Promotion of neurite outgrowth by an endogenous Nogo66 receptor antagonist LOTUS

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

P2-e21 Overstimulated NMDA receptor impairs brain development Tomomi Aida 1 , Yoshimasa Ito 1 , Shuichi Maeda 1 , Yuko Takahashi 1 , Toshiko R. Matsugami 1 , Masami Mishina 2 , Kohichi Tanaka 1 1

Lab. Mol. Neurosci., Med. Res. Instit., Tokyo Medical and Dental Univ., Tokyo 2 Lab. Mol. Neurobiol. and Pharmacol., Univ. of Tokyo, Tokyo The construction of the brain during embryonic development has thought to be largely independent of its electrical activity. This is because loss-offunction mouse models of glutamatergic signaling that are generated by genetic deletion of glutamate receptors or glutamate release show normal brain assembly, whereas previous in vitro studies have shown that the neurotransmitter glutamate is important in brain development. However, recent findings demonstrate that activity plays essential roles in early development of the nervous system. Previously we showed the overstimulation of glutamate receptors in mice embryo by deleting glutamate transporters both GLAST and GLT1, leading to multiple brain defects including cortical, hippocampal, and olfactory bulb disorganization due to impaired stem cell proliferation, radial migration, neuronal differentiation, and survival of subplate neurons. The abnormal stratification of the mutant cerebral cortex and hippocampus were partially reversed by pharmacological administration of glutamate receptor antagonists to NMDA and AMPA receptors, although mutant cerebral cortex remained less organized than in control mice. Here, we show the deletion of NMDA receptor 1 (NR1), a critical subunit of NMDA receptors, almost totally reverses the multiple defects in this mutant. These results provide direct in vivo evidence that embryonic NMDA receptors are functional and excess stimulation of NMDA receptors, not non-NMDA ionotropic and metabotropic glutamate receptors, impairs brain development. Research fund: This work was supported by a Grant-in-Aid for Science Research (22700328 to T.A) from Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT), and a grant to T.A. from The Moritani Scholarship Foundation. A part of this study is the result of “Understanding of molecular and environmental bases for brain health” carried out under the Strategic Research Program for Brain Sciences by MEXT. doi:10.1016/j.neures.2011.07.560

P2-f01 The role of Notch-pathway in the monolayer formation of Bergmann glial cells Yuichi Hiraoka 1 Tanaka 1

, Okiru

Komine 1 , Mai

Nagaoka 2 , Kohichi

1 School of Biomedical Science Tokyo Medical and Dental University, Tokyo, Japan 2 Department of Physiology, Saitama Medical University, Saitama, Japan

The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. Bergmann glial processes provide contact guidance for granule cell soma to migrate across the molecular layer. Thus Bergmann glia is very important in developing and developed cerebellum. But the mechanism of Bergmann glia development is still almost unknown. Previously we reported that the Notch1/2-RBP-J signaling plays an important role in the monolayer formation of Bergmann glia. In the present study, we aimed to identify the physiological ligand of Notch in Bergmann glial development. The in vivo function of the Notch ligands Delta like 1 (Dll1) and Jagged1 were assessed in mice with Bergmann glia-specific gene targeting (GFAP-Cre mice). Inactivation of Dll1 resulted in poor radial fiber extension and defective positioning of Bergmann glia without effects of progenitor proliferation. In contrast, deletion of Jagged1 resulted in the decreased Bergmann glial proliferation. These results suggest that Dll1 plays the important role in the monolayer formation of Bergmann glia via Notch-RBP-J pathway, whereas Jagged1 is involved in Bergmann glial proliferation, independent of Notch-RBP-J pathway. doi:10.1016/j.neures.2011.07.561

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P2-f02 Prolyl hydroxylase (PHD) inhibitors prevent cortical neurite elongation by a mechanism dependent on RhoA/Rho-associated protein kinase (ROCK) Shuzo Miyake 1,2 , Rieko Muramatsu 1,2 , Toshihide Yamashita 1,2 1 Department of Molecular Neuroscience, Graduate school of Medicine, Osaka University, Osaka, Japan 2 Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Tokyo, Japan

Prolyl 4-hydroxylases (PHDs: PHD1, PHD2, PHD3), a part of an established oxygen sensor system, regulate transcription of hypoxia/stress-regulated genes and thus are involved in cell protection against oxygen, iron or 2-oxoglutarate deprivation. PHD2 knockdown resulted in activation of RhoA/Rho-associated protein kinase (ROCK) pathway. RhoA and its downstream effector protein ROCK are key regulators of neurite retraction. In this study, we report that PHD2 plays a role in neurite elongation in vitro. We examined the expression of PHD2 in cultured mouse neurons obtained from cerebral cortex by immunocytochemical and western blot analysis. We investigated the effects of PHD inhibitors on neurite length of cultured cortical neurons. Treatment with PHD inhibitors, desfferrioxamine (DFO) or ethyl-3,4-dihydroxybenzoate (EDHB), inhibited neurite elongation in a dose-dependent manner. We next investigated the involvement of RhoA in PHD2-regulated neurite elongation. Treatment with Y27632, an inhibitor of ROCK, prevented PHD inhibitor-mediated suppression of neurite elongation. Then, we investigated the RhoA expression in the cortical neurons which were treated with DFO or EDHB. Treatment with DFO or EDHB increased the expression of RhoA protein. These data suggested that PHD2 regulated neurite elongation by a mechanism dependent on RhoA/ROCK pathway. doi:10.1016/j.neures.2011.07.562

P2-f03 Dopaminergic innervation of striatal neurons through integrin ␣5␤1 Seiko Wakita , Yasuhiko Izumi, Toshiaki Kume, Akinori Akaike Dept. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ., Kyoto The main pathological feature of Parkinson disease is a selective degeneration of the nigrostriatal pathway, and regeneration of the neuroprojection is a potential therapeutic approach. However, the precise mechanisms promoting striatal innervation of dopaminergic neurons are still unclear. Integrins, heterodimers of ␣ and ␤ subunits, play an important role in development of central nervous system. In this study, we investigated the involvement of integrins in dopaminergic innervation of striatal neurons. We evaluated dopaminergic innervation of striatal neurons by using primary dissociated culture system. Mecencephalic and striatal cells were dissected from rat embryos, then a mecencephalic cell region were formed adjacent to a striatal cell region on a plastic coverslips. After paired-culturing for 11 days, dopaminergic neurons in the mesencephalic cell region extended their neurites to the striatal cell region. Immunofluorescent microscopy showed that dopaminergic axons elongated along striatal neurons and formed synapses with striatal neurons. Next, we examined the role of RGD-binding integrins in dopaminergic innervation of striatal neurons. Inhibition of RGD-binding integrins by the small peptide RGDS suppressed dopaminergic neurite outgrowth to the striatal cell region. In addition, dopaminergic neurite outgrowth was enhanced by coating with fibronectin, an RGD ligand. To identify the RGDbinding integrin subunits, we used neutralizing integrin ␤1 antibody and an integrin ␣5␤1/␣v␤3-blocking peptide ATN-161 (Ac-PHSCN-NH2 ). Both inhibitors suppressed dopaminergic innervation of striatal neurons. Furthermore, an integrin ␣5␤1-blocking peptide A5-1 (VILVIF) attenuated the dopaminergic innervation, while an integrin ␣v␤3-blocking peptide cycloRGDfV had no effect. These results suggest that dopaminergic innervation of striatal neruons required signaling via RGD-binding integrins, particularly of the ␣5␤1 subtype. Research fund: KAKENHI21650089. doi:10.1016/j.neures.2011.07.563

P2-f04 Promotion of neurite outgrowth by an endogenous Nogo66 receptor antagonist LOTUS Hiromu Ito , Yuji Kurihara, Masumi Iketani, Kuniyuki Nishiyama, Fumio Nakamura, Yoshio Goshima, Kohtaro Takei Dept. of Mol. Pharmacol. & Neurobiol., Grad. Sch. of Med., Yokohama City Univ., Yokohama, Japan Lateral olfactory tract usher substance (LOTUS) was identified as a novel key molecule for LOT formation. As a LOTUS binding protein, we further identified Nogo66 receptor (NgR1), which was a common receptor of myelin-

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

derived axon growth inhibitors, such as Nogo. Accumulating line of evidence suggests that LOTUS functions as an endogenous NgR1 antagonist. In this study, we found that the neurite outgrowth was remarkably promoted in retinal ganglion cell (RGC) and dorsal root ganglion (DRG) neurons cultured on LOTUS substrate. Inactivation of LOTUS substrate by fluorophore-assisted light inactivation reduced neurite outgrowth. Furthermore, RGC neurons in ngr1-deficient mice also showed the similar promoting effect of LOTUS on neurite outgrowth. These findings suggest that LOTUS promotes neurite outgrowth in RGC and DRG neurons and its promoting effect may be mediated by unidentified LOTUS binding molecule. doi:10.1016/j.neures.2011.07.564

P2-f05 Upregulation of an actin binding protein, Coactosin during maturation of parvalbumin-positive interneurons in mouse visual cortex Xubin Hou , Sayaka Sugiyama Lab. of Neuronal Development, Graduate School of Medical and Dental Sciences, Niigata Univ., Niigata, Japan Binocular vision is established in the primary visual cortex (V1) through an activity-dependent competition during early postnatal life. Experiencedependent circuit rewiring is triggered by a balanced excitation-inhibition via distinct GABAergic connections within V1. The previous work identified that maturation of this parvalbumin (PV)-cell network and hence plasticity onset is regulated by a non-cell autonomous accumulation of the Otx2 homeoprotein (Sugiyama et al., Cell 2008), which persists into adulthood (>P60). Postnatal recapture of Otx2 predicts this homeodomain transcription factor induces a genetic cascade for critical period plasticity, which is largely unknown yet. Here, we found that an actin binding protein, Coactosin, was extremely down-regulated in cortical Otx2 reduction. Although broadly present in visual cortex at pre-critical period ages (P60). Interestingly, the emergence of Coactosin within these cells was closely similar to the onset of Otx2, suggesting that Coactosin is served as a downstream effector for cortical plasticity. Importantly, Coactosin plays a role in remodeling of actin cytoskeleton and inhibits F-actin depolymerization (Hou et al., in submission). Thus, Coactosininducible cytoskeletal reorganization may effect on both frameworks for mechanical flexibility of synaptic structure and for scaffold of perineuronal net (PNN)-components through other actin binding proteins on PV-cells. Research fund: Supported by Founding Program for Next Generation for World-Leading Researchers, KAKENHI (22700330) and JST PRESTO program. doi:10.1016/j.neures.2011.07.565

P2-f06 ERK2 phosphorylates Par3 and controls its transport in axons Yasuhiro Funahashi 1,2 , Shinichi Nakamuta 1 , Takashi Namba 1,2 , Kozo Kaibuchi 1,2 1 Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 2 JST, CREST, Tokyo

Neurons are highly polarized and comprised of two structurally and functionally distinct parts, an axon and dendrites. Axon specification is one of the important events for neuronal polarization, and is regulated by signaling molecules involved in cytoskeletal rearrangement and in protein transport. We previously found that partition defective 3 (Par3) is associated with KIF3A (kinesin-2), and is transported into the nascent axon. Par3 interacts with Tiam1/2, Rac1 GEF, activates Rac1, and participates in axon specification. However, the regulatory mechanism of Par3–KIF3A interaction is not well understood. Here, we identified extracellular signal-regulated kinase 2 (ERK2) as novel Par3-interacting protein by affinity column chromatography and mass spectrometry. Immunoprecipitation assay showed that ERK2 interacted with Par3 in vivo. ERK2 phosphorylated the C-terminal domain of Par3 (Par3-C-term) both in vitro and in vivo. Phosphorylation of Par3C-term by ERK2 canceled the interaction of Par3 with KIF3A. Par3-C-term served as a dominant negative mutant, which inhibited the association of KIF3A with Par3. The expression of Par3-C-term perturbed neuronal polarity, whereas the phosphomimic mutant did not. These results suggest that ERK2 phosphorylates Par3 and inhibits its binding activity to KIF3A, thereby controlling Par3 transport. doi:10.1016/j.neures.2011.07.566

P2-f07 Lamellipodin, a novel effector of M-Ras, regulates dendrite development by reconstructing actin network Genichi Tasaka , Izumi Oinuma, Manabu Negishi Lab. of Mol. Neurobiol., Grad. Sch. of Biostudies, Kyoto Univ., Kyoto, Japan Neurons are highly polarized cells with two distinct processes, a single long axon and multiple dendrites. Dendrite development is a very important step because the pattern of dendritic maturation determines neuronal functions. However, the molecular mechanisms of dendrite development are still not well understood. M-Ras, a member of Ras family GTPases, is predominantly expressed in the central nervous system. Recent reports have shown that M-Ras is a key molecule of dendritic maturation. However, the downstream signaling of M-Ras in dendritic maturation still remains obscure. Here, we report that Lamellipodin (Lpd), a binding partner with Ena/VASP, is a novel effector of M-Ras involved in dendritic maturation downstream of M-Ras in cultured cortical neurons. Lpd bound directly to M-Ras through its Ras association (RA) domain in vitro, and the interaction of endogenous Lpd with M-Ras was also observed in cortical neurons. Knockdown of endogenous Lpd in cortical neurons suppressed the dendrite outgrowth and branching promoted by constitutively active form of M-Ras. Interestingly, we found that M-Ras translocated Lpd from cytosolic fraction to the membrane fraction. Wild-type Lpd, Lpd-CAAX (harboring membrane targeting signal) and RA-Lpd-CAAX (lacking RA domain), but not RA-Lpd promoted dendrite outgrowth and branching. Furthermore, Lpd-CAAX and RA-Lpd-CAAX but not wild-type Lpd and RA-Lpd overcame the suppression of dendrite outgrowth and branching induced by knockdown of endogenous M-Ras. We also found that Lpd colocalized with F-actin at the tips of dendrites and knockdown of endogenous Lpd caused disappearance of F-actin in the dendrites. Overexpression of wild-type Lpd, but not C-Lpd, lacking Ena/VASP binding motifs, promoted dendrite outgrowth and branching. These results suggest that M-Ras promotes the membrane translocation of Lpd, leading to dendrite development by reconstructing actin network. doi:10.1016/j.neures.2011.07.567

P2-f08 The role of cadherin in barrel net formation in the mouse somatosensory cortex Mayu Wakimoto 1,2 , Keisuke Sehara 1,2 , Yoshio Hoshiba 1,2 , Kaori Tanno 1,2 , Masatoshi Takeichi 3 , Hiroshi Kawasaki 1,2,4 1 Dept. of Mol. & Sys. Neurobiol., Grad. Sch. of Med., Univ. of Tokyo, Tokyo, Japan 2 GCOE, Univ. of Tokyo, Tokyo, Japan 3 Cell Adhesion and Tissue Patterning, RIKEN-CDB, Kobe, Japan 4 PRESTO, JST, Tokyo, Japan

The molecular mechanisms underlying the formation of intracortical circuitries are of great interest. We recently identified “barrel nets”, which are novel intracortical axonal trajectories of layer 2/3 neurons in the primary somatosensory cortex. Barrel nets surround barrels showing whisker-related patterns. We noticed that the expression pattern of cadherin-8 mRNA was well-correlated spatiotemporally with barrel nets during development. To inhibit cadherin functions, we overexpressed dominant-negative cadherin cN390 in layer 2/3 neurons using in utero electroporation, and found that barrel net formation was disrupted. Consistent result was obtained by using another dominant-negative hDN-cadherin. Overexpression of dominantnegative cadherins did not inhibit callosal projections of layer 2/3 neurons, suggesting that cadherins are selectively involved in barrel net formation. Research fund: KAKENHI, GCOE, HFSP, PRESTO. doi:10.1016/j.neures.2011.07.568

P2-f09 Novel screening of molecules deciding synaptic connections using behavior induced by Channelrhodopsin2 in C. elegans Sayaka Hori , Shohei Mitani Department of Physiol., Tokyo Women’s Med. Univ. Sch. of Med., Tokyo, Japan Mechanism of selective synapse formation is important for a basic science to understand normal brain development and medical applications. Following steps forms synapses: 1. axon guidance by guidance cues and their receptors, 2. physical binding by cell adhesion molecules, and 3. selection and maintenance by neurotrophic factors. Although each neuron is thought to express own factors, upstream factors determining its specificity have not been identified. Then, we report a novel, simple, and comprehensive screening method for identification of the genes deciding the specificity of synapses using C. elegans, which is the only model animal that their all neurons (118 types, 302 cells) and all synapses (approx. 5000) have been already identified. In this