Neurofly 2008 abstracts : the 12th European Drosophila neurobiology conference 6-10 September 2008 Wuerzburg, Germany

This volume consists of a collection of conference abstracts

Targeted mutagenesis of the Sap47 gene of Drosophila: Flies lacking the synapse associated protein of 47 kDa are viable and fertile

BACKGROUND: Conserved proteins preferentially expressed in synaptic terminals of the nervous system are likely to play a significant role in brain function. We have previously identified and molecularly characterized the Sap47 gene which codes for a novel synapse associated protein of 47 kDa in Drosophila. Sequence comparison identifies homologous proteins in numerous species including C. elegans, fish, mouse and human. First hints as to the function of this novel protein family can be obtained by generating mutants for the Sap47 gene in Drosophila. RESULTS: Attempts to eliminate the Sap47 gene through targeted mutagenesis by homologous recombination were unsuccessful. However, several mutants were generated by transposon remobilization after an appropriate insertion line had become available from the Drosophila P-element screen of the Bellen/Hoskins/Rubin/Spradling labs. Characterization of various deletions in the Sap47 gene due to imprecise excision of the P-element identified three null mutants and three hypomorphic mutants. Null mutants are viable and fertile and show no gross structural or obvious behavioural deficits. For cell-specific over-expression and "rescue" of the knock-out flies a transgenic line was generated which expresses the most abundant transcript under the control of the yeast enhancer UAS. In addition, knock-down of the Sap47 gene was achieved by generating 31 transgenic lines expressing Sap47 RNAi constructs, again under UAS control. When driven by a ubiquitously expressed yeast transcription factor (GAL4), Sap47 gene suppression in several of these lines is highly efficient resulting in residual SAP47 protein concentrations in heads as low as 6% of wild type levels. CONCLUSION: The conserved synaptic protein SAP47 of Drosophila is not essential for basic synaptic function. The Sap47 gene region may be refractory to targeted mutagenesis by homologous recombination. RNAi using a construct linking genomic DNA to anti-sense cDNA in our hands is not more effective than using a cDNA-anti-sense cDNA construct. The tools developed in this study will now allow a detailed analysis of the molecular, cellular and systemic function of the SAP47 protein in Drosophila

The conserved protein kinase-A target motif in synapsin of Drosophila is effectively modified by pre-mRNA editing.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Synapsins are abundant synaptic vesicle associated phosphoproteins that are involved in the fine regulation of neurotransmitter release. The Drosophila member of this protein family contains three conserved domains (A, C, and E) and is expressed in most or all synaptic terminals. Similar to mouse mutants, synapsin knock-out flies show no obvious structural defects but are disturbed in complex behaviour, notably learning and memory. RESULTS: We demonstrate that the N-terminal phosphorylation consensus motif RRxS that is conserved in all synapsins investigated so far, is modified in Drosophila by pre-mRNA editing. In mammals this motif represents the target site P1 of protein kinase A (PKA) and calcium/calmodulin dependent protein kinase I/IV. The result of this editing, by which RRFS is modified to RGFS, can be observed in cDNAs of larvae and adults and in both isolated heads and bodies. It is also seen in several newly collected wild-type strains and thus does not represent an adaptation to laboratory culture conditions. A likely editing site complementary sequence is found in a downstream intron indicating that the synapsin pre-mRNA can form a double-stranded RNA structure that is required for editing by the adenosine deaminase acting on RNA (ADAR) enzyme. A deletion in the Drosophila Adar gene generated by transposon remobilization prevents this modification, proving that the ADAR enzyme is responsible for the pre-mRNA editing described here. We also provide evidence for a likely function of synapsin editing in Drosophila. The N-terminal synapsin undeca-peptide containing the genomic motif (RRFS) represents an excellent substrate for in-vitro phosphorylation by bovine PKA while the edited peptide (RGFS) is not significantly phosphorylated. Thus pre-mRNA editing by ADAR could modulate the function of ubiquitously expressed synapsin in a cell-specific manner during development and adulthood. CONCLUSION: Similar to several other neuronal proteins of Drosophila, synapsin is modified by ADAR-mediated recoding at the pre-mRNA level. This editing likely reduces or abolishes synapsin phosphorylation by PKA. Since synapsin in Drosophila is required for various forms of behavioural plasticity, it will be fascinating to investigate the effect of this recoding on learning and memory.Published versio

Identification and Structural Characterization of Interneurons of the Drosophila Brain by Monoclonal Antibodies of the Würzburg Hybridoma Library

Several novel synaptic proteins have been identified by monoclonal antibodies (mAbs) of the Würzburg hybridoma library generated against homogenized Drosophila brains, e.g. cysteine string protein, synapse-associated protein of 47 kDa, and Bruchpilot. However, at present no routine technique exists to identify the antigens of mAbs of our library that label only a small number of cells in the brain. Yet these antibodies can be used to reproducibly label and thereby identify these cells by immunohistochemical staining. Here we describe the staining patterns in the Drosophila brain for ten mAbs of the Würzburg hybridoma library. Besides revealing the neuroanatomical structure and distribution of ten different sets of cells we compare the staining patterns with those of antibodies against known antigens and GFP expression patterns driven by selected Gal4 lines employing regulatory sequences of neuronal genes. We present examples where our antibodies apparently stain the same cells in different Gal4 lines suggesting that the corresponding regulatory sequences can be exploited by the split-Gal4 technique for transgene expression exclusively in these cells. The detection of Gal4 expression in cells labeled by mAbs may also help in the identification of the antigens recognized by the antibodies which then in addition to their value for neuroanatomy will represent important tools for the characterization of the antigens. Implications and future strategies for the identification of the antigens are discussed

Synchronized brain activity during rehearsal and short-term memory disruption by irrelevant speech is affected by recall mode

EEG coherence as a measure of synchronization of brain activity was used to investigate effects of irrelevant speech. In a delayed serial recall paradigm 21 healthy participants retained verbal items over a 10-s delay with and without interfering irrelevant speech. Recall after the delay was varied in two modes (spoken vs. written). Behavioral data showed the classic irrelevant speech effect and a superiority of written over spoken recall mode. Coherence, however, was more sensitive to processing characteristics and showed interactions between the irrelevant speech effect and recall mode during the rehearsal delay in theta (4–7.5 Hz), alpha (8–12 Hz), beta (13–20 Hz), and gamma (35–47 Hz) frequency bands. For gamma, a rehearsal-related decrease of the duration of high coherence due to presentation of irrelevant speech was found in a left-lateralized fronto-central and centro-temporal network only in spoken but not in written recall. In theta, coherence at predominantly fronto-parietal electrode combinations was indicative for memory demands and varied with individual working memory capacity assessed by digit span. Alpha coherence revealed similar results and patterns as theta coherence. In beta, a left-hemispheric network showed longer high synchronizations due to irrelevant speech only in written recall mode. EEG results suggest that mode of recall is critical for processing already during the retention period of a delayed serial recall task. Moreover, the finding that different networks are engaged with different recall modes shows that the disrupting effect of irrelevant speech is not a unitary mechanism

Identification of Eps15 as Antigen Recognized by the Monoclonal Antibodies aa2 and ab52 of the Wuerzburg Hybridoma Library against Drosophila Brain

The Wuerzburg Hybridoma Library against the Drosophila brain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15) as the antigen for these two antibodies