involved in dense-core vesicle exocytosis. CAPS1/CADPS1 and CAPS2/CADPS2 have been identified in mammals. CAPS1 regulates catecholamine release from neuroendocrine cells, whereas CAPS2 is involved in the release of brain-derived neurotrophic factor and neurotrophin-3 from cerebellar granule cells. CAPS1 and CAPS2 are predominantly expressed in brain. Here we show the immunohistochemical localization of the CAPS family proteins in various mouse tissues. In the pituitary gland, CAPS1 and CAPS2 were localized to the pars nervosa and the pars intermedia, respectively. In non-neural tissues, CAPS1 was observed in the islets of Langerhans, minor cell types of the spleen and stomach, and medullary cells of the adrenal gland, whereas CAPS2 was present in bronchial epithelial cells, thyroid parafollicular cells, chief cells of the stomach, ductal epithelium of the salivary gland, kidney proximal tubules, and minor cell types of the thymus, spleen, and colon. These results suggest that secretion from distinct cell types in various tissues involves either or both members of the CAPS family.

The Ca2+-dependent activator protein for secretion (CAPS/Cadps) family consists of two members, CAPS1 and CAPS2, and plays an important role in secretory granule exocytosis. It has been shown that CAPS1 regulates catecholamine release from neuroendocrine cells, whereas CAPS2 is involved in the release of two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), from parallel fibers of cerebellar granule cells. Although both CAPS proteins are expressed predominantly in the brain, their cellular and regional distributions in the brain are largely unknown. In this study we analyzed the immunohistochemical distributions of the CAPS family proteins in the mouse brain. In most areas of the embryonic nervous system CAPS1 and CAPS2 proteins were complementarily expressed. In the postnatal brain, CAPS1 was widespread at different levels. On the other hand, CAPS2 was localized to distinct cell types and fibers of various brain regions, including the olfactory bulb, cerebrum, hippocampal formation, thalamus, mesencephalic tegmentum, cerebellum, medulla, and spinal cord, except for some regions that overlapped with CAPS1. These CAPS2 cellular distribution patterns had the marked feature of coinciding with those of BDNF in various brain regions. Immunolabels for CAPS2 were also colocalized with those for some proteins related to exocytosis (VAMP and SNAP-25) and endocytosis (Dynamin I) in the cell soma and processes of the mesencephalic tegmentum and cerebellum, suggesting that these proteins might be involved in the dynamics of CAPS2-associated vesicles, although their colocalization on vesicles remains elusive. These results demonstrate that the CAPS family proteins are involved in the secretion of different secretory substances in developing and postnatal brains, and that CAPS2 is probably involved in BDNF secretion in many brain areas. J. Comp. Neurol. 495:735–753, 2006. © 2006 Wiley-Liss, Inc.

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.

The recent identification of some of the components involved in regulated and constitutive exocytotic pathways has yielded important insights into the mechanisms of membrane trafficking and vesicle secretion. To understand precisely the molecular events taking place during vesicle exocytosis, we must identify all of the proteins implicated in these pathways. In this paper we describe the full-length cloning and characterization of human CADPS and CADPS2, two new homologs of the mouse Cadps protein involved in large dense-core vesicle (LDCV)-regulated exocytosis. We show that these two genes have disparate RNA expression patterns, with CADPS restricted to neural and endocrine tissues and CADPS2 expressed ubiquitously. We also identify a C2 domain, a known protein motif involved in calcium and phospholipid interactions, in both CADPS and CADPS2. We propose that CADPS functions as a calcium sensor in regulated exocytosis, whereas CADPS2 acts as a calcium sensor in constitutive vesicle trafficking and secretion. CADPS and CADPS2 were determined to span 475 kb and 561 kb on human chromosomes 3p21.1 and 7q31.3, respectively. The q31-q34 of human chromosome 7 has recently been identified to contain a putative susceptibility locus for autism (AUTS1). The function, expression profile, and location of CADPS2 make it a candidate gene for autism, and thus we conducted mutation screening for all 28 exons in 90 unrelated autistic individuals. We identified several nucleotide substitutions, including only one that would affect the amino acid sequence. No disease-specific variants were identified.

The regulated secretory pathway is activated by elevated cytoplasmic Ca 2+ ; however, the components mediating Ca 2+ regulation have not been identified. In semi-intact neuroendocrine cells, Ca 2+ -activated secretion is ATP- and cytosol protein-dependent. We have identified a novel brain protein, p145, as a cytosolic factor that reconstitutes Ca 2+ -activated secretion in two neuroendocrine cell types. The protein is a dimer of 145 kd subunits, exhibits Ca 2+ -dependent interaction with a hydrophobic matrix, and binds phospholipid vesicles, suggesting a membrane-associated function. A p145-specific antibody inhibits the reconstitution of Ca 2+ -activated secretion by cytosol, indicating an essential role for p145. The restricted expression of p145 in tissues exhibiting a regulated secretory pathway suggests a key role for this protein in the transduction of Ca 2+ signals into vectorial membrane fusion events.