The analysis of the role of Disc1 in the hippocampal layer formation in vivo

Abstracts / Neuroscience Research 71S (2011) e108–e415

Research fund: KAKENHI(21500330), Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects. doi:10.1016/j.neures.2011.07.1005

P3-e11 Intramolecular regulation of Dab1 protein function Satoshi Kikkawa Toshio Terashima

, Yoshimi Takahashi, Tomohiro Namikawa,

Div of Dev Neurobiol, Kobe Univ Grad Sch of Med, Kobe, Japan

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P3-e13 Functional balance between a microtubuleassociated protein RP1 and ciliary kinase Mak regulates ciliary length and survival of retinal photoreceptor cells Taro Chaya 1,2,3 , Yoshihiro Omori 1,2,4 , Kimiko Katoh 1 , Takahisa Furukawa 1,2 1

Dept Dev Biol, Osaka Biosci Inst, Suita, Osaka, Japan 2 JST, CREST, Japan 3 Dept Mol Dev, Grad Sch Med, Kyoto Univ, Kyoto, Japan 4 JST, PRESTO, Japan

In mammalians, the Reelin signal pathway plays important roles to coordinate neuronal migration during the brain development. When Reelin binds to its receptors, ApoER2 and VLDLR, the intracellular adaptor protein Disabled-1 (Dab1) is recruited to the NPXY domain of receptors and tyrosinephosphorylated by Src family kinases (SFKs). The downstream pathways seem to vary depending on brain areas, neuronal types, and/or developmental stages. We have previously identified two zebrafish orthologs of Dab1, Dab1a and Dab1b. These two isoforms showed spatially different expression patterns. When zebrafish Dab1s are overexpressed in CHO cells, the cells transfected with Dab1a or b alone showed a normal fusiform shape while the cells transfected with Dab1a/b and the Reelin receptors showed an abnormally round shape. Even when tyrosine phosphorylation by SFKs was blocked with the selective inhibitor PP2, the cells transfected with Dab1a/b and receptors showed the same round shape. We next transfected the CHO cells with various truncated-forms of Dab1a/b. The C-terminal polypeptides which lack all four putative tyrosine phosphorylation sites and the PTB domain failed to induce morphological change even when transfected with the receptors. In contrast, the N-terminal polypeptides containing the PTB domain induced morphological change in the expressed cells without co-expression of the receptors. Interestingly, when the N-terminal polypeptides were co-expressed with their complementary C-terminal polypeptides, the transfected cells showed the normal shape. These results suggest an intramolecular regulation mechanism between the N- and C-terminal domains of the Dab1 protein.

Cilia are evolutionally conserved microtubule-based organelles that extend from basal bodies and form on the apical surface of cells. In humans, ciliary dysfunction is associated with various diseases that can be broadly classified as “ciliopathies”, including polydactyly, obesity, polycystic kidney, and retinal degeneration. However, regulatory molecules which control protein modification to realize ciliary formation and maintenance have been unidentified so far. By analyzing retinal photoreceptor-enriched genes using Otx2-deficient mice, we identified that an uncharacterized kinase, male germ cell-associated kinase (Mak), is a down-stream gene of Otx2 and expressed specifically in photoreceptor cells in the retina. Mak was localized both in the connecting cilia and outer segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibited elongated cilia and progressive degeneration. We observed accumulation of IFT88 and IFT57, expansion of Kif3a and acetylated ␣-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at P14. In addition, overexpression of RP1, a microtubule-associated protein localized in outer segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induced both ciliary elongation and microtubule acetylation, and was phosphorylated by Mak. These results suggest that Mak is essential for regulation of ciliary length, ciliary protein transport, acetylation of ciliary microtubules, and the long-term survival of photoreceptors. To investigate the molecular function of Mak in the regulation of ciliary length and/or protein transport, we are in progress to identify Mak interacting proteins by proteomic analysis.

doi:10.1016/j.neures.2011.07.1006

doi:10.1016/j.neures.2011.07.1008

P3-e12 Expressions of tricellulin, claudin-19 and junctional adhesion molecule-C in myelinating mouse Schwann cells

P3-e14 Ninein is essential for the interkinetic nuclear migration of cortical progenitor cells anchoring the centrosome to microtubules

Shin Kikuchi 1 , Takafumi Ninomiya 1 , Takashi Kojima 2 , Haruyuki Tatsumi 1 1

Dept. of Anatomy, Sapporo Medical Univ. Sch. of Med., Sapporo, Japan 2 Dept. of Pathology, Sapporo Medical Univ. Sch. of Med., Sapporo, Japan Autotypic tight junctions, tight junctions connecting between same Schwann cell membranes, in mouse myelin are observed in noncompact myelin such as paranodes, Schmidt–Lanterman incisures and mesaxons. In noncompact myelin, some tight junctional (TJ) proteins are detected and thought to contribute to the integrity of myelin function. However, the role of TJ proteins in noncompact myelin is not clear. To determine in part the role of TJ proteins in noncompact myelin, we examined expressions of TJ proteins during development in mouse sciatic nerves and cultured dorsal root ganglion cells. Analyzed TJ proteins were Claudin-19 (Cldn19), junctional adhesion molecule C (JAM-C) and tricellulin (TRIC). Myelin protein zero (MPZ) was used as a marker of compact myelin. Sciatic nerves of the C57/BL mice at postnatal days (P)1, P3, P5, P7, P14, P21 and P35, and cultured dorsal root ganglion cells in 2 and 4 weeks of culture were carried out to analyze the TJ proteins in myelination by immunocytochemical staining or Western blot. In sciatic nerves, MPZ was detected at P3. Cldn19 and JAM-C were observed at P5 in paranode and TRIC was at P21. In the incisures, the expression of TRIC was observed earlier than Cldn19 or JAM-C. The expression or concentration patterns in noncompact myelin differed among the researched TJ proteins. In cultured Schwann cells, TRIC, Cldn19, JAM-C and MPZ were detected by immunostaining or Western blot in 4 weeks culture, but no they were detected in 2 weeks culture. TRIC was expressed only in mesaxon, not paranodes and the incisures. On the other hands, Cldn19 and JAM-C immunoreactivities existed at both paranodes and incisures like in vivo. TRIC was not concentrated in paranodal loops and Schmidt–Lanterman incisures under the culture condition. These results suggest that Cldn19 and JAMC play a structural maintaining role in myelination and TRIC has not only structural role but also functional one for maintaining the myelin sheath. doi:10.1016/j.neures.2011.07.1007

Hiroshi Shinohahra 1 , Nobuyuki Sakayori 2 , Masanori Takahashi 2 , Tatsunori Seki 1 , Noriko Osumi 2 1

Histology and Neuroanatomy, Tokyo Med Univ., Tokyo, Japan 2 Div. of Dev. Neurosci., Tohoku Univ. School of Med., Sendai, Japan The mammalian cerebral cortex develops from proliferative apical progenitor cells (APs) that exhibit a cell cycle-dependent nuclear movement (interkinetic nuclear migration; INM), which might be important for the efficient and continuous production of neurons. The Pax6 transcription factor plays a major role in APs by regulating various downstream molecules. We have previously observed abnormal INM and unstable localization of the centrosome in APs of the Pax6 homozygous mutant rat embryo. To understand the mechanisms of INM, we focused on the centrosome of APs. One of the centrosomal proteins, ninein, shows specific localization in the centrosome of APs. We observed a dramatic downregulation of ninein in APs of the Pax6 mutant. Moreover, knockdown of ninein by RNAi induced the ectopic distribution and the decreased number of BrdU-positive (S-phase) and PH3positive (M-phase) cells. Furthermore, by time-lapse imaging, knockdown of ninein in vivo induced abnormal INM. Finally, we observed impaired microtubule regrowth in neural progenitors taken from Pax6 homozygous mutant rat embryos, which was recovered by ninein overexpression. Our results suggest that ninein is part of the molecular machinery essential for INM by connecting microtubules to the centrosome. Research fund: Global COE Program from MEXT Japan. doi:10.1016/j.neures.2011.07.1009

P3-e15 The analysis of the role of Disc1 in the hippocampal layer formation in vivo Ken-ichiro Kubo , Kenji Tomita, Kazuhiro Ishii, Kazunori Nakajima Dep. of Anatomy, Keio Univ. Sch. of Med., Tokyo, Japan The hippocampus has a highly ordered structure and is composed of distinct layers. Neuronal migration is an essential part of the process of the layer formation because neurons are primarily generated near the ventricle and must

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Abstracts / Neuroscience Research 71S (2011) e108–e415

migrate to arrive at their final locations during brain development. Impairment of brain development is thought to underlie the etiology of psychiatric disorders. Consistent with this idea, many genetic risk factors for psychiatric disorders play critical roles during brain development. As one example, Disrupted-in-Schizophrenia-1 (DISC1) is a genetic risk factor for major psychiatric disorders and plays various roles during neurodevelopment. The DISC1 gene is disrupted by the chromosome 1 breakpoint of a balanced t(1;11) translocation in a Scottish family with a 47% prevalence of major mental illnesses including schizophrenia. Recent association studies also indicate that DISC1 has a role in psychiatric disorders in general populations. We have recently shown that Disc1 plays important roles in the development of the mouse cerebral cortex by utilizing the in utero gene transfer system. Here, we investigated the role of Disc1 in the layer formation of the mouse hippocampus. Disc1 suppression was found to impair migration of the hippocampal pyramidal neurons. Our findings indicate that Disc1 is required for migration and layer formation by the hippocampal pyramidal neurons during development. Research fund: Strategic Research Program for Brain Sciences, MEXT. doi:10.1016/j.neures.2011.07.1010

P3-e16 Proteolytic cleavage of Reelin within its C-terminal region regulates its ability to induce growth cone collapse Takao Kohno , Ayaka Tsuchiya, Saori Matsumaru, Mai Takayanagi, Mitsuharu Hattori Dept. of Biomed. Sci., Grad. Sch. of Pharm. Sci., Nagoya City Univ., Nagoya, Japan Reelin is a large secreted glycoprotein that regulates variety of events in mammalian brain, including neuronal migration, neuronal dendritic formation, and synaptic plasticity. However, the primary role of Reelin in the developing brain and its underlying molecular mechanism remain largely unknown. Reelin protein is composed of the N-terminal domain, Reelin repeats, and the highly basic C-terminal region (CTR). The primary sequence of CTR is highly conserved in most vertebrates, suggesting its important function. In this study, we found that Reelin is proteolytically cleaved within CTR by a certain protease of proprotein convertase family. This cleavage presumably occurs between Arg3455 and Ser3456 (CTR-site), and releases the peptide of six residues from full-length Reelin (3461 amino acids). We established monoclonal antibodies that specifically recognize Reelin with uncleaved CTR (the genuine full-length Reelin). By utilizing these monoclonal antibodies, we found that the CTR-site cleavage of Reelin is developmentally regulated in vivo. The CTR-site cleavage of Reelin does not affect its Dab1phosphorylating bioactivity. However, it modulates the interaction between Reelin and the neuronal cell membrane. We also found that Reelin with uncleaved CTR, but not with cleaved one, has the ability to induce growth cone collapse in dorsal root ganglion neurons. These results suggested that Reelin plays roles in neuronal axon guidance through specific proteolysis. We recently found a candidate receptor that binds to Reelin with uncleaved CTR and are now trying to clarify the physiological significance of its interaction. Research fund: KAKENHI(22890155). doi:10.1016/j.neures.2011.07.1011

P3-e17 Functional analysis of shootin2 in the formation and extension of the leading process of cultured inhibitory neurons derived from the ganglionic eminence Hirotaka S. Shibata , Kazuhiro Katsuta, Michinori Toriyama, Seinichi Kanemura, Kazuhiro Horinouchi, Naoyuki Inagaki Grad. Sch. of Biol. Sci., NAIST, Nara, Japan Most of the inhibitory neurons in the rodent forebrain are born in the ganglionic eminence (GE) and migrate to their destinations through defined streams during embryogenesis. The migratory routes, chemoattractants and intracellular regulators, involved in the migration of inhibitory neurons, have been studied extensively in the past years. Although the leading process (LP) is thought to play a key role in neuronal migration, it is unclear how the mechanical forces are generated in the LP for the movement of neurons. Recently, we reported that shootin1 mediated the linkage between actin retrograde flow and L1-CAM as a clutch molecule to promote axon outgrowth in cultured hippocampal neurons (Shimada et al., 2008). Here, we are focusing on shootin2 to analyze the formation and the outgrowth of the LP in migratory GE neurons. Shootin2, an alternative splice variant of shootin1, was mainly expressed in the GE and cerebral cortex during migration of inhibitory neurons, and was also concentrated in the LP of cultured GE neurons. Time-lapse imag-

ing showed that accumulation of EGFP-shootin2 at the leading edge appears to occur prior to elongation of the LP. In addition, overexpression of mycshootin2 in GE neurons induced the formation of multiple LPs. These data suggest that shootin2 is involved in the formation and extension of the LP in GE neurons. Dot-like immunoreactivities of shootin2 were co-localized with the actin filaments at the edge of the LP, and single molecule imaging of EGFP-shootin2 showed that shootin2 molecules moved retrogradely similarly to actin filaments. In addition, the disruption of actin filament networks in filopodia and lamellipodia by cytochalasin D treatment led to disturbance of shootin2 retrograde flow. These data indicate that shootin2 interacts with actin retrograde flow. Taken together, our data suggest a possibility that shootin2 functions as a clutch molecule to extend the LP. Research fund: KAKENHI10J09106. doi:10.1016/j.neures.2011.07.1012

P3-e18 Developmental shift of NMDA receptor subunits is essential for closing the critical period in corticospinal synapse elimination Takae Ohno 1 , Mutsumi Isowaki 1 , Noriko Isoo 1 , Naoyuki Murabe 1 , Hitoshi Maeda 1 , Satoshi Fukuda 1 , Noboru Yoshioka 1 , Masayoshi Mishina 2 , Masaki Sakurai 1 1

Dept Physiol, Teikyo Univ Sch Med, Tokyo 2 Dept Mol Neurobiol & Pharmacol, Grad Sch Med, Univ Tokyo, Tokyo

Immature brain has high modifiability, which often is lost with maturation. There are relatively narrow time windows, so called critical periods, during which extrinsic events of short duration have life-long influence on the neuronal circuits. Because GluN2B(2B) subunit-containing NMDA receptors (NMDAR) show much longer time course with larger Ca influx than 2A, shift of dominant NMDAR subunit from 2B to 2A during development is hypothesized to be essential for regulating this time window. However, this is still controversial. We previously showed in in vitro slice co-cultures of mice cerebral cortex and spinal cord that corticospinal (CS) synapses, once formed throughout the spinal cord, were eliminated from the ventral side during development in an NMDAR-dependent manner. This synapse elimination was regulated selectively by 2B subunit and had critical period from 6 to 11 DIV. Both optically recorded corticospinal EPSPs and NMDA current kinetics of the EPSCs suggest that 2B subunits replaced by the other type, possibly 2A, near the end of the critical period in wild type (WT) spinal cords. These data suggest that 2B-2A shift determines the end of critical period of this type. On the other hand, in 2A knockout mice (KO), NMDAR subunit composition remains 2B-dominant even after the critical period: averaged amplitude of 2B current at this point was 73.3 ± 10.9 pA, which was only 10% lower than that of WT during the critical period (89.1 ± 11.1 pA). Thus, we first applied APV until the end of critical period to block the synapse elimination, and thereafter APV was removed in WT or 2AKO spinal cords co-cultured with WT cortex. The synapse elimination was no longer seen in WT spinal cords. However, the CS synapses on the ventral side were eliminated in 2AKO spinal cords: i.e. the critical period was not closed. These data suggest that closing the critical period plasticity window is determined by the shift of NMDA subunit composition in the corticospinal synapse formation. Research fund: KAKENHI 2080047, KAKENHI 20300138. doi:10.1016/j.neures.2011.07.1013

P3-e19 Antagonistic action of LOTUS, an endogenous antagonist of Nogo receptor, to B lymphocyte stimulatorinduced axon growth inhibition Kuniyuki Nishiyama , Yuji Kurihara, Masumi Iketani, Hiromu Itoh, Yoshio Goshima, Kohtaro Takei Dept. of Mol. Pharmacol. & Neurobiol., Yokohama City Univ. Grad. Sch. of Med., Yokohama, Japan Axonal regeneration after injury in the adult central nervous system (CNS) is limited by myelin-derived axon growth inhibitors (MDAGIs), such as Nogo. Recently, B lymphocyte stimulator (BLyS), a tumor necrosis factor family protein essential for B cell development, was identified as a functional ligand for Nogo66 receptor (NgR1). NgR1 is a common receptor of MDAGIs. We have found that lateral olfactory tract usher substance (LOTUS) functions as an endogenous NgR1 antagonist. In this study, we examined whether LOTUS exerts antagonistic action on BLyS-induced axon growth inhibition. Exogenously applied BLyS-induced growth cone collapse in cultured E13 chick dorsal root ganglion (DRG) neurons in which NgR1 is expressed, whereas the DRG neurons exposed to pre-incubation of purified LOTUS did not show inducible growth cone collapse, thus suggesting that exogenous binding of