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In the last decades, the strong increase in the proportion of older people worldwide, and the increased prevalence of age associated degenerative diseases, have put a stronger focus on aging biology. In spite of important progresses in our understanding of the aging process, an integrative view is still lacking and there is still need for efficient anti-aging interventions that could improve healthspan, reduce incidence of age-related disease and, eventually, increase the lifespan. Interestingly, some compounds from traditional medicine have been found to possess anti-oxidative and anti-inflammatory properties, suggesting that they could play a role as anti-aging compounds, although in depth in vivo investigations are still scarce. In this study we used one the major aging model organisms, Drosophila melanogaster, to investigate the ability of four herb extracts (HEs: Dendrobium candidum, Ophiopogon japonicum, Ganoderma sinense and Panax notoginseng) widely used in traditional Chinese medicine (TCM). to slow down aging and improve healthspan of aged animals. Combining multiple approaches (stress resistance assays, lifespan and metabolic measurements, functional heart characterizations and behavioral assays), we show, that these four HEs provide in vivo protection from various insults, albeit with significant compound-specific differences. Importantly, extracts of P. notoginseng and G. sinense increase the healthspan of aging animals, as shown by increased activity during aging and improved heart function. In addition, these two compounds also provide protection in a Drosophila model of Huntington’s disease (HD), suggesting that, besides their anti-aging properties in normal individuals, they could be also efficient in the protection against age-related diseases. Published version This work was supported by a grant from Infinitus Ltd Guangzhou (China).

International audience; Glia are important modulators of neural activity, yet few studies link glia to sleep regulation. We find that blocking activity of the endocytosis protein, dynamin, in adult Drosophila glia increases sleep and enhances sleep need, manifest as resistance to sleep deprivation. Surface glia comprising the fly equivalent of the blood-brain barrier (BBB) mediate the effect of dynamin on sleep. Blocking dynamin in the surface glia causes ultrastructural changes, albeit without compromising the integrity of the barrier. Supporting a role for endocytic trafficking in sleep, a screen of Rab GTPases identifies sleep-modulating effects of the recycling endosome Rab11 in surface glia. We also find that endocytosis is increased in BBB glia during sleep and reflects sleep need. We propose that endocytic trafficking through the BBB represents a function of sleep.

Glia are important modulators of neural activity, yet few studies link glia to sleep regulation. We find that blocking activity of the endocytosis protein, dynamin, in adult Drosophila glia increases sleep and enhances sleep need, manifest as resistance to sleep deprivation. Surface glia comprising the fly equivalent of the blood-brain barrier (BBB) mediate the effect of dynamin on sleep. Blocking dynamin in the surface glia causes ultrastructural changes, albeit without compromising the integrity of the barrier. Supporting a role for endocytic trafficking in sleep, a screen of Rab GTPases identifies sleep-modulating effects of the recycling endosome Rab11 in surface glia. We also find that endocytosis is increased in BBB glia during sleep and reflects sleep need. We propose that endocytic trafficking through the BBB represents a function of sleep.

Amyloid-β protein precursor (AβPP) and the microtubule-associated protein tau (MAPT) are the two key players involved in Alzheimer's disease (AD) and are associated with amyloid plaques and neurofibrillary tangles respectively, two key hallmarks of the disease. Besides vertebrate models, Drosophila models have been widely used to understand the complex events leading to AD in relation to aging. Drosophila benefits from the low redundancy of the genome which greatly simplifies the analysis of single gene disruption, sophisticated molecular genetic tools, and reduced cost compared to mammals. The aim of this review is to describe the recent advances in modeling AD using fly and to emphasize some limits of these models. Genetic studies in Drosophila have revealed some key aspects of the normal function of Appl and Tau, the fly homologues of AβPP and MAPT that may be disrupted during AD. Drosophila models have also been useful to uncover or validate several pathological pathways or susceptibility genes, and have been readily implemented in drug screening pipelines. We discuss some limitations of the current models that may arise from differences in structure of Appl and Tau compared to their human counterparts or from missing AβPP or MAPT protein interactors in flies. The advent of new genome modification technologies should allow the development of more realistic fly models and to better understand the relationship between AD and aging, taking advantage of the fly's short lifespan.

Friedreich ataxia (FA), the most common inherited autosomal-recessive ataxia in Caucasians, is characterized by progressive degeneration of the central and peripheral nervous system, hypertrophic cardiomyopathy and increased incidence of diabetes. FA is caused by a GAA repeat expansion in the first intron of the gene encoding frataxin, an evolutionarily conserved mitochondrial protein, which results in decreased gene expression. Ubiquitous inactivation of the fly frataxin ortholog dfh blocks the transition from larval to pupal stages. In this study, we show that this phenotype is due to ecdysteroid deficiency and that feeding larvae with the 20-hydroxyecdysone steroid hormone rescues this developmental blockage. In mammals, adrenodoxin, the ferredoxin FDX1, is an Fe-S-containing protein essential for the synthesis of various steroid hormones. We show here that the two fly ferredoxins, Fdxh and Fdxh2 (encoded by CG1319), are also involved in steroidogenesis. This provides a potent mechanism by which frataxin, known to be involved in Fe-S cluster biosynthesis, could affect steroidogenesis through reduced ferredoxin activity. Finally, we show that frataxin inactivation decreases progesterone synthesis in human KGN ovarian granulosa cells. Thus, the involvement of frataxin in steroid synthesis appears to be a conserved function of the protein from flies to human and our data suggest that steroidogenesis could be affected in FA patients.

Aging’s most obvious characteristic is the time dependent increase of an individual’s probability to die. This lifelong process is accompanied by a large number of molecular and physiological changes. Although numerous genes involved in aging have been identified in the past decades its leading factors have yet to be determined. To identify the very processes driving aging we have developed in the past years an assay to identify physiologically old individuals in a synchronized population of Drosophila melanogaster. Those individuals show an age-dependent increase of intestinal permeability followed by a high risk of death. Here we show that this physiological marker of aging is conserved in 3 invertebrate species Drosophila mojavensis, Drosophila virilis, Caenorhabditis elegans as well as in 1 vertebrate species Danio rerio. Our findings suggest that intestinal barrier dysfunction may be an important event in the aging process conserved across a broad range of species, thus raising the possibility that it may also be the case in Homo sapiens.

Summary Ubiquitin proteases remove ubiquitin monomers or polymers to modify the stability or activity of proteins and thereby serve as key regulators of signal transduction. Here, we describe the function of the Drosophila ubiquitin-specific protease 36 (dUSP36) in negative regulation of the immune deficiency (IMD) pathway controlled by the IMD protein. Overexpression of catalytically active dUSP36 ubiquitin protease suppresses fly immunity against Gram-negative pathogens. Conversely, silencing dUsp36 provokes IMD-dependent constitutive activation of IMD-downstream Jun kinase and NF-κB signaling pathways but not of the Toll pathway. This deregulation is lost in axenic flies, indicating that dUSP36 prevents constitutive immune signal activation by commensal bacteria. dUSP36 interacts with IMD and prevents K63-polyubiquitinated IMD accumulation while promoting IMD degradation in vivo. Blocking the proteasome in dUsp36 -expressing S2 cells increases K48-polyubiquitinated IMD and prevents its degradation. Our findings identify dUSP36 as a repressor whose IMD deubiquitination activity prevents nonspecific activation of innate immune signaling.

The steroid hormone ecdysone influences Drosophila lifespan. Longevity is extended in mutants deficient for ecdysone synthesis or mutants of the ecdysone receptor (EcR). However, the underlying mechanisms remain unclear. Here we conditionally inactivated EcR by RNA interference or expression of dominant negative forms, using the RU486 inducible system. A mild ubiquitous inactivation of EcR during adulthood was sufficient to slow the aging of male flies, whereas a stronger EcR inactivation decreased longevity. Surprisingly, ubiquitous inactivation of EcR strongly decreased female lifespan. This deleterious effect was suppressed in sterile ovo D1 mutant females, suggesting that EcR represses a negative signal for lifespan produced in ovaries. These results reveal a complex adult and sex-specific control of lifespan by steroid signalling in Drosophila.

Septic injury triggers a rapid and widespread response in Drosophila adults that involves the up-regulation of many genes required to combat infection and for wound healing. Genome-wide expression profiling has already demonstrated that this response is controlled by signaling through the Toll, Imd, JAK-STAT and JNK pathways. Using oligonucleotide microarrays, we now demonstrate that the MAPKKK Mekk1 regulates a small subset of genes induced by septic injury including Turandot (Tot) stress genes. Our analysis indicates that Tot genes show a complex regulation pattern including signals from both the JAK-STAT and Imd pathways and Mekk1. Interestingly, Mekk1 flies are resistant to microbial infection but susceptible to paraquat, an inducer of oxidative stress. These results point to a role of Mekk1 in the protection against tissue damage and/or protein degradation and indicate complex interactions between stress and immune pathways in Drosophila.

An original apparatus based on laser-induced fluorescence detection is presented. One lane migration combined to four equidistant detection points allows the study of the dynamics of DNA bands during electrophoresis. We focus this article on the study of the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T, 5% C and 4.3% T, 5% C cross-linked polyacrylamide gels at an electric field of 45 V/cm [T=(g acrylamide+g N,N'-methylenebisacrylamide)/100 ml solution; C=g N,N'-methylenebisacrylamide/% T]. Activation energy has been investigated under these experimental conditions with a temperature varying from 25 to 50 degrees C. The activation energy for migration through the cross-linked polyacrylamide gel decreases with fragment length under our experimental conditions and it varies along the migration.

International audience; : Friedreich's ataxia (FRDA), the most common hereditary ataxia, is characterized by progressive degeneration of the central and peripheral nervous system, hypertrophic cardiomyopathy and a high risk of diabetes. FRDA is caused by abnormally low levels of frataxin, a highly conserved mitochondrial protein. Drosophila has been previously successfully used to model FRDA in various cell types, including neurons and glial cells. Here, we report the development of a Drosophila cardiac model of FRDA. In vivo heart imaging revealed profound impairments in heart function in frataxin-depleted Drosophila, including a strong increase in end-systolic and end-diastolic diameters and a decrease in fractional shortening (FS). These features, reminiscent of pathological phenotypes in humans, are fully rescued by complementation with human frataxin, suggesting conserved cardiac functions of frataxin between the two organisms. Oxidative stress is not a major factor of heart impairment in frataxin-depleted flies, suggesting the involvement of other pathological mechanisms notably mitochondrial respiratory chain (MRC) dysfunction. Accordingly, we report that methylene blue (MB), a compound known to act as an alternative electron carrier that bypasses mitochondrial complexes I-III, was able to prevent heart dysfunction. MB also partially rescued the phenotype when administered post-symptomatically. Analysis of MB derivatives demonstrates that only compounds with electron carrier properties are able to prevent the heart phenotype. Thus MB, a compound already used for several clinical applications, appears promising for the treatment of the heart dysfunctions that are a major cause of death of FRDA patients. This work provides the grounds for further evaluation of MB action in mammals.

The segment polarity gene fused (fu) is involved in specification of positional information inside embryonic segments in Drosophila melanogaster (Dm). The predicted Fused (Fu) protein contains a serine/threonine kinase domain and a second domain with unknown function. We cloned and sequenced the fu homologous gene from Drosophila virilis (Dv) and made an interspecific DNA sequence comparison to identify regions that have been conserved during evolution. Comparison of the predicted amino acid (aa) sequences reveals two regions of strong homology, one corresponding to the kinase domain (268 aa), the other located in the third exon of the Dm fu gene, suggesting a functional importance for this region. Stretches of significantly conserved sequences are also observed in the 5' and 3' untranslated regions. Weak homology is seen in the intronic sequences although the adjacent exonic sequences are mostly conserved. These findings indicate a high conservation of the predicted Fu protein during the evolution of Drosophila.

International audience; Late onset neurodegenerative diseases represent a major public health concern as the population in many countries ages. Both frequent diseases such as Alzheimer disease (AD, 14% incidence for 80-84 year old Europeans) or Parkinson disease (PD, 1.4% prevalence for > 55 years old) share, with other low-incidence neurodegenerative pathologies such as spinocerebellar ataxias (SCAs, 0.01% prevalence) and frontotemporal lobar degeneration (FTLD, 0.02% prevalence), a lack of efficient treatment in spite of important research efforts. Besides significant progress, studies with animal models have revealed unexpected complexities in the degenerative process, emphasizing a need to better understand the underlying pathological mechanisms. Recently, microRNAs, a class of small regulatory non-coding RNAs, have been implicated in some neurodegenerative diseases. The current data supporting a role of miRNAs in PD, tauopathies, dominant ataxias and FTLD will first be discussed to emphasize the different levels of the pathological processes which may be affected by miRNAs. To investigate a potential involvement of miRNA dysregulation in the early stages of these neurodegenerative diseases we have used Drosophila models for 7 diseases (PD, 3 FTLD, 3 dominant ataxias) that recapitulate many features of the human diseases. We performed deep sequencing of head small RNAs after 3 days of pathological protein expression in the fly head neurons. We found no evidence for a statistically significant difference in miRNA expression in this early stage of the pathological process. In addition, we could not identify small non coding CAG repeat RNAs (sCAG) in polyQ disease models. Thus our data suggest that transcriptional deregulation of miRNAs or sCAG is unlikely to play a significant role in the initial stages of neurodegenerative diseases.

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo., Author Summary Spinocerebellar ataxias (SCAs) are a group of inherited neurodegenerative diseases with around 30 subtypes, which are characterized by a progressive loss of cerebellar neurons. Neuronal death has been linked to the aggregation of mutated disease-causing proteins, such as ataxin-1 (ATXN1). Pathogenic ATXN1 contains an elongated glutamine stretch, which triggers spontaneous misfolding and self-assembly of the protein into aggregates. Earlier studies in lower organisms have discovered many non-human proteins that alter aggregation and/or toxicity of mutant ATXN1. Here, we combine an experimental screening approach with bioinformatics to find human proteins that modulate aggregation and toxicity of ATXN1. We identified 21 proteins affecting mutant ATXN1 in mammalian cells. Further characterization revealed that enhancers of ATXN1-mediated toxicity contain α-helical coiled-coil domains as structural motifs, while suppressors do not. Detailed studies with the ATXN1 interacting proteins MED15 and Pum1 finally demonstrated that coiled-coil domains are indeed critical for the aggregation and toxicity promoting effects of human proteins. Our study contributes to a deeper understanding of ATXN1 aggregation and SCA1 pathogenesis and highlights potential therapeutic targets for further investigations.

Cardiac aging is a complex process, which is influenced by both environmental and genetic factors. Deciphering the mechanisms involved in heart senescence therefore requires identifying the molecular pathways that are affected by age in controlled environmental and genetic conditions. We describe a functional genomic investigation of the genetic control of cardiac senescence in Drosophila. Molecular signatures of heart aging were identified by differential transcriptome analysis followed by a detailed bio-informatic analysis. This approach implicated the JNK/dJun pathway and the transcription factor Vri/dNFIL3 in the transcription regulatory network involved in cardiac senescence and suggested the possible involvement of oxidative stress (OS) in the aging process. To validate these predictions, we developed a new in vivo assay to analyze heart performance in various contexts of adult heart-specific gene overexpression and inactivation. We demonstrate that, as in mammals, OS plays a central role in cardiac senescence, and we show that pharmacological interventions impinging on OS slow heart senescence. These observations strengthen the idea that cardiac aging is controlled by evolutionarily conserved mechanisms, further validating Drosophila as a model to study cardiac senescence. In addition, we demonstrate that Vri, the ortholog of the vertebrate NFIL3/E4B4 transcription factor, is a major genetic regulator of cardiac aging. Vri overexpression leads to major heart dysfunctions, but its loss of function significantly reduces age-related cardiac dysfunctions. Furthermore, we unambiguously show that the JNK/AP1 pathway, the role of which in cardiac aging in mammals is controversial, is activated during cardiac aging and has a detrimental effect on cardiac senescence. This data-driven functional genomic analysis therefore led to the identification of key components of the Gene Regulatory Network of cardiac aging in Drosophila and may prompt to investigate the involvement of their counterparts in the cardiac aging process in mammals., Author Summary Age-associated changes in cardiac structure and function have been implicated in the markedly increased risk of cardiovascular disease, but the molecular basis of these processes is ill-defined. It is difficult to study the genetics of heart aging in mammalian models because of their long life spans and their complexity, involving notably genetic redundancy. Here, we address this issue through identification of molecular signatures of cardiac aging in Drosophila, a model organism in which heart senescence occurs within 2 months. Tissue-specific transcriptome comparison of young and aging fly hearts were performed followed by in silico predictions of the regulatory networks involved. This analysis implicated oxidative stress (OS), the JNK/dJun pathway, and Vri/dNFIL3 in the gene regulatory network that drives cardiac senescence. Measuring heart variables in vivo following heart-specific genetic and pharmacological manipulations confirmed these predictions. We show that OS has a central role in the aging of the fly heart. Moreover, heart-specific partial knockdown of dJun and Vri prevented cardiac senescence, demonstrating that they are essential regulators of cardiac aging. Thus, our results uncover two major genetic determinants of Drosophila cardiac aging whose activities enhance heart senescence. It may therefore be valuable to investigate their involvement in the cardiac aging process in mammals.

The Drosophila segment-polarity gene fused (fu) is required for pattern formation within embryonic segments and imaginal discs. We previously reported that the 5′ part of the fused gene is homologous to the catalytic domain of serine/threonine kinases. We present here the sequence of the complete transcription unit, which predicts a 805 amino acid long protein. The kinase domain actually corresponds to 268 amino acids in the N-terminal part, and no known function can be attributed to the rest of the putative FUSED protein. Transcripts from the fused gene have been characterized: a unique 3.2 kb fused transcript is produced in nurse cells, in low abundance, from stage 8 of oogenesis, and persistently through the rest of oogenesis. In embryos, this transcript is evenly distributed in all embryonic cells until the extended germ band stage, after which its amount strongly decreases. Ubiquitous expression is detected later in imaginal wing and leg discs. Possible roles of the FUSED protein in signal transduction pathways required for intercellular communication at different stages of development are discussed.

Since the initial identification of reactive oxygen species (ROS) as the major factor in aging, many studies have provided evidence for the central role of mitochondria in longevity. A few years ago, an unexpected finding showed that the inactivation of the mitochondrial respiratory chain (MRC) in Caenorhabditis elegans, during the developmental stages only, extended lifespan. Activation of this mitochondrial pathway affecting aging (MIT) is associated with several phenotypic features: increased longevity, increased time of development, decreased fertility/fecundity and reduced adult size. Here, we investigated this pathway in another model organism, Drosophila melanogaster. To assess the role of mitochondrial activity in the Drosophila aging process, we partially inactivated the MRC using RNA interference (RNAi) during larval stages. Developmental perturbation of the respiratory process prolonged development, increased lethality during developmental stage, reduced both fecundity and fertility and slightly reduced individual weight. However, in contrast to the nematode, this genetic intervention either shortened or had no effect on lifespan, depending on the level of gene inactivation. Thus, the effects of MRC disruption during development on aging differ between species. We discuss the possible origins of such differences.

International audience; Drosophila Gcn5 is the catalytic subunit of the SAGA and ATAC histone acetylase complexes. Here, we show that mutations in Gcn5 and the ATAC component Ada2a induce a decondensation of the male X chromosome, similar to that induced by mutations in the Iswi and Nurf301 subunits of the NURF nucleosome remodelling complex. Genetic studies as well as transcript profiling analysis indicate that ATAC and NURF regulate overlapping sets of target genes during development. In addition, we find that Ada2a chromosome binding and histone H4-Lys12 acetylation are compromised in Iswi and Nurf301 mutants. Our results strongly suggest that NURF is required for ATAC to access the chromatin and to regulate global chromosome organization.

We have developed a high-resolution β− position detector which combines a thin scintillator sheet and an intensified CCD. This device has been designed for molecular biology purposes (“in situ” hybridization). Therefore, the absolute detection efficiency, S/N ratio and spatial resolution have been studied in particular. With the first version of our detector, we achieved an intrinsic resolution of 10 μm with a relative efficiency for 35S of 99% (i.e. 18% absolute efficiency). We now present results in which another scintillator and a new triggering system have been adopted. The main result is that the absolute efficiency has been improved (up to ≈ 60% for 35S) and the background influence has been significantly reduced. These improvements can be observed on the biological image displayed in this paper.

Spinocerebellar ataxia 7 (SCA7) is a neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the ataxin 7 (ATXN7) protein, a member of a multiprotein complex involved in histone acetylation. We have created a conditionalDrosophilamodel of SCA7 in which expression of truncated ATXN7 (ATXN7T) with a pathogenic polyQ expansion is induced in neurons in adult flies. In this model, mutant ATXN7T accumulated in neuronal intranuclear inclusions containing ubiquitin, the 19S proteasome subunit, and HSP70 (heat shock protein 70), as in patients. Aggregation was accompanied by a decrease in locomotion and lifespan but limited neuronal death. Disaggregation of the inclusions, when expression of expanded ATXN7T was stopped, correlated with improved locomotor function and increased lifespan, suggesting that the pathology may respond to treatment. Lifespan was then used as a quantitative marker in a candidate gene approach to validate the interest of the model and to identify generic modulators of polyQ toxicity and specific modifiers of SCA7. Several molecular pathways identified in this focused screen (proteasome function, unfolded protein stress, caspase-dependent apoptosis, and histone acetylation) were further studied in primary neuronal cultures. Sodium butyrate, a histone deacetylase inhibitor, improved the survival time of the neurons. This model is therefore a powerful tool for studying SCA7 and for the development of potential therapies for polyQ diseases.

International audience; In human, the myeloid leukemia factor 1 (hMLF1) has been shown to be involved in acute leukemia, and mlf related genes are present in many animals. Despite their extensive representation and their good conservation, very little is understood about their function. In Drosophila, dMLF physically interacts with both the transcription regulatory factor DREF and an antagonist of the Hedgehog pathway, Suppressor of Fused, whose over-expression in the fly suppresses the toxicity induced by polyglutamine. No connection between these data has, however, been established. Here, we show that dmlf is widely and dynamically expressed during fly development. We isolated and analyzed the first dmlf mutants: embryos lacking maternal dmlf product have a low viability with no specific defect, and dmlf(-)- adults display weak phenotypes. We monitored dMLF subcellular localization in the fly and cultured cells. We were able to show that, although generally nuclear, dMLF can also be cytoplasmic, depending on the developmental context. Furthermore, two differently spliced variants of dMLF display differential subcellular localization, allowing the identification of regions of dMLF potentially important for its localization. Finally, we demonstrate that dMLF can act developmentally and postdevelopmentally to suppress neurodegeneration and premature aging in a cerebellar ataxia model.

Background During the last two decades progress in the genetics of aging in invertebrate models such as C. elegans and D. melanogaster has clearly demonstrated the existence of regulatory pathways that control the rate of aging in these organisms, such as the insulin-like pathway, the Jun kinase pathway and the Sir2 deacetylase pathway. Moreover, it was rapidly shown that some of these pathways are conserved from yeast to humans. In parallel to genetic studies, genomic expression approches have given us significant information on the gene expression modifications that occur during aging either in wild type or long-lived mutant animals. But most of the genomic studies of invertebrate models have been performed so far on whole animals, while several recent studies in mammals have shown that the effects of aging are tissue specific. Results We used oligonucleotide microarrays to address the specificities of transcriptional responses in aging Drosophila in head, thorax or whole body. These fly parts are enriched in transcripts that represent different and complementary sets of genes. We present evidence for both specific and common transcriptional responses during the aging process in these tissues. About half of the genes described as downregulated with age are linked to reproduction and enriched in gonads. Greater downregulation of mitochondrial genes, activation of the JNK pathway and upregulation of proteasome subunits in the thorax of aged flies all suggest that muscle may be particularly sensitive to aging. Simultaneous age-related impairment of synaptic transmission gene expression is observed in fly heads. In addition, a detailed comparison with other microarray data indicates that in aged flies there are significant deviations from the canonical responses to oxidative stress and immune stress. Conclusion Our data demonstrates the advantages and value of regionalized and comparative analysis of gene expression in aging animals. Adding to the age-regulated genes already identified in whole animal studies, it provides lists of new regionalized genes to be studied for their functional role in the aging process. This work also emphasizes the need for such experiments to reveal in greater detail the consequences of the transcriptional modifications induced by aging regulatory pathways.

Background During their life, multicellular organisms are challenged with oxidative stress. It is generated by several reactive oxygen species (ROS), may limit lifespan and has been related to several human diseases. ROS can generate a wide variety of defects in many cellular components and thus the response of the organism challenged with oxidative stress may share some features with other stress responses. Conversely, in spite of recent progress, a complete functional analysis of the transcriptional responses to different oxidative stresses in model organisms is still missing. In addition, the functional significance of observed transcriptional changes is still elusive. Results We used oligonucleotide microarrays to address the specificities of transcriptional responses of adult Drosophila to different stresses induced by paraquat and H2O2, two oxidative stressors, and by tunicamycin which induces an endoplasmic reticulum (ER) stress. Both specific and common responses to the three stressors were observed and whole genome functional analysis identified several important classes of stress responsive genes. Within some functional classes, we observed that isozymes do not all behave similarly, which may reflect unsuspected functional specificities. Moreover, genetic experiments performed on a subset of lines bearing mutations in genes identified in microarray experiments showed that a significant number of these mutations may affect resistance of adult Drosophila to oxidative stress. Conclusions A long term common stress response to paraquat- or H2O2-induced oxidative stresses and ER stress is observed for a significant number of genes. Besides this common response, the unexpected complexity of the stress responses to oxidative and ER stresses in Drosophila, suggest significant specificities in protective properties between genes associated to the same functional classes. According to our functional analysis, a large part of the genome may play a role in protective mechanisms against oxidative stress in Drosophila.

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0925-4773 doi: DOI: 10.1016/S0925-4773(98)00130-0; The Fused (Fu) serine–threonine kinase and the Suppressor of fused (Su(fu)) product are part of the Hedgehog (Hh) signalling pathway both in embryos and in imaginal discs. In wing imaginal discs, the Hh signal induces Cubitus interruptus (Ci) accumulation and activates patched (ptc) and decapentaplegic (dpp) expression along the anterior/posterior (A/P) boundary. In this paper, we have examined the role of the Fu and Su(fu) proteins in the regulation of Hh target gene expression in wing imaginal discs, by using different classes of fu alleles and an amorphic Su(fu) mutation. We show that, at the A/P boundary, Fu kinase activity is involved in the maintenance of high ptc expression and in the induction of late anterior engrailed (en) expression. These combined effects can account for the modulation of Ci accumulation and for the precise localization of the Dpp morphogen stripe. In contrast, in more anterior cells which do not receive Hh signal, we show that Fu plays a role independent of its kinase function in the regulation of Ci accumulation. In these cells, Fu may be involved in the stabilization of a large protein complex which is probably responsible for the regulation of Ci cleavage and/or targeting to nucleus. We propose that the Fused function is necessary for the activation of full-length Ci and counteracts the negative Su(fu) effect on the pathway, leading to en, ptc and dpp expression. î 1998 Elsevier Science Ireland Ltd. All rights reserved

Used initially and extensively for medical diagnosis, nuclear imaging has been progressively extended to other applications like molecular genetics, neurosciences and surgical aids. This review article covers new types of imaging apparatus resulting from this diversification. Far from being exhaustive, we limit ourselves to the three applications cited above, in which our research group has focused its interest. In an extensive first part, we describe three types of detectors dedicated to the three complementary areas of research in genetics at the molecular level: in situ hybridization, gene cartography and DNA sequencing. In addition, we discuss the current limits of these methods and the efforts that we propose to progress further. Then, after recalling some general aspects of in vivo micro-imaging, we present our new technical strategy to investigate in vivo cerebral mechanisms in rodents. Finally, we describe our current development of mini-cameras for assisting surgeons during operations.

We have developed a program to evaluate the influence of DNA stiffness on molecular mobility and conformation during electrophoresis. This (currently) two-dimensional numerical study models DNA as a chain of uniformly charged beads connected to one another by elastic segments, of finite mean size, in the presence of fixed obstacles representing gel fibers. Contrary to the standard biased reptation model (BRM), our Langevin-type dynamics for the chain are microscopic and warrant studies of fine effects such as inner chain orientation. Using this model, we show that the introduction of a persistence length decreases the (saturated) mobility at high electric fields, providing strong evidence that the gel generates a friction force and not only a (dissipation-free) constraint force. We also show that the persistence length leads to an increase of the chain orientation in the field direction. This suggests that DNA stiffness causes the saturation plateau value to be reached for smaller chain sizes than those predicted by the BRM model.

The Fused protein is a putative serine/threonine PK, thought to be involved in signal transduction pathways at different stages of development. The fused gene belongs to the segment–polarity class of Drosophila. melanogaster segmentation genes. It is required for pattern formation within embryonic segments and imaginal discs and during oogenesis. With regard to domain structure, the function of the C-terminal region is not known but it appears to be essential for fused activity and/or regulation. A hydrophilic region in the center could correspond to a hinge region between catalytic and regulatory domains.

Suppressor of fused, Su(fu), was identified as a semi-dominant suppressor of the putative serine/threonine kinase encoded by the segment polarity gene fused in Drosophila melanogaster. The amorphic Su(fu) mutation is viable, shows a maternal effect and displays no phenotype by itself. Su(fu) mutations are often found associated to karmoisin (kar) mutations but two complementation groups can be clearly identified. By using a differential hybridization screening method, we have cloned the Su(fu) region and identified chromosomal rearrangements associated with Su(fu) mutations. Two classes of cDNAs with similar developmental patterns, including a maternal contribution, are detectable in the region. Transformation experiments clearly assigned the Su(fu)+ function to one of these transcription units while the other one can be most likely assigned to the kar+ function. Surprisingly the 5' end of the kar RNA mapped within the 3' untranslated region of the Su(fu) transcribed sequence. The Su(fu) gene encodes a 53-kD protein, which contains a PEST sequence and shows no significant homologies with known proteins. Genetic analysis shows that proper development requires a fine tuning of the genetic doses of fu and Su(fu) both maternally and zygotically. These results, together with previous genetic and molecular data, suggest that fused and Suppressor of fused could act through a competitive posttraductionnal modification of a common target in the hedgehog signaling pathway.

We are developing a new device based on the results reported previously of the successful coincidence detection of β− particles with a high spatial resolution. The novelty of the device consists in triggering and intensified CCD, i.e. a CCD coupled to an image intensifier (II), by an electrical signal collected from the II itself. This is a suitable procedure for detecting low light and rare events with high efficiency and high resolution. The trigger pulse is obtained from the secondary electrons produced by multiplication in a double microchannel plate (MCP) and collected on the aluminized layer protecting the phosphor screen in the II. Triggering efficiencies up to 80% have already been achieved.

A systematic search for X chromosome loci showing a dominant maternal interaction with the segmentation genes Krüppel, hunchback, knirps and hairy was performed using deficiencies spanning 65% of the X chromosome. No interaction with the knirps gene was observed, but five regions of the X chromosome showed a maternal dominant interaction with the Krüppel gene. Two of these regions also show a maternal dominant interaction with either hunchback (region 10A7-10A8) or hairy (region 10E1-10F3). In all of these interactions dead embryos were observed which showed the same defects as embryos homozygous for the segmentation gene tested. These results suggest that a complex repartition of maternal products necessary for subsequent segmentation may occur in the Drosophila egg.

We present a detector which associates a charge coupled device to a light amplifier. The image sensor must detect weak β − activity, with a 10 μ resolution and should replace the autoradiographic films used for molecular hybridization. The best results are obtained with the 35 S emitter, for which the resolution and the efficiency are respectively 20 μm and 100% (relative to the measured standard source).

We present characteristics of a charged particle detector which combines optical fibers with a multianode photomultiplier. Our first prototype allows detection of 32P emitters on a 26 × 26 cm2 surface with 1.5 mm and 5 mm resolutions, respectively for X and Y directions, and a 0.3% efficiency. Improvements, presently under study, in both fiber qualities and photocathode efficiency should increase the device efficiency to 40% and its resolution up to the optical fiber diameter (500 μm for the time being). Such characteristics would allow SOFI to replace autoradiographic films in biology and make it a convenient position sensitive detector in nuclear and particle physics.

fused (fu) is a segment polarity gene that encodes a putative serine/threonine kinase. A complete suppressor of the embryonic and adult phenotypes of fu mutants, Suppressor of fused (Su(fu)), was previously described. The amorphic Su(fu) mutation is viable and displays no phenotype by itself. We have used this suppressor as a tool to perform a genetic dissection of the fu gene. Analysis of the interaction between Su(fu) and 33 fu alleles shows that they belong to three different classes. Defects due to class I fu alleles are fully suppressed by Su(fu). Class II fu alleles lead to a new segment polarity phenotype in interaction with Su(fu). This phenotype corresponds to embryonic and adult anomalies similar to those displayed by the segment polarity mutant costal-2 (cos-2). Class II alleles are recessive to class I alleles in a fu[I]/fu[II];Su(fu)/Su(fu) combination. Class 0 alleles, like class I alleles, confer a normal segmentation phenotype in interaction with Su(fu). However class II alleles are dominant over class 0 alleles in a fu[0]/fu[II];Su(fu)/Su(fu) combination. Alleles of class I and II correspond to small molecular events, which may leave part of the Fu protein intact. On the contrary, class 0 alleles correspond to large deletions. Several class I and class II fu mutations have been mapped, and three mutant alleles were sequenced. These data suggest that class I mutations affect the catalytic domain of the putative Fu kinase and leave the carboxy terminal domain intact, whereas predicted class II proteins have an abnormal carboxy terminal domain. Su(fu) enhances the cos-2 phenotype and cos-2 mutations interact with fu in a way similar to Su(fu). All together these results suggest that a close relationship might exist between fu, Su(fu) and cos-2 throughout development. We thus propose a model where the Fu+ kinase is a posterior inhibitor of Costal-2+ while Su(fu)+ is an activator of Costal-2+. The expression pattern of wingless and engrailed in fu and fu;Su(fu) embryos is in accordance with this interpretation.

fused (fu) is a segment-polarity gene encoding a putative serine-threonine kinase. In a wild-type context, all fu mutations display the same set of phenotypes. Nevertheless, mutations of the Suppressor of fused [Su(fu)] gene define three classes of alleles (fu0, fuI, fuII). Here, we report the molecular analysis of known fu mutations and the generation of new alleles by in vitro mutagenesis. We show that the Fused (Fu) protein functions in vivo as a kinase. The N-terminal kinase and the extreme C-terminal domains are necessary for Fu+ activity while a central region appears to be dispensable. We observe a striking correlation between the molecular lesions of fu mutations and the phenotype displayed in their interaction with Su(fu). Indeed, fuI alleles which are suppressed by Su(fu) mutations are defined by inframe alterations of the N-terminal catalytic domain whereas the C-terminal domain is missing or altered in all fuII alleles. An unregulated FuII protein, which can be limited to the 80 N-terminal amino acids of the kinase domain, would be responsible for the neomorphic costal-2 phenotype displayed by the fuII-Su(fu) interaction. We propose that the Fu C-terminal domain can differentially regulate the Fu catalytic domain according to cell position in the parasegment.