神経化学教育口演

神経化学教育口演 Neurite Outgrowth・Network Formation

1G1-01 Extracellular vimentin interacts with insulin-like growth factor 1 receptor to promote axonal growth Shigyo Michiko¹，Kuboyama Tomoharu¹，Sawai Yusuke²，Umezaki Masahito²，Tohda Chihiro¹ ¹Division of Neuromedical Sciences, Institute of Natural Medicine, University of Toyama，²Division of Chemo-Bioinfomatics,, Institute of Natural Medicine, University of Toyama Vimentin, an intermediate filament protein, is generally recognised as an intracellular protein. Previously, we reported that vimentin was secreted from astrocytes and promoted axonal growth. The effect of extracellular vimentin in neurons was a new finding, but its signalling pathway was unknown. In this study, we aimed to determine the signalling mechanism of extracellular vimentin that facilitates axonal growth. We first identified insulin-like growth factor 1 receptor（IGF1R）as a receptor that is highly phosphorylated by vimentin stimulation. IGF1R blockades diminished vimentin- or IGF1-induced axonal growth in cultured cortical neurons. IGF1, IGF2 and insulin were not detected in the neuron culture medium after vimentin treatment. The combined drug affinity responsive target stability method and western blotting analysis showed that vimentin and IGF1 interacted with IGF1R directly. In addition, immunoprecipitation and western blotting analyses confirmed that recombinant IGF1R bound to vimentin. The results of a molecular dynamics simulation revealed that C-terminal residues（residue number 330-407）in vimentin are the most appropriate binding sites with IGF1R. Thus, extracellular vimentin may be a novel ligand of IGF1R that promotes axonal growth in a similar manner to IGF1. Our results provide novel findings regarding the role of extracellular vimentin and IGF1R in axonal growth.		1G1-02 GRAB, a GEF of Rab8, regulates axonal outgrowth in a Cdk5 phosphorylation-dependent manner Furusawa Kotaro¹，Asada Akiko¹，Fukuda Mitsunori²，Hisanaga Shin-ichi¹ ¹Dept. of Biol. Sci, Grad. Sch. of Sci, Tokyo Metropolitan Univ.，²Dept. of Dev. Biol. and Neurosci, Grad. Sch. of Life Sci, Tohoku Univ. Cyclin-dependent kinase 5（Cdk5）is a neuron specific Ser/Thr protein kinase that is activated by binding a p35 regulatory subunit. It plays an important role in a variety of neuronal functions including neurite elongation through the supply of membrane components to neurite tip. However, it is not fully understood how Cdk5-p35 regulates the membrane transport in growing axons. Membrane transport is regulated by Rab small GTPases, whose activity is in turn regulated by guanine nucleotide exchange factors（GEFs）. Among many GEFs, we were interested in GRAB, which is a GEF for Rab8A and also known as a binding protein for Rab11A/B, because GRAB could be a potential Cdk5 substrate with（S/T）PX（R/K）consensus phosphorylation sequences. Here we show that GRAB was phosphorylated at Ser169 and Ser180 by Cdk5-p35 and their phosphorylation inhibited the interaction with dominant negative Rab8A-T22N, indicating that the phosphorylation of GRAB suppresses its Rab8 activation ability. Phosphorylated GRAB colocalized with Rab11A but not with Rab8A in primary culture neurons. Live image analysis revealed that GRAB was transported on Rab11A-positive endosomes in axon. We examined axonal elongation activity of Rab8A and GRAB. Both of them stimulated axonal outgrowth when overexpressed. Further, the nonphosphorylation mutant of GRAB（GRAB-S169/180A）promoted axonal outgrowth more than its phosphomimic mutant（GRAB-S169/180D）. We would like to propose a novel Rab cascade, Rab11-GRAB-Rab8, in regulation of axonal outgrowth, in which GRAB activity is controlled by phosphorylation with Cdk5.

1G1-03 Dephosphorylation of CRMP2 enhanced recovery after spinal cord injury Nagai Jun¹，Kitamura Yoshiteru¹，Owada Kazuki¹，Goshima Yoshio²，Ohshima Toshio¹ ¹Dept. Life Sci. Med. Biosci., Grad. Sch. Adv. Sci. Eng., Waseda University，²Dept. Mol. Pharmacol. Neurobiol., Grad. Sch. Med., Yokohama City University. The presence of inhibitory molecules and the lack of neurotrophic factor are two major difficulties to central nervous system（CNS）regeneration. Common mediator of these two has been not found yet. Collapsin response mediator protein 2（CRMP2）was originally identified as a mediator of Semaphorin3A-induced repulsive response. CRMP2 directly binds and stabilizes cytoskeletal microtubule polymerization and transport tyrosine kinase B（TrkB）to axonal tip, the receptor for brain-derived neurotrophic factor（BDNF）, to promote axonal elongation. Meanwhile, inhibitory molecules-induced signals phosphorylate CRMP2 to decrease its affinity to cytoskeleton proteins, leading to axonal growth inhibition. However, the role of CRMP2 phosphorylation after CNS injury in vivo remains unknown. Here we investigate the role of CRMP2 phosphorylation after spinal cord injury（SCI）using CRMP2 knock-in（KI）mouse where CRMP2 phosphorylations by Cdk5 and GSK3β are eliminated by replacing serine522 with alanine residue. Elevated level of pCRMP2 was observed in injured spinal cord. Inhibition of CRMP2 phosphorylation exhibited neuroprotective effect against SCI by suppressing depolymerization of microtubules and fibrous scar formation. This permissive environment for enhanced axon growth of 5-HT-positive raphe-spinal tract induced locomotor recovery in CRMP2KI mice. To examine the signaling cascades involving CRMP2 phosphorylation, we cultured dorsal root ganglion（DRG）neurons. Suppressed axonal growth inhibition by chondroitin sulfate proteoglycan（CSPG）and enhanced axonal elongation with BDNF were observed in CRMP2KI neurons. Therefore, dephosphorylation of CRMP2 could be a unique approach to repair injured CNS by reduced inhibitory responses and enhanced sensitivity to neurotrophin.		1G1-04 Involvement of SRF cofactors in BDNF-induced Arc gene expression. Kikuchi Keietsu¹，Fukuchi Mamoru¹，Ishibashi Yuta¹，Tsujii Junya¹，Ishikawa Mitsuru¹，Tsuda Masaaki¹，Okuno Hiroyuki²，Bito Haruhiko³，Tabuchi Akiko¹ ¹Lab. of Mol. Neurobio., Grad. Sch. of Med. ＆ Pharm. Sci., Univ. of Toyama，²Grad. Sch. of Med. Medical Innovation Center, Kyoto Univ.，³Dep. of Neurochem., Grad. Sch. of Med., Univ. of Tokyo One of the neuronal immediate early genes, Arc（activity-regulated cytoskeleton-associated protein）, is a representative SRF（serum response factor）-target gene which plays a key role in the endocytosis of AMPA receptors and regulation of dendritic spine morphology. Arc gene is rapidly activated in response to various stimuli, such as synaptic activity and BDNF（brain-derived neurotrophic factor）. SARE（synaptic activity-responsive element）, which contains the binding sites for transcription factor CREB（cAMP-response element binding protein）and MEF2（myocyte enhancer factor 2）and SRF, is located at -7 kbp upstream of the Arc gene transcription start site. The SARE has been shown to be deeply involved in synaptic activity-regulated Arc gene expression in rat cortical neurons. However, little is known about how SRF cofactors regulate Arc expression.In this study, we have demonstrated the functional roles of SRF cofactor MKL（megakaryoblastic leukemia）on Arc gene expression after BDNF stimulation in rat cortical neurons. Arc mRNA and protein were immediately and transiently increased after BDNF stimulation. The mutation of SRF-binding site but not the mutation of SRF cofactor, ternary complex factor（TCF）-binding site, on the SARE decreased BDNF-induced Arc gene transcriptional activity. Overexpression of dominant negative SRF mutant inhibited BDNF-induced Arc gene transcription. Knockdown of MKL2 but not MKL1 inhibited minimum promoter activation of SARE induced by BDNF. On the other hand, double knockdown of MKL1 and MKL2 increased BDNF-induced gene promoter activity including the 7 kbp promoter region. Taken together, the complex of MKL2-SRF-SRE on SARE might be important to Arc gene activation. In contrast, the TCF-binding site of SARE might be involved in the repression of Arc gene activity at the basal level. We speculate that the regulation of Arc expression is highly complicated because of the involvement of not only SRF cofactors but also other transcription factors.