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

Arc 3ʹ UTR splicing leads to dual and antagonistic effects in fine-tuning arc expression upon BDNF signaling

Activity-regulated cytoskeletal associated protein (Arc) is an immediate-early gene critically involved in synaptic plasticity and memory consolidation. Arc mRNA is rapidly induced by synaptic activation and a portion is locally translated in dendrites where it modulates synaptic strength. Being an activity-dependent effector of homeostatic balance, regulation of Arc is uniquely tuned to result in short-lived bursts of expression. Cis-Acting elements that control its transitory expression post-transcriptionally reside primarily in Arc mRNA 3′ UTR. These include two conserved introns which distinctively modulate Arc mRNA stability by targeting it for destruction via the nonsense mediated decay pathway. Here, we further investigated how splicing of the Arc mRNA 3′ UTR region contributes to modulate Arc expression in cultured neurons. Unexpectedly, upon induction with brain derived neurotrophic factor, translational efficiency of a luciferase reporter construct harboring Arc 3′ UTR is significantly upregulated and this effect is dependent on splicing of Arc introns. We find that, eIF2α dephosphorylation, mTOR, ERK, PKC, and PKA activity are key to this process. Additionally, CREB-dependent transcription is required to couple Arc 3′ UTR-splicing to its translational upregulation, suggesting the involvement of de novo transcribed trans-acting factors. Overall, splicing of Arc 3′ UTR exerts a dual and unique effect in fine-tuning Arc expression upon synaptic signaling: while inducing mRNA decay to limit the time window of Arc expression, it also elicits translation of the decaying mRNA. This antagonistic effect likely contributes to the achievement of a confined yet efficient burst of Arc protein expression, facilitating its role as an effector of synapse-specific plasticity

Arc 3′ UTR Splicing Leads to Dual and Antagonistic Effects in Fine-Tuning Arc Expression Upon BDNF Signaling

Activity-regulated cytoskeletal associated protein (Arc) is an immediate-early gene critically involved in synaptic plasticity and memory consolidation. Arc mRNA is rapidly induced by synaptic activation and a portion is locally translated in dendrites where it modulates synaptic strength. Being an activity-dependent effector of homeostatic balance, regulation of Arc is uniquely tuned to result in short-lived bursts of expression. Cis-Acting elements that control its transitory expression post-transcriptionally reside primarily in Arc mRNA 3′ UTR. These include two conserved introns which distinctively modulate Arc mRNA stability by targeting it for destruction via the nonsense mediated decay pathway. Here, we further investigated how splicing of the Arc mRNA 3′ UTR region contributes to modulate Arc expression in cultured neurons. Unexpectedly, upon induction with brain derived neurotrophic factor, translational efficiency of a luciferase reporter construct harboring Arc 3′ UTR is significantly upregulated and this effect is dependent on splicing of Arc introns. We find that, eIF2α dephosphorylation, mTOR, ERK, PKC, and PKA activity are key to this process. Additionally, CREB-dependent transcription is required to couple Arc 3′ UTR-splicing to its translational upregulation, suggesting the involvement of de novo transcribed trans-acting factors. Overall, splicing of Arc 3′ UTR exerts a dual and unique effect in fine-tuning Arc expression upon synaptic signaling: while inducing mRNA decay to limit the time window of Arc expression, it also elicits translation of the decaying mRNA. This antagonistic effect likely contributes to the achievement of a confined yet efficient burst of Arc protein expression, facilitating its role as an effector of synapse-specific plasticity

Image_1_Arc 3′ UTR Splicing Leads to Dual and Antagonistic Effects in Fine-Tuning Arc Expression Upon BDNF Signaling.PDF

<p>Activity-regulated cytoskeletal associated protein (Arc) is an immediate-early gene critically involved in synaptic plasticity and memory consolidation. Arc mRNA is rapidly induced by synaptic activation and a portion is locally translated in dendrites where it modulates synaptic strength. Being an activity-dependent effector of homeostatic balance, regulation of Arc is uniquely tuned to result in short-lived bursts of expression. Cis-Acting elements that control its transitory expression post-transcriptionally reside primarily in Arc mRNA 3′ UTR. These include two conserved introns which distinctively modulate Arc mRNA stability by targeting it for destruction via the nonsense mediated decay pathway. Here, we further investigated how splicing of the Arc mRNA 3′ UTR region contributes to modulate Arc expression in cultured neurons. Unexpectedly, upon induction with brain derived neurotrophic factor, translational efficiency of a luciferase reporter construct harboring Arc 3′ UTR is significantly upregulated and this effect is dependent on splicing of Arc introns. We find that, eIF2α dephosphorylation, mTOR, ERK, PKC, and PKA activity are key to this process. Additionally, CREB-dependent transcription is required to couple Arc 3′ UTR-splicing to its translational upregulation, suggesting the involvement of de novo transcribed trans-acting factors. Overall, splicing of Arc 3′ UTR exerts a dual and unique effect in fine-tuning Arc expression upon synaptic signaling: while inducing mRNA decay to limit the time window of Arc expression, it also elicits translation of the decaying mRNA. This antagonistic effect likely contributes to the achievement of a confined yet efficient burst of Arc protein expression, facilitating its role as an effector of synapse-specific plasticity.</p

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

Kek-6 and Toll-6 interact for NMJ structural homeostasis.

<p>(A) Toll-6GAL4>mCD8-GFP is distributed in FasII+ motoneuron axons (arrows) at the muscle 6/7 NMJ terminal. (B) Muscle 6/7 NMJs (left) and box-plot graphs (right) showing: <i>Toll-6</i>^{<i>MIO2127</i>}<i>/Df(3L)BSC578</i> mutants had fewer 1b boutons. <i>Toll-6</i>^{<i>–/–</i>}and <i>Toll-6</i>^{<i>MIO2127</i>}<i>Df(3R)6361/kek6</i>^<i>35</i> <i>Df(3L)BSC578</i> double mutants had smaller NMJs (HRP, Kruskal-Wallis p = 0.0001) with reduced branching, and reduced active zones (Brp, Kruskal-Wallis p = 0.0055), post-hoc Dunn for both *p<0.05, ***p<0.001. Pre-synaptic over-expression of <i>kek-6</i> in motoneurons in Toll-6^-/-mutants (<i>w; UASkek-6/+; Toll-6</i>^{<i>MIO2127</i>}<i>GAL4/ Df(3L)BSC578</i>) did not rescue NMJ size, but upregulated Brp+. Over-expression of activated <i>Toll-6</i>^<i>CY</i> did not affect NMJ size (HRP) but increased active zones. N = 34–46 hemisegments. (C) Co-immunoprecitation from co-transfected S2 cells: Precipitating Toll-6 and Toll-7 with anti-Flag brought down Kek-6 detected with anti-HA. IP: immuno-precipitation; WB: western blot; asterisk: co-IP. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. <i>MN = D42GAL4</i>. </p

Kek-6 and DNT2 can induce active zones and NMJ growth.

<p>Confocal images of NMJs from A3-4 muscle 6/7 (left, higher magnification deail of areas indicated by asterisks), and box-plot graphs (right), showing: (A) Over-expression of <i>kek6</i> in motoneurons had no effect on HRP+ NMJ size, but it increased Brp+ active zones. HRP: Student t test n.s. p = 0.07; Brp: Mann-Whitney U test ***p<0.001.(B) Over-expression of full-length DNT2 in muscle increased NMJ size (HRP) and active zones (Brp), revealing a retrograde function. HRP: Mann-Whitney U test *p<0.05; Brp: Student t test **p<0.01. (C) Over-expression of both full-length DNT2 and mature DNT2-CK in motoneurons induced active zone formation. Brp DNT2-CK: Student t test **p<0.01, and Brp DNT2-FL: Mann-Whitney U test ***p<0.001. n = 29–66 hemisegments. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. <i>MN = D42GAL4; Muscle = MhcGAL4</i>.</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

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

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