RESUMO
Huntington's disease (HD) is a progressive and fatal autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in the first exon of the huntingtin gene (HTT). In spite of considerable efforts, there is currently no treatment to stop or delay the disease. Although HTT is expressed ubiquitously, most of our knowledge has been obtained on neurons. More recently, the impact of mutant huntingtin (mHTT) on other cell types, including glial cells, has received growing interest. It is currently unclear whether new pathological pathways could be identified in these cells compared to neurons. To address this question, we performed an in vivo screen for modifiers of mutant huntingtin (HTT-548-128Q) induced pathology in Drosophila adult glial cells and identified several putative therapeutic targets. Among them, we discovered that partial nej/dCBP depletion in these cells was protective, as revealed by strongly increased lifespan and restored locomotor activity. Thus, dCBP promotes the HD pathology in glial cells, in contrast to previous opposite findings in neurons. Further investigations implicated the transcriptional activator Foxo as a critical downstream player in this glial protective pathway. Our data suggest that combinatorial approaches combined to specific tissue targeting may be required to uncover efficient therapies in HD.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Neuroglia/metabolismo , Transdução de Sinais , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Biomarcadores , Cálcio/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Metabolismo Energético , Testes Genéticos , Doença de Huntington/diagnóstico , Doença de Huntington/etiologia , Doença de Huntington/metabolismo , Neurônios/metabolismoRESUMO
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.
RESUMO
Friedreich ataxia (FA) is caused by GAA repeat expansions in the first intron of FXN, the gene encoding frataxin, which results in decreased gene expression. Thanks to the high degree of frataxin conservation, the Drosophila melanogaster fruitfly appears as an adequate animal model to study this disease and to evaluate therapeutic interventions. Here, we generated a Drosophila model of FA with CRISPR/Cas9 insertion of approximately 200 GAA in the intron of the fly frataxin gene fh. These flies exhibit a developmental delay and lethality associated with decreased frataxin expression. We were able to bypass preadult lethality using genetic tools to overexpress frataxin only during the developmental period. These frataxin-deficient adults are short-lived and present strong locomotor defects. RNA-Seq analysis identified deregulation of genes involved in amino-acid metabolism and transcriptomic signatures of oxidative stress. In particular, we observed a progressive increase of Tspo expression, fully rescued by adult frataxin expression. Thus, Tspo expression constitutes a molecular marker of the disease progression in our fly model and might be of interest in other animal models or in patients. Finally, in a candidate drug screening, we observed that N-acetyl cysteine improved the survival, locomotor function, resistance to oxidative stress and aconitase activity of frataxin-deficient flies. Therefore, our model provides the opportunity to elucidate in vivo, the protective mechanisms of this molecule of therapeutic potential. This study also highlights the strength of the CRISPR/Cas9 technology to introduce human mutations in endogenous orthologous genes, leading to Drosophila models of human diseases with improved physiological relevance.
Assuntos
Acetilcisteína/farmacologia , Sistemas CRISPR-Cas/genética , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Animais , Modelos Animais de Doenças , Drosophila melanogaster/genética , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/patologia , Humanos , Íntrons/genética , Estresse Oxidativo/genética , RNA-Seq , Expansão das Repetições de Trinucleotídeos/genética , FrataxinaRESUMO
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.
Assuntos
Drosophila/fisiologia , Endocitose , Neuroglia/fisiologia , Sono , Animais , Barreira Hematoencefálica/fisiologia , Proteínas de Drosophila/metabolismo , Dinaminas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Friedreich's ataxia (FA) is caused by reduced levels of frataxin, a highly conserved mitochondrial protein. There is currently no effective treatment for this disease, which is characterized by progressive neurodegeneration and cardiomyopathy, the latter being the most common cause of death in patients. We previously developed a Drosophila melanogaster cardiac model of FA, in which the fly frataxin is inactivated specifically in the heart, leading to heart dilatation and impaired systolic function. Methylene Blue (MB) was highly efficient to prevent these cardiac dysfunctions. Here, we used this model to screen in vivo the Prestwick Chemical Library, comprising 1280 compounds. Eleven drugs significantly reduced the cardiac dilatation, some of which may possibly lead to therapeutic applications in the future. The one with the strongest protective effects was paclitaxel, a microtubule-stabilizing drug. In parallel, we characterized the histological defects induced by frataxin deficiency in cardiomyocytes and observed strong sarcomere alterations with loss of striation of actin fibers, along with full disruption of the microtubule network. Paclitaxel and MB both improved these structural defects. Therefore, we propose that frataxin inactivation induces cardiac dysfunction through impaired sarcomere assembly or renewal due to microtubule destabilization, without excluding additional mechanisms. This study is the first drug screening of this extent performed in vivo on a Drosophila model of cardiac disease. Thus, it also brings the proof of concept that cardiac functional imaging in adult Drosophila flies is usable for medium-scale in vivo pharmacological screening, with potent identification of cardioprotective drugs in various contexts of cardiac diseases.
Assuntos
Cardiotônicos/análise , Cardiotônicos/uso terapêutico , Drosophila melanogaster/fisiologia , Avaliação Pré-Clínica de Medicamentos , Ataxia de Friedreich/tratamento farmacológico , Actinas/metabolismo , Animais , Cardiotônicos/farmacologia , Modelos Animais de Doenças , Ataxia de Friedreich/patologia , Proteínas de Ligação ao Ferro/metabolismo , Azul de Metileno/farmacologia , Azul de Metileno/uso terapêutico , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miosinas/metabolismo , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Reprodutibilidade dos Testes , Sarcômeros/metabolismo , Bibliotecas de Moléculas Pequenas , FrataxinaRESUMO
Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene leading to a polyglutamine expansion in the ataxin-3 protein. The nuclear presence and aggregation of expanded ataxin-3 are critical steps in disease pathogenesis. To identify novel therapeutic targets, we investigated the nucleocytoplasmic transport system by screening a collection of importins and exportins that potentially modulate this nuclear localization. Using cell, Drosophila, and mouse models, we focused on three transport proteins, namely, CRM1, IPO13, KPNA3, and their respective Drosophila orthologs Emb, Cdm, and Kap-α3. While overexpression of CRM1/Emb demonstrated positive effects in Drosophila, KPNA3/Kap-α3 emerged as the most promising target, as knockdown via multiple RNAi lines demonstrated its ability to shuttle both truncated and full-length expanded ataxin-3, rescue neurodegeneration, restore photoreceptor formation, and reduce aggregation. Furthermore, KPNA3 knockout in SCA3 mice resulted in an amelioration of molecular and behavioral disturbances such as total activity, anxiety, and gait. Since KPNA3 is known to function as an import protein and recognize nuclear localization signals (NLSs), this work unites ataxin-3 structure to the nuclear pore machinery and provides a link between karyopherins, NLS signals, and polyglutamine disease, as well as demonstrates that KPNA3 is a key player in the pathogenesis of SCA3.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Ataxina-3/genética , Doença de Machado-Joseph/genética , alfa Carioferinas/genética , Animais , Ataxina-3/metabolismo , Expansão das Repetições de DNA , Modelos Animais de Doenças , Drosophila , Feminino , Células HEK293 , Humanos , Doença de Machado-Joseph/metabolismo , Masculino , Camundongos , Camundongos Knockout , Peptídeos , alfa Carioferinas/metabolismoRESUMO
In mammals, both sterile wounding and infection induce inflammation and activate the innate immune system, and the combination of both challenges may lead to severe health defects, revealing the importance of the balance between the intensity and resolution of the inflammatory response for the organism's fitness. Underlying mechanisms remain however elusive. Using Drosophila, we show that, upon infection with the entomopathogenic bacterium Pseudomonas entomophila (Pe), a sterile wounding induces a reduced resistance and increased host mortality. To identify the molecular mechanisms underlying the susceptibility of wounded flies to bacterial infection, we analyzed the very first steps of the process by comparing the transcriptome landscape of infected (simple hit flies, SH), wounded and infected (double hit flies, DH) and wounded (control) flies. We observed that overexpressed genes in DH flies compared to SH ones are significantly enriched in genes related to stress, including members of the JNK pathway. We demonstrated that the JNK pathway plays a central role in the DH phenotype by manipulating the Jra/dJun activity. Moreover, the CrebA/Creb3-like transcription factor (TF) and its targets were up-regulated in SH flies and we show that CrebA is required for mounting an appropriate immune response. Drosophila thus appears as a relevant model to investigate interactions between trauma and infection and allows to unravel key pathways involved.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , MAP Quinase Quinase 4/metabolismo , Infecções por Pseudomonas/metabolismo , Ferimentos e Lesões/metabolismo , Animais , Drosophila melanogaster , Transdução de Sinais , Transcriptoma , Ferimentos e Lesões/microbiologiaRESUMO
Aging is commonly described as being a continuous process affecting progressively organisms as time passes. This process results in a progressive decrease in individuals fitness through a wide range of both organismal-decreased motor activity, fertility, resistance to stress-and molecular phenotypes-decreased protein and energy homeostasis, impairment of insulin signaling. In the past 20 years, numerous genes have been identified as playing a major role in the aging process, yet little is known about the events leading to that loss of fitness. We recently described an event characterized by a dramatic increase of intestinal permeability to a blue food dye in aging flies committed to die within a few days. Importantly, flies showing this so called 'Smurf' phenotype are the only ones, among a population, to show various age-related changes and exhibit a high-risk of impending death whatever their chronological age. Thus, these observations suggest that instead of being one continuous phenomenon, aging may be a discontinuous process well described by at least two distinguishable phases. In this paper we addressed this hypothesis by implementing a new 2 Phases of Aging mathematiCal model (2PAC model) to simulate longevity curves based on the simple hypothesis of two consecutive phases of lifetime presenting different properties. We first present a unique equation for each phase and discuss the biological significance of the 3 associated parameters. Then we evaluate the influence of each parameter on the shape of survival curves. Overall, this new mathematical model, based on simple biological observations, is able to reproduce many experimental longevity curves, supporting the existence of 2 phases of aging exhibiting specific properties and separated by a dramatic transition that remains to be characterized. Moreover, it indicates that Smurf survival can be approximated by one single constant parameter for a broad range of genotypes that we have tested under our environmental conditions.
Assuntos
Envelhecimento/fisiologia , Modelos Biológicos , Animais , Drosophila melanogasterRESUMO
Friedreich's ataxia (FA) is a rare neurodegenerative disease which is very debilitating for the patients who progressively lose their autonomy. The lack of efficient therapeutic treatment of the disease strongly argues for urgent need to search for new active compounds that may stop the progression of the disease or prevent the appearance of the symptoms when the genetic defect is diagnosed early enough. In the present study, we used a yeast strain with a deletion of the frataxin homologue gene as a model of FA cells in a primary screen of two chemical libraries, a fraction of the French National Chemical Library (5500 compounds) and the Prestwick collection (880 compounds). We ran a secondary screen on Drosophila melanogaster flies expressing reduced levels of frataxin during larval development. Half of the compounds selected in yeast appeared to be active in flies in this developmental paradigm, and one of the two compounds with highest activities in this assay partially rescued the heart dilatation phenotype resulting from heart specific depletion of frataxin. The unique complementarity of these two frataxin-deficient models, unicellular and multicellular, appears to be very efficient to select new compounds with improved selectivity, bringing significant perspectives towards improvements in FA therapy.
Assuntos
Drosophila/metabolismo , Proteínas de Ligação ao Ferro/genética , Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/química , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Coração/efeitos dos fármacos , Proteínas de Ligação ao Ferro/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Microscopia de Vídeo , Rafinose/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , FrataxinaRESUMO
BACKGROUND: Huntington's disease (HD) is a Polyglutamine disease caused by the presence of CAG repeats in the first exon of Huntingtin (Htt), a large protein with multiple functions. In addition to neurodegeneration of specific brain regions, notably the striatum, HD also shows alterations in peripheral tissues, such as the heart, skeletal muscles or peripheral endocrine glands. Mutant Huntingtin (mHtt)-driven mitochondrial impairment may underlie some of the CNS and peripheral tissues dysfunctions, especially in tissues with high energy demand such as the heart. OBJECTIVE: The aim of this study is to characterize two new inducible Drosophila HD heart models and to assay the therapeutic potential of methylene blue in these HD models. METHODS: We report the construction of inducible Drosophila HD heart models, expressing two Nter fragments of the protein encompassing either exon 1 or the first 171 amino acids and the characterization of heart phenotypes in vivo. RESULTS: We show that both mHtt fragments are able to impair fly cardiac function with different characteristics. Additionally, expression of mHtt, which was limited to adulthood only, leads to mild heart impairment, as opposed to a strong and age-dependent phenotype observed when mHtt expression was driven during both developmental and adult stages. We report that treatment with methylene blue (MB), a protective compound in mitochondria-related diseases, partially protects the fly's heart against mHtt-induced toxicity, but does not rescue neuronal or glial phenotypes in other fly models of HD. This may be linked to its low penetration through the fly's blood-brain barrier. CONCLUSIONS: Our data suggest that improvement of mitochondrial function by MB, or related compounds, could be an efficient therapeutic strategy to prevent cardiac failure in HD patients.
Assuntos
Modelos Animais de Doenças , Cardiopatias Congênitas/genética , Doença de Huntington/genética , Azul de Metileno/administração & dosagem , Proteínas Associadas aos Microtúbulos/genética , Animais , Drosophila , Proteínas de Drosophila/genética , Cardiopatias Congênitas/prevenção & controle , Proteína Huntingtina , Doença de Huntington/complicações , Proteínas Associadas aos Microtúbulos/metabolismo , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacosRESUMO
Amyloid-ß protein precursor (AßPP) 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 AßPP 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 AßPP 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.
Assuntos
Doença de Alzheimer , Modelos Animais de Doenças , Drosophila , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
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.
Assuntos
Inativação Gênica , Estudos de Associação Genética , Proteínas de Ligação ao Ferro/genética , Ovário/citologia , Ovário/metabolismo , Esteroides/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Dípteros , Ecdisteroides/deficiência , Ecdisterona/administração & dosagem , Feminino , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Fenótipo , Progesterona/biossíntese , Interferência de RNA , FrataxinaRESUMO
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.
Assuntos
Cardiotônicos/farmacologia , Ataxia de Friedreich/tratamento farmacológico , Azul de Metileno/farmacologia , Animais , Cardiotônicos/uso terapêutico , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Avaliação Pré-Clínica de Medicamentos , Ataxia de Friedreich/patologia , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Masculino , Azul de Metileno/uso terapêutico , Interferência de RNA , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia , FrataxinaRESUMO
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.
Assuntos
Envelhecimento , Proteínas de Drosophila , Drosophila melanogaster , Coração/fisiologia , Sistema de Sinalização das MAP Quinases/genética , Fatores de Transcrição , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica , Humanos , Estresse Oxidativo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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 (miRNAs), 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 seven 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.
RESUMO
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.
Assuntos
Complexo Mediador/química , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Peptídeos/química , Proteínas de Ligação a RNA/química , Animais , Ataxina-1 , Ataxinas , Células COS , Chlorocebus aethiops , Escherichia coli/genética , Humanos , Complexo Mediador/genética , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genética , Plasmídeos , Polimerização , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-Atividade , TransfecçãoRESUMO
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.
Assuntos
Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Longevidade/genética , Mitocôndrias/metabolismo , Animais , Drosophila melanogaster/fisiologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Crescimento e Desenvolvimento/fisiologiaRESUMO
Organismal stress responses to oxidative stress are relevant to ageing and disease and involve key cell-/tissue-specific signal transduction mechanisms. Using Drosophila, an established in vivo model for stress studies, we show that cell-specific inositol phosphate signalling specifically via inositol 1,4,5 trisphosphate 3-kinase (InsP(3) 3-K, IP(3)K), negatively regulates organismal responses to oxidative stress. We demonstrate that the Drosophila Malpighian tubule (equivalent to vertebrate kidney and liver) is a key epithelial sensor for organismal oxidative stress responses: precise targeting of either gain-of-function constructs of Drosophila IP(3)Ks (IP(3)K-1 and IP(3)K-2), or loss-of-function (RNAi) constructs to only one cell type in tubule reversibly modulates survival of stress-challenged adult flies. In vivo, targeted IP(3)K-1 directly increases H(2)O(2) production, pro-apoptotic caspase-9 activity and mitochondrial membrane potential. The mitochondrial calcium load in tubule principal cells-assessed by luminescent and fluorescent genetically-encoded mitochondrial calcium reporters-is significantly increased by IP(3)K-1 under oxidative stress conditions, leading to apoptosis. The Drosophila orthologues of human apoptotic bcl-2 genes include debcl and buffy. Oxidative stress challenge does not modulate gene expression of either debcl or buffy in tubules; and altered debcl expression does not influence survival rates under oxidative stress challenge. Finally, targeted over-expression of either debcl or buffy to tubule principal cells does not impact on tubule caspase-9 activity. Thus, IP(3)K-1 modulates epithelial cell apoptosis without involvement of bcl-2-type proteins.
Assuntos
Apoptose , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Estresse Oxidativo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/metabolismo , Caspase 9/metabolismo , Peróxido de Hidrogênio/metabolismo , Túbulos de Malpighi/citologia , Túbulos de Malpighi/enzimologia , Potencial da Membrana Mitocondrial , Mitocôndrias/enzimologia , Especificidade de Órgãos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Análise de SobrevidaRESUMO
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-kappaB 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.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/imunologia , Endopeptidases/fisiologia , Regulação da Expressão Gênica , Transdução de Sinais , Animais , Dosagem de Genes , Inativação Gênica , Vida Livre de Germes/imunologia , Bactérias Gram-Negativas/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , NF-kappa B/biossíntese , Mapeamento de Interação de ProteínasRESUMO
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.