Yves Grau

Shaggy, the Homolog of Glycogen Synthase Kinase 3, Controls Neuromuscular Junction Growth in Drosophila

A protein-trap screen using the Drosophila neuromuscular junction (NMJ) as a model synapse was performed to identify genes that control synaptic structure or plasticity. We found that Shaggy (Sgg), the Drosophila homolog of the mammalian glycogen synthase kinases 3 α and β, two serine-threonine kinases, was concentrated at this synapse. Using various combinations of mutant alleles of shaggy, we found that Shaggy negatively controlled the NMJ growth. Moreover, tissue-specific expression of a dominant-negative Sgg indicated that this kinase is required in the motoneuron, but not in the muscle, to control NMJ growth. Finally, we show that Sgg controlled the microtubule cytoskeleton dynamics in the motoneuron and that Futsch, a microtubule-associated protein, was required for Shaggy function on synaptic growth.

The Drosophila metabotropic glutamate receptor DmGluRA regulates activity-dependent synaptic facilitation and fine synaptic morphology.

In vertebrates, several groups of metabotropic glutamate receptors (mGluRs) are known to modulate synaptic properties. In contrast, the Drosophila genome encodes a single functional mGluR (DmGluRA), an ortholog of vertebrate group II mGluRs, greatly expediting the functional characterization of mGluR-mediated signaling in the nervous system. We show here that DmGluRA is expressed at the glutamatergic neuromuscular junction (NMJ), localized in periactive zones of presynaptic boutons but excluded from active sites. Null DmGluRA mutants are completely viable, and all of the basal NMJ synaptic transmission properties are normal. In contrast, DmGluRA mutants display approximately a threefold increase in synaptic facilitation during short stimulus trains. Prolonged stimulus trains result in very strongly increased (∼10-fold) augmentation, including the appearance of asynchronous, bursting excitatory currents never observed in wild type. Both defects are rescued by expression of DmGluRA only in the neurons, indicating a specific presynaptic requirement. These phenotypes are reminiscent of hyperexcitable mutants, suggesting a role of DmGluRA signaling in the regulation of presynaptic excitability properties. The mutant phenotypes could not be replicated by acute application of mGluR antagonists, suggesting that DmGluRA regulates the development of presynaptic properties rather than directly controlling short-term modulation. DmGluRA mutants also display mild defects in NMJ architecture: a decreased number of synaptic boutons accompanied by an increase in mean bouton size. These morphological changes bidirectionally correlate with DmGluRA levels in the presynaptic terminal. These data reveal the following two roles for DmGluRA in presynaptic mechanisms: (1) modulation of presynaptic excitability properties important for the control of activity-dependent neurotransmitter release and (2) modulation of synaptic architecture.

Glutamate and its metabotropic receptor in Drosophila clock neuron circuits

Identification of the neurotransmitters in clock neurons is critical for understanding the circuitry of the neuronal network that controls the daily behavioral rhythms in Drosophila. Except for the neuropeptide pigment‐dispersing factor, no neurotransmitters have been clearly identified in the Drosophila clock neurons. Here we show that glutamate and its metabotropic receptor, DmGluRA, are components of the clock circuitry and modulate the rhythmic behavior pattern of Drosophila. The dorsal clock neurons, DN1s in the larval brain and some DN1s and DN3s in the adult brain, were immunolabeled with antibodies against Drosophila vesicular glutamate transporter (DvGluT), suggesting that they are glutamatergic. Because the DN1s may communicate with the primary pacemaker neurons, s‐LNvs, we tested glutamate responses of dissociated larval s‐LNvs by means of calcium imaging. Application of glutamate dose dependently decreased intracellular calcium in the s‐LNvs. Pharmacology of the response suggests the presence of DmGluRA on the s‐LNvs. Antibodies against DmGluRA labeled dissociated s‐LNvs and the LNv dendrites in the intact larval and adult brain. The role of metabotropic glutamate signaling was tested in behavior assays in transgenic larvae and flies with altered DmGluRA expression in the LNvs and other clock neurons. Larval photophobic behavior was enhanced in DmGluRA mutants. For adults, we could induce altered activity patterns in the dark phase under LD conditions and increase the period during constant darkness by knockdown of DmGluRA expression in LNvs. Our results suggest that a glutamate signal from some of the DNs modulates the rhythmic behavior pattern via DmGluRA on the LNvs in Drosophila. J. Comp. Neurol. 505:32–45, 2007.

Divergent Evolution in Metabotropic Glutamate Receptors A NEW RECEPTOR ACTIVATED BY AN ENDOGENOUS LIGAND DIFFERENT FROM GLUTAMATE IN INSECTS

The metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors involved in the regulation of glutamatergic synapses. Surprisingly, the evolution-arily distant Drosophila mGluR shares a very similar pharmacological profile with its mammalian orthologues (mGlu2R and mGlu3R). Such a conservation in ligand recognition indicates a strong selective pressure during evolution to maintain the ligand recognition selectivity of mGluRs and suggests that structural constraints within the ligand binding pocket (LBP) would hinder divergent evolution. Here we report the identification of a new receptor homologous to mGluRs found in Anopheles gambiae, Apis mellifera, and Drosophila melanogaster genomes and called AmXR, HBmXR, and DmXR, respectively (the mXRs group). Sequence comparison associated with three-dimensional modeling of the LBP revealed that the residues contacting the amino acid moiety of glutamate (the α-COO- and batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{NH}_{3}^{+}) end{document} groups) were conserved in mXRs, whereas the residues interacting with the γ-carboxylic group were not. This suggested that the mXRs evolved to recognize an amino acid different from glutamate. The Drosophila cDNA encoding DmXR was isolated and found to be insensitive to glutamate or any other standard amino acid. However, a chimeric receptor with the heptahelical and intracellular domains of DmXR coupled to G-protein. We found that the DmX receptor was activated by a ligand containing an amino group, which was extracted from Drosophila head and from other insects (Anopheles and Schistocerca). No orthologue of mXR could be detected in Caenorhabditis elegans or human genomes. These data indicate that the LBP of the mGluRs has diverged in insects to recognize a new ligand.

Distribution of metabotropic glutamate receptor DmGlu‐A in Drosophila melanogaster central nervous system

L‐glutamate is the excitatory neurotransmitter at neuromuscular junctions in insects. It may also be involved in neurotransmission within the central nervous system (CNS), but its function therein remains elusive. The roles of glutamatergic synapses in the Drosophila melanogaster CNS were investigated, with focus on the study of DmGluRA, a G‐protein‐coupled glutamate receptor. In a first attempt to determine the function of this receptor, we describe its distribution in the larval and adult Drosophila CNS, using a polyclonal antibody raised against the C‐terminal sequence of the protein. DmGluRA is expressed in a reproducible pattern both in the larva and in the adult. In particular, DmGluRA can be found in the antennal lobes, the optic lobes, the central complex, and the median bundle in the adult CNS. However, DmGluRA‐containing neurons represented only a small fraction of all CNS neurons. DmGluRA immunoreactivity was not detected at the larval neuromuscular junction nor in the body wall muscles. The correlations between DmGluRA distribution and previously described glutamate‐like immunoreactivity patterns, as well as the implications of these observations concerning the possible functions of DmGluRA in the Drosophila CNS, are discussed. J. Comp. Neurol. 438:213–225, 2001.

Widespread brain distribution of the Drosophila metabotropic glutamate receptor

Glutamate is the predominant excitatory neurotransmitter in the vertebrate brain, whereas acetylcholine has been considered to play the same role in insects. Recent studies have, however, questioned the latter view by showing a rather general distribution of glutamate transporters. Here, we describe the expression pattern of the receptor DmGlu-A (DmGluRA), the unique homolog of vertebrate metabotropic glutamate receptors. Metabotropic glutamate receptors play important roles in the regulation of glutamatergic neurotransmission. Using a specific antibody, we report DmGluRA expression in most neuropile areas in both larvae and adults, but not in the lobes of the mushroom bodies. These observations suggest a key role for glutamate in the insect brain.

DmGluRA, a Drosophila metabotropic glutamate receptor, activates G-protein inwardly rectifying potassium channels in Xenopus oocytes.

Xenopus oocytes were coinjected with cDNAs encoding the Drosophila melanogaster metabotropic glutamate receptor (DmGluRA) and two mammalian G-protein inwardly rectifying potassium channel subunits (GIRK1 and GIRK2). Glutamate and two vertebrate group II mGluR agonists (order of potency: LY 354740 > glutamate > DCG IV) elicited inwardly rectifying potassium currents. These inward currents were sensitive to cesium and barium. They were also blocked by two group II specific antagonists MCCG and APICA (IC50s 97.5 and 200 microM, respectively) and not affected by a group I antagonist (AIDA). Finally, the A-protomer of PTX reduced the glutamate-induced GIRK currents. This study is the first characterization of an invertebrate mGluR-mediated GIRK currents via a PTX-sensitive G protein.

Identification of DmTTLL5 as a Major Tubulin Glutamylase in the Drosophila Nervous System

Microtubules (MTs) play crucial roles during neuronal life. They are formed by heterodimers of alpha and beta-tubulins, which are subjected to several post-translational modifications (PTMs). Amongst them, glutamylation consists in the reversible addition of a variable number of glutamate residues to the C-terminal tails of tubulins. Glutamylation is the most abundant MT PTM in the mammalian adult brain, suggesting that it plays an important role in the nervous system (NS). Here, we show that the previously uncharacterized CG31108 gene encodes an alpha-tubulin glutamylase acting in the Drosophila NS. We show that this glutamylase, which we named DmTTLL5, initiates MT glutamylation specifically on alpha-tubulin, which are the only glutamylated tubulin in the Drosophila brain. In DmTTLL5 mutants, MT glutamylation was not detected in the NS, allowing for determining its potential function. DmTTLL5 mutants are viable and we did not find any defect in vesicular axonal transport, synapse morphology and larval locomotion. Moreover, DmTTLL5 mutant flies display normal negative geotaxis behavior and their lifespan is not altered. Thus, our work identifies DmTTLL5 as the major enzyme responsible for initiating neuronal MT glutamylation specifically on alpha-tubulin and we show that the absence of MT glutamylation is not detrimental for Drosophila NS function.