Dystrophin Is Required for Appropriate Retrograde Control of Neurotransmitter Release at theDrosophilaNeuromuscular Junction

Abstract

Mutations in the human dystrophin gene cause the Duchenne and Becker muscular dystrophies. The Dystrophin protein provides a structural link between the muscle cytoskeleton and extracellular matrix to maintain muscle integrity. Recently, Dystrophin has also been found to act as a scaffold for several signaling molecules, but the roles of dystrophin-mediated signaling pathways remain unknown. To further our understanding of this aspect of the function of dystrophin, we have generated Drosophila mutants that lack the large dystrophin isoforms and analyzed their role in synapse function at the neuromuscular junction. In expression and rescue studies, we show that lack of the large dystrophin isoforms in the postsynaptic muscle cell leads to elevated evoked neurotransmitter release from the presynaptic apparatus. Overall synapse size, the size of the readily releasable vesicle pool as assessed with hypertonic shock, and the number of presynaptic neurotransmitter release sites (active zones) are not changed in the mutants. Short-term synaptic facilitation of evoked transmitter release is decreased in the mutants, suggesting that the absence of dystrophin results in increased probability of release. Absence of the large dystrophin isoforms does not lead to changes in muscle cell morphology or alterations in the postsynaptic electrical response to spontaneously released neurotransmitter. Therefore, postsynaptic glutamate receptor function does not appear to be affected. Our results indicate that the postsynaptically localized scaffolding protein Dystrophin is required for appropriate control of neuromuscular synaptic homeostasis.

The RhoGAPcrossveinless-cInteracts withDystrophinand Is Required for Synaptic Homeostasis at theDrosophilaNeuromuscular Junction

“…The pcv phenotype is caused by the homozygous loss of the large isoforms; the phenotype is observed in the mutant allele Dys E6 that lacks only these isoforms (van der Plas et al, 2006). The observation that the large isoforms are expressed in the developing wing disk (data not shown) is consistent with their roles in wing vein formation.…”

“…The following mutant alleles were used in the genetic enhancer screen and obtained from the Bloomington Drosophila 1118 , the genetic background in which the Dys mutation was generated, served as the wild-type control genotype for stainings, electrophysiology, and transmission electron microscopy analyses. The Dys DLP2 isoform null mutant allele Dys E6 was described previously (van der Plas et al, 2006). The cv-c mutant alleles cv-c 1 , cv-c C524 , UAS-cv-c-RNA interference flies (Billuart et al, 2001) and Cdc42 4 were obtained from the Bloomington Drosophila Stock Center; cv-c M62 and UAS-cv-c-RA (Denholm et al, 2005) were gifts from R. Ray (University of Sussex, Brighton, UK).…”

“…The upstream activating sequence (UAS)-bearing GS12472 P-element insertion (Toba et al, 1999), 1.9 kb upstream of the DLP2 ATG initiator codon, was used to overexpress DLP2 (van der Plas et al, 2006).…”

“…Electrophysiology. Electrophysiological recordings were performed as described previously (van der Plas et al, 2006). Briefly, a microelectrode filled with 3 M KCl was inserted into muscle 6 (segments A3-A4) of dissected third-instar female larvae bathed in HL3 containing 0.6 mM Ca 2ϩ unless described otherwise (Stewart et al, 1994).…”

“…Recent studies have demonstrated the utility of the fruit fly Drosophila melanogaster, with its single Dystrophin (Dys) gene (Roberts and Bobrow, 1998), as a model for exploration of the roles of Dys at both extrasynaptic (Shcherbata et al, 2007;van der Plas et al, 2007;Christoforou et al, 2008;Kucherenko et al, 2008;Taghli-Lamallem et al, 2008) and synaptic (van der Plas et al, 2006;Fradkin et al, 2008) sites. We reported previously that the Dys DLP2 isoform is enriched at the postsynaptic side of the larval neuromuscular junction (NMJ) (van der Plas et al, 2006). Its absence from the muscle results in increased evoked neurotransmitter release by the presynaptic motoneuron.…”