細胞内シグナル Axonal regulation
O3-9-2-1 アミロイドベータによるエンドサイトーシスを介した軸索成長円錐の変性 Endocytosis-mediated axonal growth cone degeneration induced by amyloid β ○久保山友晴1, 西子裕章1, 東田千尋1 ○Tomoharu Kuboyama1, Hiroaki Nishiko1, Chihiro Tohda1 富山大学和漢医薬学総合研究所神経機能学分野1 Div of Neuromedical Science, Inst of Natural Medicine, Univ of Toyama, Toyama1 In the brain of Alzheimer's disease (AD), axons are degenerated nearby amyloid β (Aβ) deposits and shown as swollen or atrophied structures. The axon degeneration probably results in breakage of neuronal networks and memory impairment in AD. However, it has not been clarified how axons are degenerated by Aβ. In this study, we aimed to elucidate the mechanism of Aβ-induced degeneration of axonal growth cone. In cultured mouse cortical neurons (E14), shapes of axonal growth cones were changed to collapse-like structures within 1 h after the treatment with Aβ25-35 (10 μM, active partial fragment) or Aβ1-42 (0.5 μM, full length). BAPTA-AM (1 μM, Ca2+ chelator) or memantine (50 μM, NMDA receptor antagonist) inhibited the degeneration of growth cones induced by Aβ25-35, but NiCl2 (100 μM, voltage-dependent Ca2+ channel blocker) or thapsigargin (0.2 μM, inhibitor of Ca2+ release from endoplasmic reticulum) did not. Then several factors triggered by Ca2+ entry are investigated. MDL28170 (1 μM, calpain inhibitor), FK506 (2 μM) or deltamethrin (10 nM, calcineurin inhibitors) inhibited the growth cone degeneration, but KN-93 (10 μM, Ca2+/calmodulin-dependent protein kinase inhibitor) or Go6973 (0.1 μM, PKC inhibitor) did not. In addition, monodansylcadaverine (1 μM, endocytosis inhibitor) inhibited the growth cone degeneration. We therefore supposed that endocytosis might be a key event causing axonal degeneration. Endocytosis was evaluated by incorporation of FM1-43 into growth cones. Aβ1-42 increased endocytosis of FM1-43 in growth cones. The Aβ1-42-induced endocytosis was significantly inhibited by BAPTA-AM, memantine, MDL28170, FK506 or monodansylcadaverine. These results indicate that Aβ induces Ca2+ influx, possibly via NMDA receptor, activates calpain and calcineurin and then facilitates endocytosis. Endocytosis of plasma membrane and its components perhaps leads to the growth cone degeneration. Regulation of endocytosis would be a novel target of therapy for AD.	O3-9-2-2 マリネスコ・シェーグレン症候群における大脳皮質形成障害の分子病態機構 SIL1, a causative gene of Marinesco-Sjogren syndrome, plays an essential role in establishing the architecture of the developing cerebral cortex ○永田浩一1, 稲熊裕1, 浜田奈々子1, 田畑秀典1, 西村嘉晃1, 伊東秀記1, 水野誠1, 岩本郁子1, 森下理香1, 鈴木基正2, 熊谷俊幸2 ○Koichi Nagata1, Yutaka Inaguma1, Nanako Hamada1, Hidenori Tabata1, Yoshiaki Nishimura1, Hidenori Ito1, Makoto Mizuno1, Ikuko Iwamoto1, Rika Morishita1, Motomasa Suzuki2, Toshiyuki Kumagai2 愛知県心身障害者コロニー発達障害研究所 神経制御学部1, 愛知県心身障害者コロニー中央病院2 Dept Mol Neurobiol, Inst for Dev Res, Aichi Human Service Center1, Central Hospital, Aichi Human Service Center2 Marinesco-Sjogren syndrome (MSS) is a rare autosomal recessively inherited neurodegenerative disorder characterized by mental retardation, cerebellar ataxia, cataracts and progressive myopathy. Recently, mutations in SIL1 gene, which encodes an endoplasmic reticulum resident cochaperone regulating the heat-shock protein family chaperone HSPA5 function, were identified as a major cause of MSS. We here report a novel in-frame 15 bp-deletion mutation in the SIL1 coding region, resulting in 5 amino acids deletion of the protein. To understand the pathophysiological significance of SIL1 in MSS, we examined the role of SIL1 and three MSS-causing SIL1 mutants including the newly identified one during brain development. Knockdown experiments using in utero electroporation method revealed that reduction of SIL1 causes inhibition of neuronal migration during corticogenesis. While RNAi-resistant SIL1 rescued the migration defect, the MSS-causing SIL1 mutants tested did not. We then found that HSPA5 is also involved in neuronal migration during corticogenesis. Biochemical analyses revealed that MSS-causing SIL1 mutants had lower affinities to HSPA5, and inhibition of SIL1-HSPA5 interaction induced impaired neuronal migration in vivo. Further analyses revealed that the neuronal migration defect by MSS-causing SIL1 mutations is at least partly due to abnormal morphology of the neuron. These data suggest that the MSS-causing mutations abrogate the ability of SIL1 to interact with and regulate HSPA5 and that impaired HSPA5 function may cause abnormal neuronal migration, which may contribute to the mental retardation observed in MSS.
O3-9-2-3 逆行性軸索輸送を介したセマフォリン3Aシグナル伝達はTrkAにより仲介される A retrograde axonal transport signal of Semaphorin3A is mediated through TrkA ○山根昌之1, 山下直也1, 佐々木幸生1, 中村史雄1, 五嶋良郎1 ○Masayuki Yamane1, Naoya Yamashita1, Yukio Sasaki1, Fumio Nakamura1, Yoshio Goshima1 横浜市立大学大学院　医学研究科　分子薬理神経生物学1 Dept. Mol Pharmacol & Neurobio, Yokohama City Univ. Grad., Yokohama, Japan1 Semaphorin3A (Sema3A), a repulsive axon guidance molecule, facilitates fast axonal transport. However, little is known about the mechanism underlying Sema3A-induced facilitation and its functional implications. To identify molecules involved in the retrograde Sema3A signaling, we performed time-lapse imaging of EGFP-fused proteins expressed in cultured dorsal root ganglion (DRG) neurons. Sema3A facilitated transport of Neuropilin-1, Plexin-A4 (PlexA4), and Fyn which are the components of Sema3A signaling. Sema3A also facilitated the transport of TrkA, a nerve growth factor (NGF) receptor. Double imaging of PlexA4 and TrkA showed that Sema3A induced colocalization of PlexA4 with TrkA at the growth cone and these co-localized particles were then retrogradely transported toward the cell body. TrkA K537A, kinase-dead mutant, suppressed Sema3A-induced PlexA4 transport. Introduction of TrkA mutant, lacking dynein binding domain, into DRG neurons attenuated Sema3A-induced PlexA4 transport. NGF facilitated transport of TrkA but not PlexA4, implicating distinct mechanisms of retrograde transport-driven by Sema3A and NGF signaling. These results suggest that TrkA is essential for Sema3A to facilitate axonal transport of its signaling components.	O3-9-2-4 Sema3A情報伝達におけるCRMP1とFアクチン結合蛋白質Filamin-Aの相互作用 Collapsin Response Mediator Protein 1 augments Filamin-A and F-actin interaction in Sema3A-signaling ○中村史雄1, 五嶋良郎1 ○Fumio Nakamura1, Yoshio Goshima1 横浜市立大学・医学部・分子薬理神経生物学1 Dept Mol Pharmacol, Sch Med, Yokohama City Univ, Yokohama1 Collapsin Response Mediator Proteins (CRMPs) and their C. elegans homologue UNC-33 are intracellular axon guidance signaling molecules. We here found that UNC-33 interacts with an actin-binding protein Filamin-1 in C. elegans (Fln-1). Guidance defect of commissural DD/VD neurons was observed in the null mutant of fln-1(tm545) as well as in a weak allele mutant of unc-33(e204). Double mutant of fln-1(tm545) and unc-33(e204) augmented this guidance failure, suggesting the genetic interaction. The interaction of CRMPs/UNC-33 and Filamin-A is preserved beyond species. Filamin-A was co-immunoprecipitated with CRMP1 from rat brain. CRMP1 bound to the N-terminal actin binding domain (ABD) and the C-terminal immunoglobulin-like repeat (Ig24) of Filamin-A. RNAi knockdown of Filamin-A, or the overexpression of Filamin-A(His93Ala-Arg96Ala) mutant in dorsal root ganglion (DRG) neurons suppressed Sema3A-induced growth cone collapse response. Mutational analysis of CRMP1 revealed that Arg245 to Asn247 residues are involved in ABD binding. Overexpression of CRMP1(Ile246Ala) or CRMP1(Asn247Ala) mutant suppressed Sema3A-response in DRG neurons. In contrast, the introduction of CRMP1(Ser522Asp) mutant, a cyclin-dependent kinase 5-phosphorylated mimicking form of CRMP1, enhanced Sema3A-induced collapse response. CRMP1(Ser522Asp) showed higher affinity to Filamin-A and enhanced the binding of Filamin-A to actin-filament. Taken together, phosphorylated CRMP1 by Cdk5 may augment the interaction of Filamin-A and actin-filament in turn to facilitate the reorganization of actin-cytoskeleton in Sema3A signaling.

上部に戻る

前に戻る