DIVISION OF MOLECULAR NEUROBIOLOGY

Professor: NODA, Masaharu
Research Associates: SHINTANI, Takafumi
  SAKUTA, Hiraki
  HIYAMA, Takeshi
Technical Staff: TAKEUCHI, Yasushi
NIBB Research Fellow: TAKAHASHI, Hiroo
JSPS Postdoctoral Fellows: SUZUKI, Ryoko
  TAKAO, Motoharu
Postdoctoral Fellows: FUJIKAWA, Akihiro 1)
  NAKAMURA, Takahiro 1)
  YAMAMOTO, Yasunori 1)
  FUKADA, Masahide 1) (Apr. ’04 -)
Graduate Students: OHKAWARA, Takeshi (- Dec. ’04)
  IHARA, Masaru
  ETANI, Kazuma
  SHIMIZU, Hidetada
  TANAKA, Rumi (- Nov. ’04)
  YONEHARA, Keisuke
  NAKAMURA, Kayo (Apr. ’04 -)
Visiting Scientist: TAMURA, Hiroshi
Secretary: KODAMA, Akiko
Technical Assistant: KATAYAMA, Tomomi (Dec. ’04 -)
ST Technical Staffs: MIZOGUCHI, Masae
  GOTOH, Megumi
  YAMADA, Kaoru
  AYABE, Yuko
 
1 CREST, JST
 

We have been studying the molecular and cellular mechanisms underlying the development of the vertebrate central nervous system, mainly using the visual system. It covers all the major events including the patterning of the nervous system, neuronal differentiation, axonal navigation and targeting, synapse formation and plasticity, and neuronal regeneration. The scope of our interests also encompasses various functions of the matured brain, including sensation, behavior, learning and memory.

I. Regional specification in the retina

Topographic maps are a fundamental feature of neural networks in the nervous system. Understanding the molecular mechanisms by which topographically ordered neuronal connections are established during development has long been a major challenge in developmental neurobiology. The retinotectal projection of lower vertebrates including birds has been used as a readily accessible model system. In this projection, the temporal (posterior) retina is connected to the rostral (anterior) part of the contralateral optic tectum, the nasal (anterior) retina to the caudal (posterior) tectum, and likewise the dorsal and ventral retina to the ventral and dorsal tectum, respectively. Thus, images received by the retina are precisely projected onto the tectum in a reversed manner.

Regional specification along the nasotemporal and dorsoventral axes precedes the topographic retinotectal projection in the developing retina. To understand the molecular basis of topographic retinotectal projection, an overall view of the asymmetrically expressed molecules in the developing retinas is needed. Since 1992, we have been devoting our efforts to searching for molecules with asymmetrical distribution in the embryonic chick retina, and to characterization of their roles in the topographic retinotectal projection.

We performed a large-scale screening using restriction landmark cDNA scanning (RLCS) in the embryonic day 8 (E8) chick retina several years ago. RLCS is a cDNA display system, in which a large number of cDNA species are displayed as two-dimensional spots with intensities reflecting their expression levels as mRNA. We detected about 200 spots that gave different signal intensities between the nasal and temporal retinas or between the dorsal and ventral retinas. The asymmetric expression of each gene was verified by Northern blotting and in situ hybridization. By subsequent analyses using molecular cloning, DNA sequencing, and database searching, 33 asymmetric molecules along the nasotemporal (N-T) axis and 20 along the dorsoventral (D-V) axis were finally identified.

This year, we published the whole of results as one paper. These included transcription factors, secretory factors, transmembrane proteins, and intracellular proteins with various putative functions. Their expression profiles revealed by in situ hybridization are highly diverse and individual (Fig. 1). Moreover, many of them begin to be expressed in the retina from the early developmental stages, suggesting that they are implicated in the establishment and maintenance of regional specificity in the developing retina. We have already described on several molecules in published papers, but the study to know their hierarchical order to establish the regional specification in the retina is still in progress. The molecular repertoire revealed by this work will provide candidates for future studies to elucidate the molecular mechanisms of topographic retinotectal map formation.

Fig.1. In situ hybridization analysis of the expression of identified genes in E8 retina. Dig-labeled cRNA probes were hybridized to horizontal sections (Nasal and Temporal) or coronal sections (Dorsal,Ventral and Double). Horizontal sections are oriented with nasal up and temporal down. Coronal sections are oriented with dorsal up and ventral down. The double gradient expression of some genes at E8 was verified in (Double). Scale bars: 500 μm.

II. Topographic retinotectal projection

In the chick embryos, the first retinal ganglion cells become postmitotic at day E2 and their axons leave the retina at E3. The earliest axons arrive at the most anterior part of the tectum at day E6 and advance over its surface in the posterior direction. These retinal axons form the stratum opticum (SO), which covers the entire tectum at E12. At the onset the chick retinotectal projection has a general topographic order, and axonal sprouting begin predominantly at the vicinity and towards the site of normal terminal zone. In all vertebrates studied, however, the developing axons make trial branches, many of which are not entirely within the retinotopic area, and elimination of ectopic branches and elaboration of appropriate branches is shaped in an activity dependent manner. We have already identified some molecules among the region-specific molecules in the retina that show abnormal targeting, branching or synaptic arborization when their expression was experimentally elevated or suppressed in vivo.

III. Physiological roles of protein tyrosine phosphatase receptor type Z (Ptprz)

Protein tyrosine phosphorylation plays crucial roles in various biological aspects including brain development and brain functions. PTPζ/RPTPβ/Ptprz, a nervous system-rich receptor-type PTP, is expressed as a chondroitin sulfate proteoglycan in the brain from the early developmental stage to the adulthood in neurons as well as astrocytes. This suggests that this molecule plays variegated roles in the brain development and brain function.

Ptprz binds various cell adhesion molecules (Nr-CAM, L1, contactin, NCAM and TAG-1) and extracellular matrix molecules (tenascin-C/R). We revealed that Ptprz binds pleiotrophin (PTN)/HB-GAM and midkine (MK), closely related heparin-binding growth factors which share many biological activities. The chondroitin sulfate portion of Ptprz is essential for the high affinity binding to these growth factors.

It is well known that the vacuolating cytotoxin VacA produced by Helicobacter pylori causes massive cellular vacuolation in vitro and gastric tissue damage in vivo, leading to gastric ulcers, when administered intragastrically. In 2002, we found that Ptprz expressed in the gastric epithelial cells functions as a receptor for VacA: Mice deficient in Ptprz do not show mucosal damage by VacA, indicating that erroneous Ptprz signaling induces gastric ulcers.

Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We published a genetic method to screen for PTP substrates which we named the “yeast substrate-trapping system”. This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait (Fig. 2). This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. By using this method, we successfully identified several substrate molecules, together with many interacting molecules for Ptprz. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.

Fig. 2. Bait and prey vectors and tyrosine phosphorylation by the PTK induction in the yeast. (A,a) Structure of the pBridgeLexA/v-src vector. The PTP substrate-trap mutant is inserted into the multiple cloning site (MCS) in-frame with LexA. (A,b) DNA sequence of the MCS region in the vector. Unique restriction sites are shown in bold. The SmaI and PstI sites are not shown in bold, because both sites exist in the v-src sequence. (B) Structure of the pACT2 vector (CLONTECH). (C) Cellular proteins in the yeast are highly tyrosine phosphorylated when v-src is induced in the absence of methionine (-Met), while almost no tyrosine phosphorylation was observed in the presence of methionine (+Met). L40 cells containing pBridgeLexA/v-src were cultured in the medium both with and without 1 mM methionine for 24 h, and the tyrosine phosphorylation of cellular proteins was analyzed by Western blotting with anti-phosphotyrosine 4G10 antibody.

IV. Na+-level sensing in the brain

Dehydration causes an increase in the sodium (Na) concentration and osmolarity of body fluid in mammals. For Na homeostasis of the body, controls of Na and water intake and excretion are of prime importance. However, the system for sensing the Na level within the brain that is responsible for the control of Na- and water-intake behavior remains to be elucidated. We reported previously that the Nax channel is preferentially expressed in the circumventricular organs (CVOs) in the brain and that Nax knock-out mice ingest saline in excess under dehydrated conditions. Subsequently, we demonstrated that Nax is a Na-level-sensitive Na channel.

We revealed this year that the subfornical organ (SFO) is the principal site for the control of salt-intake behavior, where the Nax channel is the Na-level sensor. Infusion of a hypertonic Na solution into the cerebral ventricle induced extensive water intake and aversion to saline in wild-type animals but not in the knock-out mice. Importantly, the aversion to salt was not induced by the infusion of a hyperosmotic mannitol solution with physiological Na concentration in either genotype of mice. When Nax cDNA was introduced into the brain of the knock-out mice with an adenoviral expression vector, only animals that received a transduction of the Nax gene into the SFO among the CVOs recovered salt-avoiding behavior under dehydrated conditions. These results clearly show that the SFO is the center of the control of salt-intake behavior in the brain, where the Na-level-sensitive Nax channel is involved in sensing the physiological increase in the Na level of body fluids.

Fig. 3. Abnormal salt-intake behavior of Nax knock-out mice was rescued by introduction of the Nax gene to SFO. The coronal sections of the brain showing the loci infected by the expression of EGFP (left column). Time course of water and saline (0.3 M NaCl) intake by the infected mice before and after 48 hr dehydration (middle and right columns, respectively). Behavioral data are the average of six mice that were successfully infected in a specific site in the brain by an adenoviral vector encoding egfp (EGFP) or by vectors encoding Nax and egfp (Nax and EGFP).

Publication List:

Nakayama, M., Kimura, M., Wada, A., Yahiro, K., Ogushi, K., Niidome, T., Fujikawa, A., Shirasaka, D., Aoyama, N., Kurazono, H., Noda, M., Moss, J. and Hirayama, T. (2004) Helicobacter pylori VacA activates the p38/activating transcription factor 2-mediated signal pathway in AZ-521 cells. J. Biol. Chem. 279, 7024-7028.

Ohyama, K., Ikeda, E., Kawamura, K., Maeda, N. and Noda, M. (2004) Receptor-like protein tyrosine phosphatase ζ/RPTPβ is expressed on tangentially aligned neurons in early mouse neocortex. Develop. Brain Res., 148, 121-127.

Shintani, T., Kato, A., Yuasa-Kawada, J., Sakuta, H., Takahashi, M., Suzuki, R., Ohkawara, T., Takahashi, H. and Noda, M. (2004) Large-scale identification and characterization of genes with asymmetric expression patterns in the developing chick retina. J. Neurobiol., 59, 34-47.

Ohkawara, T., Shintani, T., Saegusa, C., Yuasa-Kawada, J., Takahashi, M. and Noda, M. (2004) A novel basic helix-loop-helix (bHLH) transcriptional repressor, NeuroAB, expressed in bipolar and amacrine cells in the chick retina. Mol. Brain Res. 128, 58-74.

Hiyama, T. Y., Watanabe, E., Okado, H. and Noda, M. (2004) The subfornical organ is the primary locus of sodium-level sensing by Nax sodium channels for the control of salt-intake behavior. J. Neurosci., 24, 9276-9281.

Kiyosue, K., Hiyama, T. Y., Nakayama, K., Kasai, M. and Taguchi, T. (2004) Re-expression of NR2B-containing NMDA receptors in vitro by suppression of neuronal activity. Int. J. Dev. Neurosci., 22, 59-65.

Muramatsu, H., Zou, P., Suzuki, H., Oda, Y., Chen, G-Y., Sakaguchi, N., Sakuma, S., Maeda, N., Noda, M., Takada, Y. and Muramatsu, T. (2004) α4β1- and α6β1-integrins are functional receptors for midkine, a heparin-binding growth factor. J. Cell Sci., 117, 5405-5415.

Fukada, M., Kawachi, H., Fujikawa, A. and Noda, M. (2005) Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z. Methods, 35, 54-63.

Niisato, K., Fujikawa, A., Komai, S., Shintani, T., Watanabe, E., Sakaguchi, G., Katsuura, G., Manabe, T. and Noda, M. (2005) Age-dependent enhancement of hippocampal LTP and impairment of spatial learning through the ROCK pathway in protein tyrosine phosphatase receptor type Z-deficient mice. J. Neurosci., in press.