Research Progress : Laboratory for Molecular neurogenesis RIKEN-BSI

1. Systematization of gene expression responsible for cerebellar development
　　Cerebellar Development Transcriptome Database (CDT-DB) project
(A. Sato*, C. Saruta, H. Nishibe, N. Morita**, Y. Sekine, Y. Sato, T. Sadakata, Y. Shinoda, K. Hayashi, T. Furuichi) * Present address: RIKEN-GSC, **Mimasaka Univ.


Sato et al (2008) Neural Networks 21:1056-1069. Experiments to obtain spatiotemporal gene expression data

	Fluorescent Diffrential Display (FDD): Analysis of differentail RNA expression patterns in mouse cerebella at E18, P0, P3, P7, P12, P15, P21 and P56. Developmental GeneChip: Analysis of developmental time series gene expression patterns in mouse cerebella at E18, P7, P14, P21 and P56. in situ hybridization (ISH): Analysis of spatial cellular mRNA expression patterns in mouse brains at P7 and P21. Tissue GeneChip: Analysis of tissue distribution patterns of gene expression in mouse tissues (brain, thymus, lung, heart, liver, spleen, kidney and testis) at P7 and P21.


	http://www.cdtdb.brain.riken.jp

We recently developed the Cerebellar Development Transcriptome Databse (CDT-DB), an open version of our database and publicized it through the internet on Dec. 16, 2006. The CDT-DB contains various lines of gene expression profile information (ISH brain images, RT-PCR gel images, microarray and GeneChip plots, etc.) with the functions for keyword search and links to relevant public database sites. The latest version, CDT-DB ver.4.1, was released on June 5, 2009. Our database project is supported by the Neuroinfomatics Japan Center (NIJC).


1. CDT-DB Top page; 2. Search menu page

3. Temporal, developmetal stage specific expression information page (data obtained by RT-PCR, GeneChip, & CDT array-custom made microarray analyses).


4. Spatial, cellular expression information page (ISH data). Three image viewers are provided (three panel viewer, detailed viewer and simple zoom). 5. Detailed viewer: ISH image in the view frame can be magnified and can be moved to any brain areas. Color image mode of the view frame can be converted into grayscale mode or inverted mode.

6. Tissue distribution page (RT-PCR & GeneChip data).

7. Gene Ontology search (search for GO terms; e.g., genes related to denrites, spines, plasticity, etc.).

8. Gene category graph (graph tool to analyze expression patterns of genes categorized based on their functions).

9. Gene information page with hyperlinks to many relevant bioinfomatics websites.

2. Functional analysis of brain development genes
　　Molecular mechanisms of synapse and circuit development
(T. Sadakata, Y. Shinoda, K. Hayashi, R. Katoh-Semba, J. Huang+, Y. Sato, A. Furuya, Y. Sekine, E. Kinameri, S. Motohashi, Y. Shiraishi*, F. Yoshikawa**, S. Shoji#, N. Morita##, T. Furuichi) Present address: *Nagasaki Univ, **Saitama City Hospital, #Hokubu Central Hospital, ##Mimosaka Univ, +Taiho Pharmaceutical Co.

(Y. Shinoda, K. Hayashi, Y. Shiraishi, S. Shoji, Y. Muto)
　　A scaffold protein for postsynaptic density proteins (mGluR1/5, IP3R, shank, etc.)

We isolated Cupidin (clone 13-2) by FDD analysis as a transiently-expressed gene in granule cells during the first-second postnatal weeks when various cytogenetic and morphogenetic events are actively occuring. Cupidin turned out to be identical to an isoform (Homer2/Vesl-2) of the Homer/Vesl family that interacts with many postsynaptic proteins.

The Homer gene family consists of three distinct genes. There are two major alternative-splicing variants, long-Homer and short-Homer. Long-Homer protein (Homer1b/c, Cupidin/Homer2a/b, & Homer3a/b) is composed of two functional domains, the N-terminal target binding EVH-1 domain and the C-terminal self-assembly domain containing coiled-coil/Leu zipper motifs. Long-Homers probably form a physical and functional link (or cluster) of various target proteins with a PPxxF motif, including the proteins involved in two glutamate-induced calcium signaling pathways, the "calcium-release pathway" and the "calcium-influx pathway", by binding mGluR1a/5 and IP3R in the former and by binding Shank that binds to the NMDAR complex in the latter. We found that Cupidin/Homer2 also binds two actin-cytoskeleton-related proteins, Drebrin (a dendritic actin-binding protein) and Cdc42 (a Rho small GTPase). On the other hand, short-Homer (Homer1a/Vesl-1s, & ania-3) is activity-dependently expressed and has only the N-terminal domain, suggesting that short-Homer acts as a natural dominant-negative to break a long-Homer link (or cluster) in response to neural activity. In addition to short-Homer associated declustering, we found the declustering of Cupidin/Homer2, which was triggered by the calcium influx via NMDAR activated by glutamate. The molecular mechanism of glutamate-induced Cupidin/Homer2 declustering remains elusive.



		Cupidin/Homer2 inhibits Cdc42-induced microspike formation in HeLa cells
Homer1 and Homer2 are expressed in granule cells, whereas Homer3 is restricted to Purkinje cells.

			Punctate distribution of Cupidin/Homer2 at dendritic spines of cultured hippocampal neurons.

Differential localization of Cupidin/Homer2 and Homer3 at postsynapses of granule cells (A, B, D) and Purkinje cells (C), respectively.
	Overexpressed GFP-Cupidin/Homer2 is accumulated in spine heads of cultured hippocampal neurons.

We suggest that Cupidin has two states, clustering and declustering, in response to synaptic activity and acts as a "mobile adaptor protein" in developing excitatory postsynapses, regulating molecular organization of postsynaptic receptors (e.g., regulation of glutamate-activated calcium signaling or receptor targeting) and actin-cytoskeletal reorganization (e.g., remodeling of postsynapse morphology). We also revealed a coincidence of the timing of clustering and synaptic-targeting between Cupidin and NMDA receptor complex such as NMDAR2B and PSD95 during development of hippocampal neurons.

(T. Sadakata, Y. Shinoda, R. Katoh-Semba, Y. Sekine, E. Kinameri)
　　A secretory vesicle associated protein that is involved in BDNF and NT-3 release

The second clone CAPS2 (Ca²⁺-dependent activator protein for secretion 2) is another transient gene with an expression peak around P12. Its mRNA was densely localized in the post-mitotic external germinal layer (EGL) and the internal granular layer (IGL) granule cells, while its protein was predominantly localized in the molecular layer (ML), indicating that its localization was restricted in granule cell axons (parallel fibers).

Structure of CAPS2. CAPS2 has about 70% amino-acid identity with CAPS1 which was shown to regulate Ca²⁺-triggered dense-core vesicle exocytosis at the ATP-dependent primint step by binding both PIP2 and vesicle membrane (Grichanin et al., Neuron 43:551, 2004).

Sadakata, T., et al. (2004) J. Neurosci. 24:43-52.
Sadakata, T., et al. (2006) J. Comp. Neurol. 495:735-753.
Sato, Y., et al. (2004) J. Neurosci. Methods 137:111-121 (adenovirus vector-mediated gene delivery to cerebellar cells).
Sadakata et al., (2007) J. Histochem Cytochem 55:301-311.
Sadakata et al., (2007) J. Neurosci. 27:2472-2482.
Sadakata and Furuichi (2008) Cerebellum in press.

CAPS2 Ca²⁺-dependent activator protein for secretion 2


CAPS2 mRNA shows a differential expression pattern with a peak in the postnatal first-second weeks in cerebellar granule cells, whereas CAPS2 protein is densely localized in the molecular layer and is concentrated as punctate patterns around dendrites of Purkinje cells in cerebellar primary cell cultures. These results indicate that CAPS2 proteins are specifically localized at presynaptic sites of paralle fibers, axons of granule cells, connecting postsynaptic Purkinje cell dendrites.	The immuno-electron microscopic study showed that CAPS2 immunogolds are localized at vesicular structures and extra-synaptic membrane of parallel fiber terminals connecting with postsynaptic Purkinje cell spines. Biochemical analyses indicated that CAPS2 is associated with vesicles containing BDNF (brain-derived neurotrophic factor) and NT-3 (neurotrophin-3), but not synaptic vesicle marker proteins.

Overexpression of CAPS2 in granule cells by using adenovirus vectors (AdV) enhanced high KCl-induced NT-3 release and promotes survival of Purkinje cells in primary cerebellar cultures. These results indicate that CAPS2 is the molecule involved in activity-dependent neurotrophin release and may play an important role in neuronal differentiation and survival.
	CAPS2 proteins are also differentially localized in various brain areas including cerebrum and hippocampus. Interestingly, CAPS2 immunoreactivity is largely colocalized with BDNF immunoreactivity in many areas. These results suggest that CAPS2 plays a role in release of neurotrophins (and possibly other secretory substances) over the mouse brain regions, although which step(s) in the secretory pathway (biosynthesis, trafficking, and exocytosis) are directly regulated by CAPS2 awaites further investigation.

(K. Hayashi, J. Huang, A. Furuya)
　　A docking protein that links Src family tyrosine kinase signaling molecules

We found that p130Cas (Crk associated substrate) is highly expressed in developing mouse cerebellum. Interestingly, tyrosine-phosphorylated Cas (YP-Cas) is enriched in growth cones of cerebellar granule cells and Purkinje cells. YP-Cas interacts with Crk, Src family tyrosine kinases, NCAM, N-cadherin, etc. in the cerebellum. Dominant negative and RNAi experiments revealed that p130Cas is involved in neurite extension of cerebellar neurons.

	In fibroblasts (left), Cas acts as an adaptor for various cell signaling proteins including Integrin-Src family kinases-actin reorganizing molecules and plays a role in cell migration and morphology. In cerebellar granule cells (right), Cas is associated with cell surface molecules, Src family kinases (SFKs), Crk, and JNK, and is involved in neurite elongation and possibly cell migration during the postnatal development. Huang, JH., et al. (2006) Mol. Biol. Cell 17:3187-3196.

p130Cas protein level is up-regulated during postnatal development of mouse cerebellum. Interestingly, tyrosine-phosphrylated Cas (YP-Cas), however, shows a transiently-regulated pattern with a peak at P7. YP-Cas is predominantly localized in the post-mitotic inner EGL and in distal part of Purkinje cell dendrites at early stage.

YP-Cas is fractionated into the growth cone fraction and is immunocytochemically co-localized with F-actin and Crk in growth cones of cultured granule cells.

Dominant negative (deletion of substrate domain [SD]) and RNAi experiments revealed that p130Cas is involved in neurite extension of cultured granule cells.

(K. Hayashi, J. Huang, A. Furuya, Y. Shinoda)
　　A brain specific, two KIND domain containing RasGEF (Ras guanine nucleotide exchange factor)

v-KIND is a brain specific, two-KIND domain-containing RasGEF. We have recently demonstrated that v-KIND controls dendrite growth of both cerebellar granule cells and hippocampal neurons.

The v-KIND mRNA is up-regulated during the mouse cerebellar development and is predominantly expressed in the brain. The mRNA is predominantly localized in cerebellar granule cells and is also localized at lower levels in other areas of P21 mouse brains. The protein is concentrated in glomeruli with which granule cell dendrites are extended to form excitatory synapses with mossy fiber terminals and inhibitory synapses with Golgi cell axons.

Huang, J., et al. (2007) J. Cell Biol. 179:539-552.

We are also studying the function and expression of two phosphorylation-related proteins, AATYK (apoptosis-activated protein tyrosine kinase; Tomomura et al., 2003, 2005, 2007) and Ebr kinase (embryonic brain related protein kinase; Matsuki et al., 2005), and two novel oligodendrocyte-related proteins, Opalin (a transmembrane sialylglycoprotein lcocalized in CNS myelin paranodal loop membrane; Aruga et al., 2007; Yoshikawa et al., 2008) and FD0130 (a differentiating OLG specific protein), in developing brains.

Oplain is a sialylglyco-transmembrane protein that is localized in paranodal loop membrane of the central myelin and is specific to mammals. Opalin immunoreactivity is observed in myelin wrapping axons of Purkinje cells in the white matter of cerebellar cortex.

As part of this project to understand the molecular mechanism underlying the longitudinal compartmentalization of cerebellar cortex, we studied the gene expression restricted to parasagittal zones (or stripes) of Purkinje cell subpopulation. We generated a transgenic mouse 1NM13 line that showed a characteristic zonal expression pattern of the lacZ transgene in parasagittally-oriented subsets of developing Purkinje cells (i.e., lacZ-positive and -negative Purkinje cell zones). By using the 1NM13 mice, we analyzed generation of lacZ-positive Purkinje cell population. We showed that migrating and post-migrating Purkinje cells weakly expressed lacZ as early as embryonic day 14.5, and that the lacZ-positive and -negative zones were already distinguishable at embryonic day 15.5. The boundary between the positive and negative zones could be visible just over a stack with a few cells close to the ventricular zone.

Differential expression of the lacZ transgene in subsets of developing Purkinje cells


		Left two panels, the zonal expression pattern of the lacZ transgene in parasagittally-oriented subsets of developing Purkinje cells (Top, X-gal staining of whole cerebellum; bottom, X-gal staining of the coronal section) ; right two panels, the differential expression in Purkinje cell subsets between 1NM13-lacZ genes (X-gal staining, blue color) and zebrin II gene (immunological HRP-DAB staining, brown color). (Collaboration with Dr. R. Hawks, Calgary)

There were only some Purkinje cells positive to both lacZ and zebrin II, but the overall pattern of zone distribution was different between them. These data suggested that heterogeneous subsets of Purkinje cells having different properties in transcriptional regulation are already generated by cellular stages after born at, or beginning to migrate from the ventricular zone.We are continuing to analyze the zonal lacZ expression at embryonic developmental stages. By microdissecting the positive and negative Purkinje cells and comparing their differential gene expression, we will search whether there is any other molecular heterogeneity between them. We have also begun to study on gene expression in forming the anatomical division sand compartments (e.g., vermis, hemispheres, flocculi, fissures, lobules, folia, zonal projections) of cerebellum.

3. Disturbed CAPS2-mediated secretion pathway and autism susceptibility

Deficits in neuronal development and impaired social behaviors in CAPS2 knockout mice

CAPS2 KO mice and autism

(T. Sadakata, Y. Shinoda, R. Katoh-Semba, Y. Sato, E. Kinameri, S. Motohashi)

We recently showed that CAPS2 knockout mice have impairments in BDNF release activity and also exhibit autistic-like cellular and behavioral phenotypes.
1) Cerebellar deficits: decrease in NT-3 release and Purkinje cell number in cerebellar cultures, increased granule cell apoptosis, impaired dendritic arborization of Purkinje cells, aberrant morphology of lobules VI-VII, impaired structure and plasticity of parallel fiber-Purkinje synapses, impaired eye movement adaptation.


1) CAPS2 KO mice show delayed development of CGCs, increased apoptosis of EGL cells, decreased dendrite growth of PCs, and hypoplasia of vermal region. In addition, PF-PC synapses have aberrant morphology and reduced paired-pulse facilitation, which are especially marked in cerebellar vermis (lobules VI-VII). Sadakata, T., et al. (2007) J. Neurosci. 27:2472-2482.

2) Cortical and hippocampal deficits: decreased BDNF release in cortical cell cultures, decreased number of parvalbumin-positive interneurons in neocortex and hippocampus. Sadakata, T., et al. (2007) J. Clinical Investigation 117:931-943.

	3) Imapired behavioral phenotypes: decreased reciprocal interactions, hyperactivity, impaired response to a novel enviroment, aberrant circadian rhythm, impaired maternal behavior, impaired Morris water-maze spatial learning, etc. Sadakata, T., et al. (2007) J. Clinical Investigation 117:931-943.


These phenotypes of CAPS2 KO mice are reminiscent of those charaterized in patients with autism, a neurodevelopmental disorder. Sadakata, T., et al. (2007) J. Clinical Investigation 117:931-943.	The human CAPS2 gene is located within the autism susceptibility locus 1 (AUTS1) of chromosome 7.

Moreover, we demonstrated that some of autistic patients express an increased amount of a rare splicing variant of CAPS2 mRNA that specifically lacks exon 3. CAPS2-delta exon3 retains BDNF release activity but fails to bind dynactin1, resulting in the impairment in axonal distribution in cultured cortical and cerebellar neurons, suggesting a deficit in local presynaptic secretion of BDNF. BDNF released from presynaptic terminals plays an indispensable role in synaptic plasticity and connectivity.
	We also identified seven nonsynonymous SNPs within the CAPS2 genome of autistic patients.

In conclusion, CAPS2 likely regulates local BDNF secretion. We suggest that impaired CAPS2 function (deficits in local BDNF release activity, altered copy number, or other mutations such as SNPs) may contribute to autism susceptibility.


Aruga, J., et al. (2007) J. Neurochem. 102:1533-1547 Yoshikawa, F., et al. (2008) J. Biol. Chem. 283:20830-20840