Mitofusin gain and loss of function drive pathogenesis in Drosophila models of CMT2A neuropathy.

Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by dominant alleles of the mitochondrial pro-fusion factor Mitofusin 2 (MFN2). To address the consequences of these mutations on mitofusin activity and neuronal function, we generate Drosophila models expressing in neurons the two most frequent substitutions (R94Q and R364W, the latter never studied before) and two others localizing to similar domains (T105M and L76P). All alleles trigger locomotor deficits associated with mitochondrial depletion at neuromuscular junctions, decreased oxidative metabolism and increased mtDNA mutations, but they differently alter mitochondrial morphology and organization. Substitutions near or within the GTPase domain (R94Q, T105M) result in loss of function and provoke aggregation of unfused mitochondria. In contrast, mutations within helix bundle 1 (R364W, L76P) enhance mitochondrial fusion, as demonstrated by the rescue of mitochondrial alterations and locomotor deficits by over-expression of the fission factor DRP1. In conclusion, we show that both dominant negative and dominant active forms of mitofusin can cause CMT2A-associated defects and propose for the first time that excessive mitochondrial fusion drives CMT2A pathogenesis in a large number of patients.

Glial lipid droplets and neurodegeneration in a Drosophila model of complex I deficiency.

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.

A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila.

Long-term exposure to environmental oxidative stressors, like the herbicide paraquat (PQ), has been linked to the development of Parkinson's disease (PD), the most frequent neurodegenerative movement disorder. Paraquat is thus frequently used in the fruit fly Drosophila melanogaster and other animal models to study PD and the degeneration of dopaminergic neurons (DNs) that characterizes this disease. Here, we show that a D1-like dopamine (DA) receptor, DAMB, actively contributes to the fast central nervous system (CNS) failure induced by PQ in the fly. First, we found that a long-term increase in neuronal DA synthesis reduced DAMB expression and protected against PQ neurotoxicity. Secondly, a striking age-related decrease in PQ resistance in young adult flies correlated with an augmentation of DAMB expression. This aging-associated increase in oxidative stress vulnerability was not observed in a DAMB-deficient mutant. Thirdly, targeted inactivation of this receptor in glutamatergic neurons (GNs) markedly enhanced the survival of Drosophila exposed to either PQ or neurotoxic levels of DA, whereas, conversely, DAMB overexpression in these cells made the flies more vulnerable to both compounds. Fourthly, a mutation in the Drosophila ryanodine receptor (RyR), which inhibits activity-induced increase in cytosolic Ca(2+), also strongly enhanced PQ resistance. Finally, we found that DAMB overexpression in specific neuronal populations arrested development of the fly and that in vivo stimulation of either DNs or GNs increased PQ susceptibility. This suggests a model for DA receptor-mediated potentiation of PQ-induced neurotoxicity. Further studies of DAMB signaling in Drosophila could have implications for better understanding DA-related neurodegenerative disorders in humans.

The Drosophila inner-membrane protein PMI controls crista biogenesis and mitochondrial diameter.

Cristae are mitochondrial inner-membrane structures that concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling crista morphogenesis are poorly understood and few crista determinants have been identified. Among them are the Mitofilins that are required to establish crista junctions and ATP-synthase subunits that bend the membrane at the tips of the cristae. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondrial ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI overexpression is sufficient to reduce crista length in vivo. Finally, these crista defects are associated with impaired respiratory chain activity and increases in the level of reactive oxygen species. Since PMI and its human orthologue TMEM11 are regulators of mitochondrial morphology, our data suggest that, by controlling crista length, PMI influences mitochondrial diameter and tubular shape.

Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways.

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.

M1BP is an essential transcriptional activator of oxidative metabolism during Drosophila development.

Oxidative metabolism is the predominant energy source for aerobic muscle contraction in adult animals. How the cellular and molecular components that support aerobic muscle physiology are put in place during development through their transcriptional regulation is not well understood. Using the Drosophila flight muscle model, we show that the formation of mitochondria cristae harbouring the respiratory chain is concomitant with a large-scale transcriptional upregulation of genes linked with oxidative phosphorylation (OXPHOS) during specific stages of flight muscle development. We further demonstrate using high-resolution imaging, transcriptomic and biochemical analyses that Motif-1-binding protein (M1BP) transcriptionally regulates the expression of genes encoding critical components for OXPHOS complex assembly and integrity. In the absence of M1BP function, the quantity of assembled mitochondrial respiratory complexes is reduced and OXPHOS proteins aggregate in the mitochondrial matrix, triggering a strong protein quality control response. This results in isolation of the aggregate from the rest of the matrix by multiple layers of the inner mitochondrial membrane, representing a previously undocumented mitochondrial stress response mechanism. Together, this study provides mechanistic insight into the transcriptional regulation of oxidative metabolism during Drosophila development and identifies M1BP as a critical player in this process.

Tobramycin disposition in ICU patients receiving a once daily regimen: population approach and dosage simulations.

UNLABELLED: WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT? It is well known that tobramycin given as an once daily dose according to the usual recommendations needs therapeutic drug monitoring by measurement of peak and trough concentrations. In the literature, there are only few published studies on the population pharmacokinetics of once daily tobramycin in critically ill patients. Glomerular filtration rate and bodyweight were identified as covariates contributing to the inter-individual variability in the disposition of aminoglycosides. The study, by Peris-Marti et al. [24], only evaluated the pharmacodynamic effectiveness of a 4 mg kg(-1) dose of tobramycin given once daily in critically ill patients. The authors concluded with a simulation showing that for a theoretical MIC of 1 or 2 mg l(-1) , a 7 mg kg(-1) dose was required. WHAT THIS STUDY ADDS: Our results confirm the high variability of tobramycin disposition in intensive care patients and consequently the possible lack of effectiveness. By using a population pharmacokinetic approach, two explicative covariates (height and Cockcroft creatinine clearance) added to a two-compartment model with proportional error, explained much of the inter-individual variability of tobramycin disposition in the critically ill patient population. In a median ICU patient, simulations were performed at various dosage regimens and peak and AUC pharmacodynamic targets could not be reached simultaneously in more than 45% of the ICU patient population. Drug monitoring is required to manage efficacy and toxicity. AIM: The aim of this study was to evaluate the disposition of tobramycin (TOB) in critically ill patients (ICU) by a population pharmacokinetic approach, to determine the covariates involved, and to simulate tobramycin dosage regimens. METHODS: Forty-nine adult ICU patients received TOB (5 mg kg(-1) ) once daily. NonMem modelling was performed on 32 patients. The 17 other patients were used for the qualification process by normalized prediction distribution error. Then Monte Carlo simulations (MCS) were performed. RESULTS: A two-compartment model with a proportional error best fitted the data. TOB total clearance (CL(TOB) ) was significantly correlated with Cockcroft creatinine clearance (COCK) and height. TOB clearance was 4.8 ± 1.9 l h(-1) (range 1.22-8.95), the volume of distribution of the central compartment was 24.7 ± 3.7 l (range 17.34-32.83) and that of the peripheral compartment and the inter-compartmental clearance were 30.6 l and 4.74 l h(-1) , respectively. Only 29% of the patients presented a target AUC between 80 and 125 mg l(-1) h and 61% were lower than 80 mg l(-1) h. After considering COCK and height, MCS showed that only 50% of the population could achieve the target AUC for the 375 and 400 mg dosages. CONCLUSION: Even after taking into account COCK and height, for strains with an MIC ≤ 1 mg l(-1) , MCS doses evidenced that peak and AUC pharmacodynamic targets could not be reached simultaneously in more than 45% of the ICU patient population. Combination therapy in addition to drug monitoring are required to manage efficacy and toxicity.

Myofibril and mitochondria morphogenesis are coordinated by a mechanical feedback mechanism in muscle.

Complex animals build specialised muscles to match specific biomechanical and energetic needs. Hence, composition and architecture of sarcomeres and mitochondria are muscle type specific. However, mechanisms coordinating mitochondria with sarcomere morphogenesis are elusive. Here we use Drosophila muscles to demonstrate that myofibril and mitochondria morphogenesis are intimately linked. In flight muscles, the muscle selector spalt instructs mitochondria to intercalate between myofibrils, which in turn mechanically constrain mitochondria into elongated shapes. Conversely in cross-striated leg muscles, mitochondria networks surround myofibril bundles, contacting myofibrils only with thin extensions. To investigate the mechanism causing these differences, we manipulated mitochondrial dynamics and found that increased mitochondrial fusion during myofibril assembly prevents mitochondrial intercalation in flight muscles. Strikingly, this causes the expression of cross-striated muscle specific sarcomeric proteins. Consequently, flight muscle myofibrils convert towards a partially cross-striated architecture. Together, these data suggest a biomechanical feedback mechanism downstream of spalt synchronizing mitochondria with myofibril morphogenesis.

TP53INP1 exerts neuroprotection under ageing and Parkinson's disease-related stress condition.

TP53INP1 is a stress-induced protein, which acts as a dual positive regulator of transcription and of autophagy and whose deficiency has been linked with cancer and metabolic syndrome. Here, we addressed the unexplored role of TP53INP1 and of its Drosophila homolog dDOR in the maintenance of neuronal homeostasis under chronic stress, focusing on dopamine (DA) neurons under normal ageing- and Parkinson's disease (PD)-related context. Trp53inp1-/- mice displayed additional loss of DA neurons in the substantia nigra compared to wild-type (WT) mice, both with ageing and in a PD model based on targeted overexpression of α-synuclein. Nigral Trp53inp1 expression of WT mice was not significantly modified with ageing but was markedly increased in the PD model. Trp53inp2 expression showed similar evolution and did not differ between WT and Trp53inp1-/- mice. In Drosophila, pan-neuronal dDOR overexpression improved survival under paraquat exposure and mitigated the progressive locomotor decline and the loss of DA neurons caused by the human α-synuclein A30P variant. dDOR overexpression in DA neurons also rescued the locomotor deficit in flies with RNAi-induced downregulation of dPINK1 or dParkin. Live imaging, confocal and electron microscopy in fat bodies, neurons, and indirect flight muscles showed that dDOR acts as a positive regulator of basal autophagy and mitophagy independently of the PINK1-mediated pathway. Analyses in a mammalian cell model confirmed that modulating TP53INP1 levels does not impact mitochondrial stress-induced PINK1/Parkin-dependent mitophagy. These data provide the first evidence for a neuroprotective role of TP53INP1/dDOR and highlight its involvement in the regulation of autophagy and mitophagy in neurons.

Fenton chemistry and oxidative stress mediate the toxicity of the beta-amyloid peptide in a Drosophila model of Alzheimer's disease.

The mechanism by which aggregates of the beta-amyloid peptide (Abeta) mediate their toxicity is uncertain. We show here that the expression of the 42-amino-acid isoform of Abeta (Abeta(1-42)) changes the expression of genes involved in oxidative stress in a Drosophila model of Alzheimer's disease. A subsequent genetic screen confirmed the importance of oxidative stress and a molecular dissection of the steps in the cellular metabolism of reactive oxygen species revealed that the iron-binding protein ferritin and the H(2)O(2) scavenger catalase are the most potent suppressors of the toxicity of wild-type and Arctic (E22G) Abeta(1-42). Likewise, treatment with the iron-binding compound clioquinol increased the lifespan of flies expressing Arctic Abeta(1-42). The effect of iron appears to be mediated by oxidative stress as ferritin heavy chain co-expression reduced carbonyl levels in Abeta(1-42) flies by 65% and restored the survival and locomotion function to normal. This was achieved despite the presence of elevated levels of the Abeta(1-42). Taken together, our data show that oxidative stress, probably mediated by the hydroxyl radical and generated by the Fenton reaction, is essential for Abeta(1-42) toxicity in vivo and provide strong support for Alzheimer's disease therapies based on metal chelation.

Decreasing glutamate buffering capacity triggers oxidative stress and neuropil degeneration in the Drosophila brain.

L-glutamate is both the major brain excitatory neurotransmitter and a potent neurotoxin in mammals. Glutamate excitotoxicity is partly responsible for cerebral traumas evoked by ischemia and has been implicated in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). 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, 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.

Alteration of plasma phospholipid fatty acid profile in patients with septic shock.

In septic shock patients, alterations of plasma phospholipid fatty acid profile have never been described. The purpose of this monocentric, non-interventional, observational prospective study was to describe this fatty acid profile in the early phase of septic shock in intensive care unit. Thirty-seven adult patients with septic shock were included after the first day of stay in intensive care unit, before any form of artificial nutritional support. Plasma phospholipid fatty acid composition was determined by gas chromatography. All biological data from patients with septic shock were compared with laboratory reference values. Patients presented hypocholesterolemia and hypertriglyceridemia. They had low concentrations of phospholipid fatty acids specifically n-6 and n-3 polyunsaturated fatty acids (PUFAs) with a high n-6/n-3 ratio. Plasma phospholipid PUFA concentrations were strongly correlated with cholesterolemia. PUFAs/SFAs (saturated fatty acids) and PUFAs/MUFAs (monounsaturated fatty acids) ratios were low because of low percentage of n-6 and n-3 PUFAs and high percentage of SFAs and MUFAs. Low levels of plasma long chain PUFAs (≥20 carbons) were significantly associated with mortality at 28th day. In conclusion, plasma phospholipid FA profile of septic patients is very characteristic, close to that of acute respiratory distress syndrome and mortality is associated with long chain PUFA decrease. This profile could be explained by numerous non-exclusive physio-pathological processes 1) an activation of hepatic de novo lipogenesis that could contribute to hepatic steatosis, 2) an elevated adipose tissue lipolysis, 3) an increased free radical attack of FA by oxidative stress, 4) an over-production of inflammatory lipid mediators.

Bacterial detection by Drosophila peptidoglycan recognition proteins.

The mechanisms and molecular effectors of pathogen recognition systems in diverse hosts are highly conserved. Both plant and animal recognition of pathogens relies on sensing of Pathogen-Associated Molecular Patterns (PAMPs) by Pattern Recognition Molecules (PRMs). To detect bacteria, these sensor molecules can recognize a wide array of molecules ranging from lipopolysaccharides (LPS) to peptidoglycan (PGN) or proteins. In contrast to that of mammals, the repertoire of bacterial motifs recognized by the immune system of the fruit fly seems to be much narrower. Works published so far indicate that it is limited to bacterial PGN and its derivatives. The mode of detection of PGN by host proteins is also simpler in the fly immune system than it is in the mammalian counterpart. Although PGN can be detected by Toll-like receptors, Nucleotide-binding oligomerization domain proteins and Peptidoglycan Recognition proteins (PGRPs) in vertebrates, PGRP family members are, so far, the only PGN sensors identified in Drosophila. Interactions between PGN and PGRPs induce multiple processes required to mount a specific and is implicated in multiple processes require to induce a specific and fine-tuned bacterial immune response in fly. Here, we present an overview of our current knowledge of PGRP and their bacterial detection in Drosophila.

Physiological requirement for the glutamate transporter dEAAT1 at the adult Drosophila neuromuscular junction.

L-glutamate is the major excitatory neurotransmitter in the mammalian brain. Specific proteins, the Na+/K+-dependent high affinity excitatory amino acid transporters (EAATs), are involved in the extracellular clearance and recycling of this amino acid. Type I synapses of the Drosophila neuromuscular junction (NMJ) similarly use L-glutamate as an excitatory transmitter. However, the localization and function of the only high-affinity glutamate reuptake transporter in Drosophila, dEAAT1, at the NMJ was unknown. Using a specific antibody and transgenic strains, we observed that dEAAT1 is present at the adult, but surprisingly not at embryonic and larval NMJ, suggesting a physiological maturation of the junction during metamorphosis. We found that dEAAT1 is not localized in motor neurons but in glial extensions that closely follow motor axons to the adult NMJ. Inactivation of the dEAAT1 gene by RNA interference generated viable adult flies that were able to walk but were flight-defective. Electrophysiological recordings of the thoracic dorso-lateral NMJ were performed in adult dEAAT1-deficient flies. The lack of dEAAT1 prolonged the duration of the individual responses to motor nerve stimulation and this effect was progressively increased during physiological trains of stimulations. Therefore, glutamate reuptake by glial cells is required to ensure normal activity of the Drosophila NMJ, but only in adult flies.

Expanded polyglutamine peptides disrupt EGF receptor signaling and glutamate transporter expression in Drosophila.

Huntington's disease (HD) is a late onset heritable neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) sequence in the protein huntingtin (Htt). Transgenic models in mice have suggested that the motor and cognitive deficits associated to this disease are triggered by extended neuronal and possibly glial dysfunction, whereas neuronal death occurs late and selectively. Here, we provide in vivo evidence that expanded polyQ peptides antagonize epidermal growth factor receptor (EGFR) signaling in Drosophila glia. We targeted the expression of the polyQ-containing domain of Htt or an extended polyQ peptide alone in a subset of Drosophila glial cells, where the only fly glutamate transporter, dEAAT1, is detected. This resulted in formation of nuclear inclusions, progressive decrease in dEAAT1 transcription and shortened adult lifespan, but no significant glial cell death. We observed that brain expression of dEAAT1 is normally sustained by the EGFR-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, suggesting that polyQ could act by antagonizing this pathway. We found that the presence of polyQ peptides indeed abolished dEAAT1 upregulation by constitutively active EGFR and potently inhibited EGFR-mediated ERK activation in fly glial cells. Long polyQ also limited the effect of activated EGFR on Drosophila eye development. Our results further indicate that the polyQ acts at an upstream step in the pathway, situated between EGFR and ERK activation. This suggests that disruption of EGFR signaling and ensuing glial cell dysfunction could play a direct role in the pathogenesis of HD and other polyQ diseases in humans.

Terminal glial differentiation involves regulated expression of the excitatory amino acid transporters in the Drosophila embryonic CNS.

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.