Kek-6: A truncated-Trk-like receptor for Drosophila neurotrophin 2 regulates structural synaptic plasticity

Neurotrophism, structural plasticity, learning and long-term memory in mammals critically depend on neurotrophins binding Trk receptors to activate tyrosine kinase (TyrK) signaling, but Drosophila lacks full-length Trks, raising the question of how these processes occur in the fly. Paradoxically, truncated Trk isoforms lacking the TyrK predominate in the adult human brain, but whether they have neuronal functions independently of full-length Trks is unknown. Drosophila has TyrK-less Trk-family receptors, encoded by the kekkon (kek) genes, suggesting that evolutionarily conserved functions for this receptor class may exist. Here, we asked whether Keks function together with Drosophila neurotrophins (DNTs) at the larval glutamatergic neuromuscular junction (NMJ). We tested the eleven LRR and Ig-containing (LIG) proteins encoded in the Drosophila genome for expression in the central nervous system (CNS) and potential interaction with DNTs. Kek-6 is expressed in the CNS, interacts genetically with DNTs and can bind DNT2 in signaling assays and co-immunoprecipitations. Ligand binding is promiscuous, as Kek-6 can also bind DNT1, and Kek-2 and Kek-5 can also bind DNT2. In vivo, Kek-6 is found presynaptically in motoneurons, and DNT2 is produced by the muscle to function as a retrograde factor at the NMJ. Kek-6 and DNT2 regulate NMJ growth and synaptic structure. Evidence indicates that Kek-6 does not antagonise the alternative DNT2 receptor Toll-6. Instead, Kek-6 and Toll-6 interact physically, and together regulate structural synaptic plasticity and homeostasis. Using pull-down assays, we identified and validated CaMKII and VAP33A as intracellular partners of Kek-6, and show that they regulate NMJ growth and active zone formation downstream of DNT2 and Kek-6. The synaptic functions of Kek-6 could be evolutionarily conserved. This raises the intriguing possibility that a novel mechanism of structural synaptic plasticity involving truncated Trk-family receptors independently of TyrK signaling may also operate in the human brain.


Introduction
Brain plasticity, neurotrophism in development, structural and synaptic plasticity during learning and long-term memory in humans critically depend on the receptor TrkB binding its neurotrophin (NT) ligand BDNF [1,2]. During development, NTs and Trks regulate neuronal number and connectivity; subsequently BDNF and TrkB establish a reinforcing positive feedback loop promoting synaptic potentiation, and regulate the dynamic generation and elimination of synaptic boutons and dendritic spines in response to activity [1,[3][4][5]. Thus, NTs and Trks are fundamental to linking structure and function in the brain, and this is thought to depend mostly on the tyrosine kinase (TyrK) function of Trks. Through its intracellular TyrK domain, TrkB activates the Ras/MAPKinase, PI3Kinase/ AKT and PLCγ signaling pathways downstream [1,2]. Pre-synaptic targets include Synapsin, to trigger vesicle release [5]. Postsynaptic targets include NMDAR and CREB, essential for long-term potentiation, learning and long-term memory [1,5]. Paradoxically, full-length TrkB decays postnatally and TrkB homodimers are not formed in the adult mammalian brain [6][7][8][9][10]. Instead, the most abundant adult isoform is truncated TrkB-T1 lacking the TyrK [8][9][10]. Mutant mice lacking TrkB-T1 have anxiety, and in humans alterations in TrkB-T1 are linked to severe mental health disorders [11][12][13]. However, the neuronal functions of the truncated Trk isoforms are poorly understood.
No canonical, bona fide, full-length, TyrK Trk receptors have been found in Drosophila. However, neurotrophism, structural and synaptic plasticity, learning and long-term memory all occur in fruit-flies, implying that either in the course of evolution insects and humans found different molecular solutions to elicit equivalent functions, or that undiscovered mechanisms contribute to brain plasticity in both humans and fruit-flies. Finding out what happened to the Trks in Drosophila is important, as it could uncover novel fundamental mechanisms of structure-function relationships in any brain.
Trk receptors have long been searched for in Drosophila. Trks (TrkA,B,C) bear the unique combination of Cysteine Rich Repeats (CRR), Leucine Rich Repeats (LRR) and Immunoglobulin (Ig) domains extracellularly, and an intracellular TyrK domain (Fig 1A). Original searches focused on the TyrK domain, and identified DTrk and Dror as candidate Drosophila Trk homologues, but these are unlikely to bind neurotrophins. DTrk, also known as Off-Track (Otk) [14,15] lacks the LRR and CRR modules, it is kinase-dead and binds Semaphorins ( Fig  1A). Dror/Dnrk, like all Ror-family receptors, has an extracellular Frizzled/Kringle module instead [16][17][18] (Fig 1A). Subsequent proteomic analyses found no full-length, canonical Trk orthologues with the combination of LRR, CRR and Ig modules extracellularly and a Trk-family TyrK intracellularly in Drosophila [19][20][21]. However, phylogenetic analysis of the Trkreceptor superfamily identified the Drosophila Kekkons (Keks), lacking an intracellular TyrK domain, as closely related to the Trks [22] (Fig 1A and 1B). Trks and Keks both belong to the LIG family of proteins that contain extracellular ligand-binding LRR and Ig motifs [22,23]. There are 38 LIGs in humans, and amongst these are transmembrane proteins with a divergent intracellular domain lacking a TyrK or any conserved motifs [22]. There are 9 LIGs in Drosophila. Phylogenetic analysis clusters mammalian AMIGO, LINGO, LRIG and LRRC4 in one clade with Drosophila Lambik, and mammalian Trks in a separate clade together with Drosophila Keks (Kek1-6) [22] (Fig 1B). Keks are more similar to the Trks than all other vertebrate LIGs are to each other [22]. Keks have only been found in insects, and thus are remnant, conserved Trk-like receptors in fruit-flies.
There are 6 Keks in Drosophila, with a cytoplasmic tail lacking any remarkable conservation, except that Kek-1, -2, -5 and -6 have a PDZ domain that could bind cytoplasmic effectors [24]. Kek-1, -2, -5 and -6 are highly conserved in insects [24]. At least kek-1, kek-2 and kek-5 are expressed in the CNS [25][26][27]. Only Kek2 has been investigated in the CNS, where it functions as a neuronal activity dependent modulator of synaptic growth and activity [27]. The ligands for the Keks have not been identified.

Kek-6 is a truncated-Trk-like receptor that binds DNTs
To investigate if Drosophila LIGs might function as DNT receptors in the CNS, we first looked at their expression in embryos. CG15744 did not reveal expression above background in the ventral nerve cord (VNC) (Fig 1C); lambik and CG16974 mRNAs were absent from the VNC, but might be expressed in the Peripheral Nervous System (PNS) or muscle, respectively ( Fig  1C and 1D). kek1 and kek2 were expressed in VNC cells, and possibly in PNS cells too ( Fig  1D), as previously shown [25,27]. kek-3 and kek-4 transcripts were not detected in embryonic CNS. kek-5 expressed in the VNC [26], and we confirmed this with a kek5-GAL4 reporter, which also revealed PNS expression (Fig 1D). kek-6 transcripts were abundant in the VNC ( Fig 1D). Thus, amongst the 9 LIGs, Kek-1, -2, -5, and -6 could function in the CNS.
To test whether Keks could function downstream of DNTs in vivo, we took advantage of the cold semi-lethality of DNT1 41 DNT2 e03444 double mutants [38], and asked whether it could be rescued with the over-expression of keks in neurons. Over-expression of kek-1 and kek-4 in neurons (with elavGAL4) did not rescue, and kek-2 and kek-6 did most prominently (Fig 1E,  S1 Table). Thus, Kek-2 and Kek-6 could function downstream of DNTs.
To ask whether DNT ligands could bind Keks and induce signaling, as Keks lack the TyrK, to enable a signaling readout, we generated chimaeric receptors formed of the extracellular and transmembrane domains of the Keks fused to the intracellular domain of Toll-6 ( Fig 2A). Toll-6 uses a conserved TIR domain to activate Dif/NFκB signaling downstream, which can be measured through the activation of drosomicin-luciferase (dros-luc) [38]. We used S2 cells stably transfected with dros-luc, transfected them with kek-Toll-6 chimaeric receptors, and tested whether stimulation with purified cleaved DNT2 (DNT2-CK) could induce Dif/NFκB signaling. The chimaeric receptors targeted correctly to the S2 cell membrane (Fig 2A). Stimulation with DNT2-CK in pDONR transfected controls induced dros-luc, as S2 cells express multiple Toll receptors [36,38] (Fig 2A). Stimulation with DNT2-CK of cells transfected with kek3,4,5,6--Toll-6 chimaeras had an effect comparable to the induction by stimulated full-length Toll-6, and Kek3-Toll-6 and Kek6-Toll-6 chimaeras responded more robustly (Fig 2A, S1 Table). We could not generate Kek-1 and Kek-2 chimeric receptors, thus a potential interaction with them cannot be ruled out. Thus, DNT2 can interact physically with at least Kek-3 and Kek-6.
As Kek-6 was widely expressed in the CNS, rescued the semi-lethality of DNT1 DNT2 double mutants, and bound DNT ligands in both a signaling assay and in co-immunoprecipitations, from this point we focused on Kek-6. We asked whether Kek-6 could function as a DNT receptor. Kek-6 in motoneurons and DNT2 from the muscle function together at the NMJ For a functional in vivo analysis we focused on Kek-6. kek-6 CNS expression was examined in larvae using kek6 MIMIC13953 flies bearing a GFP insertion into the kek-6 coding region (hereafter named Kek6 GFP ). Kek6 GFP was found in HB9+( Fig 3A) and Eve+(S1A Fig) inter-neurons and motoneurons, and excluded from Repo+ glia (S1B Fig) in third instar larval VNCs. Kek6 GFP was present in motoneuron terminals at the neuro muscular junction (NMJ) of third instar larvae and not in the muscle (Fig 3B and 3C), revealing NMJ6/7 synaptic boutons, surrounded by post-synaptic Dlg (i.e. Drosophila PSD95) ( Fig 3C). To verify this, we swapped MIMIC for GAL4 using RMCE, and drove expression of the membrane tethered FlyBow reporter. kek6>FlyBow revealed abundant signal in CNS neurons, axons and dendrites, and in the cell body clusters of RP3,4,5 motoneurons ( Fig 3D). kek6>FlyBow was also present in motoraxons reaching the NMJ, and in synaptic boutons ( Fig 3F). No prominent signal was detected in the muscle above background levels. However, data do not exclude potential postsynaptic distribution in the CNS. To conclude, kek-6 is expressed pre-synaptically in motoneurons ( Fig 3E).
DNT2 transcripts are expressed in the larval body wall muscles, and localised to post-synaptic boutons [39]. To test if DNT2 could function retrogradely, we generated a tagged form of full-length DNT2 with GFP at the C-terminus, and over-expressed DNT2-FL-GFP in the muscle with MhcGAL4. DNT2-FL-GFP would result in secretion of mature DNT2-CK-GFP [36], although not all protein might get cleaved and secreted. Over-expression of DNT2-FL-GFP in muscle resulted in the localization of GFP pre-synaptically in boutons, surrounded by the post-synaptic marker Dlg ( Fig 3G). DNT2-FL-GFP also colocalised with the motoneuron marker FasII in boutons (Fig 3H, 3I and 3K) and motoraxons ( Fig 3I, 3J and 3K). Thus, DNT2 produced in muscle could get distributed to the motoneuron, consistent with a retrograde function.
Locomotion phenotypes suggested the NMJ might be affected, so we looked at the muscle 6/7 NMJs, which require DNT1 and 2 [39]. Targeting at the embryonic muscle 6/7 NMJ was affected in kek6 -/mutants and upon kek-6 over-expression in neurons (S3 Fig). In wandering third instar larvae, kek6 -/and DNT2 -/single mutant larvae, and kek6 -/-DNT2 -/double mutants, had smaller NMJs than controls, with fewer Ib boutons and shorter axonal terminal length (Fig 4F, S1 Table). All mutant genotypes also had reduced NMJ branching ( Fig 4F). Thus, Kek-6 and DNT2 are required for normal NMJ growth. kek6 -/and DNT2 -/single mutant NMJs had higher active zone density, visualized with anti-Brp (Drosophila ELKs) and quantified automatically throughout the NMJ stack of images using DeadEasy Synapse software [39] (Fig 4F, S1 Table). Since the NMJs were smaller, this suggested that the increase in active zones was a homeostatic compensation of defective synaptic function, to enable adequate behaviour. Homeostatic adjustments in active zones are a common manifestation of structural plasticity at the NMJ [47]. Remarkably, the increase in active zone density did not occur in kek-6 -/-DNT2 -/double mutants ( Fig 4F, S1 Table), meaning that compensation fails in the double mutants. To further test how Kek-6 affects the synapse, we also visualised Synapsin, which phosphorylates components of the SNARE complex to promote neurotransmitter vesicle release [48], and quantified it automatically using DeadEasy Synapse software [39]. kek6 -/mutants had reduced Synapsin production at synaptic locations ( Fig 4F, S1 Table), revealing defective synapse composition. These data show that: (1) Kek-6 is required for appropriate synaptic structure; (2) that in kek-6 -/mutants homeostatic compensation regulates Brp but not Synapsin; (3) and that DNT2 is involved in the compensation mechanism, as it fails in its absence.
Over-expression of kek-6 in motoneurons alone did not alter NMJ size, but increased branching ( Table). It also induced ghost boutons, albeit not significantly (similarly in kek-6 -/mutants, S4C-S4E Fig, S1 Table). Ghost boutons are presynaptic, HRP+ Dlg-negative, immature boutons that fail to get stabilized, and are correlates of increased neuronal activity. Together, these data are consistent with Kek-6 influencing synaptic structure and/or function. Over-expression of DNT2-FL in muscle increased axonal terminal size and branching (Figs 5B and 6B). Furthermore, overexpression of full-length DNT2-FL in muscle, and either full-length DNT2-FL or mature DNT2-CK in motoneurons (with D42GAL4), also increased active zones (Fig 5B and 5C, S1 Table). Thus, DNT2 can affect NMJ size and synaptic structure.
Data suggested that DNT2 may function as a retrograde ligand for Kek-6. To further test this, we used epistasis analysis. Over-expression of kek6 in motoneurons rescued the NMJ mutant phenotypes of bouton number and axonal length of kek6 34 /Df(3R)6361 mutants ( Fig  6A, S1 Table), demonstrating that the kek6 -/mutant phenotypes were specific. Over-expression of kek6 in motoneurons rescued the NMJ phenotypes of DNT2 37 /Df(3L)6092 mutants and kek6 -/-DNT2 -/double mutants (Fig 6B and 6C, S1 Table), demonstrating that Kek-6 functions Speed distribution classified into arbitrary categories. (C) Mutants spend more time at the lowest speeds than controls, generally do not crawl at the higher speeds (pale grey, left), but like controls can reach the highest speeds for a small fraction of time. (D) Wild-type larvae are hardly at speed = 0, contrary to the mutants. (E) All genotypes can achieve the highest speeds, but none spend much time crawling at these speeds. (F) NMJs (left, with higher magnification details of areas indicated by asterisks) and box-plot graphs (right) showing: kek-6 -/and DNT2 -/single mutants and kek-6 -/-DNT2 -/double mutants have fewer Dlg+ boutons, smaller HRP+ axonal terminals (normalized to muscle area, MSA), and less complex NMJs with reduced axonal branching. Dlg: Kruskal-Wallis p<0.0001, and *p<0.05, **p<0.01, ***p<0.001 post-hoc Dunn; HRP: One Way ANOVA p<0.0001, and **p<0.01, ***p<0.001 post-hoc Dunnett. kek-6 and DNT2 single mutants, but not the double mutants, have increased active zone density (Brp+/HRP +axonal length). Brp: Kruskal-Wallis p = 0.0012, and **p<0.01, ***p<0.001 Dunn's post-hoc. Kek-6 mutants have reduced Synapsin, Mann-Whitney U test ***p<0.001. For statistical details, see S1 Table. Table). Since Kek-6 is expressed in motoneurons and functions pre-synaptically, and DNT2 was over-expressed in muscle, this demonstrates that DNT2 is a retrograde ligand for Kek-6. Altogether, our data demonstrate that DNT2 is a retrograde ligand for Kek-6 at the NMJ and that DNT2 and Kek-6 are required together for synaptic structure and NMJ growth. Intriguingly, our data also suggested that DNT2 had additional functions compared to kek-6. Firstly, the homeostatic compensation of active zones seen in kek6 -/mutants did not occur in kek-6 -/-DNT2 -/double mutants ( Fig 4F). This suggests that homeostatic compensation depends on an alternative mechanism downstream of DNT2. Secondly, over-expression of DNT2 but not kek-6 increased NMJ size, as both HRP+ axonal terminal length ( Fig 5B) and bouton number (Fig 6B) increased when DNT2-FL was over-expressed from muscle. This suggests that a second mechanism downstream of DNT2 can influence NMJ growth. DNT2 is a known ligand of Toll-6 [38], and Toll-6 and -8 are required in motoneurons for NMJ growth and active zone formation [37,43]. So this raised two questions: do Toll-6 and Kek-6 interact functionally as DNT2 receptors at the NMJ, and why?
Toll-6 was shown to function at the larval NMJ [43], but its expression here had not been reported. Using a MIMIC insertion into the intronless coding region of Toll-6, we generated Toll-6GAL4 flies by RMCE. Toll-6>mCD8-GFP was distributed along the motoraxon of the muscle 6/7 NMJ ( Fig 7A). Like kek-6 -/and DNT2 -/mutants, Toll-6 MIO2127 / Df(3L)BSC578 mutants had smaller NMJs, with fewer 1b boutons (Fig 7B), shorter HRP+ axonal length (Fig 7B, S1 Table) and reduced branching ( Fig 7B) than controls. This confirms that Toll-6 is required for NMJ growth. Contrary to kek-6 -/mutants, Toll-6 -/mutants had reduced Brp + active zones compared to controls (Fig 7B), meaning that Toll-6 is required for active zone formation. kek-6 -/-Toll-6 -/double mutant larvae also had small NMJs (Fig 7B, S1 Table), with reduced branching complexity (Fig 7B), both consistently more pronounced than the single Toll-6 mutants when compared to controls. Importantly, Brp+ active zones remained lower than in controls and comparable to the levels of Toll-6 -/single mutants ( Fig 7B). Thus, the compensatory increase in active zones observed in kek-6 -/single mutants did not occur in the absence of Toll-6. Conversely, over-expression of Toll-6 did not alter NMJ size, but increased active zones (Fig 7B). Thus, Toll-6 can induce Brp+ active zone formation. Together, these data show that active zone formation and homeostasis depend on Toll-6.
Evidence indicated that Brp depends on Toll-6 and not Kek-6, but both Kek-6 and Toll-6 could induce Brp+ active zones. This could mean that Kek-6 might induce Brp+ indirectly via Toll-6. To test this, we asked whether Kek-6 could induce active zone formation in the absence of Toll-6. Over-expression of kek-6 presynaptically in motoneurons in Toll-6 -/mutants increased Brp+ (Fig 7B, yellow). This demonstrates that Kek-6 can induce active zone formation independently of Toll-6.
These results revealed that: 1) Loss of both kek-6 -/and Toll-6 -/more robustly reduced NMJ size and complexity than loss of Toll-6 -/alone, suggesting that Kek-6 contributes to NMJ growth via an alternative mechanism independently of Toll-6. 2) NMJ growth requires DNT2, Kek-6 and Toll-6. However, over-expression of DNT2 had a much stronger effect than overexpressing either kek-6 or Toll-6 alone. This means that NMJ growth is regulated by the concerted functions of both DNT2 receptors, Toll-6 and Kek-6. 3) Synaptic structure requires Toll-6 and Kek-6, but via distinct parallel mechanisms. 4) The compensatory increase in active zones observed in kek-6 -/and DNT2 -/single mutants did not occur in kek-6 -/-DNT2 -/or kek-6 -/-Toll-6 -/double mutants, meaning that the concerted functions of Toll-6 and Kek-6 are required for NMJ structural synaptic homeostasis. 5) Since Toll-6 is required for active zone formation and NMJ growth, and over-expression of kek-6 increased active zones and did not compromise NMJ growth, Kek-6 does not function as a ligand sink, an inhibitor or dominant negative co-receptor to abrogate Toll-6 function.
The data suggested that Kek-6 and Toll-6 can function together forming a receptor complex for DNT2. To test whether Kek-6 and Toll-6 might physically interact, we carried out coimmunoprecipitations. S2 cells were co-transfected with the active forms Toll-6 CY -Flag and Kek6-HA, and we found that precipitating Toll-6 CY with anti-Flag brought down Kek6 detected with anti-HA (Fig 7C). Similarly, precipitating Toll-7 CY also brought down Kek6 ( Fig  7C). Thus, Kek6 can bind Toll-6, and also Toll-7.
Altogether, these data showed that retrograde DNT2 can bind Toll-6 and Kek-6, which can interact pre-synaptically to form a receptor complex. They cooperatively promote NMJ growth and regulate synaptic structure. This raised a new question: if Kek-6 could influence NMJ growth and synaptic structure independently of Toll-6, and without a TyrK, how might it function?
Kek-6 functions via CaMKII and VAP33A To find out how Kek-6 might function, we carried out pull-down assays to isolate candidate factors binding its intracellular domain. S2 cells were transfected with kek6-Flag, and anti-Flag coated beads were used to expose Kek-6 to cell lysates from either S2 cells or wild-type adult fly heads, and bound proteins were isolated by SDS-PAGE followed by mass spectrometry (Fig  8A and 8B). Candidates were identified as proteins present in Kek6-Flag samples and absent from non-transfected mock controls, and if identified from multiple peptides (S2 and S3 Tables). Prevalent amongst these were proteins involved in vesicle trafficking, axonogenesis, dendrite morphogenesis and synaptic function (Fig 8C). Amongst the top hits were CaMKinase II identified from fly heads, and VAP33A from both S2 cells and heads (Fig 8C). CaMKII functions both as a kinase and a scaffolding protein, to promote structural synaptic plasticity. Post-synaptically, it phosphorylates and recruits AMPAR and NMDAR to the post-synaptic density, leading to post-synaptic potentiation, and pre-synaptically it localizes to active zones and phosphorylates Synapsin and other SNARE complex proteins, triggering neurotransmitter release [48][49][50][51]. VAP33A is a Vamp Associated Protein, with evolutionarily conserved functions in exocytosis and vesicle trafficking, at synapses and in wider contexts [52]. To validate these two candidates as downstream effectors of Kek-6, we carried out co-immunoprecipitations. We co-transfected S2 cells with Kek6-Flag and either CaMKII or VAP33A tagged with HA. Precipitating Kek-6-Flag, brought down CaMKII-HA ( Fig 8D). Similarly, precipitating Kek-6-Flag also brought down VAP33A-HA ( Fig 8D). Thus, Kek-6 can bind CaMKII and VAP33A.
To validate the functional relationship between Kek-6 and CaMKII in vivo, we asked whether altering Kek-6 function would affect CaMKII activation in brain. We tested for the constitutively active state of CaMKII, corresponding to phosphorylation at Thr286-T287 in Drosophila-using antibodies that detect pCaMKII T287 . In the heads of kek6 34 /Df(3R)ED6361 mutant adult flies the relative levels of pCaMKII T287 , normalised over non-phosphorylated CaMKII, decreased (Fig 8E, S1 Table). Conversely, over-expression of kek-6 in retina (with GMRGAL4) increased CaMKII phosphorylation (Fig 8E, S1 Table). Over-expressing DNT2-FL had the same effect ( Fig 8E). Thus, Kek-6 is required for CaMKII activation, and both Kek-6 and DNT2 can activate CaMKII downstream.
Next we asked whether CaMKII and VAP33A might function downstream of Kek6 at the NMJ. Unfortunately, we were not able to get antibodies to inactive CaMKII to work reliably at the NMJ for normalisation, so we visualised constitutively activated CaMKII with anti-pCaM-KII T287 and quantified it automatically, using DeadEasy Synapse [39]. DeadEasy Synapse  S5A and S5C Fig). Over-expression of CaMKII T287 presynaptically in motoneurons increased axonal length, Ib bouton number and active zone density (S5A and S5C Fig, S1 Table). Conversely, inhibiting CaMKII only pre-synaptically by over-expressing the CaMKII phosphorylation inhibitor Ala [53] in motoneurons resulted in smaller NMJs and reduced active zones (S5A and S5B Fig, S1 Table), as previously reported [54]. Thus we asked whether Kek-6 influenced CaMKII at the NMJ. Pre-synaptic over-expression of kek-6 in motoneurons increased pCaMKII T287 levels (D42>kek6 Fig 9A, S1 Table). This increase was rescued by the pre-synaptic over-expression of Ala together with kek-6 (D42>kek6, Ala) showing that this phenotype was specific (Fig 9A, S1 Table). However, pre-synaptic over-expression in motoneurons of Ala alone (D42>Ala) did not result in a detectable reduction in pCaMKII in this case (Fig 9A, S1 Table). To try an alternative approach, we knocked-down pre-synaptic CaMKII expression with RNAi in motoneurons, and this decreased overall pCaMKII T287 levels, causing a stronger effect than Ala (D42>CaMKII-RNAi, Fig 9B S1 Table). Like Ala, pre-synaptic CaMKII-RNAi knock-down together with kek-6 overexpression in motoneurons also rescued pCaMKII T287 levels, and reduced them further than controls (D42>kek6, CaMKII-RNAi, Fig 9B, S1 Table). This demonstrates that CaMKII functions downstream of Kek-6 in motoneurons. Together, these data show that Kek-6 function results in the activation of CaMKII pre-synaptically.
Pre-synaptic activated CaMKII localizes active zones [49,55], so next we asked whether CaMKII was required for the increased active zones caused by kek-6 gain of function. Pre-synaptic CaMKII inhibition with Ala or knock-down with RNAi in motoneurons decreased active zones (S5A and S5B Fig and Fig 9D, S1 Table) and over-expression of activated CaMKII in motoneurons increased active zones (S5A and S5C Fig, S1 Table). Remarkably, pre-synaptic over-expression in motoneurons of Ala or CaMKII-RNAi together with kek-6, rescued the increase in active zones caused by kek-6 over-expression (Fig 9C and 9D, S1 Table). These showed that CaMKII is required downstream of Kek-6 for active zone formation. Furthermore, in kek6 -/mutants and kek6 -/-DNT2 -/double mutants the levels of pCaMKII T287 decreased (Fig 10A, S1 Table), showing that Kek-6 is required for CaMKII activation at the NMJ. We found no significant effect of DNT2 loss or gain of function on pCaMKII T287 levels at the NMJ. Importantly, over-expressing activated CaMKII T287D pre-synaptically rescued the phenotypes of decreased axonal length and reduced Ib boutons of kek6 -/- (Fig 10B) and DNT2 -/- (Fig 10C) single mutants, and kek6 -/-DNT2 -/double mutants (Fig 10D, S1 Table). This means that the mutant phenotypes were caused, at least partly, by decreased CaMKII activation. Together, these data demonstrate that Kek-6 and DNT2 function in concert upstream of CaMKII to regulate NMJ size and active zones.
To conclude, altogether these data show that CaMKII and VAP33A function downstream of DNT2 and Kek-6 in motoneurons.

Discussion
Drosophila homologues of the Trk receptor family had long been sought. This was important to find fundamental principles linking structure and function in any brain. Here, we show that Keks are Trk-family receptors lacking a TyrK, for Drosophila neurotrophin ligands (DNTs) (Fig 12A). Kek-2, -5 and -6 are expressed in the CNS, and genetic rescues, a signaling assay and co-immunoprecipitations demonstrate that they bind DNTs promiscuously. For in vivo analyses we focused on Kek-6. We demonstrate that motoneurons express kek-6 pre-synaptically, and Kek-6 binds DNT2, which is produced by the muscle from post-synaptic boutons (Fig 12B). kek-6 -/and DNT2 -/share mutant phenotypes, and using genetics we demonstrate they are functionally linked in vivo. Kek-6 can interact physically with the alternative DNT2 receptor, Toll-6. Most likely Kek-6 and Toll-6 function as a DNT2 receptor complex (Fig 12C). We show that: (1) The concerted functions of Toll-6 and Kek-6 are required to regulate NMJ growth. All single mutants-kek-6 -/-, Toll-6 -/and DNT2 -/--decreased NMJ size and complexity (Fig 12D and 12E), but only over-expression of DNT2 could induce a dramatic increase in NMJ size (Fig 12F). This shows that the compound activation of both receptors by DNT2 has a stronger effect on NMJ growth that activating each alone (Fig 12F). (2) Kek-6 and Toll-6 can each regulate active zone formation via alternative pathways, as Kek-6 can promote active zone formation independently of Toll-6 ( Fig 12C). The ability of both Kek-6 and Toll-6 to regulate active zones enables homeostatic compensation in single mutants (Fig 12D). We show that Kek-6 functions by recruiting CaMKII and VAP33A to a pre-snaptic downstream complex and induces CaMKII activation. Epistasis analysis demonstrated that Kek-6 functions in motoneurons downstream of DNT2 and upstream of CaMKII and VAP33A to promote both NMJ growth, and active zone formation (Fig 12C). We conclude that at the NMJ, Kek-6 is a pre-synaptic receptor for DNT2, regulating structural synaptic plasticity (Fig 12A-12C).

DNT2 is a retrograde factor at the NMJ
Despite abundant evidence that retrograde signals and positive feedback loops regulate structural synaptic plasticity at the Drosophila glutamatergic NMJ, the identification of retrograde factors had been scarce [45,56]. This created a void for the general understanding of structural synaptic plasticity across different brain types. BDNF is a key retrograde ligand in mammals, and together with its receptor TrkB they form a positive feedback loop that reinforces synaptic function [1,4]. We have shown that the neurotrophin DNT2 is a retrograde ligand for Kek-6 ( Fig 12B). DNT2 is expressed in muscle and both its receptors kek-6 and Toll-6 are expressed in motoneurons. Over-expression of DNT2 in muscle leads to its distribution in pre-synaptic boutons; over-expression of DNT2 in muscle induces NMJ growth and active zone formation pre-synaptically; DNT2 mutants have smaller NMJs; pre-synaptic over-expression of either kek-6, CaMKII or VAP33A can rescue the DNT2 loss of function phenotype; and conversely, kek6 loss of function rescues the phenotype caused by DNT2 over-expression in muscle. Thus, DNT2 is an evolutionarily conserved retrograde factor that regulates structural synaptic plasticity in Drosophila. This finding will have an impact bridging the universal understanding of synaptic plasticity and potentiation.

Keks are the Trk-family receptors in Drosophila
We demonstrate that keks are the Drosophila Trk-family homologues (Fig 12A). Previous searches for Trk-family receptors in fruitflies had focused on the Tryrosine Kinase domain, and did not identify any bona fide candidates [14][15][16]18]. Subsequent proteomic analyses confirmed the absence of full-length Trk receptors with a conserved tyrosine kinase in fruitflies [19][20][21]. Since those earlier searches, Trks in mammals have been found to encode multiple isoforms, most of which lack the tyrosine kinase domain [6,57]. Human TrkB has 100 isoforms, which produce 36 different proteins, of which four are abundant. Importantly, the most abundant isoform in the adult human brain is truncated TrkB-T1, which lacks the tyrosine kinase [6,57]. Paradoxically, the function of TrkB-T1 in neurons in the mammalian brain is unknown. Our findings suggest that truncated Trk family receptors could have an evolutionarily conserved function regulating structural synaptic plasticity.
We demonstrate that Kek-6 can activate CaMKII downstream (Fig 12B and 12C). However, we do not yet know how this may come about. CaMKII activation depends on the intracellular increase in Ca 2+ levels, either through Ca 2+ channels, or Ca 2+ stores through the PLC and IP3 pathways [50,55]. In mammals, TrkB-T1 regulates Ca 2+ levels in glia and in the heart, two contexts expressing high levels of TrkB-T1 and no TrkB-FL [58][59][60]. However, the mechanism by which TrkB-T1 raises Ca 2+ is unknown [58,59]. Furthermore, whether this results in the activation of CaMKII is unclear or may be context dependent [58,60]. CaMKII activation in neurons could depend on the increase in Ca 2+ with neuronal activity. A potential link of Kek-6 to neuronal activity was revealed by the increase in ghost boutons upon alterations in kek-6 levels (S4 Fig). Furthermore, kek-2 expression is modulated by neuronal activity, appropriate Kek-2 levels at the NMJ are required for normal synaptic structure, and DNT2 can modulate the Na +/K+ ATPase [27,61]. Thus, Kek-6 could function via a PLCβ pathway or by influencing membrane channels, to increase Ca 2+ levels and induce CaMKII activation.
The finding that Trk-like receptors can regulate neuronal plasticity independently of kinase signaling is very important. Activation of PLCγ by TrkB TyrK signaling is generally seen as the key mechanism of Trk-depedent plasticity [1,5]. Yet CaMKII is necessary and sufficient for synaptic structural plasticity, LTP and long-term memory, and has wide functions in synaptic organization and homeostasis [49][50][51]. CaMKII can function as a frequency detector, and cause long lasting changes in synaptic strength, structure and brain plasticity [50]. Thus, the regulation of CaMKII and Ca 2+ by truncated Trk-family receptors and Keks means that they could regulate structural synaptic plasticity independently of the canonical TyrK-dependent PLCγ pathway.
Keks are not identical to truncated Trk receptors and may carry out further functions that could be implemented via other routes in mammals. TrkB-T1 has a very short intracellular domain, whereas Kek-2, -5 and -6 have longer intracellular fragments that include a PSD95/ Dlg/ZO1 (PDZ) motif [22,24,25]. PDZ domains are involved in scaffolding, and assembly of post-synaptic complexes that regulate the size and strength of synapses [62]. Through its PDZ domain, Kek-6 might recruit synaptic partners and/or Ca 2+ channels. Interestingly, truncated TrkC-T1 binds the PDZ-containing protein Tamalin to induce cellular protrusions via Rac [63]. Exploring further signaling mechanisms of Keks vs. truncated Trks could uncover novel mechanisms of synaptic plasticity, perhaps also in the human brain.
Novel mechanism of structural synaptic plasticity involving concerted functions of DNTs, Trk and Toll receptor families LIG proteins and truncated Trk isoforms can function as 'dominant negative' co-receptors or 'ligand sinks'. TrkB-T1 can form dimers with TrkB-FL and abrogate signaling, and bind BDNF rendering it unavailable to TrkB-FL [6,7]. This is thought to modulate kinase-signaling levels by TrkB [64]. Similarly, in Drosophila, Kek-5 is an inhibitor of BMP signaling [26] and Kek-1 an inhibitor of EGFR signaling [65]. Thus, each Kek could function as a specific inhibitor of different signaling pathways. By contrast, our evidence shows that Kek-6 does not functions via CaMKII and VAP33A downstream, the mechanism downstream of Toll-6 at the NMJ has not been investigated in this work. Red arrows: positive regulation by Kek-6; blue arrows: positive regulation by Toll-6. (D-F) Summary of the experimental evidence provided, green arrows indicate upor down-regulation as a result or loss or gain function genotypes. Altering the levels of DNT2, Kek-6 and Toll-6 affects locomotion, NMJ growth and synaptic structure. Importantly, loss of both kek-6 and Toll-6 prevents homeostatic compensation of active zones, and whereas gain of function for kek-6 or Toll-6 is not sufficient to increase NMJ size, over-expression of DNT2 can. The data suggest that Kek-6 and Toll-6 function in concert as a receptor complex for DNT2, to regulate structural synaptic plasticity.
https://doi.org/10.1371/journal.pgen.1006968.g012 function as an inhibitor of Toll-6. Instead, it indicates that Kek-6 and Toll-6 function in concert to regulate NMJ structure and growth (Fig 12C). Firstly, kek-6 -/-, Toll-6 -/and DNT2 -/single mutants, and kek-6 -/-Toll-6 -/double mutants, all have smaller NMJs, ruling out antagonistic functions between the two receptor types (Fig 12D and 12E). Secondly, whereas over-expression of kek-6 or Toll-6 alone did not increase NMJ size, over-expression of DNT2 did, showing that the activation of both receptor types at once has a stronger effect than each alone (Fig 12F). This also rules out an inhibitory function. Thirdly, Toll-6 is required for active zone formation, and over-expression of either Toll-6 or kek-6 increases active zones (Fig 12C-12F). This implies that Kek-6 does not inhibit Toll-6. Instead, DNT2 can modulate the homeostatic compensation of active zones, via its two receptors (Fig 12D and 12E). Indeed, compensation fails in kek6 -/ -DNT2 -/and kek-6 -/-Toll-6 -/double mutants (Fig 12D and 12E). The data suggest that Kek-6 and Toll-6 influence synaptic structure via alternative, independent pathways. Toll-6 generally functions in neurons via NFκB and JNK [36], and at the NMJ Toll-6 and Toll-8 regulate structural synaptic plasticity through Sarm, JNK and dFOXO [37,43]. Here, we show that instead, Kek-6 functions via CaMKII and VAP33A to regulate active zones and synaptic structure ( Fig  12B and 12C). Kek-6 functions independently of Toll-6, as it can induce active zone formation in Toll-6 mutants; the induction of active zones downstream of Kek-6 depends on CaMKII; and there is no evidence that Toll-6 can activate CaMKII on its own. Furthermore, Kek-6 also affects other synaptic factors, like Synapsin. We have shown that Kek-6 and Toll-6 can interact physically, thus could form a receptor complex for DNT2. We conclude that DNT2 regulates NMJ size and active zone formation, by engaging a receptor complex that drives distinct parallel pathways downstream (Fig 12C).
An intriguing observation is that DNTs can bind various Tolls [38] and Keks (this work) promiscuously, and similarly multiple Tolls can bind Kek-6. The biological significance of such promiscuity is not understood. Whether this reflects redundancy will depend on the spatial, cell-type specific and temporal distribution of each ligand and receptor. Redundancy can serve to compensate for deficits, thus conferring robustness to the CNS and resulting behaviour. For instance, homeostatic compensation of active zones vs. NMJ size, which was also observed in Toll-8 mutants [37]. Either way, our findings show that DNTs, Keks and Tolls working together constitute a novel molecular mechanism for synaptic structural plasticity and homeostasis. Whether this could be mirrored by the mammalian NTs, Truncated Trks and TLRs, will be intriguing and important to find out.
To conclude, evolution may have resolved how to implement structural synaptic plasticity through distinct mechanisms in fruit-flies and humans, or perhaps common molecular principles are shared by both, even if not in every detail. We found that in fruitflies, truncated-Trklike receptors encoded by the Keks bind neurotrophin ligands to regulate structural synaptic plasticity via CaMKII and VAP33A, and the receptor complex also includes Tolls. It is compelling to consider whether such a non-canonical mechanism of neuronal plasticity, downstream of neurotrophins and Trk receptors but independently of kinase signaling, may also operate in humans, as it could uncover novel mechanisms of brain function and brain disease.

Luciferase signaling in S2 cells
For Dif signaling assays using chimaeric kek-Toll-6 receptors, S2 cells stably transfected with drosomycin-luciferase were maintained at 27˚C in air in Insect-Xpress medium (Lonza) supplemented with penicillin/streptomycin/L-glutamine mix (Lonza) and 10% foetal bovine serum (Lonza). 1ml suspended cells were passaged every two days into 4ml fresh medium. 3x10 6 cells in 2ml media were seeded per well of a 6-well plate 24 hours prior to transfection. Per well of experiment, 250μl serum-free media, 3μl TransIT-2020 (Mirus), 2μg of HA tagged chimaeric receptors kek-toll6-HA plus 1μg of pAct-renilla-luciferase were incubated at room temperature for 30 minutes, supplemented with 350μl serum-free media and added to aspirated cells. After 4 hours, transfection mixture was removed and 2ml supplemented medium added. All experiments were conducted 48 hours after transfection; for imaging membrane targeting of Kek-Toll6-HA protein S2 cells were starved for 6h prior to fixation.
For signaling assays, S2 cells were stimulated with 50nM/well of purified baculovirus DNT2 protein (generated as previously described [38]) and Dif signaling quantified by luminescence. DNT2 was added 48 hours after transfection and luminescence quantified 24 hours after DNT stimulation. Transfected and stimulated cells were pelleted from single wells, resuspended in 400μl media and separated into three 50μl aliquots in an opaque 96-well plate. 40μl of Firefly Luciferase Substrate (Dual-Glo Luciferase Assay System; Promega) was added per 50μl aliquot, incubated for 10 minutes, and luminescence measured using a Mithras LB 940 Multimode Microplate Reader (Berthold). 40μl Stop & Glo substrate (Dual-Glo Luciferase Assay System; Promega) was added to quench Dif signal and activate Renilla Luciferase. Renilla luminescence data was used to normalize Firefly Luciferase data.

Co-immunoprecipitations
Coimmunoprecipitations were carried out as previously described [38], after transfecting standard S2 cells with the following constructs: (1)  Pull-down assays and proteomics S2 cells were transfected with 4 μg pAct5C-Kek6-3xFlag expression construct. For controls mock-transfected cells (i.e. no construct) were used. After transfection cells were processed as described above for co-immunoprecipitation. 600 μL of cell lysates were incubated with 20 μL of anti-Flag conjugated magnetic beads overnight at 4˚C. Beads were washed thoroughly in PBS, then proteins were eluted in 40 μL 2x Laemmli-buffer for 10 min at room temperature. Eluted proteins were loaded onto 10% polyacryalamide gels. Alternatively, proteins were not eluted after overnight incubation with anti-Flag beads, but they were re-incubated with whole fly (OregonR) head lysates. Here, 60 heads were lysed in 600 μL of NP-40 buffer. After overnight incubation at 4˚C, proteins were eluted and analysed as for S2 cell lysate proteins. Thus, eluted proteins were loaded onto 10% polyacryalamide gels, and the gels were stained with Coomassie Blue and cut into several pieces. Gel pieces were subjected to in-gel digestion with trypsin using a standard protocol. Tryptic peptides were analysed by LC-MS/MS using Ultimate 3000 HPLC coupled to a LTQ Orbitrap Velos ETD mass-spectrometer. Peptide separation, mass spectrometric analysis and database search were carried out as specified at the University of Birmingham Proteomics Facility. Candidate binding proteins were identified on these criteria: (1) Proteins were accepted only if they were identified with at least two high confident peptides. (2) Mock-transfected controls and Kek6-3xFlag samples were compared, and only proteins identified from Kek6-3xFlag lysates or heads, but absent form controls, were considered as possible interacting partners for Kek6.

Western blotting
Western blot was carried out following standard procedures using antibodies listed here:

NMJ dissections and preparation
NMJ preparations were carried out according to [67]. For GFP stainings in MhcGAL4>UAS-DNT2-FL-GFP, L3 larvae were placed in agar plates (2%) and left at 29˚C for 90 minutes to potentiate the NMJ before dissections [68]. A hundred and twenty female flies and 50 males were placed in a cage with a removable agar and grape juice plate. On the second day, plates were changed for new ones in the morning and evening, and discarded. On the third day, the plates were changed every 1h 30min and discarded. On the fourth day, the plates were changed every 1h 30min and kept in a 25˚C incubator for larvae collection. The next day, hatched L1 larvae from each plate were transferred to a vial, and exactly 40 larvae were placed in each vial. When larvae reached L3 stage (5 days after egg laying), they were dissected in low calcium saline as previously described [67], and fixed for 10 minutes in Bouin's solution (HT10132 SIGMA). Samples were washed 6 times for 10 minutes in PBT (0.1% of Triton in 1M PBS) to remove fixative solution and kept overnight in blocking solution (10% normal goat serum in 0.1% Triton in PBS 1M). Primary antibodies were incubated overnight at 4˚C and samples were washed the following day 8 times for 10 minutes in PBT. Secondary antibodies were incubated for 2h at room temperature and samples were washed 8 times for 10 minutes in PBT. Samples were mounted in Vectashield anti-bleacing medium (Vector Labs) in No.1 coverslips. NMJs were analysed for muscle 6/7 only; segments A3 and A4, left and right, were analysed in each larva.

Microscopy and imaging
Wide-field Nomarski optics images were taken with a Zeiss Axioplan microscope, 63x lens and JVC 3CCD camera and Image Grabber graffics card (Neotech) and a Zeiss AxioCam HRc camera and Zen software. Laser scanning confocal microscopy was carried out using inverted Leica SP2 AOBS, upright Leica SP8 or inverted Zeiss LSM710 laser scanning confocal microscopes, with 40x lens, 1024 x 1024 resolution, 0.25μm step for anti-Brp, and 0.5 μm step for the rest. Images were compiled using ImageJ, Adobe Photoshop and Illustrator.
For NMJ data, all images provided in the figures are projections from Z stacks; all the quantitative anlyses were carried out in the raw stacks, not in projections. Muscle surface area was measured from bright field images using ImageJ. Anti-HRP was used to measure total terminal axonal length using ImageJ, and branching points. Boutons were visualized with anti-Dlg, and total boutons as well as separately Is and Ib boutons, were counted manually with the aid of the ImageJ Cell Counter plug-in. Automatic quantification of anti-Brp and anti-pCaMKII T286 were carried out in 3D throughout the stacks of images using the DeadEasy Synapse ImageJ plug-in that we previously validated [39], and anti-Synapsin was analysed with a slightly modified version.
DeadEasy Synapse has already been described and validated [39]. DeadEasy Synapse first reduces the Poisson noise, characteristic of confocal microscopy images, of each slice in the stack using a median filter. Subsequently, in order to separate signal (e.g. Brp+ active zones) from the background, images are segmented. Since the intensity of the staining varied within each image and from image to image, the maximum entropy threshold method [69] was used to find a local optimal threshold value for each pixel. For this, we used a square window of size 15x15, sufficient to find the local optimal threshold around each pixel in an image. In this way, each pixel is considered part of an active zone if the value of the pixel is higher than the local threshold, otherwise assigned to the background. Since this method is computationally expensive and very low intensity pixels correspond to background, it was possible to reduce the computation time by assigning pixels whose intensity was lower or equal than 20 directly as part of the background and only applying the thresholding method to pixels whose intensity was higher than 20. Finally the volume of the active zones is measured. This method worked just as accurately with anti-Brp, anti-pCaMKII T286 and anti-Synapsin stainings. Data were normalized to muscle surface area or axonal length.

Locomotion assays
L3 wandering larvae were placed one at a time on an agar plate (2%) and left it crawl for 40 seconds. Larvae were filmed crawling across the agar plate and then discarded. Plates were cleared before placing another larva. At least 50 larvae were filmed per genotype, and the test was always done in the morning. Films of 400 frames per larva were analysed using FlyTracker software developed in our lab to obtain the trajectory and speed, as previously described [38].

Statistical analysis
Data were analysed in SPSS Statistics 21 (IBM) and GraphPad Prism 6. Confidence interval was 95% (p<0.05). Categorical data were tested using χ 2 , and a Bonferroni correction was applied for multiple comparisons. Continuous data were first tested for normality by determining the kurtosis and skewness, and a Levene's Test was applied to test for homogeneity of variance. Data were considered not normally distributed if absolute kurtosis and skewness values for each genotype were greater than 1.96 x standard error of kurtosis/skewness. Variance of the populations of different samples were considered unequal if Levene's test for homogeneity of variance gave a p value of <0.05. If samples were normally distributed and variances were equal, Student-t tests were applied for 2-sample type graphs, and One-Way ANOVA was used to compare means from >2 samples. When data were normally distributed, but variances were unequal, Welch ANOVA was used instead. Multiple comparison corrections to normal data were applied using a post-hoc Dunnett test of comparisons to a control, Games-Howell or Bonferroni post-hoc comparing all samples. Non-parametric continuous data were compared using a Mann-Whitney U-test when 2 sample types were being analysed, and a Kruskal-Wallis test for >2 samples, and multiple comparison corrections were applied using a posthoc Dunn test. See S1 Table for all statistical sample sizes, applied tests and p values.  [36] where we provided evidence that a construct generated from the sequences downstream of the breakpoint to the terminal stop codon, tagged at the 3' with HA, did not translate into a protein product in transfected S2 cells. Together, these data demonstrate that DNT2 37 is a null allele. (TIF) S3 Fig. Altered kek-6 function affects motoraxon targeting at embryonic NMJ. The motoneuron marker FasII reveals motoraxon targeting phenotypes at muscle 6,7,12,13 in stage 17 embryos, in kek-6 mutants and upon over-expression of kek-6 in all neurons (with elavGAL4). Arrows indicate stereotypic projections in wild-type, and mistargeting in other genotypes. Chi-square p<0.0001 and ÃÃÃ p<0.001 Boferroni corrections, see S1 Table.