InteractiveFly: GeneBrief

methuselah : Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

A screen for gene mutations that extend life-span in Drosophila melanogaster was performed in order to provide a genetic dissection of the processes involved in aging (see Drosophila as a Model for Human Diseases: Aging and Lifespan). The mutant line methuselah (mth) displays approximately 35% increase in average life-span and enhanced resistance to various forms of stress, including starvation, high temperature, and food supplemented with paraquat, a free-radical generator. The mth gene encodes a protein with homology to several guanosine triphosphate-binding protein-coupled seven-transmembrane domain receptors. Thus, the organism may use signal transduction pathways to modulate stress response and life-span (Lin, 1998).

The effect of genes on life-span in Drosophila has been established by selective breeding. However, the participation of multiple genes with additive, quantitative effects can be difficult to unravel. A direct search for life-extension mutants could identify individual genes that regulate biological aging. Indeed, in the nematode Caenorhabditis elegans, several mutations, for example, age-1, daf-2, and clk-1, have been described that can increase the worm's life-span (see Regulation of the insulin-like developmental pathway of Caenorhabditis elegans by a homolog of the PTEN tumor suppressor gene for related literature). The corresponding genes have been cloned and are involved in various aspects of development and metabolism (Lin, 1998 and references therein).

Life-span and stress response are closely associated. In C. elegans, the age-1 mutant displays elevated resistance to thermal exposure and to oxidative stress. In Drosophila, laboratory stocks selected for postponed senescence also show increased tolerance to heat, starvation, desiccation, and oxidative damage. Tandem overexpression of Cu-Zn superoxide dismutase (SOD) and catalase genes in Drosophila increase life-span by 30% (Orr, 1994). Similar observations were made in flies expressing the human SOD1 transgene in motor neurons (Parkes, 1998). However, the physiological and molecular events involved in life-span determination and stress resistance have remained largely elusive (Lin, 1998 and references therein).

A set of P-element insertion lines were generated and screened for lines that outlived a parent strain. methuselah was isolated by its increase in life-span at 29°C. The life extension was confirmed at 25°C. At both temperatures, flies homozygous for the P-element live, on the average, 35% longer than the parent strain (Lin, 1998).

The ability of mth flies to resist stress was assayed. mth mutant flies are more resistant to dietary paraquat, which, upon intake by the cell, generates superoxide anion. At a concentration of 20 mM, paraquat renders normal males sluggish by 12 hours; at 48 hours, nearly 90% are dead. In contrast, mth males are still active at 24 hours, and at 48 hours more than 50% are still alive. In a long-lived strain of Drosophila derived by selection, life-span extension also accompanies increased paraquat resistance (Arking, 1991). Transgenic Drosophila carrying extra copies of SOD and catalase, two primary components of the defense system against reactive oxygen species, also have increased life-span (Orr, 1994). Flies transgenic for the human SOD1 gene display increased life-span and paraquat resistance, the degree of effect correlating with dosage of the transgene (Parkes, 1998). Thus, mth may have a higher capacity of the free-radical defense system (Lin, 1998).

In the starvation test, mth shows a greater than 50% increase in average survival time over the parent strain. Females are consistently more resistant than males, suggesting that their larger body weight may contribute to resistance. Indeed, mth males and females weighed 20% to 30% more than their w¹¹¹⁸ counterparts. In a Drosophila stock selectively bred for postponed senescence, resistance to starvation and lipid content are higher than the baseline stock. In C. elegans, the mutant daf-2, which exhibits marked increase in longevity, has extensive fat accumulation when grown at 25°C, suggesting a coupling of its metabolism with longevity (Lin, 1998 and references therein).

Exposure to high temperature was tested. At 36°C, mth mutants survived longer than the parent strain. Heat shock proteins, a class of molecular chaperones, are thought to counter stress-induced detrimental effects during aging. In a transgenic fly harboring 12 additional copies of the heat-inducible hsp70 gene, there is a positive correlation between life expectancy and elevated Hsp70 protein expression. Correspondingly, in daf-2 and age-1 mutant worms, resistance to thermal stress is higher than in control animals. The increased thermotolerance of mth may result from higher expression of heat shock proteins and related molecular chaperones (Lin, 1998 and references therein).

Because life-span and stress response are closely related, genetic screening by stress resistance provides an effective alternative to the much slower direct screening for lifetime. The ability of the mth fly to resist various kinds of stress is notable because there are likely to exist differences in pathways of response to individual forms of stress (Lin, 1998).

G protein-coupled receptors are involved in a remarkably diverse array of biological activities including neurotransmission, hormone physiology, drug response, and transduction of stimuli such as light and odorants. The data suggest that Mth is a GPCR involved in stress response and biological aging. By regulating an associated G protein and thus its downstream pathway, the normal mth gene may maintain homeostasis and metabolism, playing a central role in modulating molecular events in response to stress. The pre-adult lethality of the null alleles demonstrates that at least some activity of the mth gene is essential for survival. When mutated, the intermediate level of expression of a hypomorphic allele might adjust response to stress in a way that is more favorable for survival, whereas full expression of the normal gene exceeds the optimum value. The delicate balance among the embryonic lethality of a null allele, enhanced longevity of a hypomorphic allele, and the normal wild phenotype, suggests that the level of mth gene expression is an important component of the system controlling life-span (Lin, 1998).

Dramatic expansion and developmental expression diversification of the methuselah gene family during recent Drosophila evolution

Functional studies of the methuselah/methuselah-like (mth/mthl) gene family have focused on the founding member mth, but little is known regarding the developmental functions of this receptor or any of its paralogs. A comprehensive analysis of developmental expression and sequence divergence in the mth/mthl gene family members was undertaken. Using in situ hybridization techniques, expression was detected of six genes (mthl1, 5, 9, 11, 13, and 14) in the embryo during gastrulation and development of the gut, heart, and lymph glands. Four receptors (mthl3, 4, 6, and 8) are expressed in the larval central nervous system, imaginal discs, or both, and two receptors (mthl10 and mth) are expressed in both embryos and larvae. Phylogenetic analysis of all mth/mthl genes in five Drosophila species, mosquito and flour beetle structured the mth/mthl family into several subclades. mthl1, 5, and 14 are present in most species, each forming a separate clade. A newly identified Drosophila mthl gene (CG31720; herein mthl15) formed another ancient clade. The remaining Drosophila receptors, including mth, are members of a large “superclade” that diversified relatively recently during dipteran evolution, in many cases within the melanogaster subgroup. Comparing the expression patterns of the mth/mthl “superclade” paralogs to the embryonic expression of the singleton ortholog in Tribolium suggests both subfunctionalization and acquisition of novel functionalities. Taken together, these findings shed novel light on mth as a young member of an adaptively evolving developmental gene family (Patel, 2012).

Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor

Animals adapt their growth rate and body size to available nutrients by a general modulation of insulin-insulin-like growth factor signaling. In Drosophila, dietary amino acids promote the release in the hemolymph of brain insulin-like peptides (Dilps), which in turn activate systemic organ growth. Dilp secretion by insulin-producing cells involves a relay through unknown cytokines produced by fat cells. This study identified Methuselah (Mth) as a secretin-incretin receptor subfamily member required in the insulin-producing cells for proper nutrient coupling. Using genetic and ex vivo organ culture experiments, it was shown that the Mth ligand Stunted is a circulating insulinotropic peptide produced by fat cells. Therefore, Sun and Mth define a new cross-organ circuitry that modulates physiological insulin levels in response to nutrients (Delanoue, 2016).

Environmental cues, such as dietary products, alter animal physiology by acting on developmental and metabolic parameters like growth, longevity, feeding, and energy storage or expenditure. The systemic action of this control suggests that intermediate sensor tissues evaluate dietary nutrients and trigger hormonal responses. Previous work in Drosophila melanogaster established that a specific organ called the fat body translates nutritional information into systemic growth-promoting signals. The leptinlike Janus kinase-signal transducers and activators of transcription (JAK-STAT) ligand unpaired 2 and the CCHamid2 peptide are produced by fat cells in response to both sugar and fat and trigger a metabolic response. Dietary amino acids activate TORC1 signaling in fat cells and induce the production of relay signals that promote the release of insulin-like peptides (Dilps) by brain insulin-producing cells (IPCs). Two fat-derived peptides (GBP1 and GBP2) activate insulin secretion in response to a protein diet, although their receptor and neural targets remain uncharacterized. To identify critical components of this organ crosstalk, a genetic screen was conducted in Drosophila larvae. The gene methuselah (mth), which encodes a heterotrimeric GTP-binding protein (G protein)-coupled receptor belonging to the subfamily of the secretin-incretin receptor subfamily came out as a strong hit. Impairing mth function in the IPCs reduces larval body growth, whereas silencing mth in a distinct set of neurons or in the larval fat body had no impact on pupal volume. Larvae in which expression of the mth gene is reduced by RNA interference (RNAi), specifically in the IPCs (hereafter, dilp2>mth-Ri), present an accumulation of Dilp2 and Dilp5 in the IPCs, whereas dilp2 gene expression remains unchanged, a phenotype previously described as impaired Dilp secretion. Indeed, forced depolarization of the IPCs rescues pupal volume and Dilp2 accumulation upon IPC-specific mth depletion. Therefore, Mth is required for Dilps secretion and larval body growth (Delanoue, 2016).

Two peptides encoded by the stunted (sun) gene, SunA and SunB, serve as bona fide ligands for Mth and activate a Mth-dependent intracellular calcium response. Silencing sun in fat cells, but no other larval tissue, of well-fed larvae mimics the mth loss-of-function phenotype with no effect on the developmental timing. Conversely, overexpression of sun in the larval fat body (lpp>sun) partially rescues the systemic growth inhibition observed upon feeding larvae a diet low in amino acids or upon 'genetic starvation' [silencing of the slimfast (slif) gene in fat cells. This growth rescue is abolished in mth¹ homozygous mutants. This shows that Sun requires Mth to control growth. However, sun overexpression has no effect in animals fed a normal diet. A modification of sun expression does not prevent fat body cells from responding to amino acid deprivation as seen by the level of TORC1 signaling, general morphology, and lipid droplet accumulation but affects the ability of larvae to resist to starvation (Delanoue, 2016).

Dilp2-containing secretion granules accumulate in the IPCs following starvation and are rapidly released upon refeeding. Mth is required in the IPCs to promote Dilp secretion after refeeding, and forced membrane depolarization of IPCs using a bacterial sodium channel (dilp2>NaChBac) is dominant over the blockade of Dilp2 secretion in dilp2>mth-Ri animals. This dominance indicates that Mth acts upstream of the secretion machinery. In addition, Dilp2 secretion after refeeding is abrogated in lpp>sun-Ri animals, and overexpression of sun in fat cells prevents Dilp2 accumulation upon starvation. Altogether, these findings indicate that Mth and its ligand Sun are two components of the systemic nutrient response controlling Dilp secretion (Delanoue, 2016).

Hemolymph from fed animals triggers Dilp2 secretion when applied to brains dissected from starved larvae. This insulinotropic activity requires the function of Mth in the IPCs and the production of Sun by fat body cells. Conversely, overexpressing sun in the fat body (lpp>sun) is sufficient to restore insulinotropic activity to the hemolymph of starved larvae. A 2-hour incubation with a synthetic peptide corresponding to the Sun isoform A (Sun-A) is also sufficient to induce Dilp secretion from starved brains. A similar effect is observed with an N-terminal fragment of Sun (N-SUN) that contains the Mth-binding domain but not with a C-terminal fragment (C-SUN) that does not bind Mth. The insulinotropic effect of N-SUN is no longer observed in brains from larvae of the mth allele, mth¹ . This absence of effect indicates that N-SUN action requires Mth in the brain. In addition, preincubation of control hemolymph with antiserum containing Sun antibodies specifically suppresses its insulinotropic function. These results indicate that Sun is both sufficient and necessary for insulinotropic activity in the hemolymph of protein-fed animals (Delanoue, 2016).

To directly quantify the amount of circulating Sun protein, Western blot experiments wee performed on hemolymph using antibodies against Sun. A 6-kD band was detected in hemolymph collected from fed larvae, and size was confirmed using Schneider 2 (S2) cell extracts. The band intensity was reduced upon sun knockdown in fat body cells but not in gut cells. Therefore, circulating Sun peptide appears to be mostly contributed by fat cells, as suggested by functional experiments. The levels of circulating Sun are strongly reduced upon starvation. In line with this, sun transcripts are drastically reduced after 4 hours of protein starvation and start increasing after 1 hour of refeeding, whereas expression of the sun homolog CG31477 is not modified. sun transcription is not affected by blocking TORC1, the main sensor for amino acids in fat body cells. However, adipose-specific TORC1 inhibition induces a dramatic reduction of circulating Sun, indicating that TORC1 signaling controls Sun peptide translation or secretion from fat cells. PGC1-Spargel is a transcription activator, the expression of which relies on nutritional input. PGC1 was found to be required for sun transcription, and fat body silencing of PGC1 and sun induce identical larval phenotypes. Although PGC1 expression is strongly suppressed upon starvation, blocking TORC1 activity in fat cells does not reduce PGC1 expression. Conversely, knocking down PGC1 does not inhibit TORC1 activity. This finding suggests that PGC1 and TORC1 act in parallel. Therefore, Sun production by fat cells in response to nutrition is controlled at two distinct levels by PGC1 and TORC1 (Delanoue, 2016).

The Sun peptide is identical to the ε subunit of the mitochondrial F₁F₀-adenosine triphosphatase (F₁F₀-ATPase) synthase (complex V). Indeed, both endogenous Sun and Sun labeled with a hemagglutinin tag (Sun-HA) colocalize with mitochondrial markers in fat cells , and the Sun peptide cofractionates with mitochondrial complex V in blue native polyacrylamide gel electrophoresis. In addition, silencing sun in fat cells decreases mitochondrial Sun staining and the amounts of adenosine triphosphate (ATP). However, recent evidence indicates that an ectopic (ecto) form of the F₁F₀-ATP synthase is found associated with the plasma membrane in mammalian and insect cells. In addition, coupling factor 6, a subunit of complex V, is found in the plasma. Therefore, Stunted could participate in two separate functions carried by distinct molecular pools. To address this possibility, a modified form of Stunted carrying a green fluorescent protein (GFP) tag at its N terminus (GFP-Sun), next to the mitochondria-targeting signal (MTS), was used. When expressed in fat cells, GFP-Sun does not localize to the mitochondria, contrarily to a Sun peptide tagged at its C-terminal end (Sun-GFP). This suggests that addition of the N-terminal tag interferes with the MTS and prevents mitochondrial transport of Sun. However, both GFP-Sun and Sun-GFP are found in the hemolymph and rescue pupal size and Dilp2 accumulation in larvae fed a low-amino acid diet as efficiently as wild-type Sun (wt-Sun) and do so in a mth-dependent manner. This indicates that the growth-promoting function of Sun requires its secretion but not its mitochondrial localization and suggests the existence of one pool of Sun peptide located in the mitochondria devoted to F₁F₀-ATP synthase activity and ATP production and another pool released in the hemolymph for coupling nutrient and growth control. In this line, although fat body levels of Sun are decreased upon starvation, its mitochondrial localization is not reduced. This finding indicates that starvation affects a nonmitochondrial pool of Sun. In support of this, starved fat bodies contain normal levels of ATP and lactate, indicating that mitochondrial oxidative phosphorylation is preserved in fat cells in poor nutrient conditions. Last, other subunits from complex V (ATP5a) or complex I (NdufS3) were not detected in circulating hemolymph. Therefore, the release of Sun in the hemolymph relies on a specific mechanism (Delanoue, 2016).

In conclusion, this study has provided evidence for a molecular cross-talk between fat cells and brain IPCs involving the ligand Stunted and its receptor Methuselah. Stunted is a moonlighting peptide present both in the mitochondria as part of the F₁F₀-ATP synthase complex and as an insulinotropic ligand circulating in the hemolymph. The mechanism of Stunted release remains to be clarified. The beta subunit of the ectopic form of F₁F₀-ATP synthase is a receptor for lipoproteins, which serve as cargos for proteins and peptides. In addition, Drosophila lipid transfer particle-containing lipoproteins were shown to act on the larval brain to control systemic insulin signaling in response to nutrition. This suggests that Sun could be loaded on lipoproteins for its transport. Given the role of insulin-insulin-like growth factor (IGF) signaling in aging, the current findings could help in understanding the role of Sun/Mth in aging adult flies (Delanoue, 2016).

The same genetic screen previously identified the fly tumor necrosis factor α Eiger (Egr) as an adipokine necessary for long-term adaptation to protein starvation, and recent work pointed to other adipose factors, illustrating the key role of the larval fat body in orchestrating nutrient response. The multiplicity of adipose factors and their possible redundancy could explain the relatively mild starvation-like phenotype obtained after removal of only one of them. Overall, these findings suggest a model whereby partially redundant fat-derived signals account for differential response to positive and negative valence of various diet components, as well as acute versus long-term adaptive responses (Delanoue, 2016).

REGULATION

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 (see Drosophila as a Model for Human Diseases: Aging and Lifespan). 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).

Protein Interactions

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. This study reports 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. It is concluded that the Stunted-Methuselah system is involved in the control of animal ageing (Cvejic, 2004).

Extension of Drosophila melanogaster life span with a GPCR peptide inhibitor

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).

The predicted binding site and dynamics of peptide inhibitors to the Methuselah GPCR from Drosophila melanogaster

Peptide inhibitors of Methuselah (Mth), a G protein-coupled receptor (GPCR), were reported that can extend the life span of Drosophila melanogaster. Mth is a class B GPCR, which is characterized by a large, N-terminal ectodomain that is often involved with ligand recognition. The crystal structure of the Mth ectodomain, which binds to the peptide inhibitors with high affinity, was previously determined. This study reports the predicted structures for RWR motif peptides in complex with the Mth ectodomain. Representatives were studied of both Pro-class and Arg-class RWR motif peptides and identified ectodomain residues Asp139, Phe130, Asp127, and Asp78 as critical in ligand binding. To validate these structures, the effects were predicted of various ligand mutations on the structure and binding to Mth. The binding of five mutant peptides to Mth was characterized experimentally by surface plasmon resonance, revealing measured affinities that are consistent with predictions. The electron density map calculated from the MD structure compares well with the experimental map of a previously determined peptide/Mth crystal structure and could be useful in refining the current low-resolution data. The elucidation of the ligand binding site may be useful in analyzing likely binding sites in other class B GPCRs (Heo, 2008).

The Drosophila G protein-coupled receptor, Methuselah, exhibits a promiscuous response to peptides

Methuselah (Mth) is a G protein-coupled receptor (GPCR) associated with longevity in Drosophila melanogaster. Previously, Stunted (Sun) was identified as a peptide agonist of Mth. This study identified two additional activators of Mth signaling: Drosophila Sex Peptide (SP) and a novel peptide (Serendipitous Peptide Activator of Mth, SPAM). Minimal functional sequences and key residues were identified from Sun and SPAM by studying truncation and alanine-scanning mutations. These peptide agonists share little sequence homology and illustrate the promiscuity of Mth for activation. mth mutants exhibit no defects in behaviors controlled by SP, casting doubt on the biological significance of Mth activation by any of these agonists, and illustrating the difficulty in applying in vitro studies to their relevance in vivo. Future studies of Mth ligands will help further understanding of the functional interaction of agonists and GPCRs (Ja, 2009).

EFFECTS OF MUTATION

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 1_B 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 1_B boutons of Drosophila NMJs are a bit larger than in 1_S 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).

Functional significance of allelic variation at methuselah, an aging gene in Drosophila

Longevity and age-specific patterns of mortality are complex traits that vary within and among taxa. Multiple candidate genes for aging have been identified in model systems by extended longevity mutant phenotypes, including the G-protein coupled receptor methuselah (mth) in Drosophila. These genes offer important insights into the mechanisms of lifespan determination and have been major targets of interest in the biology of aging. However, it is largely unknown whether these genes contribute to genetic variance for lifespan in natural populations, and consequently contribute to lifespan evolution. For a gene to contribute to genetic variance for a particular trait, it must meet two criteria: natural allelic variation and functional differences among variants. Previous work showed that mth varies significantly among wild populations; this study assessed the functional significance of wild-derived mth alleles on lifespan, fecundity and stress resistance using a quantitative complementation scheme. The results demonstrate that mth alleles segregating in nature have a functional effect on all three traits. These results suggest that allelic variation at mth contributes to observed differences in lifespan and correlated phenotypes in natural populations, and that evaluation of genetic diversity at candidate genes for aging can be a fruitful approach to identifying loci contributing to lifespan evolution (Paaby, 2008).

A mutation in Drosophila methuselah resists paraquat induced Parkinson-like phenotypes

Parkinson's disease (PD) is a prevalent and devastating neurodegenerative disorder having limited cure options and strong association with the loss of dopaminergic neurons in the substantia nigra region of the mid brain. Etiology of PD includes both genetic and environmental factors. Paraquat (PQ), a widely used herbicide, is known to be associated with pathogenesis of PD. This study reports that a mutation in Drosophila methuselah (mth1), which is associated with aging, has a role in preventing dopaminergic neuronal cell death in PQ-exposed organism. Exposed mth1 flies exhibit significant resistance against PQ-induced Parkinson's phenotypes and behavior in terms of oxidative stress, dopaminergic neuronal degeneration, locomotor performance, dopamine content, phosphorylated JNK, pFOXO, Hid, and cleaved caspase-3 levels. Conversely, over-expression of mth in dopaminergic neurons makes the exposed organism more vulnerable to oxidative stress, neuronal cell death, and behavioral deficit. The study suggests that lesser activation of JNK-mediated apoptosis in dopaminergic neurons of exposed mth1 flies protects the organism from PQ-induced damage, which may be causally linked to a common mechanism for PQ-induced neurodegeneration (Shukla, 2014).

REFERENCES

Search PubMed for articles about Drosophila methuselah

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Delanoue, R., Meschi, E., Agrawal, N., Mauri, A., Tsatskis, Y., McNeill, H. and Leopold, P. (2016). Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor. Science 353: 1553-1556. PubMed ID: 27708106

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Gimenez, L. E., Ghildyal, P., Fischer, K. E., Hu, H., Ja, W. W., Eaton, B. A., Wu, Y., Austad, S. N. and Ranjan, R. (2013). Modulation of methuselah expression targeted to Drosophila insulin-producing cells extends life and enhances oxidative stress resistance. Aging Cell 12: 121-129. Pubmed: 23121290

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Ja, W. W., Carvalho, G. B., Madrigal, M., Roberts, R. W. and Benzer, S. (2009). The Drosophila G protein-coupled receptor, Methuselah, exhibits a promiscuous response to peptides. Protein Sci 18: 2203-2208. Pubmed: 19672878

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Shukla, A. K., Pragya, P., Chaouhan, H. S., Patel, D. K., Abdin, M. Z. and Kar Chowdhuri, D. (2014). A mutation in Drosophila methuselah resists paraquat induced Parkinson-like phenotypes. Neurobiol Aging [Epub ahead of print]. PubMed ID: 24819147

Song, W., et al. (2002). Presynaptic regulation of neurotransmission in Drosophila by the G protein-coupled receptor Methuselah. Neuron 36: 105-119. 12367510

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date revised: 3 July 2014

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