B8 - Rappold

The aim of this study is to gain insight into the function of the Mental disorder associated GAP protein, MEGAP, in neuronal development. To analyse and understand MEGAP evoked cellular processes leading to cognitive impairment, we use a mouse model developed in the last funding period with Megap gene ablation, mimicking the genetic defect observed in patients with severe mental retardation. Human and mouse MEGAP genes are highly homologous and strongly expressed throughout embryonic and postnatal development from E9.5 until adulthood. Expression is restricted to neural tissues in fore-, mid- and hindbrain and spinal cord. MEGAP, which is also termed Slit-Robo GTPase activating protein 3 (srGAP3), constitutes a downstream signaling component of the neuronal guidance factor Slit and its receptor Robo. Due to its GAP activity, MEGAP downregulates active levels of the small RhoGTPase Rac1 and affects actin polymerization by inhibiting the WASP- related protein WAVE1. During the last funding period, we identified c-Abl, Lamellipodin and mDia1 as novel MEGAP interacting partners. We have shown that c-Abl regulates the function of MEGAP through phosphorylation of a distinct tyrosine residue within the SH3 domain, thereby affecting its binding to WAVE1 and Lamellipodin.

Overexpression of MEGAP in neuroblastoma or fibroblast cells has been shown to lead to a reduction of actin-rich lamellipodia; gain-of-function in hippocampal neurons reduces axon lengths. In contrast, hippocampal neurons derived from homozygous Megap^(-/-) animals show a dramatic increase in lamellipodia formation along neurites and cell bodies. These striking effects may be caused by an increase in active Rac1, the main regulator of lamellipodia formation. We will use livecell imaging of fibroblasts and Xenopus spinal cord neurons to gain insight into the involvement of MEGAP (and its novel interaction partners) in the dynamics of cytoskeletal remodelling at the leading edge, also in response to guidance factors. To investigate potential defects in the neuronal actin cytoskeleton, we will use immunohistochemistry and in situ hybridisation on sections of embryonic, postnatal and adult  Megap^(-/-)brains. After examining for gross morphhological differences, we will expand our analyses using markers for specific neuronal subpopulations. For example, we want to study GABAergic interneurons. A subset of these inhibitory interneurons originates from a region known as the lateral ganglionic eminence, from where they migrate towards the cerebral cortex. Megap, as well as Robo1 are expressed inside the ganglionic eminences, while Slit is expressed in the adjacent ventricular zone. In addition, Slit promotes branching and elongation of the neurites of GABAergic interneurons, Furthermore, we want to study axonal pathfinding deficits, especially midline crossing defects in the Megap knock-out mouse. We will focus on two axonal populations, first commissural neurons in the spinal cord and secondly retinal ganglion cells, which both cross the midline and are prevented from recrossing by the action of Slit1/2.

In den letzten Jahren wurde eine Vielzahl an Genen im Zusammenhang mit mentaler Retardierung identifiziert, doch blieb deren Funktion für die Gehirnentwicklung weitgehend unverstanden. Im Rahmen dieses Projektantrages soll mithilfe eines in der letzen Antragsperiode etablierten Mausmodells die Funktion des Mental disorder-associated GAP proteins MEGAP auf zellulärer Ebene untersucht werden. Bisherige Ergebnisse deuten darauf hin, daß Megap eine Rolle in der neuronalen Migration sowie bei der Projektion axonaler Wachstumskegel spielt. Eine Überexpression von Megap konnte mit verlangsamter Aktindynamik und eingeschränkter Zellmigration in Verbindung gebracht werden. Ein Verlust von Megap dagegen (in hippocampalen Neuronen) führt zur Entstehung einer riesigen Zahl von migrationsfördernden Lamellipodien. Neben dem Fokus der Analyse von Migrationsdefekten in den Knock-out Mäusen soll die Interaktion von MEGAP mit den drei von uns identifizierten Bindepartnern c-Abl, Lamellipodin und mDia1 weiter untersucht werden.

Own project-related publications within the SFB since 2005

Yang, Y., Marcello, M., Endris, V., Saffrich, R., Fischer, R., Trendelenburg, M.F., Sprengel, R., Rappold, G. (2006) MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation. Experimental Cell Research 312: 2379-2393

Own project-related publications (submitted / in preparation)

Endris, V., Rappold, G. (2008) Abl-dependent phosphorylation of MEGAP/srGAP3 affects its binding to WAVE1. Journal of Biological Chemistry, in revision

Bacon, C., Endris, V., Haussmann, L., Rappold, G. (2008) Expression of the Slit-Robo GAP family members in the developing mouse embryo, submitted

Haussmann, L., Endris, V., Engel, U., Pinheiro, E.M., Gertler, F., and Rappold, G. (2008) Interaction of of MEGAP with Lamellipodin exerts an inhibitory effect on lamellipodia dynamics, in preparation

Own project-related publications prior to 2005

Endris, V., Wogatzky, B., Leimer, U., Bartsch, D., Zatyka, M., Latif, F., Maher, E. R., Tariverdian, G., Kirsch, S., Karch, D., and Rappold, G. A. (2002) The novel Rho-GTPase activating gene MEGAP/ srGAP3 has a putative role in severe mental retardation. Proc Natl Acad Sci USA 99: 11754-11759

Selected publications from other projects outside the SFB since 2005

Kirsch, S., Weiss, B., Miner, T.L., Waterston, R.H., Clark, R.A., Eichler, E.E., Münch, C., Schempp, W., and Rappold, G. (2005) Interchromosomal segmental duplications of the pericentromeric region on the human Y chromosome. Genome Res. 15: 195-204

Schneider, K.U., Marchini, A., Sabherwal, N., Röth, R., Niesler, B., Marttila, T., Blaschke, R.J., Lawson, M., Dumic, M., and Rappold, G. (2005) Alteration of DNA binding, dimerization, and nuclear translocation of SHOX homeodomain mutations identified in idiopathic short stature and Leri-Weill dyschondrosteosis. Hum Mutat. 26: 44-52

Schneider, K.U., Sabherwal, N., Jantz, K., Röth, R., Muncke, N., Blum, W.F., Cutler, G.B. Jr, and Rappold, G. (2005) Identification of a major recombination hotspot in patients with short stature and SHOX deficiency. Am J Hum Genet. 77: 89-96

Marchini, A., Daeffler, L., Marttila, T., Schneider, K.U., Blaschke, R.J., Schnölzer, M., Rommelaere, J.,and Rappold, G. (2006) Phosphorylation on Ser106 modulates the cellular functions of the SHOX homeodomain protein. J Mol Biol. 355: 590-603

Tiecke, E., Bangs, F., Blaschke, R., Farrell, E.R., Rappold, G., and Tickle, C. (2006) Expression of the short stature homeobox gene Shox is restricted by proximal and distal signals in chick limb buds and affects the length of skeletal elements. (Joint last authors) Dev Biol. 298: 585-96

Blaschke, R.J., and Rappold, G.(2006) The pseudoautosomal regions, SHOX and disease. Curr. Opin. Genet. Dev. 16: 233-239

Blaschke, R.J.,Hahurij, N.D., Kuijper, S., Just, S., Wisse, L.J., Deissler, K., Maxelon, T., Anastassiadis, K., Spitzer, J., Hardt, S.E., Schöler, H., Feitsma, H., Rottbauer, W., Blum, M., Meijlink, F., Rappold, G., and Gittenberger-de Groot, A.C. (2007) Targeted mutation reveals essential functions of the homeodomain transcription factor Shox2 in sinoatrial and pacemaking development. (Joint last authors) Circulation 115: 1830-1838

Sabherwal, N., Bangs, F.,Röth, R., Weiss, B., Jantz, K., Tiecke, E., Hinkel, G.K.,Spaich, C., Hauffa, B.P., van der Kamp, H., Kapeller, J., Tickle, C., and Rappold, G. (2007) Long-range conserved non-coding SHOX sequences regulate expression in developing chicken limb and are associated with short stature phenotypes in human patients. Hum Mol Genet. 16:210-222

Niesler, B., Walstab, J., Combrink, S., Möller, D., Kapeller, J., Rietdorf, J., Bönisch, H., Göthert, M., Rappold, G. and Brüss, M. (2007) Characterization of the Novel Human Serotonin Receptor Subunits 5-HT_3C, 5-HT_3D, and 5-HT_3E.. Mol Pharmacol 72: 8-17

Marchini, A., Häcker, B., Marttila, T., Hesse, V., Emons, J., Weiss, B., Karperien, M., and Rappold, G. (2007) BNP is a transcriptional target of the short stature homeobox gene SHOX. Hum Mol Genet. 16: 3081-3087

Kapeller, J., Houghton, L.A., Mönnikes, H., Walstab, J., Möller, D., Bönisch, H., Burwinkel, B., Autschbach, F., Funke, B., Lasitschka, F., Gassler, N., Fischer, C., Whorwell, P.J., Atkinson, W., Fell, C., Buchner, K., Schmidtmann, M., van der Voort, I., Wisser, A.–S., Berg, T., Rappold, G., and Niesler, B.:(2008) A functional variant in the miR-510 target site of the serotonin receptor type 3E gene is associated with diarrhea predominant irritable bowel syndrome, submitted

Gudrun Rappold

Institut für Humangenetik
Universität Heidelberg
Im Neuenheimer Feld 366
69120 Heidelberg

Phone: 06221-565153
Fax:     06221-565155
email: gudrun_rappold(at)med.uni-heidelberg.de