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

Function of Kek-6 and DNT2 in structural synaptic plasticity in DrosophilaDrosophila

The $Drosophila$ nervous system undergoes structural synaptic plasticity, however, the mechanisms that govern such event are little understood. Structural synaptic plasticity in mammals is regulated by the neurotrophin brain derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase full length (TrkB-FL). TrkB-FL has a tyrosine kinase domain (TyrK) intracellularly, that is required for its function in structural synaptic plasticity. Trk receptors have long been sought in $Drosophila$ to verify mechanisms of structural synaptic plasticity, but they have not been found. Later, the Kek receptor family was identified as the kinaseless-Trk homologues in flies (Mandai et al., 2009, Bishop, 2013). Here, I validated that Kek-6 is a neurotrophin receptor for DNT2. DNT2 is a novel retrograde factor at the neuromuscular junction (NMJ), and both DNT2 and Kek-6 regulate structural synaptic plasticity. Kek-6 functions in concert with Toll-6. DNT2 and Kek-6 function upstream of CaMKII and Vap33A at the NMJ synapse. Finally, I show that Kek-6 can regulate intracellular levels of calcium in larval motorneurons. In conclusion, I identified a novel mechanism of structural synaptic plasticity in flies that is independent of a TyrK domain. If there are conserved mechanisms, this may also shed light on how truncated Trks function in the adult mammalian brain

Use of fibrin sealant associated with mononuclear stem cells to repair dorsal roots at CNS and PNS interface

Orientador: Alexandre Leite Rodrigues de OliveiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Lesões nas raízes dorsais da medula espinal são frequentes e muitas vezes decorrentes de acidentes automobilísticos. Devido à possibilidade de geração de dor neuropática, os procedimentos cirúrgicos não priorizam o reparo do componente aferente, sendo reparado apenas o componente motor. Adicionalmente, a perda das informações sensoriais gera parestesia ou anestesia do membro lesado, bem como descoordenação motora. Nesse contexto, novas terapias precisam ser desenvolvidas para o reparo das raízes dorsais. Uma substância capaz de conectar tecidos por adesão e que promova a hemostase e estabilidade do tecido, como o selante de fibrina (SF), pode ser uma alternativa a ser empregada no reparo de raízes lesadas. Além disso, o emprego conjunto do SF com células-tronco mononuclares de medula óssea (CTMMO) pode potencializar uma eventual regeneração tecidual. Assim, o presente estudo avaliou a resposta glial, a reorganização sináptica, a morfologia das fibras sensoriais e a coordenação motora após reparo com SF e terapia celular. Para isso, foram empregados ratos Lewis fêmeas (6-8 semanas), sendo divididos em três grupos: rizotomia (RZ, n=20), rizotomia reparada com SF (RZ+SF; n=22) e rizotomia reparada com SF e CTMMO (RZ+SF+CT; n=20). O tempo de sobrevida pós-cirúrgico foi de até 8 semanas. Para imunoistoquímica, foram utilizados anticorpos anti-VGLUT1 (terminais pré-sinápticos glutamatérgicos), GAD65 (terminais pré-sinápticos gabaérgicos), sinaptofisina (terminais sinápticos), GFAP (astrócitos), Iba1 (microglia) e BDNF (fator neurotrófico). Além disso, foram realizados citoquímica com Sudan black (coloração para lipídeos) e os testes comportamentais von-Frey eletrônico e walking track test (sistema CatWalk). Os resultados demonstraram regeneração das aferências nos grupos RZ+SF e RZ+SF+CT. Porém, apenas no segundo grupo, houve crescimento axonal até lâminas mais profundas da medula espinal, o que resultou em melhor desempenho nos testes comportamentais. Concluímos que o reimplante de raízes sensitivas com SF e CTMMO pode ser uma alternativa terapêutica para o reparo de lesões dorsais na interface do SNC e SNPAbstract: Dorsal root lesions are common and often occur in automobile accidents. Due to the possibility of generating neuropathic pain, surgical procedures do not prioritize the repair of the afferent component, focusing on the motor output instead. Moreover, the loss of sensory inputs triggers paresthesis or anesthesia of the injured limb, and motor impairments. In this context, new therapies have to be developed for dorsal root repair. A substance that can promote tissue adhesion and stability and tissue haemostasis, such as fibrin sealant (FS), could be an alternative for the repair of damaged roots. Furthermore, the combined use of FS plus bone marrow mononuclear stem cells (BMSC) may enhance tissue regeneration. Thus, the present study evaluated the glial response, synaptic changes, the cytoarchitecture of the sensory fibers and motor coordination with FS with or without cell therapy for root replantation. Female Lewis rats (6-8 weeks old) were divided into three groups: rhizotomy (RZ, n = 20), rhizotomy repaired with FS (RZ+FS, n = 22) and rhizotomy repaired with SF and BMSC (RZ+FS+SC, n = 20). The survival time after surgery was up to 8 weeks. For immunohistochemistry VGLUT 1 (presynaptic glutamatergic terminals), GAD65 (GABAergic presynaptic terminals), synaptophysin (synaptic terminals), GFAP (astrocytes), Iba1 (microglia) and BDNF (neurotrophic factor) antibodies were used. Also, cytochemistry with Sudan black (lipid staining) and the behavioral tests electronic von-Frey and Walking track test (CatWalk system) were carried out. The results showed regeneration of afferent inputs in groups RZ+FS and RZ+FS+SC. However, only in the group with BMSC, the axonal growth was able to reach deeper laminae of the spinal cord, resulting in a better performance in behavioral tests. We conclude that the sensory root replantation with FS and BMSC may be an alternative therapy for the repair of dorsal root injuries in the CNS and PNS interfaceMestradoBiologia CelularMestra em Biologia Celular e Estrutura

Structural circuit plasticity by a neurotrophin with a Toll modifies dopamine-dependent behaviour

Experience shapes the brain, as neural circuits can be modified by neural stimulation or the lack of it. How this modifies behaviour, and the underlying molecular mechanisms, are poorly understood, but could reveal how the brain works, in health and disease. Subjective experience requires dopamine, a neuromodulator that assigns a value to stimuli, and it also controls behaviour, including locomotion, learning and memory. In Drosophila, Toll receptors are ideally placed to translate experience into structural brain change. Toll-6 is expressed in dopaminergic neurons (DANs), raising the intriguing possibility that Toll-6 could regulate structural plasticity in dopaminergic circuits. Drosophila neurotrophin-2 (DNT-2) is the ligand for Toll-6, but whether it is required for circuit structural plasticity was unknown. Here, we show that DNT-2 expressing neurons are connected with DANs and they modulate each other. Loss of function for DNT-2 or its receptors Toll-6 and kinase-less Trk-like kek-6 caused DAN and synapse loss, impaired dendrite growth and caused locomotion deficits. By contrast, over-expressed DNT-2 increased dendrite complexity and promoted synaptogenesis. Neuronal activity increased the levels of DNT-2 and its maturation, and induced synaptic remodelling, which required DNT-2, Toll-6 and kek-6. Altering the levels of DNT-2 or Toll-6 could also modify dopamine-dependent behaviours, including locomotion and long-term memory. We conclude that an activity-dependent feedback loop involving dopamine and DNT-2 labelled the circuits engaged, and DNT-2 with Toll-6 and Kek-6 induced structural plasticity in this circuit, modifying brain function

<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

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