RESUMO
BACKGROUND: Inherited mutations in the LRRK2 protein are common causes of Parkinson's disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. OBJECTIVE: To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies. METHODS: LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern ('sleep'), and courtship memory determined in aged flies. RESULTS: Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons. CONCLUSION: We conclude that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson's disease.
Assuntos
Neurônios Dopaminérgicos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Proteínas rab de Ligação ao GTP , Animais , Neurônios Dopaminérgicos/metabolismo , Drosophila/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
We investigated in larval and adult Drosophila models whether loss of the mitochondrial chaperone Hsc70-5 is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that we studied an early stage of pathogenesis. At this stage, we noted a loss of synaptic vesicle proteins and active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of dj-1ß, Lrrk, DCTN2-p50, DCTN1-p150, Atg1, Atg101, Atg5, Atg7, and Atg12. The knockdown of components of the macroautophagy/autophagy machinery or overexpression of human HSPA9 broadly rescued larval and adult phenotypes, while disease-associated HSPA9 variants did not. Overexpression of Pink1 or promotion of autophagy exacerbated defects.Abbreviations: AEL: after egg laying; AZ: active zone; brp: bruchpilot; Csp: cysteine string protein; dlg: discs large; eEJPs: evoked excitatory junctional potentials; GluR: glutamate receptor; H2O2: hydrogen peroxide; mEJP: miniature excitatory junctional potentials; MT: microtubule; NMJ: neuromuscular junction; PD: Parkinson disease; Pink1: PTEN-induced putative kinase 1; PSD: postsynaptic density; SSR: subsynaptic reticulum; SV: synaptic vesicle; VGlut: vesicular glutamate transporter.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Peróxido de Hidrogênio , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Proteína Desglicase DJ-1/metabolismo , Proteínas Serina-Treonina QuinasesRESUMO
LRRK2 mutations cause Parkinson's, but the molecular link from increased kinase activity to pathological neurodegeneration remains undetermined. Previous in vitro assays indicate that LRRK2 substrates include at least 8 Rab GTPases. We have now examined this hypothesis in vivo in a functional, electroretinogram screen, expressing each Rab with/without LRRK2-G2019S in selected Drosophila dopaminergic neurons. Our screen discriminated Rab10 from Rab3. The strongest Rab/LRRK2-G2019S interaction is with Rab10; the weakest with Rab3. Rab10 is expressed in a different set of dopaminergic neurons from Rab3. Thus, anatomical and physiological patterns of Rab10 are related. We conclude that Rab10 is a valid substrate of LRRK2 in dopaminergic neurons in vivo We propose that variations in Rab expression contribute to differences in the rate of neurodegeneration recorded in different dopaminergic nuclei in Parkinson's.
Assuntos
Drosophila , Doença de Parkinson , Animais , Neurônios Dopaminérgicos/metabolismo , Drosophila/genética , Drosophila/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutação , Doença de Parkinson/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoAssuntos
Esclerose Lateral Amiotrófica , Doença de Parkinson , Humanos , Ocupações , Percepção , Estados UnidosRESUMO
There is increasing evidence for a strong genetic basis for autism, with many genetic models being developed in an attempt to replicate autistic symptoms in animals. However, current animal behaviour paradigms rarely match the social and cognitive behaviours exhibited by autistic individuals. Here, we instead assay another functional domain-sensory processing-known to be affected in autism to test a novel genetic autism model in Drosophila melanogaster. We show similar visual response alterations and a similar development trajectory in Nhe3 mutant flies (total n = 72) and in autistic human participants (total n = 154). We report a dissociation between first- and second-order electrophysiological visual responses to steady-state stimulation in adult mutant fruit flies that is strikingly similar to the response pattern in human adults with ASD as well as that of a large sample of neurotypical individuals with high numbers of autistic traits. We explain this as a genetically driven, selective signalling alteration in transient visual dynamics. In contrast to adults, autistic children show a decrease in the first-order response that is matched by the fruit fly model, suggesting that a compensatory change in processing occurs during development. Our results provide the first animal model of autism comprising a differential developmental phenotype in visual processing.
Assuntos
Transtorno Autístico/patologia , Transtorno Autístico/fisiopatologia , Drosophila melanogaster , Animais , Modelos Animais de Doenças , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Modelos Genéticos , Percepção VisualRESUMO
The excitotoxic theory of Parkinson's disease (PD) hypothesizes that a pathophysiological degeneration of dopaminergic neurons stems from neural hyperactivity at early stages of disease, leading to mitochondrial stress and cell death. Recent research has harnessed the visual system of Drosophila PD models to probe this hypothesis. Here, we investigate whether abnormal visual sensitivity and excitotoxicity occur in early-onset PD (EOPD) Drosophila models DJ-1αΔ72, DJ-1ßΔ 93, and PINK15. We used an electroretinogram to record steady-state visually evoked potentials driven by temporal contrast stimuli. At 1 day of age, all EOPD mutants had a twofold increase in response amplitudes compared with wÌ controls. Furthermore, we found that excitotoxicity occurs in older EOPD models after increased neural activity is triggered by visual stimulation. In an additional analysis, we used a linear discriminant analysis to test whether there were subtle variations in neural gain control that could be used to classify Drosophila into their correct age and genotype. The discriminant analysis was highly accurate, classifying Drosophila into their correct genotypic class at all age groups at 50-70% accuracy (20% chance baseline). Differences in cellular processes link to subtle alterations in neural network operation in young flies, all of which lead to the same pathogenic outcome. Our data are the first to quantify abnormal gain control and excitotoxicity in EOPD Drosophila mutants. We conclude that EOPD mutations may be linked to more sensitive neuronal signaling in prodromal animals that may cause the expression of PD symptomologies later in life. NEW & NOTEWORTHY Steady-state visually evoked potential response amplitudes to multivariate temporal contrast stimuli were recorded in early-onset PD Drosophila models. Our data indicate that abnormal gain control and a subsequent visual loss occur in these PD mutants, supporting a broader excitotoxicity hypothesis in genetic PD. Furthermore, linear discriminant analysis could accurately classify Drosophila into their correct genotype at different ages throughout their lifespan. Our results suggest increased neural signaling in prodromal PD patients.
Assuntos
Potenciais Evocados Visuais , Doença de Parkinson/fisiopatologia , Retina/fisiopatologia , Animais , Animais Geneticamente Modificados , Análise Discriminante , Modelos Animais de Doenças , Genótipo , Masculino , Neurônios/fisiologia , Estimulação Luminosa , Células Fotorreceptoras/fisiologiaRESUMO
In a number of Drosophila models of genetic Parkinson's disease (PD) flies climb more slowly than wild-type controls. However, this assay does not distinguish effects of PD-related genes on gravity sensation, "arousal", central pattern generation of leg movements, or muscle. To address this problem, we have developed an assay for the fly proboscis extension response (PER). This is attractive because the PER has a simple, well-identified reflex neural circuit, in which sucrose sensing neurons activate a pair of "command interneurons", and thence motoneurons whose activity contracts the proboscis muscle. This circuit is modulated by a single dopaminergic neuron (TH-VUM). We find that expressing either the G2019S or I2020T (but not R1441C, or kinase dead) forms of human LRRK2 in dopaminergic neurons reduces the percentage of flies that initially respond to sucrose stimulation. This is rescued fully by feeding l-DOPA and partially by feeding kinase inhibitors, targeted to LRRK2 (LRRK2-IN-1 and BMPPB-32). High-speed video shows that G2019S expression in dopaminergic neurons slows the speed of proboscis extension, makes its duration more variable, and increases the tremor. Testing subsets of dopaminergic neurons suggests that the single TH-VUM neuron is likely most important in this phenotype. We conclude the Drosophila PER provides an excellent model of LRRK2 motor deficits showing bradykinesia, akinesia, hypokinesia, and increased tremor, with the possibility to localize changes in neural signaling.
RESUMO
An organism's biological day is characterized by a pattern of anticipatory physiological and behavioral changes that are governed by circadian clocks to align with the 24-h cycling environment. Here, we used flash electroretinograms (ERGs) and steady-state visually evoked potentials (SSVEPs) to examine how visual responsiveness in wild-type Drosophila melanogaster and the circadian clock mutant ClkJrk varies over circadian time. We show that the ERG parameters of wild-type flies vary over the circadian day, with a higher luminance response during the subjective night. The SSVEP response that assesses contrast sensitivity also showed a time-of-day dependence, including 2 prominent peaks within a 24-h period and a maximal response at the end of the subjective day, indicating a tradeoff between luminance and contrast sensitivity. Moreover, the behaviorally arrhythmic ClkJrk mutants maintained a circadian profile in both luminance and contrast sensitivity, but unlike the wild-types, which show bimodal profiles in their visual response, ClkJrk flies show a weakening of the bimodal character, with visual responsiveness tending to peak once a day. We conclude that the ClkJrk mutation mainly affects 1 of 2 functionally coupled oscillators and that the visual system is partially separated from the locomotor circadian circuits that drive bouts of morning and evening activity. As light exposure is a major mechanism for entrainment, our work suggests that a detailed temporal analysis of electrophysiological responses is warranted to better identify the time window at which circadian rhythms are most receptive to light-induced phase shifting.
Assuntos
Proteínas CLOCK/genética , Ritmo Circadiano , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Potenciais Evocados Visuais , Visão Ocular , Animais , Eletrorretinografia , MasculinoRESUMO
Myoglianin, the Drosophila homolog of the secreted vertebrate proteins Myostatin and GDF-11, is an important regulator of neuronal modeling, and synapse function and morphology. While Myoglianin suppression during development elicits positive effects on the neuromuscular system, genetic manipulations of myoglianin expression levels have a varied effect on the outcome of performance tests in aging flies. Specifically, Myoglianin preserves jumping ability, has no effect on negative geotaxis, and negatively regulates flight performance in aging flies. In addition, Myoglianin exhibits a tissue-specific effect on longevity, with myoglianin upregulation in glial cells increasing the median lifespan. These findings indicate complex role for this TGF-ß-like protein in governing neuromuscular signaling and consequent behavioral outputs and lifespan in adult flies.
Assuntos
Envelhecimento/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Fator de Crescimento Transformador beta/metabolismo , Animais , Drosophila/metabolismo , Drosophila/fisiologia , Regulação da Expressão Gênica , Músculos/fisiologia , Miostatina/metabolismo , Transdução de Sinais , Asas de Animais/fisiologiaRESUMO
Saposin deficiency is a childhood neurodegenerative lysosomal storage disorder (LSD) that can cause premature death within three months of life. Saposins are activator proteins that promote the function of lysosomal hydrolases that mediate the degradation of sphingolipids. There are four saposin proteins in humans, which are encoded by the prosaposin gene. Mutations causing an absence or impaired function of individual saposins or the whole prosaposin gene lead to distinct LSDs due to the storage of different classes of sphingolipids. The pathological events leading to neuronal dysfunction induced by lysosomal storage of sphingolipids are as yet poorly defined. We have generated and characterised a Drosophila model of saposin deficiency that shows striking similarities to the human diseases. Drosophila saposin-related (dSap-r) mutants show a reduced longevity, progressive neurodegeneration, lysosomal storage, dramatic swelling of neuronal soma, perturbations in sphingolipid catabolism, and sensory physiological deterioration. Our data suggests a genetic interaction with a calcium exchanger (Calx) pointing to a possible calcium homeostasis deficit in dSap-r mutants. Together these findings support the use of dSap-r mutants in advancing our understanding of the cellular pathology implicated in saposin deficiency and related LSDs.
Assuntos
Modelos Animais de Doenças , Proteínas de Drosophila/deficiência , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Saposinas/deficiência , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Animais Geneticamente Modificados , Antiporters/genética , Antiporters/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Cálcio/metabolismo , Ceramidas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Homeostase/fisiologia , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/patologia , Doenças Neurodegenerativas/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Saposinas/genética , Esfingosina/metabolismo , Análise de SobrevidaRESUMO
This study describes how the application of evolutionary algorithms (EAs) can be used to study motor function in humans with Parkinson's disease (PD) and in animal models of PD. Human data is obtained using commercially available sensors via a range of non-invasive procedures that follow conventional clinical practice. EAs can then be used to classify human data for a range of uses, including diagnosis and disease monitoring. New results are presented that demonstrate how EAs can also be used to classify fruit flies with and without genetic mutations that cause Parkinson's by using measurements of the proboscis extension reflex. The case is made for a computational approach that can be applied across human and animal studies of PD and lays the way for evaluation of existing and new drug therapies in a truly objective way.
Assuntos
Algoritmos , Antiparkinsonianos/uso terapêutico , Diagnóstico por Computador/métodos , Doença de Parkinson/diagnóstico , Doença de Parkinson/tratamento farmacológico , Animais , Drosophila melanogaster , Feminino , Humanos , Masculino , Peixe-ZebraRESUMO
Electrophysiological studies indicate altered contrast processing in some Parkinson's Disease (PD) patients. We recently demonstrated that vision is altered in Drosophila PD models and hypothesised that different types of genetic and idiopathic PD may affect dopaminergic visual signalling pathways differently. Here we asked whether visual responses in Drosophila could be used to identify PD mutations. To mimic a clinical setting a range of flies was used. Young flies from four control lines were compared to three early-onset PD mutations (PINK1, DJ-1α and DJ-1ß), and to two other neurodegenerative mutations, one in the fly LRRK2 orthologue (dLRRK) the other in eggroll, a model of general neurodegeneration in Drosophila. Stimuli were contrast reversing gratings spanning 64 spatiotemporal frequency combinations. We recorded the steady-state visually-evoked response amplitude across all combinations. We found that the pattern of neuronal responses differed between genotypes. Wild-type and early-onset PD flies formed separate clusters; the late-onset mutation is an outlier. Neuronal responses in early-onset PD flies were stronger than in wild-types. Multivariate pattern analysis grouped flies by PD/non-PD genotype with an accuracy >85%. We propose that machine learning algorithms may be useful in increasing the diagnostic specificity of human electrophysiological measurements in both animal models and PD patients.
Assuntos
Genótipo , Doença de Parkinson/diagnóstico , Animais , Drosophila melanogaster , Potenciais Evocados Visuais , Humanos , Masculino , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Sensibilidade e Especificidade , Análise Espaço-Temporal , Percepção VisualRESUMO
We provide an insight into the role Drosophila has played in elucidating neurophysiological perturbations associated with Parkinson's disease- (PD-) related genes. Synaptic signalling deficits are observed in motor, central, and sensory systems. Given the neurological impact of disease causing mutations within these same genes in humans the phenotypes observed in fly are of significant interest. As such we observe four unique opportunities provided by fly nervous system models of Parkinson's disease. Firstly, Drosophila models are instrumental in exploring the mechanisms of neurodegeneration, with several PD-related mutations eliciting related phenotypes including sensitivity to energy supply and vesicular deformities. These are leading to the identification of plausible cellular mechanisms, which may be specific to (dopaminergic) neurons and synapses rather than general cellular phenotypes. Secondly, models show noncell autonomous signalling within the nervous system, offering the opportunity to develop our understanding of the way pathogenic signalling propagates, resembling Braak's scheme of spreading pathology in PD. Thirdly, the models link physiological deficits to changes in synaptic structure. While the structure-function relationship is complex, the genetic tractability of Drosophila offers the chance to separate fundamental changes from downstream consequences. Finally, the strong neuronal phenotypes permit relevant first in vivo drug testing.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Transtornos da Visão/genética , Animais , Benzodiazepinonas/farmacologia , Drosophila/efeitos dos fármacos , Proteínas de Drosophila/efeitos dos fármacos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação , Doença de Parkinson/complicações , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Pirimidinas/farmacologia , Transtornos da Visão/etiologiaRESUMO
Class IIa histone deacetylases (HDACs) regulate the activity of many transcription factors to influence liver gluconeogenesis and the development of specialized cells, including muscle, neurons, and lymphocytes. Here, we describe a conserved role for class IIa HDACs in sustaining robust circadian behavioral rhythms in Drosophila and cellular rhythms in mammalian cells. In mouse fibroblasts, overexpression of HDAC5 severely disrupts transcriptional rhythms of core clock genes. HDAC5 overexpression decreases BMAL1 acetylation on Lys-537 and pharmacological inhibition of class IIa HDACs increases BMAL1 acetylation. Furthermore, we observe cyclical nucleocytoplasmic shuttling of HDAC5 in mouse fibroblasts that is characteristically circadian. Mutation of the Drosophila homolog HDAC4 impairs locomotor activity rhythms of flies and decreases period mRNA levels. RNAi-mediated knockdown of HDAC4 in Drosophila clock cells also dampens circadian function. Given that the localization of class IIa HDACs is signal-regulated and influenced by Ca(2+) and cAMP signals, our findings offer a mechanism by which extracellular stimuli that generate these signals can feed into the molecular clock machinery.
Assuntos
Fatores de Transcrição ARNTL/genética , Relógios Circadianos/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Histona Desacetilases/genética , RNA Mensageiro/genética , Fatores de Transcrição ARNTL/metabolismo , Acetilação , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Cálcio/metabolismo , Sequência Conservada , AMP Cíclico , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genes Reporter , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Células NIH 3T3 , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Our understanding of Parkinson's disease (PD) has been revolutionized by the discovery of disease-causing genetic mutations. The most common of these is the G2019S mutation in the LRRK2 kinase gene, which leads to increased kinase activity. However, the link between increased kinase activity and PD is unclear. Previously, we showed that dopaminergic expression of the human LRRK2-G2019S transgene in flies led to an activity-dependent loss of vision in older animals and we hypothesized that this may have been preceded by a failure to regulate neuronal activity correctly in younger animals. To test this hypothesis, we used a sensitive measure of visual function based on frequency-tagged steady-state visually evoked potentials. Spectral analysis allowed us to identify signals from multiple levels of the fly visual system and wild-type visual response curves were qualitatively similar to those from human cortex. Dopaminergic expression of hLRRK2-G2019S increased contrast sensitivity throughout the retinal network. To test whether this was due to increased kinase activity, we fed Drosophila with kinase inhibitors targeted at LRRK2. Contrast sensitivity in both day 1 and day 14 flies was normalized by a novel LRRK2 kinase inhibitor 'BMPPB-32'. Biochemical and cellular assays suggested that BMPPB-32 would be a more specific kinase inhibitor than LRRK2-IN-1. We confirmed this in vivo, finding that dLRRK(-) null flies show large off-target effects with LRRK2-IN-1 but not BMPPB-32. Our data link the increased Kinase activity of the G2019S-LRRK2 mutation to neuronal dysfunction and demonstrate the power of the Drosophila visual system in assaying the neurological effects of genetic diseases and therapies.
Assuntos
Drosophila melanogaster/fisiologia , Doença de Parkinson/fisiopatologia , Visão Ocular/fisiologia , Animais , Sensibilidades de Contraste/efeitos dos fármacos , Modelos Animais de Doenças , Potenciais Evocados Visuais/efeitos dos fármacos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Modelos Biológicos , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Mascaramento Perceptivo , Células Fotorreceptoras de Invertebrados/efeitos dos fármacos , Células Fotorreceptoras de Invertebrados/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Visão Ocular/efeitos dos fármacosRESUMO
Flies expressing the most common Parkinson disease (PD)-related mutation, LRRK2-G2019S, in their dopaminergic neurons show loss of visual function and degeneration of the retina, including mitochondrial abnormalities, apoptosis and autophagy. Since the photoreceptors that degenerate are not dopaminergic, this demonstrates nonautonomous degeneration, and a spread of pathology. This provides a model consistent with Braak's hypothesis on progressive PD. The loss of visual function is specific for the G2019S mutation, implying the cause is its increased kinase activity, and is enhanced by increased neuronal activity. These data suggest novel explanations for the variability in animal models of PD. The specificity of visual loss to G2019S, coupled with the differences in neural firing rate, provide an explanation for the variability between people with PD in visual tests.
Assuntos
Neurônios Dopaminérgicos/patologia , Mutação/genética , Degeneração Neural/patologia , Doença de Parkinson/enzimologia , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/genética , Trifosfato de Adenosina/metabolismo , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/enzimologia , Drosophila melanogaster/genética , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Degeneração Neural/enzimologia , Degeneração Retiniana/enzimologia , Degeneração Retiniana/patologiaRESUMO
Parkinson's disease (PD) is associated with loss of dopaminergic signalling, and affects not just movement, but also vision. As both mammalian and fly visual systems contain dopaminergic neurons, we investigated the effect of LRRK2 mutations (the most common cause of inherited PD) on Drosophila electroretinograms (ERGs). We reveal progressive loss of photoreceptor function in flies expressing LRRK2-G2019S in dopaminergic neurons. The photoreceptors showed elevated autophagy, apoptosis and mitochondrial disorganization. Head sections confirmed extensive neurodegeneration throughout the visual system, including regions not directly innervated by dopaminergic neurons. Other PD-related mutations did not affect photoreceptor function, and no loss of vision was seen with kinase-dead transgenics. Manipulations of the level of Drosophila dLRRK suggest G2019S is acting as a gain-of-function, rather than dominant negative mutation. Increasing activity of the visual system, or of just the dopaminergic neurons, accelerated the G2019S-induced deterioration of vision. The fly visual system provides an excellent, tractable model of a non-autonomous deficit reminiscent of that seen in PD, and suggests that increased energy demand may contribute to the mechanism by which LRRK2-G2019S causes neurodegeneration.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Proteínas de Drosophila/genética , Expressão Gênica , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/genética , Degeneração Retiniana/genética , Animais , Apoptose/genética , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Eletrorretinografia , Feminino , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mutação , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologiaRESUMO
Cytoplasmic accumulation and nuclear clearance of TDP-43 characterize familial and sporadic forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, suggesting that either loss or gain of TDP-43 function, or both, cause disease formation. Here we have systematically compared loss- and gain-of-function of Drosophila TDP-43, TAR DNA Binding Protein Homolog (TBPH), in synaptic function and morphology, motor control, and age-related neuronal survival. Both loss and gain of TBPH severely affect development and result in premature lethality. TBPH dysfunction caused impaired synaptic transmission at the larval neuromuscular junction (NMJ) and in the adult. Tissue-specific knockdown together with electrophysiological recordings at the larval NMJ also revealed that alterations of TBPH function predominantly affect pre-synaptic efficacy, suggesting that impaired pre-synaptic transmission is one of the earliest events in TDP-43-related pathogenesis. Prolonged loss and gain of TBPH in adults resulted in synaptic defects and age-related, progressive degeneration of neurons involved in motor control. Toxic gain of TBPH did not downregulate or mislocalize its own expression, indicating that a dominant-negative effect leads to progressive neurodegeneration also seen with mutational inactivation of TBPH. Together these data suggest that dysfunction of Drosophila TDP-43 triggers a cascade of events leading to loss-of-function phenotypes whereby impaired synaptic transmission results in defective motor behavior and progressive deconstruction of neuronal connections, ultimately causing age-related neurodegeneration.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Degeneração Neural/genética , Envelhecimento , Esclerose Lateral Amiotrófica/patologia , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/patologia , Larva , Degeneração Neural/metabolismo , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Transmissão Sináptica/genéticaRESUMO
Parkinson's disease (PD) is characterized by movement disorders, including bradykinesia. Analysis of inherited, juvenile PD, identified several genes linked via a common pathway to mitochondrial dysfunction. In this study, we demonstrate that the larva of the Drosophila parkin mutant faithfully models the locomotory and metabolic defects of PD and is an excellent system for investigating their inter-relationship. parkin larvae displayed a marked bradykinesia that was caused by a reduction in both the frequency of peristalsis and speed of muscle contractions. Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle. Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit. This was supported by our observations in parkin larvae that the resting potential was depolarized, oxygen consumption and ATP concentration were drastically reduced while lactate was increased. These findings suggest that neuronal mitochondrial respiration is severely compromised and there is a compensatory switch to glycolysis for energy production. parkin mutants also possessed overgrown neuromuscular synapses, indicative of oxidative stress, which could be rescued by overexpression of parkin or scavengers of reactive oxygen species (ROS). Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes. We therefore propose that mitochondrial dysfunction in parkin mutants induces Parkinsonian bradykinesia via a neuronal energy deficit and resulting synaptic failure, rather than as a consequence of downstream oxidative stress.