RESUMO
Members of the Ski/Sno protein family are classified as proto-oncogenes and act as negative regulators of the TGF-ß/BMP-pathways in vertebrates and invertebrates. A newly identified member of this protein family is fussel (fuss), the Drosophila homologue of the human functional Smad suppressing elements (fussel-15 and fussel-18). We and others have shown that Fuss interacts with SMAD4 and that overexpression leads to a strong inhibition of Dpp signaling. However, to be able to characterize the endogenous Fuss function in Drosophila melanogaster, we have generated a number of state of the art tools including anti-Fuss antibodies, specific fuss-Gal4 lines and fuss mutant fly lines via the CRISPR/Cas9 system. Fuss is a predominantly nuclear, postmitotic protein, mainly expressed in interneurons and fuss mutants are fully viable without any obvious developmental phenotype. To identify potential target genes or cells affected in fuss mutants, we conducted targeted DamID experiments in adult flies, which revealed the function of fuss in bitter gustatory neurons. We fully characterized fuss expression in the adult proboscis and by using food choice assays we were able to show that fuss mutants display defects in detecting bitter compounds. This correlated with a reduction of gustatory receptor gene expression (Gr33a, Gr66a, Gr93a) providing a molecular link to the behavioral phenotype. In addition, Fuss interacts with Rpd3, and downregulation of rpd3 in gustatory neurons phenocopies the loss of Fuss expression. Surprisingly, there is no colocalization of Fuss with phosphorylated Mad in the larval central nervous system, excluding a direct involvement of Fuss in Dpp/BMP signaling. Here we provide a first and exciting link of Fuss function in gustatory bitter neurons. Although gustatory receptors have been well characterized, little is known regarding the differentiation and maturation of gustatory neurons. This work therefore reveals Fuss as a pivotal element for the proper differentiation of bitter gustatory neurons acting within a chromatin modifying complex.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Histona Desacetilase 1/genética , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Superfície Celular/genética , Animais , Animais Geneticamente Modificados , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Genes de Insetos , Histona Desacetilase 1/metabolismo , Masculino , Mutação , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular/metabolismo , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais , Paladar/genéticaRESUMO
In Drosophila, female development is governed by a single RNA-binding protein, Sex-lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Sxl expression in female flies is maintained by an auto-regulatory, positive feedback loop with Sxl controlling splicing of its own mRNA. Until now, it remained unclear how males prevent accidental triggering of the Sxl expression cascade and protect themselves against runaway protein production. Here, we identify the protein Sister-of-Sex-lethal (Ssx) as an inhibitor of Sxl auto-regulatory splicing. Sxl and Ssx have a comparable RNA-binding specificity and compete for binding to RNA regulatory elements present in the Sxl transcript. In cultured Drosophila cells, Sxl-induced changes to alternative splicing can be reverted by the expression of Ssx. Moreover, in adult male flies ablation of the ssx gene results in a low level of productive Sxl mRNA splicing and Sxl protein production in isolated, clonal cell populations. In sum, this demonstrates that Ssx safeguards male animals against Sxl protein production to reinforce a stable, male-specific gene expression pattern.
Assuntos
Processamento Alternativo/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Caracteres Sexuais , Animais , Células Cultivadas , Proteínas de Drosophila/biossíntese , Éxons/genética , Feminino , Perfilação da Expressão Gênica , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/biossíntese , Sequências Reguladoras de Ácido Ribonucleico/genéticaRESUMO
Friedreich ataxia (FRDA) is the most important autosomal recessive ataxia in the Caucasian population. FRDA patients display severe neurological and cardiac symptoms that reflect a strong cellular and axonal degeneration. FRDA is caused by a loss of function of the mitochondrial protein frataxin which impairs the biosynthesis of iron-sulfur clusters and in turn the catalytic activity of several enzymes in the Krebs cycle and the respiratory chain leading to a diminished energy production. Although FRDA is due to frataxin depletion, overexpression might also be very helpful to better understand cellular functions of frataxin. In this work, we have increased frataxin expression in neurons to elucidate specific roles that frataxin might play in these tissues. Using molecular, biochemical, histological and behavioral methods, we report that frataxin overexpression is sufficient to increase oxidative phosphorylation, modify mitochondrial morphology, alter iron homeostasis and trigger oxidative stress-dependent cell death. Interestingly, genetic manipulation of mitochondrial iron metabolism by silencing mitoferrin successfully improves cell survival under oxidative-attack conditions, although enhancing antioxidant defenses or mitochondrial fusion failed to ameliorate frataxin overexpression phenotypes. This result suggests that cell degeneration is directly related to enhanced incorporation of iron into the mitochondria. Drosophila frataxin overexpression might also provide an alternative approach to identify processes that are important in FRDA such as changes in mitochondrial morphology and oxidative stress induced cell death.
Assuntos
Morte Celular/genética , Proteínas de Drosophila/genética , Proteínas de Ligação ao Ferro/genética , Ferro/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/genética , Animais , Animais Geneticamente Modificados , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Homeostase/genética , Proteínas de Ligação ao Ferro/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fosforilação Oxidativa , FrataxinaRESUMO
Rhodopsin 7 (Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000 and thought to encode for a functional Rhodopsin protein. Indeed, Rh7 exhibits most hallmarks of the known Rhodopsins, except for the G-protein-activating QAKK motif in the third cytoplasmic loop that is absent in Rh7. Here, we show that Rh7 can partially substitute Rh1 in the outer receptor cells (R1-6) for rhabdomere maintenance, but that it cannot activate the phototransduction cascade in these cells. This speaks against a role of Rh7 as photopigment in R1-6, but does not exclude that it works in the inner photoreceptor cells.
Assuntos
Drosophila melanogaster/fisiologia , Rodopsina/metabolismo , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Células Fotorreceptoras de Invertebrados/química , Células Fotorreceptoras de Invertebrados/metabolismoRESUMO
The GTPase Ras can either promote or inhibit cell survival. Inactivating mutations in Drosophila RasGAP (encoded by vap), a Ras GTPase-activating protein, lead to age-related brain degeneration. Genetic interactions implicate the epidermal growth factor receptor (EGFR)-Ras pathway in promoting neurodegeneration but the mechanism is not known. Here, we show that the Src homology 2 (SH2) domains of RasGAP are essential for its neuroprotective function. By using affinity purification and mass spectrometry, we identify a complex containing RasGAP together with Sprint, which is a Ras effector and putative activator of the endocytic GTPase Rab5. Formation of the RasGAP-Sprint complex requires the SH2 domains of RasGAP and tyrosine phosphorylation of Sprint. RasGAP and Sprint colocalize with Rab5-positive early endosomes but not with Rab7-positive late endosomes. We demonstrate a key role for this interaction in neurodegeneration: mutation of Sprint (or Rab5) suppresses neuronal cell death caused by the loss of RasGAP. These results indicate that the long-term survival of adult neurons in Drosophila is crucially dependent on the activities of two GTPases, Ras and Rab5, regulated by the interplay of RasGAP and Sprint.
Assuntos
Drosophila/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Sobrevivência Celular/fisiologia , Drosophila/genética , Endocitose , Feminino , Masculino , Fosforilação , Transdução de SinaisRESUMO
Drosophila melanogaster has contributed significantly to the understanding of disease mechanisms in Parkinson's disease (PD) as it is one of the very few PD model organisms that allow the study of age-dependent behavioral defects, physiology and histology, and genetic interactions among different PD-related genes. However, there have been contradictory results from a number of recent reports regarding the loss of dopaminergic neurons in different PD fly models. In an attempt to re-evaluate and clarify this issue, we have examined three different genetic (α-synuclein, Pink1, parkin) and two toxin-based (rotenone and paraquat) models of the disease for neuronal cell loss. Our results showed no dopaminergic neuronal loss in all models tested. Despite this surprising result, we found additional phenotypes showing the dysfunctional status of the dopaminergic neurons in most of the models analyzed. A common feature found in most models is a quantifiable decrease in the fluorescence of a green-fluorescent protein reporter gene in dopaminergic neurons that correlates well with other phenotypes found for these models and can be reliably used as a hallmark of the neurodegenerative process when modeling diseases affecting the dopaminergic system in Drosophila. Analyzing three genetic and two toxin-based Drosophila models of Parkinson's disease (PD) through green fluorescent protein reporter and α-tyrosine hydroxylase staining, we have found the number of dopaminergic neurons to remain unchanged. Despite the lack of neuronal loss, we have detected a remarkable decrease in a reporter green-fluorescent protein (GFP) signal in dopaminergic neurons, suggesting an abnormal neuronal status that correlates with the phenotypes associated with those PD fly models.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Drosophila/fisiologia , Doença de Parkinson Secundária/patologia , Doença de Parkinson/patologia , Animais , Contagem de Células , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Mutação/genética , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Doença de Parkinson/genética , Doença de Parkinson Secundária/induzido quimicamente , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética , alfa-Sinucleína/biossíntese , alfa-Sinucleína/genéticaRESUMO
The dynactin p150glued subunit, encoded by the gene DCTN1 is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport. This subunit is a candidate modifier for neurodegenerative diseases, in particular motoneuron and extrapyramidal diseases. Based on an extensive screening effort of all 32 exons in more than 2,500 ALS/MND patients, patients suffering from Parkinsonian Syndromes and controls, we investigated 24 sequence variants of p150 in cell-based studies. We used both non-neuronal cell lines and primary rodent spinal motoneurons and report on cell biological abnormalities in five of these sequence alterations and also briefly report on the clinical features. Our results suggest the presence of biological changes caused by some p150 mutants pointing to a potential pathogenetic significance as modifier of the phenotype of the human disease.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas Associadas aos Microtúbulos/genética , Neurônios Motores/metabolismo , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , Proteínas Adaptadoras de Transdução de Sinal , Esclerose Lateral Amiotrófica/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Complexo Dinactina , Embrião de Mamíferos , Feminino , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neurônios Motores/patologia , Neurônios Motores/ultraestrutura , Mutação/genética , Gravidez , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Estudos Retrospectivos , Medula Espinal/citologia , Fatores de TempoRESUMO
Friedreich's ataxia (FRDA) is the most common form of autosomal recessive ataxia caused by a deficit in the mitochondrial protein frataxin. Although demyelination is a common symptom in FRDA patients, no multicellular model has yet been developed to study the involvement of glial cells in FRDA. Using the recently established RNAi lines for targeted suppression of frataxin in Drosophila, we were able to study the effects of general versus glial-specific frataxin downregulation. In particular, we wanted to study the interplay between lowered frataxin content, lipid accumulation and peroxidation and the consequences of these effects on the sensitivity to oxidative stress and fly fitness. Interestingly, ubiquitous frataxin reduction leads to an increase in fatty acids catalyzing an enhancement of lipid peroxidation levels, elevating the intracellular toxic potential. Specific loss of frataxin in glial cells triggers a similar phenotype which can be visualized by accumulating lipid droplets in glial cells. This phenotype is associated with a reduced lifespan, an increased sensitivity to oxidative insult, neurodegenerative effects and a serious impairment of locomotor activity. These symptoms fit very well with our observation of an increase in intracellular toxicity by lipid peroxides. Interestingly, co-expression of a Drosophila apolipoprotein D ortholog (glial lazarillo) has a strong protective effect in our frataxin models, mainly by controlling the level of lipid peroxidation. Our results clearly support a strong involvement of glial cells and lipid peroxidation in the generation of FRDA-like symptoms.
Assuntos
Modelos Animais de Doenças , Drosophila , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Transtornos do Metabolismo dos Lipídeos/complicações , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Sobrevivência Celular/genética , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Ácidos Graxos/sangue , Ataxia de Friedreich/complicações , Ataxia de Friedreich/metabolismo , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Ligação ao Ferro/fisiologia , Transtornos do Metabolismo dos Lipídeos/genética , Transtornos do Metabolismo dos Lipídeos/metabolismo , Transtornos do Metabolismo dos Lipídeos/patologia , Peroxidação de Lipídeos/genética , Peroxidação de Lipídeos/fisiologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Sistema Nervoso/metabolismo , Sistema Nervoso/patologia , Neuroglia/metabolismo , Neuroglia/fisiologia , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , FrataxinaRESUMO
The stability of proteins in solution poses a great challenge for both technical applications and molecular biology, including neurodegenerative diseases. In this work, a phosphorylated resveratrol material was examined for its anti-aggregation properties in vitro and in vivo. Here, an anti-fibrillation effect could be measured for amyloid beta and human insulin in vitro and general anti-aggregation properties for crude chicken egg white in solution. Using a drosophila fly model for the overexpression of amyloid beta protein, changes in physiological protein aggregation and improved locomotor abilities could be observed in the presence of dietary phosphorylated resveratrol.
RESUMO
Over the years Ski and Sno have been found to be involved in cancer progression e.g. in oesophageal squamous cell carcinoma, melanoma, oestrogen receptor-positive breast carcinoma, colorectal carcinoma, and leukaemia. Often, their prooncogenic features have been linked to their ability of inhibiting the anti-proliferative action of TGF-ß signalling. Recently, not only pro-oncogenic but also anti-oncogenic functions of Ski/Sno proteins have been revealed. Besides Ski and Sno, which are ubiquitously expressed other members of Ski/Sno proteins exist which show highly specific neuronal expression, the SKI Family Transcriptional Corepressors (Skor). Among others Skor1 and Skor2 are involved in the development of Purkinje neurons and a mutation of Skor1 has been found to be associated with restless legs syndrome. But neither Skor1 nor Skor2 have been reported to be involved in cancer progression. Using overexpression studies in the Drosophila eye imaginal disc, we analysed if the Drosophila Skor homologue Fuss has retained the potential to inhibit differentiation and induce increased proliferation. Fuss expressed in cells posterior to the morphogenetic furrow, impairs photoreceptor axon pathfinding and inhibits differentiation of accessory cells. However, if its expression is induced prior to eye differentiation, Fuss might inhibit the differentiating function of Dpp signalling and might maintain proliferative action of Wg signalling, which is reminiscent of the Ski/Sno protein function in cancer.
Assuntos
Proteínas de Drosophila/metabolismo , Discos Imaginais/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Aciltransferases/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Discos Imaginais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Proteínas Nucleares/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Cellular and organismal iron storage depends on the function of the ferritin protein complex in insects and mammals alike. In the central nervous system of insects, the distribution and relevance of ferritin remain unclear, though ferritin has been implicated in Drosophila models of Alzheimers' and Parkinsons' disease and in Aluminum-induced neurodegeneration. Here we show that transgene-derived expression of ferritin subunits in glial cells of Drosophila melanogaster causes a late-onset behavioral decline, characterized by loss of circadian rhythms in constant darkness and impairment of elicited locomotor responses. Anatomical analysis of the affected brains revealed crystalline inclusions of iron-loaded ferritin in a subpopulation of glial cells but not significant neurodegeneration. Although transgene-induced glial ferritin expression was well tolerated throughout development and in young flies, it turned disadvantageous at older age. The flies we characterize in this report contribute to the study of ferritin in the Drosophila brain and can be used to assess the contribution of glial iron metabolism in neurodegenerative models of disease.
Assuntos
Sintomas Comportamentais/metabolismo , Ferritinas/biossíntese , Distúrbios do Metabolismo do Ferro/metabolismo , Ferro/metabolismo , Neuroglia/metabolismo , Lobo Óptico de Animais não Mamíferos/metabolismo , Animais , Animais Geneticamente Modificados , Sintomas Comportamentais/genética , Sintomas Comportamentais/patologia , Ritmo Circadiano/genética , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Ferritinas/genética , Distúrbios do Metabolismo do Ferro/genética , Distúrbios do Metabolismo do Ferro/patologia , Masculino , Atividade Motora/genética , Neuroglia/citologia , Lobo Óptico de Animais não Mamíferos/patologiaRESUMO
Parkinson's disease has been found to be caused by both, genetic and environmental factors. Despite the diversity of causes involved, a convergent pathogenic mechanism might underlie the special vulnerability of dopaminergic neurons in different forms of Parkinsonism. In recent years, a number of reports have proposed dopamine as a common player responsible in the loss of dopaminergic neurons independent of its etiology. Using RNAi lines we were able to generate flies with drastically reduced dopamine levels in the dopaminergic neurons. Combining these flies with a chemically induced Parkinson model (rotenone) and a familial form of Parkinson (mutant alpha-synuclein) we were able to show a strong reduction of neurotoxicity and a protection of the dopaminergic neurons when cellular dopamine levels were reduced. These results show that dopamine homeostasis plays a central role for the susceptibility of dopaminergic neurons to environmental and genetic factors in in vivo models of Parkinson disease.
Assuntos
Dopamina/fisiologia , Drosophila melanogaster/genética , Modelos Genéticos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Humanos , Degeneração Neural/induzido quimicamente , Doença de Parkinson/etiologia , Rotenona/toxicidadeRESUMO
Friedreich ataxia (FRDA) is a neurodegenerative disorder characterized by neuromuscular and neurological manifestations. It is caused by mutations in the FXN gene, which results in loss of the mitochondrial protein frataxin. Endoplasmic Reticulum-mitochondria associated membranes (MAMs) are inter-organelle structures involved in the regulation of essential cellular processes, including lipid metabolism and calcium signaling. In the present study, we have analyzed in both, unicellular and multicellular models of FRDA, calcium management and integrity of MAMs. We observed that function of MAMs is compromised in our cellular model of FRDA, which was improved upon treatment with antioxidants. In agreement, promoting mitochondrial calcium uptake was sufficient to restore several defects caused by frataxin deficiency in Drosophila Melanogaster. Remarkably, our findings describe for the first time frataxin as a member of the protein network of MAMs, where interacts with two of the main proteins implicated in endoplasmic reticulum-mitochondria communication. These results suggest a new role of frataxin, indicate that FRDA goes beyond mitochondrial defects and highlight MAMs as novel therapeutic candidates to improve patient's conditions.
Assuntos
Ataxia de Friedreich , Animais , Cálcio/metabolismo , Drosophila melanogaster/genética , Retículo Endoplasmático , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Mitocôndrias/metabolismo , Estresse OxidativoRESUMO
The conserved Hedgehog signaling pathway has well-established roles in development. However, its function during adulthood remains largely unknown. Here, we investigated whether the Hedgehog signaling pathway is active during adult life in Drosophila melanogaster, and we uncovered a protective function for Hedgehog signaling in coordinating correct proteostasis in glial cells. Adult-specific depletion of Hedgehog reduces lifespan, locomotor activity, and dopaminergic neuron integrity. Conversely, increased expression of Hedgehog extends lifespan and improves fitness. Moreover, Hedgehog pathway activation in glia rescues the lifespan and age-associated defects of hedgehog mutants. The Hedgehog pathway regulates downstream chaperones, whose overexpression in glial cells was sufficient to rescue the shortened lifespan and proteostasis defects of hedgehog mutants. Finally, we demonstrate the protective ability of Hedgehog signaling in a Drosophila Alzheimer's disease model expressing human amyloid beta in the glia. Overall, we propose that Hedgehog signaling is requisite for lifespan determination and correct proteostasis in glial cells.
Assuntos
Drosophila melanogaster/metabolismo , Proteínas Hedgehog/metabolismo , Longevidade , Neuroglia/metabolismo , Proteostase , Transdução de Sinais , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Sobrevivência Celular , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Homeostase , Humanos , Modelos Biológicos , Mutação/genética , Neurônios/metabolismo , Neuroproteção , Análise de SobrevidaRESUMO
Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Some of the inherited forms of the disease are caused by mutations in the alpha-synuclein gene and the triplication of its locus. Oxidative stress has been proposed as a central mechanism for the progression of the disease although its relation with alpha-synuclein toxicity remains obscure. Targeted expression of human alpha-synuclein has been effectively used to recreate the pathology of PD in Drosophila melanogaster and it has been proved an excellent tool for the study of testable hypothesis in relation to the disease. We show that dopaminergic neurons are specifically sensitive to hyperoxia induced oxidative stress and that mutant forms of alpha-synuclein show an enhanced toxicity under these conditions suggesting synergic interactions. In addition, the co-expression of Cu/Zn superoxid dismutase protects against the dopaminergic neuronal loss induced by mutant alpha-synuclein overexpression thus identifying oxidative stress as an important causative factor in the pathology of autosomal-dominant Parkinsonism.
Assuntos
Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios/metabolismo , Doença de Parkinson/patologia , Substância Negra/patologia , Superóxido Dismutase/metabolismo , Fatores Etários , Animais , Animais Geneticamente Modificados , Sobrevivência Celular/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hipóxia/fisiopatologia , Mutação/fisiologia , Estresse Oxidativo/genética , Doença de Parkinson/genética , Superóxido Dismutase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/genéticaRESUMO
Friedreich's ataxia (FRDA) is the most important recessive ataxia in the Caucasian population. It is caused by a deficit of the mitochondrial protein frataxin. Despite its pivotal effect on biosynthesis of iron-sulfur clusters and mitochondrial energy production, little is known about the influence of frataxin depletion on homeostasis of the cellular mitochondrial network. We have carried out a forward genetic screen to analyze genetic interactions between genes controlling mitochondrial homeostasis and Drosophila frataxin. Our screen has identified silencing of Drosophila mitofusin (Marf) as a suppressor of FRDA phenotypes in glia. Drosophila Marf is known to play crucial roles in mitochondrial fusion, mitochondrial degradation and in the interface between mitochondria and endoplasmic reticulum (ER). Thus, we have analyzed the effects of frataxin knockdown on mitochondrial morphology, mitophagy and ER function in our fly FRDA model using different histological and molecular markers such as tetramethylrhodamine, ethyl ester (TMRE), mitochondria-targeted GFP (mitoGFP), p62, ATG8a, LAMP1, Xbp1 and BiP/GRP78. Furthermore, we have generated the first Drosophila transgenic line containing the mtRosella construct under the UAS control to study the progression of the mitophagy process in vivo. Our results indicated that frataxin-deficiency had a small impact on mitochondrial morphology but enhanced mitochondrial clearance and altered the ER stress response in Drosophila. Remarkably, we demonstrate that downregulation of Marf suppresses ER stress in frataxin-deficient cells and this is sufficient to improve locomotor dysfunction, brain degeneration and lipid dyshomeostasis in our FRDA model. In agreement, chemical reduction of ER stress by means of two different compounds was sufficient to ameliorate the effects of frataxin deficiency in three different fly FRDA models. Altogether, our results strongly suggest that the protection mediated by Marf knockdown in glia is mainly linked to its role in the mitochondrial-ER tethering and not to mitochondrial dynamics or mitochondrial degradation and that ER stress is a novel and pivotal player in the progression and etiology of FRDA. This work might define a new pathological mechanism in FRDA, linking mitochondrial dysfunction due to frataxin deficiency and mitofusin-mediated ER stress, which might be responsible for characteristic cellular features of the disease and also suggests ER stress as a therapeutic target.
RESUMO
A growing body of evidence suggests that oxidative stress is a common underlying mechanism in the pathogenesis of neurodegenerative disorders such as Alzheimer's, Huntington's, Creutzfeld-Jakob and Parkinson's diseases. Despite the increasing number of reports finding a causal relation between oxidative stress and neurodegeneration, little is known about the genetic elements that confer protection against the deleterious effects of oxidation in neurons. We have isolated and characterized the Drosophila melanogaster gene sniffer, whose function is essential for preventing age-related neurodegeneration. In addition, we demonstrate that oxidative stress is a direct cause of neurodegeneration in the Drosophila central nervous system and that reduction of sniffer activity leads to neuronal cell death. The overexpression of the gene confers neuronal protection against oxygen-induced apoptosis, increases resistance of flies to experimental normobaric hyperoxia, and improves general locomotor fitness. Sniffer belongs to the family of short-chain dehydrogenase/reductase (SDR) enzymes and exhibits carbonyl reductase activity. This is the first in vivo evidence of the direct and important implication of this enzyme as a neuroprotective agent in the cellular defense mechanisms against oxidative stress.
Assuntos
Oxirredutases do Álcool/genética , Apoptose/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Degeneração Neural/genética , Neurônios/fisiologia , Fatores Etários , Oxirredutases do Álcool/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/genética , Western Blotting , Encéfalo/fisiopatologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Histocitoquímica , Marcação In Situ das Extremidades Cortadas , Dados de Sequência Molecular , Neurônios/metabolismo , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Oxigênio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
Ras signaling has been shown to play an important role in promoting cell survival in many different tissues. Here we show that upregulation of Ras activity in adult Drosophila neurons induces neuronal cell death, as evident from the phenotype of vacuolar peduncle (vap) mutants defective in the Drosophila RasGAP gene, which encodes a Ras GTPase-activating protein. These mutants show age-related brain degeneration that is dependent on activation of the EGF receptor signaling pathway in adult neurons, leading to autophagic cell death (cell death type 2). These results provide the first evidence for a requirement of Egf receptor activity in differentiated adult Drosophila neurons and show that a delicate balance of Ras activity is essential for the survival of adult neurons.
Assuntos
Envelhecimento/fisiologia , Encéfalo/fisiologia , Drosophila/fisiologia , Receptores ErbB/metabolismo , Transdução de Sinais/fisiologia , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Morte Celular/fisiologia , Drosophila/genética , Mutação , Regulação para Cima , Vacúolos/genética , Vacúolos/metabolismoRESUMO
Maintenance of metal homeostasis is crucial for many different enzymatic activities and in turn for cell function and survival. In addition, cells display detoxification and protective mechanisms against toxic accumulation of metals. Perturbation of any of these processes normally leads to cellular dysfunction and finally to cell death. In the last years, loss of metal regulation has been described as a common pathological feature in many human neurodegenerative diseases. However, in most cases, it is still a matter of debate whether such dyshomeostasis is a primary or a secondary downstream defect. In this review, we will summarize and critically evaluate the contribution of Drosophila to model human diseases that involve altered metabolism of metals or in which metal dyshomeostasis influence their pathobiology. As a prerequisite to use Drosophila as a model, we will recapitulate and describe the main features of core genes involved in copper and zinc metabolism that are conserved between mammals and flies. Drosophila presents some unique strengths to be at the forefront of neurobiological studies. The number of genetic tools, the possibility to easily test genetic interactions in vivo and the feasibility to perform unbiased genetic and pharmacological screens are some of the most prominent advantages of the fruitfly. In this work, we will pay special attention to the most important results reported in fly models to unveil the role of copper and zinc in cellular degeneration and their influence in the development and progression of human neurodegenerative pathologies such as Parkinson's disease, Alzheimer's disease, Huntington's disease, Friedreich's Ataxia or Menkes, and Wilson's diseases. Finally, we show how these studies performed in the fly have allowed to give further insight into the influence of copper and zinc in the molecular and cellular causes and consequences underlying these diseases as well as the discovery of new therapeutic strategies, which had not yet been described in other model systems.
RESUMO
Circadian rhythms can be entrained by light to follow the daily solar cycle. In Drosophila melanogaster a pair of extraretinal eyelets expressing immunoreactivity to Rhodopsin 6 each contains four photoreceptors located beneath the posterior margin of the compound eye. Their axons project to the region of the pacemaker center in the brain with a trajectory resembling that of Bolwig's organ, the visual organ of the larva. A lacZ reporter line driven by an upstream fragment of the developmental gap gene Krüppel is a specific enhancer element for Bolwig's organ. Expression of immunoreactivity to the product of lacZ in Bolwig's organ persists through pupal metamorphosis and survives in the adult eyelet. We thus demonstrate that eyelet derives from the 12 photoreceptors of Bolwig's organ, which entrain circadian rhythmicity in the larva. Double labeling with anti-pigment-dispersing hormone shows that the terminals of Bolwig's nerve differentiate during metamorphosis in close temporal and spatial relationship to the ventral lateral neurons (LN(v)), which are essential to express circadian rhythmicity in the adult. Bolwig's organ also expresses immunoreactivity to Rhodopsin 6, which thus continues in eyelet. We compared action spectra of entrainment in different fly strains: in flies lacking compound eyes but retaining eyelet (so(1)), lacking both compound eyes and eyelet (so(1);gl(60j)), and retaining eyelet but lacking compound eyes as well as cryptochrome (so(1);cry(b)). Responses to phase shifts suggest that, in the absence of compound eyes, eyelet together with cryptochrome mainly mediates phase delays. Thus a functional role in circadian entrainment first found in Bolwig's organ in the larva is retained in eyelet, the adult remnant of Bolwig's organ, even in the face of metamorphic restructuring.