methuselah
Stress and Lifespan in Drosophila
A long-lived (L) strain of Drosophila melanogaster, derived from a normal-lived (R) strain by artificial selection, has a significantly different adult longevity. The two strains age in the same manner; the major genes responsible for much of the L strain's extended longevity are located on the 3rd chromosome, and the extended longevity phenotype is significantly modulated by the larval environment. The resistance of the L and R strains to the lethal effects of dietary paraquat was examined. Within the limitations of described chromosomal and environmental manipulations, the extended longevity phenotype always accompanies the phenotype of elevated paraquat resistance. In addition, reversed selection applied to the L strain results in the simultaneous decrease of both life span and paraquat resistance. Thus, the presence or absence of the latter phenotype may be used as a bioassay for the presence or absence of the extended longevity phenotype, without any necessary implication of causality. Use of this bioassay should greatly speed up the genetic analysis of this system by allowing identification of long-lived animals at a young age. The age-related loss of elevated paraquat resistance in both strains precedes all the other age-related functional decrements that have been noted in this system (Arking, 1991).
Superoxide dismutases (SODs) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eukaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (O2-) to H2O2 and O2. Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of O2- and oxidative injury to biological structures, transgenic strains of Drosophila melanogaster overproducing CuZn SOD were generated. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin 5c gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an O2(-)-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines (Reveillaud, 1991).
Several oxidative and non-oxidative stresses were applied to two transgenic strains of Drosophila melanogaster (designated P(bSOD)5 and P(bSOD)11) that express superoxide dismutase (SOD) at elevated levels, and control strains that express normal SOD levels. Transgenic strain P(bSOD)5 exposed to paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride), a redox cycling agent that generates superoxide anion when metabolized in vivo, was significantly more resistant to this xenobiotic than control flies. When test flies were subjected to 100% oxygen for 20 min each day, the mean lifespan was 3.62 days for control strain 25, but 4.35 days for both transgenic strains. The mortality curves of strains fed 1% H2O2 were similar, but the median lifespan of 72 h for controls and 64 h for transgenics suggests that the transgenic flies were slightly more sensitive to H2O2. The activity of catalase was the same for all strains. Using starvation resistance as a non-oxidative stress, flies maintained on water without any food had identical survival curves; for all strains, the median lifespan was 72 h. Throughout the lifespan, no statistically significant difference in physical activity was displayed for transgenic versus control flies. Collectively, these data suggest that the increased lifespan previously observed in SOD transgenics is specifically related to resistance to oxidative stresses (Reveillaud, 1992).
Tests for the causal involvement of specific physiological mechanisms in the control of aging require evidence that these mechanisms can be used to increase longevity or reproductive lifespan. Selection for later reproduction in Drosophila has been shown to lead to increased longevity, as well as increased resistance to starvation and desiccation stresses. Selection for increased resistance to starvation and desiccation in Drosophila melanogaster is shown to lead to increased longevity, indicating that alleles that increase stress resistance also may increase longevity. The responses of desiccation and starvation resistance to selection are partly independent of each other, indicating a multiplicity of physiological mechanisms involved in selectively postponed aging, and thus aging in general (Rose, 1992).
The hypothesis that oxygen free radicals are causally involved in the aging process was tested by a study of the effects of simultaneous overexpression of copper-zinc superoxide dismutase and catalase. As compared to diploid controls, transgenic flies carrying three copies of each of these genes exhibit as much as a one-third extension of life-span, a longer mortality rate doubling time, a lower amount of protein oxidative damage, and a delayed loss in physical performance. Results provide direct support for the free radical hypothesis of aging (Orr, 1994).
Reactive oxygen (RO) has been identified as an important effector in ageing and lifespan determination. The specific cell types, however, in which oxidative damage acts to limit lifespan of the whole organism have not been explicitly identified. The association between mutations in the gene encoding the oxygen radical metabolizing enzyme CuZn superoxide dismutase (SOD1) and loss of motorneurons in the brain and spinal cord that occurs in the life-shortening paralytic disease, Familial Amyotrophic Lateral Sclerosis (FALS), suggests that chronic and unrepaired oxidative damage occurring specifically in motor neurons could be a critical causative factor in ageing. To test this hypothesis, transgenic Drosophila were generated which express human SOD1 specifically in adult motorneurons. Overexpression of a single gene, SOD1, in a single cell type, the motorneuron, extends normal lifespan by up to 40% and rescues the lifespan of a short-lived Sod null mutant. Elevated resistance to oxidative stress suggests that the lifespan extension observed in these flies is due to enhanced RO metabolism. These results show that SOD activity in motorneurons is an important factor in ageing and lifespan determination in Drosophila (Parkes, 1998).
Mutations in human CuZn superoxide dismutase (SOD) have been associated with familial amyotrophic lateral sclerosis (FALS). Although leading to many experimental advances, this finding has not yet led to a clear understanding of the biochemical mechanism by which mutations in SOD promote the degeneration of motorneurons that causes this incurable paralytic disease. To explore the biochemical mechanism of FALS SOD-mediated neuropathogenesis, transgenic methodology was used to target the expression of a human FALS SOD to motorneurons of Drosophila, an organism known for its phenotypic sensitivity to genetic manipulation of SOD. Targeted expression of human SOD in motorneurons of Drosophila causes a dramatic extension of adult lifespan (>40%) and rescues most of the phenotypes of SOD-null mutants. Using the same genetic system, it was asked if targeted expression of a mutant allele of human SOD that is associated with FALS causes paralysis and premature death, or is otherwise injurious in Drosophila as it is in humans and transgenic mice. High-level expression of a human FALS SOD in motorneurons is not detrimental and does not promote paralysis and premature death when expressed in motorneurons of Drosophila. In sharp contrast, the expression of FALS SOD in Drosophila actually extends lifespan, augments resistance to oxidative stress and partially rescues SOD-null mutants in a manner predicted by studies on the expression of wildtype human SOD in Drosophila motorneurons (Elia, 1999).
G protein-coupled receptors (GPCRs) mediate signaling from extracellular ligands to intracellular signal transduction proteins. Methuselah (Mth) is a class B (secretin-like) GPCR, a family typified by their large, ligand-binding, N-terminal extracellular domains. Downregulation of mth increases the life span of flies; inhibitors of Mth signaling should therefore enhance longevity. mRNA display selection was used to identify high-affinity (Kd = 15 to 30 nM) peptide ligands that bind to the N-terminal ectodomain of Mth. The selected peptides are potent antagonists of Mth signaling, and structural studies suggest that they perturb the interface between the Mth ecto- and transmembrane domains. Flies constitutively expressing a Mth antagonist peptide have a robust life span extension, which suggests that the peptides inhibit Mth signaling in vivo. This work thus provides new life span-extending ligands for a metazoan and a general approach for the design of modulators of this important class of GPCRs (Ja, 2007).
By Southern (DNA) blot analysis of mth genomic DNA, it was confirmed that mth carries a single P-element insertion in the genome. Genetic mapping indicated that the P-element is inserted in the third chromosome. By crossing mth to flies harboring a transposase, lines were generated in which the P-element was precisely excised from the insertion site (as determined by polymerase chain reaction). Eight lines obtained in this manner have life-spans reverted to that of the parent strain, indicating that the phenotype in mth is specifically caused by the P-element insertion. The precise-excision strains were used as controls throughout the study; they behave similarly to the parental strain in stress resistance as well (Lin, 1998).
Two other lines isolated had imprecise excisions of the P-element, resulting in deletion of DNA adjacent to the insertion site. Both of these lines, which likely represent null alleles of the mth gene, display pre-adult lethality in homozygotes, suggesting that the gene also plays an essential role in development. Flies heterozygous for the P-element over an imprecise excision allele are more resistant to stress than those homozygous for the P-element, indicating that the mutation created by the P-element insertion is a hypomorphic allele. The P-element insertion in the third intron of the mth gene may reduce the level of gene expression by interfering with RNA splicing, without eliminating the gene function (Lin, 1998).
Examination of the phenotypic effects of specific mutations has been extensively used to identify candidate genes affecting traits of interest. However, such analyses do not reveal anything about the evolutionary forces acting at these loci, or whether standing allelic variation contributes to phenotypic variance in natural populations. The Drosophila gene methuselah has been proposed as having major effects on organismal stress response and longevity phenotype. In this study, patterns of polymorphism and divergence at mth have been studied in population level samples of Drosophila melanogaster, D. simulans, and D. yakuba. Mth has experienced an unusually high level of adaptive amino acid divergence concentrated in the intra- and extra-cellular loop domains of the receptor protein, suggesting the historical action of positive selection on those regions of the molecule that modulate signal transduction. Further analysis of single nucleotide polymorphisms (SNPs) in D. melanogaster provided evidence for contemporary and spatially variable selection at the mth locus. In ten surveyed populations, the most common mth haplotype exhibited a 40% cline in frequency that coincided with population level differences in multiple life-history traits including lifespan. This clinal pattern was not associated with any particular SNP in the coding region, indicating that selection is operating at a closely linked site that may be involved in gene expression. Together, these consistently nonneutral patterns of inter- and intra-specific variation suggest adaptive evolution of a signal transduction pathway that may modulate lifespan in nature (Schmidt, 2000).
Comparisons between the sibling species D. melanogaster and D. simulans and an outgroup, D. yakuba, reveal that mth is one of the fastest evolving genes in Drosophila. Twenty-eight replacement fixed differences were observed between D. melanogaster and D. simulans, and approximately one in every six amino acids is different between these species and D. yakuba. This extreme rate of amino acid evolution is not the result of increased mutational pressure, because the level of silent divergence at mth between D. melanogaster and D. simulans is similar to that reported for other Drosophila genes. The mth gene is clearly under selective constraints, because the level of interspecific divergence for replacement sites remains much lower than that for synonymous sites (Schmidt, 2000).
If the elevated level of amino acid replacements at mth relative to other Drosophila genes was solely a consequence of low functional constraints, neutral theory predicts that the ratio of silent to replacement substitutions should be the same for polymorphisms within species and fixed differences between species. For the complete mth coding region, the total number of mutational events observed across all three species indicates a significant excess of replacement fixed differences. This pattern is in sharp contrast to that exhibited by other rapidly evolving Drosophila genes, for which the high level of amino acid divergence appears to result from a combination of reduced constraints and fixation of slightly deleterious variants. Given the level of silent divergence at mth between D. yakuba and D. melanogaster/D. simulans, less than 5% of silent sites would be expected to have mutated more than once (Schmidt, 2000).
To examine which portions of the mth molecule had experienced adaptive evolution, the coding region was divided into functional domains based on homology of mth to other GPCRs and Kyte-Doolittle hydropathy profiles. The large N-terminal segment of the mth protein (codons 1-219) projects outside the cell membrane and may be involved in ligand binding. Tests for this region fail to reject the null hypothesis of neutrality. The C-terminal region of the molecule (codons 220-514) comprises the more highly conserved seven transmembrane domains that characterize GPCRs and the intervening EC and IC loops. The evolutionary history of these two functionally disparate regions has been quite distinct. Whereas a neutral pattern of evolution was observed in the transmembrane domains, a significant excess of replacement fixed differences was evident in the loops for both the D. melanogaster/D. simulans comparison as well as the total number of mutational events across all three species. The EC and IC loops are responsible for the correct insertion and geometrical arrangement of the transmembrane domains in other GPCRs, and also determine the activation state of both the receptor and the coupled G-protein. Thus, the adaptive amino acid divergence appears localized to those regions of the protein that modulate signal transduction (Schmidt, 2000).
Signal transduction through GPCRs may be modulated in two primary ways: either by variation in the effective number of receptors available to the ligand or by structural modifications of various interactions between the receptor and other molecules that determine signal amplification and termination. These interactions are determined by the GPCR loop domains, which are the portions of the molecule accessible for posttranslational modification. The data suggest adaptive differentiation of the mth signal transduction pathway among Drosophila species. It remains to be seen whether other GPCR loci, in particular the various potential mth paralogs, evolve in a similar manner. Variation in the expression of mth may be a crucial determinant of the organismal response to stress and may greatly affect lifespan. In natural populations, selection on mth may involve a pleiotropic tradeoff between longevity and other fitness-associated traits that are negatively affected. These trade-offs may vary in a latitudinal fashion. The observations are consistent with this hypothesis and suggest that variation in cis-acting regulatory regions may be driving the mth cline in D. melanogaster. However, the link between existing variation at mth and genetic variance for longevity in natural populations has not been addressed. Functional studies of mth alleles identified in this study and further examination of the association between variation in mth allele frequency and longevity phenotype among natural populations should provide additional insights regarding the role of this gene in aging and senescence (Schmidt, 2000).
Regulation of synaptic strength is essential for neuronal information processing, but the molecular mechanisms that control changes in neuroexocytosis are only partially known. The putative G protein-coupled receptor Methuselah (Mth) is required in the presynaptic motor neuron to acutely upregulate neurotransmitter exocytosis at larval Drosophila NMJs. Mutations in the mth gene reduce evoked neurotransmitter release by ~50%, and decrease synaptic area and the density of docked and clustered vesicles. Pre- but not postsynaptic expression of normal Mth restores normal release in mth mutants. Conditional expression of Mth restores normal release and normal vesicle docking and clustering but not the reduced size of synaptic sites, suggesting that Mth acutely adjusts vesicle trafficking to synaptic sites (Song, 2002).
This analysis provides insights into a significant regulatory role of Mth in neurotransmitter release at excitatory glutamatergic NMJs of Drosophila. The ~50% reduction of synaptic transmission at Mth-deficient synapses could have been caused by an abnormal pre- or a postsynaptic mechanism. In mth mutants, the defect in synaptic transmission is intrinsic to evoked neurotransmitter release for two reasons: (1) the defect must be presynaptic, as indicated by normal amplitudes of unitary quantal release events (mEJPs) and normal electric properties of the muscle; (2) the defect must be downstream of action potential excitation or propagation because decreased release elicited by action potentials is similar to that observed for electrotonically elicited release (Song, 2002).
The lack of any significant gross morphological abnormalities at mth mutant NMJs and the embryonic nervous system points toward the possibility that the presynaptic defect might be due to a physiological but not to developmental abnormality. This idea was confirmed by a conditional rescue of EJP amplitudes in mth mutants 60 min after heat shock-induced expression of normal Mth protein; this experiment excludes the possibility that Mth is required within a fixed time frame during early synaptogenesis (Song, 2002).
The presynaptic defect at Mth-deficient synapses is clearly due to a loss of Mth protein activity in the presynaptic motor neuron because pre- but not postsynaptic expression of normal Mth protein at mth mutant NMJs restores normal evoked release. This rescue not only correlates the abnormal release of neurotransmitter with mutations in the mth gene but also suggests that endogenous Mth protein is normally expressed in the presynaptic motor neuron and acts cell autonomously. The rescue also refutes the possibility that Mth might signal from postsynaptic muscle and/or glia. The localization of GFP-tagged Mth in the presynaptic membrane of synaptic boutons at NMJs supports a putative localization of Mth in presynaptic terminals. However, whether Mth is localized intra- and/or extrasynaptically as well as whether Mth acts directly and/or indirectly on the synaptic machinery remains to be resolved (Song, 2002).
To define the presynaptic function of Mth, various mechanisms of neurotransmitter release were systematically examined in mth mutants. Fluo4 imaging of relative cytosolic Ca2+ levels in presynaptic boutons of mth mutant NMJs revealed normal levels of Ca2+ entry/extrusion, indirectly indicating that an impaired step of exocytosis but not of Ca2+ entry might reduce release. Since Ca2+ ionophores induce release by bypassing voltage-gated Ca2+ channels, the ~50% reduction of Ca2+ ionophore-induced release in mth mutants further supports the idea that Mth regulates a step of exocytosis. The impaired step could have been a step in the Ca2+ signaling pathway, like the Ca2+ sensor, or a step in vesicle trafficking and/or maturation. The Ca2+ sensor of vesicle fusion is apparently a major determinant for the Ca2+ cooperativity of release. Mutations in mth do not impair the Ca2+ cooperativity and are thus unlikely to impair the Ca2+ sensor of evoked release (Song, 2002).
To address a possible defect in vesicle trafficking, an ultrastructural analysis was conducted using serial sections to spatially reconstruct synapses in presynaptic type 1B boutons of larval NMJs. This analysis revealed that the size of synaptic areas and the density of associated docked (0 nm) and clustered (50–300 nm) vesicles are reduced in mth. The reduction in docked and clustered vesicles, as well as the reduction in synaptic size, correlates well with the reduction in evoked release, raising the question, which of these ultrastructural defects is causal for impaired neurotransmitter release (Song, 2002).
Three considerations suggested that the vital effects of Mth on transmitter release are likely related to vesicle docking and clustering rather than to synaptic size. (1) Changes in the density of vesicle docking and clustering may easily occur within minutes while changes in synaptic size likely occur on a slower time scale (probably hours). 2) Phorbol 12-myristate 13-acetate (PMAP signaling, which appears to be associated with Mth signaling, increases the size and the refilling of the readily releasable vesicle pool. Changes in vesicle docking are likely to affect such a pool. (3) There is no strong correlation between synaptic size and vesicular release. Synapses in type 1B boutons of Drosophila NMJs are a bit larger than in 1S boutons, but release less transmitter per synapse at low frequency (Song, 2002).
To test the above assumption, the synaptic ultrastructure of mth mutants was examined after conditional rescue of transmitter release; the defect in vesicle docking and clustering but not the defect in synaptic size is restored 60 min after heat shock induction of normal Mth protein. Consequently, normal quantal release can occur despite a reduction in synaptic size, and synaptic size is not tightly linked to transmitter release. In regard to Mth function, the conditional rescue of transmitter release together with the rescue of docked and clustered vesicle densities suggests that a smaller number of docked and/or clustered vesicles attenuates neurotransmitter release at mth mutant NMJs. Thus, Mth-mediated adjustments in the size of the docked and/or clustered vesicle pool but not the regulation of synaptic size are vital for normal transmitter release (Song, 2002).
Since a smaller number of docked and/or clustered vesicles attenuates neurotransmitter release at mth mutant NMJs, the question arose whether the reduced number of vesicles is caused by an impaired mechanism of docking and/or clustering per se, or alternatively, by a defect in vesicle recycling. A recycling defect is expected to reduce the overall number of vesicles in a synaptic bouton, which then might gradually reduce the number of docked and clustered vesicles. However, the significant decrease of docked and clustered vesicles (0–300 nm) in mth mutants is unlikely a consequence of a defect in vesicle recycling for several reasons: (1) no dramatic decrease was observed in the density of the total vesicle pool per bouton; (2) in the case of a recycling defect, the reduction of cytoplasmic vesicles is expected to be actually worse than the reduction of docked vesicles. However, in mth mutants, the opposite is the case. The density of vesicles up to 300 nm from synaptic sites is significantly reduced but not the density of vesicles further away (300–500 nm). (3) The p values for the observed vesicle densities are lowest for the density of docked vesicles and highest for vesicles furthest away. (4) No further ultrastructural abnormalities were observed that are typically seen in association with a defect in synaptic vesicle recycling at larval NMJs, including large membrane invaginations, 'coated pits,' increased numbers of cisternae, and coated vesicles. (5) Evoked release did not abnormally fatigue during prolonged repetitive stimulation at 10–30 Hz, a frequency at which the rate of endocytosis becomes rate-limiting for exocytosis. Consequently, it is suggested that Mth regulates a mechanism for docking and/or clustering synaptic vesicles at presynaptic release sites (Song, 2002).
The genetic analysis of Mth function suggests that presynaptically localized Mth acutely upregulates the number of docked and clustered vesicles at active sites, and in turn upregulates the efficiency of evoked release. This interpretation is mainly supported by the reduction of release in loss-of-function mutants, the rescue of release by presynaptic and conditional Mth expression in loss-of-function mutants, and the increase of evoked release by presynaptic and conditional expression of Mth in otherwise wild-type larvae. Since Mth is a member of the GPCR superfamily (Brody, 2000), it is likely to regulate but not mediate steps of release. But what is then the signaling cascade associated with Mth signaling (Song, 2002)?
Mth signaling was examined by testing second messenger systems that could be downstream of Mth function. DAG was an apparent candidate because phorbol esters, like phorbol 12-myristate 13-acetate (PMA), have been shown to regulate the size of a readily releasable vesicle pool, which correlates with the pool of docked vesicles. PMA-induced facilitation of release is essentially blocked at mth mutant nerve terminals in the presence of low [Ca2+]e and greatly attenuated at higher [Ca2+]es (referring to Ca2+ supplementation of the recording solution). The Ca2+-dependent block of PMA-induced facilitation at mth mutant NMJs suggests an acute requirement of Mth in a PMA/DAG-mediated signaling pathway that controls neurotransmitter exocytosis. This finding is consistent with the proposed synaptic function of Mth and the suggested synaptic function of PMA/DAG-mediated signaling, which besides other functions has been shown to control the size and refilling of a readily releasable vesicle pool. Moreover, the proposed role of Mth adjusting the density of docked and clustered vesicles at synaptic sites is consistent with the proposed effects of PMA/DAG on the readily releasable vesicle pool, which apparently correlates with the pool of docked vesicles. However, in contrast to other secretion systems, it seems unlikely that PKC has a major role in DAG-mediated facilitation of release at fly NMJs, since commonly used PKC inhibitors, like BIS and sphingosine, do not block PMA-induced facilitation (Song, 2002).
The Ca2+-dependent block of PMA-induced facilitation associates DAG signaling with Mth function but is nevertheless unconventional. Since DAG signaling occurs downstream of GPCR-mediated signaling, one might expect that PMA would counteract the defect induced by loss of Mth activity. Since PMA-induced facilitation is largely attenuated in mth, it is thus possible that a DAG signaling pathway could essentially control the activity of Mth-mediated signaling. In this scenario, DAG signaling would be located upstream of Mth activity, which is reasonable given the divergence and convergence of cross-talk between GPCR-mediated signaling pathways. However, many GPCR-mediated signaling cascades require not only physical coupling of associated G proteins, but also the clustering of the targeted downstream effector protein as, for example, in phototransduction. This opens the possibility that proper Mth signaling might require clustering of an unidentified downstream DAG-effector protein in a common protein complex (Song, 2002).
Does Mth link neurosecretion and life span? Mutations in mth raise an intriguing but perplexing relationship between excitatory neurotransmission and aging. Behaviorally, the mth1 mutation extends life span and increases stress resistance in Drosophila. Synaptically, mth1 reduces excitatory neurosecretion at larval NMJs by about half. However, the connection between the cellular deficit and the behavioral gain remains a puzzle. This study provides no evidence whether the reduction of synaptic activity in larval motor neurons has bearing on extending life span in the adult fly (Song, 2002).
In general, Mth could have multiple functions regulating neurosecretion, stress resistance, and life span by independent pathways. Alternatively, Mth might directly regulate only one or two mechanisms; the regulation of neurosecretion and/or stress resistance could then passively reflect on life span. Stress resistance in motor neurons apparently is a critical determinant of aging, as indicated by the overexpression of human Superoxide Dismutase in motor neurons, extending life span in Drosophila. In addition, there is also evidence that neurosecretion can affect life span, since hypomorphic mutations in C. elegans genes of the neurosecretory proteins unc-13, unc-64 (Syntaxin), and unc-31 (CAPS) promote longevity. Thus, impaired neurosecretion could theoretically increase the life span of mth mutants. However, expression of Mth with the neuron-specific elav promoter does not affect the increased longevity of adult mth mutant flies. Since the proper levels of Mth expression for normal life span are not known, this result neither supports nor excludes a relation between synaptic transmission and life span (Song, 2002).
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date revised: 15 October 2007
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