Cell Death and Differentiation (2012) 19, 194–208 & 2012 Macmillan Publishers Limited All rights reserved 1350-9047/12 www.nature.com/cdd Kidins220/ARMS mediates the integration of the neurotrophin and VEGF pathways in the vascular and nervous systems F Cesca*,1,9, A Yabe2,9, B Spencer-Dene3, J Scholz-Starke1, L Medrihan1, CH Maden4, H Gerhardt5, IR Orriss6, P Baldelli1, M Al-Qatari7, M Koltzenburg7, RH Adams8, F Benfenati1,10 and G Schiavo2,10 Signaling downstream of receptor tyrosine kinases controls cell differentiation and survival. How signals from different receptors are integrated is, however, still poorly understood. In this work, we have identified Kidins220 (Kinase Dinteracting substrate of 220 kDa)/ARMS (Ankyrin repeat-rich membrane spanning) as a main player in the modulation of neurotrophin and vascular endothelial growth factor (VEGF) signaling in vivo, and a primary determinant for neuronal and cardiovascular development. Kidins220À/À embryos die at late stages of gestation, and show extensive cell death in the central and peripheral nervous systems. Primary neurons from Kidins220À/À mice exhibit reduced responsiveness to brain-derived neurotrophic factor, in terms of activation of mitogen-activated protein kinase signaling, neurite outgrowth and potentiation of excitatory postsynaptic currents. In addition, mice lacking Kidins220 display striking cardiovascular abnormalities, possibly due to impaired VEGF signaling. In support of this hypothesis, we demonstrate that Kidins220 constitutively interacts with VEGFR2. These findings, together with the data presented in the accompanying paper, indicate that Kidins220 mediates the integration of several growth factor receptor pathways during development, and mediates the activation of distinct downstream cascades according to the location and timing of stimulation. Cell Death and Differentiation (2012) 19, 194–208; doi:10.1038/cdd.2011.141; published online 3 November 2011 Companion paper of: doi:10.1038/cddis.2011.108 In the central (CNS) and peripheral (PNS) nervous systems, a reduction in lumbar motor neurons (MNs)5 as well as increased neurons rely on several trophic pathways, such as neuro- perinatal cell death in several brain regions.6 However, the CNS trophin (NT) signaling via tropomyosin-related kinase (Trk) is largely unaffected by the deletion of single NTs or Trk receptors,1 and glial cell-derived neurotrophic factor signaling receptors, suggesting that during development different growth via the Ret tyrosine kinase/glial cell-derived neurotrophic factors compensate for each other.4,7 In the PNS, neuronal loss factor family receptor a,2 to regulate axonal growth, dendritic is seen in specific ganglia, such as the vestibular and cochlear branching, synapse assembly and plasticity.1,3 The non- (BDNFÀ/À,NT3À/À,TrkBÀ/À and TrkCÀ/À),8,9 or sympathetic catalytic p75 NT receptor (p75NTR) functions in complex with superior cervical ganglia (NGFÀ/À11), as well as in dorsal root Trks to induce survival responses. In addition, p75NTR in ganglia (DRGs), where specific neuronal pools are lost in mice association with sortilin binds pro-NTs, such as pro-nerve lacking distinct NTs or their receptors.7 Non-neuronal pheno- growth factor (NGF), to trigger apoptotic pathways.3 types have also been observed. For example, both NT3À/À and Several mutant mouse lines lacking NT receptors or their TrkCÀ/À mice display cardiac phenotypes such as septal and ligands have been generated. NT impairment in vivo often valve malformations, whereas BDNFÀ/À and TrkBÀ/À mice results in perinatal lethality, reflecting the prominent role played display ventricular wall hemorrhages and abnormal vasculature. by these factors in development.4 Some of these mutant mice In addition, mice lacking p75NTR display perinatal lethality due to display CNS defects. In particular, TrkB-deficient animals show the rupture of blood vessels.11 1Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, via Morego 30, 16163 Genoa, Italy; 2Molecular Neuropathology Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK; 3Experimental Pathology Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK; 4Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK; 5Vascular Biology Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK; 6Department of Cell and Developmental Biology, University College of London, Gower Street, London WC1E 6BT, UK; 7Institute of Child Health, University College of London, 30 Guildford Street, London WC1N 1EH, UK and 8Max Planck Institute for Molecular Biomedicine, Roentgenstrasse 20, 48149 Muenster, Germany *Corresponding author: F Cesca, Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, via Morego 30, 16163 Genova, Italy. Tel: þ 3901071781788; Fax: þ 3901071781230; E-mail: [email protected] 9These authors contributed equally to this work. 10These authors contributed equally to this work. Keywords: Kidins220/ARMS; Neurotrophin signaling; Synaptic plasticity; VEGF receptor Abbreviations: BDNF, brain-derived neurotrophic factor; CNS, central nervous system; DIV, days in vitro; DRG, dorsal root ganglia; EPSC, excitatory postsynaptic current; ES, embryonic stem; Kidins220/ARMS, kinase D interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning; MAPK/Erk, mitogen-activated protein kinase/extracellular signal-activated kinase; MN, motor neuron; NGF, nerve growth factor; Nrp1, neuropilin-1; NT, neurotrophin; p75NTR, p75 neurotrophin receptor; PNS, peripheral nervous system; Trk, tropomyosin-related kinase receptor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor Received 08.3.11; revised 13.9.11; accepted 20.9.11; Edited by G Melino; published online 03.11.11 Kidins220 in NT/VEGF signaling during development F Cesca et al 195 Kidins220 (Kinase Dinteracting substrate of 220 kDa)/ Results ARMS (Ankyrin repeat-rich membrane spanning), referred to hereafter as Kidins220, is a transmembrane protein contain- Generation of Kidins220 knockout mice. To delete the ing cytoplasmic domains involved in protein–protein interac- Kidins220 gene in mice, we inserted a Kidins220 cDNA- tions.12,13 Several partners for Kidins220 have been polyA cassette flanked by two loxP sites in the unique NruI identified, including the NT receptors Trks and p75NTR,13,14 site within exon 16 of the mKidins220 gene (Figure 1A). In the Rho-GEF Trio,15 NMDA and AMPA receptors.16,17 Kidins220lox/lox mice, Kidins220 mRNA is transcribed under Kidins220 participates in the activation of the mitogen- the control of its endogenous promoter, generating the full- activated protein kinase/extracellular signal-activated kinase length protein. Upon crossing these mice with strains (MAPK/Erk),14 Jak/Stat18 and NF-kB19 pathways, down- expressing Cre recombinase, the floxed region is removed, stream of NT and ephrin signaling. It modulates basal synaptic yielding an mRNA encoding the N-terminal portion of activity17,20 and regulates neuronal polarity and dendrite Kidins220 fused to b-galactosidase (lacZ). Recombinant specification both in vitro21 and in vivo.22 Furthermore, clones were screened for the 50 and 30 recombination sites Kidins220 has a critical role in the control of cell survival, as (Figure 1Bb). Three clones showed the expected it mediates the neuroprotective effect of brain-derived hybridization pattern by Southern blot, confirming that a neurotrophic factor (BDNF) in cortical neurons.19 single integration had occurred (Supplementary Figure S1). Here and in the accompanying paper,23 we defined a major Kidins220lox/ þ animals were generated by blastocyst in vivo function of Kidins220 in the development of the nervous injection, back-crossed to C57Bl6/J and then crossed with and cardiovascular systems. Using Kidins220 knockout mice, we males expressing Cre recombinase under the ubiquitous found that Kidins220 has a crucial role in the integration of several phosphoglycerate kinase promoter, which is active from early growth factor receptor pathways in different tissues, and mediates stages of embryogenesis.24 Kidins220 þ /À animals were the activation of distinct downstream signaling cascades accord- viable and fertile, and did not show any overt behavioral ingtothelocationandtimingofgrowthfactorstimulation. phenotype. Kidins220 þ /À crosses, however, yielded only TM1TM2 TM3-4 9 1011 12 13 14 15 1617 18 1920 21 22 wt N-term 3' UTR IRES-lacZ-polyA Neo MW 12 13 14 15 16 cDNA-polyA 16 18 lox (bp) +/+ +/- -/- +/+ +/- -/- loxP loxP Frt Frt 1314 15 16IRES-lacZ-polyA 18 19 20 21 22 7000 ko loxP Frt 3000 short arm long arm (5' recomb) (3' recomb) 5000 cDNA-polyAIRES-lacZ-polyA Neo 12 13 14 15 16 16 18 3000 loxP loxP Frt Frt 2000 5’ recomb 3' recomb MW (kDa) +/+ +/- -/- MW MW 2F8 4E10 4B12 wt 2F8 4E10 4B12 wt (bp) (bp) 250 Kidins220 400 1600 lox lox 150 300 wt TrkB 200 100 150 wt -/- Kidins220 TrkC 100 50 tubulin E18.5 Figure 1 Generation of Kidins220 knockout mice. (A) The pFlrt construct recombined with the wild-type allele (wt; exon 9–22 of the Kidins220 gene), generating the lox allele (lox). The knockout allele (ko) is generated upon Cre excision. TM, transmembrane domain. (Ba) Probes for the PCR screen (red arrows) are indicated. (b) PCR for 50 and 30 recombination sites in a wild-type and three recombinant ES clones. Chimeras were derived