The haplolethality paradox of the wupA gene in Drosophila.

Haplolethals (HL) are regions of diploid genomes that in one dose are fatal for the organism. Their biological meaning is obscure because heterozygous loss-of-function mutations result in dominant lethality (DL) and, consequently, should be under strong negative selection. We report an in depth study of the HL associated to the gene wings up A (wupA). It encodes 13 transcripts (A-M) that yield 11 protein isoforms (A-K) of Troponin I (TnI). They are functionally diverse in their control of muscle contraction, cell polarity and cell proliferation. Isoform K transfers to the nucleus where it increases transcription of the cell proliferation related genes CDK2, CDK4, Rap and Rab5. The nuclear translocation of isoform K is prevented by the co-expression of A or B isoforms, which illustrates isoform interactions. The corresponding DL mutations are, either DNA rearrangements clustered towards the gene 3' end, thus affecting the genomic organization of all transcripts, or CRISPR-induced mutations in one of the two ATG sites which eliminate a subset of wupA products. The joint elimination of isoforms C, F, G and H, however, do not cause DL phenotypes. Genetically driven expression of single isoforms rescue neither DL nor any of the mutants known in the gene, suggesting that normal function requires properly regulated expression of specific combinations, rather than single, TnI isoforms. We conclude that the wupA associated HL results from the combined haploinsufficiency of a large set of TnI isoforms. The qualitative and quantitative normal expression of which, requires the chromosomal integrity of the wupA genomic region. Since all fly TnI isoforms are encoded in the same gene, its HL condition becomes unavoidable. These wupA features are comparable to those of dpp, the only other HL studied to some extent, and reveal a scenario of strict dosage dependence with implications for gene expression regulation and splitting.

The ubiquitin ligase Ariadne-1 regulates neurotransmitter release via ubiquitination of NSF.

Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified 16 candidates that met the established criteria: a significant change of at least twofold increase on ubiquitination, with at least two unique peptides identified. Among those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pull-down approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss-of-function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the presynaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared with controls in a Ca2+-dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous and evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.

CCB is Involved in Actin-Based Axonal Transport of Selected Synaptic Proteins.

Synapse formation, maturation, and turnover require a finely regulated transport system that delivers selected cargos to specific synapses. However, the supporting mechanisms of this process are not fully understood. The present study unravels a new molecular system for vesicle-based axonal transport of proteins in male and female flies (Drosophila melanogaster). Here, we identify the gene CG14579 as the transcription unit corresponding to the regulatory mutations known as central complex broad (ccb). These mutations were previously isolated for their morphological phenotype in R-neurons of the ellipsoid body, a component of the central complex. Mutant axons from R-neurons fail to cross the midline, which is indicative of an aberrant composition of the growth cone. However, the molecular mechanism remained to be deciphered. In this manuscript, we show that CCB is involved in axonal trafficking of FasII and synaptobrevin, but not syntaxin. These results suggest that axonal transport of certain proteins is required for the correct pathfinding of R-neurons. We further investigated the molecular network supporting the CCB system and found that CCB colocalizes and coimmunoprecipitates with Rab11. Epistasis studies indicated that Rab11 is positioned downstream of CCB within this axonal transport system. Interestingly, ccb also interacts with actin and the actin nucleator spire The data revealed that this interaction plays a key role in the development of axonal connections within the ellipsoid body. We propose that the CCB/Rab11/SPIRE system regulates axonal trafficking of synaptic proteins required for proper connectivity and synaptic function.SIGNIFICANCE STATEMENT Proper function of the nervous system requires the establishment of mature, functional synapses. Differential protein composition in the synapse enables optimal performance of cognitive tasks. Therefore, it is critical to have a finely regulated transport system to deliver selected synaptic proteins to synapses. Remarkably, impairments in cytoskeleton-based protein-transport systems often underlie cognitive deficits, such as those associated with aging and neurodegenerative diseases. This study reveals that CCB is part of a novel transport system that delivers certain synaptic proteins via the actin cytoskeleton within the Rab11-related domain of slow recycling endosomes.

Troponin-I mediates the localization of selected apico-basal cell polarity signaling proteins.

Beyond its role in muscle contraction, Drosophila Troponin I (TnI; also known as Wings up A) is expressed in epithelial cells where it controls proliferation. TnI traffics between nucleus and cytoplasm through a sumoylation-dependent mechanism. We address here the role of TnI in the cytoplasm. TnI accumulates apically in epidermal cells and neuroblasts. TnI co-immunoprecipitates with Bazooka (also known as Par3) and Discs large (Dlg1, hereafter Dlg), two apico-basal polarity components. TnI depletion causes Baz and Dlg mislocalization; by contrast, the basolateral localization of Scribbled is not altered. In neuroblasts, TnI contributes to the polar localization of Miranda, while non-polar Dlg localization is not affected. Vertebrate phosphoinositide 3-kinase (PI3K) contributes to the apico-basal polarity of epithelia, but we find that Drosophila PI3K depletion alters neither the apical localization of TnI or Bazooka, nor the basal localization of Dlg. Nevertheless, overexpressing PI3K prevents the defects seen upon TnI depletion. TnI loss-of-function disrupts cytoskeletal ß-Catenin, E-Cadherin and γ-Tubulin, and causes an increase in DNA damage, as revealed by analyzing γH2Av. We have previously shown that TnI depletion leads to apoptosis that can be suppressed by upregulating Sparc or downregulating Dronc. However, TnI-depleted cells expressing Sparc or downregulating Dronc, as well as those expressing p35 (also known as Cdk5α), that do not undergo apoptosis, still show DNA damage. This indicates that DNA damage is mechanistically independent of apoptosis induction. Thus, TnI binds certain apico-basal polarity signaling proteins in a cell type-dependent context, and this unveils a previously unsuspected diversity of mechanisms to allocate cell polarity factors.

Interference of the complex between NCS-1 and Ric8a with phenothiazines regulates synaptic function and is an approach for fragile X syndrome.

The protein complex formed by the Ca2+ sensor neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor protein Ric8a coregulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses, such as fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 interferes with NCS-1/Ric8a binding, and it restores normal synapse number and associative learning in a Drosophila FXS model. The synaptic effects elicited by FD44 feeding are consistent with the genetic manipulation of NCS-1. The crystal structure of NCS-1 bound to FD44 and the structure-function studies performed with structurally close analogs explain the FD44 specificity and the mechanism of inhibition, in which the small molecule stabilizes a mobile C-terminal helix inside a hydrophobic crevice of NCS-1 to impede Ric8a interaction. Our study shows the drugability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 for development of additional drugs of potential use on FXS and related synaptic disorders.

Molecular mechanisms that change synapse number.

Synapses are the functional units of the nervous system, and their number and protein composition undergo changes over a wide time scale. These synaptic changes manifest into differential behavioural outputs and, in turn, changes in the external conditions to the individual may elicit changes in synapses. We review here publications appeared during the last 10 years in which advances on molecular and cellular mechanisms for synapse changes have been reported. We focus on synaptic changes occurring in the time range of minutes to hours, mainly.

The guanine-exchange factor Ric8a binds to the Ca²âº sensor NCS-1 to regulate synapse number and neurotransmitter release.

The conserved Ca(2+)-binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and Gαs regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2-Ric8a-Gαs, which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.

One plus one makes more than two.

Pleasant memories rejuvenate and this letter is written to that purpose. Although dedicated to Barry Ganetzky, I hope it will please also those that shared their times with us in the lab of Seymour Benzer. For those too young to have so distant memories, I hope this text will teach them the same lessons we learnt then.

Cell types and coincident synapses in the ellipsoid body of Drosophila.

Cellular ultrastructures for signal integration are unknown in any nervous system. The ellipsoid body (EB) of the Drosophila brain is thought to control locomotion upon integration of various modalities of sensory signals with the animal internal status. However, the expected excitatory and inhibitory input convergence that virtually all brain centres exhibit is not yet described in the EB. Based on the EB expression domains of genetic constructs from the choline acetyl transferase (Cha), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) genes, we identified a new set of neurons with the characteristic ring-shaped morphology (R neurons) which are presumably cholinergic, in addition to the existing GABA-expressing neurons. The R1 morphological subtype is represented in the Cha- and TH-expressing classes. In addition, using transmission electron microscopy, we identified a novel type of synapse in the EB, which exhibits the precise array of two independent active zones over the same postsynaptic dendritic domain, that we named 'agora'. This array is compatible with a coincidence detector role, and represents ~8% of all EB synapses in Drosophila. Presumably excitatory R neurons contribute to coincident synapses. Functional silencing of EB neurons by driving genetically tetanus toxin expression either reduces walking speed or alters movement orientation depending on the targeted R neuron subset, thus revealing functional specialisations in the EB for locomotion control.

Central adaptation to odorants depends on PI3K levels in local interneurons of the antennal lobe.

We have previously shown that driving PI3K levels up or down leads to increases or reductions in the number of synapses, respectively. Using these tools to assay their behavioral effects in Drosophila melanogaster, we showed that a loss of synapses in two sets of local interneurons, GH298 and krasavietz, leads to olfaction changes toward attraction or repulsion, while the simultaneous manipulation of both sets of neurons restored normal olfactory indexes. We show here that olfactory central adaptation also requires the equilibrated changes in both sets of local interneurons. The same genetic manipulations directed to projection (GH146) or mushroom body (201Y, MB247) neurons did not affect adaptation. Also, we show that the equilibrium is a requirement for the glomerulus-specific size changes which are a morphological signature of central adaptation. Since the two sets of local neurons are mostly, although not exclusively, inhibitory (GH298) and excitatory (krasavietz), we interpret that the normal phenomena of sensory perception, measured as an olfactory index, and central adaptation rely on an inhibition/excitation ratio.

Synapse loss in olfactory local interneurons modifies perception.

Synapse loss correlates with cognitive decline in aging and most neurological pathologies. Sensory perception changes often represent subtle dysfunctions that precede the onset of a neurodegenerative disease. However, a cause-effect relationship between synapse loss and sensory perception deficits is difficult to prove and quantify due to functional and structural adaptation of neural systems. Here we modified a PI3K/AKT/GSK3 signaling pathway to reduce the number of synapses--without affecting the number of cells--in five subsets of local interneurons of the Drosophila olfactory glomeruli and measured the behavioral effects on olfactory perception. The neuron subsets were chosen under the criteria of GABA or ChAT expression. The reduction of one subset of synapses, mostly inhibitory, converted the responses to all odorants and concentrations tested as repulsive, while the reduction of another subset, mostly excitatory, led to a shift toward attraction. However, the simultaneous reduction of both synapse subsets restored normal perception. One group of local interneurons proved unaffected by the induced synapse loss in the perception of some odorants, indicating a functional specialization of these cells. Using genetic tools for space and temporal control of synapse number decrease, we show that the perception effects are specific to the local interneurons, rather than the mushroom bodies, and are not based on major structural changes elicited during development. These findings demonstrate that synapse loss cause sensory perception changes and suggest that normal perception is based on a balance between excitation and inhibition.

Phosphoinositide-3-kinase activation controls synaptogenesis and spinogenesis in hippocampal neurons.

The possibility of changing the number of synapses may be an important asset in the treatment of neurological diseases. In this context, the synaptogenic role of the phosphoinositide-3-kinase (PI3K) signaling cascade has been previously demonstrated in Drosophila. This study shows that treatment with a PI3K-activating transduction peptide is able to promote synaptogenesis and spinogenesis in primary cultures of rat hippocampal neurons, as well as in CA1 hippocampal neurons in vivo. In culture, the peptide increases synapse density independently of cell density, culture age, dendritic complexity, or synapse type. The induced synapses also increase neurotransmitter release from cultured neurons. The synaptogenic signaling pathway includes PI3K-Akt. Furthermore, the treatment is effective on adult neurons, where it induces spinogenesis and enhances the cognitive behavior of treated animals in a fear-conditioning assay. These findings demonstrate that functional synaptogenesis can be induced in mature mammalian brains through PI3K activation.

Frequenin/NCS-1 and the Ca2+-channel alpha1-subunit co-regulate synaptic transmission and nerve-terminal growth.

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca(2+)-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca(2+) entry and enhanced nerve-terminal growth. The impaired Ca(2+) entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca(2+) channels, or that it regulates the PI4Kbeta pathway as described in other organisms. To determine whether Frq interacts with PI4Kbeta with consequent effects on Ca(2+) channels, we first characterized a PI4Kbeta-null mutant and found that PI4Kbeta was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4Kbeta-null background. We conclude that the effects of Frq are not due to an interaction with PI4Kbeta. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the alpha(1)-subunit of voltage-gated Ca(2+) channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca(2+) entry through a functional interaction with the alpha(1) voltage-gated Ca(2+)-channel subunit; this interaction regulates neurotransmission and nerve-terminal growth.

Two frequenins in Drosophila: unveiling the evolutionary history of an unusual neuronal calcium sensor (NCS) duplication.

BACKGROUND: Drosophila frequenin (Frq), the homolog of the mammalian neuronal calcium sensor-1 (NCS-1), is a high affinity calcium-binding protein with ubiquitous expression in the nervous system. This protein has an important role in the regulation of neurotransmitter release per synapse, axonal growth and bouton formation. In D. melanogaster, frequenin is encoded by two genes (frq1 and frq2), a very unexpected feature in the Frq/NCS-1 subfamily. These genes are located in tandem in the same genomic region, and their products are 95% identical in their amino acid sequence, clearly indicating their recent origin by gene duplication. Here, we have investigated the factors involved in this unusual feature by examining the molecular evolution of the two frq genes in Drosophila and the evolutionary dynamics of NCS family in a large set of bilaterian species. RESULTS: Surprisingly, we have found no amino acid replacements fixed across the twelve Drosophila species surveyed. In contrast, synonymous substitutions have been prevalent in the evolution of the coding region of frq1 and frq2, indicating the presence of strong functional constraints following gene duplication. Despite that, we have detected that significant evolutionary rate acceleration had occurred in Frq1 in early times from the duplication, in which positive selection (likely promoting functional diversification) had probably an important role. The analysis of sequence conservation and DNA topology at the non-coding regions of both genes has allowed the identification of DNA regions candidates to be cis-regulatory elements. The results reveal a possible mechanism of regulatory diversification between frq1 and frq2. CONCLUSIONS: The presence of two frequenins in Drosophila and the rapid accumulation of amino acid substitutions after gene duplication are very unusual features in the evolution of the Frq/NCS-1 subfamily. Here we show that the action of positive selection in concordance with some extent of regulatory diversification might explain these findings. Selected amino acid substitutions in Frq1 likely contributed to the functional divergence between the two duplicates, which, in turn, should have diverged in their regulation by ecdysone-induced early genes.

PI3K activation prevents Aß42-induced synapse loss and favors insoluble amyloid deposit formation.

Excess of Aß42 peptide is considered a hallmark of the disease. Here we express the human Aß42 peptide to assay the neuroprotective effects of PI3K in adult Drosophila melanogaster. The neuronal expression of the human peptide elicits progressive toxicity in the adult fly. The pathological traits include reduced axonal transport, synapse loss, defective climbing ability and olfactory perception, as well as lifespan reduction. The Aß42-dependent synapse decay does not involve transcriptional changes in the core synaptic protein encoding genes bruchpilot, liprin and synaptobrevin. All toxicity features, however, are suppressed by the coexpression of PI3K. Moreover, PI3K activation induces a significant increase of 6E10 and thioflavin-positive amyloid deposits. Mechanistically, we suggest that Aß42-Ser26 could be a candidate residue for direct or indirect phosphorylation by PI3K. Along with these in vivo experiments, we further analyze Aß42 toxicity and its suppression by PI3K activation in in vitro assays with SH-SY5Y human neuroblastoma cell cultures, where Aß42 aggregation into large insoluble deposits is reproduced. Finally, we show that the Aß42 toxicity syndrome includes the transcriptional shut down of PI3K expression. Taken together, these results uncover a potential novel pharmacological strategy against this disease through the restoration of PI3K activity.

Age-independent synaptogenesis by phosphoinositide 3 kinase.

Synapses are specialized communication points between neurons, and their number is a major determinant of cognitive abilities. These dynamic structures undergo developmental- and activity-dependent changes. During brain aging and certain diseases, synapses are gradually lost, causing mental decline. It is, thus, critical to identify the molecular mechanisms controlling synapse number. We show here that the levels of phosphoinositide 3 kinase (PI3K) regulate synapse number in both Drosophila larval motor neurons and adult brain projection neurons. The supernumerary synapses induced by PI3K overexpression are functional and elicit changes in behavior. Remarkably, PI3K activation induces synaptogenesis in aged adult neurons as well. We demonstrate that persistent PI3K activity is necessary for synapse maintenance. We also report that PI3K controls the expression and localization of synaptic markers in human neuroblastoma cells, suggesting that PI3K synaptogenic activity is conserved in humans. Thus, we propose that PI3K stimulation can be applied to prevent or delay synapse loss in normal aging and in neurological disorders.

Transcription of Drosophila troponin I gene is regulated by two conserved, functionally identical, synergistic elements.

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.

Drosophila enhancer-Gal4 lines show ectopic expression during development.

In Drosophila melanogaster the most widely used technique to drive gene expression is the binary UAS/Gal4 system. We show here that a set of nervous system specific enhancers (elav, D42/Toll-6, OK6/RapGAP1) display ectopic activity in epithelial tissues during development, which is seldom considered in experimental studies. This ectopic activity is variable, unstable and influenced by the primary sequence of the enhancer and the insertion site in the chromosome. In addition, the ectopic activity is independent of the protein expressed, Gal4, as it is reproduced also with the expression of Gal80. Another enhancer, LN2 from the sex lethal (Sxl) gene, shows sex-dependent features in its ectopic expression. Feminization of LN2 expressing males does not alter the male specific pattern indicating that the sexual dimorphism of LN2 expression is an intrinsic feature of this enhancer. Other X chromosome enhancers corresponding to genes not related to sex determination do not show sexual dimorphism in their ectopic expressions. Although variable and unstable, the ectopic activation of enhancer-Gal4 lines seems to be regulated in terms of tissue and intensity. To characterize the full domain of expression of enhancer-Gal4 constructs is relevant for the design of transgenic animal models and biotechnology tools, as well as for the correct interpretation of developmental and behavioural studies in which Gal4 lines are used.

Amyloid ß42 peptide is toxic to non-neural cells in Drosophila yielding a characteristic metabolite profile and the effect can be suppressed by PI3K.

The human Aß42 peptide is associated with Alzheimer's disease through its deleterious effects in neurons. Expressing the human peptide in adult Drosophila in a tissue- and time-controlled manner, we show that Aß42 is also toxic in non-neural cells, neurosecretory and epithelial cell types in particular. This form of toxicity includes the aberrant signaling by Wingless morphogen leading to the eventual activation of Caspase 3. Preventing Caspase 3 activation by means of p53 keeps epithelial cells from elimination but maintains the Aß42 toxicity yielding more severe deleterious effects to the organism. Metabolic profiling by nuclear magnetic resonance (NMR) of adult flies at selected ages post Aß42 expression onset reveals characteristic changes in metabolites as early markers of the pathological process. All morphological and most metabolic features of Aß42 toxicity can be suppressed by the joint overexpression of PI3K.