Drosophila Homolog of the Human Carpenter Syndrome Linked Gene, MEGF8, Is Required for Synapse Development and Function.

Drosophila multiple epidermal growth factor-like domains 8 (dMegf8) is a homolog of human MEGF8 MEGF8 encodes a multidomain transmembrane protein which is highly conserved across species. In humans, MEGF8 mutations cause a rare genetic disorder called Carpenter syndrome, which is frequently associated with abnormal left-right patterning, cardiac defects, and learning disabilities. MEGF8 is also associated with psychiatric disorders. Despite its clinical relevance, MEGF8 remains poorly characterized; and although it is highly conserved, studies on animal models of Megf8 are also very limited. The presence of intellectual disabilities in Carpenter syndrome patients and association of MEGF8 with psychiatric disorders indicate that mutations in MEGF8 cause underlying defects in synaptic structure and functions. In this study, we investigated the role of Drosophila dMegf8 in glutamatergic synapses of the larval neuromuscular junctions (NMJ) in both males and females. We show that dMegf8 localizes to NMJ synapses and is required for proper synaptic growth. dMegf8 mutant larvae and adults show severe motor coordination deficits. At the NMJ, dMegf8 mutants show altered localization of presynaptic and postsynaptic proteins, defects in synaptic ultrastructure, and neurotransmission. Interestingly, dMegf8 mutants have reduced levels of the Type II BMP receptor Wishful thinking (Wit). dMegf8 displays genetic interactions with neurexin-1 (dnrx) and wit, and in association with Dnrx and Wit plays an essential role in synapse organization. Our studies provide insights into human MEGF8 functions and potentially into mechanisms that may underlie intellectual disabilities observed in Carpenter syndrome as well as MEGF8-related synaptic structural and/or functional deficits in psychiatric disorders.SIGNIFICANCE STATEMENT Carpenter syndrome, known for over a century now, is a genetic disorder linked to mutations in Multiple Epidermal Growth Factor-like Domains 8 (MEGF8) gene and associated with intellectual disabilities among other symptoms. MEGF8 is also associated with psychiatric disorders. Despite the high genetic conservation and clinical relevance, the functions of MEGF8 remain largely uncharacterized. Patients with intellectual disabilities and psychiatric diseases often have an underlying defect in synaptic structure and function. This work defines the role of the fly homolog of human MEGF8, dMegf8, in glutamatergic synapse growth, organization, and function and provide insights into potential functions of MEGF8 in human central synapses and synaptic mechanisms that may underlie psychiatric disorders and intellectual disabilities seen in Carpenter syndrome.

TM2D genes regulate Notch signaling and neuronal function in Drosophila.

TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each model organism species that has been sequenced. Rare variants in TM2D3 are associated with Alzheimer's disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. We knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the γ-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive motor and electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD.

Activity-Dependent Global Downscaling of Evoked Neurotransmitter Release across Glutamatergic Inputs in Drosophila.

Within mammalian brain circuits, activity-dependent synaptic adaptations, such as synaptic scaling, stabilize neuronal activity in the face of perturbations. Stability afforded through synaptic scaling involves uniform scaling of quantal amplitudes across all synaptic inputs formed on neurons, as well as on the postsynaptic side. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. We tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. We used motoneuron-specific genetic manipulations to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. We discovered an adaptation which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This "presynaptic downscaling" maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. Our results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side.SIGNIFICANCE STATEMENT To date, compensatory adaptations which stabilise target cell activity through activity-dependent global scaling have been observed only within central circuits, and on the postsynaptic side. Considering that maintenance of stable activity is imperative for the robust function of the nervous system as a whole, we tested whether activity-dependent global scaling could also manifest within peripheral circuits. We uncovered a compensatory adaptation which causes global scaling within a peripheral circuit and on the presynaptic side through uniform downscaling of evoked neurotransmitter release. Unlike in central circuits, uniform scaling maintains functionality over a wide, rather than a narrow, operational range, affording robust and stable activity. Activity-dependent global scaling therefore operates on both the presynaptic and postsynaptic sides to maintain target cell activity.

PGC1α Controls Sucrose Taste Sensitization in Drosophila.

Perceived palatability of food controls caloric intake. Sweet taste is the primary means of detecting the carbohydrate content of food. Surprisingly, sweet taste sensitivity is responsive to extrinsic factors like diet, and this occurs by unknown mechanisms. Here, we describe an unbiased proteomic investigation into sweet taste sensitivity in the fruit fly. We identify a dopamine/cyclic AMP (cAMP)/CREB axis acting within sweet taste neurons that controls taste perception but is largely dispensable for acute taste transduction. This pathway modulates sweet taste perception in response to both sensory- and nutrient-restricted diets and converges on PGC1α, a critical regulator of metabolic health and lifespan. By electrophysiology, we found that enhanced sucrose taste sensitivity was the result of heightened sweet taste intensity and that PGC1α was both necessary and sufficient for this effect. Together, we provide the first molecular insight into how diet-induced taste perception is regulated within the sweet taste neuron.

Nerve injury drives a heightened state of vigilance and neuropathic sensitization in Drosophila.

Injury can lead to devastating and often untreatable chronic pain. While acute pain perception (nociception) evolved more than 500 million years ago, virtually nothing is known about the molecular origin of chronic pain. Here we provide the first evidence that nerve injury leads to chronic neuropathic sensitization in insects. Mechanistically, peripheral nerve injury triggers a loss of central inhibition that drives escape circuit plasticity and neuropathic allodynia. At the molecular level, excitotoxic signaling within GABAergic (γ-aminobutyric acid) neurons required the acetylcholine receptor nAChRα1 and led to caspase-dependent death of GABAergic neurons. Conversely, disruption of GABA signaling was sufficient to trigger allodynia without injury. Last, we identified the conserved transcription factor twist as a critical downstream regulator driving GABAergic cell death and neuropathic allodynia. Together, we define how injury leads to allodynia in insects, and describe a primordial precursor to neuropathic pain may have been advantageous, protecting animals after serious injury.

Tubulin Polymerization Promoting Protein, Ringmaker, and MAP1B Homolog Futsch Coordinate Microtubule Organization and Synaptic Growth.

Drosophila Ringmaker (Ringer) is homologous to the human Tubulin Polymerization Promoting Proteins (TPPPs) that are implicated in the stabilization and bundling of microtubules (MTs) that are particularly important for neurons and are also implicated in synaptic organization and plasticity. No in vivo functional data exist that have addressed the role of TPPP in synapse organization in any system. Here, we present the phenotypic and functional characterization of ringer mutants during Drosophila larval neuromuscular junction (NMJ) synaptic development. ringer mutants show reduced synaptic growth and transmission and display phenotypic similarities and genetic interactions with the Drosophila homolog of vertebrate Microtubule Associated Protein (MAP)1B, futsch. Immunohistochemical and biochemical analyses show that individual and combined loss of Ringer and Futsch cause a significant reduction in MT loops at the NMJs and reduced acetylated-tubulin levels. Presynaptic over-expression of Ringer and Futsch causes elevated levels of acetylated-tubulin and significant increase in NMJ MT loops. These results indicate that Ringer and Futsch regulate synaptic MT organization in addition to synaptic growth. Together our findings may inform studies on the close mammalian homolog, TPPP, and provide insights into the role of MTs and associated proteins in synapse growth and organization.

High Dietary Sugar Reshapes Sweet Taste to Promote Feeding Behavior in Drosophila melanogaster.

Recent studies find that sugar tastes less intense to humans with obesity, but whether this sensory change is a cause or a consequence of obesity is unclear. To tackle this question, we study the effects of a high sugar diet on sweet taste sensation and feeding behavior in Drosophila melanogaster. On this diet, fruit flies have lower taste responses to sweet stimuli, overconsume food, and develop obesity. Excess dietary sugar, but not obesity or dietary sweetness alone, caused taste deficits and overeating via the cell-autonomous action of the sugar sensor O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in the sweet-sensing neurons. Correcting taste deficits by manipulating the excitability of the sweet gustatory neurons or the levels of OGT protected animals from diet-induced obesity. Our work demonstrates that the reshaping of sweet taste sensation by excess dietary sugar drives obesity and highlights the role of glucose metabolism in neural activity and behavior.

Neuronal Lamin regulates motor circuit integrity and controls motor function and lifespan.

Neuronal aging involves a progressive decline in cognitive abilities and loss of motor function. Mutations in human Lamin genes (LMNA, LMNB1, LMNB2) lead to a wide-range of diseases including muscular dystrophy, peripheral neuropathy and progeria. Here we investigate the role of neuronal Lamin in regulating age-related phenotypes. Neuronal targeting of Lamin led to shortened lifespan, progressive impairment of motor function and loss of dopaminergic (DA) neurons within the protocerebral anterior medial (PAM) cluster in the Drosophila melanogaster brain. Loss of neuronal Lamin caused an age-related decline in neural physiology, with slower neurotransmission and increased chance of motor circuit failure with age. Unexpectedly, Lamin-dependent decline in motor function was specific for the chemical synapses of the dorsal longitudinal muscle (DLM). Together these findings highlight a central role for Lamin dysfunction in regulating neuronal survival and motor circuit physiology during aging.

A simple high throughput assay to evaluate water consumption in the fruit fly.

Water intake is essential for survival and thus under strong regulation. Here, we describe a simple high throughput system to monitor water intake over time in Drosophila. The design of the assay involves dehydrating fly food and then adding water back separately so flies either eat or drink. Water consumption is then evaluated by weighing the water vessel and comparing this back to an evaporation control. Our system is high throughput, does not require animals to be artificially dehydrated, and is simple both in design and implementation. Initial characterisation of homeostatic water consumption shows high reproducibility between biological replicates in a variety of experimental conditions. Water consumption was dependent on ambient temperature and humidity and was equal between sexes when corrected for mass. By combining this system with the Drosophila genetics tools, we could confirm a role for ppk28 and DopR1 in promoting water consumption, and through functional investigation of RNAseq data from dehydrated animals, we found DopR1 expression in the mushroom body was sufficient to drive consumption and enhance water taste sensitivity. Together, we provide a simple high throughput water consumption assay that can be used to dissect the cellular and molecular machinery regulating water homeostasis in Drosophila.

The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission.

Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.

Sucralose Promotes Food Intake through NPY and a Neuronal Fasting Response.

Non-nutritive sweeteners like sucralose are consumed by billions of people. While animal and human studies have demonstrated a link between synthetic sweetener consumption and metabolic dysregulation, the mechanisms responsible remain unknown. Here we use a diet supplemented with sucralose to investigate the long-term effects of sweet/energy imbalance. In flies, chronic sweet/energy imbalance promoted hyperactivity, insomnia, glucose intolerance, enhanced sweet taste perception, and a sustained increase in food and calories consumed, effects that are reversed upon sucralose removal. Mechanistically, this response was mapped to the ancient insulin, catecholamine, and NPF/NPY systems and the energy sensor AMPK, which together comprise a novel neuronal starvation response pathway. Interestingly, chronic sweet/energy imbalance promoted increased food intake in mammals as well, and this also occurs through an NPY-dependent mechanism. Together, our data show that chronic consumption of a sweet/energy imbalanced diet triggers a conserved neuronal fasting response and increases the motivation to eat.

A TRPV channel in Drosophila motor neurons regulates presynaptic resting Ca2+ levels, synapse growth, and synaptic transmission.

Presynaptic resting Ca(2+) influences synaptic vesicle (SV) release probability. Here, we report that a TRPV channel, Inactive (Iav), maintains presynaptic resting [Ca(2+)] by promoting Ca(2+) release from the endoplasmic reticulum in Drosophila motor neurons, and is required for both synapse development and neurotransmission. We find that Iav activates the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin, which is essential for presynaptic microtubule stabilization at the neuromuscular junction. Thus, loss of Iav induces destabilization of presynaptic microtubules, resulting in diminished synaptic growth. Interestingly, expression of human TRPV1 in Iav-deficient motor neurons rescues these defects. We also show that the absence of Iav causes lower SV release probability and diminished synaptic transmission, whereas Iav overexpression elevates these synaptic parameters. Together, our findings indicate that Iav acts as a key regulator of synaptic development and function by influencing presynaptic resting [Ca(2+)].

Mitochondrial fusion but not fission regulates larval growth and synaptic development through steroid hormone production.

Mitochondrial fusion and fission affect the distribution and quality control of mitochondria. We show that Marf (Mitochondrial associated regulatory factor), is required for mitochondrial fusion and transport in long axons. Moreover, loss of Marf leads to a severe depletion of mitochondria in neuromuscular junctions (NMJs). Marf mutants also fail to maintain proper synaptic transmission at NMJs upon repetitive stimulation, similar to Drp1 fission mutants. However, unlike Drp1, loss of Marf leads to NMJ morphology defects and extended larval lifespan. Marf is required to form contacts between the endoplasmic reticulum and/or lipid droplets (LDs) and for proper storage of cholesterol and ecdysone synthesis in ring glands. Interestingly, human Mitofusin-2 rescues the loss of LD but both Mitofusin-1 and Mitofusin-2 are required for steroid-hormone synthesis. Our data show that Marf and Mitofusins share an evolutionarily conserved role in mitochondrial transport, cholesterol ester storage and steroid-hormone synthesis.

Syncrip/hnRNP Q influences synaptic transmission and regulates BMP signaling at the Drosophila neuromuscular synapse.

Synaptic plasticity involves the modulation of synaptic connections in response to neuronal activity via multiple pathways. One mechanism modulates synaptic transmission by retrograde signals from the post-synapse that influence the probability of vesicle release in the pre-synapse. Despite its importance, very few factors required for the expression of retrograde signals, and proper synaptic transmission, have been identified. Here, we identify the conserved RNA binding protein Syncrip as a new factor that modulates the efficiency of vesicle release from the motoneuron and is required for correct synapse structure. We show that syncrip is required genetically and its protein product is detected only in the muscle and not in the motoneuron itself. This unexpected non-autonomy is at least partly explained by the fact that Syncrip modulates retrograde BMP signals from the muscle back to the motoneuron. We show that Syncrip influences the levels of the Bone Morphogenic Protein ligand Glass Bottom Boat from the post-synapse and regulates the pre-synapse. Our results highlight the RNA-binding protein Syncrip as a novel regulator of synaptic output. Given its known role in regulating translation, we propose that Syncrip is important for maintaining a balance between the strength of presynaptic vesicle release and postsynaptic translation.

Crag is a GEF for Rab11 required for rhodopsin trafficking and maintenance of adult photoreceptor cells.

Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.

Drosophila neuroligin 2 is required presynaptically and postsynaptically for proper synaptic differentiation and synaptic transmission.

Trans-synaptic adhesion between Neurexins (Nrxs) and Neuroligins (Nlgs) is thought to be required for proper synapse organization and modulation, and mutations in several human Nlgs have shown association with autism spectrum disorders. Here we report the generation and phenotypic characterization of Drosophila neuroligin 2 (dnlg2) mutants. Loss of dnlg2 results in reduced bouton numbers, aberrant presynaptic and postsynaptic development at neuromuscular junctions (NMJs), and impaired synaptic transmission. In dnlg2 mutants, the evoked responses are decreased in amplitude, whereas the total active zone (AZ) numbers at the NMJ are comparable to wild type, suggesting a decrease in the release probability. Ultrastructurally, the presynaptic AZ number per bouton area and the postsynaptic density area are both increased in dnlg2 mutants, whereas the subsynaptic reticulum is reduced in volume. We show that both presynaptic and postsynaptic expression of Dnlg2 is required to restore synaptic growth and function in dnlg2 mutants. Postsynaptic expression of Dnlg2 in dnlg2 mutants and wild type leads to reduced bouton growth whereas presynaptic and postsynaptic overexpression in wild-type animals results in synaptic overgrowth. Since Nlgs have been shown to bind to Nrxs, we created double mutants. These mutants are viable and display phenotypes that closely resemble those of dnlg2 and dnrx single mutants. Our results provide compelling evidence that Dnlg2 functions both presynaptically and postsynaptically together with Neurexin to determine the proper number of boutons as well as the number of AZs and size of synaptic densities during the development of NMJs.

Tilting the balance between facilitatory and inhibitory functions of mammalian and Drosophila Complexins orchestrates synaptic vesicle exocytosis.

SNARE-mediated synaptic exocytosis is orchestrated by facilitatory and inhibitory mechanisms. Genetic ablations of Complexins, a family of SNARE-complex-binding proteins, in mice and Drosophila cause apparently opposite effects on neurotransmitter release, leading to contradictory hypotheses of Complexin function. Reconstitution experiments with different fusion assays and Complexins also yield conflicting results. We therefore performed cross-species rescue experiments to compare the functions of murine and Drosophila Complexins in both mouse and fly synapses. We found that murine and Drosophila Complexins employ conserved mechanisms to regulate exocytosis despite their strikingly different overall effects on neurotransmitter release. Both Complexins contain distinct domains that facilitate or inhibit synaptic vesicle fusion, and the strength of each facilitatory or inhibitory function differs significantly between murine and Drosophila Complexins. Our results show that a relative shift in the balance of facilitatory and inhibitory functions results in differential regulation of neurotransmitter release by murine and Drosophila Complexins in vivo, reconciling previous incompatible findings.

A synaptic vesicle-associated Ca2+ channel promotes endocytosis and couples exocytosis to endocytosis.

Synaptic vesicle (SV) exo- and endocytosis are tightly coupled to sustain neurotransmission in presynaptic terminals, and both are regulated by Ca(2+). Ca(2+) influx triggered by voltage-gated Ca(2+) channels is necessary for SV fusion. However, extracellular Ca(2+) has also been shown to be required for endocytosis. The intracellular Ca(2+) levels (<1 microM) that trigger endocytosis are typically much lower than those (>10 microM) needed to induce exocytosis, and endocytosis is inhibited when the Ca(2+) level exceeds 1 microM. Here, we identify and characterize a transmembrane protein associated with SVs that, upon SV fusion, localizes at periactive zones. Loss of Flower results in impaired intracellular resting Ca(2+) levels and impaired endocytosis. Flower multimerizes and is able to form a channel to control Ca(2+) influx. We propose that Flower functions as a Ca(2+) channel to regulate synaptic endocytosis and hence couples exo- with endocytosis.

Importin 13 regulates neurotransmitter release at the Drosophila neuromuscular junction.

In an unbiased genetic screen designed to isolate mutations that affect synaptic transmission, we have isolated homozygous lethal mutations in Drosophila importin 13 (imp13). Imp13 is expressed in and around nuclei of both neurons and muscles. At the larval neuromuscular junction (NMJ), imp13 affects muscle growth and formation of the subsynaptic reticulum without influencing any presynaptic structural features. In the absence of imp13, the probability of release of neurotransmitter and quantal content is increased, yet the abundance of the postsynaptic receptors and the amplitude of miniature excitatory junctional potentials are not affected. Interestingly, imp13 is required in the muscles to control presynaptic release. Thus, imp13 is a novel factor that affects neurotransmitter release at the fly NMJ. Its role in the context of synaptic homeostasis is discussed.

Fos and Jun potentiate individual release sites and mobilize the reserve synaptic vesicle pool at the Drosophila larval motor synapse.

In all nervous systems, short-term enhancement of transmitter release is achieved by increasing the weights of unitary synapses; in contrast, long-term enhancement, which requires nuclear gene expression, is generally thought to be mediated by the addition of new synaptic vesicle release sites. In Drosophila motor neurons, induction of AP-1, a heterodimer of Fos and Jun, induces cAMP- and CREB-dependent forms of presynaptic enhancement. Light and electron microscopic studies indicate that this synaptic enhancement is caused by increasing the weight of unitary synapses and not through the insertion of additional release sites. Electrophysiological and optical measurements of vesicle dynamics demonstrate that enhanced neurotransmitter release is accompanied by an increase in the actively cycling synaptic vesicle pool at the expense of the reserve pool. Finally, the observation that AP-1 mediated enhancement eliminates tetanus-induced forms of presynaptic potentiation suggests: (i) that reserve-pool mobilization is required for tetanus-induced short-term synaptic plasticity; and (ii) that long-term synaptic plasticity may, in some instances, be accomplished by stable recruitment of mechanisms that normally underlie short-term synaptic change.