Analysis of two P-element enhancer-trap insertion lines that show expression in the giant fibre neuron of Drosophila melanogaster.

The giant fibre system (GFS) of Drosophila is a simple neural circuit that mediates escape responses in adult flies. Here we report the initial characterization of two genes that are preferentially expressed in the GFS. Two P-element insertion lines, carrying the GAL4 transcriptional activator, were identified that exhibited pronounced expression in elements of the GFS and relatively low levels elsewhere within the adult central nervous system. Genomic DNA flanking the P-element insertion site was recovered from each of these lines, sequenced, and nearby transcripts identified and confirmed to exhibit GFS expression by in situ hybridization. This analysis revealed that these P-elements were in previously characterized genes. Line P[GAL4]-A307 has an insert in the gene short stop for which we have identified a novel transcript, while line P[GAL4]-141 has an insert in the transcription factor ken and barbie. Here we show that ken and barbie mutants have defects in escape behaviour, behavioural responses to visual stimuli and synaptic functions in the GFS. We have therefore revealed a neural role for a transcription factor that previously had no implicated neural function.

GSK-3beta inhibition reverses axonal transport defects and behavioural phenotypes in Drosophila.

The tauopathies are a group of disorders characterised by aggregation of the microtubule-associated protein tau and include Alzheimer's disease (AD) and the fronto-temporal dementias (FTD). We have used Drosophila to analyse how tau abnormalities cause neurodegeneration. By selectively co-expressing wild-type human tau (0N3R isoform) and a GFP vesicle marker in motorneurons, we examined the consequences of tau overexpression on axonal transport in vivo. The results show that overexpression of tau disrupts axonal transport causing vesicle aggregation and this is associated with loss of locomotor function. All these effects occur without neuron death. Co-expression of constitutively active glycogen-synthase kinase-3beta (GSK-3beta) enhances and two GSK-3beta inhibitors, lithium and AR-A014418, reverse both the axon transport and locomotor phenotypes, suggesting that the pathological effects of tau are phosphorylation dependent. These data show that tau abnormalities significantly disrupt neuronal function, in a phosphorylation-dependent manner, before the classical pathological hallmarks are evident and also suggest that the inhibition of GSK-3beta might have potential therapeutic benefits in tauopathies.

The C(2)B Ca(2+)-binding motif of synaptotagmin is required for synaptic transmission in vivo.

Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca(2+) sensor for fast, evoked neurotransmitter release. Deleting the gene for synaptotagmin (syt(null)) strongly suppresses synaptic transmission in every species examined, showing that synaptotagmin is central in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin contains two C(2) domains, C(2)A and C(2)B. Five, highly conserved, acidic residues in both the C(2)A and C(2)B domains of synaptotagmin coordinate the binding of Ca(2+) ions, and biochemical studies have characterized several in vitro Ca(2+)-dependent interactions between synaptotagmin and other nerve terminal molecules. But there has been no direct evidence that any of the Ca(2+)-binding sites within synaptotagmin are required in vivo. Here we show that mutating two of the Ca(2+)-binding aspartate residues in the C(2)B domain (D(416,418)N in Drosophila) decreased evoked transmitter release by >95%, and decreased the apparent Ca(2+) affinity of evoked transmitter release. These studies show that the Ca(2+)-binding motif of the C(2)B domain of synaptotagmin is essential for synaptic transmission.

Development of the giant fiber neuron of Drosophila melanogaster.

The giant fiber system (GFS) of Drosophila melanogaster provides a convenient system in which to study neural development. It mediates escape behaviour through a small number of neurons, including the giant fibers (GFs), to innervate the tergotrochantral jump muscle (TTM) and the dorsal longitudinal flight muscles. The GFS has been intensively studied physiologically in both wild-type and mutant flies, and is often used as a system to study the effects of neural mutations on the physiology of the adult nervous system. Recently, much information has been gleaned as to how and when synaptogenesis, with its major target neurons, is achieved. However, little is known of the earlier development of this neuron. Here we have used an enhancer-trap, marking parts of the GFS, in conjunction with BrdU labelling, to attempt to follow the birth, axonogenesis, and the early morphological meeting of the GFs with their target neurons. From these anatomical observations we propose that the GF cell is not born during the larval or pupal stages and, therefore. appears to be a persistent embryonic cell. The axons of the GFs develop during the third instar. During the early pupal stages the GFs contact other identified neurons of the GFS. In addition, we see some aberrant development of the network, with some flies carrying only one GF, and yet others with extended axons. We present a model for the initial joining of the GFs and tergotrochanteral motorneurons (TTMns).