ADAR-mediated RNA editing suppresses sleep by acting as a brake on glutamatergic synaptic plasticity.

It has been postulated that synaptic potentiation during waking is offset by a homoeostatic reduction in net synaptic strength during sleep. However, molecular mechanisms to support such a process are lacking. Here we demonstrate that deficiencies in the RNA-editing gene Adar increase sleep due to synaptic dysfunction in glutamatergic neurons in Drosophila. Specifically, the vesicular glutamate transporter is upregulated, leading to over-activation of NMDA receptors, and the reserve pool of glutamatergic synaptic vesicles is selectively expanded in Adar mutants. Collectively these changes lead to sustained neurotransmitter release under conditions that would otherwise result in synaptic depression. We propose that a shift in the balance from synaptic depression towards synaptic potentiation in sleep-promoting neurons underlies the increased sleep pressure of Adar-deficient animals. Our findings provide a plausible molecular mechanism linking sleep and synaptic plasticity

The Proteome of BLOC-1 Genetic Defects Identifies the Arp2/3 Actin Polymerization Complex to Function Downstream of the Schizophrenia Susceptibility Factor Dysbindin at the Synapse

Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. We tested this idea by determining the proteome sensitive to genetic defects in a locus encoding dysbindin, a protein required for synapse biology and implicated in schizophrenia risk. We applied quantitative mass spectrometry to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). We found 491 proteins sensitive to dysbindin and BLOC-1 loss of function. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex, as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, we demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. Our results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects

A Gateway MultiSite Recombination Cloning Toolkit

The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduce a series of entry clones and a destination vector for use in two, three, and four fragment Gateway MultiSite recombination cloning whose advantages include increased flexibility and versatility. In contrast to Gateway single-fragment cloning approaches where variations are typically incorporated into model system-specific destination vectors, our Gateway MultiSite cloning strategy incorporates variations in easily generated entry clones that are model system-independent. In particular, we present entry clones containing insertions of GAL4, QF, UAS, QUAS, eGFP, and mCherry, among others, and demonstrate their in vivo functionality in Drosophila by using them to generate expression clones including GAL4 and QF drivers for various trp ion channel family members, UAS and QUAS excitatory and inhibitory light-gated ion channels, and QUAS red and green fluorescent synaptic vesicle markers. We thus establish a starter toolkit of modular Gateway MultiSite entry clones potentially adaptable to any model system. An inventory of entry clones and destination vectors for Gateway MultiSite cloning has also been established (www.gatewaymultisite.org)

synaptojanin1 Is Required for Temporal Fidelity of Synaptic Transmission in Hair Cells

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses

Profiling Synaptic Proteins Identifies Regulators of Insulin Secretion and Lifespan

Cells are organized into distinct compartments to perform specific tasks with spatial precision. In neurons, presynaptic specializations are biochemically complex subcellular structures dedicated to neurotransmitter secretion. Activity-dependent changes in the abundance of presynaptic proteins are thought to endow synapses with different functional states; however, relatively little is known about the rules that govern changes in the composition of presynaptic terminals. We describe a genetic strategy to systematically analyze protein localization at Caenorhabditis elegans presynaptic specializations. Nine presynaptic proteins were GFP-tagged, allowing visualization of multiple presynaptic structures. Changes in the distribution and abundance of these proteins were quantified in 25 mutants that alter different aspects of neurotransmission. Global analysis of these data identified novel relationships between particular presynaptic components and provides a new method to compare gene functions by identifying shared protein localization phenotypes. Using this strategy, we identified several genes that regulate secretion of insulin-like growth factors (IGFs) and influence lifespan in a manner dependent on insulin/IGF signaling

Synaptic Dysbindin-1 Reductions in Schizophrenia Occur in an Isoform-Specific Manner Indicating Their Subsynaptic Location

Background: An increasing number of studies report associations between variation in DTNBP1, a top candidate gene in schizophrenia, and both the clinical symptoms of the disorder and its cognitive deficits. DTNBP1 encodes dysbindin-1, reduced levels of which have been found in synaptic fields of schizophrenia cases. This study determined whether such synaptic reductions are isoform-specific. Methodology/Principal Findings: Using Western blotting of tissue fractions, we first determined the synaptic localization of the three major dysbindin-1 isoforms (A, B, and C). All three were concentrated in synaptosomes of multiple brain areas, including auditory association cortices in the posterior half of the superior temporal gyrus (pSTG) and the hippocampal formation (HF). Tests on the subsynaptic tissue fractions revealed that each isoform is predominantly, if not exclusively, associated with synaptic vesicles (dysbindin-1B) or with postsynaptic densities (dysbindin-1A and -1C). Using Western blotting on pSTG (n = 15) and HF (n = 15) synaptosomal fractions from schizophrenia cases and their matched controls, we discovered that synaptic dysbindin-1 is reduced in an isoform-specific manner in schizophrenia without changes in levels of synaptophysin or PSD-95. In pSTG, about 92% of the schizophrenia cases displayed synaptic dysbindin-1A reductions averaging 48% (p = 0.0007) without alterations in other dysbindin-1 isoforms. In the HF, by contrast, schizophrenia cases displayed normal levels of synaptic dysbindin-1A, but 67% showed synaptic reductions in dysbindin-1B averaging 33% (p = 0.0256), while 80% showed synaptic reductions in dysbindin-1C averaging 35% (p = 0.0171). Conclusions/Significance: Given the distinctive subsynaptic localization of dysbindin-1A, -1B, and -1C across brain regions, the observed pSTG reductions in dysbindin-1A are postsynaptic and may promote dendritic spine loss with consequent disruption of auditory information processing, while the noted HF reductions in dysbindin-1B and -1C are both presynaptic and postsynaptic and could promote deficits in spatial working memory

Gut microbiota and sirtuins in obesity-related inflammation and bowel dysfunction

Obesity is a chronic disease characterized by persistent low-grade inflammation with alterations in gut motility. Motor abnormalities suggest that obesity has effects on the enteric nervous system (ENS), which controls virtually all gut functions. Recent studies have revealed that the gut microbiota can affect obesity and increase inflammatory tone by modulating mucosal barrier function. Furthermore, the observation that inflammatory conditions influence the excitability of enteric neurons may add to the gut dysfunction in obesity. In this article, we discuss recent advances in understanding the role of gut microbiota and inflammation in the pathogenesis of obesity and obesity-related gastrointestinal dysfunction. The potential contribution of sirtuins in protecting or regulating the circuitry of the ENS under inflamed states is also considered

Epsin 1 Promotes Synaptic Growth by Enhancing BMP Signal Levels in Motoneuron Nuclei

We thank Carl-Henrik Heldin (Uppsala University, Sweden) for his generous gift of the PS1 pMad antibody, Hugo Bellen, Corey Goodman, Janis Fischer, Graeme Davis, Guillermo Marques, Michael O'Connor, Kate O'Connor-Giles, and the Bloomington Drosophila Stock Center for flies strains, the Developmental Studies Hybridoma Bank at the University of Iowa for antibodies to Wit and CSP; Marie Phillips for advice on membrane fractionation; Avital Rodal, Kate O'Connor-Giles, Ela Serpe, Kristi Wharton, Mojgan Padash-Barmchi for discussions or comments on the manuscript. We also thank Jody Summers at OUHSC for her generosity in letting us to use her confocal microscope.Conceived and designed the experiments: PAV TRF LRC BZ. Performed the experiments: PAV TRF LRC SMR HB NER BZ. Analyzed the data: PAV TRF LRC SMR HB NER BZ. Wrote the paper: PAV TRF BZ.Bone morphogenetic protein (BMP) retrograde signaling is crucial for neuronal development and synaptic plasticity. However, how the BMP effector phospho-Mother against decapentaplegic (pMad) is processed following receptor activation remains poorly understood. Here we show that Drosophila Epsin1/Liquid facets (Lqf) positively regulates synaptic growth through post-endocytotic processing of pMad signaling complex. Lqf and the BMP receptor Wishful thinking (Wit) interact genetically and biochemically. lqf loss of function (LOF) reduces bouton number whereas overexpression of lqf stimulates bouton growth. Lqf-stimulated synaptic overgrowth is suppressed by genetic reduction of wit. Further, synaptic pMad fails to accumulate inside the motoneuron nuclei in lqf mutants and lqf suppresses synaptic overgrowth in spinster (spin) mutants with enhanced BMP signaling by reducing accumulation of nuclear pMad. Interestingly, lqf mutations reduce nuclear pMad levels without causing an apparent blockage of axonal transport itself. Finally, overexpression of Lqf significantly increases the number of multivesicular bodies (MVBs) in the synapse whereas lqf LOF reduces MVB formation, indicating that Lqf may function in signaling endosome recycling or maturation. Based on these observations, we propose that Lqf plays a novel endosomal role to ensure efficient retrograde transport of BMP signaling endosomes into motoneuron nuclei.Yeshttp://www.plosone.org/static/editorial#pee