Giant fiber activation of flight muscles in Drosophila: asynchrony in latency of wing depressor fibers

In Drosophila, brain stimulation of the giant fiber pathway brings about highly stereotyped electrical responses in target muscles involved in the escape response. Both the order of muscle response and the latency of that response are predictable in wild-type flies. The neuronal circuit to the targets is well defined and has been used in the analysis of a number of mutant phenotypes, including induced anomalies in temperature-sensitive (ts) mutations such as shibire (shi). It has been assumed that the stereotyped response includes simultaneous activation of all six fibers of the wing depressor muscle, DLM, resulting in equal latencies for all fibers. We report here a small, but distinct, inherent difference in latency between two sets of DLM fibers in a proportion of two wild-type strains as well as in a strain carrying the ts mutation shi. This difference may occur on one or both sides of an individual, is stable over time, and persists when the motor axon is stimulated peripherally. These results, due to the circuit leading to the target, suggest that the difference in latency arises peripherally. In flies reared at the shi permissive temperature (22 degrees C), the difference is more common in shi than in wild-type flies; however, in shi flies reared at 18 degrees C, the prevalence resembles that of wild-type flies. This indicates a subtle expression of the shi defect even at the presumed permissive temperature of 22 degrees C. The difference in latency is similar to that induced in shi flies whose development is affected by pupal heat pulse. Thus, correct interpretation of differences in latency, e.g., in shi/wild-type mosaic flies or in flies with mutations affecting the GF pathway, requires recognition of the inherent asynchrony that can occur between DLM fibers.     

The effects of age on radiation resistance and oxidative stress in adult Drosophila melanogaster

Drosophila melanogaster (fruit fly) is a well-established model organism for genetic studies of development and aging. We examined the effects of lethal ionizing radiation on male and female adult Drosophila of different ages, using doses of radiation from 200 to 1500 Gy. Fifty percent lethality 2 days postirradiation (LD(50/2)) in wild-type 1-day-old adult fruit flies was approximately 1238 Gy for males and 1339 Gy for females. We observed a significant age-dependent decline in the radiation resistance of both males and females. Radiation damage is postulated to occur by the generation of oxygen radicals. An age-related decline in the ability of flies to resist an agent that induces oxygen radicals, paraquat, was observed when comparing 10- and 20-day adults. Female flies are more resistant to paraquat than male flies. Oxidative stress mediated by paraquat was additive with sublethal exposures to radiation in young adults. Therefore, the ability to repair the damage caused by oxygen radicals seems to decline with the age of the flies. Because Drosophila adults are largely post-mitotic, our data suggest that adult Drosophila melanogaster can serve as an excellent model to study the factors responsible for radiation resistance in post-mitotic tissue and age-dependent changes in this resistance.     

Relationships between the Circadian System and Alzheimer's Disease-Like Symptoms in Drosophila

Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimer''s disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, we utilized transgenic Drosophila expressing various Amyloid-β (Aβ) peptides, which are prone to form aggregates characteristic of AD pathology in humans. We compared development of AD-like symptoms in adult flies expressing Aβ peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per⁰¹). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Aβ peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. We report that rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Aβ peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Aβ peptides causes rhythm degradation downstream from the central clock mechanism.     

Dietary restriction affects lifespan but not cognitive aging in Drosophila melanogaster

Dietary restriction extends lifespan in a wide variety of animals, including Drosophila, but its relationship to functional and cognitive aging is unclear. Here, we study the effects of dietary yeast content on fly performance in an aversive learning task (association between odor and mechanical shock). Learning performance declined at old age, but 50‐day‐old dietary‐restricted flies learned as poorly as equal‐aged flies maintained on yeast‐rich diet, even though the former lived on average 9 days (14%) longer. Furthermore, at the middle age of 21 days, flies on low‐yeast diets showed poorer short‐term (5 min) memory than flies on rich diet. In contrast, dietary restriction enhanced 60‐min memory of young (5 days old) flies. Thus, while dietary restriction had complex effects on learning performance in young to middle‐aged flies, it did not attenuate aging‐related decline of aversive learning performance. These results are consistent with the hypothesis that, in Drosophila, dietary restriction reduces mortality and thus leads to lifespan extension, but does not affect the rate with which somatic damage relevant for cognitive performance accumulates with age.     

Electrophysiological correlates of rapid escape reflexes in intact earthworms,Eisenia foetida. II. Effects of food deprivation on the functional development of giant nerve fibers

Noninvasive electrophysiological recording methods were used to study the effects of prolonged food deprivation on the postembryonic patterns of giant fiber growth, as indicated by age-dependent changes in giant fiber conduction velocity and diameter, in the earthworm, Eisenia foetida. In addition, giant fiber growth was compared to patterns of somatic growth, as indicated by increases in body weight. Within a wide range of food deprivation levels, normal age-dependent increases in conduction velocity and diameter occurred in spite of marked stunting of somatic growth. Stunting of giant fiber velocity and diameter occurred only during severe food deprivation, but giant fiber spikes and associated rapid escape responses were still readily evoked. The stunting effects of prolonged and severe food deprivation upon giant fiber conduction velocity and diameter were readily reversed by replenishing food. The results demonstrate the persistence of rapid escape reflex functioning, as well as the priority of giant fiber growth relative to somatic growth, during severe and prolonged food deprivation. As a consequence of the priority of giant fiber growth during limited food availability, giant fiber conduction velocity appears to be a more reliable predictor of animal age than body size.     

Expression of human FUS protein in Drosophila leads to progressive neurodegeneration

Mutations in the Fused in sarcoma/Translated in liposarcoma gene (FUS/TLS, FUS) have been identified among patients with amyotrophic lateral sclerosis (ALS). FUS protein aggregation is a major pathological hallmark of FUS proteinopathy, a group of neurodegenerative diseases characterized by FUS-immunoreactive inclusion bodies. We prepared transgenic Drosophila expressing either the wild type (Wt) or ALS-mutant human FUS protein (hFUS) using the UAS-Gal4 system. When expressing Wt, R524S or P525L mutant FUS in photoreceptors, mushroom bodies (MBs) or motor neurons (MNs), transgenic flies show age-dependent progressive neural damages, including axonal loss in MB neurons, morphological changes and functional impairment in MNs. The transgenic flies expressing the hFUS gene recapitulate key features of FUS proteinopathy, representing the first stable animal model for this group of devastating diseases.     

Poor geotaxis correlated with haematoporphyrin‐induced peroxidation of brain lipids as a predictor of medfly longevity reduction

Under illumination conditions, porphyrins generate cytotoxic radicals in cells. Our study evaluated the effects of haematoporphyrin IX (HP IX) in a laboratory population of male Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) during exposure to a low fluence rate (39 μE m^?2 s^?1) of light. We found that exposing flies to HP IX for at least 5 days was sufficient to cause irreversible damage that led to anticipated death, as also provoked by chronic exposure to the same concentration. To identify early indicators of the accelerated senescence, we analysed both in vitro and in vivo parameters. The thiobarbituric acid reactive substances content in the heads of treated flies revealed a significant increase in lipid hydroperoxides at day 10, whereas this occurred several days later in controls. In addition, a significant decrease in glycogen content was observed at 15 days of age, 5 days before the reduction observed in the control group. This decrease has been associated with a decline in locomotor activity. Differences in the distribution of flies in the rearing flasks were observed, reflecting an impairment of the motility and climbing capacity of HP IX‐treated flies. This finding was also corroborated by a geotactic response assay (a rapid iterative negative geotaxis or RING assay). The results presented here demonstrate that low‐lethal oxidative stress can anticipate the senescence of flies, which can be predicted using a simple and fast behavioural test, such as the RING assay.     

Spermidine-triggered autophagy ameliorates memory during aging

The aging process drives the progressive deterioration of an organism and is thus subject to a complex interplay of regulatory and executing mechanisms. Our understanding of this process eventually aims at the delay and/or prevention of age-related pathologies, among them the age-dependent decrease in cognitive performance (e.g., learning and memory). Using the fruit fly Drosophila melanogaster, which combines a generally high mechanistic conservation with an efficient experimental access regarding aging and memory studies, we have recently unveiled a protective function of polyamines (including spermidine) against age-induced memory impairment (AMI). The flies’ age-dependent decline of aversive olfactory memory, an established model for AMI, can be rescued by both pharmacological treatment with spermidine and genetic modulation that increases endogenous polyamine levels. Notably, we find that this effect strictly depends on autophagy, which is remarkable in light of the fact that autophagy is considered a key regulator of aging in other contexts. Given that polyamines in general and spermidine in particular are endogenous metabolites, our findings place them as candidate target substances for AMI treatment.     

Calorie restriction delays age-dependent loss in androgen responsiveness of the rat liver

We have shown that restricted calorie intake retards age-associated loss in androgen responsiveness of the rat liver. Sustained androgen receptivity delays age-dependent decline in the synthesis of the androgen-inducible alpha 2u globulin and derepression of the androgen-repressible senescence marker protein (SMP-2). Quantitation of mRNAs for alpha 2u globulin and SMP-2 in the liver of animals of various ages maintained on either ad libitum or restricted diets revealed that, although the 27-month-old ad libitum-fed rat had only 5% as much alpha 2u mRNA as the 6-month-old rat, the mRNA level was as high as 45% in the 27-month-old food-restricted rat. Conversely, the 27-month-old food-restricted rat had a much reduced amount (45%) of SMP-2 mRNA compared to the age-matched control that was allowed unlimited access to food. Furthermore, we have correlated the effect of dietary restriction on age-dependent changes in specific gene expression with the hepatic level of the immunoreactive cytoplasmic androgen-binding (CAB) protein. We observed that senescence in the male causes a substantial decrease in the circulating level of testosterone. However, dietary restriction does not retard the rate of decline in the plasma level of the male hormone during aging. These results indicate that age-dependent changes in the expression of androgen-responsive genes (alpha 2u globulin and SMP-2) reflect changing androgen sensitivity and that food restriction may directly influence the androgen receptivity of the liver.     

Nerve-muscle interactions regulate motor terminal growth and myoblast distribution during muscle development

Interactions between motoneurons and muscles influence many aspects of neuromuscular development in all animals. These interactions can be readily investigated during adult muscle development in holometabolous insects. In this study, the development of the dorsolongitudinal flight muscle (DLM) and its innervation is investigated in the moth, Manduca sexta, to address the specificity of neuromuscular interactions. The DLM develops from an anlage containing both regressed larval template fibers and imaginal myoblasts. In the adult, each fiber bundle (DLM1-5) is innervated by a single motoneuron (MN1-MN5), with the dorsal-most fiber bundle (DLM5) innervated by a mesothoracic motoneuron (MN5). The DLM failed to develop following complete denervation because myoblasts failed to accumulate in the DLM anlage. After lesioning MN1-4, MN5 retained its specificity for the DLM5 region of the anlage and failed to rescue DLM1-4. Thus specific innervation of the DLM fiber bundles does not depend on interactions among motoneurons. Myoblast accumulation, but not myonuclear proliferation, increased around the MN5 terminals, producing a hypertrophied adult DLM5. Therefore, motoneurons compete for uncommitted myoblasts. MN5 terminals subsequently grew more rapidly over the hypertrophied DLM5 anlage, indicating that motoneuron terminal expansion is regulated by the size of the target muscle anlage.     

Destabilization of the neuromuscular junction by proteolytic cleavage of agrin results in precocious sarcopenia

Etiology and pathogenesis of sarcopenia, the progressive decline in skeletal muscle mass and strength that occurs with aging, are still poorly understood. We recently found that overexpression of the neural serine protease neurotrypsin in motoneurons resulted in the degeneration of their neuromuscular junctions (NMJ) within days. Therefore, we wondered whether neurotrypsin-dependent NMJ degeneration also affected the structure and function of the skeletal muscles. Using histological and functional analyses of neurotrypsin-overexpressing and neurotrypsin-deficient mice, we found that overexpression of neurotrypsin in motoneurons installed the full sarcopenia phenotype in young adult mice. Characteristic muscular alterations included a reduced number of muscle fibers, increased heterogeneity of fiber thickness, more centralized nuclei, fiber-type grouping, and an increased proportion of type I fibers. As in age-dependent sarcopenia, excessive fragmentation of the NMJ accompanied the muscular alterations. These results suggested the destabilization of the NMJ through proteolytic cleavage of agrin at the onset of a pathogenic pathway ending in sarcopenia. Studies of neurotrypsin-deficient and agrin-overexpressing mice revealed that old-age sarcopenia also develops without neurotrypsin and is not prevented by elevated levels of agrin. Our results define neurotrypsin- and age-dependent sarcopenia as the common final outcome of 2 etiologically distinct entities.     

Dynamics of the male germline stem cell population during aging of Drosophila melanogaster

Drosophila melanogaster has emerged as an important model system for the study of both stem cell biology and aging. Much is known about how molecular signals from the somatic niche regulate adult stem cells in the germline, and a variety of environmental factors as well as single point mutations have been shown to affect lifespan. Relatively little is known, however, about how aging affects specific populations of cells, particularly adult stem cells that may be susceptible to aging-related damage. Here we show that male germline stem cells (GSCs) are lost from the stem cell niche during aging, but are efficiently replaced to maintain overall stem cell number. We also find that the division rate of GSCs slows significantly during aging, and that this slowing correlates with a reduction in the number of somatic hub cells that contribute to the stem cell niche. Interestingly, slowing of stem cell division rate was not observed in long-lived methuselah mutant flies. We finally investigated whether two mechanisms that are thought to be used in other adult stem cell types to minimize the effects of aging were operative in this system. First, in many adult tissues stem cells exhibit markedly fewer cell cycles relative to transit-amplifying cells, presumably protecting the stem cell pool from replication-associated damage. Second, at any given time not all stem cells actively cycle, leading to 'clonal succession' from the reserve pool of initially quiescent stem cells. We find that neither of these mechanisms is used in Drosophila male GSCs.     

Acquired temperature-sensitive paralysis as a biomarker of declining neuronal function in aging Drosophila

General locomotor activity decreases with normal aging in animals and could be partially explained by decreases in neuronal function. Voltage-gated Na⁺ channels are essential in initiating and propagating rapid electrical impulses underlying normal locomotor activity and behavior in animals. Isolation of mutations conferring temperature-sensitive (ts) paralysis has been an extremely powerful paradigm for identifying genes involved in neuronal functions, such as membrane excitability and synaptic transmission. For instance, decreased expression of wild-type Na⁺ channels in flies harboring the no-action-potential ( nap ) mutant allele ( mle^napts ) confers rapid and reversible ts paralysis, because of failure of action potential propagation. Here, we report that aging wild-type Drosophila gradually develops an acquired susceptibility to ts paralysis that is indistinguishable from that seen in young ts paralytic mle^napts mutants. Moreover, we show that this general age-dependent susceptibility is also present in mle^napts flies, although the effects are shifted to lower temperature regimes. The mle^napts flies also exhibit decreased lifespan and increased frailty. Paralysis and decreased lifespan of mle^napts flies were partially rescued by increasing the dosage of para , the structural gene for the major action potential Na⁺ channel in central nervous system of Drosophila . Lastly, we show a dramatic scaling of ts paralysis susceptibility with chronological age in short-lived and long-lived mutant flies, further demonstrating that this age-dependent risk is independent of genetic background. Thus, decreased neural transmission, a hallmark of which is ts paralysis, is a biomarker of aging.     

Drosophila couch potato mutants exhibit complex neurological abnormalities including epilepsy phenotypes

RNA-binding proteins play critical roles in regulation of gene expression, and impairment can have severe phenotypic consequences on nervous system function. We report here the discovery of several complex neurological phenotypes associated with mutations of couch potato (cpo), which encodes a Drosophila RNA-binding protein. We show that mutation of cpo leads to bang-sensitive paralysis, seizure susceptibility, and synaptic transmission defects. A new cpo allele called cpo(EG1) was identified on the basis of a bang-sensitive paralytic mutant phenotype in a sensitized genetic background (sda/+). In heteroallelic combinations with other cpo alleles, cpo(EG1) shows an incompletely penetrant bang-sensitive phenotype with approximately 30% of flies becoming paralyzed. In response to electroconvulsive shock, heteroallelic combinations with cpo(EG1) exhibit seizure thresholds less than half that of wild-type flies. Finally, cpo flies display several neurocircuit abnormalities in the giant fiber (GF) system. The TTM muscles of cpo mutants exhibit long latency responses coupled with decreased following frequency. DLM muscles in cpo mutants show drastic reductions in following frequency despite exhibiting normal latency relationships. The labile sites appear to be the electrochemical GF-TTMn synapse and the chemical PSI-DLMn synapses. These complex neurological phenotypes of cpo mutants support an important role for cpo in regulating proper nervous system function, including seizure susceptibility.     

Modulation of tension production by proctolin in the dorsal longitudinal muscles of the cricket,Teleogryllus oceanicus

High-frequency electrical stimulation (～20 Hz) of the lateral nerve in abdominal segments of the cricket, Teleogryllus oceanicus, caused an increase in tonus of the abdominal dorsal longitudinal muscle (DLM). This effect persisted for 1–5 min following stimulation. Application of the pentapeptide proctolin (threshold 1–10 nM) mimicked the increase in muscle tonus produced by electrical stimulation. Individual twitches were unaffected or slightly reduced by proctolin. Low-frequency electrical stimulation (<7 Hz) of the lateral nerve counteracted a previously induced increase in muscle tonus, apparently by activation of an inhibitory motoneuron. γ-Aminobutyric acid (GABA) mimicked the effect of low-frequency stimulation and reduced muscle tonus. Octopamine, in concentrations of ≤0.1 mM, was inactive on the abdominal DLM when stimulated at low frequencies (0.5–2 Hz). Application of proctolin to the metathoracic DLM caused an increase in twitch amplitude but had little effect on basal tonus. In conjunction with the previously described responses of the metathoracic DLM to octopamine, these results show that the serially homologous abdominal and metathoracic DLMs have dissimilar responses to the modulators proctolin and octopamine.     

Stem cell aging is controlled both intrinsically and extrinsically in the Drosophila ovary

It is widely postulated that tissue aging could be, at least partially, caused by reduction of stem cell number, activity, or both. However, the mechanisms of controlling stem cell aging remain largely a mystery. Here, we use Drosophila ovarian germline stem cells (GSCs) as a model to demonstrate that age-dependent decline in the functions of stem cells and their niche contributes to overall stem cell aging. BMP signaling activity from the niche significantly decreases with age, and increasing BMP signaling can prolong GSC life span and promote their proliferation. In addition, the age-dependent E-cadherin decline in the stem cell-niche junction also contributes to stem cell aging. Finally, overexpression of SOD, an enzyme that helps eliminate free oxygen species, in either GSCs or their niche alone can prolong GSC life span and increase GSC proliferation. Therefore, this study demonstrates that stem cell aging is controlled extrinsically and intrinsically in the Drosophila ovary.     

Organelle aging:Lessons from model organisms

Most cellular processes descend into failure during aging. While a large collection of longevity pathways has been identified in the past decades, the mechanism for age-related decline of cellular homeostasis and organelle function remains largely unsolved. It is known that many organelles undergo structural and functional changes during normal aging, which significantly contributes to the decline of tissue function at old ages. Since recent studies have revealed an emerging role of organelles as regulatory hubs in maintaining cellular homeostasis, understanding of organelle aging will provide important insights into the cellular basis of organismal aging. Here we review current progress on the characterization of age-dependent structural and functional alterations in the more well-studied organelles, as well as the known mechanisms governing organelle aging in model organisms, with a special focus on the fruit fly Drosophila melanogaster.     

Rapamycin slows aging in mice

Rapamycin increases lifespan in mice, but whether this represents merely inhibition of lethal neoplastic diseases, or an overall slowing in multiple aspects of aging is currently unclear. We report here that many forms of age-dependent change, including alterations in heart, liver, adrenal glands, endometrium, and tendon, as well as age-dependent decline in spontaneous activity, occur more slowly in rapamycin-treated mice, suggesting strongly that rapamycin retards multiple aspects of aging in mice, in addition to any beneficial effects it may have on neoplastic disease. We also note, however, that mice treated with rapamycin starting at 9 months of age have significantly higher incidence of testicular degeneration and cataracts; harmful effects of this kind will guide further studies on timing, dosage, and tissue-specific actions of rapamycin relevant to the development of clinically useful inhibitors of TOR action.     

The endogenous ligand Stunted of the GPCR Methuselah extends lifespan in Drosophila

Many extracellular signals are transmitted to the interior of the cell by receptors with seven membrane-spanning helices that trigger their effects by means of heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins). These G-protein-coupled receptors (GPCRs) control various physiological functions in evolution from pheromone-induced mating in yeast to cognition in humans. The potential role of the G-protein signalling system in the control of animal ageing has been highlighted by the genetic revelation that mutation of a GPCR encoded by methuselah extends the lifespan of adult Drosophila flies. How methuselah functions in controlling ageing is not clear. A first essential step towards the understanding of methuselah function is to determine the ligands of Methuselah. Here we report the identification and characterization of two endogenous peptide ligands of Methuselah, designated Stunted A and B. Flies with mutations in the gene encoding these ligands show an increase in lifespan and resistance to oxidative stress. We conclude that the Stunted-Methuselah system is involved in the control of animal ageing.     

Synaptic proteome changes in a DNA repair deficient ercc1 mouse model of accelerated aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used Ercc1 mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged Ercc1 mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and Ercc1 mutant hippocampal neurons displayed normal outgrowth and synapse formation in vitro. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline.