Regulation of neurite outgrowth by the neurite-outgrowth multiadaptor RhoGAP protein, NOMA-GAP

Regulation of neurite outgrowth by the neurite-outgrowth multiadaptor RhoGAP protein, NOMA-GAP

Poster Session 1 Abstracts 2 June 2008 / Int. J. Devl Neuroscience 26 (2008) 841–866 ncam-1. A strain combining mutations in all eight genes is viabl...

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Poster Session 1 Abstracts 2 June 2008 / Int. J. Devl Neuroscience 26 (2008) 841–866

ncam-1. A strain combining mutations in all eight genes is viable and shows no additional defects in the neurons that were analyzed, suggesting that there is only limited genetic redundancy among members of the IgCAM superfamily in C. elegans. doi: 10.1016/j.ijdevneu.2008.09.102 [P1.53] Characterization of the interaction between Mmp2 and Frac during axon guidance C.M. Miller 1,*, A. Page-McCaw 2, H.T. Broihier 1 1

Case Western Reserve University, USA Rensselaer Polytechnic Institute, USA *Corresponding author. 2

Keywords: MMP; Axon guidance; Drosophila; Motorneuron

Motor axon guidance is thought to be regulated by the balance of attractive and repulsive cues that serve to guide axons along their migratory path. In this way, defined axon pathways are created and connections within the adult nervous system are ultimately established. The two Drosophila MMPs, Mmp1 and Mmp2, have recently been reported to promote fasciculation during motor axon guidance in the Drosophila embryo, however, the mechanism by which these proteases function is unknown. In order to identify Mmp2 substrates and interacting proteins important for axon guidance, a yeast two-hybrid screen was performed using full-length Mmp2. From the identified candidate proteins, we have focused on Fatal Attraction (Frac) as the most likely Mmp2 substrate to function in neuronal development. frac is a previously uncharacterized gene predicted to encode an adhesion molecule containing EGF-like calcium binding domains, matrilin repeats, and a Hyalin repeat domain. frac cDNA is expressed strongly throughout the muscle tissue consistent with a role in axon guidance and with modulation by Mmp2. Overexpression of frac in Drosophila embryos results in a phenotype in which branch ‘‘b’’ of the intersegmental nerve (ISNb) exhibits loosely bundled branches, extra projections, and a fraying of nerve bundles. Interestingly, this phenotype is indistinguishable from that observed in Mmp2 loss-of-function (LOF) mutants and suggests that the balance of attractive and repulsive cues regulating pathfinding has been disrupted. Based on this similarity, we propose that Frac acts in muscle to promote defasciculation by attracting motor axons to their muscle targets. Mmp2 would then promote fasciculation by cleaving Frac and downregulating this adhesive interaction. Mmp2 may deactivate Frac by cleaving a particular domain or abrogating an adhesive interaction, a role consistent with the Mmp2 mutant phenotype. We will present our continued progress toward characterizing the interaction between Mmp2 and Frac including analysis of frac LOF embryos. doi: 10.1016/j.ijdevneu.2008.09.103 [P1.54] Regulation of neurite outgrowth by the neurite-outgrowth multiadaptor RhoGAP protein, NOMA-GAP M. Rosa´rio *, W. Birchmeier Max Delbru¨ck Center for Molecular Medicine, Japan *Corresponding author. Keywords: NOMA-GAP; NGF; Differentiation; SHP2

Introduction: Neuronal differentiation involves the formation and extension of neuronal processes. This is controlled by signaling by growth factors such as the Neurotrophin family member, NGF.

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Methods: We have used expression profiling of dissected mouse embryonic spinal cords to identify novel regulators of neuronal differentiation. This has led to the identification of NeuriteOutgrowth MultiAdaptor, NOMA-GAP, which belongs to a new family of multiadaptor proteins with RhoGAP activity (Rosa´rio et al., 2007). We have addressed the biological and molecular role of NOMA-GAP in the NGF-stimulated differentiation of PC12 neuronal precursor cells and in the developing spinal cord of chick embryos. Results: NOMA-GAP is expressed throughout the nervous system during stages of active neuronal differentiation. We show, using siRNA, that NOMA-GAP is essential for NGF-stimulated neuronal differentiation and for the regulation of the ERK5 MAP kinase and the Cdc42 signaling pathways downstream of NGF. NOMA-GAP binds directly to the NGF receptor, TrkA, and becomes tyrosine-phosphorylated upon receptor activation, thus enabling recruitment and activation of the tyrosine phosphatase SHP2. Furthermore we demonstrate that recruitment of SHP2 downstream of NOMA-GAP is required for the stimulation of neuronal process extension, in PC12 cells and in the chick spinal cord, and for sustained activation of ERK5. In addition, we show that NOMA-GAP has GAP activity specifically for Cdc42 and that NOMA-GAPcatalysed tempering of the Cdc42/PAK signaling pathway is essential for successful neurite outgrowth in response to NGF. Discussion: We have therefore identified a new regulator of neuronal differentiation, which provides the first molecular link between the NGF receptor TrkA and the sustained activation of ERK5 and whose role in the negative regulation of Cdc42 provides a novel insight into the mechanisms promoting neurite extension.

doi: 10.1016/j.ijdevneu.2008.09.104 [P1.55] Effect of the chemotherapeutic agent, 5-fluorouracil, on memory and neurogenesis in the adult hippocampus M. EL-Beltagy *, S. Mustafa, G. Bennett, P. Wigmore University of Nottingham, UK *Corresponding author. Keywords: 5-Fluorouracil; Neurogenesis; Hippocampus; Memory

Deteriorations in cognitive function have been reported by cancer patients undertaking chemotherapy. These include problems with memory and may indicate that chemotherapy directly affects the hippocampus. However numerous confounding variables have made interpretation of patient studies difficult. This study is part of the development of an animal model to investigate the effects of chemotherapy on cognition and neurogenesis within the hippocampus. The dentate gyrus of the hippocampus is one of two brain regions to maintain adult neurogenesis. In most situations changes in the formation of new neurons in the hippocampus correlate with changes in memory ability. Our hypothesis is that the cognitive deficits experienced by patients on chemotherapy result from a decrease in neurogenesis within the dentate gyrus. To test this, the chemotherapeutic agent, 5-flurouracil (5-FU), was administered to adult hooded lister rats (dose 25 mg/kg). Cognitive behaviour was tested using the conditioned emotional response test (CER) and the object location test (OLR). In addition the number of dividing cells in the subgranular zone of the dentate gyrus was quantified by staining for the cell proliferative marker (Ki67). Preliminary results (10 controls and 8 drug treated) showed a significant decrease in the measured freezing time of the CER test