Institut de
Génomique
Fonctionnelle

I- Drosophila larval neuromuscular junction : role of different components in growth and activity of this synapse

II- Insect metabotropic glutamate receptors : structure, functions and evolution

I- Drosophila larval neuromuscular junction : role of different components in growth and activity of this synapse

A- Situation of the research area :

The Drosophila neuromuscular junction has been instrumental in revealing mechanisms of glutamate synapse development, transmission, and activity-dependent plasticity. The larval Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse, easily accessible. It contains large synaptic varicosities (1 to 5 µm in diameter), which can be easily studied (Figure 2). We have developped this model in the lab, to study the function of the metabotropic glutamate receptor DmGluA and also to find and study new molecular components involved in the growth and plasticity of glutamatergic synapses.

Fig2 : Muscle 13 NMJ immunolabelled against the microtubule associated protein Futsch (green), Discs- Large, the PSD95 homologue (blue) and a neuronal epitope (HRP) in red.

B- Previous data from this team :

B1) Function of DmGluAR at the NMJ (Bogdanik et al, 2004, JNeuroSci) We have shown, by immunocytochemistry, that DmGluAR is localised at the larval NMJ, in periphery of the active zones. We obtained a loss of function mutation of DmGluAR, DmGluAR112B. DmGluAR112B mutants are hyperexcitables when synaptic activity is registered in specific conditions.This phenotype can be rescued when the receptor is expressed in the presynaptic compartment of the mutant synapse. This indicates that DmGluAR presynaptically controls synapse excitability. In addition, DmGluAR also controls synaptic morphologye since DmGluAR mutants display less but bigger synaptic boutons that control larvae. To our knowledge, this is the first report of the role of a group II mGluR in modifying the morphology of synapses.

B2) Protein-trap screen to find new proteins enriched at the NMJ

We undertook a protein-trap screen to find new proteins enriched at the NMJ. Thanks to this screen, we found that proteins like Gelded (unknown function in insects) or Gilgamesh (casein kinase) are concentrated in the postsynaptic side of the NMJ. We have also shown that Shaggy, the homologue of vertebrate glycogen synthase kinases, is concentrated in the presynaptic compartment of the NMJ (N110). We have demonstrated that shaggy mutants have an hyperdevelopped NMJ. In addition, microtubules form many loops compared to the controls, which suggests changes in the stability of these polymers (Franco et al., 2004, J NeuroSci).

C- Research project (methodology, techniques, planned milestones)

C1 Function of shaggy and the wingless pathway in the dynamics of presynaptic microtubules.

Actin filaments and microtubules play an essential role in the growth cone mobility and in the axonal growth. We want to understand how are controlled the expansion and the integrity of the microtubular axis during the development and the growth of the NMJ on its target muscle. We will test if partners known to act on shaggy activity, like proteins of the wingless pathway (Wg, Frizzled, Dishevelled) also play a role on microtubular cytoskeleton stability and on the NMJ growth. We have obtained preliminary data showing a clear decrease of the amount of Futsch (microtubule associated protein) associated to the microtubules, when a dominant negative form of the frizzled2 receptor is expressed presynaptically.

C2 Role of Dystroglycan at the NMJ

In mammals, Dystroglycan (DG) is a key molecule of the DGC (Dystrophin Glycoprotein Complex), linking the extracellular matrix and the intracellular cytoskeleton in the muscle cells. DG is concentrated at the NMJ and can interact directly with utophin or dystrophin (Figure 3). Loss of dystroglycan function via glycosylation defects of the extracellular DG-a subunit leads to muscular dystrophies, like the Walker Warburg syndrome (Winder, 2001). Despite the interaction between DG and utrophin, rapsyn and the adpatator protein Grb2 (Winder, 2001), we still do not know exhaustively what are the intracellular partners of dystroglycan. In Drosophila, the function of DG at the NMJ, like the function of other DGC members, has not been studied yet.

Figure 3 : The DGC complex, involved in the stabilization of nAchR clusters at the ostsynaptic side of the mammalian NMJ. This complex can interplay with nAchRs via rapsyn, known to interact with β- DG. Several congenital myastenic syndromes (Walker Warburg syndrome, Fukuyama disease, Muscle-eye-brain disease) are due to glycosylation defects of α-DG and loss of interaction with extracellular matric components. DG= Dystroglycan, SG= Sarcoglycan, SYN = Syntrophin

An anti-DG antibody labelled the NMJ and the signal colocalized with the postsynaptic protein Discs-Large, the homologue of PSD95. Our data show that the genetic decrease of postsynaptic amount of DG leads to smaller clusters of postsynaptic glutamate channel receptors. Postsynaptic overexpression of DG leads to an increase in the size of these clusters. We will test the electrophysiological consequences of these modifications. We will also look for genetic interactors of DG at the NMJ. We will take advantage of the lethality induced by strong overexpression of DG in the muscles (at 29°C). We will undertake a screen for suppressors of this lethality, using a deficiency kit containing 200 deletions, covering about 80% of the fly genome. We will study the effect of the selected deletions on other phenotypes due to DG overexpression (increase in the amount of postsynaptic glutamate receptors). If these deletions also interact with the synaptic effects of DG, we will look for the interesting genes within these deletions.

D - Expected consequences for fundamental research and for disease therapy

This project will provide a way to better know the transduction pathways controlling microtubular dynamics, which is essential for a correct growth of this model synapse, the Drosophila larval NMJ. We think that the Wnt/Wg pathway is implicated . Recently, the presence of different Wnt ligands in the mammalian brain (cerebellum) was described. Understanding the role of this signalling pathways on the nervous system development, and also in the adult, may provide a way to understand still orphan brain pathologies. In addition, the protein trap screen will allow to find new components involved in the differentiation and the stability of the postsynapse. These new components could be the targets of drugs, acting on synaptic plasticity. Finally, the genetic study of dystroglycan interactors will provide a way to find new enzymes modifying DG (like glycosylases, which are affected in several syndromic pathologies of the NMJ), hence to find new candidate genes responsible for orphan neuromuscular pathologies.