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International audience; Mitochondrial defects associated with respiratory chain complex I deficiency lead to heterogeneous fatal syndromes. While the role of NDUFS8, an essential subunit of the core assembly of the complex I, is established in mitochondrial diseases, the mechanisms underlying neuropathology are poorly understood. We developed a Drosophila model of NDUFS8 deficiency by knocking down the expression of its fly homologue in neurons or in glial cells. Downregulating ND23 in neurons resulted in shortened lifespan, and decreased locomotion. Although total brain ATP levels were decreased, histological analysis did not reveal any signs of neurodegeneration except for photoreceptors of the retina. Interestingly, ND23 deficiency-associated phenotypes were rescued by overexpressing the glucose transporter hGluT3 demonstrating that boosting glucose metabolism in neurons was sufficient to bypass altered mitochondrial functions and to confer neuroprotection. We then analyzed the consequences of ND23 knockdown in glial cells. In contrast to neuronal knockdown, loss of ND23 in glia did not lead to significant behavioral defects nor to reduced lifespan, but induced brain degeneration, as visualized by numerous vacuoles found all over the nervous tissue. This phenotype was accompanied by the massive accumulation of lipid droplets at the cortex-neuropile boundaries, suggesting an alteration of lipid metabolism in glia. These results demonstrate that complex I deficiency triggers metabolic alterations both in neurons and glial cells which may contribute to the neuropathology.

International audience; Huntington's disease (HD) is a polyglutamine (polyQ) disease caused by an expanded CAG tract within the coding region of Huntingtin protein. Mutant Huntingtin (mHtt) is ubiquitously expressed, abundantly in neurons but also significantly in glial cells. Neuron-intrinsic mechanism and alterations in glia-to-neuron communication both contribute to the neuronal dysfunction and death in HD pathology. However, it remains to be determined the role of glial cells in HD pathogenesis. In recent years, development of Drosophila models facilitated the dissection of the cellular and molecular events in polyQ-related diseases. By using genetic approaches in Drosophila, we manipulated the expression levels of mitochondrial uncoupling proteins (UCPs) that regulate production of both ATP and reactive oxygen species in mitochondria. We discovered that enhanced levels of UCPs alleviated the HD phenotype when mHtt was selectively expressed in glia, including defects in locomotor behavior and early death of Drosophila. In contrast, UCPs failed to prevent the HD toxicity in neurons. Increased oxidative stress defense was found to rescue neuron but not glia-induced pathology. Evidence is now emerging that UCPs are fundamental to adapt the energy metabolism in order to meet the metabolic demand. Thus, we propose that UCPs are glioprotective by rescuing energy-dependent functions in glia that are challenged by mHtt. In support of this, increasing glucose entry in glia was found to alleviate glia-induced pathology. Altogether, our data emphasize the importance of energy metabolism in the glial alterations in HD and may lead to a new therapeutic avenue.

The Drosophila excitatory amino acid transporters dEAAT1 and dEAAT2 are nervous-specific transmembrane proteins that mediate the high affinity uptake of L-glutamate or aspartate into cells. Here, we demonstrate by colocalization studies that both genes are expressed in discrete and partially overlapping subsets of differentiated glia and not in neurons in the embryonic central nervous system (CNS). We show that expression of these transporters is disrupted in mutant embryos deficient for the glial fate genes glial cells missing (gcm) and reversed polarity (repo). Conversely, ectopic expression of gcm in neuroblasts, which forces all nerve cells to adopt a glial fate, induces an ubiquitous expression of both EAAT genes in the nervous system. We also detected the dEAAT transcripts in the midline glia in late embryos and dEAAT2 in a few peripheral neurons in head sensory organs. Our results show that glia play a major role in excitatory amino acid transport in the Drosophila CNS and that regulated expression of the dEAAT genes contributes to generate the functional diversity of glial cells during embryonic development.

In vertebrates, excitatory amino acid transporters (EAATs) are believed to mediate the removal of glutamate released at excitatory synapses and to maintain extracellular concentrations of this neurotransmitter below excitotoxic levels. Glutamate is also used in insects as an excitatory neurotransmitter at the neuromuscular junction and probably in the central nervous system where its role remains to be established. We report the molecular characterization and developmental expression pattern of two Drosophila cDNAs: dEAAT1, which has recently been identified as a high affinity glutamate transporter [1], and dEAAT2, a novel protein sharing strong homology to dEAAT1 and to the mammalian EAAT protein family. The developmental expression pattern of the two Drosophila EAAT genes has been compared by Northern blot analysis and whole-mount in situ hybridizations. The two transporters are transcribed in distinct cell types of the nervous system and are strongly expressed in the adult visual system.

A cDNA clone isolated from the wing discs at the metamorphosis of Bombyx mori during the period of morphogenesis has been characterized. The amino acid sequence predicted for the putative protein is highly homologous to the Drosophila alpha1-tubulin. This is the first alpha-tubulin gene isolated in Bombyx mori and other isotype sequences are present in the Bombyx genome. The transcript is detected in the wing discs at every postembryonic stage examined, and is also expressed in other tissues, but at different levels. Although the mRNA level is maximum when the 20-hydroxyecdysone titre is high, its accumulation is independent of the hormone level both in vivo and in vitro. Significance of the accumulation of the mRNA of an ubiquitous alpha-tubulin in developing wing discs is discussed by comparison with our knowledge of the alpha-tubulin family in Drosophila and in other organisms.

International audience; Huntington's disease (HD) is a genetic neurodegenerative disease characterized by movement disorders, cognitive decline and neuropsychiatric symptoms. HD is caused by expanded CAG tract within the coding region of Huntingtin protein. Despite major insights into the molecular mechanisms leading to HD, no effective cure is yet available. Mutant Huntingtin (mHtt) has been reported to alter the stability and levels of β-Catenin, a key molecule in cell adhesion and signal transduction in Wingless (Wg)/Wnt pathway. However it remains to establish whether manipulation of Wg/Wnt signaling can impact HD pathology. We here investigated the phenotypic interactions between mHtt and Wg/Wnt signaling by using the power of Drosophila genetics. We provide compelling evidence that reducing Armadillo/β-Catenin levels confers protection and that this beneficial effect is correlated with the inactivation of the canonical Wg/Wnt signaling pathway. Knockdowns of Wnt ligands or of the downstream transcription factor Pangolin/TCF both ameliorate the survival of HD flies. Similarly, overexpression of one Armadillo/β-Catenin destruction complex component (Axin, APC2 or Shaggy/GSK-3β) increases the lifespan of HD flies. Loss of functional Armadillo/β-Catenin not only abolishes neuronal intrinsic but also glia-induced alterations in HD flies. Our findings highlight that restoring canonical Wg/Wnt signaling may be of therapeutic value.

Excitatory amino acid transporters (EAATs) are structurally related plasma membrane proteins known to mediate the Na+/K+-dependent uptake of the amino acids l-glutamate and dl-aspartate. In the nervous system, these proteins contribute to the clearance of glutamate from the synaptic cleft and maintain excitatory amino acid concentrations below excitotoxic levels. Two homologues exist in Drosophila melanogaster, dEAAT1 and dEAAT2, which are specifically expressed in the nervous tissue. We previously reported that dEAAT2 shows unique substrate discrimination as it mediates high affinity transport of aspartate but not glutamate. We now show that dEAAT2 can also transport the amino acid taurine with high affinity, a property that is not shared by two other transporters of the same family, Drosophila dEAAT1 and human hEAAT2. Taurine transport by dEAAT2 was efficiently blocked by an EAAT antagonist but not by inhibitors of the structurally unrelated mammalian taurine transporters. Taurine and aspartate are transported with similar Km and relative efficacy and behave as mutually competitive inhibitors. dEAAT2 can mediate either net uptake or the heteroexchange of its two substrates, both being dependent on the presence of Na+ ions in the external medium. Interestingly, heteroexchange only occurs in one preferred substrate orientation, i.e. with taurine transported inwards and aspartate outwards, suggesting a mechanism of transinhibition of aspartate uptake by intracellular taurine. Therefore, dEAAT2 is actually an aspartate/taurine transporter. Further studies of this protein are expected to shed light on the role of taurine as a candidate neuromodulator and cell survival factor in the Drosophila nervous system.

L-glutamate is both the major brain excitatory neurotransmitter [1, 2] and a potent neurotoxin [3, 4] in mammals. Glutamate excitotoxicity is partly responsible for cerebral traumas evoked by ischemia [5, 6] and has been implicated in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) [7–9]. In contrast, very little is known about the function or potential toxicity of glutamate in the insect brain. Here, we show that decreasing glutamate buffering capacity is neurotoxic in Drosophila . We found that the only Drosophila high-affinity glutamate transporter, dEAAT1 [10–13], is selectively addressed to glial extensions that project ubiquitously through the neuropil close to synaptic areas. Inactivation of dEAAT1 by RNA interference led to characteristic behavior deficits that were significantly rescued by expression of the human glutamate transporter hEAAT2 or the administration in food of riluzole, an anti-excitotoxic agent used in the clinic for human ALS patients. Signs of oxidative stress included hypersensitivity to the free radical generator paraquat and rescue by the antioxidant melatonin. Inactivation of dEAAT1 also resulted in shortened lifespan and marked brain neuropil degeneration characterized by widespread microvacuolization and swollen mitochondria. This suggests that the dEAAT1-deficient fly provides a powerful genetic model system for molecular analysis of glutamate-mediated neurodegeneration.

The main features of epidermal cell cycle during the metamorphosis of Tenebrio molitor have been investigated by cell density study, [3H]thymidine incorporation, and cytophotometry. G1, S, and M phases are restricted to short periods during each larval stage. The pupal mitotic wave is relatively longer than the larval ones. Outside of each mitotic period, 95 to 100% of the epidermal cells are blocked in G2 (4C DNA content). The cell density study shows that during the penultimate larval instar, numerous cells bypass the mitotic period without dividing while during the mitotic period of the last larval instar and of the pupal stage, each cell divides once and twice for some cells. After the pupal mitotic period, numerous adult cells are definitely blocked in G2 but 20 to 30% of cells present in old adults are in G1. Adult epidermal oenocytes differentiate during the last larval mitotic period, becoming 8 to 32C. From these results, it can be concluded that the G2 block and the release of G2-blocked cells into M are two critical events of each molting cycle. A possible hormonal control of these events is discussed.