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

Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote cell survival via their Trk and p75 receptors and cell death via p75 and Sortilin. NTs (DNTs) bind Toll receptors instead to promote neuronal survival, but whether they can also regulate cell death is unknown. In this study, we show that DNTs and Tolls can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88-NF-κB and cell death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate in wider contexts

Kek-6 is expressed pre-synaptically in motoneurons and binds post-synaptic DNT2.

<p>(A) In Kek-6^GFP larval VNCs, GFP colocalises with the neuronal marker HB9 (arrows show examples). (B) Kek-6^GFP was found in third instar larval muscle 6/7 NMJ and synaptic boutons (dotted rectangle: higher magnification, right). (C) Kek-6^GFP was found in the motoneuron axonal terminal (arrows), and in pre-synaptic bouton lumen (dotted rectangle: higher magnification, right), not colocalising with the post-synaptic marker anti-Dlg (arrows).(D) Kek-6>FlyBow was localized to CNS axons and dendrites (arrows), and cell bodies of the RP3,4,5 motoneuron clusters (ventral and transverse views, arrows). (E) Illustration. (F) Kek-6>FlyBow was also distributed along the motoneuron axons, NMJ terminal (arrow) and synaptic boutons (arrows). (G-K) Over-expression of GFP tagged full-length DNT2 in muscle <i>(MhcGAL4>UAS-DNT2-FL-GFP)</i> revealed: (G) DNT2-GFP distribution within the pre-synaptic bouton lumen (arrows), boutons labeled post-synaptically with anti-Dlg; (H-K) DNT2-GFP along the motoraxon (labeled with anti-FasII) and within the pre-synaptic bouton lumen (arrows).</p

Keks are Trk-like receptors expressed in the CNS.

<p>(A) Modular composition of TrkB, TrkB-T1, Dror, Otk and <i>Drosophila</i> LIGs. (B) Amongst the LIGs, Keks are closer to the Trks than any other mammalian or <i>Drosophila</i> LIGs, adapted from the phylogeny of Mandai et al.[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.ref022" target="_blank">22</a>]. (C,D) mRNA distribution in embryos: <i>CG15744</i>, <i>lambik</i> and <i>CG16974</i> are not expressed in the VNC (arrows) above background, but <i>lambik</i> is in PNS and <i>CG16974</i> in muscle precursors (arrowheads); <i>kek-1</i>, <i>kek-2</i> and <i>kek-6</i> transcripts are found in the VNC, and <i>kek5GAL4>tdTomato</i> drives expression in VNC and PNS (right) neurons. (E) Over-expression of <i>keks</i>– most prominently <i>kek2</i> and <i>6</i> -in all neurons with <i>elavGAL4</i> rescued the cold semi-lethality of <i>DNT1</i>^<i>41</i> <i>DNT2</i>^{<i>e03444</i>} double mutants, n = 52–313 pupae. Chi-square and Bonferroni multiple comparisons correction. *p<0.05, ***p<0.001. For statistical details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>.</p

Retrograde DNT2 binds pre-synaptic Kek-6 activating CaMKII and regulating structural synaptic plasticity.

<p>(A) Illustration of Kek-6 compared to Trk isoforms. DNT2 binds Kek-6, which functions via CaMKII and VAP33A downstream. (B) Pre-synaptic motoneuron terminal at the NMJ: DNT2 is produced at the muscle and secreted, binds pre-synaptic Kek-6, functioning via CaMKII and VAP33A downstream. DNT2 also binds Toll-6 which can interact with Toll-6. (C) The concerted functions of DNT2 and its two receptors Kek-6 and Toll-6 regulates NMJ growth and synaptic structure. Kek-6 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 up- or 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.</p

Kek-6 functions downstream of DNT2.

<p>Confocal images of NMJs from A3-4 muscle 6/7 (left), and box-plot graphs (right), showing: (A) Over-expression of <i>kek-6</i> in motoneurons rescued the phenotypes of <i>kek-6</i> mutants. Dlg: One Way ANOVA p<0.0001 and post-hoc Bonferroni *p<0.05, ***p<0.001. HRP: One Way ANOVA p<0.001 and post-hoc Bonferroni **p<0.01, **p<0.01. (B) Left: Over-expression of <i>kek-6</i> in neurons rescued the phenotype of <i>DNT2</i> mutants. Dlg: Kruskal-Wallis p = 0.001, and post-hoc Dunn test **p<0.01, ***p<0.001. Right: <i>kek-6</i> loss of function rescued the increase in boutons caused by the muscle over-expression of <i>DNT2</i>. Dlg: Welch ANOVA p<0.01 and post-hoc Games-Howell *p<0.05, **p<0.01. (C) Over-expression of <i>kek-6</i> in motoneurons rescued the phenotypes of <i>kek-6 DNT-2</i> double mutants. Dlg: Kruskal-Wallis p = 0.001 and post-hoc Dunn’s test *p<0.05, **p<0.01. HRP: Welch ANOVA p = 0.000, post-hoc Games Howell **p<0.01. n = 29–101 hemisegments. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. GAL4 drivers: Muscle: <i>MhcGAL4</i>; Neurons: <i>elavGAL4;</i> MN: <i>D42 or Toll-7GAL4</i>. Controls: white boxes: yw/+; grey boxes: GAL4/+; mutant genotypes as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.g004" target="_blank">Fig 4</a>. Rescue genotypes: (A) <i>w; UASkek6RFP/+; Df(3R)6361/kek6</i>^<i>34</i><i>D42GAL4</i>. (B) <i>w; UASkek6RFP/+; elavGAL4 Df(3L)6092/ DNT2</i>^<i>37</i>; and <i>w; UASDNT2-FL/+; Df(3R)6361/kek6</i>^<i>34</i><i>D42GAL4</i>. (C) <i>w; Toll-7GAL4/UASkek6RFP; kek6</i>^<i>34</i><i>Df(3L)6092/ Df(3R)6361 DNT2</i>^<i>37</i>.</p

VAP33A functions downstream of Kek-6.

<p>(A) Confocal images of NMJs from segments A3-4, muscle 6/7. (B-E) Box-plot graphs. (B) <i>VAP33A</i>^<i>G0231</i> mutants have reduced bouton number, Mann-Whitney U test ***p<0.001. (C,D) Pre-synaptic over-expression of <i>VAP33A</i> rescues bouton number in (C) <i>kek-6</i>^{<i>–/–</i>}mutants and (D) <i>DNT2</i>^{<i>–/–</i>}single mutants, Kruskal-Wallis p<0.0001 and *p<0.05, ***p<0.001 post-hoc Dunn for both. (E) <i>kek-6</i>^{<i>–/–</i>}<i>DNT2</i>^{<i>–/–</i>}double mutants rescue the bouton number phenotype caused by <i>VAP33A</i> gain of function, Kruskal-Wallis p<0.0001 and **p<0.01, ***p<0.001 post-hoc Dunn. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. N = 23–48 hemisegments. MN = motoneuron, <i>D42GAL4</i> (D) or <i>Toll-7GAL4</i> (E); Neurons = <i>elavGAL4</i>. Rescue genotypes: (C) <i>UASVAP33A/+; D42GAL4 kek6</i>^<i>34</i><i>/Df(3R)6361</i>. (D) <i>UASVAP33A/+; elavGAL4 Df(3L)6092/DNT2</i>^<i>37</i>.. (E) <i>UASVAP33A/Toll-7GAL4; kek6</i>^<i>34</i><i>Df(3L)6092/ Df(3R)6361 DNT2</i>^<i>37</i>.</p

<i>kek6</i> and <i>DNT2</i> mutants have smaller NMJs and impaired locomotion.

<p>(A) Plotted trajectories of filmed larvae, and (B) histograms of percentage frames at each speed analysed with FlyTracker. Kruskal-Wallis p<0.0001 and ***p<0.001 post-hoc Dunn test, n≥22344 frames. (C-E) 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: <i>kek-6</i>^{<i>–/–</i>}and <i>DNT2</i>^{<i>–/–</i>}single mutants and <i>kek-6</i>^{<i>–/–</i>}<i>DNT2</i>^<i>-/-</i>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. <i>kek-6</i> and <i>DNT2</i> 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. <i>Kek-6</i> mutants have reduced Synapsin, Mann-Whitney U test ***p<0.001. For statistical details, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. N = 30–113 hemisegments. Mutant genotypes throughout figures: Control: <i>yw/+;</i> Mutants: <i>kek-6</i>^{<i>–/–</i>}: <i>kek6</i>^<i>34</i><i>/Df(3R)6361; DNT2</i>^{<i>–/–</i>}: <i>DNT2</i>^<i>37</i><i>/Df(3L)6092; kek-6</i>^{<i>–/–</i>}<i>DNT2</i>^{<i>–/–</i>}: <i>kek6</i>^<i>34</i><i>Df(3L)6092/</i> Df(3R)6361 <i>DNT2</i>^<i>37</i>.</p