Ruediger Klein

Receptor-Specific Regulation of Phosphatidylinositol 3′-Kinase Activation by the Protein Tyrosine Phosphatase Shp2

ABSTRACT Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. We have found that the protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3′-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1 compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. Our results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner.

Neuron-glia communication via EphA4/ephrin-A3 modulates LTP through glial glutamate transport

Astrocytes are critical participants in synapse development and function, but their role in synaptic plasticity is unclear. Eph receptors and their ephrin ligands have been suggested to regulate neuron-glia interactions, and EphA4-mediated ephrin reverse signaling is required for synaptic plasticity in the hippocampus. Here we show that long-term potentiation (LTP) at the CA3–CA1 synapse is modulated by EphA4 in the postsynaptic CA1 cell and by ephrin-A3, a ligand of EphA4 that is found in astrocytes. Lack of EphA4 increased the abundance of glial glutamate transporters, and ephrin-A3 modulated transporter currents in astrocytes. Pharmacological inhibition of glial glutamate transporters rescued the LTP defects in EphA4 (Epha4) and ephrin-A3 (Efna3) mutant mice. Transgenic overexpression of ephrin-A3 in astrocytes reduces glutamate transporter levels and produces focal dendritic swellings possibly caused by glutamate excitotoxicity. These results suggest that EphA4/ephrin-A3 signaling is a critical mechanism for astrocytes to regulate synaptic function and plasticity.

Developmental changes in NT3 signalling via TrkA and TrkB in embryonic neurons.

Neurotrophins promote neuronal survival by signalling through Trk receptor tyrosine kinases: nerve growth factor signals through TrkA, brain‐derived neurotrophic factor (BDNF) and neurotrophin (NT)4 through TrkB and NT3 through TrkC. Although studies in some, but not all, cell lines indicate that NT3 can also signal through TrkA and TrkB, it is not known if such signalling can occur in neurons. We show that NT3 can promote the in vitro survival of sensory and sympathetic neurons isolated from embryos that are homozygous for a null mutation in the trkC gene. During the mid‐embryonic period, NT3 promoted the survival of as many trigeminal and nodose neurons as the preferred neurotrophins, NGF and BDNF. However, later in development, these neurons lost their ability to respond to NT3. NT3 also promoted the survival of almost all sympathetic neurons, but no decrease in effectiveness was observed during development. Trigeminal neurons from trkC−/− trkA−/− embryos did not respond to NT3 and nodose neurons from trkB−/− embryos likewise failed to respond to NT3. These results show that NT3 can signal through TrkA and TrkB in neurons at certain stages of development and may explain why the phenotype of NT3−/− mice is more severe than that of trkC−/− mice.

Eph/ephrin signalling during development

Eph receptors and their membrane-tethered ligands have important functions in development. Trans interactions of Eph receptors with ephrins at cell-cell interfaces promote a variety of cellular responses, including repulsion, attraction and migration. Eph-ephrin signalling can be bi-directional and controls actin cytoskeleton dynamics, thereby leading to changes in cellular shape. This article provides an overview of the general structures and signalling mechanisms, and of typical developmental functions along with cell biological principles.

FLRT2 and FLRT3 act as repulsive guidance cues for Unc5‐positive neurons

Netrin‐1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A–Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin‐1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine‐rich transmembrane protein‐3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5‐positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2‐expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ‐derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5‐positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.

Met Signaling Is Required for Recruitment of Motor Neurons to PEA3-Positive Motor Pools

Motor neurons in the spinal cord are grouped into motor pools, each of which innervates a single muscle. The ETS transcription factor PEA3 is a marker of a few such motor pools. Here, we show that pea3 is first induced by GDNF in a caudal subset of the motor neurons that will constitute the pea3+ population. Expansion of the pea3 domain subsequently occurs by recruitment of neurons from more anterior segments. Signaling by Met, the HGF receptor, is required for the rostral expansion of the pea3 domain, while the onset of pea3 expression is independent of met function. met expression is observed in pioneer neurons but does not precede that of pea3 in recruited neurons. We provide genetic evidence for a non-cell-autonomous function of met during the recruitment process. We propose the presence of a relay mechanism allowing cells induced by peripheral signals to recruit more anterior neurons to adopt the same motor pool-related phenotype.

EphA4-Mediated Ipsilateral Corticospinal Tract Misprojections Are Necessary for Bilateral Voluntary Movements But Not Bilateral Stereotypic Locomotion

In this study, we took advantage of the reported role of EphA4 in determining the contralateral spinal projection of the corticospinal tract (CST) to investigate the effects of ipsilateral misprojections on voluntary movements and stereotypic locomotion. Null EphA4 mutations produce robust ipsilateral CST misprojections, resulting in bilateral corticospinal tracts. We hypothesize that a unilateral voluntary limb movement, not a stereotypic locomotor movement, will become a bilateral movement in EphA4 knock-out mice with a bilateral CST. However, in EphA4 full knock-outs, spinal interneurons also develop bilateral misprojections. Aberrant bilateral spinal circuits could thus transform unilateral corticospinal control signals into bilateral movements. We therefore studied mice with conditional forebrain deletion of the EphA4 gene under control by Emx1, a gene expressed in the forebrain that affects the developing CST but spares brainstem motor pathways and spinal motor circuits. We examined two conditional knock-outs targeting forebrain EphA4 during performance of stereotypic locomotion and voluntary movement: adaptive locomotion over obstacles and exploratory reaching. We found that the conditional knock-outs used alternate stepping, not hopping, during overground locomotion, suggesting normal central pattern generator function and supporting our hypothesis of minimal CST involvement in the moment-to-moment control of stereotypic locomotion. In contrast, the conditional knock-outs showed bilateral voluntary movements under conditions when single limb movements are normally produced and, as a basis for this aberrant control, developed a bilateral motor map in motor cortex that is driven by the aberrant ipsilateral CST misprojections. Therefore, a specific change in CST connectivity is associated with and explains a change in voluntary movement.

EphA4 Receptor Shedding Regulates Spinal Motor Axon Guidance

BACKGROUND Proteolytic processing of axon guidance receptors modulates their expression and functions. Contact repulsion by membrane-associated ephrins and Eph receptors was proposed to be facilitated by ectodomain cleavage, but whether this phenomenon is required for axon guidance in vivo is unknown. RESULTS In support of established models, we find that cleavage of EphA4 promotes cell-cell and growth cone-cell detachment in vitro. Unexpectedly, however, a cleavage resistant isoform of EphA4 is as effective as a wild-type EphA4 in redirecting motor axons in limbs. Mice in which EphA4 cleavage is genetically abolished have motor axon guidance defects, suggesting an important role of EphA4 cleavage in nonneuronal tissues such as the limb mesenchyme target of spinal motor neurons. Indeed, we find that blocking EphA4 cleavage increases expression of full-length EphA4 in limb mesenchyme, which-via cis-attenuation-apparently reduces the effective concentration of ephrinAs capable of triggering EphA4 forward signaling in the motor axons. CONCLUSIONS We propose that EphA4 cleavage is required to establish the concentration differential of active ephrins in the target tissue that is required for proper axon guidance. Our study reveals a novel mechanism to regulate guidance decision at an intermediate target based on the modulation of ligand availability by the proteolytic processing of the receptor.

Protein tyrosine phosphatase receptor type O inhibits trigeminal axon growth and branching by repressing TrkB and Ret signaling.

Axonal branches of the trigeminal ganglion (TG) display characteristic growth and arborization patterns during development. Subsets of TG neurons express different receptors for growth factors, but these are unlikely to explain the unique patterns of axonal arborizations. Intrinsic modulators may restrict or enhance cellular responses to specific ligands and thereby contribute to the development of axon growth patterns. Protein tyrosine phosphatase receptor type O (PTPRO), which is required for Eph receptor-dependent retinotectal development in chick and for development of subsets of trunk sensory neurons in mouse, may be such an intrinsic modulator of TG neuron development. PTPRO is expressed mainly in TrkB-expressing (TrkB+) and Ret+ mechanoreceptors within the TG during embryogenesis. In PTPRO mutant mice, subsets of TG neurons grow longer and more elaborate axonal branches. Cultured PTPRO−/− TG neurons display enhanced axonal outgrowth and branching in response to BDNF and GDNF compared with control neurons, indicating that PTPRO negatively controls the activity of BDNF/TrkB and GDNF/Ret signaling. Mouse PTPRO fails to regulate Eph signaling in retinocollicular development and in hindlimb motor axon guidance, suggesting that chick and mouse PTPRO have different substrate specificities. PTPRO has evolved to fine tune growth factor signaling in a cell-type-specific manner and to thereby increase the diversity of signaling output of a limited number of receptor tyrosine kinases to control the branch morphology of developing sensory neurons. The regulation of Eph receptor-mediated developmental processes by protein tyrosine phosphatases has diverged between chick and mouse.

Axonal growth and pathfinding : from phenomena to molecules

A long-standing goal of developmental neurobiology is to characterize the molecular mechanisms underlying the formation of functional connections during the development of the nervous system. These connections are primarily established by the elongation of axons along restricted pathways. A specialized tip of an extending axon, the so-called growth cone, continuously explores the local environment for guidance information and makes choices in certain places. The growth cone displays a peripheral motile region from which filopodia protrude. These filopodia can extend tens of microns from the central region of the growth cone and are therefore able to survey a large area in a relatively short period. Filopodia are thus important sensors of the growth cone. The detection of local guidance cues depends on the expression of receptor complexes that lie on the surface of the extending growth cone and that probably transduce extracellular signals into intracellular events to direct the growth cone. Cell biological studies have shown that the molecular signals that might affect the behavior of growth cones include diffusible and substrate-bound factors that act as at-tractants or repellents. Research over the past decade has focused on the identification of molecular components expressed on the surface of extending axons and found in the environment of axons. These investigations have led to the identification of a variety of components that influence extending ax-ons; these components are reviewed in this Special Issue of Cell and Tissue Research. A relatively recently discovered family of short-range repulsive guidance cues is represented by the family of Eph receptors and their cell-surface ligands. Although the ligands have only been discovered during the last three years, remarkable progress has been made in elucidating the functions of these molecules. The Eph family represents by far the largest family of receptor tyrosine kinases, comprising at present 14 members. Eight ligands, termed ephrins, can be subdivided into two classes, based on the mode of membrane anchorage, viz., Ephrin A (gly-cosyl-phosphatidyl-inositol-anchored) and Ephrin B (transmembrane ligands). Because of their binding spec-ificities, the Eph receptors can also be subdivided into two classes, EphA (interacting with A-Ephrins) and EphB (interacting with B-Ephrins). Some of the special features of these families include: (1) considerable promiscuity in binding within the EphA and EphB subclasses, in that nearly every ligand of a certain subclass can interact with almost all receptors of the corresponding subclass; (2) li-gands can activate their receptors only in membrane-bound form; (3) when stimulated, …