A novel Fibroblast Growth Factor Receptor family member promotes neuronal outgrowth and synaptic plasticity in Aplysia
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Fibroblast Growth Factor (FGF) Receptors (FGFRs) regulate essential biological processes, including embryogenesis, angiogenesis, cellular growth and memory-related long-term synaptic plasticity. Whereas canonical FGFRs depend exclusively on extracellular Immunoglobulin (Ig)-like domains for ligand binding, other receptor types, including members of the tropomyosin-receptor-kinase (Trk) family, use either Ig-like or Leucine-Rich Repeat (LRR) motifs, or both. Little is known, however, about the evolutionary events leading to the differential incorporation of LRR domains into Ig-containing tyrosine kinase receptors. Moreover, although FGFRs have been identified in many vertebrate species, few reports describe their existence in invertebrates. Information about the biological relevance of invertebrate FGFRs and evolutionary divergences between them and their vertebrate counterparts is therefore limited. Here, we characterized ApLRRTK, a neuronal cell-surface protein recently identified in Aplysia. We unveiled ApLRRTK as the first member of the FGFRs family deprived of Ig-like domains that instead contains extracellular LRR domains. We describe that ApLRRTK exhibits properties typical of canonical vertebrate FGFRs, including promotion of FGF activity, enhancement of neuritic outgrowth and signaling via MAPK and the transcription factor CREB. ApLRRTK also enhanced the synaptic efficiency of neurons known to mediate in vivo memory-related defensive behaviors. These data reveal a novel molecular regulator of neuronal function in invertebrates, provide the first evolutionary linkage between LRR proteins and FGFRs and unveil an unprecedented mechanism of FGFR gene diversification in primeval central nervous systems.
KeywordsFibroblast Growth Factor Receptor Leucine-Rich Repeat Nervous system Neuronal outgrowth Evolutionary divergence Synaptic plasticity
Fibroblast Growth Factors
Fibroblast Growth Factor Receptors
Fibroblast Growth Factor Receptors (FGFRs) are cell-surface tyrosine kinase proteins that regulate a variety of essential biological functions, including embryological development, cellular growth, nervous system formation, adult neurogenesis and learning-related long-term synaptic plasticity (Itoh and Ornitz 2004; Itoh 2007; Coulier et al. 1997; Lessmann 1998; Reuss and von Bohlen und Halbach O 2003; Stevens et al. 2012; Zhao et al. 2007). Canonical FGFRs have been classically characterized by two major features: First, the distinctive presence of an extracellular region containing two to three Ig-like domains implicated in the interaction and signaling by Fibroblast Growth Factors (FGFs). And second, the presence of a highly conserved intracellular tyrosine kinase domain responsible for their phosphorylative catalytic activity (Eswarakumar et al. 2005; Coulier et al. 1997; Powers et al. 2000; Itoh and Ornitz 2004). We here studied ApLRRTK, a previously uncharacterized Aplysia receptor recently identified by Kassabov et al. (GenBank: FJ969839.1 and GenBank: ADB97918.1 (Kassabov et al. 2013)). We used a cutting age bioinformatical approach to learn about the biological identity of ApLRRTK and identified ApLRRTK as a member of the FGFRs gene family. Combined molecular biological and functional electrophysiological studies corroborated the bioinformatics predictions and unveiled ApLRRTK as an enhancer of FGF signaling and a promoter of neuronal outgrowth and memory-related synaptic plasticity. These data reveal a completely novel molecular regulator in invertebrate nervous systems. Moreover, we provide the first direct evolutionary link between LRR proteins and FGFRs, thus offering a novel molecular perspective for the evolutionary origins and diversification of FGFRs and LRR tyrosine kinase receptors.
ApLRRTK is a member of the FGFRs gene family
ApLRRTK is expressed in nervous system neurons
Immunohistochemical, immunocytochemical, single-cell RT-PCR and in situ hybridization analyses indicated that ApLRRTK is expressed in the Aplysia nervous system and localizes at the cell-surface plasma membrane (Fig. 1b–d, Supplementary Data and Supplementary Fig. 3), in agreement with the bioinformatics predictions (Fig. 1a). Overexpression experiments using an ApLRRTK plasmidic construct (in fusion with innocuous Green Fluorescent Protein (GFP)) substantiated localization of ApLRRTK to the plasma membrane of sensory neurons, matching the expression pattern of FGFRs (Fig. 1b).
ApLRRTK promotes neuritic outgrowth and synaptic strengthening
Given the robust morphological phenotype induced by ApLRRTK, we sought to determine whether this structural remodeling was also accompanied by functional electrical changes in the synaptic strength of the sensory-motor neuron synapses. We therefore electrophysiologically measured presynaptically evoked excitatory postsynaptic potentials (EPSPs) as an index of synaptic strength (Kandel 2001) before and 24 h after presynaptic overexpression of ApLRRTK. Overexpression of ApLRRTK resulted in a significant increase of the basal amplitude of the evoked postsynaptic electrical response as compared to control levels (Fig. 2d). Taken together, these observations suggest that ApLRRTK may act in vivo as a regulator of neuritogenesis and synaptic strengthening in the Aplysia central nervous system.
On the basis of the remarkable capability of ApLRRTK to promote synaptic strengthening by itself, we next explored whether ApLRRTK overexpression would attenuate or saturate the enhancement in synaptic strengthening typically induced by serotonin (5-HT) in sensory-motor neuron co-cultures (Kandel 2001). 5HT is a neurotransmitter that acts as a promoter of both neuritogenesis and synaptic strengthening and mediates the sensitization of the gill-withdrawal reflex in Aplysia (Kandel 2001). We found that ApLRRTK acted as an enhancer of 5-HT activity, as presynaptic overexpression of ApLRRTK significantly augmented the long-term synaptic strengthening typically induced after stimulation with five pulses of 5-HT (Fig. 2d).
ApLRRTK shares conserved signaling features of FGFRs
We moreover examined electrophysiologically the effects of recombinant FGFs on ApLRRTK overexpressed in sensory-motor synapses reconstituted in vitro and found that ApLRRTK enhanced the effects of FGF on synaptic strengthening (Supplementary Data; Supplementary Fig. 4b). These observations are in agreement with previous reports relating LRR proteins with the actions of FGFs (Bottcher et al. 2004; Maretto et al. 2008) (see also Supplementary Data). Further observations (Supplementary Data) also suggested that ApLRRTK might act in vivo as a neuronal receptor for putative Aplysia FGF-like peptides thus sharing most of the highly conserved signaling features typical of canonical vertebrate FGFRs.
ApLRRTK promotes synaptic strengthening
To substantiate these hypotheses, we examined the effects of ApLRRTK inhibition via intracellular microinjection of two different ApLRRTK-specific antisense-oligonucleotides (As1 and As2) following methods that we previously described (Puthanveettil et al. 2008). Inhibition of ApLRRTK hampered the typical 5-HT-induced long-term synaptic facilitation and increased number of presynaptic varicosities growing on the target motor neuron. No effects were observed in experiments injecting control sense-oligonucleotides (Fig. 4a–c).
We next examined whether ApLRRTK function was also required for the 5-HT-induced short-term facilitation, a paradigmatic model for short-term memory storage. We intracellularly microinjected ApLRRTK antisense-oligonucleotides into sensory neurons as described before (Puthanveettil et al. 2008); and 4 h later stimulated the sensory-motor co-cultures with a single 5 min pulse of 5-HT to induce short-term facilitation as previously established (Kandel 2001). We observed that short-term facilitation was not affected by depleting the levels of ApLRRTK following protocols previously described (Puthanveettil et al. 2008) (Fig. 4d).
We moreover examined whether the postsynaptic inhibition of ApLRRTK could also impair 5-HT-induced long-term synaptic plasticity. We found that postsynaptic microinjection of antisense-oligonucleotides specific for ApLRRTK did not impair the 5-HT-induced long-term facilitation (Fig. 4e), thus indicating that ApLRRTK has a selective presynaptic role.
FGFRs are known to promote structural presynaptic reorganization, neuronal outgrowth, and memory-related neuronal synaptic strengthening (Eswarakumar et al. 2005; Coulier et al. 1997; Powers et al. 2000; Itoh and Ornitz 2004; Reuss and von Bohlen und Halbach 2003; Stevens et al. 2012; Zhao et al. 2007). Here, we revealed ApLRRTK as the first known member of the FGFRs family that contains extracellular LRR motifs. In addition, we unveiled a role of ApLRRTK as an enhancer of ligand signaling and as promoter of presynaptic reorganization, neuronal outgrowth and behaviorally relevant synaptic strengthening.
The most striking results deriving from the bioinformatics analysis are the establishment of ApLRRTK as a phylogenetic member of the FGFRs gene family and the fact that the extracellular domain of ApLRRTK does not contain the distinctive peptide-binding Ig domains present in all canonical vertebrate and invertebrate FGFRs identified so far. The basic question emerging is whether absence of Ig motifs would preclude ApLRRTK of having peptide-binding properties. Although ApLRRTK has no Ig domains in its ectodomain, bioinformatics analysis indicates that ApLRRTK contains instead several extracellular LRR motifs. While Ig domains are prominently known for the ability to mediate the binding of signaling peptides, also LRR motifs have been described to act as adaptor molecules for the binding of distinct types of signaling ligand peptides in a variety of proteins (Kobe and Deisenhofer 1994; Buchanan and Gay 1996), including Tyrosine Kinase Receptors (Windisch et al. 1995a; Windisch et al. 1995b). For example, LRR motifs can be found in the extracellular domain of vertebrate TrkA and TrkB receptors (Schneider and Schweiger 1991; Windisch et al. 1995a; Windisch et al. 1995c) were they mediate the binding of associated neurotrophins with affinities in the nanomolar range (Windisch et al. 1995c). The functional relevance of ligand-binding LRR motifs in tyrosine kinase receptors is further supported by reports on the transcription of TrkB receptor genes into endogenous splice variants of TrkB in which some -or all- the LRR motifs are eliminated, resulting in responsive receptors, incapable of binding their related ligands (Ninkina et al. 1997). Moreover, in invertebrate central nervous systems, a previous study by van Kesteren et al. reported the existence of neurotrophin-binding receptors that belong to the vertebrate Trk gene family of Tyrosine Kinase Receptors and that although constitutively lack Ig motifs, they contain LRR domains associated to the binding of neurotrophins (van Kesteren et al. 1998). All these observations demonstrate that Ig domains are not a requisite for the binding of signaling peptides in LRR-containing Tyrosine Kinase Receptors.
We hypothesize that the observed synaptic strengthening induced by ApLRRTK is mediated through the enhanced outgrowth of filopodial structures, which are known to allow the formation of synaptic contacts (Mattila and Lappalainen 2008) that are important for learning-related long-term synaptic strengthening (Cingolani and Goda 2008; Toni et al. 1999). In agreement with this hypothesis and supporting all the structural and functional commonalities between ApLRRTK and FGFRs, the promotion of filopodial growth and synaptogenesis induced by ApLRRTK in co-culture neurons resembles the presynaptic filopodial structures and synaptogenesis induced by FGFRs in co-cultured cells (Li et al. 2011). In a physiological context, ApLRRTK could also act as a plasticity-related inducible gene, since we further found that the levels of ApLRRTK mRNAs are increased in response to 5-HT (Supplementary Data and Supplementary Fig. 4f), which is in agreement with our previously published observations for neuronal proteins regulating synaptic functions (Puthanveettil et al. 2008).
Various models about the emergence of immunoglobulin-like domains of tyrosine kinase receptors have been proposed (Itoh and Ornitz 2004; Grassot et al. 2006). Similarly, different groups have addressed the evolutionary origins of FGFRs using both molecular and bioinformatics approaches (Agnes et al. 1997; Itoh and Ornitz 2004; Rebscher et al. 2009; Huang and Stern 2005; D’Aniello et al. 2008; Bertrand et al. 2014). However, in invertebrates, only few organisms have been described to have FGFRs (Coulier et al. 1997; Itoh and Ornitz 2004; Huang and Stern 2005; Agnes et al. 1997). For example, in the case of the Aplysia genus, despite abundant information on the genome, transcriptome (Moroz et al. 2006) and proteome (Monje et al. 2012), no canonical Ig-like-containing FGFRs have been identified thus far, to the best of our knowledge. Regulation of FGFs and FGFRs signaling by LRR proteins has proven to be important for several physiological processes, including embryogenesis, migration or axonal growth (Bottcher et al. 2004; Maretto et al. 2008; Morris et al. 2007; Skjerpen et al. 2002; Zhao et al. 2008; Zhen et al. 2012; Wang et al. 2003). Moreover, both LRR proteins and FGFs/FGFRs have been independently implicated in Alzheimer’s disease (Majercak et al. 2006; Tatebayashi et al. 1999), thereby highlighting the importance of clarifying the functional and evolutionary interrelationships between FGFs/FGFRs and LRR proteins. Previous studies have shown that diverse repertoires of immune-related receptors can be generated either through the rearrangement of immunoglobulin domains in some organisms, or exclusively through the recombinatorial assemblage of LRR modular units in other organisms lacking the immunoglobulin-based mechanism (Pancer and Cooper 2006). All these observations suggest the possibility that during evolution some primeval organisms might have become deprived of genes encoding for Ig-like containing FGFRs. Instead, those organisms might have diverged into alternative lineages in which transmembrane tyrosine kinase proteins belonging to the FGFRs gene family underwent the recombinatorial assemblage of genetic LRR modules resulting in the formation of FGFRs-family receptors serving functions analogue to those of canonical Ig-like containing FGFRs.
A large degree of attention has been given to Ig-like domains during the study of the evolutionary nature of tyrosine kinase receptors (Grassot et al. 2006; Rousset et al. 1995; Agnes et al. 1997; Itoh and Ornitz 2004; Itoh 2007; Coulier et al. 1997). Nevertheless, the importance of LRR in tyrosine kinase receptors evolution remains poorly investigated. Our work provides an unprecedented direct genetic linkage between LRR domains and FGFRs and highlights the biological relevance of LRR-containing receptors from the FGFRs gene family as critical regulators of neuronal function. These findings shed new light onto the evolutionary mechanisms of diversification of the FGFRs gene family based on the alternative presence of LRR motifs instead of Ig-like domains.
Materials and methods
See Supplementary Data for image analysis of structural changes; In Situ Hybridization; Semi-Quantitative RT-PCR; HEK-293 Cell Culture and Transfection; Western Blotting; MAPK and CREB inhibition; Immunoprecipitation; Immunocytochemistry; Immunohistochemistry; Protein Tyrosine kinase Assay.
We retrieved the tyrosine kinase-containing protein sequences (FGFR, NTRK, RET, and ROR) from the NCBI database. Sequences were identified by BLASTP searches using ApLRRTK (NP_001232922) and ApNTRK (NP_001232923) sequences as query. We downloaded the hidden markov model (HMM) for the TK domain from the Pfam database (Punta et al. 2012) and used hmmsearch3 (Eddy 2011) to extract the tyrosine kinase domains for all protein sequences. We then constructed the multiple amino acid sequence alignment for the tyrosine kinase domain sequences using MAFFT (Katoh and Standley 2013). Finally, we reconstructed the maximum likelihood phylogenetic tree and assessed clade supports using the IQ-TREE ultrafast bootstrap approximation (Minh et al. 2013). For tree reconstruction, we applied the LG + I + G + F substitution model as the best-fit model according to the Bayesian information criterion (Schwarz 1978). The resulting alignment and tree were visualized by Jalview (Waterhouse et al. 2009) and Figtree (http://tree.bio.ed.ac.uk/software/figtree/), respectively.
Aplysia neuronal culture, protein expressions and electrophysiology
ApLRRTK-GFP and ApLRRTK-ΔK-GPF cDNA constructs cloned into the pNEX3 expression vector (Kaang 1996) were a generous gift from Dr. Stefan Kassabov. Aplysia sensory-motor neuron co-cultures, electrophysiological and antisense-oligonucleotide inhibition experiments were carried out as previously described (Kandel 2001; Puthanveettil et al. 2008).
5-HT treatment of sensory neurons
Sensory neuron containing pleural ganglia were isolated and connective tissue removed after 2 h of protease (Sigma, St. Louis, MO, U.S.A.) treatment. Ganglia were plated in L-15 culture media overnight to allow recovery from protease treatment. The next day, culture media was washed out and ganglia were treated with five pulses of 5-HT, as previously described (Monje et al. 2012). RNA was extracted 1 and 6 h after 5-HT treatment and RT-PCR analysis was carried out as described above.
Differences between groups were analyzed by one-way ANOVA tests followed by Tukey–Kramer Multiple Comparisons Tests. An α-level of 0.05 was adopted in all instances. All analyses were carried out using BioStat 2009 professional software (AnalystSoft Inc).
We thank Dr. Stefan Kassabov, Dr. Sathya Puthanveettil, Kevin Karl, Dr. Craig H. Bailey and Prof. Eric Kandel at Columbia University for providing the ApLRRTK cDNA clone, expression constructs, antibodies and other reagents as well as their helpful comments during the preparation of this manuscript. We are also in debt to Mary Ann Gawinowicz at the Protein Chemistry Core Facility of Columbia University for carrying out mass spectrometric analysis. D.D.P. is supported by the Austrian Science Fund (FWF): P22424 and member of the special research network (SFB) 35. B.Q.M. was supported by the Austrian Science Fund FWF (I760-B17).
Conflict of interest
No conflict of interest declared.
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