Signaling by Neuronal Tyrosine Kinase ReceptorsRelevance for Development and Regeneration.код для вставкиСкачать
THE ANATOMICAL RECORD 292:1976–1985 (2009) Signaling by Neuronal Tyrosine Kinase Receptors: Relevance for Development and Regeneration BARBARA HAUSOTT,1 ISIL KURNAZ,2 SRECKO GAJOVIC,3 1 AND LARS KLIMASCHEWSKI * 1 Division of Neuroanatomy, Medical University Innsbruck, Innsbruck, Austria 2 Department of Genetics and Bioengineering, Yeditepe University, Kayisdagi-Istanbul, Turkey 3 Croatian Institute for Brain Research, School of Medicine University of Zagreb, Zagreb, Croatia ABSTRACT Receptor tyrosine kinase activation by binding of neurotrophic factors determines neuronal morphology and identity, migration of neurons to appropriate destinations, and integration into functional neural circuits as well as synapse formation with appropriate targets at the right time and at the right place. This review summarizes the most important aspects of intraneuronal signaling mechanisms and induced gene expression changes that underlie morphological and neurochemical consequences of receptor tyrosine kinase activation in central and peripheral C 2009 Wiley-Liss, Inc. neurons. Anat Rec, 292:1976–1985, 2009. V Key words: signaling; receptor; neurotrophic Neurons receive, process, and deliver information to other neurons and to non-neuronal cells. During development the receiving part of the neuron increases its surface by extending dendrites and protruding spines; the outgoing single process, the axon, carries information to distant targets and forms synaptic boutons at its ending. This morphological level of complexity is brought about by the inﬂuence of neurotrophins, such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), or neurotrophin 3 (NT3) that are all crucial for neuronal development, but also for maintenance, adaptation, and plasticity in the adult nervous system (Bibel and Barde, 2000; Kaplan and Miller, 2000; Huang and Reichardt, 2001). Neurotrophins and other growth factors are expressed in limiting amounts in the central and peripheral nervous system, thereby controlling the Abbreviations used: ABP ¼ actin binding protein; ATF ¼ activating transcription factor; CREB ¼ cAMP response element binding protein; CGRP ¼ calcitonin gene-related peptide; BDNF ¼ brain derived neurotrophic factor; DRG ¼ dorsal root ganglia; EGF ¼ epidermal growth factor; ERK ¼ extracellular signalregulated kinase; FGF ¼ ﬁbroblast growth factor; GDNF ¼ glial cell line-derived neurotrophic factor; GF ¼ growth factor; GSK-3 ¼ glycogen synthase 3-kinase; IAP ¼ inhibitor of apoptosis; IEG ¼ immediate-early gene; LMC ¼ Lateral Motor Column; MAP ¼ microtubule associated protein; MAP kinase ¼ mitogen activated protein kinase; MBP ¼ microtubule binding proteins; MEK ¼ MAPK kinase; MKP ¼ MAP kinase phosphatase; MVBs ¼ multivesicular bodies; NF-H ¼ neuroﬁlament heavy chain; NGF ¼ nerve growth factor; NT3 ¼ Neurotrophin 3; p75NTR ¼ p75 neurotrophin receptor; PDGF ¼ Platelet-derived growth factor; PI3K ¼ phosphatidylinositol-3 kinase; PLC ¼ phospholipase C; PS1 ¼ Presenilin 1; RTK ¼ receptor tyrosine kinase; ROCK ¼ Rho-associated kinase; SCG ¼ superior cervical ganglion; SOS ¼ Son of sevenless; SRF ¼ Serum Response Factor; TRAF6 ¼ tumor necrosis factor receptor-associated factor 6; Trk ¼ tropomyosin related kinase; VEGF ¼ vascular epithelial growth factor Grant sponsor: COST (Action B30 ‘‘Neural Regeneration and Plasticity—NEREPLAS’’), the Daniel-Swarowski-Fonds, the Austrian Science Foundation (FWF); Grant sponsor: TUBITAK COST; Grant number: SBAG106S247; Grant sponsor: the Ministry of Science, Education and Sports of the Republic of Croatia; Grant number: 108-1081870-1902; Grant sponsor: International Center for Genetic Engineering and Biotechnology; Grant number: CRP/CRO06-02. *Correspondence to: Lars Klimaschewski, Muellerstr 59, Innsbruck 6020, Austria. E-mail: firstname.lastname@example.org Received 8 April 2009; Accepted 9 June 2009 DOI 10.1002/ar.20964 Published online in Wiley InterScience (www.interscience.wiley. com). SIGNALING BY NEURONAL TYROSINE KINASE RECEPTORS number and processes of neurons required for a suitable density of dendritic ﬁelds and target innervation. Neurotrophins bind to tropomyosinrelated kinases (Trk) receptors mainly at axonal endings. These subsequently convey a signal to the cell body and to the nucleus (Ginty and Segal, 2002). Together with receptors for other factors exhibiting neurotrophic activity, e.g. ﬁbroblast growth factor (FGF) or epidermal growth factor (EGF), they form the receptor tyrosine kinase (RTK) family which has been demonstrated to be associated with a plethora of different intraneuronal responses necessary for survival, differentiation, regeneration, and plasticity (e.g. required for learning). Here, we present a summary of the most relevant neuronal RTKs (mainly Trks, FGF, and EGF receptors) with regard to their ability to inﬂuence intrinsic neuronal signaling pathways and gene expression in order to shape neuronal morphology and determine the neurochemical phenotype. For speciﬁc aspects of RTK signaling, we cite recent in-depth reviews and refer to the literature therein. OVERALL FUNCTIONS OF RTKs IN NEURONAL DEVELOPMENT The in vivo relevance of RTKs in the nervous system has been mainly investigated in transgenic animals lacking RTKs in the whole body from fertilization onwards. From most of these studies it became obvious that inducible and/or conditional mouse mutants would be required to alter the expression of the respective RTK receptor in neuronal subpopulations at speciﬁc stages of development. Only detailed analyses of the phenotypes of these mice will provide deﬁnite evidence for the speciﬁc role of RTKs in the regulation of neuronal development and regenerative axon growth in the future. Currently, we rely on studies of conventional knockout mice which suggest that the neurotrophin receptors play a major role in the development of peripheral sensory, motor, and sympathetic neurons. It is important to note that, although sympathetic and sensory neurons depend on neurotrophins during embryogenesis and early postnatal life, they are not absolutely required for adult peripheral neurons. Even upon removal of any possible source of neurotrophic factors, such as non-neuronal cells, about 80% of adult neurons survive and extend long processes in vitro (Lindsay, 1988). The speciﬁc role of Trk receptors has been investigated in detail in the superior cervical ganglion (SCG) of rodents in vivo and in vitro. In this sympathetic neuron model, TrkC is expressed early in development, before neurons coalesce to form the SCG. TrkB receptors do not play a role in postganglionic neuron development, but are required for preganglionic neurons projecting to the adrenal gland (Schober et al., 1998). The lack of TrkA receptors does not inﬂuence neurogenesis, neuronal marker expression, or initial axonal growth in the SCG. In contrast, TrkA is absolutely necessary for the survival and target innervation of sympathetic neurons later in the development (Fagan et al., 1996). Mice lacking TrkA receptors have not only sympathetic, but also severe sensory neuropathies and most die within 1 month of birth (Smeyne et al., 1994). Neuronal cell death is detected in the SCG and in trigeminal and dorsal root ganglia (DRG) as well. The expression and activity of many survival-related proteins, such as caspase-9, Bax or Bcl-xL, 1977 have been shown to be regulated by Trk signaling. Furthermore, a decrease in cholinergic basal forebrain projections to the hippocampus and cortex is observed in animals lacking TrkA (Smeyne et al., 1994). All peripheral sensory neurons exhibit one of the three Trk receptors at different stages of development. TrkA/ mice are devoid of nociceptive and thermoceptive neurons, whereas TrkC knockout animals lack the largesized proprioceptive neuron subpopulation. TrkA-positive sensory neurons extend unmyelinated C ﬁbers or thinly myelinated Ad ﬁbers and are primarily nociceptive. Their neurochemical phenotype is characterized by the expression of substance P, calcitonin gene-related peptide (CGRP), the capsaisin receptor and by Naþ channel gene products suggesting that Trk receptors play not only a permissive, but also an instructive role during neuronal development. This is corroborated by the observation that lack of pro-apoptotic Bax has been shown to rescue dorsal root ganglion neurons in TrkA knockouts, but the surviving neurons do not make correct peripheral connections and lose their nociceptive markers (Patel et al., 2000). With regard to other RTKs, it has been demonstrated that ﬁbroblast growth factor receptors (FGFRs) regulate neural tube development of the CNS and proliferation of cortical progenitors (Hasegawa et al., 2004). The latter require FGFR, in particular FGFR3, for conferring migratory properties on nascent neuronal progeny. Moreover, oligodendrocytes develop under the inﬂuence of FGFR signaling. Epidermal growth factor receptor (EGFR) null mice reveal massive degeneration in various parts of the brain in the early postnatal period exhibiting neuronal loss but also delays in GFAP expression (Kornblum et al., 1998). The brains of EGFR knockout mice are smaller but cytoarchitecturally normal at birth. Therefore, EGFR (ErbB1) expression is critical for the maintenance of the postnatal mouse forebrain as well as for the normal development of astrocytes, whereas ErbB4 has been demonstrated to be required for myelin formation, neural crest migration, and development of dopaminergic functions (Roy et al., 2007). These various effects of RTKs have now been analyzed in detail at the cellular and molecular level in different cell types in vitro and in vivo. ACTIVATION, ENDOCYTOSIS, AND TRAFFICKING OF RTKs The major signaling pathways regulated by growth factor binding have been mostly elucidated and provide our basic knowledge about the role of RTKs in neurons. The speciﬁc shape of the neuron requires that receptor signaling information is transported along long distances. Upon binding of the ligand to the receptor, the complex is internalized by endocytosis and transported via early and late endosomes/multivesicular bodies (MVBs) toward the lysosomes, where the ﬁnal degradation of receptors occurs (Romanelli and Wood, 2008). Alternatively, receptors recycle back to the plasma membrane. The general function of this vesicular pathway is to control cellular functions by downregulating the signal. The ligand-receptor complex actually continues signaling even after internalization by endocytosis, but it stops as a consequence of increasing acidiﬁcation of the vesicles 1978 HAUSOTT ET AL. achieved by the activity of the proton pump, vacuolar Hþ-ATPase (V-ATPase), which ﬁnally causes a dissociation of the ligand from its receptor (Nishi and Forgac, 2002; Saroussi and Nelson, 2009). The signaling endosome hypothesis indicates the ability of endosomes not only to attenuate the signal along the degradation or recycling pathway, but also, in contrast to carry the signal along considerable distances, in particular, during retrograde axonal transport in neurons bringing the information to the cell body and the nucleus (Grimes et al., 1996). The whole complex including the vesicle, the ligand-receptor complex, and eventually the downstream signaling molecules is carried by the motor protein dynein along microtubules and upon arrival in the cell body the signaling molecules are released, pass the nuclear pore, and regulate neuronal gene expression (Wu et al., 2007). In case of NGF, signaling starts with binding at the axon terminal to TrkA receptors or, alternatively, to the common receptor for all neurotrophins, p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor receptor superfamily. Following endocytosis and internalization, they could independently be sorted for recycling or for retrograde transport and subsequent degradation. Still, it is not clear which type of endosome travels retrogradely along the axon. It seems that the early endosomes (marked by small GTPase Rab5) are located within terminal buttons, where the sorting for recycling occurs, whereas GTPase Rab7 positive late endosomes/MVBs are transported retrogradely along the axon (Deinhardt et al., 2006; Moises et al., 2007). Nevertheless, the other types of endosomes, i.e. early and macroendosomes (the latter resulting from clathrin independent macropinocytosis) are suggested to carry cargo along the axon as well (Wu et al., 2008). Ubiquitination emerged to be crucial for the decision of how RTKs are sorted along the endosome pathway (Acconcia et al., 2008). TrkA requires NGF binding to be both endocytosed and ubiquitinated. It becomes polyubiquitinated by action of tumor necrosis factor receptor-associated factor 6 (TRAF6), which catalyzes the formation of K63-linked polyubiquitin chains. The action of TRAF6 and polyubiquitination is necessary for internalization of the ligand-receptor complex (Geetha et al., 2005). TRAF6 causes polyubiqutination of p75NTR as well and together with Presenilin 1 (PS1) inﬂuences its regulated intramembrane proteolysis (Powell et al., 2009). Multi-monoubiquitination of TrkA is mediated speciﬁcally by Nedd4-2, another E3 ubiquitin ligase, which does not act on other Trks (Arevalo et al., 2006). Therefore, both monoubiquitination and polyubiquitination of receptors can occur, but the relation of these two ways of ubiquitination remains unclear. Moreover, for the ﬁnal degradation of TrkA a step involving the proteasome seems to be necessary prior to its entry into the lysosome. The involvement of the proteasome in deubiquitination before targeting cargo toward the lysosome represents a novel concept in receptor trafﬁcking and degradation (Geetha and Wooten, 2008). The disturbed trafﬁcking of the signaling complexes via signaling endosomes is suggested to be one of the causes for human neurodegenerative diseases. Indeed, defects of the vesicular transport accompanied by reduced growth factor signaling were indicated in Alz- Fig. 1. Receptor tyrosine kinase (RTK) signaling pathways involved in neuronal survival and neurite outgrowth and its inhibition by negative feedback inhibitors. Ras/Raf/MEK/ERK and PI3K/Akt represent two major pathways for survival and neurite outgrowth induced by RTKs. Induction of ERK via Ras/Raf/MEK promotes survival and neurite outgrowth by transcriptional control. The survival pathway of PI3K includes Akt which phosphorylates BAD and prevents its inhibition of anti-apoptotic proteins. Inhibition of GSK-3 induces neurite outgrowth by regulation of microtubule dynamics via microtubule binding proteins (MAPs). Rac and Cdc42 regulate actin dynamics via actin binding proteins (ABP). RhoA inﬂuences actin dynamics via actin-based motor protein myosin II. PI3K transiently inactivates RhoA which decreases its ability to associate down-stream with ROCK. The negative feedback inhibitors Sprouty and Sef attenuate RTK signalling. Sprouty speciﬁcally inhibits MAP kinase-signalling upstream of Ras at the level of Grb-2 and downstream of Ras at the level of Raf. Sef-a blocks phosphorylation of the receptor as a transmembrane protein and cytoplasmic Sef-b prevents nuclear translocation of ERK by inhibition of the dissociation of the ERK-MEK complex. heimer’s disease, Down syndrome, Huntington disease, amyotrophic lateral sclerosis, Niemann Pick disease type C, and Charcot-Marie-Tooth neuropathies (Bronfman et al., 2007). Moreover, defective ubiquitination contributes to neuronal loss, but it is still difﬁcult to distinguish whether this is due to the inadequate signaling, inadequate degradation, in particular, inadequate autophagic degradation, or a combination of those (Filimonenko et al., 2007; Tamai et al., 2008). RTK SIGNALING PATHWAYS IN NEURONS Ligand binding induced activation and autophosphorylation results in recruitment of several adapter molecules to tyrosine residues present in the intracellular domain of RTKs (Kouhara et al., 1997; Meakin et al., 1999). Adapter proteins like FRS2 or Shc then trigger the activation of several intracellular signaling pathways. The main cascades activated by RTKs include the Ras/Raf/MEK/ERK, the PI3K/Akt and the phospholipase C (PLC) pathway (Fig. 1). ERK- and Akt-signaling is involved in both, neuronal survival and axon growth. PLC mainly regulates intracellular Ca2þ-levels and protein kinase C activity via cleavage of PIP2 to DAG and IP3. This mechanism appears to enhance MEK activity and is involved in neurotrophin release (Canossa et al., 1997). SIGNALING BY NEURONAL TYROSINE KINASE RECEPTORS The ERK- and PI3K-Pathways are Central for RTK Signaling Activation of PI3K/Akt and their downstream targets represents the major survival promoting pathway in neurons (Dudek et al., 1997; Crowder and Freeman, 1998). In contrast, the ERK pathway plays a predominant role in survival following cellular insult (Hetman et al., 1999). PI3K and its target Akt are also necessary for NGFdependent survival of sympathetic (Nobes et al., 1996) and sensory neurons (Klesse and Parada, 1998). Induction of ERK induces neuronal survival via transcriptional processes by expression of anti-apoptotic proteins like Bcl-2 (Liu et al., 1999). Akt phosphorylates Bcl-2 antagonist of cell death (BAD) and prevents it from inactivating anti-apoptotic Bcl-2 proteins (Bonni et al., 1999). The transcription factor Forkhead, which induces apoptosis by increasing the levels of Fas ligand, is inhibited by Akt (Brunet et al., 1999). Furthermore, Akt indirectly prevents apoptosis by suppression of glycogen synthase 3-kinase (GSK-3) (Pap and Cooper, 1998). Besides activation of Akt, PI3K promotes survival via activation of a group of caspase inhibitors, the inhibitors of apoptosis (IAPs; LeCasse et al., 1998), which participate in neurotrophin-mediated survival in sensory and sympathetic neurons (Wiese et al., 1999). Enhanced RTK signaling promotes axon elongation not only by development but also by adult peripheral neurons as well (Hausott et al., 2008). The ERK- and the PI3Kpathway are both required for neurotrophin-mediated axon growth (Markus et al, 2002). In PC12 cells, activation of PI3K, Akt, Ras, Raf, Mek, and ERK are necessary for NGF-induced neurite outgrowth, and overexpression of Ras, Raf and MEK induces neuritogenesis in the absence of NGF (Wood et al., 1993; Klesse et al., 1999). Sustained ERK activation is required for neurite outgrowth by PC12 cells, whereas transient ERK activation leads to proliferation (Traverse et al., 1992), suggesting that the duration of the ERK signal determines differentiation of this cell line. In primary cultures, active Ras induces neurite outgrowth by nodose ganglion neurons in the absence of neurotrophins (Borasio et al., 1989), whereas in sympathetic neurons ERK-signaling is required for BDNF/TrkB-mediated axon elongation (Atwal et al., 2000). Moreover, ERK phosphorylation is required for retrograde transport of an ‘‘injury signal’’ as discussed earlier (Perlson et al., 2005) and for local axon assembly induced by neurotrophins (Atwal et al., 2000). ERK regulates polymerization of axonal microtubules and actin ﬁlaments (Goold and Gordon-Weeks, 2005). Vice versa, inhibition of ERK induces actin depolymerization and growth cone collapse (Atwal et al., 2003). PI3K is necessary for NGF-induced axon growth by sympathetic neurons (Kuruvilla et al., 2000) and for the growth cone response to NGF (Ming et al., 1999). Activated Akt has been shown to accelerate motor axon regeneration in vivo (Namikawa et al., 2000). Local assembly of the cytoskeleton which regulates axon morphogenesis is also under the control of PI3K signaling. PI3K inﬂuences the activity of various small GTPases of the Rho family such as Rac, Cdc42, and RhoA, thereby regulating actin cytoskeletal dynamics (Higgs and Pollard, 2001; Smith and Li, 2004). In ﬁbroblasts, activation of cdc42 induces ﬁlopodia and activation of Rac lamellipodia formation, whereas activation of RhoA results in stress ﬁber formation (Mackay 1979 et al., 1995; Nobes and Hall, 1995). Neurites can spread by extension of ﬁlopodia and subsequent generation of lamellipodia between ﬁlopodia. In PC12 cells, both Cdc42 and Rac are regulated by PI3K (Yamaguchi et al., 2001; Aoki et al., 2005) and lamellipodia formation is dependent on Rac activity (Posern et al., 2000). In primary spinal cord neurons, overexpression of active Cdc42 promotes growth cone ﬁlopodia formation and axon elongation (Brown et al., 2000). RhoA activates Rho kinase (ROCK), which leads to phosphorylation of various target proteins including myosin light chain that in turn activates myosin (Zhou and Snider, 2006). In contrast to Rac and Cdc42 which promote neurite outgrowth, Rho causes growth cone collapse and neurite retraction (Kozma et al., 1997). Pharmacological inhibition of RhoA induces neurite outgrowth by PC12 cells (Welsh and Assoian, 2000). Conversely, PC12 cell differentiation is blocked by an active dominant mutant of RhoA (Sebök et al., 1999). In adult DRG neurons, inhibition of RhoA stimulates neurite outgrowth even if exposed to inhibitory substrates (Fu et al., 2007). NGF-induced PI3K activation transiently inactivates RhoA via Rac1, upon which RhoA translocates from the membrane to the cytoplasm and its ability to associate down-stream with ROCK is decreased (Nusser et al., 2002). Integrins inactivate RhoA via activation of RhoGAP (Arthur and Burridge, 2001). Activation of PI3K/ Akt inactivates GSK-3ß (Cross et al., 1995), which induces axon growth by stimulating microtubule dynamics via microtubule binding proteins (MBPs; Zhou et al., 2004). GSK-3b regulates all aspects of microtubule assembly like microtubule polymerization, plus end binding and maintenance of microtubule dynamics by different MBPs. In adult DRG neurons, inhibition of GSK-3b results in enhanced neurite growth (Jones et al., 2003). The ERK- and the PI3K-pathway display distinct functions in axon growth. In embryonic DRG cultures (Liu and Snider, 2001) and in adult DRG explants, which are not dependent on neurotrophic factors for survival (Edström and Ekström, 2003), inhibition of PI3K results in diminished axon growth. PI3K inhibitors block spontaneous and NGF-induced neurite outgrowth. Inhibitors of the Ras/ ERK pathway only transiently arrest spontaneous axonal outgrowth from DRG explants, while NGF-increased outgrowth is signiﬁcantly impaired. In Bax knockout mice, overexpression of Ras and Raf-1 (c-Raf) induces axonal elongation by embryonic sensory neurons, whereas overexpression of Akt or PI3K increased axon caliber and axonal branching. Co-transfection with Raf and Akt resulted in long, thick, and moderately branched axons and the effects of Raf and Akt appear to be independent of each other (Markus et al., 2002). In adult DRG neurons, FGF-2 treatment induces prominent ERK phosphorylation, whereas Akt phosphorylation is slightly increased only. In contrast, NGF treatment causes phosphorylation of both, ERK and Akt, to a similar extent (Hausott et al., submitted). It is therefore not surprising that FGF-2, but not NGF, signiﬁcantly improves elongative axon growth by adult sensory neurons in response to a preconditioning sciatic nerve lesion (Klimaschewski et al., 2004). Negative Feedback Inhibitors Regulate RTK Signaling RTK signaling is attenuated by degradation of the receptors or by negative feedback inhibitors such as 1980 HAUSOTT ET AL. Sprouty or Sef (Tsang and Dawid, 2004; Mason et al., 2006). Sprouty proteins were ﬁrst discovered in 1998 (Hacohen et al., 1998). They represent a major class of negative feedback inhibitors that limit the intensity and duration of activation of tyrosine kinases, thereby controlling growth and differentiation processes. Mammals exhibit four Sprouty isoforms (Sprouty1–4) and their expression is regulated by growth factors (Ozaki et al., 2001; Sasaki et al., 2001). Activation of Sprouty speciﬁcally inhibits the Ras/Raf/ERK pathway (Yusoff et al., 2002; Hanafusa et al., 2002; Tefft et al., 2002) induced by FGF (Impagnatiello et al., 2001), brain-derived neurotrophic factor (BDNF, Gross et al., 2007), glial cell linederived neurotrophic factor (GDNF, Ishida et al., 2007), platelet-derived growth factor (PDGF, Gross et al., 2001), or vascular epithelial growth factor (VEGF, Impagnatiello et al., 2001; Sasaki et al., 2001). In response to growth factor stimulation, Sprouty1 and 2 bind Grb2 and inhibit the recruitment of the Grb2-SOScomplex to FRS2 or Shp2, thereby preventing downstream Ras/ERK activation (Hanafusa et al., 2002; Tefft et al., 2002). Sprouty2 and 4 bind Raf and interfere with the activation of the Ras/ERK pathway downstream of Ras (Yusoff et al., 2002; Sasaki et al., 2003). In PC12 cells, Ras, Raf, Mek, and ERK are required for neurite outgrowth (Wood et al., 1993; Klesse et al., 1999). Overexpression of Sprouty1 and 2 blocks PC12 cell neurite outgrowth induced by NGF or FGF-2 (Gross et al., 2001), conversely, non-phosphorylated dominant negative Sprouty1, 2 (Hanafusa et al., 2002) or 4 (Sasaki et al., 2001) promote FGF-induced neurite outgrowth. In cultures of immature cerebellar granule neurons, overexpression of Sprouty2 blocks neurite formation and inhibition of Sprouty2 by a dominant-negative mutant or by siRNAs promotes neurite outgrowth. In mature neurons that already exhibit an extensive neurite network, overexpression of Sprouty2 induces cell death whereas its inhibition promotes survival (Gross et al., 2007), which suggests a developmental difference in the effects of enhanced or reduced Ras/ERK signaling affecting neurite outgrowth or survival depending on age. Sprouty2 is highly expressed in adult DRG neurons and its down-regulation induces elongative axon growth by adult DRG in vitro through enhanced Ras/ERK signaling (Hausott et al., submitted). The negative feedback inhibitor Sef (similar expression of FGF genes) was ﬁrst identiﬁed in zebraﬁsh (Fürthauer et al., 2002; Tsang et al., 2002). Sef is synexpressed with Sprouty2 and Sprouty4 and similarities between the expression patterns of these genes indicate that they could be co-regulated. The protein interferes with FGFR signaling upstream and downstream of Ras. Depending on the isoform, it blocks phosphorylation of the receptor (Kovalenko et al., 2003) as a transmembrane protein by inhibition of FRS2 phosphorylation (Sef-a) or it prevents nuclear translocation of ERK by inhibition of the dissociation of the ERK–MEK complex as a cytoplasmatic isoform without inﬂuencing the cytoplasmic activity of ERK (Sef-b, Torii et al., 2004). Overexpression of Sef inhibits FGF-2- and NGFinduced neurite outgrowth by PC12 cells (Xiong et al., 2003). Sef is expressed in the brain, the spinal cord, and in DRGs and it is up-regulated in response to a sciatic nerve crush at the lesion site (Grothe et al., 2008). TRANSCRIPTIONAL EVENTS INDUCED BY RTKs IN NEURONS The main function of RTK signaling is to maintain the level of proteins required for survival, axon growth, and guidance via changes in gene transcription, protein synthesis, and degradation. The overall effects of Raf-mediated signaling were analyzed using conditional targeting of B-Raf and C-Raf, two downstream effectors of RTK signaling. Defects in Raf lead to growth retardation, partly due to reduced survival, but largely due to decreased levels of the GDNF Receptor, Ret, as well as reduced levels of Brn-2 and Klf-7, both transcriptional regulators of TrkA expression (Zhong et al., 2007). In these Raf-defective animals, NGF-induced axon growth, which is under the control of Runx1 and CBF, is also affected. Runx1/AML1 is involved in axonal outgrowth by neural crest stem cell-derived nociceptive neurons and regulates TrkA signaling in combination with an existing neurogenin2 transcriptional program in these cells (Marmigere et al., 2006). In one study, the global effects of RTK signaling during differentiation into neuronal lineages were analyzed using an Affymetrix microarray involving over 30,000 genes. Pollard et al. (2008) have investigated the transcriptional proﬁle of neural stem cells upon FGF-mediated differentiation. They have identiﬁed a set of 64 genes that appears to be a signature for NSC populations, including transcriptional regulators such as Mef2c, Nkx2.2, Olig1, Pou3, and Sox9, among others. ERK1/2 and JNK pathways play a role in differentiation upon NGF stimulation, by regulating either the expression or phosphorylation of various target proteins such as c-Jun, Elk-1, activating transcription factor (ATF), neuroﬁlament heavy chain (NF-H), or MAP kinase phosphatase (MKP, Waetzig and Herdegen, 2003). Elk-1 is a protein that belongs to the ETS domain superfamily of transcription factors, the founding father of which is the Ets-1 proto-oncogene initially identiﬁed from the E-26 virus (Sharrocks, 2001). Elk-1 is typically known as a mitogen-activated transactivator, involved in the regulation of proliferation in cells (Sharrocks, 2001), however, Elk-1 has been shown to be localized to axons and dendrites in largely post-mitotic adult rat brain (Sgamboto et al., 1998; Demir et al., 2009). Stimulation of primary neural or neuroblastoma cells results in translocation of the phosphorylated Elk-1 to the neuronal nucleus (Demir et al., 2009) and this was shown to suppress the expression of pro-apoptotic genes in neuroblastoma cells (Demir and Aksan Kurnaz, 2008). In mice deﬁcient for Elk-1, however, there appears to be only a mild neuronal impairment, most probably due to redundancy (Cesari et al., 2004). Therefore, Elk-1 is likely to serve a function beyond proliferation in neurons, probably in regulating survival (Ginty and Segal, 2002; Vickers et al., 2004) or neuronal apoptotis (Barrett et al., 2006a,b). Another transcription factor regulated by neurotrophins and ERK signaling is CREB (cAMP response element binding protein). In CREB/Bax double null mice (to prevent apoptosis), DRG and sympathetic neurons extend much shorter axons than wild type cells in the presence of NGF (Lonze et al., 2002). Elk-1 and CREB are phosphorylated in the dentate gyrus upon long-term potentiation (LTP) induction as a result of RTK activation (Davies et al., 2000). SIGNALING BY NEURONAL TYROSINE KINASE RECEPTORS Fig. 2. Transcriptional regulators inﬂuenced by receptor tyrosine kinase (RTK) signalling. RTK signaling pathways mainly operate through Ras/Raf/MAPK signal transduction or the PI3K signaling pathways, and create a wide range of responses in the cell, from neuronal survival to motor neuron positioning, from axon extension to learning and memory. Many transcription factors are targeted by RTKs to mediate those responses as well as feedback regulation, such as ETS domain proteins, MADS box proteins, Zn ﬁnger or Islet proteins, among many others. Also targeted by the RTKs are the MAPKAP kinase family members, most notably RSK2 histone kinase. Serum response factor (SRF), a transcriptional partner for Elk-1, plays an important role in neurons as well. SRF mediates neuronal survival via the PI3K/Akt pathway and was shown to rescue cells from apoptosis on serum withdrawal (Chang et al., 2004). Furthermore, SRF protects cortical neurons from DNA damage-induced apoptosis. Whether Elk-1 and SRF cooperate on these survival functions in neuronal systems is yet to be investigated. SRF is also necessary and sufﬁcient for NGFdependent axonal extension and branching by DRG sensory neurons (Wickramashinghe et al., 2008). Furthermore, SRF mediates activity-dependent gene expression and possibly morphological plasticity in adult neurons. Selective depletion of SRF in adult CA1 neurons, for instance, decreases LTP as well as immediateearly gene (IEG) induction upon novelty stimulation (which normally activates place cells of the hippocampus) without affecting survival of these neurons (Ramanan et al., 2005). Therefore, plasticity in the nervous system may not necessarily require a different set of transcriptional programs but rather utilize the existing survival, axon extension, and related mechanisms to adapt to changing conditions in the environment. An interesting group of ETS domain transcription factors regulated by RTK signaling is the Pea3 subfamily (Fig. 2), comprising Pea3, ERM, and Er81 (Sharrocks, 2001). Er81 is coexpressed with TrkC and Pea3 with TrkA (Arber et al., 2000). Pea3 is expressed in many tissues with branching morphogenesis and plays a role in cell body positioning and axonal connectivity. It is induced in motor neurons in response to GDNF and thus modiﬁes motor neuron positioning (Haase et al., 2002). Met signaling is important for expression of pea3 in brachial motor neurons and for their recruitment. In turn met expression is affected by GDNF and Pea3, 1981 thereby providing a positive feedback regulation to the system (Helmbacher et al., 2003). IGF-1 is yet another signal that is implicated in the regulation of ERM but not ER81 in PC12 neuronal differentiation model system. In proprioceptive sensory neurons, late onset of ETS and Er81 signaling is crucial for normal development of sensory afferent projections to the spinal cord, normally regulated by NGF or NT3-like signals (Hippenmeyer et al., 2005). However, precocious expression of EWS-Pea3 can lead to axonal projection defects—indicating tightly regulated temporal control for axonal growth at this stage of development. Er81 and Pea3 are not crucial for early stages of neural differentiation, because in Er81 mutant mice motor neurons did develop. However, group Ia proprioceptice afferents fail to form a discrete termination zone in the ventral spinal cord, thus a defect in motor coordination developed in mutant mice (Arber et al, 2000). It was further observed that Er81 and Pea3 expression are mutually exclusive in different motor neuron subpopulations of the lateral motor column (LMC) and these subpopulations appear to connect to developing DRGs that express the same proteins. The LIM homeodomain proteins Islet-1 and 2 (Fig. 2) that comprise functional partners for Pea3 proteins are expressed in different subsets of motor and interneurons, essentially regulating axonal pathﬁnding of motor neurons as well as neurotransmitter production. Interestingly, mice lacking Isl-1 exhibit a loss of neurons due to apoptosis (Thor and Thomas, 1997). Apoptosis and survival are two sides of the same coin, and many survival proteins such as Bcl2, Mcl2, Elk-1, ATF3 protect neurons against apoptotic stimuli during development. C-Jun itself participates in neuronal apoptosis upon NGF withdrawal, with c-Jun activation resulting in cytochrome release and subsequent apoptosis (Whitﬁeld et al., 2001). The POU (Pit-Oct-Unc) domain family member Brn-3 was shown to be required not for the initial speciﬁcation of sensory neurons, but rather for their normal differentiation and survival (Wang et al., 2002, and references therein). In Brn-3b/-3c double knockout mice, defects in axon growth go along with the elimination of optic ﬁbers; very few correctly routed ﬁbers were detected, and cell numbers were reduced in the ganglion cell layer (Wang et al., 2002), indicating that apoptotic stimuli could not be bypassed in the absence of Brn-3 transcription factors. In differentiated PC12 cells as well as in medullablastoma cells Brn-3a regulates the expression of TrkA receptors (Valderrama and Misra, 2008), providing a feedback control to the system (Fig. 2). Thus, a range of different transcriptional regulators work in concert to regulate differentiation, migration, cell body positioning, axonal growth, and survival of neurons during development. Some of them are crucial in the adult neuron as well, for example, CREB acts as an integration point for many GPCRs, Trks, NMDA receptors, and L-type calcium channels, and thus is implicated in a variety of different processes including learning, memory, addiction, depression, anxiety, and many other higher cognitive functions, as well as plasticity associated with these processes (for a detailed review on CREB, Carlezon et al., 2005). CREB also regulates the expression of proteins such as c-Fos, BDNF, or glutamate receptor GluR1, thereby providing yet another feedback control to the system. Similarly, another target 1982 HAUSOTT ET AL. of MAPK signaling implicated in LTP, learning and memory is the ribosomal S6 kinase, RSK2 (Sharma and Carew, 2004). RSK2 is a histone kinase (Merienne et al., 2001), and mutations in the rsk2 gene result in CofﬁnLowry syndrome (CLS), an X-linked disorder with mild mental retardation and facial abnormalities (Delaunoy et al., 2001). In summary, although each neuronal subtype is unique in its function, there are common themes in all of those subtypes both during embryonic development as well as in adult plasticity, and similar groups of transcriptional regulators and other proteins act as effectors of RTK signaling in almost all types of neurons to generate a plethora of responses and higher cognitive abilities. CONCLUSIONS AND FUTURE DIRECTIONS Despite signiﬁcant progress in our understanding of the molecular and cellular events induced by RTK activation, several key issues remain to be addressed before we can claim a comprehensive understanding of the neuronal functions of RTKs. Different cellular levels are now in the focus of many laboratories, aiming to elucidate the activation and recycling of RTKs, the binding and modiﬁcation of signaling molecules, and the regulation of nuclear events leading to changes in gene expression. Many of these studies ﬁnally aim at developing clinical treatments based on activation of RTKs to promote neuronal survival and axonal regeneration. In fact, neuroprotective effects could be achieved by treating traumatic or ischemic lesions, neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Huntington’s or Parkinson’s disease and various peripheral neuropathies with large amounts of RTK ligands in animal models. As soon as neurotrophins and other growth factors became available in recombinant form, immediate therapeutic success was expected similar to the success seen with erythropoietin in the hematopoietic system. However, early clinical trials with neurotrophins given to patients with neurodegenerative diseases did not fulﬁll the promise (Thoenen and Sendtner, 2002). Consequently, based on more detailed knowledge of the signal transduction pathways and transcriptional changes induced by RTK activation, a new effort to search for endogenous or pharmacological modulators of RTK signaling has been started. Exciting results of recent preclinical studies suggest a promising therapeutic potential of these small molecules activating RTK signaling in slowing the progression of neurodegenerative diseases. ACKNOWLEDGMENTS Authors apologize to all contributors to the ﬁeld whose work has not been cited due to space limitations. LITERATURE CITED Acconcia F, Sigismund S, Polo S. 2009. Ubiquitin in trafﬁcking: The network at work. Exp Cell Res 315:1610–1618. Aoki K, Nakamura T, Fujikawa K, Matsuda M. 2005. Local phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells. Mol Biol Cell 16: 2207–2217. 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