Yukiko Sekine

Autistic-like phenotypes in Cadps2-knockout mice and aberrant CADPS2 splicing in autistic patients

Autism, characterized by profound impairment in social interactions and communicative skills, is the most common neurodevelopmental disorder, and its underlying molecular mechanisms remain unknown. Ca(2+)-dependent activator protein for secretion 2 (CADPS2; also known as CAPS2) mediates the exocytosis of dense-core vesicles, and the human CADPS2 is located within the autism susceptibility locus 1 on chromosome 7q. Here we show that Cadps2-knockout mice not only have impaired brain-derived neurotrophic factor release but also show autistic-like cellular and behavioral phenotypes. Moreover, we found an aberrant alternatively spliced CADPS2 mRNA that lacks exon 3 in some autistic patients. Exon 3 was shown to encode the dynactin 1-binding domain and affect axonal CADPS2 protein distribution. Our results suggest that a disturbance in CADPS2-mediated neurotrophin release contributes to autism susceptibility.

Impaired cerebellar development and function in mice lacking CAPS2, a protein involved in neurotrophin release

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 that CAPS2 knock-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 the CAPS2 gene in the cerebellar deficits of autistic patients is discussed.

2008 Special Issue: Cerebellar development transcriptome database (CDT-DB): Profiling of spatio-temporal gene expression during the postnatal development of mouse cerebellum

A large amount of genetic information is devoted to brain development and functioning. The neural circuit of the mouse cerebellum develops through a series of cellular and morphological events (including neuronal proliferation and migration, axogenesis, dendritogenesis, synaptogenesis and myelination) all within three weeks of birth. All of these events are controlled by specific gene groups, whose temporal and spatial expression profiles must be encoded in the genome. To understand the genetic basis underlying cerebellar circuit development, we analyzed gene expression (transcriptome) during the developmental stages on a genome-wide basis. Spatio-temporal gene expression data were collected using in situ hybridization for spatial (cellular and regional) resolution and fluorescence differential display, GeneChip, microarray and RT-PCR for temporal (developmental time series) resolution, and were annotated using Gene Ontology (controlled terminology for genes and gene products) and anatomical context (cerebellar cell types and circuit structures). The annotated experimental data were integrated into a knowledge resource database, the Cerebellar Development Transcriptome Database (CDT-DB http://www.cdtdb.brain.riken.jp), with seamless links to the relevant information at various bioinformatics database websites. The CDT-DB not only provides a unique informatics tool for mining both spatial and temporal pattern information on gene expression in developing mouse brains, but also opens up opportunities to elucidate the transcriptome for cerebellar development.

Differential distributions of the Ca2+-dependent activator protein for secretion family proteins (CAPS2 and CAPS1) in the mouse brain

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.

Reduced axonal localization of a Caps2 splice variant impairs axonal release of BDNF and causes autistic-like behavior in mice

Ca2+-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.

Opalin, a Transmembrane Sialylglycoprotein Located in the Central Nervous System Myelin Paranodal Loop Membrane

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1–30), a transmembrane domain (residues 31–53), and a long C-terminal intracellular domain (residues 54–143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.

Expression of the IP3R1 promoter‐driven nls‐lacZ transgene in Purkinje cell parasagittal arrays of developing mouse cerebellum

The cerebellar Purkinje cell monolayer is organized into heterogeneous Purkinje cell compartments that have different molecular compositions. Here we describe a transgenic mouse line, 1NM13, that shows heterogeneous transgene expression in parasagittal Purkinje cell arrays. The transgene consists of a nuclear localization signal (nls) fused to the β‐galactosidase (lacZ) composite gene driven by the type 1 inositol 1,4,5‐trisphosphate receptor (IP3R1) gene promoter. IP3R1‐nls‐lacZ transgene expression was detected at a single Purkinje cell level over the surface of a whole‐mount X‐gal‐stained cerebellum because of nuclear accumulation of the nls‐lacZ activity. Developing cerebella of 1NM13 mice showed stripe‐like X‐gal staining patterns of parasagittal Purkinje cell subsets. The X‐gal stripe pattern was likely determined by an intrinsic property as early as E15 and showed increasing complexity with cerebellar development. The X‐gal stripe pattern was reminiscent of, but not identical to, the stripe pattern of zebrin II immunoreactivity. We designated the symmetrical X‐gal‐positive (transgene‐positive, Tg+) Purkinje cell stripes about the midline as vermal Tg1+, Tg2(a, b)+ and Tg3(a, b)+ stripes and hemispheric Tg4(a, b)+, Tg5(a, b)+, Tg6(a, b, c)+, and Tg7(a, b)+ stripes, where a, b, and c indicate substripes. We also assigned three parafloccular substripes Tg8(a, b, c)+. The boundaries of X‐gal stripes at P5 were consistent with raphes in the Purkinje cell layer through which granule cells migrate, suggesting a possible association of the X‐gal stripes with raphe formation. Our results indicate that 1NM13 is a good mouse model with a reproducible and clear marker for the compartmentalization of Purkinje cell arrays.

Interaction of Calcium-dependent Activator Protein for Secretion 1 (CAPS1) with the Class II ADP-ribosylation Factor Small GTPases Is Required for Dense-core Vesicle Trafficking in the trans-Golgi Network

Ca2+-dependent activator protein for secretion (CAPS) regulates exocytosis of catecholamine- or neuropeptide-containing dense-core vesicles (DCVs) at secretion sites, such as nerve terminals. However, large amounts of CAPS protein are localized in the cell soma, and the role of somal CAPS protein remains unclear. The present study shows that somal CAPS1 plays an important role in DCV trafficking in the trans-Golgi network. The anti-CAPS1 antibody appeared to pull down membrane fractions, including many Golgi-associated proteins, such as ADP-ribosylation factor (ARF) small GTPases. Biochemical analyses of the protein-protein interaction showed that CAPS1 interacted specifically with the class II ARF4/ARF5, but not with other classes of ARFs, via the pleckstrin homology domain in a GDP-bound ARF form-specific manner. The pleckstrin homology domain of CAPS1 showed high affinity for the Golgi membrane, thereby recruiting ARF4/ARF5 to the Golgi complex. Knockdown of either CAPS1 or ARF4/ARF5 expression caused accumulation of chromogranin, a DCV marker protein, in the Golgi, thereby reducing its DCV secretion. In addition, the overexpression of CAPS1 binding-deficient ARF5 mutants induced aberrant chromogranin accumulation in the Golgi and consequently reduced its DCV secretion. These findings implicate a functional role for CAPS1 protein in the soma, a major subcellular localization site of CAPS1 in many cell types, in regulating DCV trafficking in the trans-Golgi network; this activity occurs via protein-protein interaction with ARF4/ARF5 in a GDP-dependent manner.

Calcium‐dependent activator protein for secretion 2 interacts with the class II ARF small GTPases and regulates dense‐core vesicle trafficking

The Ca2+‐dependent activator protein for secretion (CAPS) family consists of two members (CAPS1 and CAPS2) and regulates the exocytosis of catecholamine‐containing or neuropeptide‐containing dense‐core vesicles (DCVs) at secretion sites such as nerve terminals. A large fraction of CAPS1, however, is localized in the cell soma, and we have recently shown the possible involvement of somal CAPS1 in DCV trafficking in the trans‐Golgi network. CAPS1 and CAPS2 are differentially expressed in various regions of the mouse brain but exhibit similar expression patterns in other tissues, such as the spleen. Thus, in the present study we analyzed whether CAPS2 displays similar subcellular localization and functional roles in the cell soma as CAPS1. We found that somal CAPS2 is associated with the Golgi membrane, and mediates binding and recruitment of the GDP‐bound form of ARF4 and ARF5 (members of the membrane‐trafficking small GTPase family) to the Golgi membrane. CAPS2 knockdown and overexpression of CAPS2‐binding‐deficient ARF4/ARF5 both induced accumulation of the DCV resident protein chromogranin A around the Golgi apparatus. CAPS2 knockout mice have dilated trans‐Golgi structures when viewed by electron microscopy. These results for CAPS2 strongly support our idea that the CAPS family proteins exert dual roles in DCV trafficking, mediating trafficking at both the secretion site for exocytosis and at the Golgi complex for biogenesis.

CAPS1 Deficiency Perturbs Dense-Core Vesicle Trafficking and Golgi Structure and Reduces Presynaptic Release Probability in the Mouse Brain

Ca2+-dependent activator protein for secretion 1 (CAPS1) plays a regulatory role in the dense-core vesicle (DCV) exocytosis pathway, but its functions at the cellular and synaptic levels in the brain are essentially unknown because of neonatal death soon after birth in Caps1 knock-out mice. To clarify the functions of the protein in the brain, we generated two conditional knock-out (cKO) mouse lines: 1) one lacking Caps1 in the forebrain; and 2) the other lacking Caps1 in the cerebellum. Both cKO mouse lines were born normally and grew to adulthood, although they showed subcellular and synaptic abnormalities. Forebrain-specific Caps1 cKO mice showed reduced immunoreactivity for the DCV marker secretogranin II (SgII) and the trans-Golgi network (TGN) marker syntaxin 6, a reduced number of presynaptic DCVs, and dilated trans-Golgi cisternae in the CA3 region. Cerebellum-specific Caps1 cKO mice had decreased immunoreactivity for SgII and brain-derived neurotrophic factor (BDNF) along the climbing fibers. At climbing fiber–Purkinje cell synapses, the number of DCVs was markedly lower and the number of synaptic vesicles was also reduced. Correspondingly, the mean amplitude of EPSCs was decreased, whereas paired-pulse depression was significantly increased. Our results suggest that loss of CAPS1 disrupts the TGN–DCV pathway, which possibly impairs synaptic transmission by reducing the presynaptic release probability.