α-Synuclein E46K Mutation and Involvement of Oxidative Stress in a Drosophila Model of Parkinson's Disease.

Parkinson's disease (PD) is an age-associated neurodegenerative condition in which some genetic variants are known to increase disease susceptibility on interaction with environmental factors inducing oxidative stress. Different mutations in the SNCA gene are reported as the major genetic contributors to PD. E46K mutation pathogenicity has not been investigated as intensive as other SNCA gene mutations including A30P and A53T. In this study, based on the GAL4-UAS binary genetic tool, transgenic Drosophila melanogaster flies expressing wild-type and E46K-mutated copies of the human SNCA gene were constructed. Western blotting, immunohistochemical analysis, and light and confocal microscopy of flies' brains were undertaken along with the survival rate measurement, locomotor function assay, and ethanol and paraquat (PQ) tolerance to study α-synuclein neurotoxicity. Biochemical bioassays were carried out to investigate the activity of antioxidant enzymes and alterations in levels of oxidative markers following damages induced by human α-synuclein to the neurons of the transgenic flies. Overexpression of human α-synuclein in the central nervous system of these transgenic flies led to disorganized ommatidia structures and loss of dopaminergic neurons. E46K α-synuclein caused remarkable climbing defects, reduced survivorship, higher ethanol sensitivity, and increased PQ-mediated mortality. A noticeable decline in activity of catalase and superoxide dismutase enzymes besides considerable increase in the levels of lipid peroxidation and reactive oxygen species was observed in head capsule homogenates of α-synuclein-expressing flies, which indicates obvious involvement of oxidative stress as a causal factor in SNCA E46K neurotoxicity. In all the investigations, E46K copy of the SNCA gene was found to impose more severe defects when compared to wild-type SNCA. It can be concluded that the constructed Drosophila models developed PD-like symptoms that facilitate comparative studies of molecular and cellular pathways implicated in the pathogenicity of different α-synuclein mutations.

Oxidative stress resistance as a factor in aging: evidence from an extended longevity phenotype of Drosophila melanogaster.

Longevity of a species is a multifactorial quantitative trait influenced by genetic background, sex, age and environment of the organism. Extended longevity phenotypes (ELP) from experimental evolution in the laboratory can be used as model systems to investigate the mechanisms underlying aging and senescence. ELPs of Drosophila are correlated with various life history attributes such as resistance to environmental stressors (starvation, desiccation, cold and paraquat), developmental time, biochemical defenses, etc. The association between oxidative stress resistance and longevity is not clear and ELPs offer an opportunity to examine the role of oxidative stress resistance in longevity. Here, we have investigated the hypothesis that enhanced oxidative stress resistance and elevated antioxidant defense system play a positive role in longevity using an ELP of Drosophila melanogaster. An ELP of D. melanogaster isolated and characterized in our laboratory through artificial selection (inbred laboratory strain of Oregon K) is employed in this study. Our ELP, named as long lifespan (LLS) flies, shows marked extension in lifespan when compared to the progenitor population (normal lifespan, NLS) and makes a suitable model to study the role of mitochondrial genome in longevity because of its least heterogeneity. In this study, sensitivity to ethanol with age was employed as a measure of resistance to oxidative stress in NLS and LLS flies. Effect of age and oxidative stress on longevity was examined by employing NLS and LLS flies of different age groups against ethanol-induced oxidative stress. Results show that the lower mortality against ethanol was associated with enhanced oxidative stress resistance, higher antioxidant defenses, lower reactive oxygen species (ROS) levels, enhanced alcohol dehydrogenase activity and better locomotor ability attributes of LLS flies. In addition, age-related changes like locomotor impairments, decreased antioxidant defenses, higher ROS levels and sensitivity to oxidative stress were delayed in LLS flies when compared to NLS. Our study supports the hypothesis that higher oxidative stress resistance and enhanced antioxidant defenses are significant factors in extending longevity.

Correction to: Paraquat-Induced Movement Disorder in Relation to Oxidative Stress-Mediated Neurodegeneration in the Brain of Drosophila melanogaster.

The original version of this article unfortunately contained a mistake. The entries missing in the reference list are given below and their corresponding citations are provided in the Discussion section text.

Is Longevity a Heritable Trait? Evidence for Non-genomic Influence from an Extended Longevity Phenotype of Drosophila melanogaster.

BACKGROUND: Although genetic variations are heritable, some quantitative traits like longevity may have non-genomic influence on heritability. Laboratory-selected inbred strains of extended longevity phenotype of Drosophila offer an opportunity to study the inheritance of longevity. OBJECTIVE: The aim of the study was to examine the heritability of longevity in an extended longevity phenotype of Drosophila melanogaster using reciprocal cross effects in F1 and F2 generations. METHODS: Lifespan variations of virgin and mated flies in parent, F1 and F2 generations were investigated using reciprocal crosses between normal and long lifespan lines of inbred population of D. melanogaster. Heterosis, narrow-sense heritability, recombination loss, maternal effect and overdominance with respect to survivorship in virgin and mated flies were analyzed. RESULTS: Virgin flies lived longer than mated flies. There was no significant effect of mid-parent heterosis, recombination loss and overdominance on variations in longevity, whereas, significant maternal effect and narrow-sense heritability were observed in mated and virgin flies, respectively. CONCLUSION: Absence of heterosis in our study population of Drosophila phenotypes could be due to the lack of genetic heterogeneity. The heritability of the longevity trait in an inbred extended longevity phenotype depends on the variations in genetic and environmental factors.

Paraquat-Induced Movement Disorder in Relation to Oxidative Stress-Mediated Neurodegeneration in the Brain of Drosophila melanogaster.

Exposure to pesticides like paraquat (PQ) is considered as a risk factor for Parkinson's disease (PD). PQ has been shown to induce PD-like phenotype in experimental animals. Drosophila, a valuable laboratory model organism, is widely used to study neurodegenerative disorders including PD. The acute (single dose) PQ model of PD in Drosophila is associated with high mortality as well as reversibility of locomotor deficits and, therefore, does not replicate the disease phenotype. We have investigated the relevance of the acute and multiple (sublethal) dose of PQ to induce PD-like symptoms in Drosophila and shown that multiple-dose of PQ induces irreversible locomotor impairment without significant mortality. Our study has provided ultrastructural evidence for neurodegeneration involving mitochondrial damage in the brain caused by free radical-induced oxidative stress, which leads to locomotor impairment in Drosophila. The multiple (sublethal) dose of PQ could be an appropriate Drosophila model to induce PD-like symptoms of movement disorder associated with neurodegeneration, which could be useful to evaluate neuroprotective compounds.

Sex differences in oxidative stress resistance in relation to longevity in Drosophila melanogaster.

Gender differences in lifespan and aging are known across species. Sex differences in longevity within a species can be useful to understand sex-specific aging. Drosophila melanogaster is a good model to study the problem of sex differences in longevity since females are longer lived than males. There is evidence that stress resistance influences longevity. The objective of this study was to investigate if there is a relationship between sex differences in longevity and oxidative stress resistance in D. melanogaster. We observed a progressive age-dependent decrease in the activity of SOD and catalase, major antioxidant enzymes involved in defense mechanisms against oxidative stress in parallel to the increased ROS levels over time. Longer-lived females showed lower ROS levels and higher antioxidant enzymes than males as a function of age. Using ethanol as a stressor, we have shown differential susceptibility of the sexes to ethanol wherein females exhibited higher resistance to ethanol-induced mortality and locomotor behavior compared to males. Our results show strong correlation between sex differences in oxidative stress resistance, antioxidant defenses and longevity. The study suggests that higher antioxidant defenses in females may confer resistance to oxidative stress, which could be a factor that influences sex-specific aging in D. melanogaster.

Life History Traits of an Extended Longevity Phenotype of Drosophila melanogaster.

BACKGROUND: Aging or senescence is a complex biological phenomenon. Artificially selected Drosophila for extended longevity is one of the experimental models used to understand the mechanisms involved in aging and to test various theories. OBJECTIVE: To examine the life history traits and biochemical defenses in relation to aging in an extended longevity phenotype of Drosophila melanogaster. METHODS: Life history traits viz., survivability, fecundity, development time, dry weight, wing size, lipid content, starvation, desiccation and cold resistances, locomotory ability, antioxidant enzyme activities and reactive oxygen species level between control and selected lines of D. melanogaster were investigated. RESULTS: In our model of Drosophila, extended longevity is associated with no trade-off in fecundity and shows variable resistance to environmental stress such as starvation, cold and desiccation. Enhanced biochemical defense involving the antioxidant enzymes was positively correlated with longevity. CONCLUSION: Extended longevity phenotypes of Drosophila represent genomic plasticity associated with variable life history traits attributed to the genetic background of the progenitor population and the environment of selection. Oxidative stress resistance seems to be a significant factor in longevity.

4-Hydroxyisophthalic acid from Decalepis hamiltonii rescues the neurobehavioral deficit in transgenic Drosophila model of taupathies.

Oxidative stress is one of the major etiological factors implicated in pathogenesis of neurodegenerative diseases. Since neurons are more sensitive to oxidative damage there is an increasing interest in developing novel antioxidant therapies, especially herbal preparations due to their safety profile and high efficiency. In this regard, the neuroprotective potential of a novel antioxidant compound, 4-hydroxyisophthalic acid (4-HIPA) isolated from aqueous extract of Decalepis hamiltonii roots was examined using transgenic Drosophila model of taupathy expressing wild-type and mutant forms of 2N4R isoform of human microtubule associated protein tau (MAPT). Taupathy model flies showed cognitive deficits in olfactory memory and deteriorated circadian rhythm of locomotory activities. Administration of 0.1 mg/ml 4-HIPA, markedly enhanced their olfactory memory performance and restored circadian rhythmicity of the transgenic flies locomotory behavior to the normal range. The mechanism of action that underlies 4-HIPA neuroprotection involves enhancement in efficiency of cellular antioxidant defense system by means of elevation in antioxidant enzyme activities and attenuation of oxidative stress. The molecule could positively affect the activity of neurotransmitter enzymes, which in turn enhances neuronal function and ameliorates the Tau-induced neurobehavioral deficits. Our findings showed that 4-HIPA can be considered as a suitable therapeutic candidate for drug development towards treatment of neurodegenerative disorders.

Biochemical and Behavioral Evaluation of Human MAPT Mutations in Transgenic Drosophila melanogaster.

Mutations in the human microtubule-associated protein tau (hMAPT) gene including R406W and V337M result in autosomal dominant neurodegenerative disorder. These mutations lead to hyperphosphorylation and aggregation of Tau protein which is a known genetic factor underlying development of Alzheimer's disease (AD). In the present study, transgenic Drosophila models of AD expressing wild-type and mutant forms of hMAPT exhibit a progressive neurodegeneration which was manifested in the form of early death and impairment of cognitive ability. Moreover, they were also found to have significantly decreased activity of neurotransmitter enzymes accompanied by decreased cellular endogenous antioxidant profile. The extent of neurodegeneration, memory impairment, and biochemical profiles was different in the tau transgenic strains which indicate multiple molecular and cellular responses underlie each particular form of hMAPT.

Transgenic Drosophila model to study apolipoprotein E4-induced neurodegeneration.

The ε4 isoform of apolipoprotein E (ApoE4) that is involved in neuron-glial lipid metabolism has been demonstrated as the main genetic risk factor in late-onset of Alzheimer's disease. However, the mechanism underlying ApoE4-mediated neurodegeneration remains unclear. We created a transgenic model of neurodegenerative disorder by expressing ε3 and ε4 isoforms of human ApoE in the Drosophila melanogaster. The genetic models exhibited progressive neurodegeneration, shortened lifespan and memory impairment. Genetic interaction studies between amyloid precursor protein and ApoE in axon pathology of the disease revealed that over expression of hApoE in Appl-expressing neurons of Drosophila brain causes neurodegeneration. Moreover, acute oxidative damage in the hApoE transgenic flies triggered a neuroprotective response of hApoE3 while chronic induction of oxidative damage accelerated the rate of neurodegeneration. This Drosophila model may facilitate analysis of the molecular and cellular events implicated in hApoE4 neurotoxicity.

Attenuation of neuromotor deficits by natural antioxidants of Decalepis hamiltonii in transgenic Drosophila model of Parkinson's disease.

Oxidative stress is believed to be a major factor for the onset of Parkinson's disease (PD). In this study, we have investigated oxidative status in transgenic Drosophila model of PD. Our results revealed elevated levels of reactive oxygen species (ROS) and lipid peroxidation (LPO) in A30P and A53T α-synuclein PD model flies compared to control. We have demonstrated for the first time the ameliorating potential of natural antioxidants characterized from the roots of Dh in A30P and A53T α-synuclein PD model flies. Feeding of transgenic flies with aqueous Dh root extract for 21 days significantly improved their climbing ability and circadian rhythm of locomotor activity which was associated with reduction in levels of ROS and LPO and enhancement in the activities of catalase (CAT) and superoxide dismutase (SOD). Dh protected against paraquat (PQ) sensitivity in α-synuclein transgenic flies and delayed the onset of PD-like symptoms which appears to be mediated by suppression of oxidative stress.

Modulatory effect of Decalepis hamiltonii on ethanol-induced toxicity in transgenic Drosophila model of Parkinson's disease.

Overexpression of human α-synuclein gene in Drosophila can reduce lifespan, and we have performed lifespan assay for A30P and A53Tα-synuclein transgenic and control (elav-GAL4, UAS-A30P, UAS-A53T) flies. Our results showed reduced lifespan of transgenic flies compared to controls. We have also investigated behavioral responses, levels of reactive oxygen species (ROS) and lipid peroxidation (LPO) and activities of catalase (CAT) and superoxide dismutase (SOD) in a combined genetic-toxin model (Ethanol-A30P or A53Tα-synuclein models) and controls. Our results showed that sedation time (ST50) of A30P or A53Tα-synuclein PD model flies was significantly lower while recovery time (RC50) of them was remarkably higher compared to control flies. The levels of oxidative markers (ROS and LPO) were significantly higher and the activities of CAT and SOD were lower in transgenic flies that underwent ethanol exposure compared to control. Based on our earlier studies on antioxidant properties of isolated and characterized molecules from Decalepis hamiltonii (Dh) root extract, its protective effect in this combined toxicity model has been investigated. Surprisingly, Dh treatment increased ST50 and decreased RC50 values of transgenic flies. Moreover, we showed that Dh pre-treatment could decrease the levels of ROS and LPO and increase the activities of CAT and SOD in the ethanol-α-synuclein model. This is the first report on protective effects of natural antioxidants in A30P or A53Tα-synuclein PD model flies against oxidative stress induced by ethanol.

Brain aging, memory impairment and oxidative stress: a study in Drosophila melanogaster.

Memory impairment during aging is believed to be a consequence of decline in neuronal function and increase in neurodegeneration. Accumulation of oxidative damage and reduction of antioxidant defense system play a key role in organismal aging and functional senescence. In our study, we examined the age-related memory impairment (AMI) in relation to oxidative stress using Drosophila model. We observed a decline in cognitive function in old flies with respect to both short-lived and consolidated forms of olfactory memory. Light and electron microscopy of mushroom bodies revealed a reduction in the number of synapses and discernible architectural defects in mitochondria. An increase in neuronal apoptosis in Kenyon cells was also evident in aged flies. Biochemical investigations revealed a comparable age-associated decrease in the activity of antioxidant enzymes such as catalase and superoxide dismutase as well as the GSH level, accompanied by an increase in the level of lipid peroxidation and generation of reactive oxygen species in the brain. There was no significant difference in the activity level of AChE and BChE enzymes between different age groups while immunohistochemical studies showed a significant decrease in the level of ChAT in 50-day-old flies. RNAi-mediated silencing of cat and sod1 genes caused severe memory impairment in 15-day-old flies, whereas, over-expression of cat gene could partially rescue the memory loss in the old flies. We demonstrated that a Drosophila long-lived strain, possessing enhanced activity of antioxidant enzymes and higher rate of resistance to oxidative stress, shows lower extent of AMI compared to normal lifespan strain. Present study provides evidence for involvement of oxidative stress in AMI in Drosophila.

Neuroprotective effect of Decalepis hamiltonii in paraquat-induced neurotoxicity in Drosophila melanogaster: biochemical and behavioral evidences.

In this paper, we have demonstrated for the first time, the antioxidant and neuroprotective effects of Decalepis hamiltonii (Dh) root extract against paraquat (PQ)-induced oxidative stress and neurotoxicity in Drosophila melanogaster. Exposure of adult D. melanogaster (Oregon K) to PQ induced oxidative stress as evidenced by glutathione depletion, lipid peroxidation and enhanced activities of antioxidant enzymes such as catalase, superoxide dismutase as well as elevated levels of acetylcholine esterase. Pretreatment of flies by feeding with Dh extract (0.1, 0.5 %) for 14 days boosted the activities of antioxidant enzymes and prevented the PQ-induced oxidative stress. Dietary feeding of Dh extract prior to PQ exposure showed a lower incidence of mortality and enhanced motor activities of flies in a negative geotaxis assay; both suggesting the neuroprotective potential of Dh. Based on the results, we contemplate that the roots of Dh might prevent and ameliorate the human diseases caused by oxidative stress. The neuroprotective action of Dh can be attributed to the antioxidant constituents while the precise mechanism of its action needs further investigations.

Decalepis hamiltonii root extract attenuates the age-related decline in the cognitive function in Drosophila melanogaster.

Age-associated accumulation of oxidative damage linked to decline of antioxidant defense mechanism, leads to impairment of cognitive function in many organisms. These damages can pass through generations and affect the cognitive quality of progenies. In Drosophila, classical olfactory conditioning results in the formation of different types of memory. Age-related memory impairment (AMI) causes reduction in middle term memory (MTM) and parental senescence causes decline in short-term memory (STM) of the offspring. We have further examined the neuromodulatory effect of Decalepis hamiltonii (Dh) root extract, which is a cocktail of novel antioxidant molecules, on the biochemical oxidative defenses in relation to cognitive ability of the aged flies and their offspring. There is a strong correlation between the age-related decline in the activity of the antioxidant enzymes and the lower cognitive ability of the aged flies and their offspring. Feeding of aged flies in the diet containing 0.1% Dh, markedly enhances the cognitive ability of both aged flies and their offspring which is associated with enhanced antioxidant defenses as evident for the activity of superoxide dismutase (SOD) and catalase. Our findings, for the first time, show that the antioxidant-rich Dh root extract attenuates the age-related decline in cognitive ability of Drosophila, and also shows ameliorative effect on the memory of the offspring.

Influence of mating histories and age on female remating behaviour in a few closely related species of Drosophila nasuta subgroup.

Female remating with more than one male leads to coexistence of sperm from different males in the same female, thus creating a selection pressure on sperm. To understand the extent of divergence in the reproductive behaviour among closely related species, in the present study, the influence of first mating histories like mating latency, duration of copulation and age of flies have been analysed on female remating behaviour in closely related Drosophila nasuta subgroup species with varying levels of reproductive isolation. The time taken for the once mated females to remate varied from 7 days in D. s. sulfurigaster to 19 days in D. s. neonasuta after first mating. The female remating frequency varied from a minimum of 29% in D. s. neonasuta to a maximum of 95% in D. s. sulfurigaster. The younger flies, which had remating latency of three times less than aged flies, show 100% remating frequency. In addition, it was observed that the duration of copulation in the first mating influences the remating behaviour among the nasuta subgroup members. The results revealed that D. nasuta subgroup members despite being closely related differ in their reproductive behaviour.

Male accessory gland secretory protein polymorphism in natural populations of Drosophila nasuta nasuta and Drosophila sulfurigaster neonasuta.

Male accessory gland secretory protein polymorphism was analysed in natural populations of Drosophila nasuta nasuta and D. sulfurigaster neonasuta for the first time, using SDS-PAGE to score polymorphism of these proteins in 2788 individuals of D. n. nasuta and 2232 individuals of D. s. neonasuta from 12 different populations from southern India. A total of 25 and 18 variant protein phenotypes were identified in D. n. nasuta and D. s. neonasuta, respectively. Protein fractions of group III were more polymorphic than those from groups I and II. The results show that accessory gland secretory proteins show high levels of polymorphism, irrespective of species or habitat. Moreover, we have used the variation in the accessory gland proteins to assess the extent of divergence between the species and to infer their population structure. The study suggests that though both D. n. nasuta and D. s. neonasuta belong to the same subgroup, they differ in population structure, as far as accessory gland protein polymorphism is concerned.

Male accessory gland proteins in Drosophila: a multifaceted field [corrected].

Male accessory gland in Drosophila is a secretory tissue of the reproductive system. The proteins synthesized in the accessory gland are tissue specific, stage specific-seen only during the adult stage and sex specific in the sense of male limited expression. These secretions that form a component of the seminal fluid are transferred to the female at the time of copulation and play an important role in reproduction. In conjunction with sperm, these secretory proteins assure reproductive success by reducing the female's receptivity to mating and escalating the rate of egg laying. Some of these proteins are antibacterial in nature with a likely function of protecting the female's genital tract against microbial infection during/after mating. Most of the genes involved in the synthesis of accessory gland proteins are autosomal but a few are still X-linked. Their male specific expression is achieved at the time of sex determination. The level of expression of these genes is dose dependent and they follow Mendelian pattern of segregation. Further, majority of these proteins are rapidly evolving with high rates of non-synonymous substitutions. In this review, by considering the work carried out in different fields, we have tried to generate a comprehensive picture about the male accessory gland and the role of its proteins in the reproduction of Drosophila.