Vesicular Axonal Transport is Modified In Vivo by Tau Deletion or Overexpression in Drosophila.

Structural microtubule associated protein Tau is found in high amount in axons and is involved in several neurodegenerative diseases. Although many studies have highlighted the toxicity of an excess of Tau in neurons, the in vivo understanding of the endogenous role of Tau in axon morphology and physiology is poor. Indeed, knock-out mice display no strong cytoskeleton or axonal transport phenotype, probably because of some important functional redundancy with other microtubule-associated proteins (MAPs). Here, we took advantage of the model organism Drosophila, which genome contains only one homologue of the Tau/MAP2/MAP4 family to decipher (endogenous) Tau functions. We found that Tau depletion leads to a decrease in microtubule number and microtubule density within axons, while Tau excess leads to the opposite phenotypes. Analysis of vesicular transport in tau mutants showed altered mobility of vesicles, but no change in the total amount of putatively mobile vesicles, whereas both aspects were affected when Tau was overexpressed. In conclusion, we show that loss of Tau in tau mutants not only leads to a decrease in axonal microtubule density, but also impairs axonal vesicular transport, albeit to a lesser extent compared to the effects of an excess of Tau.

A presynaptic role of microtubule-associated protein 1/Futsch in Drosophila: regulation of active zone number and neurotransmitter release.

Structural microtubule-associated proteins (MAPs), like MAP1, not only control the stability of microtubules, but also interact with postsynaptic proteins in the nervous system. Their presynaptic role has barely been studied. To tackle this question, we used the Drosophila model in which there is only one MAP1 homolog: Futsch, which is expressed at the larval neuromuscular junction, presynaptically only. We show that Futsch regulates neurotransmitter release and active zone density. Importantly, we provide evidence that this role of Futsch is not just the consequence of its microtubule-stabilizing function. Using high-resolution microscopy, we show that Futsch and microtubules are almost systematically present in close proximity to active zones, with Futsch being localized in-between microtubules and active zones. Using proximity ligation assays, we further demonstrate the proximity of Futsch, but not microtubules, to active zone components. Altogether our data are in favor of a model by which Futsch locally stabilizes active zones, by reinforcing their link with the underlying microtubule cytoskeleton.

Drosophila Nesprin-1 controls glutamate receptor density at neuromuscular junctions.

Nesprin-1 is a core component of a protein complex connecting nuclei to cytoskeleton termed LINC (linker of nucleoskeleton and cytoskeleton). Nesprin-1 is anchored to the nuclear envelope by its C-terminal KASH domain, the disruption of which has been associated with neuronal and neuromuscular pathologies, including autosomal recessive cerebellar ataxia and Emery-Dreifuss muscular dystrophy. Here, we describe a new and unexpected role of Drosophila Nesprin-1, Msp-300, in neuromuscular junction. We show that larvae carrying a deletion of Msp-300 KASH domain (Msp-300 (∆KASH) ) present a locomotion defect suggestive of a myasthenia, and demonstrate the importance of muscle Msp-300 for this phenotype, using tissue-specific RNAi knock-down. We show that Msp-300 (∆KASH) mutants display abnormal neurotransmission at the larval neuromuscular junction, as well as an imbalance in postsynaptic glutamate receptor composition with a decreased percentage of GluRIIA-containing receptors. We could rescue Msp-300 (∆KASH) locomotion phenotypes by GluRIIA overexpression, suggesting that the locomotion impairment associated with the KASH domain deletion is due to a reduction in junctional GluRIIA. In summary, we found that Msp-300 controls GluRIIA density at the neuromuscular junction. Our results suggest that Drosophila is a valuable model for further deciphering how Nesprin-1 and LINC disruption may lead to neuronal and neuromuscular pathologies.

Engrailed homeoprotein acts as a signaling molecule in the developing fly.

Homeodomain transcription factors classically exert their morphogenetic activities through the cell-autonomous regulation of developmental programs. In vertebrates, several homeoproteins have also been shown to have direct non-cell-autonomous activities in the developing nervous system. We present the first in vivo evidence for homeoprotein signaling in Drosophila. Focusing on wing development as a model, we first demonstrate that the homeoprotein Engrailed (En) is secreted. Using single-chain anti-En antibodies expressed under the control of a variety of promoters, we delineate the wing territories in which secreted En acts. We show that En is a short-range signaling molecule that participates in anterior crossvein development, interacting with the Dpp signaling pathway. This report thus suggests that direct signaling with homeoproteins is an evolutionarily conserved phenomenon that is not restricted to neural tissues and involves interactions with bona fide signal transduction pathways.

The influence of environmental crisis perception and trait anxiety on the level of eco-worry and climate anxiety.

Eco-anxiety, which refers to the anxiety experienced in response to worsening environmental conditions, is a growing global phenomenon. Climate change anxiety, due to the escalating impact of ongoing climate change, stands out as the most commonly recognized form of eco-anxiety. Nevertheless, numerous uncertainties persist regarding the relationship of this eco-anxiety response to pro-environmental behaviors, as well as its connection with trait anxiety and the perception of the environmental crisis. In this study, we conducted an analysis with a sample size of 431 participants to elucidate the respective implications of these factors, delving into the different facets of the eco-anxiety response: worry and anxiety-related impairments. We measured eco-worry using a brief 5-item scale and assessed climate anxiety-related impairments using the Climate Change Anxiety Scale (CCAS). Our findings reveal that eco-worry acts as a mediator between the perception of the environmental crisis and the manifestation of climate anxiety-related impairments. Furthermore, eco-worry plays a constructive role in relation to the commitment to pro-environmental behaviors, with no additional contribution from the climate anxiety reaction involving impairments. In summary, our findings underscore the existence of distinct constructs within the anxiety response to climate change and environmental issues, each with different contributing factors.

[Unconventional protein secretion - new perspectives in protein trafficking]. / Sécrétion non conventionnelle - Nouvelles perspectives dans le trafic des protéines.

The characterization of the structural and functional organization of eukaryotic cells has revealed the membrane compartments and machinery required for vesicular protein transport. Most proteins essential for intercellular communication contain an N-terminal signal sequence enabling them to be incorporated into the biosynthetic or conventional secretory pathway, in which proteins are sequentially transported through the endoplasmic reticulum (ER) and the Golgi apparatus. However, major research studies have shown the existence of alternative secretory routes that are independent of the ER-Golgi and designated as unconventional secretory pathways. These pathways involve a large number of players that may divert specific compartments from their primary function in favor of secretory roles. The comprehensive description of these processes is therefore of utmost importance to unveil how proteins secreted through these alternative pathways control cell homeostasis or contribute to disease development.

Title: Sécrétion non conventionnelle - Nouvelles perspectives dans le trafic des protéines. Abstract: L'étude de l'organisation structurale et fonctionnelle des cellules eucaryotes a révélé les compartiments membranaires ainsi que la machinerie nécessaires au trafic vésiculaire des protéines. La plupart des protéines essentielles à la communication intercellulaire contiennent une séquence signal leur permettant d'être incorporées dans la voie de sécrétion conventionnelle, par laquelle les protéines sont transportées séquentiellement dans le réticulum endoplasmique (RE) puis l'appareil de Golgi. Cependant, les cellules eucaryotes sont également dotées de voies de sécrétion alternatives ou voies de sécrétion non conventionnelles, qui mettent en jeu de nombreux acteurs susceptibles de détourner certains compartiments de leurs fonctions principales au profit de fonctions sécrétoires.

Important neuronal toxicity of microtubule-bound Tau in vivo in Drosophila.

The microtubule-associated protein Tau is found in large amount in axons of neurons and is involved in human neurodegenerative diseases called tauopathies, which include Alzheimer's disease. In these diseases, the Tau protein is abnormally hyperphosphorylated and one therapeutic strategy currently under consideration consists in inhibiting Tau phosphorylation. However, the consequences of an excess of hypophosphorylated Tau onto neuronal physiology have not been investigated in vivo. Here we studied how important is Tau phosphorylation for axonal transport and neurohormone release in vivo, using the Drosophila model. Surprisingly, our results demonstrate a stronger toxicity of hypophosphorylated Tau for neuronal function, when compared with normal or pseudophosphorylated Tau. This reveals a potential limit of the current therapeutic strategy aimed at inhibiting Tau phosphorylation.

Mushroom body neuronal remodelling is necessary for short-term but not for long-term courtship memory in Drosophila.

The remodelling of neurons during their development is considered necessary for their normal function. One fundamental mechanism involved in this remodelling process in both vertebrates and invertebrates is axon pruning. A well-documented case of such neuronal remodelling is the developmental axon pruning of mushroom body γ neurons that occurs during metamorphosis in Drosophila. The γ neurons undergo pruning of larval-specific dendrites and axons at metamorphosis, followed by their regrowth as adult-specific dendrites and axons. We recently revealed a molecular cascade required for this pruning. The nuclear receptor ftz-f1 activates the expression of the steroid hormone receptor EcR-B1, a key component for γ remodelling, and represses expression of Hr39, an ftz-f1 homologous gene. If ectopically expressed in the γ neurons, HR39 inhibits normal pruning, probably by competing with endogenous FTZ-F1, which results in decreased EcR-B1 expression. The mushroom bodies are a bilaterally symmetric structure in the larval and adult brain and are involved in the processing of different types of olfactory memory. How memory is affected in pruning-deficient adult flies that possess larval-stage neuronal circuitry will help to explain the functional role of neuron remodelling. Flies overexpressing Hr39 are viable as adults and make it possible to assess the requirement for wild-type mushroom body pruning in memory. While blocking mushroom body neuron remodelling impaired memory after short-term courtship conditioning, long-term memory was normal. These results show that larval pruning is necessary for adult memory and that expression of courtship short-term memory and long-term memory may be parallel and independent.

Plant insecticide L-canavanine repels Drosophila via the insect orphan GPCR DmX.

For all animals, the taste sense is crucial to detect and avoid ingesting toxic molecules. Many toxins are synthesized by plants as a defense mechanism against insect predation. One example of such a natural toxic molecule is L-canavanine, a nonprotein amino acid found in the seeds of many legumes. Whether and how insects are informed that some plants contain L-canavanine remains to be elucidated. In insects, the taste sense relies on gustatory receptors forming the gustatory receptor (Gr) family. Gr proteins display highly divergent sequences, suggesting that they could cover the entire range of tastants. However, one cannot exclude the possibility of evolutionarily independent taste receptors. Here, we show that L-canavanine is not only toxic, but is also a repellent for Drosophila. Using a pharmacogenetic approach, we find that flies sense food containing this poison by the DmX receptor. DmXR is an insect orphan G-protein-coupled receptor that has partially diverged in its ligand binding pocket from the metabotropic glutamate receptor family. Blockade of DmXR function with an antagonist lowers the repulsive effect of L-canavanine. In addition, disruption of the DmXR encoding gene, called mangetout (mtt), suppresses the L-canavanine repellent effect. To avoid the ingestion of L-canavanine, DmXR expression is required in bitter-sensitive gustatory receptor neurons, where it triggers the premature retraction of the proboscis, thus leading to the end of food searching. These findings show that the DmX receptor, which does not belong to the Gr family, fulfills a gustatory function necessary to avoid eating a natural toxin.

Roads and hubs of unconventional protein secretion.

In eukaryotes, there is now compelling evidence that in addition to the conventional endoplasmic reticulum-Golgi secretory pathway, there are additional routes for the export of cytoplasmic proteins with a critical role in numerous physio-pathological conditions. These alternative secretory pathways or unconventional protein secretion (UPS) start now to be molecularly dissected, and while UPS landscape appears to be governed by a striking diversity and heterogeneity of mechanisms, common principles are emerging. We review here the role of key molecular determinants as well as the role of central hubs for UPS, highlighting the plasticity and dynamic properties of membrane-bound compartments. We also describe recent findings that position UPS as an integral component of adaptive responses to cope with particular cellular needs and stresses.

Protective role of Engrailed in a Drosophila model of Huntington's disease.

Huntington's disease (HD) is caused by the expansion of the polyglutamine (polyQ) tract in the human Huntingtin (hHtt) protein (polyQ-hHtt). Although this mutation behaves dominantly, htt loss of function may also contribute to HD pathogenesis. Using a Drosophila model of HD, we found that Engrailed (EN), a transcriptional activator of endogenous Drosophila htt (dhtt), is able to prevent aggregation of polyQ-hHtt. To interpret these findings, we tested and identified a protective role of N-terminal fragments of both Drosophila and Human wild-type Htt onto polyQ-hHtt-induced cellular defects. In addition, N-terminal parts of normal hHtt were also able to rescue eye degeneration due to the loss of Drosophila endogenous dhtt function. Thus, our data indicate that Drosophila and Human Htt share biological properties, and confirm a model whereby EN activates endogenous dhtt, which in turn prevents polyQ-hHtt-induced phenotypes. The protective role of wild-type hHtt N-terminal parts, specifically onto polyQ-hHtt-induced cellular toxicity suggests that the HD may be considered as a dominant negative disease rather than solely dominant.

Widespread brain distribution of the Drosophila metabotropic glutamate receptor.

Glutamate is the predominant excitatory neurotransmitter in the vertebrate brain, whereas acetylcholine has been considered to play the same role in insects. Recent studies have, however, questioned the latter view by showing a rather general distribution of glutamate transporters. Here, we describe the expression pattern of the receptor DmGlu-A (DmGluRA), the unique homolog of vertebrate metabotropic glutamate receptors. Metabotropic glutamate receptors play important roles in the regulation of glutamatergic neurotransmission. Using a specific antibody, we report DmGluRA expression in most neuropile areas in both larvae and adults, but not in the lobes of the mushroom bodies. These observations suggest a key role for glutamate in the insect brain.

Glutamate and its metabotropic receptor in Drosophila clock neuron circuits.

Identification of the neurotransmitters in clock neurons is critical for understanding the circuitry of the neuronal network that controls the daily behavioral rhythms in Drosophila. Except for the neuropeptide pigment-dispersing factor, no neurotransmitters have been clearly identified in the Drosophila clock neurons. Here we show that glutamate and its metabotropic receptor, DmGluRA, are components of the clock circuitry and modulate the rhythmic behavior pattern of Drosophila. The dorsal clock neurons, DN1s in the larval brain and some DN1s and DN3s in the adult brain, were immunolabeled with antibodies against Drosophila vesicular glutamate transporter (DvGluT), suggesting that they are glutamatergic. Because the DN1s may communicate with the primary pacemaker neurons, s-LN(v)s, we tested glutamate responses of dissociated larval s-LN(v)s by means of calcium imaging. Application of glutamate dose dependently decreased intracellular calcium in the s-LN(v)s. Pharmacology of the response suggests the presence of DmGluRA on the s-LN(v)s. Antibodies against DmGluRA labeled dissociated s-LN(v)s and the LN(v) dendrites in the intact larval and adult brain. The role of metabotropic glutamate signaling was tested in behavior assays in transgenic larvae and flies with altered DmGluRA expression in the LN(v)s and other clock neurons. Larval photophobic behavior was enhanced in DmGluRA mutants. For adults, we could induce altered activity patterns in the dark phase under LD conditions and increase the period during constant darkness by knockdown of DmGluRA expression in LN(v)s. Our results suggest that a glutamate signal from some of the DNs modulates the rhythmic behavior pattern via DmGluRA on the LN(v)s in Drosophila.

A New Behavioral Test and Associated Genetic Tools Highlight the Function of Ventral Abdominal Muscles in Adult Drosophila.

The function of the nervous system in complex animals is reflected by the achievement of specific behaviors. For years in Drosophila, both simple and complex behaviors have been studied and their genetic bases have emerged. The neuromuscular junction is maybe one of the prototypal simplest examples. A motor neuron establishes synaptic connections on its muscle cell target and elicits behavior: the muscle contraction. Different muscles in adult fly are related to specific behaviors. For example, the thoracic muscles are associated with flight and the leg muscles are associated with locomotion. However, specific tools are still lacking for the study of cellular physiology in distinct motor neuron subpopulations. Here we decided to use the abdominal muscles and in particular the ventral abdominal muscles (VAMs) in adult Drosophila as new model to link a precise behavior to specific motor neurons. Hence, we developed a new behavioral test based on the folding movement of the adult abdomen. Further, we performed a genetic screen and identify two specific Gal4 lines with restricted expression patterns to the adult motor neurons innervating the VAMs or their precursor cells. Using these genetic tools, we showed that the lack of the VAMs or the loss of the synaptic transmission in their innervating motor neurons lead to a significant impairment of the abdomen folding behavior. Altogether, our results allow establishing a direct link between specific motor neurons and muscles for the realization of particular behavior: the folding behavior of the abdomen in Drosophila.

Identification of DmTTLL5 as a Major Tubulin Glutamylase in the Drosophila Nervous System.

Microtubules (MTs) play crucial roles during neuronal life. They are formed by heterodimers of alpha and beta-tubulins, which are subjected to several post-translational modifications (PTMs). Amongst them, glutamylation consists in the reversible addition of a variable number of glutamate residues to the C-terminal tails of tubulins. Glutamylation is the most abundant MT PTM in the mammalian adult brain, suggesting that it plays an important role in the nervous system (NS). Here, we show that the previously uncharacterized CG31108 gene encodes an alpha-tubulin glutamylase acting in the Drosophila NS. We show that this glutamylase, which we named DmTTLL5, initiates MT glutamylation specifically on alpha-tubulin, which are the only glutamylated tubulin in the Drosophila brain. In DmTTLL5 mutants, MT glutamylation was not detected in the NS, allowing for determining its potential function. DmTTLL5 mutants are viable and we did not find any defect in vesicular axonal transport, synapse morphology and larval locomotion. Moreover, DmTTLL5 mutant flies display normal negative geotaxis behavior and their lifespan is not altered. Thus, our work identifies DmTTLL5 as the major enzyme responsible for initiating neuronal MT glutamylation specifically on alpha-tubulin and we show that the absence of MT glutamylation is not detrimental for Drosophila NS function.

Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death.

In neurodegenerative diseases such as Alzheimer's disease (AD), cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown, in particular with respect to the microtubule (MT)-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of affected individuals. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for MTs, there is, to our knowledge, no indication that an excess of this protein could affect mitosis. Here, we studied the effect of an excess of human Tau (hTau) protein on cell mitosis in vivo. Using the Drosophila developing wing disc epithelium as a model, we show that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. We studied the mechanism of action of hTau and found that the MT-binding domain of hTau is responsible for these defects. We also demonstrate that the effects of hTau occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11). We finally show that this deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts) and tissues (the developing eye disc), as well as in human HeLa cells. By demonstrating that MT-bound Tau inhibits the Eg5 kinesin and cell mitosis, our work provides a new framework to consider the role of Tau in neurodegenerative diseases.

Honey Bee Allatostatins Target Galanin/Somatostatin-Like Receptors and Modulate Learning: A Conserved Function?

Sequencing of the honeybee genome revealed many neuropeptides and putative neuropeptide receptors, yet functional characterization of these peptidic systems is scarce. In this study, we focus on allatostatins, which were first identified as inhibitors of juvenile hormone synthesis, but whose role in the adult honey bee (Apis mellifera) brain remains to be determined. We characterize the bee allatostatin system, represented by two families: allatostatin A (Apime-ASTA) and its receptor (Apime-ASTA-R); and C-type allatostatins (Apime-ASTC and Apime-ASTCC) and their common receptor (Apime-ASTC-R). Apime-ASTA-R and Apime-ASTC-R are the receptors in bees most closely related to vertebrate galanin and somatostatin receptors, respectively. We examine the functional properties of the two honeybee receptors and show that they are transcriptionally expressed in the adult brain, including in brain centers known to be important for learning and memory processes. Thus we investigated the effects of exogenously applied allatostatins on appetitive olfactory learning in the bee. Our results show that allatostatins modulate learning in this insect, and provide important insights into the evolution of somatostatin/allatostatin signaling.

Deletion of Neurotrophin Signaling through the Glucocorticoid Receptor Pathway Causes Tau Neuropathology.

Glucocorticoid resistance is a risk factor for Alzheimer's disease (AD). Molecular and cellular mechanisms of glucocorticoid resistance in the brain have remained unknown and are potential therapeutic targets. Phosphorylation of glucocorticoid receptors (GR) by brain-derived neurotrophic factor (BDNF) signaling integrates both pathways for remodeling synaptic structure and plasticity. The goal of this study is to test the role of the BDNF-dependent pathway on glucocorticoid signaling in a mouse model of glucocorticoid resistance. We report that deletion of GR phosphorylation at BDNF-responding sites and downstream signaling via the MAPK-phosphatase DUSP1 triggers tau phosphorylation and dendritic spine atrophy in mouse cortex. In human cortex, DUSP1 protein expression correlates with tau phosphorylation, synaptic defects and cognitive decline in subjects diagnosed with AD. These findings provide evidence for a causal role of BDNF-dependent GR signaling in tau neuropathology and indicate that DUSP1 is a potential target for therapeutic interventions.

Shaggy, the homolog of glycogen synthase kinase 3, controls neuromuscular junction growth in Drosophila.

A protein-trap screen using the Drosophila neuromuscular junction (NMJ) as a model synapse was performed to identify genes that control synaptic structure or plasticity. We found that Shaggy (Sgg), the Drosophila homolog of the mammalian glycogen synthase kinases 3 alpha and beta, two serine-threonine kinases, was concentrated at this synapse. Using various combinations of mutant alleles of shaggy, we found that Shaggy negatively controlled the NMJ growth. Moreover, tissue-specific expression of a dominant-negative Sgg indicated that this kinase is required in the motoneuron, but not in the muscle, to control NMJ growth. Finally, we show that Sgg controlled the microtubule cytoskeleton dynamics in the motoneuron and that Futsch, a microtubule-associated protein, was required for Shaggy function on synaptic growth.

The Drosophila metabotropic glutamate receptor DmGluRA regulates activity-dependent synaptic facilitation and fine synaptic morphology.

In vertebrates, several groups of metabotropic glutamate receptors (mGluRs) are known to modulate synaptic properties. In contrast, the Drosophila genome encodes a single functional mGluR (DmGluRA), an ortholog of vertebrate group II mGluRs, greatly expediting the functional characterization of mGluR-mediated signaling in the nervous system. We show here that DmGluRA is expressed at the glutamatergic neuromuscular junction (NMJ), localized in periactive zones of presynaptic boutons but excluded from active sites. Null DmGluRA mutants are completely viable, and all of the basal NMJ synaptic transmission properties are normal. In contrast, DmGluRA mutants display approximately a threefold increase in synaptic facilitation during short stimulus trains. Prolonged stimulus trains result in very strongly increased ( approximately 10-fold) augmentation, including the appearance of asynchronous, bursting excitatory currents never observed in wild type. Both defects are rescued by expression of DmGluRA only in the neurons, indicating a specific presynaptic requirement. These phenotypes are reminiscent of hyperexcitable mutants, suggesting a role of DmGluRA signaling in the regulation of presynaptic excitability properties. The mutant phenotypes could not be replicated by acute application of mGluR antagonists, suggesting that DmGluRA regulates the development of presynaptic properties rather than directly controlling short-term modulation. DmGluRA mutants also display mild defects in NMJ architecture: a decreased number of synaptic boutons accompanied by an increase in mean bouton size. These morphological changes bidirectionally correlate with DmGluRA levels in the presynaptic terminal. These data reveal the following two roles for DmGluRA in presynaptic mechanisms: (1) modulation of presynaptic excitability properties important for the control of activity-dependent neurotransmitter release and (2) modulation of synaptic architecture.