Implications of the Sap47 null mutation for synapsin phosphorylation, longevity, climbing proficiency and behavioural plasticity in adult Drosophila.

The Sap47 gene of Drosophila melanogaster encodes a highly abundant 47 kDa synaptic vesicle-associated protein. Sap47 null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of Drosophila synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. Here, we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body and in large synapses near the lateral triangle. The effects of knockout of Sap47 or synapsin on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of Sap47 but not synapsin reduces lifespan, whereas knockout of Sap47 and synapsin, either individually or together, affects climbing proficiency, as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in Sap47 and synapsin single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.

Altering the Temporal Regulation of One Transcription Factor Drives Evolutionary Trade-Offs between Head Sensory Organs.

Size trade-offs of visual versus olfactory organs is a pervasive feature of animal evolution. This could result from genetic or functional constraints. We demonstrate that head sensory organ size trade-offs in Drosophila are genetically encoded and arise through differential subdivision of the head primordium into visual versus non-visual fields. We discover that changes in the temporal regulation of the highly conserved eyeless/Pax6 gene expression during development is a conserved mechanism for sensory trade-offs within and between Drosophila species. We identify a natural single nucleotide polymorphism in the cis-regulatory region of eyeless in a binding site of its repressor Cut that is sufficient to alter its temporal regulation and eye size. Because eyeless/Pax6 is a conserved regulator of head sensory placode subdivision, we propose that its temporal regulation is key to define the relative size of head sensory organs.

An essential role of the mouse synapse-associated protein Syap1 in circuits for spontaneous motor activity and rotarod balance.

Synapse-associated protein 1 (Syap1) is the mammalian homologue of synapse-associated protein of 47 kDa (Sap47) in Drosophila Genetic deletion of Sap47 leads to deficiencies in short-term plasticity and associative memory processing in flies. In mice, Syap1 is prominently expressed in the nervous system, but its function is still unclear. We have generated Syap1 knockout mice and tested motor behaviour and memory. These mice are viable and fertile but display distinct deficiencies in motor behaviour. Locomotor activity specifically appears to be reduced in early phases when voluntary movement is initiated. On the rotarod, a more demanding motor test involving control by sensory feedback, Syap1-deficient mice dramatically fail to adapt to accelerated speed or to a change in rotation direction. Syap1 is highly expressed in cerebellar Purkinje cells and cerebellar nuclei. Thus, this distinct motor phenotype could be due to a so-far unknown function of Syap1 in cerebellar sensorimotor control. The observed motor defects are highly specific since other tests in the modified SHIRPA exam, as well as cognitive tasks like novel object recognition, Pavlovian fear conditioning, anxiety-like behaviour in open field dark-light transition and elevated plus maze do not appear to be affected in Syap1 knockout mice.

Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons.

Synapse-associated protein 1 (Syap1/BSTA) is the mammalian homologue of Sap47 (synapse-associated protein of 47 kDa) in Drosophila. Sap47 null mutant larvae show reduced short-term synaptic plasticity and a defect in associative behavioral plasticity. In cultured adipocytes, Syap1 functions as part of a complex that phosphorylates protein kinase Bα/Akt1 (Akt1) at Ser(473) and promotes differentiation. The role of Syap1 in the vertebrate nervous system is unknown. Here, we generated a Syap1 knock-out mouse and show that lack of Syap1 is compatible with viability and fertility. Adult knock-out mice show no overt defects in brain morphology. In wild-type brain, Syap1 is found widely distributed in synaptic neuropil, notably in regions rich in glutamatergic synapses, but also in perinuclear structures associated with the Golgi apparatus of specific groups of neuronal cell bodies. In cultured motoneurons, Syap1 is located in axons and growth cones and is enriched in a perinuclear region partially overlapping with Golgi markers. We studied in detail the influence of Syap1 knockdown and knockout on structure and development of these cells. Importantly, Syap1 knockout does not affect motoneuron survival or axon growth. Unexpectedly, neither knockdown nor knockout of Syap1 in cultured motoneurons is associated with reduced Ser(473) or Thr(308) phosphorylation of Akt. Our findings demonstrate a widespread expression of Syap1 in the mouse central nervous system with regionally specific distribution patterns as illustrated in particular for olfactory bulb, hippocampus, and cerebellum.

Synapsin is required to "boost" memory strength for highly salient events.

Synapsin is an evolutionarily conserved presynaptic phosphoprotein. It is encoded by only one gene in the Drosophila genome and is expressed throughout the nervous system. It regulates the balance between reserve and releasable vesicles, is required to maintain transmission upon heavy demand, and is essential for proper memory function at the behavioral level. Task-relevant sensorimotor functions, however, remain intact in the absence of Synapsin. Using an odor-sugar reward associative learning paradigm in larval Drosophila, we show that memory scores in mutants lacking Synapsin (syn(97)) are lower than in wild-type animals only when more salient, higher concentrations of odor or of the sugar reward are used. Furthermore, we show that Synapsin is selectively required for larval short-term memory. Thus, without Synapsin Drosophila larvae can learn and remember, but Synapsin is required to form memories that match in strength to event salience-in particular to a high saliency of odors, of rewards, or the salient recency of an event. We further show that the residual memory scores upon a lack of Synapsin are not further decreased by an additional lack of the Sap47 protein. In combination with mass spectrometry data showing an up-regulated phosphorylation of Synapsin in the larval nervous system upon a lack of Sap47, this is suggestive of a functional interdependence of Synapsin and Sap47.

Synapsin function in GABA-ergic interneurons is required for short-term olfactory habituation.

In Drosophila, short-term (STH) and long-term habituation (LTH) of olfactory avoidance behavior are believed to arise from the selective potentiation of GABAergic synapses between multiglomerular local circuit interneurons (LNs) and projection neurons in the antennal lobe. However, the underlying mechanisms remain poorly understood. Here, we show that synapsin (syn) function is necessary for STH and that syn(97)-null mutant defects in STH can be rescued by syn(+) cDNA expression solely in the LN1 subset of GABAergic local interneurons. As synapsin is a synaptic vesicle-clustering phosphoprotein, these observations identify a presynaptic mechanism for STH as well as the inhibitory interneurons in which this mechanism is deployed. Serine residues 6 and/or 533, potential kinase target sites of synapsin, are necessary for synapsin function suggesting that synapsin phosphorylation is essential for STH. Consistently, biochemical analyses using a phospho-synapsin-specific antiserum show that synapsin is a target of Ca(2+) calmodulin-dependent kinase II (CaMKII) phosphorylation in vivo. Additional behavioral and genetic observations demonstrate that CaMKII function is necessary in LNs for STH. Together, these data support a model in which CaMKII-mediated synapsin phosphorylation in LNs induces synaptic vesicle mobilization and thereby presynaptic facilitation of GABA release that underlies olfactory STH. Finally, the striking observation that LTH occurs normally in syn(97) mutants indicates that signaling pathways for STH and LTH diverge upstream of synapsin function in GABAergic interneurons.

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.

The Drosophila homologue of tubulin-specific chaperone E-like protein is required for synchronous sperm individualization and normal male fertility.

The tubulin-specific chaperone E-like protein (TBCEL or E-like) of vertebrates shows sequence homology to TBCE, a component of the multimolecular complex required for tubulin heterodimer formation in all eukaryotic cells. TBCEL apparently serves more specific functions, as it is found only in animals. At the cellular level, TBCEL plays a role as a regulator of tubulin stability. It is strongly expressed in human testes, but its systemic function is not known. The gene CG12214 codes for the Drosophila homologue of the vertebrate TBCEL protein. Here we show that disruption of the Drosophila Tbcel gene causes defects in spermatid individualixation, which leads to dispersed migration of F-actin-rich investment cones. Mutations affecting the Tbcel gene cause strong reduction in male, but not female, fertility. However, mature sperm function apparently is not impaired. We generated polyclonal antisera against TBCEL to study its localization and distribution in Drosophila tissues. Immunostainings of wild-type and null mutant testes demonstrated that TBCEL is localized in testes, presumably associated with axoneme bundles prior to spermatid individualization. Molecular analysis of the transposon insertion site in the mutant mulet (mlt), for which male sterility and sperm individualization defects have previously been described, demonstrates that the mlt P-element insertion resides in the Tbcel gene. Our results show that loss of TBCEL in Drosophila is compatible with viability and normal female fertility but causes reduced male fertility. We conclude that Drosophila TBCEL is strongly expressed in testes and plays an important role in sperm individualization during spermatogenesis. The high level of Tbcel mRNA in human testes suggests a general role of TBCEL in animal spermatogenesis. However, Western blots and courtship analysis suggest that TBCEL may have additional functions in the nervous system of Drosophila that could contribute to the observed reduced male fertility. These functions now have to be investigated.

Avoidance of heat and attraction to optogenetically induced sugar sensation as operant behavior in adult Drosophila.

Animals have to perform adequate behavioral actions dependent on internal states and environmental situations, and adjust their behavior according to positive or negative consequences. The fruit fly Drosophila melanogaster represents a key model organism for the investigation of neuronal mechanisms underlying adaptive behavior. The authors are using a behavioral paradigm in which fruit flies attached to a manipulator can walk on a Styrofoam ball whose movements are recorded such that intended left or right turns of the flies can be registered and used to operantly control heat stimuli or optogenetic activation of distinct subsets of neurons. As proof of principle, the authors find that flies in this situation avoid heat stimuli but prefer optogenetic self-stimulation of sugar receptors. Using this setup it now should be possible to study the neuronal network underlying positive and negative value assessment of adult Drosophila in an operant setting.

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.

Behavioral and synaptic plasticity are impaired upon lack of the synaptic protein SAP47.

The synapse-associated protein of 47 kDa (SAP47) is a member of a phylogenetically conserved gene family of hitherto unknown function. In Drosophila, SAP47 is encoded by a single gene (Sap47) and is expressed throughout all synaptic regions of the wild-type larval brain; specifically, electron microscopy reveals anti-SAP47 immunogold labeling within 30 nm of presynaptic vesicles. To analyze SAP47 function, we used the viable and fertile deletion mutant Sap47(156), which suffers from a 1.7 kb deletion in the regulatory region and the first exon. SAP47 cannot be detected by either immunoblotting or immunohistochemistry in Sap47(156) mutants. These mutants exhibit normal sensory detection of odorants and tastants as well as normal motor performance and basic neurotransmission at the neuromuscular junction. However, short-term plasticity at this synapse is distorted. Interestingly, Sap47(156) mutant larvae also show a 50% reduction in odorant-tastant associative learning ability; a similar associative impairment is observed in a second deletion allele (Sap47(201)) and upon reduction of SAP47 levels using RNA interference. In turn, transgenically restoring SAP47 in Sap47(156) mutant larvae rescues the defect in associative function. This report thus is the first to suggest a function for SAP47. It specifically argues that SAP47 is required for proper behavioral and synaptic plasticity in flies-and prompts the question whether its homologs are required for proper behavioral and synaptic plasticity in other species as well.

Mass spectrometric analysis of synapsins in Drosophila melanogaster and identification of novel phosphorylation sites.

Synapsins are synaptic vesicle-associated phosphoproteins that play a major role in the fine regulation of neurotransmitter release. In Drosophila, synapsins are required for complex behavior including learning and memory. Synapsin isoforms were immunoprecipitated from homogenates of wild-type Drosophila heads using monoclonal antibody 3C11. Synapsin null mutants (Syn(97)) served as negative controls. The eluted proteins were separated by SDS-PAGE and visualized by silver staining. Gel pieces picked from five bands specific for wild type were analyzed by nano-LC-ESI-MS/MS following multienzyme digestion (trypsin, chymotrypsin, AspN, subtilisin, pepsin, and proteinase K). The protein was unambiguously identified with high sequence coverage (90.83%). A number of sequence conflicts were observed and the N-terminal amino acid was identified as methionine rather than leucine expected from the cDNA sequence. Several peptides from the larger isoform demonstrated that the in-frame UAG stop codon at position 582 which separates two large open reading frames is read through by tRNAs for lysine. Seven novel phosphorylation sites in Drosophila synapsin were identified at Thr-86, Ser-87, Ser-464, Thr-466, Ser-538, Ser-961, and Tyr-982 and verified by phosphatase treatment. No phosphorylation was observed at the conserved PKA/CaM kinase-I/IV site (RRFS, edited to RGFS) in domain A or a potential PKA site near domain E.

Bruchpilot in ribbon-like axonal agglomerates, behavioral defects, and early death in SRPK79D kinase mutants of Drosophila.

Defining the molecular structure and function of synapses is a central theme in brain research. In Drosophila the Bruchpilot (BRP) protein is associated with T-shaped ribbons ("T-bars") at presynaptic active zones (AZs). BRP is required for intact AZ structure and normal evoked neurotransmitter release. By screening for mutations that affect the tissue distribution of Bruchpilot, we have identified a P-transposon insertion in gene CG11489 (location 79D) which shows high homology to mammalian genes for SR protein kinases (SRPKs). SRPKs phosphorylate serine-arginine rich splicing factors (SR proteins). Since proteins expressed from CG11489 cDNAs phosphorylate a peptide from a human SR protein in vitro, we name CG11489 the Drosophila Srpk79D gene. We have characterized Srpk79D transcripts and generated a null mutant. Mutation of the Srpk79D gene causes conspicuous accumulations of BRP in larval and adult nerves. At the ultrastructural level, these correspond to extensive axonal agglomerates of electron-dense ribbons surrounded by clear vesicles. Basic synaptic structure and function at larval neuromuscular junctions appears normal, whereas life expectancy and locomotor behavior of adult mutants are significantly impaired. All phenotypes of the mutant can be largely or completely rescued by panneural expression of SRPK79D isoforms. Isoform-specific antibodies recognize panneurally overexpressed GFP-tagged SRPK79D-PC isoform co-localized with BRP at presynaptic active zones while the tagged -PB isoform is found in spots within neuronal perikarya. SRPK79D concentrations in wild type apparently are too low to be revealed by these antisera. We propose that the Drosophila Srpk79D gene characterized here may be expressed at low levels throughout the nervous system to prevent the assembly of BRP containing agglomerates in axons and maintain intact brain function. The discovery of an SR protein kinase required for normal BRP distribution calls for the identification of its substrate and the detailed analysis of SRPK function for the maintenance of nervous system integrity.

The Wuerzburg hybridoma library against Drosophila brain.

This review describes the present state of a project to identify and characterize novel nervous system proteins by using monoclonal antibodies (mAbs) against the Drosophila brain. Some 1,000 hybridoma clones were generated by injection of homogenized Drosophila brains or heads into mice and fusion of their spleen cells with myeloma cells. Testing the mAbs secreted by these clones identified a library of about 200 mAbs, which selectively stain specific structures of the Drosophila brain. Using the approach "from antibody to gene", several genes coding for novel proteins of the presynaptic terminal were cloned and characterized. These include the "cysteine string protein" gene (Csp, mAb ab49), the "synapse-associated protein of 47 kDa" gene (Sap47, mAbs nc46 and nb200), and the "Bruchpilot" gene (brp, mAb nc82). By a "candidate" approach, mAb nb33 was shown to recognize the pigment dispersing factor precursor protein. mAbs 3C11 and pok13 were raised against bacterially expressed Drosophila synapsin and calbindin-32, respectively, after the corresponding cDNAs had been isolated from an expression library by using antisera against mammalian proteins. Recently, it was shown that mAb aa2 binds the Drosophila homolog of "epidermal growth factor receptor pathway substrate clone 15" (Eps15). Identification of the targets of mAbs na21, ab52, and nb181 is presently attempted. Here, we review the available information on the function of these proteins and present staining patterns in the Drosophila brain for classes of mAbs that either bind differentially in the eye, in neuropil, in the cell-body layer, or in small subsets of neurons. The prospects of identifying the corresponding antigens by various approaches, including protein purification and mass spectrometry, are discussed.

Stimulating PACalpha increases miniature excitatory junction potential frequency at the Drosophila neuromuscular junction.

Photoactivated adenylate cyclase alpha (PACalpha) is a light-activated adenylate cyclase that was originally cloned from the eye spot of the protozoan Euglena gracilis. PACalpha has been shown to rapidly increase intracellular cyclic adenosine monophosphate (cAMP) in vivo in Xenopus oocytes and HEK293 cells, increase the spike width in Aplysia sensory neurons, and modify behavior in Drosophila. Using the GAL4 UAS system, we heterologously expressed PACalpha in motorneurons and quantified the effects of its activation at the neuromuscular junction of the Drosophila third instar wandering larva, a well-characterized model synapse. By recording from body-wall muscle 6, we show that the presynaptic activation of PACalpha with blue light significantly increased miniature excitatory junction potential (mEJP) frequency in the presence of calcium with a delay of about 1 minute. Similar effects have been observed in previous studies that utilized adenylate cyclase agonists (Forskolin) or membrane-permeable cAMP analogs [dibutyryl cAMP and 4-chlorophenylthio-(CPT)-cAMP] to increase presynaptic cAMP concentrations. PACalpha's efficacy in combination with its specificity make it an invaluable tool for the rapid regulation of cAMP in vivo and for investigating the mechanisms by which cAMP can modulate synaptic transmission and neuronal plasticity in Drosophila.

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

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.

Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae.

During classical conditioning, a positive or negative value is assigned to a previously neutral stimulus, thereby changing its significance for behavior. If an odor is associated with a negative stimulus, it can become repulsive. Conversely, an odor associated with a reward can become attractive. By using Drosophila larvae as a model system with minimal brain complexity, we address the question of which neurons attribute these values to odor stimuli. In insects, dopaminergic neurons are required for aversive learning, whereas octopaminergic neurons are necessary and sufficient for appetitive learning. However, it remains unclear whether two independent neuronal populations are sufficient to mediate such antagonistic values. We report the use of transgenically expressed channelrhodopsin-2, a light-activated cation channel, as a tool for optophysiological stimulation of genetically defined neuronal populations in Drosophila larvae. We demonstrate that distinct neuronal populations can be activated simply by illuminating the animals with blue light. Light-induced activation of dopaminergic neurons paired with an odor stimulus induces aversive memory formation, whereas activation of octopaminergic/tyraminergic neurons induces appetitive memory formation. These findings demonstrate that antagonistic modulatory subsystems are sufficient to substitute for aversive and appetitive reinforcement during classical conditioning.

Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release.

The molecular organization of presynaptic active zones during calcium influx-triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) were entirely lost, Ca2+ channels were reduced in density, evoked vesicle release was depressed, and short-term plasticity was altered. BRP-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity.