The InteractiveFly: Drosophila as a Model for Human Diseases



Aging and Lifespan
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Abstract
Methionine restriction extends the lifespan of various model organisms. Limiting S-adenosyl-methionine (SAM) synthesis, the first metabolic reaction of dietary methionine, extends longevity in Caenorhabditis elegans but accelerates pathology in mammals. This study shows that, as an alternative to inhibiting SAM synthesis, enhancement of SAM catabolism by glycine N-methyltransferase (Gnmt) extends the lifespan in Drosophila. Gnmt strongly buffers systemic SAM levels by producing sarcosine in either high-methionine or low-sams conditions. During ageing, systemic SAM levels in flies are increased. Gnmt is transcriptionally induced in a dFoxO-dependent manner; however, this is insufficient to suppress SAM elevation completely in old flies. Overexpression of gnmt suppresses this age-dependent SAM increase and extends longevity. Pro-longevity regimens, such as dietary restriction or reduced insulin signalling, attenuate the age-dependent SAM increase, and rely at least partially on Gnmt function to exert their lifespan-extending effect in Drosophila. The study suggests that regulation of SAM levels by Gnmt is a key component of lifespan extension (Obata and Miura, 2015).

Highlights

• Knockdown of SAM synthase shortens Drosophila lifespan.
• Gnmt is a dominant regulator of systemic SAM levels.
• Gnmt overexpression increases longevity.
• Gnmt is essential for lifespan-extending regimens.
• SAM levels increase during ageing despite Gnmt induction.
• SAM levels are maintained under pro-longevity regimens.

Discussion
Data from this study indicate that the enhancement of SAM catabolism by Gnmt is an essential component for lifespan extension. Although Gnmt is transcriptionally induced during ageing at a site downstream of dFoxO activity in the fat body, this seems to be insufficient to maintain SAM levels in aged flies. The reason behind the increase in SAM during ageing has yet to be elucidated; however, strengthening Gnmt activity attenuates the elevation of SAM and, importantly, extends longevity. Moreover, the data implies that DR and reduced IIS signalling (probably TOR and CncC as well) commonly target SAM metabolism to extend lifespan by inducing Gnmt. In humans, whether SAM levels increase in an age-dependent manner remains unknown, since only a few studies have tested this. However, one report suggests that serum SAM levels are higher in older individuals than in middle-aged individuals, at least in some populations (Obata and Miura, 2015).

It was found that sams-RNAi results in shorter lifespans. If present in excess, Met is a toxic compound in Drosophila. It is therefore possible that hypermethioninemia in sams knockdown flies, the MAT1A knockout mice and patients with MAT1A deficiency causes adverse health effects. However, whether sams1-RNAi in C. elegans results in the accumulation of methionine is unknown. Unexpectedly, loss of Gnmt function and subsequent SAM elevation did not have a negative effect on lifespan. The fact that the correlation of SAM levels and lifespan is not bidirectional implies a threshold in SAM levels that modulate organismal lifespan. One explanation is the biochemical character (for example, Km) of methyltransferases or other enzymes related to SAM-dependent metabolic pathways such as polyamine biosynthesis, methionine salvage pathway or trans-sulfuration pathway (TSP), as excess SAM does not always lead to elevated methylation or increased downstream metabolites (Obata and Miura, 2015).

The fact that Gnmt overexpression increases Drosophila lifespan suggests that decreases in SAM (and Met) and/or increase in SAM catabolites have a positive effect on longevity. For example, the acceleration of SAM catabolism by Gnmt may enhance the TSP, which will increase anti-oxidative capacity by upregulating cysteine, taurine and glutathione synthesis. In addition, TSP is critical for producing hydrogen sulfide, H2S, which is suggested to be the mediator of DR-induced benefits in both hepatic damage from ischaemia/reperfusion in mice and longevity in worms. A study in Drosophila also suggests that TSP mediates DR-induced longevity. Therefore, TSP or H2S might represent an underlying mechanism for Gnmt-dependent lifespan extension. In the sams-overexpressing flies, SAM and probably downstream metabolites were found to be increased. However, any effect on lifespan was not observed in these flies, suggesting that not only enhancing SAM catabolism but also reducing SAM under the threshold is required for lifespan extension. In contrast, Gnmt overexpression reduces SAM and simultaneously enhances the generation of SAH and downstream metabolites. Whether reduction of SAM without enhancing SAM catabolism is sufficient for lifespan extension is not known, although it is suggested by the fact that sams-RNAi in worms can extend lifespan. Since lifespan represents a total sum of both positive and negative effect of different pathways, it is difficult to pinpoint the SAM-related pathway(s) essential for lifespan control, until it is elucidated as to how each component affects lifespan (Obata and Miura, 2015).

It is also possible that SAM amount in host cells is recognized as a hallmark of nutrition availability. Thus, SAM reduction triggers the ‘fasting’ response. For example, in yeast, nutrient poor diet induces autophagy, which is inhibited by methionine at least partially through the regulation of SAM-dependent PP2A methylation by ppm1 methyltransferase. Autophagy, induced by MR, has been reported to be a direct cause of lifespan extension, suggesting that SAM reduction-induced autophagy extends longevity, although no orthologue of ppm1 is found in Drosophila. SAM-dependent transmethylation, including ribosomal RNA methylation, that affects lifespan through modulating translation is another possible connection between SAM and longevity. The exact molecular mechanisms behind the SAM effect on lifespan need to be investigated (Obata and Miura, 2015).

Ames dwarf mice are long-lived mutants that have defects in the production of growth hormone (GH) with consequent reductions in IGF-1 levels. Interestingly, Ames dwarf mice also show elevated GNMT expression and activity in addition to reduced SAM levels in their liver. Administration of GH to Ames dwarf reduces GNMT activity while GH receptor knockout mice show increased GNMT expression, indicating that GH signalling negatively regulates GNMT. Although the contribution of GNMT in longevity has not been not studied, MR does not further extend lifespan in Ames dwarf mice, suggesting that altered methionine metabolism is responsible for longevity in these animals. gnmt is one of seven genes commonly upregulated under DR (or resveratrol treatment) conditions in flies and mice, further demonstrating that the positive effects of enhanced gnmt activity on longevity in mammals is conserved (Obata and Miura, 2015).

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Post, S., Liao, S., Yamamoto, R., Veenstra, J. A., Nassel, D. R. and Tatar, M. (2018). Drosophila insulin-like peptide dilp1 increases lifespan and glucagon-like Akh expression epistatic to dilp2. Aging Cell: e12863. PubMed ID: 30511458

The Drosophila genome encodes eight insulin/IGF-like peptide (dilp) paralogs, including tandem-encoded dilp1 and dilp2. This study finds that dilp1 is highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Dilp1 and dilp2 single and double mutants were used to describe interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1-dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 - dilp2 double mutants. dilp1 and dilp2 interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. Repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro-longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. The data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro-longevity insulin-like peptide (Post, 2018).

Based on mutational analyses of the insulin receptor (daf-2, InR) and its associated adaptor proteins and signaling elements, numerous studies in C. elegans and Drosophila established that decreased insulin/IGF signaling (IIS) extends lifespan. Studies on how reduced IIS in Drosophila systemically slows aging also reveal systems of feedback where repressed IIS in peripheral tissue decreases DILP2 production in brain insulin-producing cells (IPC), which may then reinforce a stable state of longevity assurance. This study finds that expression of dilp1 is required for loss of dilp2 to extend longevity. This novel observation contrasts with conventional interpretations where reduced insulin ligand is required to slow aging: Elevated dilp1 is associated with longevity in dilp2 mutants, and transgene expression of dilp1 increases longevity (Post, 2018).

dilp1 and dilp2 are encoded in tandem, likely having arisen from a duplication event. Perhaps as a result, some aspects of dilp1 and dilp2 are regulated in common: Both are expressed in IPCs, are regulated by sNPF, and have strongly correlated responses to dietary composition. Nonetheless, the paralogs are differentially expressed throughout development. While dilp2 is expressed in larvae, dilp1 expression is elevated in the pupal stage when dilp2 expression is minimal. In reproductive adults, dilp1 expression decreases substantially after eclosion and dilp2 expression increases (Post, 2018).

Furthermore, DILP1 production is associated with adult reproductive diapause. IIS regulates adult reproductive diapause in Drosophila, a somatic state that prolongs survival during inclement seasons. DILP1 may stimulate these diapause pro-longevity pathways, while expression in nondiapause adults is sufficient to extend survival even in optimal environments (Post, 2018).

The current data suggest a hypothesis whereby dilp1 extends longevity in part through induction of adipokinetic hormone (AKH), which is also increased during reproductive diapause and acts as a functional homolog of mammalian glucagon. Critically, AKH secretion has been shown to increase Drosophila lifespan and to induce triacylglycerides and free fatty acid catabolism. Here, it is noted that dilp1 mutants were more sensitive to starvation than wild-type and dilp2 mutants, as might occur if DILP1 and AKH help mobilize nutrients during fasting and diapause. Mammalian insulin and glucagon inversely regulate glucose storage and glycogen breakdown, while insulin decreases glucagon mRNA expression. It is propose that DILP2 in Drosophila indirectly regulates AKH by repressing dilp1 expression, while DILP1 otherwise induces AKH (Post, 2018).

A further connection between dilp1 and diapause involves juvenile hormone (JH). In many insects, adult reproductive diapause and its accompanied longevity are maintained by the absence of JH. Furthermore, ablation of JH-producing cells in adult Drosophila is sufficient to extend lifespan, and JH is greatly reduced in long-lived Drosophila insulin receptor mutants. In each case, exogenous treatment of long-lived flies with a JH analog (methoprene) restores survival to the level of wild-type or nondiapause controls. JH is a terpenoid hormone that interacts with a transcriptional complex consisting of Met (methoprene tolerant), Taimen, and Kruppel homolog 1 (Kr-h1). As well, JH induces expression of kr-h1 mRNA, and this serves as a reliable proxy for functionally active JH. This study finds that dilp2 mutants have reduced kr-h1 mRNA, while the titer of this message is similar to that of wild-type in dilp1 - dilp2 double mutants. DILP1 may normally repress JH activity, as would occur in diapause when DILP1 is highly expressed. Such JH repression may contribute to longevity assurance during diapause as well as in dilp2 mutant flies maintained in laboratory conditions (Post, 2018).

Does DILP1 act as an insulin receptor agonist or inhibitor? Inhibitory DILP1 could directly interact with the insulin receptor to suppress IIS, potentially even in the presence of other insulin peptides. Such action could induce programs for longevity assurance that are associated with activated FOXO. Alternatively, DILP1 may act as a typical insulin receptor agonist that induces autophosphorylation and represses FOXO. In this case, to extend lifespan, DILP1 should stimulate cellular responses distinct from those produced by other insulin peptides such as DILP2 or DILP5. Through a third potential mechanism, DILP1 may interact with binding proteins such as IMPL2 or dALS to indirectly inhibit IIS output. These distinctions may be resolvednin a future study using synthetic DILP1 applied to cells in culture (Post, 2018).

A precedent exists from C. elegans where some insulin-like peptides are thought to function as antagonists. In genetic analyses, ins-23 and ins-18 stimulate larval diapause and longevity, while ins-1 promotes Dauer formation during development and longevity in adulthood. Moreover, C. elegans ins-6 acts through DAF-2 to suppress ins-7 expression in neuronal circuits to affect olfactory learning, where ins-7 expression inhibits DAF-2 signaling. These studies propose that additional amino acid residues of specific insulin peptides contribute to their distinct functions, and notably, the B-chain of DILP1 has an extended N-terminus relative to other DILP sequences (Post, 2018).

While dFOXO and DAF-16 are intimately associated with how reduced IIS regulates aging in Drosophila and C. elegans, in the current work, the behavior of FOXO does not correspond with how longevity is controlled epistatically by dilp1 and dilp2. Mutation of dilp2 did not impact FOXO activity, as measured by expression of target genes InR and 4eBP, and interactions with dilp1 did not modify this result. Some precedence suggests only a limited role for dfoxo as the mediator of reduced IIS in aging, as dfoxo only partially rescues longevity benefits of chico mutants, revealing that IIS extends lifespan through some FOXO-independent pathways. On the other hand, dilp1 expression from a transgene in the dilp1-2 double mutant background did induce FOXO targets. Differences among these results might arise if whole animal analysis of dFOXO targets obscures its role when IIS regulates aging through actions in specific tissues. In this vein, this study found that dilp2 controls thorax ERK signaling but not AKT, suggesting that dilp2 mutants may activate muscle-specific ERK/MAPK anti-aging programs (Post, 2018).

Dilp1 and dilp2 redundantly regulate glycogen levels and blood sugar, while these dilp loci interact synergistically to modulate dilp5 expression and starvation sensitivity. In contrast, dilp1 and dilp2 interact in a classic epistatic fashion to modulate longevity and AKH. Such distinct types of genetic interactions may reflect unique ways DILP1 and DILP2 stimulate different outcomes from their common tyrosine kinase insulin-like receptor, along with outcomes based on cell-specific responses. Understanding how and what is stimulated by DILP1 in the absence of dilp2 will likely reveal critical outputs that specify longevity assurance (Post, 2018).

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Rai, M., Coleman, Z., Curley, M., Nityanandam, A., Platt, A., Robles-Murguia, M., Jiao, J., Finkelstein, D., Wang, Y. D., Xu, B., Fan, Y. and Demontis, F. (2021). Proteasome stress in skeletal muscle mounts a long-range protective response that delays retinal and brain aging. Cell Metab. PubMed ID: 33773104

Neurodegeneration in the central nervous system (CNS) is a defining feature of organismal aging that is influenced by peripheral tissues. Clinical observations indicate that skeletal muscle influences CNS aging, but the underlying muscle-to-brain signaling remains unexplored. In Drosophila, this study found that moderate perturbation of the proteasome in skeletal muscle induces compensatory preservation of CNS proteostasis during aging. Such long-range stress signaling depends on muscle-secreted Amyrel amylase. Mimicking stress-induced Amyrel upregulation in muscle reduces age-related accumulation of poly-ubiquitinated proteins in the brain and retina via chaperones. Preservation of proteostasis stems from the disaccharide maltose, which is produced via Amyrel amylase activity. Correspondingly, RNAi for SLC45 maltose transporters reduces expression of Amyrel-induced chaperones and worsens brain proteostasis during aging. Moreover, maltose preserves proteostasis and neuronal activity in human brain organoids challenged by thermal stress. Thus, proteasome stress in skeletal muscle hinders retinal and brain aging by mounting an adaptive response via amylase/maltose (Rai, 2021).

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Banerjee, S., Vernon, S., Jiao, W., Choi, B. J., Ruchti, E., Asadzadeh, J., Burri, O., Stowers, R. S. and McCabe, B. D. (2021). Miniature neurotransmission is required to maintain Drosophila synaptic structures during ageing. Nat Commun 12(1): 4399. PubMed ID: 34285221

The decline of neuronal synapses is an established feature of ageing accompanied by the diminishment of neuronal function, and in the motor system at least, a reduction of behavioural capacity. This study has investigated Drosophila motor neuron synaptic terminals during ageing. Cumulative fragmentation of presynaptic structures was observed, accompanied by diminishment of both evoked and miniature neurotransmission occurring in tandem with reduced motor ability. Through discrete manipulation of each neurotransmission modality, it was found that miniature but not evoked neurotransmission is required to maintain presynaptic architecture and that increasing miniature events can both preserve synaptic structures and prolong motor ability during ageing. These results establish that miniature neurotransmission, formerly viewed as an epiphenomenon, is necessary for the long-term stability of synaptic connections (Banerjee, 2021).

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Shukla, A. K., Johnson, K. and Giniger, E. (2021). Common features of aging fail to occur in Drosophila raised without a bacterial microbiome. iScience 24(7): 102703. PubMed ID: 34235409

Lifespan is limited both by intrinsic decline in vigor with age and by accumulation of external insults. There exists a general picture of the deficits of aging, one that is reflected in a pattern of age-correlated changes in gene expression conserved across species. By comparing gene expression profiling of Drosophila raised either conventionally, or free of bacteria, this study shows that ∼70% of these conserved, age-associated changes in gene expression fail to occur in germ-free flies. Among the processes that fail to show time-dependent change under germ-free conditions are two aging features that are observed across phylogeny, declining expression of stress response genes and increasing expression of innate immune genes. These comprise adaptive strategies the organism uses to respond to bacteria, rather than being inevitable components of age-dependent decline. Changes in other processes are independent of the microbiome and can serve as autonomous markers of aging of the individual (Shukla, 2021).

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Hofer, S. J., Liang, Y., Zimmermann, A., Schroeder, S., Dengjel, J., Kroemer, G., Eisenberg, T., Sigrist, S. J. and Madeo, F. (2021). Lipophorin receptor 1 (LpR1) in Drosophila muscle influences life span by regulating mitochondrial aging. Biochem Biophys Res Commun 568: 95-102. PubMed ID: 34217014

Spermidine is a natural polyamine, central to cellular homeostasis and growth, that promotes macroautophagy/autophagy. The polyamine pathway is highly conserved from bacteria to mammals and spermidine (prominently found in some kinds of aged cheese, wheat germs, nuts, soybeans, and fermented products thereof, among others) is an intrinsic part of the human diet. Apart from nutrition, spermidine is available to mammalian organisms from intracellular biosynthesis and microbial production in the gut. Importantly, externally supplied spermidine (via drinking water or food) prolongs lifespan, activates autophagy, improves mitochondrial function, and refills polyamine pools that decline during aging in various tissues of model organisms, including mice. In two adjacent studies, how dietary spermidine supplementation enhances eEF5/EIF5A hypusination, cerebral mitochondrial function and cognition in aging Drosophila melanogaster and mice was explored (Hofer, 2021).

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Kim, A. K., Kwon, D. W., Yeom, E., Lee, K. P., Kwon, K. S., Yu, K. and Lee, K. S. (2021). Lipophorin receptor 1 (LpR1) in Drosophila muscle influences life span by regulating mitochondrial aging. Biochem Biophys Res Commun 568: 95-102. PubMed ID: 34217014

Sarcopenia is a syndrome characterized by progressive loss of muscle mass and function during aging. Although mitochondrial dysfunction and related metabolic defects precede age-related changes in muscle, their contributions to muscle aging are still not well known. This study used a Drosophila model to investigate the role of lipophorin receptors (LpRs), a Drosophila homologue of the mammalian very low-density lipoprotein receptor (VLDLR), in mitochondrial dynamics and muscle aging. Muscle-specific knockdown of LpR1 or LpR2 resulted in mitochondrial dysfunction and reduced proteostasis, which contributed to muscle aging. Activation of AMP-activated protein kinase (AMPK) ameliorated muscle dysfunction induced by LpR1 knockdown. These results suggest that LpR1/VLDLR is a novel key target that modulates age-dependent lipid remodeling and muscle homeostasis (Kim, 2021).

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Tatar, M. (2021). Aging Regulated Through a Stability Model of Insulin/Insulin Growth Factor Receptor Function. Front Endocrinol (Lausanne) 12: 649880. PubMed ID: 33776941

Mutations of the insulin-like receptor in Drosophila extend lifespan. New research suggests this receptor operates in two modes. The first extends lifespan while slowing reproduction and reducing growth. The second strongly extends lifespan without impairing growth or reproduction; it confers longevity assurance. The mutation that confers longevity assurance resides in the kinase insert domain, which contains a potential SH2 binding site for substrate proteins. A recent model for the function of receptor tyrosine kinases was applied to propose how insulin receptor structure can modulate aging. This concept hypothesizes that strong insulin-like ligands promote phosphorylation of high threshold substrate binding sites to robustly induce reproduction, which impairs survival as a consequence of trade-offs. Lower levels of receptor stimulation provide less kinase dimer stability, which reduces reproduction and extends lifespan by avoiding reproductive costs. Environmental conditions that favor diapause alter the expression of insulin ligands to further repress the stability of the interacting kinase domains, block phosphorylation of low threshold substrates and thus induce a unique molecular program that confers longevity assurance. Mutations of the insulin receptor that block low-phosphorylation site interactions, such as within the kinase insert domain, can extend lifespan while maintaining overall dimer stability. These flies are long-lived while maintaining reproduction and growth. The kinase insert domain of Drosophila provides a novel avenue from which to seek signaling of the insulin/insulin-like growth factor system of humans that modulate aging without impacting reproduction and growth, or incurring insulin resistance pathology (Tatar, 2021).

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Banerjee, S., Vernon, S., Jiao, W., Choi, B. J., Ruchti, E., Asadzadeh, J., Burri, O., Stowers, R. S. and McCabe, B. D.(2021). Miniature neurotransmission is required to maintain Drosophila synaptic structures during ageing. Nat Commun 12(1): 4399. PubMed ID: 34285221

The decline of neuronal synapses is an established feature of ageing accompanied by the diminishment of neuronal function, and in the motor system at least, a reduction of behavioural capacity. This study has investigated Drosophila motor neuron synaptic terminals during ageing. Cumulative fragmentation of presynaptic structures was observed accompanied by diminishment of both evoked and miniature neurotransmission occurring in tandem with reduced motor ability. Through discrete manipulation of each neurotransmission modality, it was found that miniature but not evoked neurotransmission is required to maintain presynaptic architecture and that increasing miniature events can both preserve synaptic structures and prolong motor ability during ageing. These results establish that miniature neurotransmission, formerly viewed as an epiphenomenon, is necessary for the long-term stability of synaptic connections (Banerjee, 2021).

Deterioration of both central and peripheral synaptic structures and alterations of neurotransmission is a conserved feature of ageing in both rodents and humans. Consistently, using a preparation that was developed to examine adult synapses in Drosophila, this study also observe age-dependent reductions of synaptic structure and neurotransmitter release. Interrogating this system, it was found that age-dependent changes in vesicular neurotransmission, rather than being a consequence of alterations in synaptic structure, may instead precede and promote the decline of synaptic architecture. This finding is surprising and contrary to the generally assumed sequence of age-dependent synapse degeneration events. Moreover, this study established that miniature neurotransmitter release is the key constituent of vesicular neurotransmission that is singularly required to maintain synaptic structures as they age (Banerjee, 2021).

Since their discovery over 60 years ago, miniature events have been observed at every chemical synapse studied, but were often dismissed as 'noise' produced as a by-product of high fidelity evoked neurotransmission. The supposition that miniature neurotransmission is an epiphenomenon has been maintained in part by the difficulty to discretely attenuate presynaptic miniature release in vivo without also simultaneously perturbing evoked neurotransmission. By employing precise amino acid mutants of neurotransmission proteins derived from the intensive investigation of the mechanisms of vesicular release, in combination with adult-specific genetic manipulations, this study has revealed an essential and exclusive function for miniature events to maintain the structural integrity of synaptic terminals. Reducing miniature events results in premature disintegration of synaptic terminals in young animals while increasing these events in older animals can not only preserve terminal morphology, but in addition retard the age-dependent decline of motor ability. Simultaneous inhibition of evoked release does not either further increase terminal degeneration when miniature neurotransmission is inhibited or limit the benefits to terminal structural integrity when miniature events are increased during ageing, indicating a unique and singular requirement for miniature events. It is speculated that miniature release may be uniquely suited for processes necessary for the long-term stability of synapses as these events are produced continuously, in contrast to the stochastic and intermittent nature of evoked release. The finding of the necessity of miniature neurotransmission for the long-term structural stability of synapses extends upon the ongoing re-evaluation of the functional importance of miniature events, in particular for synapse and circuit development (Banerjee, 2021).

While the data support a unique role for miniature neurotransmission in maintaining synaptic structural integrity, it remains to be determined how small amplitude miniature events can be discriminated from much larger evoked release to elicit their unique properties. In developing synaptic terminals, it was previously shown that the effects of postsynaptic depletion of miniature neurotransmission on presynaptic bouton maturation required the ionotropic activity of glutamate receptors. Miniature events could potentially activate spatially distinct subpopulations of postsynaptic receptors to those activated by evoked neurotransmitter release to trigger differentiating signalling cascades. In support of this, postsynaptic functional imaging with active zone resolution of Drosophila larval synaptic terminals has shown that the probability of synaptic vesicle release during evoked or miniature neurotransmission can be spatially segregated to distinct active zones. Similar observations have been made in mammalian neurons. Another possibility is that differences in the release kinetics between evoked and miniature neurotransmission could allow postsynaptic mechanisms to detect and discriminate miniature events. For example, differential activation of Calmodulin can distinguish between local or global Ca2+ signaling acting through voltage-gated Ca2+ channels. The ability to selectively inhibit either miniature or evoked neurotransmission in the presynapse, as described in this study, should enable further interrogation of these potential mechanisms (Banerjee, 2021).

Also described in this study is a Drosophila abdominal neuromuscular synapse preparation suitable to investigate mature adult synapses throughout their lifespan. Similar to intensively studied larval abdominal neuromuscular synapses, adult A2 musculi ventralis interni mediales (mvim) abdominal muscles also have both tonic and phasic innervation, though only the tonic terminal producing motor neurons expresses the HB9 transcription factor. The restriction of HB9 (and HB9 > Gal4) expression to a small subset of adult motor neurons, enables experiments, such as inhibition of neurotransmission throughout ageing as was carried out in this study, without compromising overall animal behaviour or lifespan. A voltage clamp electrophysiological approach was developed for this muscle enabling direct measurement of synaptic currents and thus accurate quantification of neurotransmission properties, particularly important during ageing where potential confounding alterations of membrane potential and electrical resistance have been documented. In this preparation, no age-dependent increase was found in quantal content or 'set-point' as has been described at Drosophila proboscis NMJ terminals. Rather a progressive age-dependent decrease was observed in evoked neurotransmission (in addition to miniature event frequency), quantal content and a reduction in the size of the ready realisable pool of synaptic vesicles. These observations at this terminal are consistent with the age-dependent decline of neurotransmission described in other Drosophila, invertebrate and vertebrate synapses (Banerjee, 2021).

As this study described in Drosophila and as others have observed in mammals, miniature events decline as animals age. Alterations of miniature neurotransmission have also been reported in the context of several neurodevelopmental, neurodegenerative and psychiatric diseases. The results suggest that, in addition to age-dependent synaptic decline, alterations of miniature events should be investigated further for a potential causal role in synaptic structural changes associated with brain disorders (Banerjee, 2021).

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Erez, N., Israitel, L., Bitman-Lotan, E., Wong, W. H., Raz, G., Cornelio-Parra, D. V., Danial, S., Flint Brodsly, N., Belova, E., Maksimenko, O., Georgiev, P., Druley, T., Mohan, R. D. and Orian, A. (2021). A Non-stop identity complex (NIC) supervises enterocyte identity and protects from premature aging. Elife 10. PubMed ID: 33629655

A hallmark of aging is loss of differentiated cell identity. Aged Drosophila midgut differentiated enterocytes (ECs) lose their identity, impairing tissue homeostasis. To discover identity regulators, an RNAi screen targeting ubiquitin-related genes was performed in ECs. Seventeen genes were identified, including the deubiquitinase Non-stop (CG4166). Lineage tracing established that acute loss of Non-stop in young ECs phenocopies aged ECs at cellular and tissue levels. Proteomic analysis unveiled that Non-stop maintains identity as part of a Non-stop identity complex (NIC) containing E(y)2, Sgf11, Cp190, (Mod) mdg4, and Nup98. Non-stop ensured chromatin accessibility, maintaining the EC-gene signature, and protected NIC subunit stability. Upon aging, the levels of Non-stop and NIC subunits declined, distorting the unique organization of the EC nucleus. Maintaining youthful levels of Non-stop in wildtype aged ECs safeguards NIC subunits, nuclear organization, and suppressed aging phenotypes. Thus, Non-stop and NIC, supervise EC identity and protects from premature aging (Erez, 2021).

Differentiated cell states are actively established and maintained through action of 'identity supervisors'. Identity supervisors safeguard the expression of genes that enable differentiated cells to respond to environmental cues and perform required physiological tasks. Concomitantly, they ensure silencing/repression of previous fate and non-relevant gene programs and reduce transcriptional noise. Inability to safeguard cell identity is a hallmark of aging and results in diseases such as neurodegeneration, diabetes, and cancer. In many cases, transcription factors (TFs) together with chromatin regulators and architectural/scaffold proteins establish and maintain large-scale chromatin and nuclear organization that is unique to the differentiated state of the cell (Erez, 2021).

In adult Drosophila midgut epithelia, the transcription factor Hey [Hairy/E(spl)-related with YRPW motif], together with Drosophila nuclear type A lamin, Lamin C (LamC), co-supervise identity of fully differentiated enterocytes (ECs). Highly similar to the vertebrate gut, Drosophila midgut epithelia intestinal stem cells (ISC) either self-renew or differentiate into progenitor cells that mature into enteroendocrine cells (EEs) or give rise to enteroblast (EB) progenitors. EBs mature into fully polyploid differentiated enterocytes (ECs) that carry out many critical physiological tasks of the intestine. Aging affects the entire midgut, and is associated with loss of EC identity, mis-differentiation of progenitors, pathological activation of the immune system, and loss of the physiological properties of the gut and its integrity. It also results in loss of intestinal compartmentalization, and microbiota-dysbiosis, all leading to reduced lifespan. During aging, the protein levels of identity supervisors such as Hey and LamC decline, resulting in inability to maintain EC-gene programs and ectopic expression of previous- and non-relevant gene programs. Indeed, continuous expression of Hey in aged ECs restores and protects EC identity, gut integrity, and tissue homeostasis (Flint Brodsly, 2019; Erez, 2021 and references therein).

Regulation of EC identity requires signaling to the nucleus to communicate physiological changes in the gut environment. An important mechanism involves changes in post-transcriptional modifications (PTMs) which may propagate, amplify, or conduct signals, ultimately leading to differential gene regulation. One type of PTM is the covalent attachment of ubiquitin or ubiquitin-like (Ub/UbL) molecules, that affect protein stability, function, localization, as well as modulatex chromatin structure. Recent works suggest an intimate link between ubiquitin, proteostasis, and aging. A RNAi screen was performed in this study to search for Ub/UbL-related genes within ECs that supervise identity. Screening of 362 genes, 17 were identified whose conditional elimination in fully differentiated ECs resulted in loss of EC identity. Further analysis revealed one of them, the deubiquitinating isopeptidase (DUB) Non-stop (Non-stop/dUSP22) is a key EC identity supervisor. Purification and proteomic analysis identified Non-stop as part of a CP190/Nup98/Sgf11/e(y)2/mdg4 protein complex, termed Non-stop identity complex (NIC), that is essential for maintenance of EC identity. In part, Non-stop protects NIC proteins from age-dependent decline, safeguarding the EC-gene expression signature, as well as large-scale nuclear organization in these cells, preventing premature aging. Over lifespan, Non-stop protein levels in ECs declined, leading to loss of NIC subunits. This decline is associated with loss of gut identity and physiology at the cellular and tissue levels. Maintaining youthful levels of Non-stop prevented loss of the NIC and prevented aging of the gut (Erez, 2021).

Based on cell-specific secondary tests, this study identified three categories of supervisors; 1. EC-specific identity regulators 2. Genes that are required for differentiated cell identity (both EEs and ECs) 3. Genes that are required for identity of all cell types (general identity regulators). Of specific interest were a group of genes (CG1490; CG2926; CG4080) whose elimination in EEs resulted in a loss of EC, but not EE, identity, acting as inductive identity regulators. Likely their effect on ECs involves cell~cell communication via diffusible factors. While loss of each individual gene identified in the screen resulted in loss of EC identity, the molecular and genetic connections between different identity regulators requires further studies (Erez, 2021).

The observation that Ub/UbL-related genes protect the differentiated identity is conserved across species. Screens in mammalian systems identified enzymes within the SUMO and ubiquitin pathways acting as a barrier against forced reprogramming of differentiated cell. Among these genes were Ubc9 (Drosophila Lesswright), the sole SUMO conjugating enzyme, that was also identified in this screen and the isopeptidase Psmd14. In addition to Non-stop, the screen identified the iso-isopeptidase UTO6-like (CG7857), Usp7, and Rpn11 as regulators of EC identity. Rpn11 is part of the lid particle of the 26S proteasome, involved in deubiquitinating proteins undergoing proteasomal degradation (Erez, 2021).

Genes regulating identity are likely to serve as a barrier to tumorigenesis having a tumor suppressive function. The human ortholog of Non-stop, USP22 has mixed oncogenic and tumor-suppressive functions. Relevant to the current study is the observation that USP22 has tumor suppressive functions in colon cancer by reducing mTor activity. Along this line it is interesting to note that 10/17 of human orthologs to genes discovered in the screen are either mutated or silenced in cancer. Thus, future studies of these human orthologs may identify potent tumor suppressors in cancer (Erez, 2021).

The proteomic, biochemical, and genetic analyses established that to maintain identity Non-stop acts as part of the NIC protein complex. Like SAGA, NIC contained the entire DUB module including Non-stop, E(y)two and Sgf11. Within SAGA, Sgf11 is critical for Non-stop activity. However, loss of Sgf11 did not result in loss of EC identity. While this may reflect intrinsic differences between the complexes it may also be possible that knock-down of Sgf11 transcripts in ECs did not reduce Sgf11 protein levels sufficiently enough to result in a phenotype (Erez, 2021).

The BTB domain of Mod(mdg4) interacts with the M domain of Cp190, and together they enable the recruitment of NIC to the promoter/enhancer regions. There are more than 30 Mod(mdg4) isoforms with different C-ends, each of which interacts with different DNA-binding TF. Cp190 can also be able to recruit different TFs and interacts with Chromo and Zinc-finger 4, that are co-localized with Nup98-96. The Mod(mdg4)/CP190 sub-complex is bound to promoters through interactions with many promoter specific C2H2 proteins including dCTCF, Su(Hw), and possibly via other TFs enriched in the ATAC-seq analysis (Erez, 2021).

Prominent phenotypes of loss of Non-stop were the reduced protein level of Nup98 and its miss-localization from the nuclear periphery to an intranuclear punctate distribution. Nup98 was shown to recruit Set1~COMPASS to enhance histone H3K4me2-3 methylations in hematopoietic progenitors. Thus, a possible function of the NIC may be the recruitment of the H3K4me2/3 COMPASS methylases to catalyze H3K9 di- and tri-methylation at enhancers and promotors, which are fundamental for gene activation. Members of the NIC complex were previously shown to form a Nuclear pore complex that enhances transcriptional memory upon exposure to ecdysone, raising the possibility that NIC is required for enhancing transcriptional memory promoting the transcription of EC-related genes and prevents Polycomb-dependent transcriptional repression (Erez, 2021).

In addition to NIC subunits the complex described by Pascual-Garcia (2017) contained Trithorax-like protein (Trl)/GAF and the boundary/architectural proteins CTCF, Suppressor of Hairy Wing (SuHw) and Mtor, but not Non-stop or E(y)2. Interestingly, the DNA binding site of Trl/GAF/ was enriched in EC genes that their expression required Non-stop for maintaining chromatin accessibility. However, Trl, CTCF, SuHw, or Mtor that interact with Cp190 were not detected as Non-stop bound proteins in the proteomic analysis, and RNAi-mediated elimination of Trl/GAF, or SuHw from ECs did not result in loss of EC identity. Thus, suggesting that these are likely separate regulatory complexes with shared subunits (Erez, 2021).

Non-stop was found to be required for the stability of NIC in ECs. RNA-seq analysis established that Non-stop did not regulate the mRNA level of NIC subunits. Suggesting that Nonstop acts by deubiquitinating NIC subunits in ECs. In this regard, it is possible that the stabilization of NIC subunits may also require the EC-related lamin, LamC. It was noticed that Lamin C protein levels (but not mRNA) decline upon loss of Non-stop. Moreover, LamC expression partially restored the protein levels of NIC subunits and their intranuclear localization, potentially by serving as a scaffold for NIC at the nuclear periphery. Therefore, additional experiments are required to establish whether NIC subunits are directly deubiquitinated and stabilized by Non-stop iso-peptidase activity and the potential contribution of LamC to stabilization of NIC subunits (Erez, 2021).

Thus, Non-stop may function at two levels; One is a direct role in transcription safeguarding chromatin accessibility at EC genes, while a second function is the stabilization of identity supervisors including NIC subunits and LamC (Erez, 2021).

Both Non-stop and the transcription factor Hey are bona-fide regulators of EC identity required for the expression of EC-related genes. A significant number of EC-related genes required both Non-stop and Hey for their expression, suggesting that Hey and Non-stop may co-regulate these genes. However, functional and epistatic tests suggest that Hey also acts upstream or in additional pathways to Non-stop. Hey binds to enhancers in lamin genes repressing the expression of the ISC-related lamin, LamDm0 and enhances the expression of LamC. In contrast, Non-stop does not regulate the accessibility, or expression of either LamDm0 or LamC at mRNA level. However, Non-stop is required for maintaining the levels of LamC protein. Therefore, loss of Non-stop results in a decline in LamC but not in the ectopic expression of LamDm0, which is observed upon acute loss of Hey or aging. This discrepancy may be due to the presence of Hey on its repressed targets in young ECs where Non-stop is targeted, and directly repressing their expression as maybe in the case of LamDm0. Moreover, EC-specific expression of Non-stop did not suppress the phenotypes associated with acute loss of Hey in young ECs further supporting for Hey-dependent, but Non-stop independent functions. However, the ECs-specific expression of either Non-stop or Hey in aging midguts restores expression of LamC and repressed ectopic LamDm0 expression (Erez, 2021).

Changes in large-scale nuclear organization are hallmarks of aging. Expression of identity supervisors can prevent age-related distortion of the nucleus EC identity and protect overall the epithelial tissue). However, to accomplish this, Non-stop or Hey were continuously expressed in ECs and temporal expression of Hey or Non-stop in already aged ECs was not sufficient to suppress aging phenotypes. Thus, if the levels of identity supervisors are kept at youthful levels, they can continue to maintain cell identity and prevent signs of aging, effectively keeping the gut organization and structure similar to young tissue (Erez, 2021).

Furthermore, it is not clear how expression of a single regulator like Non-stop has an extensive impact on the entire nucleus. Recent studies suggest that Non-stop functions in additional multiprotein complexes that may regulate large-scale cellular organization. For example, Non-stop is part of an Arp2/3 and WAVE regulatory (WRC) actin-cytoskeleton organization complex where it deubiquitinates the subunit SCAR. In this regard, a nuclear actin organizing complex, WASH, interacted with nuclear Lamin and was required for large scale nuclear organization. Thus, it is tempting to suggest that such complexes are required to maintain cell identity, and that subunits within these complexes are deubiquitinated by Non-stop (Erez, 2021).

In this regard many nuclear proteins are extremely long-lived proteins (LLPs) among them are nuclear pore complex proteins (NPCs) and core histones. The extended stability of LLPs may originate from intrinsic properties of LLPs, or due to sequestration and evading degradation. However, increased stability may be also actively maintained by constitutive de-ubiquitination. Indeed, post-translational modification by ubiquitin and SUMO were shown to regulate lamin stability and their intranuclear localization. Specifically, type-A lamin and its splice variant Progerin, the cause of Hutchinson Gilford progeria syndrome (HGPS), a premature aging syndrome, are degraded by the HECT-type E3 ligase Smurf2 via ubiquitin-dependent autophagy. The elimination of Progerin by expression of Smurf2 in HGSP-fibroblasts reduced the deformation observed in these cells. Thus, it is possible that enhancing Progerin degradation by inhibiting the human ortholog of Non-stop, USP22, will restore nuclear architecture, and suppress the premature aging phenotypes observed in HGPS cells (Erez, 2021).

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Semaniuk, U. V., Gospodaryov, D. V., Strilbytska, O. M., Kucharska, A. Z., Sokol-Lętowska, A., Burdyliuk, N. I., Storey, K. B., Bayliak, M. M. and Lushchak, O. (2022). Chili-supplemented food decreases glutathione-S-transferase activity in Drosophila melanogaster females without a change in other parameters of antioxidant system. Redox Rep 27(1): 221-229. PubMed ID: 36200601
Summary:

Many plant-derived anti-aging preparations influence antioxidant defense system. Consumption of food supplemented with chili pepper powder was found to extend lifespan in the fruit fly, Drosophila melanogaster. The present study aimed to test a connection between life-extending effect of chili powder and antioxidant defense system of D. melanogaster. Flies were reared for 15 days in the mortality cages on food with 0% (control), 0.04%, 0.12%, 0.4%, or 3% chili powder. Antioxidant and related enzymes, as well as oxidative stress indices were measured. Female flies that consumed chili-supplemented food had a 40-60% lower glutathione-S-transferase (GST) activity as compared with the control cohort. Activity of superoxide dismutase (SOD) was about 37% higher in males that consumed food with 3% chili powder in comparison with the control cohort. Many of the parameters studied were sex-dependent. It is concluded that consumption of chili-supplemented food extends lifespan in fruit fly cohorts in a concentration- and gender-dependent manner. However, this extension is not mediated by a strengthening of antioxidant defenses. Consumption of chili-supplemented food does not change the specific relationship between antioxidant and related enzymes in D. melanogaster, and does not change the linkage of the activities of these enzymes to fly gender (Semaniuk, 2022).

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Harrison, B. R., Hoffman, J. M., Samuelson, A., Raftery, D. and Promislow, D. E. L. (2021). Modular Evolution of the Drosophila Metabolome. Mol Biol Evol. PubMed ID: 34662414

Comparative phylogenetic studies offer a powerful approach to study the evolution of complex traits. While much effort has been devoted to the evolution of the genome and to organismal phenotypes, until now relatively little work has been done on the evolution of the metabolome, despite the fact that it is composed of the basic structural and functional building blocks of all organisms. This study explored variation in metabolite levels across 50 million years of evolution in the genus Drosophila, employing a common garden design to measure the metabolome within and among 11 species of Drosophila. This study found that both sex and age have dramatic and evolutionarily conserved effects on the metabolome. This study also found substantial evidence that many metabolite pairs covary after phylogenetic correction, and that such metabolome coevolution is modular. Some of these modules are enriched for specific biochemical pathways and show different evolutionary trajectories, with some showing signs of stabilizing selection. Both observations suggest that functional relationships may ultimately cause such modularity. These coevolutionary patterns also differ between sexes and are affected by age. This study also explored the relevance of modular evolution to fitness by associating modules with lifespan variation measured in the same common garden. Several modules associated with lifespan were found, particularly in the metabolome of older flies. Oxaloacetate levels in older females appear to coevolve with lifespan, and a lifespan-associated module in older females suggests that metabolic associations could underlie 50 million years of lifespan evolution (Harrison, 2021).

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Zhu, Y., Cai, Q., Zheng, X., Liu, L., Hua, Y., Du, B., Zhao, G., Yu, J., Zhuo, Z., Xie, Z. and Ji, S. (2021). Aspirin Positively Contributes to Drosophila Intestinal Homeostasis and Delays Aging through Targeting Imd. Aging Dis 12(7): 1821-1834. PubMed ID: 34631223

The intestine, a high-turnover tissue, plays a critical role in regulating aging and health in both vertebrates and invertebrates. Maintaining the epithelial barrier function of the intestine by preserving innate immune homeostasis significantly delays aging and prevents mortality. In an effort to explore effective chemicals and materials that can improve intestinal integrity, a nonbiased screen was performed utilizing Drosophila as an animal model. Long-term uptake of aspirin markedly prevented age-onset gut leakage, the over-proliferation of intestinal stem cells, and the dysbiosis of commensal microbiota in fruit flies. Mechanistically, aspirin efficiently downregulated chronic activation of intestinal immune deficiency signaling during aging. Furthermore, in vivo and in vitro biochemical analyses indicated that aspirin is a negative modulator in control of the K63-linked ubiquitination of Imd. These findings uncover a novel regulatory mechanism by which aspirin positively modulates intestinal homeostasis, thus delaying aging, in Drosophila (Zhu, 2021).

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Bouska, M. J. and Bai, H. (2021). Loxl2 is a mediator of cardiac aging in Drosophila melanogaster; genetically examining the role of aging clock genes. G3 (Bethesda). PubMed ID: 34734976

Transcriptomic, proteomic, and methylation aging clocks demonstrate that aging has a predictable preset program, while Transcriptome Trajectory Turning Points indicate that the 20 to 40 age range in humans is the likely stage at which the progressive loss of homeostatic control, and in turn aging, begins to have detrimental effects. Turning points in this age range overlapping with human aging clock genes revealed five candidates that were hypothesized could play a role in aging or age-related physiological decline. To examine these gene's effects on lifespan and health-span, this study utilized whole body and heart specific gene knockdown of human orthologs in Drosophila melanogaster. Whole body Loxl2, fz3, and Glo1 RNAi positively affected lifespan as did heart-specific Loxl2 knockdown. Loxl2 inhibition concurrently reduced age-related cardiac arrythmia and collagen (Pericardin) fiber width. Loxl2 binds several transcription factors in humans and RT-qPCR confirmed that a conserved transcriptional target CDH1 (Drosophila CadN2), has expression levels which correlate with Loxl2 reduction in Drosophila. These results point to conserved pathways and multiple mechanisms by which inhibition of Loxl2 can be beneficial to heart health and organismal aging (Bouska, 2021).

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Ebanks, B., Wang, Y., Katyal, G., Sargent, C., Ingram, T. L., Bowman, A., Moisoi, N. and Chakrabarti, L. (2021). Exercising D. melanogaster Modulates the Mitochondrial Proteome and Physiology. The Effect on Lifespan Depends upon Age and Sex. Int J Mol Sci 22(21). PubMed ID: 34769041

Ageing is a major risk factor for many of the most prevalent diseases, including neurodegenerative diseases, cancer, and heart disease. As the global population continues to age, behavioural interventions that can promote healthy ageing will improve quality of life and relieve the socioeconomic burden that comes with an aged society. Exercise is recognised as an effective intervention against many diseases of ageing, but we do not know the stage in an individual's lifetime at which exercise is most effective at promoting healthy ageing, and whether or not it has a direct effect on lifespan. This study exercised w(1118) Drosophila melanogaster, investigating the effects of sex and group size at different stages of their lifetime, and recorded their lifespan. Climbing scores at 30 days were measured to record differences in fitness in response to exercise. The mitochondrial proteome was assessed of w(1118) Drosophila that had been exercised for one week, alongside mitochondrial respiration measured using high-resolution respirometry, to determine changes in mitochondrial physiology in response to exercise. This study found that age-targeted exercise interventions improved the lifespan of both male and female Drosophila, and grouped males exercised in late life had improved climbing scores when compared with those exercised throughout their entire lifespan. The proteins of the electron transport chain were significantly upregulated in expression after one week of exercise, and complex-II-linked respiration was significantly increased in exercised Drosophila. Taken together, our findings provide a basis to test specific proteins, and complex II of the respiratory chain, as important effectors of exercise-induced healthy ageing (Ebanks, 2021).

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Azpurua, J., El-Karim, E. G., Tranquille, M. and Dubnau, J. (2021). A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia. PLoS Genet 17(11): e1009882. PubMed ID: 34723963

Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), ~40% of frontotemporal dementia (FTD) and in many cases of Alzheimer's disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, This study expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, this study conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, this screen also yielded genes involved in chromatin regulation and nuclear/import export - pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. The identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator (Azpurua, 2021).

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Parkhitko, A. A., Wang, L., Filine, E., Jouandin, P., Leshchiner, D., Binari, R., Asara, J. M., Rabinowitz, J. D. and Perrimon, N. (2021). A genetic model of methionine restriction extends Drosophila health- and lifespan. Proc Natl Acad Sci U S A 118(40). PubMed ID: 34588310

Loss of metabolic homeostasis is a hallmark of aging and is characterized by dramatic metabolic reprogramming. To analyze how the fate of labeled methionine is altered during aging, This study applied C5-Methionine labeling to Drosophila and demonstrated significant changes in the activity of different branches of the methionine metabolism as flies age. Whether targeted degradation of methionine metabolism components would "reset" methionine metabolism flux and extend the fly lifespan was tested. Specifically, this study created transgenic flies with inducible expression of Methioninase, a bacterial enzyme capable of degrading methionine and revealed methionine requirements for normal maintenance of lifespan. Also demonstrated was the microbiota-derived methionine is an alternative and important source in addition to food-derived methionine. In this genetic model of methionine restriction (MetR), this study also demonstrate that either whole-body or tissue-specific Methioninase expression can dramatically extend Drosophila health- and lifespan and exerts physiological effects associated with MetR. Interestingly, while previous dietary MetR extended lifespan in flies only in low amino acid conditions, MetR from Methioninase expression extends lifespan independently of amino acid levels in the food. Finally, because impairment of the methionine metabolism has been previously associated with the development of Alzheimer's disease, this study compared methionine metabolism reprogramming between aging flies and a Drosophila model relevant to Alzheimer's disease, and found that overexpression of human Tau caused methionine metabolism flux reprogramming similar to the changes found in aged flies. Altogether, this study highlights Methioninase as a potential agent for health- and lifespan extension (Parkhitko, 2021).

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Landis, G. N., Hilsabeck, T. A. U., Bell, H. S., Ronnen-Oron, T., Wang, L., Doherty, D. V., Tejawinata, F. I., Erickson, K., Vu, W., Promislow, D. E. L., Kapahi, P. and Tower, J. (2021). Mifepristone Increases Life Span of Virgin Female Drosophila on Regular and High-fat Diet Without Reducing Food Intake. Front Genet 12: 751647. PubMed ID: 34659367.

The synthetic steroid mifepristone is reported to have anti-obesity and anti-diabetic effects in mammals on normal and high-fat diets (HFD). Previous work showed that mifepristone blocks the negative effect on life span caused by mating in female Drosophila melanogaster. This study asked if mifepristone could protect virgin females from the life span-shortening effect of HFD. Mifepristone increased life span of virgin females on regular media, as well as on media supplemented with either 2.5 or 5% coconut oil. Food intake was not reduced in any assay, and was significantly increased by mifepristone in half of the assays. To ask if mifepristone might rescue virgin females from all life span-shortening stresses, the oxidative stressor paraquat was tested, and mifepristone produced little to no rescue. Analysis of extant metabolomics and transcriptomics data suggested similarities between effects of mifepristone in virgin and mated females, including reduced tryptophan breakdown and similarities to dietary restriction. Bioinformatics analysis identified candidate mifepristone targets, including transcription factors Paired and Extra-extra. In addition to shortening life span, mating also causes midgut hypertrophy and activation of the lipid metabolism regulatory factor SREBP. Mifepristone blocked the increase in midgut size caused by mating, but did not detectably affect midgut size in virgins. Finally, mating increased activity of a SREBP reporter in abdominal tissues, as expected, but reporter activity was not detectably reduced by mifepristone in either mated or virgin females. Mifepristone increases life span of virgin females on regular and HFD without reducing food intake. Metabolomics and transcriptomics analyses suggest some similar effects of mifepristone between virgin and mated females, however reduced midgut size was observed only in mated females (Landis, 2021).

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Goh, G. H., Blache, D., Mark, P. J., Kennington, W. J. and Maloney, S. K. (2021). Daily temperature cycles prolong lifespan and have sex-specific effects on peripheral clock gene expression in Drosophila melanogaster. J Exp Biol. PubMed ID: 33758022

Circadian rhythms optimize health by coordinating the timing of physiological processes to match predictable daily environmental challenges. The circadian rhythm of body temperature is thought to be an important modulator of molecular clocks in peripheral tissues, but how daily temperature cycles impact physiological function is unclear. This study examined the effect of constant (25°C, T(CON)) and cycling (28°C/22°C during light/dark, T(CYC)) temperature paradigms on lifespan of Drosophila melanogaster, and the expression of clock genes, Heat shock protein 83 (Hsp83), Frost (Fst), and Senescence-associated protein 30 (smp-30). Male and female Drosophila housed at T(CYC) had longer median lifespans than those housed at T(CON) T(CYC) induced robust Hsp83 rhythms and rescued the age-related decrease in smp-30 expression that was observed in flies at T(CON), potentially indicating an increased capacity to cope with age-related cellular stress. Ageing under T(CON) led to a decrease in the amplitude of expression of all clock genes in the bodies of male flies, except for cyc, which was non-rhythmic, and for per and cry in female flies. Strikingly, housing under T(CYC) conditions rescued the age-related decrease in amplitude of all clock genes, and generated rhythmicity in cyc expression, in the male flies, but not the female flies. The results suggest that ambient temperature rhythms modulate Drosophila lifespan, and that the amplitude of clock gene expression in peripheral body clocks may be a potential link between temperature rhythms and longevity in male Drosophila Longevity due to T(CYC) appeared predominantly independent of clock gene amplitude in female Drosophila (Goh, 2021).

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Schroeder, S., Hofer, S. J., et al. (2021). Dietary spermidine improves cognitive function. Cell Rep 35(2): 108985. PubMed ID: 33852843
Summary:

Decreased cognitive performance is a hallmark of brain aging, but the underlying mechanisms and potential therapeutic avenues remain poorly understood. Recent studies have revealed health-protective and lifespan-extending effects of dietary spermidine, a natural autophagy-promoting polyamine. This study shows that dietary spermidine passes the blood-brain barrier in mice and increases hippocampal eIF5A hypusination and mitochondrial function. Spermidine feeding in aged mice affects behavior in homecage environment tasks, improves spatial learning, and increases hippocampal respiratory competence. In a Drosophila aging model, spermidine boosts mitochondrial respiratory capacity, an effect that requires the autophagy regulator Atg7 and the mitophagy mediators Parkin and Pink1. Neuron-specific Pink1 knockdown abolishes spermidine-induced improvement of olfactory associative learning. This suggests that the maintenance of mitochondrial and autophagic function is essential for enhanced cognition by spermidine feeding. Finally, this study showed large-scale prospective data linking higher dietary spermidine intake with a reduced risk for cognitive impairment in humans (Schroeder, 2021).

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Liang, Y., T., Piao, C., Beuschel, C. B., Toppe, D., Kollipara, L., Bogdanow, B., Maglione, M., Lutzkendorf, J., See, J. C. K., Huang, S., Conrad, T. O. F., Kintscher, U., Madeo, F., Liu, F., Sickmann, A. and Sigrist, S. J. (2021). eIF5A hypusination, boosted by dietary spermidine, protects from premature brain aging and mitochondrial dysfunction. Cell Rep 35(2): 108941. PubMed ID: 33852845
Summary:

Recent evidence indicates that the composition of the ribosome is heterogeneous and that multiple types of specialized ribosomes regulate the synthesis of specific protein subsets. In Drosophila, expression of the ribosomal RpS28 protein variants RpS28a and RpS28-like were found to preferentially occurs in the germline, a tissue resistant to aging; it significantly declines in skeletal muscle during aging. Muscle-specific overexpression of RpS28a at levels similar to those seen in the germline decreases early mortality and promotes the synthesis of a subset of proteins with known anti-aging roles, some of which have preferential expression in the germline. These findings indicate a contribution of specialized ribosomal proteins to the regulation of the muscle proteome during aging (Liang, 2021).

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Lu, Y. X., Regan, J. C., Esser, J., Drews, L. F., Weinseis, T., Stinn, J., Hahn, O., Miller, R. A., Gronke, S. and Partridge, L. (2021). A TORC1-histone axis regulates chromatin organisation and non-canonical induction of autophagy to ameliorate ageing. Elife 10. PubMed ID: 33988501
Summary:

Age-related changes to histone levels are seen in many species. However, it is unclear whether changes to histone expression could be exploited to ameliorate the effects of ageing in multicellular organisms. This study shows that inhibition of mTORC1 by the lifespan-extending drug rapamycin increases expression of histones H3 and H4 post-transcriptionally, through eIF3-mediated translation. Elevated expression of H3/H4 in intestinal enterocytes in Drosophila alters chromatin organization, induces intestinal autophagy through transcriptional regulation, prevents age-related decline in the intestine. Importantly, it also mediates rapamycin-induced longevity and intestinal health. Histones H3/H4 regulate expression of an autophagy cargo adaptor Blue Cheese (Bchs (WDFY3 in mammals), increased expression of which in enterocytes mediates increased H3/H4-dependent healthy longevity. In mice, rapamycin treatment increases expression of histone proteins and Wdfy3 transcription, and alters chromatin organisation in the small intestine, suggesting the mTORC1-histone axis is at least partially conserved in mammals and may offer new targets for anti-ageing interventions.

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Nguyen, A. H. and Bachtrog, D. (2021). Toxic Y chromosome: Increased repeat expression and age-associated heterochromatin loss in male Drosophila with a young Y chromosome. PLoS Genet 17(4): e1009438. PubMed ID: 33886541
Summary:

Sex-specific differences in lifespan are prevalent across the tree of life and influenced by heteromorphic sex chromosomes. In species with XY sex chromosomes, females often outlive males. Males and females can differ in their overall repeat content due to the repetitive Y chromosome, and repeats on the Y might lower survival of the heterogametic sex (toxic Y effect). This study takes advantage of the well-assembled young Y chromosome of Drosophila miranda to study the sex-specific dynamics of chromatin structure and repeat expression during aging in male and female flies. Male D. miranda have about twice as much repetitive DNA compared to females, and live shorter than females. Heterochromatin is crucial for silencing of repetitive elements, yet old D. miranda flies lose H3K9me3 modifications in their pericentromere, with heterochromatin loss being more severe during aging in males than females. Satellite DNA becomes de-repressed more rapidly in old vs. young male flies relative to females. In contrast to what is observed in D. melanogaster, it was found that transposable elements (TEs) are expressed at higher levels in male D. miranda throughout their life. Epigenetic silencing via heterochromatin formation is ineffective on the TE-rich neo-Y chromosome, presumably due to active transcription of a large number of neo-Y linked genes, resulting in up-regulation of Y-linked TEs already in young males. This is consistent with an interaction between the evolutionary age of the Y chromosome and the genomic effects of aging. These data support growing evidence that "toxic Y chromosomes" can diminish male fitness and a reduction in heterochromatin can contribute to sex-specific aging.

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Gabrawy, M. M., Khosravian, N., Morcos, G. S., Morozova, T. V., Jezek, M., Walston, J. D., Huang, W., Abadir, P. M. and Leips, J. (2022). Genome-Wide Analysis in Drosophila Reveals the Genetic Basis of Variation in Age-Specific Physical Performance and Response to ACE Inhibition. Genes (Basel) 13(1). PubMed ID: 35052483
Summary:
Despite impressive results in restoring physical performance in rodent models, treatment with renin-angiotensin system (RAS) inhibitors, such as Lisinopril, have highly mixed results in humans, likely, in part, due to genetic variation in human populations. To date, the genetic determinants of responses to drugs, such as RAS inhibitors, remain unknown. Given the complexity of the relationship between physical traits and genetic background, genomic studies which predict genotype- and age-specific responses to drug treatments in humans or vertebrate animals are difficult. Using 126 genetically distinct lines of Drosophila melanogaster, this study tested the effects of Lisinopril on age-specific climbing speed and endurance. The data show that functional response and sensitivity to Lisinopril treatment ranges from significant protection against physical decline to increased weakness depending on genotype and age. Furthermore, genome-wide analyses led to identification of evolutionarily conserved genes in the WNT signaling pathway as being significantly associated with variations in physical performance traits and sensitivity to Lisinopril treatment. Genetic knockdown of genes in the WNT signaling pathway, Axin, frizzled, nemo, and wingless, diminished or abolished the effects of Lisinopril treatment on climbing speed traits. These results implicate these genes as contributors to the genotype- and age-specific effects of Lisinopril treatment and because they have orthologs in humans, they are potential therapeutic targets for improvement of resiliency. This approach should be widely applicable for identifying genomic variants that predict age- and sex-dependent responses to any type of pharmaceutical treatment.

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Cortot, J., Farine, J. P., Ferveur, J. F. and Everaerts, C. (2022). Aging-Related Variation of Cuticular Hydrocarbons in Wild Type and Variant Drosophila melanogaster. J Chem Ecol. PubMed ID: 35022940
Summary:
The cuticle of all insects is covered with hydrocarbons which have multiple functions. Cuticular hydrocarbons (CHCs) basically serve to protect insects against environmental harm and reduce dehydration. In many species, some CHCs also act as pheromones. CHCs have been intensively studied in Drosophila species and more especially in D. melanogaster. In this species, flies produce about 40 CHCs forming a complex sex- and species-specific bouquet. The quantitative and qualitative pattern of the CHC bouquet was characterized during the first days of adult life but remains unexplored in aging flies. This study characterized CHCs during the whole-or a large period of-adult life in males and females of several wild type and transgenic lines. Both types of lines included standard and variant CHC profiles. Some of the genotypes tested in this study showed very dramatic and unexpected aging-related variation based on their early days' profile. This study provides a concrete dataset to better understand the mechanisms underlying the establishment and maintenance of CHCs on the fly cuticle. It could be useful to determine physiological parameters, including age and response to climate variation, in insects collected in the wild.

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Cheng, X., Xie, M., Luo, L., Tian, Y., Yu, G., Wu, Q., Fan, X., Yang, D., Mao, X., Gaur, U. and Yang, M. (2022). Inhibitor GSK690693 extends Drosophila lifespan via reduce AKT signaling pathway. Mech Ageing Dev 202: 111633. PubMed ID: 35065134
Summary:
Aging is a process involving physiological changes that lead to the decline of biological functions of various tissues and organs of the body. Therefore, it is crucial to find anti-aging drugs that can intervene with the changes induced because of aging and slow down the degeneration of the biological functions. Among many signaling pathways linked with aging and aging-related diseases, PI3K-AKT signaling pathway has attracted major attention in aging biology. This paper demonstrates that AKT inhibitor GSK690693 can extend lifespan in Drosophila irrespective of start of the treatment from the beginning of life or the mid-life. Effect of GSK690693 for lifespan extension has been primarily related to the improvements in oxidative resistance, intestinal integrity and increased autophagy, but not in physical activity or starvation resistance. Furthermore, GSK690693 treatment reduced the activation of AKT and ERK, consequently activating FOXO, GSK-3β and apoptosis to modulate longevity of flies. Remarkably, GSK690693 did not induce hyperglycemia after treatment. The results indicate that GSK690693 may become an effective compound for anti-aging intervention.

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Wang, Y. and Westermark, G. T. (2021). The Amyloid Forming Peptides Islet Amyloid Polypeptide and Amyloid beta Interact at the Molecular Level. Int J Mol Sci 22(20). PubMed ID: 34681811
Summary:
Epidemiological studies support a connection between the two common disorders, type-2 diabetes and Alzheimer's disease. Both conditions have local amyloid formation in their pathogenesis, and cross-seeding between islet amyloid polypeptide (IAPP) and amyloid β (Aβ) could constitute the link. The bimolecular fluorescence complementation (BiFC) assay was used to investigate the occurrence of heterologous interactions between IAPP and Aβ and to compare the potential toxic effects of IAPP/Aβ, IAPP/IAPP, and Aβ/Aβ expression in living cells. Microscopy was used to confirm the fluorescence and determine the lysosomal, mitochondrial areas and mitochondrial membrane potential, and a FACS analysis was used to determine ROS production and the role for autophagy. Drosophila melanogaster expressing IAPP and Aβ was used to study their co-deposition and effects on longevity. Co-expression of IAPP and Aβ resulted in fluorophore reconstitution to the same extent as determined for homologous IAPP/IAPP or Aβ/Aβ expression. The BiFC(+)/BiFC(-) ratio of lysosomal area calculations increased in transfected cells independent of the vector combinations, while only Aβ/Aβ expression increased mitochondrial membrane potential. Expression combinations containing Aβ were necessary for the formation of a congophilic amyloid. In Drosophila melanogaster expressing IAPP/Aβ, co-deposition of the amyloid-forming peptides caused reduced longevity. The BiFC results confirmed a heterologous interaction between IAPP and Aβ, while co-deposits in the brain of Drosophila suggest mixed amyloid aggregates.

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Ngian, Z. K., Lin, W. Q. and Ong, C. T.(2021). NELF-A controls Drosophila healthspan by regulating heat-shock protein-mediated cellular protection and heterochromatin maintenance. Aging Cell: e13348. PubMed ID: 33788376
Summary:

NELF-mediated pausing of RNA polymerase II (RNAPII) constitutes a crucial step in transcription regulation. However, it remains unclear how control release of RNAPII pausing can affect the epigenome and regulate important aspects of animal physiology like aging. This study found that NELF-A dosage regulates Drosophila healthspan: Halving NELF-A level in the heterozygous mutants or via neuronal-specific RNAi depletion improves their locomotor activity, stress resistance, and lifespan significantly. Conversely, NELF-A overexpression shortens fly lifespan drastically. Mechanistically, lowering NELF-A level facilitates the release of paused RNAPII for productive transcription of the heat-shock protein (Hsp) genes. The elevated HSPs expression in turn attenuates the accumulation of insoluble protein aggregates, reactive oxidative species, DNA damage and systemic inflammation in the brains of aging NELF-A depleted flies as compared to their control siblings. This pro-longevity effect is unique to NELF-A due to its higher expression level and more efficient pausing of RNAPII than other NELF subunits. Importantly, enhanced resistance to oxidative stress in NELF-A heterozygous mutants is highly conserved such that knocking down its level in human SH-SY5Y cells attenuates hydrogen peroxide-induced DNA damage and apoptosis. Depleting NELF-A reconfigures the epigenome through the maintenance of H3K9me2-enriched heterochromatin during aging, leading to the repression of specific retrotransposons like Gypsy-1 in the brains of NELF-A mutants. Taken together, this study showed that the dosage of neuronal NELF-A affects multiple aspects of aging in Drosophila by regulating transcription of Hsp genes in the brains, suggesting that targeting transcription elongation might be a viable therapeutic strategy against age-onset diseases like neurodegeneration.

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Sasaki, A., Nishimura, T., Takano, T., Naito, S. and Yoo, S. K.(2021). White regulates proliferative homeostasis of intestinal stem cells during ageing in Drosophila. Nat Metab 3(4): 546-557. PubMed ID: 33820991
Summary:
Tissue integrity is contingent on maintaining stem cells. Intestinal stem cells (ISCs) over-proliferate during ageing, leading to tissue dysplasia in Drosophila melanogaster. This study describes a role for white, encoding the evolutionarily conserved ATP-binding cassette transporter subfamily G, with a particularly well-characterized role in eye colour pigmentation, in ageing-induced ISC proliferation in the midgut. ISCs increase expression of white during ageing. ISC-specific inhibition of white suppresses ageing-induced ISC dysregulation and prolongs lifespan. Of the proteins that form heterodimers with White, Brown mediates ISC dysregulation during ageing. Metabolomics analyses reveal previously unappreciated, profound metabolic impacts of white inhibition on organismal metabolism. Among the metabolites affected by White, tetrahydrofolate is transported by White, is accumulated in ISCs during ageing and is indispensable for ageing-induced ISC over-proliferation. Since Thomas Morgan's isolation of a white mutant as the first Drosophila mutant, white mutants have been used extensively as genetic systems and often as controls. These findings provide insights into metabolic regulation of stem cells mediated by the classic gene white.

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Hunt, L. C. and Demontis, F. (2021). Age-Related Increase in Lactate Dehydrogenase Activity in Skeletal Muscle Reduces Lifespan in Drosophila. J Gerontol A Biol Sci Med Sci. PubMed ID: 34477202
Summary:
Metabolic adaptations occur with aging but the significance and causal roles of such changes are only partially known. In Drosophila, this study found that skeletal muscle aging is paradoxically characterized by increased readouts of glycolysis (lactate, NADH/NAD+) but reduced expression of most glycolytic enzymes. This conundrum is explained by lactate dehydrogenase (LDH), an enzyme necessary for anaerobic glycolysis and whose expression increases with aging. Experimental Ldh overexpression in skeletal muscle of young flies increases glycolysis and shortens lifespan, suggesting that age-related increases in muscle LDH contribute to mortality. Similar results are also found with overexpression of other glycolytic enzymes (Pfrx/PFKFB, Pgi/GPI). Conversely, hypomorphic mutations in Ldh extend lifespan whereas reduction in PFK, Pglym78/PGAM, Pgi/GPI, and Ald/ALDO levels shorten lifespan to various degrees, indicating that glycolysis needs to be tightly controlled for optimal aging. Altogether, these findings indicate a role for muscle LDH and glycolysis in aging.

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Feng, X., Hong, X., Fan, Q., Chen, L., Li, J., Deng, J., Gong, S., Hou, F. F. and Zhang, F. (2021). dCubilin/dAMN- mediated protein reabsorption in Drosophila nephrocytes modulates longevity. Dis Model Mech. PubMed ID: 34437681
Summary:
Aging is a multi-faceted process regulated by multiple cellular pathways, including the proteostasis network. Pharmacological or genetic enhancement of the intracellular proteostasis network extends lifespan and prevents age-related diseases. However, how proteostasis is regulated in different tissues throughout the aging process remains unclear. This study shows that Drosophila homologs for Cubilin/Amnionless (dCubilin/dAMN)-mediated protein reabsorption from hemolymph (fly equivalent of blood) by nephrocytes modulates longevity through regulating proteostasis in muscle and brain tissues in Drosophila. Overexpression of dAMN receptor in nephrocytes extends lifespan, whereas nephrocyte-specific dCubilin or dAMN RNAi knockdown results in a protein reabsorption defect and shortens lifespan in flies. dCubilin/dAMN-mediated protein reabsorption in nephrocytes regulates proteostasis in hemolymph and improves healthspan. In addition, it was shown that enhanced dCubilin/dAMN-mediated protein reabsorption in nephrocytes slows down the aging process in muscle and brain by maintaining the proteostasis network in these tissues. Furthermore, this study shows that dCubilin/dAMN -mediated protein reabsorption in nephrocytes affects proteasome activity in the whole body and muscle tissues. Altogether, this work has revealed an inter-organ communication network across nephrocytes and muscle/neuronal tissue which is essential to maintain proteostasis and to delay senescence in these organs. The findings have provided insights into the role of renal protein reabsorption in the aging process via this tele-proteostasis network.

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Saito, T., Chiku, T., Oka, M., Wada-Kakuda, S., Nobuhara, M., Oba, T., Shinno, K., Abe, S., Asada, A., Sumioka, A., Takashima, A., Miyasaka, T. and Ando, K. (2021). Disulfide bond formation in microtubule-associated tau protein promotes tau accumulation and toxicity in vivo. Hum Mol Genet. PubMed ID: 34137825
Summary:
Accumulation of microtubule-associated tau protein is thought to cause neuron loss in a group of neurodegenerative diseases called tauopathies. In diseased brains, tau molecules adopt pathological structures that propagate into insoluble forms with disease-specific patterns. Several types of posttranslational modifications in tau are known to modulate its aggregation propensity in vitro, but their influence on tau accumulation and toxicity at the whole-organism level has not been fully elucidated. This study utilized a series of transgenic Drosophila models to compare systematically the toxicity induced by five tau constructs with mutations or deletions associated with aggregation, including substitutions at seven disease-associated phosphorylation sites (S7A and S7E), deletions of PHF6 and PHF6* sequences (ΔPHF6 and ΔPHF6*), and substitutions of cysteine residues in the microtubule binding repeats (C291/322A). Substitutions and deletions resulted in different patterns of neurodegeneration and accumulation, with C291/322A having a dramatic effect on both tau accumulation and neurodegeneration. These cysteines formed disulfide bonds in mouse primary cultured neurons and in the fly retina, and stabilized tau proteins. Additionally, they contributed to tau accumulation under oxidative stress. This study also found that each of these cysteine residues contributes to the microtubule polymerization rate and microtubule levels at equilibrium, but none of them affected tau binding to polymerized microtubules. Since tau proteins expressed in the Drosophila retina are mostly present in the early stages of tau filaments self-assembly, these results suggest that disulfide bond formation by these cysteine residues could be attractive therapeutic targets.

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Wang, L. J., Hsu, T., Lin, H. L. and Fu, C. Y. (2021). Modulation of mitochondrial nucleoid structure during aging and by mtDNA content in Drosophila. Biol Open 10(6). PubMed ID: 34180963
Summary:
Mitochondrial DNA (mtDNA) encodes gene products that are essential for oxidative phosphorylation. They organize as higher order nucleoid structures (mtNucleoids) that were shown to be critical for the maintenance of mtDNA stability and integrity. While mtNucleoid structures are associated with cellular health, how they change in situ under physiological maturation and aging requires further investigation. This study investigated the mtNucleoid assembly at an ultrastructural level in situ using the TFAM-Apex2 Drosophila model. Smaller and more compact TFAM-nucleoids are populated in the mitochondria of indirect flight muscle of aged flies. Furthermore, mtDNA transcription and replication were cross-regulated in the mtTFB2-knockdown flies as in the mtRNAPol-knockdown flies that resulted in reductions in mtDNA copy numbers and nucleoid-associated TFAM. Overall, this study reveals that the modulation of TFAM-nucleoid structure under physiological aging, which is critically regulated by mtDNA content.

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Wu, K., Tang, Y., Zhang, Q., Zhuo, Z., Sheng, X., Huang, J., Ye, J., Li, X., Liu, Z. and Chen, H. (2021). Aging-related upregulation of the homeobox gene caudal represses intestinal stem cell differentiation in Drosophila. PLoS Genet 17(7): e1009649. PubMed ID: 34228720
Summary:
The differentiation efficiency of adult stem cells undergoes a significant decline in aged animals, which is closely related to the decline in organ function and age-associated diseases. However, the underlying mechanisms that ultimately lead to this observed decline of the differentiation efficiency of stem cells remain largely unclear. This study investigated Drosophila midguts and identified an obvious upregulation of caudal (cad), which encodes a homeobox transcription factor. This factor is traditionally known as a central regulator of embryonic anterior-posterior body axis patterning. This study reports that depletion of cad in intestinal stem/progenitor cells promotes quiescent intestinal stem cells (ISCs) to become activate and produce enterocytes in the midgut under normal gut homeostasis conditions. However, overexpression of cad results in the failure of ISC differentiation and intestinal epithelial regeneration after injury. Moreover, this study suggests that cad prevents intestinal stem/progenitor cell differentiation by modulating the Janus kinase/signal transducers and activators of the transcription pathway and Sox21a-GATAe signaling cascade. Importantly, the reduction of cad expression in intestinal stem/progenitor cells restrained age-associated gut hyperplasia in Drosophila. This study identified a function of the homeobox gene cad in the modulation of adult stem cell differentiation and suggested a potential gene target for the treatment of age-related diseases induced by age-related stem cell dysfunction.

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Gao, Y., Zhu, C., Li, K., Cheng, X., Du, Y., Yang, D., Fan, X., Gaur, U. and Yang, M. (2020). Comparative proteomics analysis of dietary restriction in Drosophila. PLoS One 15(10): e0240596. PubMed ID: 33064752

To explore the underlying mechanism of dietary restriction (DR) induced lifespan extension in fruit flies at protein level, proteome sequencing was performed in Drosophila at day 7 (young) and day 42 (old) under DR and ad libitum (AL) conditions. A total of 18629 unique peptides were identified in Uniprot, corresponding to 3,662 proteins. Among them, 383 and 409 differentially expressed proteins (DEPs) were identified from comparison between DR vs AL at day 7 and 42, respectively. Bioinformatics analysis revealed that membrane-related processes, post-transcriptional processes, spliceosome and reproduction related processes, were highlighted significantly. In addition, expression of proteins involved in pathways such as spliceosomes, oxidative phosphorylation, lysosomes, ubiquitination, and riboflavin metabolism was relatively higher during DR. A relatively large number of DEPs were found to participate in longevity and age-related disease pathways. 20 proteins were identified that were consistently regulated during DR and some of which are known to be involved in ageing, such as mTORC1, antioxidant, DNA damage repair and autophagy. In the integration analysis, 15 genes were found that were stably regulated by DR at both transcriptional as well as translational levels. These results provided a useful dataset for further investigations on the mechanism of DR and aging (Gao, 2020).

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McCracken, A. W., Buckle, E. and Simons, M. J. P. (2020). The relationship between longevity and diet is genotype dependent and sensitive to desiccation in Drosophila melanogaster. J Exp Biol. PubMed ID: 33109715

Dietary restriction (DR) is a key focus in ageing research. Specific conditions and genotypes were recently found to negate lifespan extension by DR, questioning its universal relevance. However, the concept of dietary reaction norms explains why DR's effects might be obscured in some situations. This study tested the importance of dietary reaction norms by measuring longevity and fecundity on five diets in five genotypes, with and without water supplementation in female Drosophila melanogaster (N>25,000). Substantial genetic variation was found in the response of lifespan to diet. Flies supplemented with water rescued putative desiccation stress at the richest diets, suggesting water availability can be an experimental confound. Fecundity declined at these richest diets, but was unaffected by water, and this reduction is thus most likely caused by nutritional toxicity. These results demonstrate empirically that a range of diets need to be considered to conclude an absence of the DR longevity effect (McCracken, 2020).

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Zanco, B., Mirth, C. K., Sgro, C. M. and Piper, M. D. (2021). A dietary sterol trade-off determines lifespan responses to dietary restriction in Drosophila melanogaster females. Elife 10. PubMed ID: 33494859

Diet plays a significant role in maintaining lifelong health. In particular, lowering the dietary protein: carbohydrate ratio can improve lifespan. This has been interpreted as a direct effect of these macronutrients on physiology. Using Drosophila melanogaster, this study shows that the role of protein and carbohydrate on lifespan is indirect, acting by altering the partitioning of limiting amounts of dietary sterols between reproduction and lifespan. Shorter lifespans in flies fed on high protein: carbohydrate diets can be rescued by supplementing their food with cholesterol. Not only does this fundamentally alter the way we interpret the mechanisms of lifespan extension by dietary restriction, these data highlight the important principle that life histories can be affected by nutrient-dependent trade-offs that are indirect and independent of the nutrients (often macronutrients) that are the focus of study. This brings us closer to understanding the mechanistic basis of dietary restriction (Zanco, 2021).

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Dai, Z., Li, D., Du, X., Ge, Y., Hursh, D. A. and Bi, X. (2020). Drosophila Caliban preserves intestinal homeostasis and lifespan through regulating mitochondrial dynamics and redox state in enterocytes. PLoS Genet 16(10): e1009140. PubMed ID: 33057338

Precise regulation of stem cell activity is crucial for tissue homeostasis. In Drosophila, intestinal stem cells (ISCs) maintain the midgut epithelium and respond to oxidative challenges. However, the connection between intestinal homeostasis and redox signaling remains obscure. This study found that Caliban (Clbn), a component of the ribosome quality control complex (RQC), functions as a regulator of mitochondrial dynamics in enterocytes (ECs) and is required for intestinal homeostasis. The clbn knock-out flies have a shortened lifespan and lose the intestinal homeostasis. Clbn is highly expressed and localizes to the outer membrane of mitochondria in ECs. Mechanically, Clbn mediates mitochondrial dynamics in ECs and removal of clbn leads to mitochondrial fragmentation, accumulation of reactive oxygen species, ECs damage, activation of JNK and JAK-STAT signaling pathways. Moreover, multiple mitochondria-related genes are differentially expressed between wild-type and clbn mutated flies by a whole-genome transcriptional profiling. Furthermore, loss of clbn promotes tumor growth in gut generated by activated Ras in intestinal progenitor cells. These findings reveal an EC-specific function of Clbn in regulating mitochondrial dynamics, and provide new insight into the functional link among mitochondrial redox modulation, tissue homeostasis and longevity (Cai, 2020).

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Cui, L., Song, W., Zeng, Y., Wu, Q., Fan, Z., Huang, T., Zeng, B., Zhang, M., Ni, Q., Li, Y., Wang, T., Li, D., Mao, X., Lian, T., Yang, D., Yang, M. and Fan, X. (2020). Deubiquitinase USP7 regulates Drosophila aging through ubiquitination and autophagy. Aging (Albany NY) 12(22): 23082-23095. PubMed ID: 33221768

Ubiquitination-mediated protein degradation is the selective degradation of diverse forms of damaged proteins that are tagged with ubiquitin, while deubiquitinating enzymes reverse ubiquitination-mediated protein degradation by removing the ubiquitin chain from the target protein. The interactions of ubiquitinating and deubiquitinating enzymes are required to maintain protein homeostasis. The ubiquitin-specific protease USP7 is a deubiquitinating enzyme that indirectly plays a role in repairing DNA damage and development. However, the mechanism of its participation in aging has not been fully explored. Regarding this issue, this study found that USP7 was necessary to maintain the normal lifespan of Drosophila melanogaster, and knockdown of dusp7 shortened the lifespan and reduced the ability of Drosophila to cope with starvation, oxidative stress and heat stress. Furthermore, this study showed that the ability of USP7 to regulate aging depends on the autophagy and ubiquitin signaling pathways. Furthermore, 2,5-dimethyl-celecoxib (DMC), a derivative of celecoxib, can partially restore the shortened lifespan and aberrant phenotypes caused by dusp7 knockdown. These results suggest that USP7 is an important factor involved in the regulation of aging, and related components in this regulatory pathway may become new targets for anti-aging treatments (Cui, 2020).

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Thompson, J. B., Su, O. O., Yang, N. and Bauer, J. H. (2020). Sleep-length differences are associated with altered longevity in the fruit fly Drosophila melanogaster. Biol Open 9(9). PubMed ID: 32938639

Sleep deprivation has been shown to negatively impact health outcomes, leading to decreased immune responses, memory loss, increased activity of stress and inflammatory pathways, weight gain, and even behavioral changes. These observations suggest that sleep deprivation substantially interferes with important physiological functions, including metabolic pathways of energy utilization. Many of those phenotypes are correlated with age, suggesting that disrupted sleep may interfere with the aging process. However, little is known about how sleep disruption affects aging and longevity. This study investigated this relationship using eight representative fruit fly lines from the Sleep Inbred Panel (SIP). The SIP consists of 39 inbred lines that display extreme short- and long-sleep patterns, and constitutes a crucial Drosophila community resource for investigating the mechanisms of sleep regulation. The data show that flies with short-sleep periods have ∼16% longer life span, as well as reduced aging rate, compared to flies with long-sleep. In contrast, disrupting normal circadian rhythm reduces fly longevity. Short-sleep SIP flies moreover show slight metabolic differences to long-sleep lines, and to flies with disrupted circadian rhythm. These data suggest that the inbred SIP lines engage sleep mechanisms that are distinct from the circadian clock system (Thompson, 2020).

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Brengdahl, M. I., Kimber, C. M., Elias, P., Thompson, J. and Friberg, U. (2020). Deleterious mutations show increasing negative effects with age in Drosophila melanogaster. BMC Biol 18(1): 128. PubMed ID: 32993647

In order for aging to evolve in response to a declining strength of selection with age, a genetic architecture that allows for mutations with age-specific effects on organismal performance is required. Understanding of how selective effects of individual mutations are distributed across ages is however poor. Established evolutionary theories assume that mutations causing aging have negative late-life effects, coupled to either positive or neutral effects early in life. New theory now suggests evolution of aging may also result from deleterious mutations with increasing negative effects with age, a possibility that has not yet been empirically explored. To directly test how the effects of deleterious mutations are distributed across ages, age-specific effects on fecundity were separately measure for each of 20 mutations in Drosophila melanogaster. Deleterious mutations in general were found to have a negative effect that increases with age, and the rate of increase depends on how deleterious a mutation is early in life. These findings suggest that aging does not exclusively depend on genetic variants assumed by the established evolutionary theories of aging. Instead, aging can result from deleterious mutations with negative effects that amplify with age. If increasing negative effect with age is a general property of deleterious mutations, the proportion of mutations with the capacity to contribute towards aging may be considerably larger than previously believed (Brengdahl, 2020).

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Soule, S., Mellottee, L., Arab, A., Chen, C. and Martin, J. R. (2020). Jouvence a small nucleolar RNA required in the gut extends lifespan in Drosophila. Nat Commun 11(1): 987. PubMed ID: 32080190

Longevity is influenced by genetic and environmental factors, but the underlying mechanisms remain elusive. This study functionally characterise a Drosophila small nucleolar RNA (snoRNA), named jouvence whose loss of function reduces lifespan. The genomic region of jouvence rescues the longevity in mutant, while its overexpression in wild-type increases lifespan. Jouvence is required in enterocytes. In mutants, the epithelium of the gut presents more hyperplasia, while the overexpression of jouvence prevents it. Molecularly, the mutant lack pseudouridylation on 18S and 28S-rRNA, a function rescued by targeted expression of jouvence in the gut. A transcriptomic analysis performed from the gut reveals that several genes are either up- or down-regulated, while restoring the mRNA level of two genes (ninaD or CG6296) rescue the longevity. Since snoRNAs are structurally and functionally well conserved throughout evolution, this study identified a putative jouvence orthologue in mammals including humans, suggesting that its function in longevity could be conserved (Soule, 2020).

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Cho, L. C., Yu, C. C. and Kao, C. F. (2021). Social perception of young adults prolongs the lifespan of aged Drosophila. NPJ Aging Mech Dis 7(1): 21. PubMed ID: 34471134

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Sasaki, A., Nishimura, T., Takano, T., Naito, S. and Yoo, S. K. (2020). White regulates proliferative homeostasis of intestinal stem cells during ageing in Drosophila. Nat Metab 3(4): 546-557. PubMed ID: 33820991

Tissue integrity is contingent on maintaining stem cells. Intestinal stem cells (ISCs) over-proliferate during ageing, leading to tissue dysplasia in Drosophila melanogaster. This study describes a role for white, encoding the evolutionarily conserved ATP-binding cassette transporter subfamily G, with a particularly well-characterized role in eye colour pigmentation, in ageing-induced ISC proliferation in the midgut. ISCs increase expression of white during ageing. ISC-specific inhibition of white suppresses ageing-induced ISC dysregulation and prolongs lifespan. Of the proteins that form heterodimers with White, Brown mediates ISC dysregulation during ageing. Metabolomics analyses reveal previously unappreciated, profound metabolic impacts of white inhibition on organismal metabolism. Among the metabolites affected by White, tetrahydrofolate is transported by White, is accumulated in ISCs during ageing and is indispensable for ageing-induced ISC over-proliferation. Since Thomas Morgan's isolation of a white mutant as the first Drosophila mutant, white mutants have been used extensively as genetic systems and often as controls. These findings provide insights into metabolic regulation of stem cells mediated by the classic gene white (Sasaki, 2021).

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Wang, L. J., Hsu, T., Lin, H. L. and Fu, C. Y. (2020). Modulation of mitochondrial nucleoid structure during aging and by mtDNA content in Drosophila. Biol Open 10(6). PubMed ID: 34180963

Mitochondrial DNA (mtDNA) encodes gene products that are essential for oxidative phosphorylation. They organize as higher order nucleoid structures (mtNucleoids) that were shown to be critical for the maintenance of mtDNA stability and integrity. While mtNucleoid structures are associated with cellular health, how they change in situ under physiological maturation and aging requires further investigation. This study investigated the mtNucleoid assembly at an ultrastructural level in situ using the TFAM-Apex2 Drosophila model. Smaller and more compact TFAM-nucleoids are populated in the mitochondria of indirect flight muscle of aged flies. Furthermore, mtDNA transcription and replication were cross-regulated in the mtTFB2-knockdown flies as in the mtRNAPol-knockdown flies that resulted in reductions in mtDNA copy numbers and nucleoid-associated TFAM. Overall, this study reveals that the modulation of TFAM-nucleoid structure under physiological aging, which is critically regulated by mtDNA content (Wang, 2021).

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Long, D. M., Frame, A. K., Reardon, P. N., Cumming, R. C., Hendrix, D. A., Kretzschmar, D. and Giebultowicz, J. M. (2020). Lactate dehydrogenase expression modulates longevity and neurodegeneration in Drosophila melanogaster. Aging (Albany NY) 12. PubMed ID: 32484787

Lactate dehydrogenase (LDH) catalyzes the conversion of glycolysis-derived pyruvate to lactate. Lactate has been shown to play key roles in brain energetics and memory formation. However, lactate levels are elevated in aging and Alzheimer's disease patients, and it is not clear whether lactate plays protective or detrimental roles in these contexts. This study shows that Ldh transcript levels are elevated and cycle with diurnal rhythm in the heads of aged flies and this is associated with increased LDH protein, enzyme activity, and lactate concentrations. To understand the biological significance of increased Ldh gene expression, Ldh levels were genetically manipulated in adult neurons or glia. Overexpression of Ldh in both cell types caused a significant reduction in lifespan whereas Ldh down-regulation resulted in lifespan extension. Moreover, pan-neuronal overexpression of Ldh disrupted circadian locomotor activity rhythms and significantly increased brain neurodegeneration. In contrast, reduction of Ldh in neurons delayed age-dependent neurodegeneration. Thus, this unbiased genetic approach identified Ldh and lactate as potential modulators of aging and longevity in flies (Long, 2020).

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Yurkevych, I. S., Gray, L. J., Gospodaryov, D. V., Burdylyuk, N. I., Storey, K. B., Simpson, S. J. and Lushchak, O. (2020). Development of fly tolerance to consuming a high-protein diet requires physiological, metabolic and transcriptional changes. Biogerontology. PubMed ID: 32468146

Mortality in insects consuming high-protein-and-low-carbohydrate diets resembles a type III lifespan curve with increased mortality at an early age and few survivors that live a relatively long lifespan. A Drosophila line was selected for ability to live for a long time on an imbalanced high-protein-low-carbohydrate diet by carrying out five rounds of breeding to select for the most long-lived survivors. Adaptation to this diet in the selected line was studied at the biochemical, physiological and transcriptomic levels. The selected line of flies consumed less of the imbalanced food but also accumulated more storage metabolites: glycogen, triacylglycerides, and trehalose. Selected flies also had a higher activity of alanine transaminase and a higher urea content. Adaptation of the selected line on the transcriptomic level was characterized by down-regulation of genes encoding serine endopeptidases (Jon25i, Jon25ii, betaTry, and others) but up-regulation of genes encoding proteins related to the immune system, such as antimicrobial peptides, Turandot-family humoral factors, hexamerin isoforms, and vitellogenin. These sets of down- and up-regulated genes were similar to those observed in fruit flies with suppressed juvenile hormone signaling. These data show that the physiological adaptation of fruit flies to a high-protein-low-carbohydrate diet occurs via intuitive pathways, namely a decrease in food consumption, conversion of amino acids into ketoacids to compensate for the lack of carbohydrate, and accumulation of storage metabolites to eliminate the negative effects of excess amino acids. Nevertheless, transcriptomic adaptation occurs in a counter-intuitive way, likely via an influence of gut microbiota on food digestion (Yurkevych, 2020).

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Ruzzi, L. R., Schilman, P. E., San Martin, A., Lew, S. E., Gelb, B. D. and Pagani, M. R. (2020). The Phosphatase CSW Controls Life Span by Insulin Signaling and Metabolism Throughout Adult Life in Drosophila. Front Genet 11: 364. PubMed ID: 32457793

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Liao, S., Amcoff, M. and Nassel, D. R. (2020). Impact of high-fat diet on lifespan, metabolism, fecundity and behavioral senescence in Drosophila. Insect Biochem Mol Biol: 103495. PubMed ID: 33171202

Excess consumption of high-fat diet (HFD) is likely to result in obesity and increases the predisposition to associated health disorders. Drosophila melanogaster has emerged as an important model to study the effects of HFD on metabolism, gut function, behavior, and ageing. In this study, the effects of HFD on physiology and behavior of female flies was investigated at different time-points over several weeks. HFD was found to decrease lifespan, and also with age leads to accelerated decline of climbing ability in both virgins and mated flies. In virgins HFD also increased sleep fragmentation with age. Furthermore, long-term exposure to HFD results in elevated adipokinetic hormone (AKH) transcript levels and an enlarged crop with increased lipid stores. No long-term effects of HFD were detected on body mass, or levels of triacylglycerides (TAG), glycogen or glucose, although fecundity was diminished. However, one week of HFD resulted in decreased body mass and elevated TAG levels in mated flies. Finally, this study investigated the role of AKH in regulating effects of HFD during aging. Both with normal diet (ND) and HFD, Akh mutant flies displayed increased longevity compared to control flies. However, both mutants and controls showed shortened lifespan on HFD compared to ND. In flies exposed to ND, fecundity is decreased in Akh mutants compared to controls after one week, but increased after three weeks. However, HFD leads to a similar decrease in fecundity in both genotypes after both exposure times. Thus, long-term exposure to HFD increases AKH signaling, impairs lifespan and fecundity and augments age-related behavioral senescence (Liao, 2020).

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Bakalov, V., Reyes-Uribe, L., Deshpande, R., Maloy, A. L., Shapiro, S. D., Angus, D. C., Chang, C. H., Le Moyec, L., Wendell, S. G. and Kaynar, A. M. (2020). Dichloroacetate-induced metabolic reprogramming improves lifespan in a Drosophila model of surviving sepsis. PLoS One 15(11): e0241122. PubMed ID: 33151963

Sepsis is the leading cause of death in hospitalized patients and beyond the hospital stay and these long-term sequelae are due in part to unresolved inflammation. Metabolic shift from oxidative phosphorylation to aerobic glycolysis links metabolism to inflammation and such a shift is commonly observed in sepsis under normoxic conditions. By shifting the metabolic state from aerobic glycolysis to oxidative phosphorylation, it was hypothesized that the shift would reverse unresolved inflammation and subsequently improve outcome. This study proposes that a shift from aerobic glycolysis to oxidative phosphorylation as a sepsis therapy by targeting the pathways involved in the conversion of pyruvate into acetyl-CoA via pyruvate dehydrogenase (PDH). Chemical manipulation of PDH using dichloroacetic acid (DCA) will promote oxidative phosphorylation over glycolysis and decrease inflammation. This hypothesis was tested in a Drosophila melanogaster model of surviving sepsis infected with Staphylococcus aureus. Drosophila were divided into 3 groups: unmanipulated, sham and sepsis survivors, all treated with linezolid; each group was either treated or not with DCA for one week following sepsis. Lifespan, measured gene expression of Toll, defensin, cecropin A, and drosomycin, and levels of lactate, pyruvate, acetyl-CoA was studied as well as TCA metabolites. In this model, metabolic effects of sepsis are modified by DCA with normalized lactate, TCA metabolites, and was associated with improved lifespan of sepsis survivors, yet had no lifespan effects on unmanipulated and sham flies. While Drosomycin and cecropin A expression increased in sepsis survivors, DCA treatment decreased both and selectively increased defensin (Bakalov, 2020).

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Weigelt, C. M., Sehgal, R., Tain, L. S., Cheng, J., Esser, J., Pahl, A., Dieterich, C., Gronke, S. and Partridge, L. (2020). An insulin-sensitive circular RNA that regulates lifespan in Drosophila. Mol Cell 79(2): 268-279. PubMed ID: 32592682

Circular RNAs (circRNAs) are abundant and accumulate with age in neurons of diverse species. However, only few circRNAs have been functionally characterized, and their role during aging has not been addressed. This study uses transcriptome profiling during aging and find that accumulation of circRNAs is slowed down in long-lived insulin mutant flies. Next, the in vivo function was tested of a circRNA generated by the sulfateless gene (circSfl), which is consistently upregulated, particularly in the brain and muscle, of diverse long-lived insulin mutants. Strikingly, lifespan extension of insulin mutants is dependent on circSfl, and overexpression of circSfl alone is sufficient to extend the lifespan. Moreover, circSfl is translated into a protein that shares the N terminus and potentially some functions with the full-length Sfl protein encoded by the host gene. This study demonstrates that insulin signaling affects global circRNA accumulation and reveals an important role of circSfl during aging in vivo (Weigelt, 2020).

Circular RNAs (circRNAs) were originally identified more than 30 years ago, but for a long time they were thought to be by-products of the mRNA splicing process without a specific function; hence, they were not investigated further. Recently, circRNAs have been discovered in fungi, protists, and plants; C. elegans; Drosophila; mice; and humans. The majority of circRNA are generated by backsplicing of exons of protein-coding genes ('host genes'), and reverse complementary regions in the introns flanking circRNA-producing exons are crucial for circularization. Despite the high abundance and expression of certain circRNAs, only a few circRNAs have been functionally characterized; for instance, human CDR1as, which acts as an effective microRNA sponge. More recently, two independent reports have shown that a subset of circRNAs might be translated. circRNAs are enriched in neuronal tissues such as Drosophila heads and the mammalian brain. Furthermore, circRNAs have been shown to accumulate with age in C. elegans, in Drosophila heads and photoreceptor neurons, and in the mouse cortex and hippocampus but not in mouse heart tissue. However, a function of circRNAs in the aging process has not yet been revealed (Weigelt, 2020).

The nutrient-sensing insulin/insulin-like growth factor signaling (IIS) pathway is a key regulator of aging, metabolism, reproduction, and growth and is evolutionarily conserved from worms and flies to mice and humans. Downregulation of IIS pathway activity pharmacologically or by genetic modification extends the lifespan in C. elegans, Drosophila, and mice. In Drosophila, simultaneous knockout of three of the seven insulin-like peptides (dilp2-3,5) results in a robust lifespan extension of 30%-50% and ameliorates the age-related decline in sleep quality, suggesting that the healthspan is also extended. Proteome analysis of long-lived insulin mutants (genetic ablation of insulin-producing cells) revealed that the response to reduced insulin signaling and lifespan extension are highly tissue specific (Weigelt, 2020).

This study has characterized the functional link between circRNAs and insulin-mediated lifespan extension and aging. Tissue-specific, genome-wide, next-generation sequencing was used of wild-type and dilp2-3,5 mutant flies and hundreds of differentially expressed circRNAs were identified, including the circRNA encoded by the sulfateless (sfl) gene (hereafter referred to as circSfl). circSfl was highly upregulated in all tissues of several long-lived insulin mutants, and overexpression of circSfl alone was sufficient to extend the lifespan. Finally, evidence is provided that circSfl is translated into a small protein that may share some function with the protein encoded by the linear sfl transcripts. Importantly, overexpression of just the circSfl open reading frame (ORF) from a linear transcript was sufficient to extend longevity, implicating the protein encoded by circSfl in lifespan regulation. This study demonstrated that circRNAs are actively involved in the aging process and can influence the lifespan (Weigelt, 2020).

One of the most striking discoveries about circRNAs is the observation that they accumulate with age in neuronal tissues of diverse species. Several hypotheses regarding why circRNAs accumulate with age have been proposed. First, circRNAs are more stable compared with linear RNA molecules. Second, it has been suggested that circRNAs accumulate with age specifically in neuronal tissue because neurons are mostly post-mitotic, and, therefore, the more stable circRNAs are not degraded by proliferation or cell death. However, if this theory holds true, then circRNAs should accumulate in most tissues of the fruit fly because Drosophila is a mainly post-mitotic organism. Third, alternative splicing is increased and more error prone with age, potentially resulting in more backsplicing of circRNAs. This study showed that circRNA accumulation with age is slowed down in long-lived insulin mutants. This might point toward the third theory of why circRNAs accumulate with age and is supported by the finding that the splicing factor SFA-1 is required for dietary restriction-induced longevity in nematodes, highlighting the importance of splicing for lifespan extension upon deregulated nutrient sensing. These findings demonstrated that accumulation of circRNAs with age is malleable, suggesting that accumulation of circRNAs might be a potential aging biomarker (Weigelt, 2020).

This study identified several circRNAs that were differentially regulated in response to reduced insulin signaling in dilp 2-3,5 mutants, including circSfl. circSfl was also upregulated in two other insulin mutant flies that have an extended lifespan, and the upregulation is dependent on the dFoxo transcription factor, an essential mediator of longevity downstream of IIS. Notably, the magnitude of upregulation of circSfl in these mutants was correlated with the magnitude of the lifespan extension, with strong, up to 7-fold upregulation in the very long-lived dilp 2-3,5 mutants and only mildly upregulated in MNC-ablated flies and dFoxo overexpression flies (1.5- to 2-fold), which show a more mild lifespan extension. Similarly, the linear transcript Sfl RB was only upregulated in dilp2-3,5 mutant flies and not in the two other insulin mutants, suggesting that longevity and expression of the linear isoform can be uncoupled. Interestingly, neither circSfl nor the linear Sfl isoforms are upregulated upon rapamycin treatment or dietary restriction or in mth1 mutant flies, suggesting that upregulation of circSfl is not a general hallmark of lifespan-extending interventions in flies but specific to IIS-mediated longevity (Weigelt, 2020).

To overexpress circRNAs in vivo, different UAS constructs were tested. As expected, overexpression of the circRNA exon without its flanking introns did not lead to increased circRNA expression because flanking introns are required for biogenesis of circRNAs. In contrast, introducing reverse complementary matching flanking introns strongly increased biogenesis of circRNAs in vivo. These results are in line with previous studies that expressed circRNAs by engineering reverse complementary introns in zebrafish and in vitro. However, the first study that overexpressed a circRNA (circMbl) in vivo in Drosophila used a minigene construct including the circMbl exon and around 100 bp of the natural flanking introns but no inverted repeats. CircMbl overexpression led to a 4-fold increase in the circMbl expression level, much less than the strong overexpression achieved by engineered flanking introns. In summary, the mutants demonstrate that circRNAs can be efficiently overexpressed in Drosophila using engineered, reverse complementary matches in flanking introns that increase circRNA biogenesis in vivo (Weigelt, 2020).

Furthermore, sflΔex2 mutant flies were generated that lacked the Sfl RA-specific exon 2, and it was demonstrated that circSfl biogenesis is dependent on Sfl RA. Combination of sflΔex2 mutants with dilp 2-3,5 mutants revealed that the lifespan extension of dilp 2-3,5 mutants is partly dependent on the presence of this exon. The biogenesis of circRNAs is poorly understood, but several RNA binding proteins have been shown recently to inhibit or promote circularization, including Muscleblind, Quaking, and Adar1. It is tempting to speculate that an RNA binding protein might bind to the Sfl RA-specific exon and promote biogenesis of circSfl, which is abolished in sflΔex2 mutant flies. Because sflΔex2 mutants affect biogenesis of circSfl and expression of the linear Sfl RA isoform, it is currently not possible to formally exclude a role of the linear splice variant in insulin-mediated longevity. However, several lines of evidence suggest circSfl as the causal factor in this context. First, although Sfl RA expression was lost in sflΔex2 mutants, overall expression of linear Sfl was not affected. This is consistent with the finding that Sfl protein levels were not changed in dilp 2-3,5 mutant flies despite differential alternative splicing of the RA and RB isoforms. Thus, modifying exon 2 expression levels does not seem to affect Sfl protein levels, which can affect the lifespan. In addition, overexpression of circSfl and a linear transcript encoding the circSfl protein was sufficient to extend the lifespan, directly linking circSfl expression with longevity regulation. Given that most circRNAs are embedded in a host gene, generation of specific circRNA mutants that do not affect the host gene has been very challenging in the field. Because siRNA-mediated knockdown was not efficient in the case of circSfl, in new strategies should be tested in the future (e.g., by modification or deletion of the flanking introns that affect circRNA biogenesis), which can then be used to verify the hypothesis (Weigelt, 2020).

This study presented several lines of evidence showing that circSfl might be translated into a protein that is identical to the N terminus arising from linear Sfl transcripts. Sequence homology analysis showed that the in-frame stop codon after the circRNA-specific backsplice junction is conserved within Drosophila species that are separated by 10-20 million years of evolution, suggesting that the protein encoded by circSfl might also be conserved between these species. However, the identical stop codon could not be detected in more distantly related insect species, like honeybees or mosquitoes, which could indicate that the circlSfl protein is specific to Drosophila species or that other stop codons more downstream are used in other insects. A previous study identified 37 potentially translated circRNAs in Drosophila using ribosome footprinting on wild-type Drosophila heads; however, they failed to detect circSfl. Similarly, in the polysome profiling experiment, only very few circSfl reads were detected in wild-type fat bodies. In contrast, in dilp 2-3,5 mutant fat bodies, circSfl was one of the most abundant circRNAs, consistent with the insulin-dependent increase in circSfl transcript and protein levels. Furthermore, this study has shown that the protein encoded by circSfl and the protein arising from the linear Sfl transcripts can positively affect the lifespan of flies. This finding might indicate that both proteins affect the lifespan through overlapping mechanisms or by interacting with each other. Because the protein encoded by circSfl lacks the catalytic domain, it is unlikely that it acts as an active enzyme. Thus, circSfl may interact with proteins similar to the Sfl full-length protein in the cytoplasm or the membrane. For example, one could imagine that circSfl might interact with a repressor of the Sfl full-length protein, promoting the activity of Sfl and extending the lifespan. Alternatively, the truncated circSfl protein could also act as a dominant-negative protein because Sfl overexpression has also been suggested to cause a loss-of-function phenotype. Noteworthy is that overexpression of circSfl and Sfl caused tissue-specific effects on longevity, which could indicate that they work via different mechanisms or that different expression levels in different tissues are needed for the beneficial effects of the two proteins on lifespan. Interestingly, overexpression of circSfl and Sfl only extended the female but not the male lifespan despite upregulation of circSfl in dilp2-3,5 mutant males. This might reflect the gender bias in insulin-mediated longevity, in which females often show stronger effects than males (Weigelt, 2020).

The sfl gene in Drosophila encodes an Ndst and catalyzes synthesis of heparan sulfate (a glycosaminoglycan) by sulfation of the N and 6-O position of GlcNAc. Heparan sulfate is essential for wingless and fibroblast growth factor (FGF) receptor signaling, and full knockout of sfl is embryonic lethal. Sfl has been suggested to be localized to the Golgi apparatus and may be involved in the unfolded protein response. Furthermore, knockdown of Sfl increased the autophagy machinery and ubiquitinated proteins and reduced the climbing ability of flies, suggesting that Sfl is required for protein homeostasis and health. Similarly, this study has demonstrated that neuron-specific knockdown of Sfl is detrimental for the lifespan but that neuron-specific overexpression of Sfl extends the lifespan. Furthermore, this suty demonstrated, by genetic epistasis experiments, that Dally might contribute to the Sfl-mediated lifespan extension. Previous studies have demonstrated that overexpression of Sfl increases heparan sulfate levels and disrupts normal Wingless (Wg) and Decapentaplegic (Dpp) signaling, with the latter being controlled by Dally. However, similar to Sfl, the role of Dally has been characterized extensively during development but has not yet been implicated in aging (Weigelt, 2020).

In summary, this study demonstrated that neuronal circRNA accumulation with age is malleable and reduced in long-lived insulin mutants. Furthermore, this study established an efficient method to overexpress circRNAs in vivo by using reverse complementary introns. Interestingly, this study showed that a single circRNA (circSfl) can extend the lifespan in Drosophila. It is proposed that circSfl is translated into a protein that shares the same N terminus with the full-length protein arising from linear transcripts and potentially similar functions. This study will help to further elucidate the molecular mechanisms underlying longevity and provides unique insights into the in vivo function of circRNAs (Weigelt, 2020).

A limitation of this study is that it is currently unclear whether the circRNA-derived peptide and the full-length Sfl protein affect the lifespan by the same or by independent mechanisms. Lifespan extension by the full-length Sfl protein is dependent on its direct downstream target, the Dally protein. Thus, to address whether lifespan extension upon circSfl overexpression works in a similar way and also requires Dally, epistasis experiments were performed by co-overexpression of the circSfl protein and dally RNAi and the lifespan of these flies was measured. These experiments had to be terminated because of the current coronavirus crisis. In the future, it will be very interesting to further elucidate the mechanism of lifespan extension by circSfl and Sfl using genetic epistasis experiments (Weigelt, 2020).

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Escobedo, S. E., Stanhope, S. C., Dong, Z. and Weake, V. M. (2022). Aging and Light Stress Result in Overlapping and Unique Gene Expression Changes in Photoreceptors. Genes (Basel) 13(2). PubMed ID: 35205309
Summary:
The extended photoreceptor cell lifespan, in addition to its high metabolic needs due to phototransduction, makes it critical for these neurons to continually respond to the stresses associated with aging by mounting an appropriate gene expression response. This study sought to untangle the more general neuronal age-dependent transcriptional signature of photoreceptors with that induced by light stress. To do this, flies were aged or exposed to various durations of blue light, followed by photoreceptor nuclei-specific transcriptome profiling. Using this approach, genes were identified that are both common and uniquely regulated by aging and light induced stress. Whereas both age and blue light induce expression of DNA repair genes and a neuronal-specific signature of death, both conditions result in downregulation of phototransduction. Interestingly, blue light uniquely induced genes that directly counteract the overactivation of the phototransduction signaling cascade. Lastly, unique gene expression changes in aging photoreceptors included the downregulation of genes involved in membrane potential homeostasis and mitochondrial function, as well as the upregulation of immune response genes. It is proposed that light stress contributes to the aging transcriptome of photoreceptors, but that there are also other environmental or intrinsic factors involved in age-associated photoreceptor gene expression signatures.

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Taylor, J. R., Wood, J. G., Mizerak, E., Hinthorn, S., Liu, J., Finn, M., Gordon, S., Zingas, L., Chang, C., Klein, M. A., Denu, J. M., Gorbunova, V., Seluanov, A., Boeke, J. D., Sedivy, J. M. and Helfand, S. L. (2022). Sirt6 regulates lifespan in Drosophila melanogaster. Proc Natl Acad Sci U S A 119(5). PubMed ID: 35091469
Summary:

Sirt6 is a multifunctional enzyme that regulates diverse cellular processes such as metabolism, DNA repair, and aging. Overexpressing Sirt6 extends lifespan in mice, but the underlying cellular mechanisms are unclear. Drosophila melanogaster are an excellent model to study genetic regulation of lifespan; however, despite extensive study in mammals, very little is known about Sirt6 function in flies. This study characterized the Drosophila ortholog of Sirt6, dSirt6, and examined its role in regulating longevity; dSirt6 is a nuclear and chromatin-associated protein with NAD(+)-dependent histone deacetylase activity. dSirt6 overexpression (OE) in flies produces robust lifespan extension in both sexes, while reducing dSirt6 levels shortens lifespan. dSirt6 OE flies have normal food consumption and fertility but increased resistance to oxidative stress and reduced protein synthesis rates. Transcriptomic analyses reveal that dSirt6 OE reduces expression of genes involved in ribosome biogenesis, including many dMyc target genes. dSirt6 OE partially rescues many effects of dMyc OE, including increased nuclear size, up-regulation of ribosome biogenesis genes, and lifespan shortening. Last, dMyc haploinsufficiency does not convey additional lifespan extension to dSirt6 OE flies, suggesting dSirt6 OE is upstream of dMyc in regulating lifespan. These results provide insight into the mechanisms by which Sirt6 OE leads to longer lifespan.

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Krittika, S. and Yadav, P.. Alterations in lifespan and sleep/wake duration under selective monochromes of visible light in Drosophila melanogaster. Biol Open. PubMed ID: 35735020
Summary:

Rapid technology development, exposure to gadgets, and artificial lights (with different monochromes) have disturbed lifestyles and the circadian clock, which otherwise confers better regulation of behavioral patterns and sleep/wake cycles in most organisms including Drosophila melanogaster. This study assayed the effect of LD12:12 hr (light: dark) monochromatic lights (violet, blue, green, yellow, orange, and red) on the lifespan, activity, and sleep of the D. melanogaster. A shortened lifespan was observed under 12h of violet, blue, green, and yellow lights, while significantly reduced activity levels under the light phase of blue and green light as compared to their dark phase is observed. Significant increase in the evening anticipation index of flies under blue and green light alongside increased and decreased sleep depth during the day and night respectively suggests the light avoidance, while there is no effect of colored light on the waking time, daily active time, and sleep time. Thus, this study shows short and long-term exposure to certain colored lights in terms of reduced lifespan and locomotor activity, which cause qualitative as well as quantitative changes in the sleep of flies; probably as a sign of aversion towards a specific light.

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Du, X., Wang, Y., Wang, J., Liu, X., Chen, J., Kang, J., Yang, X. and Wang, H. (2022). d-Chiro-Inositol extends the lifespan of male Drosophila melanogaster better than d-Pinitol through insulin signaling and autophagy pathways. Exp Gerontol 165: 111856. PubMed ID: 35644418
Summary:

d-Pinitol (DP) is the methylated product of d-Chiro-Inositol (DCI), which is one of the nine isomers of inositol with optical activity. Both substances possess antioxidant activity. This study was conducted to investigate and compare the antioxidant and life-prolonging effects of DCI and DP on male Drosophila melanogaster. Results showed that DCI and DP prolonged the lifespan and improved the climbing, anti-stress, and antioxidant activities. After treatment with DCI and DP, intestinal homeostasis was improved and the abnormal proliferation of intestinal stem cells (ISCs) was attenuated. Furthermore, real-time PCR revealed downregulated expression levels of PI3K and Akt and upregulated expression levels of Dilp5 and FOXO, which consequently activated Atg1, Atg5, Atg8a, and Atg8b and increased the number of lysosomes. Altogether, DCI exerts a slightly better effect than DP based on various indicators. RNAi D. melanogaster lifespan and molecular docking results further suggested that DCI and DP could prolong longevity through insulin signaling (IIS) and autophagy pathways.

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Mlih, M. and Karpac, J. (2022). Integrin-ECM interactions and membrane-associated Catalase cooperate to promote resilience of the Drosophila intestinal epithelium. PLoS Biol 20(5): e3001635. PubMed ID: 35522719
Summary:

Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. Diedel, a secreted protein/cytokine, has been identified as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. This study shows that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, a resilience mechanism was uncovered of epithelial tissues, mediated by Integrin-ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, It was found that resilient epithelial cells, enriched for Diedel-Integrin-ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin-ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin-ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues.

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Malacrida, S., De Lazzari, F., Mrakic-Sposta, S., Vezzoli, A., Zordan, M. A., Bisaglia, M., Menti, G. M., Meda, N., Frighetto, G., Bosco, G., Dal Cappello, T., Strapazzon, G., Reggiani, C., Gussoni, M. and Megighian, A. (2022). Lifespan and ROS levels in different Drosophila melanogaster strains after 24 h hypoxia exposure. Biol Open 11(6). PubMed ID: 35616023
Summary:

During recent decades, model organisms such as Drosophila melanogaster have made it possible to study the effects of different environmental oxygen conditions on lifespan and oxidative stress. In this study, longevity and ROS levels were compared in young, unmated males of three laboratory wild-type lines (Canton-S, Oregon-R and Berlin-K) and one mutant line (Sod1n1) as a positive control of redox imbalance, under both normoxic and hypoxic (2% oxygen for 24 h) conditions. The genetic background was found to be a relevant factor involved in D. melanogaster longevity and ROS levels. Indeed, as expected, in normoxia Sod1n1 are the shortest-lived, while the wild-type strains, despite a longer lifespan, show some differences, with the Canton-S line displaying the lowest mortality rate. After hypoxic stress these variances are amplified, with Berlin-K flies showing the highest mortality rate and most evident reduction of lifespan. Moreover, this analysis highlighted differential effects of hypoxia on redox balance/unbalance. Canton-S flies had the lowest increase of ROS level compared to all the other strains, confirming it to be the less sensitive to hypoxic stress. Sod1n1 flies displayed the highest ROS levels in normoxia and after hypoxia. These results should be used to further standardize future Drosophila research models designed to investigate genes and pathways that may be involved in lifespan and/or ROS, as well as comparative studies on specific mutant strains.

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Mlih, M. and Karpac, J. (2022). Integrin-ECM interactions and membrane-associated Catalase cooperate to promote resilience of the Drosophila intestinal epithelium. PLoS Biol 20(5): e3001635. PubMed ID: 35522719
Summary:

Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. Diedel, a secreted protein/cytokine, has been identified as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. This study shows that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, a resilience mechanism was uncovered of epithelial tissues, mediated by Integrin-ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, It was found that resilient epithelial cells, enriched for Diedel-Integrin-ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin-ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin-ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues.

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Hayashi, Y., Kashio, S., Murotomi, K., Hino, S., Kang, W., Miyado, K., Nakao, M., Miura, M., Kobayashi, S. and Namihira, M. (2022). Serotonin and dopamine modulate aging in response to food odor and availability. Nat Commun 13(1): 3271. PubMed ID: 35672307
Summary:

An organism's ability to perceive and respond to changes in its environment is crucial for its health and survival. This study reveals how the most well-studied longevity intervention, dietary restriction, acts in-part through a cell non-autonomous signaling pathway that is inhibited by the presence of attractive smells. Using an intestinal reporter for a key gene induced by dietary restriction but suppressed by attractive smells, this study identified three compounds that block food odor effects in C. elegans, thereby increasing longevity as dietary restriction mimetics. These compounds clearly implicate serotonin and dopamine in limiting lifespan in response to food odor. A chemosensory neuron that likely perceives food odor, an enteric neuron that signals through the serotonin receptor 5-HT1A/SER-4, and a dopaminergic neuron that signals through the dopamine receptor DRD2/DOP-3. Aspects of this pathway are conserved in D. melanogaster. Thus, blocking food odor signaling through antagonism of serotonin or dopamine receptors is a plausible approach to mimic the benefits of dietary restriction.

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Shaposhnikov, M. V., Guvatova, Z. G., Zemskaya, N. V., Koval, L. A., Schegoleva, E. V., Gorbunova, A. A., Golubev, D. A., Pakshina, N. R., Ulyasheva, N. S., Solovev, I. A., Bobrovskikh, M. A., Gruntenko, N. E., Menshanov, P. N., Krasnov, G. S., Kudryavseva, A. V. and Moskalev, A. A. (2022). Molecular mechanisms of exceptional lifespan increase of Drosophila melanogaster with different genotypes after combinations of pro-longevity interventions. Commun Biol 5(1): 566. PubMed ID: 35681084
Summary:

Aging is one of the current global challenges. The search for new anti-aging interventions is also an issue of great actuality. This report describes the success of Drosophila melanogaster lifespan extension under the combined influence of dietary restriction, co-administration of berberine, fucoxanthin, and rapamycin, photodeprivation, and low-temperature conditions up to 185 days in w(1118) strain and up to 213 days in long-lived E(z)/w mutants. The trade-off was found between longevity and locomotion. The transcriptome analysis showed an impact of epigenetic alterations, lipid metabolism, cellular respiration, nutrient sensing, immune response, and autophagy in the registered effect.

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Delventhal, R., Wooder, E. R., Basturk, M., Sattar, M., Lai, J., Bolton, D., Muthukumar, G., Ulgherait, M. and Shirasu-Hiza, M. M (2022). Dietary restriction ameliorates TBI-induced phenotypes in Drosophila melanogaster. Sci Rep 12(1): 9523. PubMed ID: 35681073
Summary:

Traumatic brain injury (TBI) affects millions annually and is associated with long-term health decline. TBI also shares molecular and cellular hallmarks with neurodegenerative diseases (NDs), typically increasing in prevalence with age, and is a major risk factor for developing neurodegeneration later in life. While understanding of genes and pathways that underlie neurotoxicity in specific NDs has advanced, a complete understanding of early molecular and physiological changes that drive neurodegeneration, particularly as an individual ages following a TBI, is lacking. Recently Drosophila has been introduced as a model organism for studying closed-head TBI. In this paper, a TBI is delivered to flies early in adult life, and then molecular and physiological phenotypes were measured at short-, mid-, and long-term timepoints following the injury. The aim was to identify the timing of changes that contribute to neurodegeneration. Prior work is confirmed demonstrating a TBI-induced decline in lifespan, and evidence is presented of a progressive decline in locomotor function, robust acute and modest chronic neuroinflammation, and a late-onset increase in protein aggregation. Evidence is also presented of metabolic dysfunction, in the form of starvation sensitivity and decreased lipids, that persists beyond the immediate injury response, but does not differ long-term. An intervention of dietary restriction (DR) partially ameliorates some TBI-induced phenotypes, including lifespan and locomotor function, though it does not alter the pattern of starvation sensitivity of injured flies. In the future, molecular pathways identified as altered following TBI-particularly in the short-, or mid-term-could present potential therapeutic targets.

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Gautrey, S. L. and Simons, M. J. P. (2022). Amino acid availability is not essential for lifespan extension by dietary restriction in the fly. J Gerontol A Biol Sci Med Sci. PubMed ID: 35486979
Summary:

Dietary restriction (DR) is one of the most potent ways to extend health- and lifespan. Key progress in understanding the mechanisms of DR, and ageing more generally, was made when dietary protein, and more specifically essential amino acids (EAA), were identified as the dietary component to restrict to obtain DR's health and lifespan benefits. This role of dietary amino acids has influenced work on ageing mechanisms, especially in nutrient sensing, e.g. Tor and insulin(-like) signalling networks. Experimental biology in Drosophila melanogaster has been instrumental in generating and confirming the hypothesis that EAA availability is important in ageing. This study expanded on previous work testing the involvement of EAA in DR through large scale (N=6,238) supplementation experiments across four diets and two genotypes in female flies. Surprisingly, it was found that EAA are not essential to DR's lifespan benefits. Importantly, the fecundity benefits of EAA supplementation were not identified suggesting the supplemented EAA were bioavailable. Furthermore, it was found that the effects of amino acids on lifespan vary by diet and genetic line studied and that, at the most restricted diet, fecundity is constrained by other nutrients than EAA. It is suggested that DR for optimal health is a concert of nutritional effects, orchestrated by genetic, dietary and other environmental interactions. These results question the universal importance of amino acid availability in the biology of ageing and DR.

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Hodge, B. A., Meyerhof, G. T., Katewa, S. D., Lian, T., Lau, C., Bar, S., Leung, N. Y., Li, M., Li-Kroeger, D., Melov, S., Schilling, B., Montell, C. and Kapahi, P.. Dietary restriction and the transcription factor clock delay eye aging to extend lifespan in Drosophila Melanogaster. Nat Commun 13(1): 3156. PubMed ID: 35672419
Summary:

Many vital processes in the eye are under circadian regulation, and circadian dysfunction has emerged as a potential driver of eye aging. Dietary restriction is one of the most robust lifespan-extending therapies and amplifies circadian rhythms with age. This study demonstrates that dietary restriction extends lifespan in Drosophila melanogaster by promoting circadian homeostatic processes that protect the visual system from age- and light-associated damage. Altering the positive limb core molecular clock transcription factor, CLOCK, or CLOCK-output genes, accelerates visual senescence, induces a systemic immune response, and shortens lifespan. Flies subjected to dietary restriction are protected from the lifespan-shortening effects of photoreceptor activation. Inversely, photoreceptor inactivation, achieved via mutating rhodopsin or housing flies in constant darkness, primarily extends the lifespan of flies reared on a high-nutrient diet. These findings establish the eye as a diet-sensitive modulator of lifespan and indicates that vision is an antagonistically pleiotropic process that contributes to organismal aging.

Hayashi, Y., Kashio, S., Murotomi, K., Hino, S., Kang, W., Miyado, K., Nakao, M., Miura, M., Kobayashi, S. and Namihira, M. (2022). Biosynthesis of S-adenosyl-methionine enhances aging-related defects in Drosophila oogenesis. Sci Rep 12(1): 5593. PubMed ID: 35379840
Summary:

Exposing sires to various environmental manipulations has demonstrated that paternal effects can be non-trivial also in species where male investment in offspring is almost exclusively limited to sperm. Whether paternal effects also have a genetic component (i.e. paternal indirect genetic effects (PIGEs)) in such species is however largely unknown, primarily because of methodological difficulties separating indirect from direct effects of genes. PIGEs may nevertheless be important since they have the capacity to contribute to evolutionary change. This studyd used Drosophila genetics to construct a breeding design that allows testing nearly complete haploid genomes (more than 99%) for PIGEs. Using this technique, the variance was estimated in male lifespan due to PIGEs among four populations and compare this to the total paternal genetic variance (the sum of paternal indirect and direct genetic effects). These results indicate that a substantial part of the total paternal genetic variance results from PIGEs. A screen of 38 haploid genomes, randomly sampled from a single population, suggests that PIGEs also influence variation in lifespan within populations. Collectively, these results demonstrate that PIGEs may constitute an underappreciated source of phenotypic variation.

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Piper, M. D. W., Zanco, B., Sgro, C. M., Adler, M. I., Mirth, C. K. and Bonduriansky, R. (2022). Dietary restriction and lifespan: adaptive reallocation or somatic sacrifice?. Febs j. PubMed ID: 35466532
Summary:

Reducing overall food intake, or lowering the proportion of protein relative to other macronutrients, can extend the lifespan of diverse organisms. A number of mechanistic theories have been developed to explain this phenomenon, mostly assuming that the molecules connecting diet to lifespan are evolutionarily conserved. A recent study using Drosophila melanogaster females has pinpointed a single essential micronutrient that can explain how lifespan is changed by dietary restriction. This study proposes a likely mechanism for this observation, which involves a trade-off between lifespan and reproduction, but in a manner that is conditional on the dietary supply of an essential micronutrient - a sterol. Importantly, these observations argue against previous evolutionary theories that rely on constitutive resource reallocation or damage directly inflicted by reproduction. Instead, they are compatible with a model in which the inverse relationship between lifespan and food level is caused by the consumer suffering from varying degrees of malnutrition when maintained on lab food. The data also indicate that animals on different lab foods may suffer from different nutritional imbalances and that the mechanisms by which dietary restriction benefits the lifespan of different species may vary. This means that translating the mechanistic findings from lab animals to humans will not be simple and should be interpreted in light of the range of challenges that have shaped each organism's lifespan in the wild and the composition of the natural diets upon which they would feed.

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Butsch, T. J., Dubuisson, O., Johnson, A. E. and Bohnert, K. A. (2022). A meiotic switch in lysosome activity supports spermatocyte development in young flies but collapses with age. iScience 25(6): 104382. PubMed ID: 35620438
Summary:

Gamete development ultimately influences animal fertility. Identifying mechanisms that direct gametogenesis, and how they deteriorate with age, may inform ways to combat infertility. Recentl work has shown that lysosomes acidify during oocyte maturation in Caenorhabditis elegans, suggesting that a meiotic switch in lysosome activity promotes female germ-cell health. Using Drosophila melanogaster, this study reports that lysosomes likewise acidify in male germ cells during meiosis. Inhibiting lysosomes in young-male testes causes E-cadherin accumulation and loss of germ-cell partitioning membranes. Notably, analogous changes occur naturally during aging; in older testes, a reduction in lysosome acidity precedes E-cadherin accumulation and membrane dissolution, suggesting one potential cause of age-related spermatocyte abnormalities. Consistent with lysosomes governing the production of mature sperm, germ cells with homozygous-null mutations in lysosome-acidifying machinery fail to survive through meiosis. Thus, lysosome activation is entrained to meiotic progression in developing sperm, as in oocytes, and lysosomal dysfunction may instigate male reproductive aging.

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Li, Y., Pan, L., Li, P., Yu, G., Li, Z., Dang, S., Jin, F. and Nan, Y. (2022). Microbiota aggravates the pathogenesis of Drosophila acutely exposed to vehicle exhaust. Heliyon 8(9): e10382. PubMed ID: 36060467
Summary:

Vehicle exhaust (VE) is the primary cause of urban air pollution, which adversely affects the respiratory system, exacerbates lung diseases, and results in high mortality rates. However, the underlying mechanism of the pathogenesis is largely unclear. This study developed a Drosophila model to systematically investigate the effects of VE on their health and physiology. VE was found to significantly impaired life span and locomotion in Drosophila. Interestingly, there was an increase in bacterial load in the guts upon VE exposure, suggesting VE is able to induce dysbiosis in the guts. Microbiota depletion can ameliorate the impairment of life span and locomotion. VE causes permeability of intestinal epithelial cells and increases proliferation of intestinal cells, suggesting VE disrupts intestinal homeostasis. This study elucidated the underlying mechanism by which VE triggers Imd and DUOX gene expression. Taken together, this Drosophila model provides insight into the pathogenesis of Drosophila exposure to VE, enabling better understanding of the specific role of microbiota.

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Li, Y., Pan, L., Li, P., Yu, G., Li, Z., Dang, S., Jin, F. and Nan, Y. (2022). Microbiota aggravates the pathogenesis of Drosophila acutely exposed to vehicle exhaust. Heliyon 8(9): e10382. PubMed ID: 36060467
Summary:

Vehicle exhaust (VE) is the primary cause of urban air pollution, which adversely affects the respiratory system, exacerbates lung diseases, and results in high mortality rates. However, the underlying mechanism of the pathogenesis is largely unclear. This study developed a Drosophila model to systematically investigate the effects of VE on their health and physiology. VE was found to significantly impaired life span and locomotion in Drosophila. Interestingly, there was an increase in bacterial load in the guts upon VE exposure, suggesting VE is able to induce dysbiosis in the guts. Microbiota depletion can ameliorate the impairment of life span and locomotion. VE causes permeability of intestinal epithelial cells and increases proliferation of intestinal cells, suggesting VE disrupts intestinal homeostasis. This study elucidated the underlying mechanism by which VE triggers Imd and DUOX gene expression. Taken together, this Drosophila model provides insight into the pathogenesis of Drosophila exposure to VE, enabling better understanding of the specific role of microbiota.

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Hewitt, V. L., Miller-Fleming, L., Twyning, M. J., Andreazza, S., Mattedi, F., Prudent, J., Polleux, F., Vagnoni, A. and Whitworth, A. J. (2022). Decreasing pdzd8-mediated mito-ER contacts improves organismal fitness and mitigates Aβ(42) toxicity. Life Sci Alliance 5(11). PubMed ID: 35831024
Summary:

Mitochondria<>/a-ER contact sites (MERCs) orchestrate many important cellular functions including regulating mitochondrial quality control through mitophagy and mediating mitochondrial calcium uptake. This study identified and functionally characterize the Drosophila ortholog of the recently identified mammalian MERC protein, Pdzd8. Reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion, and reduces lifespan. Although depletion of pdzd8 prolongs the survival of flies fed with mitochondrial toxins, it is also sufficient to rescue locomotor defects of a fly model of Alzheimer's disease expressing Amyloid β(42) (Aβ(42)). Together, these results provide the first in vivo evidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis.

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Strilbytska, O., Strutynska, T., Semaniuk, U., Burdyliyk, N., Bubalo, V. and Lushchak, O. (2022). Dietary Sucrose Determines Stress Resistance, Oxidative Damages, and Antioxidant Defense System in Drosophila. Scientifica (Cairo) 2022: 7262342. PubMed ID: 35547569/a>
Summary:

Varied nutritional interventions affect lifespan and metabolic health. Abundant experimental evidence indicates that the carbohydrate restriction in the diet induces changes to support long-lived phenotypes. Reactive oxygen species (ROS) are among the main mechanisms that mediate the effect of nutrient consumption on the aging process. This study tested the influence of sucrose concentration in the diet on stress resistance, antioxidant defense systems, and oxidative stress markers in D. melanogaster. High sucrose concentration in the fly medium was found to lead to enhanced resistance to starvation, oxidative, heat, and cold stresses. However, flies that were raised on low sucrose food displayed increased levels of low-molecular-mass thiols, lipid peroxides in females, and higher activity of antioxidant enzymes, indicating that the consumption of a low carbohydrate diet could induce oxidative stress in the fruit fly. The consumption of sucrose-enriched diet increased protein carbonyl level, which may indicate about the activation of glycation processes. The results highlight a strong dependence of oxidative metabolism in D. melanogaster from dietary carbohydrates.

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Aiello, G., Sabino, C., Pernici, D., Audano, M., Antonica, F., Gianesello, M., Ballabio, C., Quattrone, A., Mitro, N., Romanel, A., Soldano, A. and Tiberi, L. (2022). Transient rapamycin treatment during developmental stage extends lifespan in Mus musculus and Drosophila melanogaster. EMBO Rep 23(9): e55299. PubMed ID: 35796299
Summary:

Lifespan is determined by complex and tangled mechanisms that are largely unknown. The early postnatal stage has been proposed to play a role in lifespan, but its contribution is still controversial. This show that a short rapamycin treatment during early life can prolong lifespan in Mus musculus and Drosophila melanogaster. Notably, the same treatment at later time points has no effect on lifespan, suggesting that a specific time window is involved in lifespan regulation. It was also found that sulfotransferases are upregulated during early rapamycin treatment both in newborn mice and in Drosophila larvae, and transient dST1 overexpression in Drosophila larvae extends lifespan. These findings unveil a novel link between early-life treatments and long-term effects on lifespan.

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Shit, B., Prakash, A., Sarkar, S., Vale, P. F. and Khan, I. (2022). Ageing leads to reduced specificity of antimicrobial peptide responses in Drosophila melanogaster. Proc Biol Sci 289(1987): 20221642. PubMed ID: 36382522
Summary:
Evolutionary theory predicts a late-life decline in the force of natural selection, possibly leading to late-life deregulations of the immune system. A potential outcome of such deregulations is the inability to produce specific immunity against target pathogens. This possibility was tested by infecting multiple Drosophila melanogaster lines (with bacterial pathogens) across age groups, where either individual or different combinations of Imd- and Toll-inducible antimicrobial peptides (AMPs) were deleted using CRISPR gene editing. A high degree of non-redundancy and pathogen-specificity of AMPs was demonstrated in young flies: in some cases, even a single AMP could confer complete resistance. However, ageing led to drastic reductions in such specificity to target pathogens, warranting the action of multiple AMPs across Imd and Toll pathways. Moreover, use of diverse AMPs either lacked survival benefits or even accompanied survival costs post-infection. These features were also sexually dimorphic: females required a larger repertoire of AMPs than males but extracted equivalent survival benefits. Finally, age-specific expansion of the AMP-repertoire was accompanied with ageing-induced downregulation of negative-regulators of the Imd pathway and damage to renal function post-infection, as features of poorly regulated immunity. Overall, this study highlights the potentially non-adaptive role of ageing in producing less-specific AMP responses, across sexes and pathogens.

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Hoedjes, K. M., Kostic, H., Keller, L. and Flatt, T. (2022). Natural alleles at the Doa locus underpin evolutionary changes in Drosophila lifespan and fecundity. Proc Biol Sci 289(1986): 20221989. PubMed ID: 36350205
Summary:
'Evolve and resequence' (E&R) studies in Drosophila melanogaster have identified many candidate loci underlying the evolution of ageing and life history, but experiments that validate the effects of such candidates remain rare. In a recent E&R study several alleles were identified of the LAMMER kinase Darkener of apricot (Doa) as candidates for evolutionary changes in lifespan and fecundity. This study used two complementary approaches to confirm a functional role of Doa in life-history evolution. First, transgenic RNAi was used to study the effects of Doa at the whole-gene level. Ubiquitous silencing of expression in adult flies reduced both lifespan and fecundity, indicating pleiotropic effects. Second, to characterize segregating variation at Doa, four candidate single nucleotide polymorphisms (SNPs; Doa-1, -2, -3, -4) were examined using a genetic association approach. Three candidate SNPs had effects that were qualitatively consistent with expectations based on the E&R study: Doa-2 pleiotropically affected both lifespan and late-life fecundity; Doa-1 affected lifespan (but not fecundity); and Doa-4 affected late-life fecundity (but not lifespan). Finally, the last candidate allele (Doa-3) also affected lifespan, but in the opposite direction from predicted.

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Lushchak, O., Strilbytska, O. and Storey, K. B. (2022). Gender-Specific Effects of Pro-longevity Interventions in Drosophila. Mech Ageing Dev: 111754. PubMed ID: 36375654
Summary:
Sex differences in lifespan are well recognized in the majority of animal species. For example, in male versus female Drosophila melanogaster there are significant differences in behavior and physiology. However, little is known about the underlying mechanisms of gender differences in responses to pro-longevity interventions in this model organism. This study summarized the existing data on the effects of nutritional and pharmacological anti-aging interventions such as nutrition regimens, diet and dietary supplementation on the lifespan of male and female Drosophila. Males and females have different sensitivities to interventions, and the effects are highly dependent on genetic background, mating, dose and exposure duration. Thia work highlights the importance of understanding the mechanisms that underlie the gender-specific effect of anti-aging manipulations. This will provide insight into how these benefits may be valuable for elucidating the primary physiological and molecular targets involved in aging and lifespan determination.

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G, N. L., Ko, S., Peng, O., Bognar, B., Khmelkov, M., H, S. B. and Tower, J. (2022) (2022). A screen of small molecule and genetic modulators of life span in female Drosophila identifies etomoxir, RH5849 and unanticipated temperature effects. Fly (Austin) 16(1): 397-413. PubMed ID: 36412257
Summary:
Mifepristone increases life span in female Drosophila melanogaster, and its molecular target(s) remain unclear. Small molecule and genetic interventions were tested for ability to mimic mifepristone, or to decrease life span in a way that can be rescued by mifepristone. Etomoxir inhibits lipid metabolism, and significantly increased life span in virgin and mated females, but not males, at 50 μM concentration. Pioglitazone is reported to activate both mammalian PPAR γ and its Drosophila homolog Eip75B. Pioglitazone produced minor and inconsistent benefits for female Drosophila life span, and only at the lowest concentrations tested. Ecdysone is a Drosophila steroid hormone reported to regulate responses to mating, and RH5849 is a potent mimic of ecdysone. RH5849 reduced virgin female life span, and this was partly rescued by mifepristone. Mifepristone did not compete with RH5849 for activation of an ecdysone receptor (EcR)-responsive transgenic reporter, indicating that the relevant target for mifepristone is not EcR. The conditional GAL4/GAL80ts system was used in attempt to test the effect of an Eip75B RNAi construct on female life span. However, the 29C temperature used for induction reduced or eliminated mating-induced midgut hypertrophy, the negative life span effects of mating, and the positive life span effects of mifepristone. Even when applied after mating was complete, a shift to 29°C temperature reduced mating-induced midgut hypertrophy by half, and the life span effects of mating by 4.8-fold. Taken together, these results identify promising small molecules for further analysis, and inform the design of experiments involving the GAL4/GAL80ts system.

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Wang, R., Zhang, P., Wang, J., Ma, L., E, W., Suo, S., Jiang, M., Li, J., Chen, H., Sun, H., Fei, L., Zhou, Z., Zhou, Y., Chen, Y., Zhang, W., Wang, X., Mei, Y., Sun, Z., Yu, C., Shao, J., Fu, Y., Xiao, Y., Ye, F., Fang, X., Wu, H., Guo, Q., Fang, X., Li, X., Gao, X., Wang, D., Xu, P. F., Zeng, R., Xu, G., Zhu, L., Wang, L., Qu, J., Zhang, D., Ouyang, H., Huang, H., Chen, M., Ng, S. C., Liu, G. H., Yuan, G. C., Guo, G. and Han, X.(2022). Construction of a cross-species cell landscape at single-cell level. Nucleic Acids Res. PubMed ID: 35929025
Summary:
Individual cells are basic units of life. Despite extensive efforts to characterize the cellular heterogeneity of different organisms, cross-species comparisons of landscape dynamics have not been achieved. This study applied single-cell RNA sequencing (scRNA-seq) to map organism-level cell landscapes at multiple life stages for mice, zebrafish and Drosophila. By integrating the comprehensive dataset of > 2.6 million single cells, a cross-species cell landscape was developed, and signatures and common pathways were identified that changed throughout the life span. structural inflammation and mitochondrial dysfunction were identified as the most common hallmarks of organism aging, and pharmacological activation of mitochondrial metabolism was found to alleviated aging phenotypes in mice. The cross-species cell landscape with other published datasets were stored in an integrated online portal-Cell Landscape. This work provides a valuable resource for studying lineage development, maturation and aging. How many cell types are there in nature? How do they change during the life cycle? These are two fundamental questions that researchers have been trying to understand in the area of biology. In this study, single-cell mRNA sequencing data were used to profile over 2.6 million individual cells from mice, zebrafish and Drosophila at different life stages, 1.3 million of which were newly collected. The comprehensive datasets allow investigators to construct a cross-species cell landscape that helps to reveal the conservation and diversity of cell taxonomies at genetic and regulatory levels. The resources in this study are assembled into a publicly available at website

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Ozaki, M., Le, T. D. and Inoue, Y. H. (2022). Downregulating Mitochondrial DNA Polymerase γ in the Muscle Stimulated Autophagy, Apoptosis, and Muscle Aging-Related Phenotypes in Drosophila Adults. Biomolecules 12(8). PubMed ID: 36008999
Summary:
Reactive oxygen species, generated as by-products of mitochondrial electron transport, can induce damage to mitochondrial DNA (mtDNA) and proteins. This study investigated whether the moderate accumulation of mtDNA damage in adult muscles resulted in accelerated aging-related phenotypes in Drosophila. DNA polymerase γ (Polγ) is the sole mitochondrial DNA polymerase. The muscle-specific silencing of the genes encoding the polymerase subunits resulted in the partial accumulation of mtDNA with oxidative damage and a reduction in the mtDNA copy number. This subsequently resulted in the production of abnormal mitochondria with reduced membrane potential and, consequently, a partially reduced ATP quantity in the adult muscle. Immunostaining indicated a moderate increase in autophagy and mitophagy in adults with RNA interference of Polγ (PolγRNAi) muscle cells with abnormal mitochondria. In adult muscles showing continuous silencing of Polγ, malformation of both myofibrils and mitochondria was frequently observed. This was associated with the partially enhanced activation of pro-apoptotic caspases in the muscle. Adults with muscle-specific PolγRNAi exhibited a shortened lifespan, accelerated age-dependent impairment of locomotor activity, and disturbed circadian rhythms. These findings in this Drosophila model contribute to understanding how the accumulation of mtDNA damage results in impaired mitochondrial activity and how this contributes to muscle aging.

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Ozaki, M., Le, T. D. and Inoue, Y. H. (2022). Downregulating Mitochondrial DNA Polymerase γ in the Muscle Stimulated Autophagy, Apoptosis, and Muscle Aging-Related Phenotypes in Drosophila Adults. Biomolecules 12(8). PubMed ID: 36008999
Summary:
Reactive oxygen species, generated as by-products of mitochondrial electron transport, can induce damage to mitochondrial DNA (mtDNA) and proteins. This study investigated whether the moderate accumulation of mtDNA damage in adult muscles resulted in accelerated aging-related phenotypes in Drosophila. DNA polymerase γ (Polγ) is the sole mitochondrial DNA polymerase. The muscle-specific silencing of the genes encoding the polymerase subunits resulted in the partial accumulation of mtDNA with oxidative damage and a reduction in the mtDNA copy number. This subsequently resulted in the production of abnormal mitochondria with reduced membrane potential and, consequently, a partially reduced ATP quantity in the adult muscle. Immunostaining indicated a moderate increase in autophagy and mitophagy in adults with RNA interference of Polγ (PolγRNAi) muscle cells with abnormal mitochondria. In adult muscles showing continuous silencing of Polγ, malformation of both myofibrils and mitochondria was frequently observed. This was associated with the partially enhanced activation of pro-apoptotic caspases in the muscle. Adults with muscle-specific PolγRNAi exhibited a shortened lifespan, accelerated age-dependent impairment of locomotor activity, and disturbed circadian rhythms. These findings in this Drosophila model contribute to understanding how the accumulation of mtDNA damage results in impaired mitochondrial activity and how this contributes to muscle aging.

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Hayashi, Y., Kashio, S., Murotomi, K., Hino, S., Kang, W., Miyado, K., Nakao, M., Miura, M., Kobayashi, S. and Namihira, M. (2022). Biosynthesis of S-adenosyl-methionine enhances aging-related defects in Drosophila oogenesis. Sci Rep 12(1): 5593. PubMed ID: 35379840
Summary:
Over recent decades, increased longevity has not been paralleled by extended health span, resulting in more years spent with multiple diseases in older age. As such, interventions to improve health span are urgently required. Zoledronate (Zol) is a nitrogen-containing bisphosphonate, which inhibits the farnesyl pyrophosphate synthase enzyme, central to the mevalonate pathway. It is already used clinically to prevent fractures in osteoporotic patients, who have been reported to derive unexpected and unexplained survival benefits. Using Drosophila as a model the effects were determined of Zol on life span, parameters of health span (climbing ability and intestinal dysplasia), and the ability to confer resistance to oxidative stress using a combination of genetically manipulated Drosophila strains and Western blotting. This study study shows that Zol extended life span, improved climbing activity, and reduced intestinal epithelial dysplasia and permeability with age. Mechanistic studies showed that Zol conferred resistance to oxidative stress and reduced accumulation of X-ray-induced DNA damage via inhibition of farnesyl pyrophosphate synthase. Moreover, Zol was associated with inhibition of phosphorylated AKT in the mammalian target of rapamycin pathway downstream of the mevalonate pathway and required dFOXO for its action, both molecules associated with increased longevity. Taken together, this work indicates that Zol, a drug already widely used to prevent osteoporosis and dosed only once a year, modulates important mechanisms of aging. Its repurposing holds great promise as a treatment to improve health span.

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Hayashi, Y., Kashio, S., Murotomi, K., Hino, S., Kang, W., Miyado, K., Nakao, M., Miura, M., Kobayashi, S. and Namihira, M. (2022). Biosynthesis of S-adenosyl-methionine enhances aging-related defects in Drosophila oogenesis. Sci Rep 12(1): 5593. PubMed ID: 35379840
Summary:
cAMP Responsible Element Binding Protein (CREB) is an evolutionarily conserved transcriptional factor that regulates cell growth, synaptic plasticity and so on. This study unexpectedly found proteasome inhibitors, such as MLN2238, robustly increase CREB activity in adult flies through a large-scale compound screening. Mechanistically, reactive oxidative species (ROS) generated by proteasome inhibition are required and sufficient to promote CREB activity through c-Jun N-terminal kinase (JNK). In 293 T cells, JNK activation by MLN2238 is also required for increase of CREB phosphorylation at Ser(133). Meanwhile, transcriptome analysis in fly intestine identified a group of genes involved in redox and proteostatic regulation are augmented by overexpressing CRTC (CREB-regulated transcriptional coactivator). Intriguingly, CRTC overexpression in muscles robustly restores protein folding and proteasomal activity in a fly Huntington's disease (HD) model, and ameliorates HD related pathogenesis, such as protein aggregates, motility, and lifespan. Moreover, CREB activity increases during aging, and further enhances its activity can suppress protein aggregates in aged muscles. Together, these results identified CRTC/CREB downstream ROS/JNK signaling as a conserved sensor to tackle oxidative and proteotoxic stresses. Boosting CRTC/CREB activity is a potential therapeutic strategy to treat aging related protein aggregation diseases.

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Hayashi, Y., Kashio, S., Murotomi, K., Hino, S., Kang, W., Miyado, K., Nakao, M., Miura, M., Kobayashi, S. and Namihira, M. (2022). Biosynthesis of S-adenosyl-methionine enhances aging-related defects in Drosophila oogenesis. Sci Rep 12(1): 5593. PubMed ID: 35379840
Summary:
Tissue aging is a major cause of aging-related disabilities and a shortened life span. Understanding how tissue aging progresses and identifying the factors underlying tissue aging are crucial; however, the mechanism of tissue aging is not fully understood. This study shows that the biosynthesis of S-adenosyl-methionine (SAM), the major cellular donor of methyl group for methylation modifications, potently accelerates the aging-related defects during Drosophila oogenesis. An aging-related increase in the SAM-synthetase (Sam-S) levels in the germline leads to an increase in ovarian SAM levels. Sam-S-dependent biosynthesis of SAM controls aging-related defects in oogenesis through two mechanisms, decreasing the ability to maintain germline stem cells and accelerating the improper formation of egg chambers. Aging-related increases in SAM commonly occur in mouse reproductive tissue and the brain. Therefore, these results raise the possibility suggesting that SAM is the factor related to tissue aging beyond the species and tissues.

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Lee, H. Y., Lee, S. H. and Min, K. J. (2022). The Increased Abundance of Commensal Microbes Decreases Drosophila melanogaster Lifespan through an Age-Related Intestinal Barrier Dysfunction. Insects 13(2). PubMed ID: 35206792
Summary:
Commensal microbiota live in their host with a symbiotic relationship that affects the host's health and physiology. Many studies showed that microbial load and composition were changed by aging and have observed that increasing the abundance and changing the composition of commensal microbes had detrimental effects on host lifespan. It was hypothesized that dysbiosis of the intestinal microbiota leads to systemic effects in aging flies as a result of the increased intestinal permeability. The fruit fly laboratory strain w(1118) was used as a model system. The incidence of intestinal dysfunction was increased with age, and intestinal dysfunction increased the permeability of the fly intestine to resident microbes. The lifespan of flies with an intestinal barrier dysfunction was increased by removal of the microbes. Interestingly, some bacteria were also found in the hemolymph of flies with intestinal barrier dysfunction. These findings suggest the possibility that, as the host ages, there is an increase in intestinal permeability, which leads to an increased intestinal microbial load and a reduction in the host lifespan. These data therefore indicate a connection between commensal microbes and host lifespan.

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Hayashi, Y., Kashio, S., Murotomi, K., Hino, S., Kang, W., Miyado, K., Nakao, M., Miura, M., Kobayashi, S. and Namihira, M. (2022). Biosynthesis of S-adenosyl-methionine enhances aging-related defects in Drosophila oogenesis. Sci Rep 12(1): 5593. PubMed ID: 35379840
Summary:
Tissue aging is a major cause of aging-related disabilities and a shortened life span. Understanding how tissue aging progresses and identifying the factors underlying tissue aging are crucial; however, the mechanism of tissue aging is not fully understood. This study showed that the biosynthesis of S-adenosyl-methionine (SAM), the major cellular donor of methyl group for methylation modifications, potently accelerates the aging-related defects during Drosophila oogenesis. An aging-related increase in the SAM-synthetase (Sam-S) levels in the germline leads to an increase in ovarian SAM levels. Sam-S-dependent biosynthesis of SAM controls aging-related defects in oogenesis through two mechanisms, decreasing the ability to maintain germline stem cells and accelerating the improper formation of egg chambers. Aging-related increases in SAM commonly occur in mouse reproductive tissue and the brain. Therefore, these results raise the possibility suggesting that SAM is the factor related to tissue aging beyond the species and tissues.

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Maslov, D. L., Zemskaya, N. V., Trifonova, O. P., Lichtenberg, S., Balashova, E. E., Lisitsa, A. V., Moskalev, A. A. and Lokhov, P. G. (2021). Comparative Metabolomic Study of Drosophila Species with Different Lifespans. Int J Mol Sci 22(23). PubMed ID: 34884677 The increase in life expectancy, leading to a rise in the proportion of older people, is accompanied by a prevalence of age-related disorders among the world population, the fight against which today is one of the leading biomedical challenges. Exploring the biological insights concerning the lifespan is one of the ways to provide a background for designing an effective treatment for the increase in healthy years of life. Untargeted direct injection mass spectrometry-based metabolite profiling of 12 species of Drosophila with significant variations in natural lifespans was conducted in this research. A cross-comparison study of metabolomic profiles revealed lifespan signatures of flies. These signatures indicate that lifespan extension is associated with the upregulation of amino acids, phospholipids, and carbohydrate metabolism. Such information provides a metabolome-level view on longevity and may provide a molecular measure of organism age in age-related studies.

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Pan, H. Y., Ye, Z. W., Zheng, Q. W., Yun, F., Tu, M. Z., Hong, W. G., Chen, B. X., Guo, L. Q. and Lin, J. F. (2022). Ergothioneine exhibits longevity-extension effect in Drosophila melanogaster via regulation of cholinergic neurotransmission, tyrosine metabolism, and fatty acid oxidation. Food Funct 13(1): 227-241. PubMed ID: 34877949
Summary:
Many studies have demonstrated the protective effect of ergothioneine (EGT), the unique sulfur-containing antioxidant found in mushrooms, on several aging-related diseases. Nevertheless, to date, no single study has explored the potential role of EGT in the lifespan of animal models. This study shows that EGT consistently extends fly lifespan in diverse genetic backgrounds and both sexes, as well as in a dose and gender-dependent manner. Additionally, EGT is shown to increases the climbing activity of flies, enhance acetylcholinesterase (AchE) activity, and maintain the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) of aged flies. The increase in lifespan by EGT is gut microorganism dependent. This study proposed potential mechanisms of lifespan extension in Drosophila by EGT through RNA-seq analysis: preservation of the normal status of the central nervous system via the coordination of cholinergic neurotransmission, tyrosine metabolism, and peroxisomal proteins, regulation of autophagic activity by altering the lysosomal protein CTSD, and the preservation of normal mitochondrial function through controlled substrate feeding into the tricarboxylic acid (TCA) cycle, the major energy-yielding metabolic process in cells.

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Iyengar, A., Ruan, H. and Wu, C. F. (2022). Distinct Aging-Vulnerable and -Resilient Trajectories of Specific Motor Circuit Functions in Oxidation- and Temperature-Stressed Drosophila. eNeuro 9(1). PubMed ID: 34876473
Summary:
In Drosophila, molecular pathways affecting longevity have been extensively studied. However, corresponding neurophysiological changes underlying aging-related functional and behavioral deteriorations remain to be fully explored. This study examined different motor circuits in Drosophila across the life span and uncovered distinctive age-resilient and age-vulnerable trajectories in their established functional properties. In the giant fiber (GF) and downstream circuit elements responsible for the jump-and-flight escape reflex, relatively mild deterioration was observed toward the end of the life span. In contrast, more substantial age-dependent modifications were seen in the plasticity of GF afferent processing, specifically in use dependence and habituation properties. In addition, there were profound changes in different afferent circuits that drive flight motoneuron activities, including flight pattern generation and seizure spike discharges evoked by electroconvulsive stimulation. Importantly, in high-temperature (HT)-reared flies (29°C), the general trends in these age-dependent trajectories were largely maintained, albeit over a compressed time scale, lending support for the common practice of HT rearing for expediting Drosophila aging studies. It was discovered that shortened life spans in Cu/Zn superoxide dismutase (Sod) mutant flies were accompanied by altered aging trajectories in motor circuit properties distinct from those in HT-reared flies, highlighting differential effects of oxidative versus temperature stressors. This work helps to identify several age-vulnerable neurophysiological parameters that may serve as quantitative indicators for assessing genetic and environmental influences on aging progression in Drosophila.

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Graham, L. C., Kline, R. A., Lamont, D. J., Gillingwater, T. H., Mabbott, N. A., Skehel, P. A. and Wishart, T. M. (2021). Temporal Profiling of the Cortical Synaptic Mitochondrial Proteome Identifies Ageing Associated Regulators of Stability. Cells 10(12). PubMed ID: 34943911
Summary:
Synapses are particularly susceptible to the effects of advancing age, and mitochondria have long been implicated as organelles contributing to this compartmental vulnerability. Despite this, the mitochondrial molecular cascades promoting age-dependent synaptic demise remain to be elucidated. This study sought to examine how the synaptic mitochondrial proteome (including strongly mitochondrial associated proteins) was dynamically and temporally regulated throughout ageing to determine whether alterations in the expression of individual candidates can influence synaptic stability/morphology. Proteomic profiling of wild-type mouse cortical synaptic and non-synaptic mitochondria across the lifespan revealed significant age-dependent heterogeneity between mitochondrial subpopulations, with aged organelles exhibiting unique protein expression profiles. Recapitulation of aged synaptic mitochondrial protein expression at the Drosophila neuromuscular junction has the propensity to perturb the synaptic architecture, demonstrating that temporal regulation of the mitochondrial proteome may directly modulate the stability of the synapse in vivo.

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Kanel, P., Noll, G. A., Schroedter, K., Naffin, E., Kronenberg, J., Busswinkel, F., Twyman, R. M., Klambt, C. and Prufer, D. (2022). The tobacco phosphatidylethanolamine-binding protein NtFT4 increases the lifespan of Drosophila melanogaster by interacting with the proteostasis network. Aging (Albany NY) 14. PubMed ID: 35396341
Summary:
Proteostasis reflects the well-balanced synthesis, trafficking and degradation of cellular proteins. This is a fundamental aspect of the dynamic cellular proteome, which integrates multiple signaling pathways, but it becomes increasingly error-prone during aging. Phosphatidylethanolamine-binding proteins (PEBPs) are highly conserved regulators of signaling networks and could therefore affect aging-related processes. To test this hypothesis, this study expressed PEPBs in a heterologous context to determine their ectopic activity. Heterologous expression of the tobacco (Nicotiana tabacum) PEBP NtFT4 in Drosophila melanogaster significantly increased the lifespan of adult flies and reduced age-related locomotor decline. Similarly, overexpression of the Drosophila ortholog CG7054 increased longevity, whereas its suppression by RNA interference had the opposite effect. In tobacco, NtFT4 acts as a floral regulator by integrating environmental and intrinsic stimuli to promote the transition to reproductive growth. In Drosophila, NtFT4 engaged distinct targets related to proteostasis, such as HSP26. In older flies, it also prolonged Hsp26 gene expression, which promotes longevity by maintaining protein integrity. In NtFT4-transgenic flies, deregulated genes were identified encoding proteases that may contribute to proteome stability at equilibrium. The results demonstrate that the expression of NtFT4 influences multiple aspects of the proteome maintenance system via both physical interactions and transcriptional regulation, potentially explaining the aging-related phenotypes that were observed.

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Narayan, V. P., Wilson, A. J. and Chenoweth, S. F. (2022). Genetic and social contributions to sex differences in lifespan in Drosophila serrata. J Evol Biol 35(4): 657-663. PubMed ID: 35290690
Summary:
Sex differences in lifespan remain an intriguing puzzle in evolutionary biology. While explanations range from sex differences in selection to sex differences in the expression of recessive lifespan-altering mutations (via X-linkage), little consensus has been reached. One unresolved issue is the extent to which genetic influences on lifespan dimorphism are modulated by the environment. For example, studies have shown that sex differences in lifespan can either increase or decrease depending upon the social environment. This study took an experimental approach, manipulating multiple axes of the social environment across inbred long- and short-lived genotypes and their reciprocal F1s in the fly Drosophila serrata. The results reveal strong genetic effects and subtle yet significant genotype-by-environment interactions for male and female lifespan, specifically due to both population density and mating status. Further, the data do not support the idea that unconditional expression of deleterious X-linked recessive alleles in heterogametic males accounts for lower male lifespan.

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Takeuchi, K. I., Honda, D., Okumura, M., Miura, M. and Chihara, T. (2021). Systemic innate immune response induces death of olfactory receptor neurons in Drosophila. Genes Cells PubMed ID: 34921694
Summary:
Neural functions are known to decline during normal aging and neurodegenerative diseases. However, the mechanisms of functional impairment owing to the normal aging of the brain are poorly understood. It was previously reported that caspase-3-like protease, the protease responsible for inducing apoptosis, is activated in a subset of olfactory receptor neurons (ORNs), especially in Drosophila Or42b neurons, during normal aging. This study investigated the molecular mechanism underlying age-related caspase-3-like protease activation and cell death in Or42b neurons. Gene expression profiling of young and aged fly antenna showed that the expression of antimicrobial peptides was significantly upregulated, suggesting an activated innate immune response. Consistent with this observation, inhibition or activation of the innate immune pathway caused delayed or precocious cell death, respectively, in Or42b neurons. Accordingly, autonomous cell activation of the innate immune pathway in Or42b neurons is not likely required for their age-related death, whereas the systemic innate immune response induces caspase-3-like protease activation in Or42b neurons; this indicated that the death of these neurons is regulated non-cell autonomously. A possible link between the innate immune response and the death of olfactory neurons during normal aging is proposed.

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Sheshadri, D., Onkar, A. and Ganesh, S. (2021). Alterations in brain glycogen levels influence life-history traits and reduce the lifespan in female Drosophila melanogaster. Biol Open 10(12). PubMed ID: 34817590
Summary:
Sexual dimorphism in lifespan, wherein females outlive males, is evident across all animal taxa. The longevity difference between sexes is controlled by multiple physiological processes with complex relationships to one another. In recent years, glycogen, the storage form of glucose, has been shown to cause rapid aging upon forced synthesis in healthy neurons. Glycogen in the form of corpora amylacea in the aging brain is also widely reported. While these studies did suggest a novel role for glycogen in aging, most of them have focused on pooled samples, and have not looked at sex-specific effects, if any. Given the widespread occurrence of sex-biased expression of genes and the underlying physiology, it is important to look at the sex-specific effects of metabolic processes. Using transgenic fly lines for the human glycogen synthase, this study investigated the sex-specific effects of glycogen on stress resistance, fitness, and survival. Drosophila melanogaster females with altered levels of glycogen in the brain were shown to display a shortened lifespan, increased resistance to starvation, and higher oxidative stress than male flies. The present study thus provides a novel insight into the sex-specific effect of glycogen in survival and aging and how differences in metabolic processes could contribute to sex-specific traits.

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Ortega-Arellano, H. F., Jimenez-Del-Rio, M. and Velez-Pardo, C. (2021). Melatonin Increases Life Span, Restores the Locomotor Activity, and Reduces Lipid Peroxidation (LPO) in Transgenic Knockdown Parkin Drosophila melanogaster Exposed to Paraquat or Paraquat/Iron. Neurotox Res. PubMed ID: 34339012

Parkinson's disease (PD) is a complex progressive neurodegenerative disorder involving impairment of bodily movement caused by the specific destruction of dopaminergic (DAergic) neurons. Mounting evidence suggests that PD might be triggered by an interplay between environmental neurotoxicants (e.g., paraquat, PQ), heavy metals (e.g., iron), and gene alterations (e.g., PARKIN gene). Unfortunately, there are no therapies currently available that protect, slow, delay, or prevent the progression of PD. Melatonin (Mel, N-acetyl-5-methoxy tryptamine) is a natural hormone with pleiotropic functions including receptor-independent pathways which might be useful in the treatment of PD. Therefore, as a chemical molecule, it has been shown that Mel prolonged the lifespan and locomotor activity, and reduced lipid peroxidation (LPO) in wild-type Canton-S flies exposed to PQ, suggesting antioxidant and neuroprotective properties. However, it is not yet known whether Mel can protect or prevent the genetic model parkin deficient in flies against oxidative stress (OS) stimuli. This study shows that Mel (0.5, 1, 3 mM) significantly extends the life span and locomotor activity of TH > parkin-RNAi/ + Drosophila melanogaster flies (> 15 days) compared to untreated flies. Knock-down (K-D) parkin flies treated with PQ (1 mM) or PQ (1 mM)/iron (1 mM) significantly diminished the survival index and climbing abilities (e.g., 50% of flies were dead and locomotor impairment by days 4 and 3, respectively). Remarkably, Mel reverted the noxious effect of PQ or PQ/iron combination in K-D parkin. Indeed, Mel protects TH > parkin-RNAi/  + Drosophila melanogaster flies against PQ- or PQ/iron-induced diminish survival, locomotor impairment, and LPO (e.g., 50% of flies were death and locomotor impairment by days 6 and 9, respectively). Similarly, Mel prevented K-D parkin flies against both PQ and PQ/iron. Taken together, these findings suggest that Mel can be safely used as an antioxidant and neuroprotectant agent against OS-stimuli in selective individuals at risk to suffer early-onset Parkinsonism and PD (Ortega-Arellano, 2021).

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Banerjee, S. J., Schonbrun, A., Eizadshenass, S., Benji, S., Cantor, Y. T., Eliach, L., Lubin, M., Narrowe, Z., Purow, J., Shulman, B., Wiener, L. and Steinhauer, J. (2021). iPLA2-VIA is required for healthy aging of neurons, muscle, and the female germline in Drosophila melanogaster. PLoS One 16(9): e0256738. PubMed ID: 34506510

Neurodegenerative disease (ND) is a growing health burden worldwide, but its causes and treatments remain elusive. Although most cases of ND are sporadic, rare familial cases have been attributed to single genes, which can be investigated in animal models. This study generated a new mutation in the calcium-independent phospholipase A2 (iPLA2) VIA gene CG6718, the Drosophila melanogaster ortholog of human PLA2G6/PARK14, mutations in which cause a suite of NDs collectively called PLA2G6-associated neurodegeneration (PLAN). The mutants display age-related loss of climbing ability, a symptom of neurodegeneration in flies. Although phospholipase activity commonly is presumed to underlie iPLA2-VIA function, locomotor decline in the mutants used in this study is rescued by a transgene carrying a serine-to-alanine mutation in the catalytic residue, suggesting that important functional aspects are independent of phospholipase activity. Additionally, it was found that iPLA2-VIA knockdown in either muscle or neurons phenocopies locomotor decline with age, demonstrating its necessity in both neuronal and non-neuronal tissues. Furthermore, RNA in situ hybridization shows high endogenous iPLA2-VIA mRNA expression in adult germ cells, and transgenic HA-tagged iPLA2-VIA colocalizes with mitochondria there. Mutant males are fertile with normal spermatogenesis, while fertility is reduced in mutant females. Mutant female germ cells display age-related mitochondrial aggregation, loss of mitochondrial potential, and elevated cell death. These results suggest that iPLA2-VIA is critical for mitochondrial integrity in the Drosophila female germline, which may provide a novel context to investigate its functions with parallels to PLAN.

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Webb, J. L., Moe, S. M., Bolstad, A. K. and McNeill, E. M. (2021). Identification of conserved transcriptome features between humans and Drosophila in the aging brain utilizing machine learning on combined data from the NIH Sequence Read Archive. PLoS One 16(8): e0255085. PubMed ID: 34379632

Aging is universal, yet characterizing the molecular changes that occur in aging which lead to an increased risk for neurological disease remains a challenging problem. Aging affects the prefrontal cortex (PFC), which governs executive function, learning, and memory. Previous sequencing studies have demonstrated that aging alters gene expression in the PFC, however the extent to which these changes are conserved across species and are meaningful in neurodegeneration is unknown. Identifying conserved, age-related genetic and morphological changes in the brain allows application of the wealth of tools available to study underlying mechanisms in model organisms such as Drosophila melanogaster. RNA sequencing data from human PFC and fly heads were analyzed to determine conserved transcriptome signatures of age. This analysis revealed that expression of 50 conserved genes can accurately determine age in Drosophila (R2 = 0.85) and humans (R2 = 0.46). These transcriptome signatures were also able to classify Drosophila into three age groups with a mean accuracy of 88% and classify human samples with a mean accuracy of 69%. Overall, this work identifies 50 highly conserved aging-associated genetic changes in the brain that can be further studied in model organisms and demonstrates a novel approach to uncovering genetic changes conserved across species from multi-study public databases.

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Brooks, D. S., Vishal, K., Bawa, S., Alder, A. and Geisbrecht, E. R. (2021). Integration of proteomic and genetic approaches to assess developmental muscle atrophy. Faseb j 35(10): e21914 J Exp Biol. PubMed ID: 34547132

Muscle atrophy, or a decline in muscle protein mass, is a significant problem in the aging population and in numerous disease states. Unraveling molecular signals that trigger and promote atrophy may lead to a better understanding of treatment options; however, there is no single cause of atrophy identified to date. To gain insight into this problem, changes in protein profiles during muscle atrophy were investigated in Manduca sexta and Drosophila melanogaster. The use of insect models provides an interesting parallel to probe atrophic mechanisms since these organisms undergo a normal developmental atrophy process during the pupal transition stage. Leveraging the inherent advantages of each model organism, protein signature changes during Manduca intersegmental muscle (ISM) atrophy were defubed and then genetic approaches were used to confirm their functional importance in the Drosophila dorsal internal oblique muscles (DIOMs). The data reveal an upregulation of proteasome and peptidase components and a general downregulation of proteins that regulate actin filament formation. Surprisingly, thick filament proteins that comprise the A band are increased in abundance, providing support for the ordered destruction of myofibrillar components during developmental atrophy. The actin filament regulator Ciboulot (Cib) was uncovered as a novel regulator of muscle atrophy. These insights provide a framework towards a better understanding of global changes that occur during atrophy and may lead to eventual therapeutic targets (Brooks, 2021).

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Deepashree, S., Shivanandappa, T. and Ramesh, S. R. (2021). Genetic repression of the antioxidant enzymes reduces the lifespan in Drosophila melanogaster. J Comp Physiol B. PubMed ID: 34625818

Aging is a biological process associated with gradual loss of function caused by cellular and molecular damages ultimately leading to mortality. Free radicals are implicated in oxidative damage which affects the longevity of organisms. Natural cellular defenses involving antioxidant enzymes delay or prevent oxidative damage and, therefore, influence the aging process and longevity has been shown in many species including Drosophila. Oxidative resistance has been shown to be an important mechanism in the aging process in Drosophila. Therefore, it was hypothesized that repressing endogenous antioxidant defenses shortens longevity in Drosophila. To study the influence of natural defense mechanisms against oxidative stress in aging, this study has investigated the effect of genetic repression of the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), on longevity in Drosophila using transgenic RNAi flies and in vivo inhibition of the enzymes with chemical inhibitors. RNAi lines of Drosophila viz., UAS-sod1-IR and UAS-cat-IR, are driven ubiquitously using Act5C-Gal4 and Tubulin-Gal4 to achieve the suppression of SOD1 and CAT activities, respectively. Genetic repression of SOD1 and CAT by RNAi in transgenic flies led to drastically reduced longevity (SOD1, 77%; CAT, 83%), presenting the evidence for the role of endogenous antioxidant defenses in lifespan extension in Drosophila. Further, this study shows that the enzyme inhibitors, diethyldithiocarbamate and 3-amino-1,2,4-triazole, although lower the enzyme activities in vivo in flies, but did not affect longevity, which could be attributed to the factors such as bioavailability and metabolism of the inhibitors and adaptive mechanisms involving de novo synthesis of the enzymes. This study of genetic repression using transgenic RNAi provides experimental evidence that extended longevity is associated with endogenous antioxidant defenses and aging is correlated with oxidative stress resistance (Deepashree, 2021).

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Yamashita, K., Oi, A., Kosakamoto, H., Yamauchi, T., Kadoguchi, H., Kuraishi, T., Miura, M. and Obata, F. (2021). Activation of innate immunity during development induces unresolved dysbiotic inflammatory gut and shortens lifespan. Dis Model Mech 14(9). PubMed ID: 34448472 An early-life inflammatory response is associated with risks of age-related pathologies. How transient immune signalling activity during animal development influences life-long fitness is not well understood. Using Drosophila as a model, this study found that activation of innate immune pathway Immune deficiency (Imd) signalling in the developing larvae increases adult starvation resistance, decreases food intake and shortens organismal lifespan. Interestingly, lifespan is shortened by Imd activation in the larval gut and fat body, whereas starvation resistance and food intake are altered by that in neurons. The adult flies that developed with Imd activation show sustained Imd activity in the gut, despite complete tissue renewal during metamorphosis. The larval Imd activation increases an immunostimulative bacterial species, Gluconobacter sp., in the gut microbiome, and this dysbiosis is persistent to adulthood. Removal of gut microbiota by antibiotics in the adult fly mitigates intestinal immune activation and rescues the shortened lifespan. This study demonstrates that early-life immune activation triggers long-term physiological changes, highlighted as an irreversible alteration in gut microbiota, prolonged inflammatory intestine and concomitant shortening of the organismal lifespan.

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Lee, B., Shin, C., Shin, M., Choi, B., Yuan, C. and Cho, K. S. (2021). The linear ubiquitin E3 ligase-Relish pathway is involved in the regulation of proteostasis in Drosophila muscle during aging. Biochem Biophys Res Commun 550: 184-190. PubMed ID: 33706102 Linear ubiquitination is an atypic ubiquitination process that directly connects the N- and C-termini of ubiquitin and is catalyzed by HOIL-1-interacting protein (HOIP). It is involved in the immune response or apoptosis by activating the nuclear factor-κB pathway and is associated with polyglucosan body myopathy 1, an autosomal recessive disorder with progressive muscle weakness and cardiomyopathy. However, little is currently known regarding the function of linear ubiquitination in muscles. This study investigated the role of linear ubiquitin E3 ligase (LUBEL), a Drosophila HOIP ortholog, in the development and aging of muscles. The muscles of the flies with down-regulation of LUBEL or its downstream factors, kenny and Relish, developed normally, and there were no obvious abnormalities in function in young flies. However, the locomotor activity of the LUBEL RNAi flies was reduced compared to age-matched control, while LUBEL RNAi did not affect the increased mitochondrial fusion or myofiber disorganization during aging. Interestingly, the accumulation of polyubiquitinated protein aggregation during aging decreased in muscles by silencing LUBEL, kenny, or Relish. Meanwhile, the levels of autophagy and global translation, which are implicated in the maintenance of proteostasis, did not change due to LUBEL down-regulation. In conclusion, a new role of linear ubiquitination is proposed in proteostasis in the muscle aging (Lee, 2021).

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Shi, D., Han, T., Chu, X., Lu, H., Yang, X., Zi, T., Zhao, Y., Wang, X., Liu, Z., Ruan, J., Liu, X., Ning, H., Wang, M., Tian, Z., Wei, W., Sun, Y., Li, Y., Guo, R., Wang, Y., Ling, F., Guan, Y., Shen, D., Niu, Y., Li, Y. and Sun, C. (2021).An isocaloric moderately high-fat diet extends lifespan in male rats and Drosophila. Cell Metab. PubMed ID: 33440166

The health effect of dietary fat has been one of the most vexing issues in the field of nutrition. Few animal studies have examined the impact of high-fat diets on lifespan by controlling energy intake. This study found that compared to a normal diet, an isocaloric moderately high-fat diet (IHF) significantly prolonged lifespan by decreasing the profiles of free fatty acids (FFAs) in serum and multiple tissues via downregulating FFA anabolism and upregulating catabolism pathways in rats and flies. Proteomics analysis in rats identified PPRC1 as a key protein that was significantly upregulated by nearly 2-fold by IHF, and among the FFAs, only palmitic acid (PA) was robustly and negatively associated with the expression of PPRC1. Using PPRC1 transgenic RNAi/overexpression flies and in vitro experiments, IHF was demonstrated to significantly reduced PA, which could upregulate PPRC1 through PPARG, resulting in improvements in oxidative stress and inflammation and prolonging the lifespan (Shi, 2021).

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Fabian, D. K., Melike Donertaş, H., Fuentealba, M., Partridge, L. and Thornton, J. M. (2021). Transposable element landscape in Drosophila populations selected for longevity. Genome Biol Evol. PubMed ID: 33595657

Transposable elements (TEs) inflict numerous negative effects on health and fitness as they replicate by integrating into new regions of the host genome. Even though organisms employ powerful mechanisms to demobilize TEs, transposons gradually lose repression during aging. The rising TE activity causes genomic instability and was implicated in age-dependent neurodegenerative diseases, inflammation and the determination of lifespan. It is therefore conceivable that long-lived individuals have improved TE silencing mechanisms resulting in reduced TE expression relative to their shorter-lived counterparts and fewer genomic insertions. This study tested this hypothesis by performing the first genome-wide analysis of TE insertions and expression in populations of Drosophila melanogaster selected for longevity through late-life reproduction for 50-170 generations from four independent studies. Contrary to expectation, TE families were generally more abundant in long-lived populations compared to non-selected controls. Although simulations showed that this was not expected under neutrality, little evidence was found for selection driving TE abundance differences. Additional RNA-seq analysis revealed a tendency for reducing TE expression in selected populations, which might be more important for lifespan than regulating genomic insertions. Limited evidence was found of parallel selection on genes related to TE regulation and transposition. However, telomeric TEs were genomically and transcriptionally more abundant in long-lived flies, suggesting improved telomere maintenance as a promising TE-mediated mechanism for prolonging lifespan. The results provide a novel viewpoint indicating that reproduction at old age increases the opportunity of TEs to be passed on to the next generation with little impact on longevity (Fabian, 2021).

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Akan, I., Halim, A., Vakhrushev, S. Y., Clausen, H. and Hanover, J. A. (2021). Drosophila O-GlcNAcase Mutants Reveal an Expanded Glycoproteome and Novel Growth and Longevity Phenotypes. Cells 10(5). PubMed ID: 33925313
Summary:
The reversible posttranslational O-GlcNAc modification of serine or threonine residues of intracellular proteins is involved in many cellular events from signaling cascades to epigenetic and transcriptional regulation. O-GlcNAcylation is a conserved nutrient-dependent process involving two enzymes, with O-GlcNAc transferase (OGT) adding O-GlcNAc and with O-GlcNAcase (OGA) removing it in a manner that's protein- and context-dependent. O-GlcNAcylation is essential for epigenetic regulation of gene expression through its action on Polycomb and Trithorax and COMPASS complexes. However, the important role of O-GlcNAc in adult life and health span has been largely unexplored, mainly due the lack of available model systems. Cataloging the O-GlcNAc proteome has proven useful in understanding the biology of this modification in vivo. In this study, a recently developed oga knockout fly mutant was leveraged to identify the O-GlcNAcylated proteins in adult Drosophila melanogaster. The adult O-GlcNAc proteome revealed many proteins related to cell and organismal growth, development, differentiation, and epigenetics. Many O-GlcNAcylated proteins were identified that play a role in increased growth and decreased longevity, including HCF, SIN3A, LOLA, KISMET, ATX2, SHOT, and FOXO. Interestingly, oga mutant flies are larger and have a shorter life span compared to wild type flies, suggesting increased O-GlcNAc results in increased growth. These results suggest that O-GlcNAc alters the function of many proteins related to transcription, epigenetic modification and signaling pathways that regulate growth rate and longevity. Therefore, these findings highlight the importance of O-GlcNAc in growth and life span in adult Drosophila.

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Oka, M., Suzuki, E., Asada, A., Saito, T., Iijima, K. M. and Ando, K. (2021). Increasing neuronal glucose uptake attenuates brain aging and promotes life span under dietary restriction in Drosophila. iScience 24(1): 101979. PubMed ID: 33490892

Brain neurons play a central role in organismal aging, but there is conflicting evidence about the role of neuronal glucose availability because glucose uptake and metabolism are associated with both aging and extended life span. This study analyzed metabolic changes in the brain neurons of Drosophila during aging. Using a genetically encoded fluorescent adenosine triphosphate (ATP) biosensor, decreased ATP concentration was found in the neuronal somata of aged flies, correlated with decreased glucose content, expression of glucose transporter and glycolytic enzymes and mitochondrial quality. The age-associated reduction in ATP concentration did not occur in brain neurons with suppressed glycolysis or enhanced glucose uptake, suggesting these pathways contribute to ATP reductions. Despite age-associated mitochondrial damage, increasing glucose uptake maintained ATP levels, suppressed locomotor deficits, and extended the life span. Increasing neuronal glucose uptake during dietary restriction resulted in the longest life spans, suggesting an additive effect of enhancing glucose availability during a bioenergetic challenge on aging (Oka, 2021).

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Vincow, E. S., Thomas, R. E., Merrihew, G. E., MacCoss, M. J. and Pallanck, L. J. (2021). Slowed protein turnover in aging Drosophila reflects a shift in cellular priorities. J Gerontol A Biol Sci Med Sci. PubMed ID: 33453098

The accumulation of protein aggregates and dysfunctional organelles as organisms age has led to the hypothesis that aging involves general breakdown of protein quality control. This hypothesis was tested using a proteomic and informatic approach in the fruit fly Drosophila melanogaster. Turnover of most proteins was markedly slower in old flies. However, ribosomal and proteasomal proteins maintained high turnover rates, suggesting that the observed slowdowns in protein turnover might not be due to a global failure of quality control. As protein turnover reflects the balance of protein synthesis and degradation, whether decreases in synthesis or decreases in degradation would best explain the observed slowdowns in protein turnover was investigated. It was found that while many individual proteins in old flies showed slower turnover due to decreased degradation, an approximately equal number showed slower turnover due to decreased synthesis, and enrichment analyses revealed that translation machinery itself was less abundant. Mitochondrial complex I subunits and glycolytic enzymes were decreased in abundance as well, and proteins involved in glutamine-dependent anaplerosis were increased, suggesting that old flies modify energy production to limit oxidative damage. Together, these findings suggest that age-related proteostasis changes in Drosophila represent a coordinated adaptation rather than a systems collapse (Vincow, 2021).

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Strilbytska, O. M., Zayachkivska, A., Koliada, A., Galeotti, F., Volpi, N., Storey, K. B., Vaiserman, A. and Lushchak, O. (2020). Anise Hyssop Agastache foeniculum Increases Lifespan, Stress Resistance, and Metabolism by Affecting Free Radical Processes in Drosophila. Front Physiol 11: 596729. PubMed ID: 33391017

Anise hyssop, Agastache foeniculum, is a widely used medicinal herb with known antioxidant properties. This study examined how dietary supplementation with dried A. foeniculum leaf powder affected physiological and metabolic traits as well as activities of antioxidant enzymes and markers of oxidative stress in Drosophila melanogaster. Dietary hyssop extended the lifespan in a sex and genotype independent manner over a broad range of concentrations up to 30 mg/ml. Dietary supplementation with the herb significantly increased fecundity, resistance to oxidative stress and starvation. Higher transcript levels of Drosophila insulin-like peptide (dilp2) and decreased dilp3 and dilp6 transcripts together with increased levels of glycogen and triacylglycerols support an alteration of insulin signaling by the plant extract. Increased enzymatic activities of superoxide dismutase and aconitase as well as elevated protein and low molecular mass thiols also supported an alteration of free radical process in flies treated with dietary A. foeniculum leaf powder. Thus, physiological and metabolic traits as well as free radical processed may be affected by active compounds detected in extracts of anise hyssop leaves and contribute to the increased lifespan and reproductive (egg-laying) activity observed (Strilbytska, 2020).

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Savola, E., Montgomery, C., Waldron, F. M., Monteith, K. M., Vale, P. and Walling, C. (2020). Testing evolutionary explanations for the lifespan benefit of dietary restriction in fruit flies (Drosophila melanogaster). Evolution. PubMed ID: 33320333

Dietary restriction (DR), limiting calories or specific nutrients without malnutrition, extends lifespan across diverse taxa. Traditionally, this lifespan extension has been explained as a result of diet-mediated changes in the trade-off between lifespan and reproduction, with survival favored when resources are scarce. However, a recently proposed alternative suggests that the selective benefit of the response to DR is the maintenance of reproduction. This hypothesis predicts that lifespan extension is a side effect of benign laboratory conditions, and DR individuals would be frailer and unable to deal with additional stressors, and thus lifespan extension should disappear under more stressful conditions. This was tested by rearing outbred female fruit flies (Drosophila melanogaster) on 10 different protein:carbohydrate diets. Flies were either infected with a bacterial pathogen (Pseudomonas entomophila), injured with a sterile pinprick, or unstressed. Lifespan, fecundity, and measures of aging were monitored. DR extended lifespan and reduced reproduction irrespective of injury and infection. Infected flies on lower protein diets had particularly poor survival. Exposure to infection and injury did not substantially alter the relationship between diet and aging patterns. These results do not provide support for lifespan extension under DR being a side effect of benign laboratory conditions (Savola, 2020).

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Krittika, S. and Yadav, P. (2020). Dietary protein restriction deciphers new relationships between lifespan, fecundity and activity levels in fruit flies Drosophila melanogaster. Sci Rep 10(1): 10019. PubMed ID: 32572062

Drosophila melanogaster has been used in Diet Restriction (DR) studies for a few decades now, due to easy diet implementation and its short lifespan. Since the concentration of protein determines the trade-offs between lifespan and fecundity, it is important to understand the level of protein and the extent of its influence on lifespan, fecundity and activity of fruit flies. This study intended to assess the effect of a series of protein restricted diets from age 1 day of the adult fly on these traits to understand the possible variations in trade-off across tested diets. Lifespan under different protein concentrations remains unaltered, even though protein restricted diets exerted an age-specific influence on fecundity. Interestingly, there was no difference in lifetime activity of the flies in most of the tested protein restricted (PR) diets, even though a sex-dependent influence of protein concentrations was observed. Additionally, it is reported that not all concentrations of PR diet increase activity, thereby suggesting that the correlation between lifespan and the lifetime activity can be challenged under protein-restricted condition. Therefore, the PR does not need to exert its effect on lifespan and fecundity only but can also influence activity levels of the flies, thereby emphasizing the role of nutrient allotment between lifespan, fecundity and activity (Krittika, 2020).

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Wilson, K. A., Beck, J. N., Nelson, C. S., Hilsabeck, T. A., Promislow, D., Brem, R. B. and Kapahi, P. (2020). GWAS for Lifespan and Decline in Climbing Ability in Flies upon Dietary Restriction Reveal decima as a Mediator of Insulin-like Peptide Production. Curr Biol. PubMed ID: 32502405

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Fan, X., Zeng, Y., Fan, Z., Cui, L., Song, W., Wu, Q., Gao, Y., Yang, D., Mao, X., Zeng, B., Zhang, M., Ni, Q., Li, Y., Wang, T., Li, D. and Yang, M. (2020). Dihydromyricetin promotes longevity and activates the transcription factors FOXO and AOP in Drosophila. Aging (Albany NY) 12. PubMed ID: 33291074

Drugs or compounds have been shown to promote longevity in various approaches. This study used Drosophila to explore novel natural compounds can be applied to anti-aging. A flavonoid named Dihydromyricetin can increase stress tolerance and lipid levels, slow down gut dysfunction and extend Drosophila lifespan. Dihydromyricetin can also lessen pERK and pAKT signaling, consequently activating FOXO and AOP to modulate longevity. These results suggested that DHM could be used as an effective compound for anti-aging intervention, which could likely be applied to both mammals and humans (Fan, 2020).

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Parkhitko, A. A., Ramesh, D., Wang, L., Leshchiner, D., Filine, E., Binari, R., Olsen, A. L., Asara, J. M., Cracan, V., Rabinowitz, J. D., Brockmann, A. and Perrimon, N. (2020). Downregulation of the tyrosine degradation pathway extends Drosophila lifespan. Elife 9. PubMed ID: 33319750

Lipid homeostasis is essential for insects to maintain phospholipid (PL)-based membrane integrity and to provide on-demand energy supply throughout life. Triacylglycerol (TAG) is the major lipid class used for energy production and is stored in lipid droplets, the universal cellular fat storage organelles. Accumulation and mobilization of TAG are strictly regulated since excessive accumulation of TAG leads to obesity and has been correlated with adverse effects on health- and lifespan across phyla. Little is known, however, about when during adult life and why excessive storage lipid accumulation restricts lifespan. This study used genetically obese Drosophila mutant males, which were all shown to be short-lived compared to control males and applied single fly mass spectrometry-based lipidomics to profile TAG, diacylglycerol and major membrane lipid signatures throughout adult fly life from eclosion to death. This comparative approach revealed distinct phases of lipidome remodeling throughout aging. Quantitative and qualitative compositional changes of TAG and PL species, which are characterized by the length and saturation of their constituent fatty acids, were pronounced during young adult life. In contrast, lipid signatures of adult and senescent flies were remarkably stable. Genetically obese flies displayed both quantitative and qualitative changes in TAG species composition, while PL signatures were almost unaltered compared to normal flies at all ages. Collectively, this suggests a tight control of membrane composition throughout lifetime largely uncoupled from storage lipid metabolism. Finally, evidence is presented for a characteristic lipid signature of moribund flies, likely generated by a rapid and selective storage lipid depletion close to death. Of note, the analytical power to monitor lipid species profiles combined with high sensitivity of this single fly lipidomics approach is universally applicable to address developmental or behavioral lipid signature modulations of importance for insect life (Hofbauer, 2020).

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Khor, S. and Cai, D. (2020). Control of lifespan and survival by Drosophila NF-κB signaling through neuroendocrine cells and neuroblasts. Aging (Albany NY) 12(24): 24604-24622. PubMed ID: 33232282

This paper reports a comparative analysis of the effects of immune activation in the fly nervous system using genetic activation models to target Drosophila NF-κB within Toll versus Imd pathways. Genetic gain-of-function models for either pathway pan-neuronally, as well as in discrete subsets of neural cells including neuroendocrine insulin-producing cells (IPCs) or neuroblasts, reduce fly lifespan, however, these phenotypes in IPCs and neuroblasts are stronger with Toll activation than Imd activation. Of note, while aging is influenced more by Toll/NF-κB activation in IPCs during adulthood, neuroblasts influence aging more substantially during development. The study then focused on Toll/NF-κB inhibition, revealing that IPCs or neuroblasts are important for the effects of lifespan and healthspan extension but in a life stage-dependent manner while some of these effects display sexual dimorphism. Importantly, co-inhibition of Toll/NF-κB pathway in IPCs and neuroblasts increased fly lifespan greater than either cell population, suggesting that independent mechanisms might exist. Toll/NF-κB inhibition in IPCs was also sufficient to enhance survival under various fatal stresses, supporting the additional benefits to fly healthspan. In conclusion, IPCs and neuroblasts are important for Drosophila NF-κB for controlling lifespan (Khor, 2020).

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Bjedov, I., Cocheme, H. M., Foley, A., Wieser, D., Woodling, N. S., Castillo-Quan, J. I., Norvaisas, P., Lujan, C., Regan, J. C., Toivonen, J. M., Murphy, M. P., Thornton, J., Kinghorn, K. J., Neufeld, T. P., Cabreiro, F. and Partridge, L. (2020). Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila. PLoS Genet 16(11): e1009083. PubMed ID: 33253201

Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. This paper reports that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. It was first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. Next autophagy was directly induced by over-expressing the major autophagy kinase Atg1; a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention (Bjedov, 2020).

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Parkhitko, A. A., Ramesh, D., Wang, L., Leshchiner, D., Filine, E., Binari, R., Olsen, A. L., Asara, J. M., Cracan, V., Rabinowitz, J. D., Brockmann, A. and Perrimon, N. (2020). Downregulation of the tyrosine degradation pathway extends Drosophila lifespan. Elife 9. PubMed ID: 33319750

To identify metabolic pathways associated with aging, age-dependent changes in the metabolomes of long-lived Drosophila were examined. Among the metabolites that changed, levels of tyrosine were increased with age in long-lived flies. The levels of enzymes in the tyrosine degradation pathway increase with age in wild-type flies. Whole-body and neuronal-specific downregulation of enzymes in the tyrosine degradation pathway significantly extends Drosophila lifespan, causes alterations of metabolites associated with increased lifespan, and upregulates the levels of tyrosine-derived neuromediators. Moreover, feeding wild-type flies with tyrosine increased their lifespan. Mechanistically, it was shown that suppression of ETC complex I drives the upregulation of enzymes in the tyrosine degradation pathway, an effect that can be rescued by tigecycline, an FDA-approved drug that specifically suppresses mitochondrial translation. In addition, tyrosine supplementation partially rescued lifespan of flies with ETC complex I suppression. Altogether, this study highlights the tyrosine degradation pathway as a Parkhitko, 2020).

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Sheng, L., Shields, E. J., Gospocic, J., Glastad, K. M., Ratchasanmuang, P., Berger, S. L., Raj, A., Little, S. and Bonasio, R. (2020). Social reprogramming in ants induces longevity-associated glia remodeling. Sci Adv 6(34): eaba9869. PubMed ID: 32875108

In social insects, workers and queens arise from the same genome but display profound differences in behavior and longevity. In Harpegnathos saltator ants, adult workers can transition to a queen-like state called gamergate, which results in reprogramming of social behavior and life-span extension. Using single-cell RNA sequencing, the distribution of neuronal and glial populations was compared before and after the social transition. This study found that the conversion of workers into gamergates resulted in the expansion of neuroprotective ensheathing glia. Brain injury assays revealed that activation of the damage response gene Mmp1 was weaker in old workers, where the relative frequency of ensheathing glia also declined. On the other hand, long-lived gamergates retained a larger fraction of ensheathing glia and the ability to mount a strong Mmp1 response to brain injury into old age. Molecular and cellular changes were observed suggestive of age-associated decline in ensheathing glia in Drosophila (Sheng, 2020).

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Nandakumar, S., Grushko, O. and Buttitta, L. A. (2020). Polyploidy in the adult Drosophila brain. Elife 9. PubMed ID: 32840209

Long-lived cells such as terminally differentiated postmitotic neurons and glia must cope with the accumulation of damage over the course of an animal's lifespan. How long-lived cells deal with ageing-related damage is poorly understood. This study shows that polyploid cells accumulate in the adult fly brain and that polyploidy protects against DNA damage-induced cell death. Multiple types of neurons and glia that are diploid at eclosion, become polyploid in the adult Drosophila brain. The optic lobes exhibit the highest levels of polyploidy, associated with an elevated DNA damage response in this brain region. Inducing oxidative stress or exogenous DNA damage leads to an earlier onset of polyploidy, and polyploid cells in the adult brain are more resistant to DNA damage-induced cell death than diploid cells. These results suggest polyploidy may serve a protective role for neurons and glia in adult Drosophila melanogaster brains (Nandakumar, 2020).

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Yamauchi, T., Oi, A., Kosakamoto, H., Akuzawa-Tokita, Y., Murakami, T., Mori, H., Miura, M. and Obata, F. (2020). Gut Bacterial Species Distinctively Impact Host Purine Metabolites during Aging in Drosophila. iScience 23(9): 101477. PubMed ID: 32916085

Gut microbiota impacts the host metabolome and affects its health span. How bacterial species in the gut influence age-dependent metabolic alteration has not been elucidated. This study shows in Drosophila melanogaster that allantoin, an end product of purine metabolism, is increased during aging in a microbiota-dependent manner. Allantoin levels are low in young flies but are commonly elevated upon lifespan-shortening dietary manipulations such as high-purine, high-sugar, or high-yeast feeding. Removing Acetobacter persici in the Drosophila microbiome attenuated age-dependent allantoin increase. Mono-association with A. persici, but not with Lactobacillus plantarum, increased allantoin in aged flies. A. persici increased allantoin via activation of innate immune signaling IMD pathway in the renal tubules. On the other hand, analysis of bacteria-conditioned diets revealed that L. plantarum can decrease allantoin by reducing purines in the diet. These data together demonstrate species-specific regulations of host purine levels by the gut microbiome (Yamauchi, 2020).

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Lien, W. Y., Chen, Y. T., Li, Y. J., Wu, J. K., Huang, K. L., Lin, J. R., Lin, S. C., Hou, C. C., Wang, H. D., Wu, C. L., Huang, S. Y. and Chan, C. C. (2020). Lifespan regulation in alpha/beta posterior neurons of the fly mushroom bodies by Rab27. Aging Cell: e13179. PubMed ID: 32627932

Brain function has been implicated to control the aging process and modulate lifespan. However, continuous efforts remain for the identification of the minimal sufficient brain region and the underlying mechanism for neuronal regulation of longevity. This study shows that the Drosophila lifespan is modulated by rab27 functioning in a small subset of neurons of the mushroom bodies (MB), a brain structure that shares analogous functions with mammalian hippocampus and hypothalamus. Depleting rab27 in the α/βp neurons of the MB is sufficient to extend lifespan, enhance systemic stress responses, and alter energy homeostasis, all without trade-offs in major life functions. Within the α/βp neurons, rab27KO causes the mislocalization of phosphorylated S6K thus attenuates TOR signaling, resulting in decreased protein synthesis and reduced neuronal activity. Consistently, expression of dominant-negative S6K in the α/βp neurons increases lifespan. Furthermore, the expression of phospho-mimetic S6 in α/βp neurons of rab27KO rescued local protein synthesis and reversed lifespan extension. These findings demonstrate that inhibiting TOR-mediated protein synthesis in α/βp neurons is sufficient to promote longevity (Lien, 2020).

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Yamakawa-Kobayashi, K., Ohhara, Y., Kawashima, T., Ohishi, Y. and Kayashima, Y. (2020). Loss of CNDP causes a shorter lifespan and higher sensitivity to oxidative stress in Drosophila melanogaster. Biomed Res 41(3): 131-138. PubMed ID: 32522930

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Brown, E. J., Nguyen, A. H. and Bachtrog, D. (2020). The Y chromosome may contribute to sex-specific ageing in Drosophila. Nat Ecol Evol. PubMed ID: 32313175

Heterochromatin suppresses repetitive DNA, and a loss of heterochromatin has been observed in aged cells of several species, including humans and Drosophila. Males often contain substantially more heterochromatic DNA than females, due to the presence of a large, repeat-rich Y chromosome, and male flies generally have a shorter average lifespan than females. This study shows that repetitive DNA becomes de-repressed more rapidly in old male flies relative to females, and repeats on the Y chromosome are disproportionally mis-expressed during ageing. This is associated with a loss of heterochromatin at repetitive elements during ageing in male flies, and a general loss of repressive chromatin in aged males away from pericentromeric regions and the Y. By generating flies with different sex chromosome karyotypes (XXY females and X0 and XYY males), this study shows that repeat de-repression and average lifespan is correlated with the number of Y chromosomes. This suggests that sex-specific chromatin differences may contribute to sex-specific ageing in flies (Brown, 2020).

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Huang, W., Campbell, T., Carbone, M. A., Jones, W. E., Unselt, D., Anholt, R. R. H. and Mackay, T. F. C. (2020). Context-dependent genetic architecture of Drosophila life span. PLoS Biol 18(3): e3000645. PubMed ID: 32134916

Understanding the genetic basis of variation in life span is a major challenge that is difficult to address in human populations. Evolutionary theory predicts that alleles affecting natural variation in life span will have properties that enable them to persist in populations at intermediate frequencies, such as late-life-specific deleterious effects, antagonistic pleiotropic effects on early and late-age fitness components, and/or sex- and environment-specific or antagonistic effects. This study quantified variation in life span in males and females reared in 3 thermal environments for the sequenced, inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and an advanced intercross outbred population derived from a subset of DGRP lines. Quantitative genetic analyses of life span and the micro-environmental variance of life span in the DGRP revealed significant genetic variance for both traits within each sex and environment, as well as significant genotype-by-sex interaction (GSI) and genotype-by-environment interaction (GEI). Genome-wide association (GWA) mapping in both populations implicates over 2,000 candidate genes with sex- and environment-specific or antagonistic pleiotropic allelic effects. Over 1,000 of these genes are associated with variation in life span in other D. melanogaster populations. The effects of 15 candidate genes were functionally assessed using RNA interference (RNAi): all affected life span and/or micro-environmental variance of life span in at least one sex and environment and exhibited sex-and environment-specific effects. These results implicate novel candidate genes affecting life span and suggest that variation for life span may be maintained by variable allelic effects in heterogeneous environments (Huang, 2020).

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Wu, Q., Yu, G., Cheng, X., Gao, Y., Fan, X., Yang, D., Xie, M., Wang, T., Piper, M. D. W. and Yang, M. (2020). Sexual dimorphism in the nutritional requirement for adult lifespan in Drosophila melanogaster. Aging Cell 19(3): e13120. PubMed ID: 32069521

The nutritional requirements of Drosophila have mostly been studied for development and reproduction, but the minimal requirements for adult male and female flies for lifespan have not been established. Following development on a complete diet, this study found substantial sex difference in the basic nutritional requirement of adult flies for full length of life. Relative to females, males require less of each nutrient, and for some nutrients that are essential for development, adult males have no requirement at all for lifespan. The most extreme (and surprising) sex differences were that chronic cholesterol and vitamin deficiencies had no effect on the lifespan of adult males, but they greatly decreased lifespan in females. Female oogenesis rather than chromosomal karyotype and mating status is the key cause of this gender difference in life-sustaining nutritional requirements. These data are important to the way the mechanisms are understood by which diet modifies lifespan (Wu, 2020).

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Obata, F., Fons, C. O. and Gould, A. P. (2018). Early-life exposure to low-dose oxidants can increase longevity via microbiome remodelling in Drosophila. Nat Commun 9(1): 975. PubMed ID: 29515102

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Matsuno, M., Horiuchi, J., Ofusa, K., Masuda, T. and Saitoe, M. (2019). Inhibiting glutamate activity during consolidation suppresses age-related long-term memory impairment in Drosophila. iScience 15: 55-65. PubMed ID: 31030182

In Drosophila, long-term memory (LTM) formation requires increases in glial gene expression. Klingon (Klg), a cell adhesion molecule expressed in both neurons and glia, induces expression of the glial transcription factor, Repo. However, glial signaling downstream of Repo has been unclear. This study demonstrates that Repo increases expression of the glutamate transporter, EAAT1, and EAAT1 is required during consolidation of LTM. The expressions of Klg, Repo, and EAAT1 decrease upon aging, suggesting that age-related impairments in LTM are caused by dysfunction of the Klg-Repo-EAAT1 pathway. Supporting this idea, overexpression of Repo or EAAT1 rescues age-associated impairments in LTM. Pharmacological inhibition of glutamate activity during consolidation improves LTM in klg mutants and aged flies. Altogether, the results indicate that LTM formation requires glial-dependent inhibition of glutamate signaling during memory consolidation, and aging disrupts this process by inhibiting the Klg-Repo-EAAT1 pathway (Matsuno, 2019).

Changes in glial transcription due to neuronal activity have been studied previously, but a specific role of glial transcription in LTM has been less characterized. Expression of the glial transcription factor, Repo, increases shortly after spaced training, and this increase is required for LTM formation. This report has identified Eaat1 as a Repo-regulated glial gene required for LTM consolidation. Eaat1 encodes a glial glutamate transporter that removes glutamate from synaptic sites and transports it into astrocytes. Thus, the data indicate that glutamate signaling needs to be inhibited during LTM consolidation (Matsuno, 2019).

To identify Eaat1, a screen was performed for various genes regulating glial physiology for altered expression during LTM formation. Expression of Eaat1 and crammer was found to increase after spaced training. As Eaat1, but not crammer, is expressed exclusively in glia, focus was placed on Eaat1 as a likely Repo-regulated gene. Indeed, spaced-training-induced increases in EAAT1 depend on Repo and Klg activity. Interestingly, expression of the glial gene, genderblind, which encodes another glial glutamate transporter, required Repo activity for expression, but was not affected by spaced training, suggesting that other transcriptional regulatory factors besides Repo are likely necessary to differentially regulate genes required for memory consolidation from those required for other glial functions (Matsuno, 2019).

Because only screened selected genes were screened, it is possible that Repo induces the expression of other unidentified genes after spaced training. However, somewhat unexpectedly, it was found that overexpression of Eaat1 alone in glial cells is sufficient to rescue the LTM defects of klg and repo mutants. This indicates that the major function of the Klg/Repo signaling pathway is to induce glial expression of Eaat1. It further suggests that one function of astrocytes is to decrease glutamate signaling during LTM consolidation (Matsuno, 2019).

Combined with results from previous studies, this work identifies a putative pathway linking neuronal activity to glial inhibition of glutamate signaling. In flies, the homophilic cell adhesion molecule, Klingon, is expressed in both neurons and glia, and needs to be expressed in both cell types for normal LTM. Repo expression normally increases after spaced training, whereas it fails to do so in klg mutants, indicating that Klg-mediated neuron-glia communication is necessary for this increase. Thus, it is proposed that spaced training increases neuronal activity, which induces signaling to glia via the cell adhesion molecule Klg. This results in increased Repo activity in glia, which increases Eaat1 expression, and subsequently decreases glutamate signaling (Matsuno, 2019).

Previous work from various groups including has shown that glutamate signaling through NMDA-type receptors (NRs) is necessary for learning and memory. Overexpression of NRs in mice enhances learning and memory formation, and it has been shown that glial production of D-serine, a neuromodulator that functions as a coactivator of NRs, is necessary for short-lasting memory. In the current study, focus was placed on glutamate activity specifically during memory consolidation, instead of during initial learning and memory formation. Considering the current findings with those of previous studies, it is proposed that NR-dependent glutamate signaling needs to be initially high, during formation of short-lasting memories, but low during a later phase where short-lasting memories are consolidated into LTM. This suggests that glia play at least two roles in memory. They produce D-serine that contributes to high NR activity during memory formation and also produce EAAT1 after learning, which functions to reduce glutamate signaling during memory consolidation (Matsuno, 2019).

Age-related impairments in Drosophila memory do not consist of a general decrease in all forms of learning and memory, but instead consist of decreases in two specific phases of memory, MTM and LTM. The current results suggest that both these memory effects are caused by age-related glial dysfunction. Glia in young flies are able to produce sufficient amounts of D-serine for normal MTM, whereas D-serine amounts decrease 2-fold in aged flies. This decrease is responsible for age-related impairments in 1-h memory, because increasing glial production of D-serine, or directly feeding of D-serine to aged flies, rescues this impairment. Likewise, glial dysfunction is also responsible for age-related impairments in LTM because aged glia are unable to inhibit glutamate signaling during consolidation. Thus, in contrast to young flies, aged flies are unable to modulate glutamate activity during learning and consolidation, leading to defects in the two memory phases (Matsuno, 2019).

The model that EAAT1 inhibits glutamate activity during consolidation stems from EAAT1's role in clearing glutamate from synaptic sites and transporting it into astrocytes. This model is consistent with several mammalian studies that demonstrated decreased expression of astrocytic glutamate transporters upon aging, with a consequent reduction of glutamate uptake. Further supporting this model, it was found that feeding flies memantine or MK801, NMDA receptor antagonists, after spaced training, restores normal LTM in klg mutants and restores LTM in aged flies to youthful levels. This effect requires feeding after training during the consolidation phase. Similar results were obtained by feeding riluzole, a glutamate modulator, which decreases glutamate release and increases astrocytic glutamate uptake. Riluzole has also been reported to ameliorate age-related cognitive decline in mammals, suggesting that the mechanisms of AMI may be conserved between species. In contrast, this study found that D-serine feeding, which rescues age-related declines in short-lasting (1-h) memory, does not improve declines in LTM, but rather attenuates it. This is consistent with the model wherein declines in short-lasting memory and LTM are caused by distinct or opposing mechanisms and glutamate signaling needs to be suppressed during consolidation. Somewhat unexpectedly, it was also found that (s)-4C3HPG, the mGluR1 antagonist/mGluR2 agonist, also ameliorated age-related impairments in LTM. This result indicates that glutamate activity through both ionotropic and metabotropic glutamate receptors antagonizes memory consolidation (Matsuno, 2019).

Currently, it is unclear why glutamate signaling needs to be inhibited during consolidation, but a previous study has shown that Mg2+ block mutations in NMDA-type glutamate receptors (NRs) cause specific defects in LTM in Drosophila. Although Mg2+ block mutations have various effects, one effect is to increase NR activity. Increased NR activity results in increased activity of dCREB2b, an inhibitory isoform of CREB. CREB-dependent gene expression is required during consolidation of LTM, suggesting that consolidation may be preferentially sensitive to NR activity (Matsuno, 2019).

Alternatively, it is possible that neuronal activity needs to be inhibited globally during memory consolidation. Sleep is known to be important for LTM. Sleep deprivation during consolidation prevents LTM formation, whereas artificially inducing sleep after training has been reported to improve LTM. Thus a second possibility is suggested that inhibition of glutamate signaling after spaced training may be a brain-wide phenomenon that promotes consolidation by inducing the organism to sleep. Thus far, gross alterations in sleep duration in klg and repo mutants have not been detected, although this does not preclude minor disruptions in sleep quality that may not be detectable by motion-based sleep assays. Finally, a third possibility is envisioned wherein neuronal inhibition may be required as a neuroprotective mechanism that may be necessary to prevent cell death in neurons that were extensively stimulated during spaced training (Matsuno, 2019).

Mapping the glutamatergic neurons whose activity is inhibited during consolidation will be of great interest in the future. As aversive olfactory memories are formed and stored in the Drosophila MBs, it is possible that specific glutamatergic MB output neurons (MBONs) are inhibited during consolidation. Several glutamatergic MBONs are involved in feedback networks with the lobes of the MBs, suggesting that altering the activity of these neurons may modulate memory consolidation and memory-associated behavioral responses (Matsuno, 2019).

This study has demonstrate that increased expression of Eaat1 is required for LTM consolidation. Based on numerous results from other groups, it is hypothesized that Eaat1 functions to reduce glutamate signaling, and support for this model is provided by demonstrating that pharmacological inhibition of glutamate signaling during consolidation improves LTM under various conditions. However, due to technical limitations, it was not possible to actually measure glutamate concentrations at synapses during memory consolidation and it is not known where and how much glutamate signaling has to be inhibited for optimal LTM consolidation (Matsuno, 2019).

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Dobson, A. J., Boulton-McDonald, R., Houchou, L., Svermova, T., Ren, Z., Subrini, J., Vazquez-Prada, M., Hoti, M., Rodriguez-Lopez, M., Ibrahim, R., Gregoriou, A., Gkantiragas, A., Bahler, J., Ezcurra, M. and Alic, N. (2019). Longevity is determined by ETS transcription factors in multiple tissues and diverse species. PLoS Genet 15(7): e1008212. PubMed ID: 31356597

Ageing populations pose a major public health crises. Reprogramming gene expression by altering the activities of sequence-specific transcription factors (TFs) can ameliorate deleterious effects of age. This explore how a circuit of TFs coordinates pro-longevity transcriptional outcomes, which reveals a multi-tissue and multi-species role for an entire protein family: the E-twenty-six (ETS) TFs. In Drosophila, reduced insulin/IGF signalling (IIS) extends lifespan by coordinating activation of Aop, an ETS transcriptional repressor, and Foxo, a Forkhead transcriptional activator. Aop and Foxo bind the same genomic loci, and this study shows that, individually, they effect similar transcriptional programmes in vivo. In combination, Aop can both moderate or synergise with Foxo, dependent on promoter context. Moreover, Foxo and Aop oppose the gene-regulatory activity of Pnt, an ETS transcriptional activator. Directly knocking down Pnt recapitulates aspects of the Aop/Foxo transcriptional programme and is sufficient to extend lifespan. The lifespan-limiting role of Pnt appears to be balanced by a requirement for metabolic regulation in young flies, in which the Aop-Pnt-Foxo circuit determines expression of metabolic genes, and Pnt regulates lipolysis and responses to nutrient stress. Molecular functions are often conserved amongst ETS TFs, prompting examination of whether other Drosophila ETS-coding genes may also affect ageing. This study shows that five out of eight Drosophila ETS TFs play a role in fly ageing, acting from a range of organs and cells including the intestine, adipose and neurons. This study expands the repertoire of lifespan-limiting ETS TFs in C. elegans, confirming their conserved function in ageing and revealing that the roles of ETS TFs in physiology and lifespan are conserved throughout the family, both within and between species (Dodson, 2019).

Ageing is characterised by a steady systematic decline in biological function, and increased likelihood of disease. Understanding the basic biology of ageing therefore promises to help improve the overall health of older people, who constitute an ever-increasing proportion of populations. In experimental systems, healthy lifespan can be extended by altered transcriptional regulation, coordinated by sequence-specific TFs. Thus, understanding TFs' functions can reveal how to promote health in late life. Forkhead family TFs, especially Forkhead Box O (Foxo) orthologues, have been studied extensively in this context. This effort has been driven by the association of Foxo3a alleles with human longevity; and the findings that the activation of Foxos is necessary and sufficient to explain the extension of lifespan observed following reduced insulin/IGF signalling (IIS) in model organisms. Foxos interact with additional TFs in regulatory circuits, and it is in this context that their function must be understood. For example, in Caenorhabditis elegans, the pro-longevity activity of Daf-16 is orchestrated with further TFs including Hsf, Elt-2, Skn-1, Pqm-1 and Hlh-30/Tfeb. Examining regions bound by Foxos across animals has highlighted the conserved presence of sites to bind ETS family TFs. In Drosophila, two members of this family, namely Aop (a.k.a. Yan) and Pnt, have been linked to ageing via genetic interactions with Foxo and IIS, and similar interactions are evident in C. elegans. These findings raise questions of the overall roles of ETS factors in ageing, and their relationship to the activities of Foxos (Dodson, 2019).

The ETS TFs are conserved across animals, including 28 representatives in humans. Their shared, defining feature is a core helix-turn-helix DNA-binding domain, which binds DNA on 5'-GGA(A/T)-3' ETS-binding motifs (EBMs). They are differentiated by tissue-specific expression, and variation in peripheral amino acid residues which, along with variation in nucleotides flanking the core EBM, confers DNA-binding specificity. ETS TFs generally function as transcriptional activators, but a few repress transcription. Aop is one such repressor in Drosophila. Aop and its human orthologue Tel are thought to repress transcription by competing with activators for binding sites, recruiting co-repressors, and forming homo-oligomers that limit activator access to euchromatin. Consequently, Aop's role in physiology must be explored in the context of its interactions with additional TFs, especially activators. Foxo is one such activator. Both Foxo and Aop are required for longevity by IIS inhibition, each is individually sufficient to extend lifespan, and both are recruited to the same genomic loci in vivo. Whilst activating either in the gut and fat body extends lifespan, the effect of activating both is not additive. Furthermore, if Aop is knocked down, activating Foxo not only ceases to extend lifespan, but even becomes deleterious for lifespan. Overall, these findings suggest that gene expression downstream of IIS is orchestrated by the coordinated activity of Aop and Foxo, and that there is a redundancy in the function of the two TFs, even though Foxo is a transcriptional activator and Aop a transcriptional repressor. This study started by characterising Aop and its relationship with relevant transcriptional activators, including Foxo. This led revealing that roles in ageing are widespread throughout the ETS TF family, extending across multiple fly tissues and diverse animal taxa (Dodson, 2019).

Promoting healthy ageing by transcriptional control is an attractive prospect, because targeting one specific protein can restructure global gene expression to provide broad-scale benefits. This study suggests key roles for ETS TFs in such optimisation. The results show dual roles for Aop: balancing Foxo's outputs, and opposing Pnt's outputs. These functions coordinate transcriptional changes that correspond to lifespan. Repressing transcription from the ETS site appears to be the key longevity-promoting step, and indeed lifespan was extended by limiting multiple ETS TFs, in multiple fly tissues, and in multiple taxa. Altogether, these results show that inhibiting lifespan is a general feature of ETS transcriptional activators. Presumably the expression of these TFs is maintained, despite costs in late life, because of benefits in other contexts. For example, Pnt is important during development, and expression may simply run-on into adulthood. This study now shows that Pnt is also important for adults facing nutritional variation or stress, and genomic evidence suggests equivalent functions for Ets-4 in C. elegans. In addition, Ets21C is required to mount an effective immune response, and both Ets21C and Pnt control gut homeostasis. Tissue environment appears to be another important contextual factor that determines the lifespan effects of specific ETS TFs. Differences between tissues in chromatin architecture are likely to alter the capacity of a given TF to bind a given site, and the current results show that a given TF, and also upstream RTKs, do not necessarily lead to the same lifespan effect across all tissues. The tissue-specific functions that are shown for ETS TFs, Foxo and RTKs, suggests that transcription is locally coordinated by distinct receptors and TFs in distinct tissues, but that lifespan-regulatory signalling nevertheless converges on the ETS site. This differentiation makes it all the more remarkable that roles in lifespan appear to be conserved amongst ETS family TFs, even in diverse tissue contexts (Dodson, 2019).

The structure of molecular networks and their integration amongst tissues underpins phenotype, including into old age. Unravelling the basics of these networks is a critical step in identifying precise anti-ageing molecular targets. Identifying the least disruptive perturbation of these networks, by targeting the 'correct' effector, is a key goal in order to achieve desirable outcomes without undesirable trade-offs that may ensue from broader-scale perturbation. This targeting can be at the level of specific proteins, cell types, points in the life-course, or a combination of all three. The tissue-specific expression pattern of ETS TFs, and the apparent conservation of their roles in longevity, highlights them as important regulators of tissue-specific programs that may be useful in precise medical targeting of specific senescent pathologies (Dodson, 2019).

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Suh, Y. S., Yeom, E., Nam, J. W., Min, K. J., Lee, J. and Yu, K. (2020). Methionyl-tRNA Synthetase Regulates Lifespan in Drosophila. Mol Cells. PubMed ID: 31940717

Methionyl-tRNA synthetase (MRS) is essential for translation. MRS mutants reduce global translation, which usually increases lifespan in various genetic models. However, this study found that inhibition of MRS in Drosophila reduced lifespan despite of the reduced protein synthesis. Microarray analysis with MRS inhibited Drosophila revealed significant changes in inflammatory and immune response genes. Especially, the expression of anti-microbial peptides (AMPs) genes was reduced. When the expression levels of AMP genes during aging was measured, those were getting increased in the control flies but reduced in MRS inhibition flies age dependently. Interestingly, in the germ-free condition, the maximum lifespan was increased in MRS inhibition flies compared with that of the conventional condition. These findings suggest that the lifespan of MRS inhibition flies is reduced due to the down-regulated AMPs expression in Drosophila (Suh, 2020).

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Parker, G. A., Kohn, N., Spirina, A., McMillen, A., Huang, W. and Mackay, T. F. C. (2020). Genetic Basis of Increased Lifespan and Postponed Senescence in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 31969430

Limited lifespan and senescence are near-universal phenomena. These quantitative traits exhibit variation in natural populations due to the segregation of many interacting loci and from environmental effects. Due to the complexity of the genetic control of lifespan and senescence, understanding of the genetic basis of variation in these traits is incomplete. This study analyzed the pattern of genetic divergence between long-lived (O) Drosophila melanogaster lines selected for postponed reproductive senescence and unselected control (B) lines. The productivity of the O and B lines were quantified, and reproductive senescence was found to be maternally controlled. 57 candidate genes were selected that are expressed in ovaries, 49 of which have human orthologs, and the effects of RNA interference in ovaries and accessary glands on lifespan and reproduction were assessed. All but one candidate gene affected at least one life history trait in one sex or productivity week. In addition, 23 genes had antagonistic pleiotropic effects on lifespan and productivity. Identifying evolutionarily conserved genes affecting increased lifespan and delayed reproductive senescence is the first step towards understanding the evolutionary forces that maintain segregating variation at these loci in nature and may provide potential targets for therapeutic intervention to delay senescence while increasing lifespan (Parker, 2020).

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Tonoki, A., Ogasawara, M., Yu, Z. and Itoh, M. (2020). Appetitive memory with survival benefit is robust across aging in Drosophila. J Neurosci. PubMed ID: 31992587

The formation of memory declines with advancing age. However, susceptibility to memory impairments depends on several factors, including the robustness of memory, the responsible neural circuits, and the internal state of aged individuals. How age-dependent changes in internal states and neural circuits affect memory formation remains unclear. This study showed in Drosophila melanogaster that aged flies of both sexes form robust appetitive memory conditioned with nutritious sugar, which suppresses their high mortality rates during starvation. In contrast, aging impairs the formation of appetitive memory conditioned with non-nutritious sugar that lacks survival benefits for the flies. Aging was found to enhanced the preference for nutritious sugar over non-nutritious sugar correlated with an age-dependent increase in the expression of Drosophila neuropeptide F, an ortholog of mammalian neuropeptide Y. Furthermore, a subset of dopaminergic neurons that signal the sweet taste of sugar decreases its function with aging, while a subset of dopaminergic neurons that signal the nutritional value of sugar maintains its function with age. These results suggest that aging impairs the ability to form memories without survival benefits; however, the ability to form memories with survival benefits is maintained through age-dependent changes in the neural circuits and neuropeptides (Tonoki, 2020).

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Martinez Corrales, G., Filer, D., Wenz, K. C., Rogan, A., Phillips, G., Li, M., Feseha, Y., Broughton, S. J. and Alic, N. (2020). Partial Inhibition of RNA Polymerase I Promotes Animal Health and Longevity. Cell Rep 30(6): 1661-1669. PubMed ID: 32049000

Health and survival in old age can be improved by changes in gene expression. RNA polymerase (Pol) I is the essential, conserved enzyme whose task is to generate the pre-ribosomal RNA (rRNA). Pol I is the fundamental structurally and functionally conserved eukaryotic enzyme that transcribes a single gene, ribosomal DNA (rDNA) (Vannini and Cramer, 2012). It generates the pre-rRNA that is processed into the mature 18S, 5.8S, and 28S rRNAs, the key structural and catalytic components of the ribosome. Reducing the levels of Pol I activity is sufficient to extend lifespan in the fruit fly. This effect can be recapitulated by partial, adult-restricted inhibition, with both enterocytes and stem cells of the adult midgut emerging as important cell types. In stem cells, Pol I appears to act in the same longevity pathway as Pol III, implicating rRNA synthesis in these cells as the key lifespan determinant. Importantly, reduction in Pol I activity delays broad, age-related impairment and pathology, improving the function of diverse organ systems. Hence, this study shows that Pol I activity in the adult drives systemic, age-related decline in animal health and anticipates mortality (Martinez Corrales, 2020).

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Tain, L. S., Jain, C., Nespital, T., Froehlich, J., Hinze, Y., Gronke, S. and Partridge, L. (2019). Longevity in response to lowered insulin signaling requires glycine N-methyltransferase-dependent spermidine production. Aging Cell: e13043. PubMed ID: 31721422 Reduced insulin/IGF signaling (IIS) extends lifespan in multiple organisms. Different processes in different tissues mediate this lifespan extension, with a set of interplays that remain unclear. This study shows that, in Drosophila, reduced IIS activity modulates methionine metabolism, through tissue-specific regulation of glycine N-methyltransferase (Gnmt), and that this regulation is required for full IIS-mediated longevity. Furthermore, fat body-specific expression of Gnmt was sufficient to extend lifespan. Targeted metabolomics showed that reducing IIS activity led to a Gnmt-dependent increase in spermidine levels. It was also shown that both spermidine treatment and reduced IIS activity are sufficient to extend the lifespan of Drosophila, but only in the presence of Gnmt. This extension of lifespan was associated with increased levels of autophagy. Finally, this study found that increased expression of Gnmt occurs in the liver of liver-specific IRS1 KO mice and is thus an evolutionarily conserved response to reduced IIS. The discovery of Gnmt and spermidine as tissue-specific modulators of IIS-mediated longevity may aid in developing future therapeutic treatments to ameliorate aging and prevent disease.

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Gospodaryov, D. V., Strilbytska, O. M., Semaniuk, U. V., Perkhulyn, N. V., Rovenko, B. M., Yurkevych, I. S., Barata, A. G., Dick, T. P., Lushchak, O. V. and Jacobs, H. T. (2019). Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila. Biogerontology. PubMed ID: 31749111

Mitochondrial alternative NADH dehydrogenase (aNDH) was found to extend lifespan when expressed in the fruit fly. This study has found that fruit flies expressing aNDH from Ciona intestinalis (NDX) had 17-71% lifespan prolongation on media with different protein-to-carbohydrate ratios except NDX-expressing males that had 19% shorter lifespan than controls on a high protein diet. NDX-expressing flies were more resistant to organic xenobiotics, 2,4-dichlorophenoxyacetic acid and alloxan, and inorganic toxicant potassium iodate, and partially to sodium molybdate treatments. On the other hand, NDX-expressing flies were more sensitive to catechol and sodium chromate. Enzymatic analysis showed that NDX-expressing males had higher glucose 6-phosphate dehydrogenase activity, whilst both sexes showed increased glutathione S-transferase activity (Gospodaryov, 2019).

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Nash, T. R., Chow, E. S., Law, A. D., Fu, S. D., Fuszara, E., Bilska, A., Bebas, P., Kretzschmar, D. and Giebultowicz, J. M. (2019). Daily blue-light exposure shortens lifespan and causes brain neurodegeneration in Drosophila. NPJ Aging Mech Dis 5: 8. PubMed ID: 31636947

Light is necessary for life, but prolonged exposure to artificial light is a matter of increasing health concern. Humans are exposed to increased amounts of light in the blue spectrum produced by light-emitting diodes (LEDs), which can interfere with normal sleep cycles. The LED technologies are relatively new; therefore, the long-term effects of exposure to blue light across the lifespan are not understood. This study investigated the effects of light in the model organism, Drosophila melanogaster, and determined that flies maintained in daily cycles of 12-h blue LED and 12-h darkness had significantly reduced longevity compared with flies maintained in constant darkness or in white light with blue wavelengths blocked. Exposure of adult flies to 12 h of blue light per day accelerated aging phenotypes causing damage to retinal cells, brain neurodegeneration, and impaired locomotion. Brain damage and locomotor impairments do not depend on the degeneration in the retina, as these phenotypes were evident under blue light in flies with genetically ablated eyes. Blue light induces expression of stress-responsive genes in old flies but not in young, suggesting that cumulative light exposure acts as a stressor during aging. This study also determined that several known blue-light-sensitive proteins are not acting in pathways mediating detrimental light effects. This study reveals the unexpected effects of blue light on fly brain and establishes Drosophila as a model in which to investigate long-term effects of blue light at the cellular and organismal level (Nash, 2019).

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Hoedjes, K. M., van den Heuvel, J., Kapun, M., Keller, L., Flatt, T. and Zwaan, B. J. (2019). Distinct genomic signals of lifespan and life history evolution in response to postponed reproduction and larval diet in Drosophila. Evol Lett 3(6): 598-609. PubMed ID: 31867121

Reproduction and diet are two major factors controlling the physiology of aging and life history, but how they interact to affect the evolution of longevity is unknown. Moreover, although studies of large-effect mutants suggest an important role of nutrient sensing pathways in regulating aging, the genetic basis of evolutionary changes in lifespan remains poorly understood. To address these questions, the genomes of experimentally evolved Drosophila melanogaster populations were subjected to a factorial combination of two selection regimes: reproductive age (early versus postponed), and diet during the larval stage ("low," "control," "high"), resulting in six treatment combinations with four replicate populations each. Selection on reproductive age consistently affected lifespan, with flies from the postponed reproduction regime having evolved a longer lifespan. In contrast, larval diet affected lifespan only in early-reproducing populations: flies adapted to the "low" diet lived longer than those adapted to control diet. This study found genomic evidence for strong independent evolutionary responses to either selection regime, as well as loci that diverged in response to both regimes, thus representing genomic interactions between the two. Overall, the genomic basis of longevity was found to be largely independent of dietary adaptation. Differentiated loci were not enriched for "canonical" longevity genes, suggesting that naturally occurring genic targets of selection for longevity differ qualitatively from variants found in mutant screens. Comparing the candidate loci to those from other "evolve and resequence" studies of longevity demonstrated significant overlap among independent experiments. This suggests that the evolution of longevity, despite its presumed complex and polygenic nature, might be to some extent convergent and predictable (Hoedjes, 2019).

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Erwin, A. A. and Blumenstiel, J. P. (2019). Aging in the Drosophila ovary: contrasting changes in the expression of the piRNA machinery and mitochondria but no global release of transposable elements. BMC Genomics 20(1): 305. PubMed ID: 31014230

Evolutionary theory indicates that the dynamics of aging in the soma and reproductive tissues may be distinct. Using mRNA sequencing data from late-stage egg chambers in Drosophila melanogaster, this study characterized the landscape of altered gene and transposable element expression in aged reproductive tissues. This allowed a test of the hypothesis that reproductive tissues may differ from somatic tissues in their response to aging. This study shows that age-related expression changes in late-stage egg chambers tend to occur in genes residing in heterochromatin, particularly on the largely heterochromatic 4th chromosome. However, these expression differences are seen as both decreases and increases during aging, inconsistent with a general loss of heterochromatic silencing. This study also identified an increase in expression of the piRNA machinery, suggesting an age-related increased investment in the maintenance of genome stability. A strong age-related reduction in the expression of mitochondrial transcripts was identified. However, no evidence was foud for global TE derepression in reproductive tissues. Rather, the observed effects of aging on TEs are primarily strain and family specific. These results identify unique responses in somatic versus reproductive tissue with regards to aging. As in somatic tissues, female reproductive tissues show reduced expression of mitochondrial genes. In contrast, the piRNA machinery shows increased expression during aging. Overall, these results also indicate that global loss of TE control observed in other studies may be unique to the soma and sensitive to genetic background and TE family (Erwin, 2019).

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Schinaman, J. M., Rana, A., Ja, W. W., Clark, R. I. and Walker, D. W. (2019). Rapamycin modulates tissue aging and lifespan independently of the gut microbiota in Drosophila. Sci Rep 9(1): 7824. PubMed ID: 31127145

The FDA approved drug rapamycin can prolong lifespan in diverse species and delay the onset of age-related disease in mammals. However, a number of fundamental questions remain unanswered regarding the mechanisms by which rapamycin modulates age-related pathophysiology and lifespan. Alterations in the gut microbiota can impact host physiology, metabolism and lifespan. While recent studies have shown that rapamycin treatment alters the gut microbiota in aged animals, the causal relationships between rapamycin treatment, microbiota dynamics and aging are not known. Using Drosophila as a model organism, this study shows that rapamycin-mediated alterations in microbiota dynamics in aged flies are associated with improved markers of intestinal and muscle aging. Critically, however, this study shows that the beneficial effects of rapamycin treatment on tissue aging and lifespan are not dependent upon the microbiota. Indeed, germ-free flies show delayed onset of intestinal barrier dysfunction, improved proteostasis in aged muscles and a significant lifespan extension upon rapamycin treatment. In contrast, genetic inhibition of autophagy impairs the ability of rapamycin to mediate improved gut health and proteostasis during aging. These results indicate that rapamycin-mediated modulation of the microbiota in aged animals is not causally required to slow tissue and organismal aging (Schinaman, 2019).

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Kristensen, T. N., Loeschcke, V., Tan, Q., Pertoldi, C. and Mengel-From, J. (2019). Sex and age specific reduction in stress resistance and mitochondrial DNA copy number in Drosophila melanogaster. Sci Rep 9(1): 12305. PubMed ID: 31444377

Environmental stresses such as extreme temperatures, dehydration and food deprivation may have distinct consequences for different age-classes and for males and females across species. This study investigated a natural population of the model organism Drosophila melanogaster. Males and females at ages 3, 19 and 35 days were tested for stress resistance; i.e. the ability of flies to cope with starvation and both cold and hot temperatures. Further, a measure of metabolic efficiency, namely mitochondrial DNA copy number (mtDNA CN), was tested in both sexes at all three age-classes. It was hypothesized that stress resistance is reduced at old age and more so in males, and that mtDNA CN is a biomarker for sex- and age-dependent reductions in the ability to cope with harsh environments. This study showed that: (1) males exhibit reduced starvation tolerance at old age, whereas older females are better in coping with periods without food compared to younger females, (2) heat tolerance decreases with increasing age in males but not in females, (3) cold tolerance is reduced at old age in both sexes, and (4) old males have reduced mtDNA CN whereas mtDNA CN slightly increases with age in females. In conclusion, these data provide strong evidence for trait and sex specific consequences of aging with females generally being better at coping with environmental stress at old age. The reduced mtDNA CN in old males suggests reduced metabolic efficiency and this may partly explain why males are less stress tolerant at old age than females. It is suggested that mtDNA CN might be a suitable biomarker for physiological robustness. These findings likely extend to other taxa than Drosophila and therefore the observations are discussed in relation to aging and sex specific lifespan across species (Kristensen, 2019).

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Duxbury, E. M. L. and Chapman, T. (2019) (2019). Sex-specific responses of lifespan and fitness to variation in developmental versus adult diets in D. melanogaster. J Gerontol A Biol Sci Med Sci. PubMed ID: 31362304

Nutritional variation across the lifetime can have significant and sex-specific impacts upon fitness. Using Drosophila melanogaster, these impacts were measured by testing the effects on lifespan and reproductive success of high or low yeast content in developmental versus adult diets, separately for each sex. Two hypotheses were tested: that dietary mismatches between development and adulthood are costly and that any such costs are sex-specific. Overall, the results revealed the rich and complex responses of each sex to dietary variation across the lifetime. Contrary to the first hypothesis, dietary mismatches between developmental and adult life stages were not universally costly. Where costs of nutritional variation across the life course did occur, they were sex-, context- and trait-specific, consistent with hypothesis 2. Effects of mismatches between developmental and adult diets on reproductive success were found in females but not males. Adult diet was the main determinant of survival, and lifespan was significantly longer on high yeast adult food, in comparison to low, in both sexes. Developing on a high yeast diet also benefited adult female lifespan and reproductive success, regardless of adult diet. In contrast, a high yeast developmental diet was only beneficial for male lifespan when it was followed by low yeast adult food. Adult diet affected mating frequency in opposing directions, with males having higher mating frequency on high and females on low, with no interaction with developmental diet for either sex. The results emphasize the importance of sex differences and of the directionality of dietary mismatches in the responses to nutritional variation (Duxbury, 2019).

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Castillo-Quan, J. I., Tain, L. S., Kinghorn, K. J., Li, L., Gronke, S., Hinze, Y., Blackwell, T. K., Bjedov, I. and Partridge, L. (2019). A triple drug combination targeting components of the nutrient-sensing network maximizes longevity. Proc Natl Acad Sci U S A 116(42): 20817-20819. PubMed ID: 31570569

Increasing life expectancy is causing the prevalence of age-related diseases to rise, and there is an urgent need for new strategies to improve health at older ages. Reduced activity of insulin/insulin-like growth factor signaling (IIS) and mechanistic target of rapamycin (mTOR) nutrient-sensing signaling network can extend lifespan and improve health during aging in diverse organisms. However, the extensive feedback in this network and adverse side effects of inhibition imply that simultaneous targeting of specific effectors in the network may most effectively combat the effects of aging. This study shows that the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib, the mTOR complex 1 (mTORC1) inhibitor rapamycin, and the glycogen synthase kinase-3 (GSK-3) inhibitor lithium act additively to increase longevity in Drosophila. Remarkably, the triple drug combination increased lifespan by 48%. Furthermore, the combination of lithium with rapamycin cancelled the latter's effects on lipid metabolism. In conclusion, a polypharmacology approach of combining established, prolongevity drug inhibitors of specific nodes may be the most effective way to target the nutrient-sensing network to improve late-life health (Castillo-Quan, 2019).

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Wen, D. T., Zheng, L., Li, J. X., Lu, K. and Hou, W. Q. (2019).The activation of cardiac dSir2-related pathways mediates physical exercise resistance to heart aging in old Drosophila. Aging (Albany NY) 11. PubMed ID: 31503544

Cardiac aging is notably characterized by increased diastolic dysfunction, lipid accumulation, oxidative stress, and contractility debility. The Sir2/Sirt1 gene overexpression delays cell aging and reduces obesity and oxidative stress. Exercise improves heart function and delays heart aging. However, it remains unclear whether exercise delaying heart aging is related to cardiac Sir2/Sirt1-related pathways. In this study, cardiac dSir2 overexpression or knockdown was regulated using the UAS/hand-Gal4 system in Drosophila. Flies underwent exercise interventions from 4 weeks to 5 weeks old. Results showed that either cardiac dSir2 overexpression or exercise remarkably increased the cardiac period, systolic interval, diastolic interval, fractional shortening, SOD activity, dSIR2 protein, Foxo, dSir2, Nmnat, and bmm expression levels in the aging flies; they also notably reduced the cardiac triacylglycerol level, malonaldehyde level, and the diastolic dysfunction index. Either cardiac dSir2 knockdown or aging had almost opposite effects on the heart as those of cardiac dSir2 overexpression. Therefore, this study claims that cardiac dSir2 overexpression or knockdown delayed or promoted heart aging by reducing or increasing age-related oxidative stress, lipid accumulation, diastolic dysfunction, and contractility debility. The activation of cardiac dSir2/Foxo/SOD and dSir2/Foxo/Bmm pathways may be two important molecular mechanisms through which exercise works against heart aging in Drosophila (Wen, 2019).

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Qiu, W., Chen, X., Tian, Y., Wu, D., Du, M. and Wang, S. (2019). Protection against oxidative stress and anti-aging effect in Drosophila of royal jelly-collagen peptide. Food Chem Toxicol: 110881. PubMed ID: 31622731

Dietary peptide has been of great interest because of its perspective in nutrition and health of human body. The aim of this study was to develop a dietary nutritional supplement exerting both antioxidant and anti-aging effects. Peptide, named as ERJ-CP, was prepared by mixing enzyme-treated royal jelly (ERJ) with collagen peptide (CP), showing stronger antioxidant activity in vitro. Drosophila was used as model animal to investigate anti-aging effect of ERJ-CP in vivo. ERJ-CP significantly prolonged the average life span of Drosophila treated with H2O2 and paraquat, reducing malondialdehyde (MDA) and protein carbonyl (PCO) levels in Drosophila. In addition, 3mg/mL of ERJ-CP could prolong the lifespan of natural aging Drosophila by 11.16%. ERJ-CP could up-regulate the levels of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and down-regulate the contents of MDA and PCO. Moreover, the intake of ERJ-CP increased the food consumption, weight gain and exercise capacity of Drosophila. The results showed that ERJ-CP played a protective role in both antioxidant and anti-aging effects on Drosophila, and the anti-aging effect may be achieved by alleviating oxidative damage. It suggests that ERJ-CP could be developed as a health-promoting ingredient with antioxidant and anti-aging effects for human body (Qiu, 2019).

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Xiao, C., Hull, D., Qiu, S., Yeung, J., Zheng, J., Barwell, T., Robertson, R. M. and Seroude, L. (2019). Expression of Heat shock protein 70 is insufficient to extend Drosophila melanogaster longevity. G3 (Bethesda). PubMed ID: 31624139

It has been known for over 20 years that Drosophila melanogaster flies with twelve additional copies of the hsp70 gene encoding the 70 kDa heat shock protein lives longer after a non-lethal heat treatment. Since the heat treatment also induces the expression of additional heat shock proteins, the biological effect can be due either to HSP70 acting alone or in combination. This study used the UAS/GAL4 system to determine whether hsp70 is sufficient to affect the longevity and the resistance to thermal, oxidative or desiccation stresses of the whole organism. It was observed that HSP70 expression in the nervous system or muscles has no effect on longevity or stress resistance but ubiquitous expression reduces the life span of males. It was also observed that the down-regulation of Hsp70 using RNAi did not affect longevity (Xiao, 2019).

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Li, J. Q., Duan, D. D., Zhang, J. Q., Zhou, Y. Z., Qin, X. M., Du, G. H. and Gao, L. (2019). Bioinformatic prediction of critical genes and pathways involved in longevity in Drosophila melanogaster. Mol Genet Genomics. PubMed ID: 31327054

The pursuit of longevity has been the goal of humanity since ancient times. Genetic alterations have been demonstrated to affect lifespan. As increasing numbers of pro-longevity genes and anti-longevity genes have been discovered in Drosophila, screening for functionally important genes among the large number of genes has become difficult. The aim of this study was to explore critical genes and pathways affecting longevity in Drosophila melanogaster. In this study, 168 genes associated with longevity in D. melanogaster were collected from the Human Ageing Genomic Resources (HAGR) database. Network clustering analysis, network topological analysis, and pathway analysis were integrated to identify key genes and pathways. Quantitative real-time PCR (qRT-PCR) was applied to verify the expression of genes in representative pathways and of predicted genes derived from the gene-gene sub-network. The results revealed that six key pathways might be associated with longevity, including the longevity-regulating pathway, the peroxisome pathway, the mTOR-signalling pathway, the FOXO-signalling pathway, the AGE-RAGE-signalling pathway in diabetic complications, and the TGF-beta-signalling pathway. Moreover, the results revealed that six key genes in representative pathways, including Cat, Ry, S6k, Sod, Tor, and Tsc1, and the predicted genes Jra, Kay, and Rheb exhibited significant expression changes in ageing D. melanogaster strain w(1118) compared to young ones. Overall, these results revealed that six pathways and six key genes might play pivotal roles in regulating longevity, and three interacting genes might be implicated in longevity. The results will not only provide new insight into the mechanisms of longevity, but also provide novel ideas for network-based approaches for longevity-related research (Li, 2019).

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Keebaugh, E. S., Yamada, R. and Ja, W. W. (2019). The nutritional environment influences the impact of microbes on Drosophila melanogaster life span. MBio 10(4). PubMed ID: 31289176

Microbes can extend Drosophila melanogaster life span by contributing to the nutritional value of malnourishing fly culture medium. The beneficial effect of microbes during malnutrition is dependent on their individual ability to proliferate in the fly environment and is mimicked by lifelong supplementation of equivalent levels of heat-killed microbes or dietary protein, suggesting that microbes can serve directly as a protein-rich food source. This study used nutritionally rich fly culture medium to demonstrate how changes in dietary composition influence monocolonized fly life span; microbes that extend fly life span on malnourishing diets can shorten life on rich diets. The mechanisms employed by microbes to affect host health likely differ on low- or high-nutrient diets. The results demonstrate how Drosophila-associated microbes can positively or negatively influence fly life span depending on the nutritional environment. Although controlled laboratory environments allow focused investigations on the interaction between fly microbiota and nutrition, the relevance of these studies is not straightforward, because it is difficult to mimic the nutritional ecology of natural Drosophila-microbe interactions. As such, caution is needed in designing and interpreting fly-microbe experiments and before categorizing microbes into specific symbiotic roles based on results obtained from experiments testing limited conditions (Keebaugh, 2019).

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Munkacsy, E., Chocron, E. S., Quintanilla, L., Gendron, C. M., Pletcher, S. D. and Pickering, A. M. (2019). Neuronal-specific proteasome augmentation via Prosbeta5 overexpression extends lifespan and reduces age-related cognitive decline. Aging Cell: e13005. PubMed ID: 31334599

Cognitive function declines with age throughout the animal kingdom, and increasing evidence shows that disruption of the proteasome system contributes to this deterioration. The proteasome has important roles in multiple aspects of the nervous system, including synapse function and plasticity, as well as preventing cell death and senescence. Previous studies have shown neuronal proteasome depletion and inhibition can result in neurodegeneration and cognitive deficits, but it is unclear if this pathway is a driver of neurodegeneration and cognitive decline in aging. This study reports that overexpression of the proteasome beta5 subunit enhances proteasome assembly and function. Significantly, it was shown that neuronal-specific proteasome augmentation slows age-related declines in measures of learning, memory, and circadian rhythmicity. Surprisingly, neuronal-specific augmentation of proteasome function also produces a robust increase of lifespan in Drosophila melanogaster. These findings appear specific to the nervous system; ubiquitous proteasome overexpression increases oxidative stress resistance but does not impact lifespan and is detrimental to some healthspan measures. These findings demonstrate a key role of the proteasome system in brain aging (Munkacsy, 2019).

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Hunt, L. C., Stover, J., Haugen, B., Shaw, T. I., Li, Y., Pagala, V. R., Finkelstein, D., Barton, E. R., Fan, Y., Labelle, M., Peng, J. and Demontis, F. (2019). A key role for the ubiquitin ligase UBR4 in myofiber hypertrophy in Drosophila and mice. Cell Rep 28(5): 1268-1281. PubMed ID: 31365869

Skeletal muscle cell (myofiber) atrophy is a detrimental component of aging and cancer that primarily results from muscle protein degradation via the proteasome and ubiquitin ligases. Transcriptional upregulation of some ubiquitin ligases contributes to myofiber atrophy, but little is known about the role that most other ubiquitin ligases play in this process. To address this question, RNAi screening in Drosophila was used to identify the function of > 320 evolutionarily conserved ubiquitin ligases in myofiber size regulation in vivo. Whereas RNAi for some ubiquitin ligases induces myofiber atrophy, loss of others (including the N-end rule ubiquitin ligase UBR4) promotes hypertrophy. In Drosophila and mouse myofibers, loss of UBR4 induces hypertrophy via decreased ubiquitination and degradation of a core set of target proteins, including the HAT1/RBBP4/RBBP7 histone-binding complex. Together, this study defines the repertoire of ubiquitin ligases that regulate myofiber size and the role of UBR4 in myofiber hypertrophy (Hunt, 2019).

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Andreazza, S., Samstag, C. L., Sanchez-Martinez, A., Fernandez-Vizarra, E., Gomez-Duran, A., Lee, J. J., Tufi, R., Hipp, M. J., Schmidt, E. K., Nicholls, T. J., Gammage, P. A., Chinnery, P. F., Minczuk, M., Pallanck, L. J., Kennedy, S. R. and Whitworth, A. J. (2019). Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila. Nat Commun 10(1): 3280. PubMed ID: 31337756

Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. This study describes a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research (Andreazza, 2019).

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Niraula, P. and Kim, M. S. (2019). N-Acetylcysteine extends lifespan of Drosophila via modulating ROS scavenger gene expression. Biogerontology 20(4): 533-543. PubMed ID: 31115735

N-Acetylcysteine (NAc) has been shown to play a diversity of favorable health-related roles (e.g., antioxidant, paracetamol antidote, mucolytics, neuroprotective agent). This study evaluated the health-promoting properties of NAc, particularly its ability to modulate organismal longevity. It is noted that 1 mg/ml NAc prolonged the lifespan of Drosophila. Furthermore, it was observed that NAc increased the capability of these flies to resist environmental stresses measured by starvation and paraquat stress assays. In an effort to reveal cellular mechanisms behind this interesting phenomenon, qPCR was performed, uncovering that transcript levels of catalase and phospholipid hydroperoxide glutathione peroxidase-key enzymes to fend off reactive oxygen species (ROS) assaults, were up-regulated. Correspondingly, enzyme activities of catalase and glutathione peroxidase were increased as well. Combined, it is hoped that this research helps broaden the spectrum of clinical application for NAc so that one may eventually determine if NAc is a potentially useful anti-aging agent by encouraging others to scrutinize the hidden health benefits of NAc (Niraula, 2019).

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Deepashree, S., Niveditha, S., Shivanandappa, T. and Ramesh, S. R. (2019). Oxidative stress resistance as a factor in aging: evidence from an extended longevity phenotype of Drosophila melanogaster. Biogerontology. PubMed ID: 31054025

Longevity of a species is a multifactorial quantitative trait influenced by genetic background, sex, age and environment of the organism. Extended longevity phenotypes (ELP) from experimental evolution in the laboratory can be used as model systems to investigate the mechanisms underlying aging and senescence. This study investigated the hypothesis that enhanced oxidative stress resistance and elevated antioxidant defense system play a positive role in longevity using an ELP of Drosophila melanogaster. An ELP of D. melanogaster isolated and characterized through artificial selection (inbred laboratory strain of Oregon K) was employed in this study. This ELP, designated long lifespan (LLS) flies, shows marked extension in lifespan when compared to the progenitor population (normal lifespan, NLS) and makes a suitable model to study the role of mitochondrial genome in longevity because of its least heterogeneity. In this study, sensitivity to ethanol with age was employed as a measure of resistance to oxidative stress in NLS and LLS flies. Effect of age and oxidative stress on longevity was examined by employing NLS and LLS flies of different age groups against ethanol-induced oxidative stress. Results show that the lower mortality against ethanol was associated with enhanced oxidative stress resistance, higher antioxidant defenses, lower reactive oxygen species (ROS) levels, enhanced alcohol dehydrogenase activity and better locomotor ability attributes of LLS flies. In addition, age-related changes like locomotor impairments, decreased antioxidant defenses, higher ROS levels and sensitivity to oxidative stress were delayed in LLS flies when compared to NLS. This study supports the hypothesis that higher oxidative stress resistance and enhanced antioxidant defenses are significant factors in extending longevity (Deepashree, 2019).

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Su, Y., Wang, T., Wu, N., Li, D., Fan, X., Xu, Z., Mishra, S. K. and Yang, M. (2019). Alpha-ketoglutarate extends Drosophila lifespan by inhibiting mTOR and activating AMPK. Aging (Albany NY) 11. PubMed ID: 31242135

Alpha-ketoglutarate (AKG) is a key metabolite of the tricarboxylic acid (TCA) cycle, an essential process influencing the mitochondrial oxidative respiration rate. Recent studies have shown that dietary AKG reduces mTOR pathway activation by inhibiting ATP synthase, thereby extending the lifespan of nematodes. Although AKG also extends lifespan in fruit flies, the antiaging mechanisms of AKG in these organisms remain unclear. This study explored changes in gene expression associated with the extension of Drosophila lifespan mediated by dietary AKG. Supplementation of the flies' diets with 5 &mi;M AKG extended their lifespan but reduced their reproductive performance. Dietary AKG also enhanced vertical climbing ability, but did not protect against oxidative stress or increase tolerance to starvation. AKG-reared flies were resistant to heat stress and demonstrated higher expression of heat shock protein genes (Hsp22 and Hsp70) than control flies. In addition, AKG significantly upregulated mRNA expression of cry, FoxO, HNF4, p300, Sirt1 and AMPKalpha, and downregulated expression of HDAC4, PI3K, TORC, PGC, and SREBP. The metabolic effects of AKG supplementation included a reduction in the ATP/ADP ratio and increased autophagy. Collectively, these observations indicate that AKG extends Drosophila lifespan by activating AMPK signaling and inhibiting the mTOR pathway (Su, 2019).

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Flintham, E. O., Yoshida, T., Smith, S., Pavlou, H. J., Goodwin, S. F., Carazo, P. and Wigby, S. (2018). Interactions between the sexual identity of the nervous system and the social environment mediate lifespan in Drosophila melanogaster. Proc Biol Sci 285(1892). PubMed ID: 30487307

Sex differences in lifespan are ubiquitous, but the underlying causal factors remain poorly understood. Inter- and intrasexual social interactions are well known to influence lifespan in many taxa, but it has proved challenging to separate the role of sex-specific behaviours from wider physiological differences between the sexes. To address this problem, the sexual identity of the nervous system - and hence sexual behaviour - was genetically manipulated in Drosophila melanogaster, and lifespan was measured under varying social conditions. Consistent with previous studies, masculinization of the nervous system in females induced male-specific courtship behaviour and aggression, while nervous system feminization in males induced male-male courtship and reduced aggression. Control females outlived males, but masculinized female groups displayed male-like lifespans and male-like costs of group living. By varying the mixture of control and masculinized females within social groups, male-specific behaviours were shown to be costly to recipients, even when received from females. However, consistent with recent findings, the data suggest courtship expression to be surprisingly low cost. Overall, this study indicates that nervous system-mediated expression of sex-specific behaviour per se-independent of wider physiological differences between the sexes, or the receipt of aggression or courtship-plays a limited role in mediating sex differences in lifespan (Flintham, 2018).

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Pacifico, R., MacMullen, C. M., Walkinshaw, E., Zhang, X. and Davis, R. L. (2018). Brain transcriptome changes in the aging Drosophila melanogaster accompany olfactory memory performance deficits. PLoS One 13(12): e0209405. PubMed ID: 30576353

Cognitive decline is a common occurrence of the natural aging process in animals and studying age-related changes in gene expression in the brain might shed light on disrupted molecular pathways that play a role in this decline. The fruit fly is a useful neurobiological model for studying aging due to its short generational time and relatively small brain size. This study investigated age-dependent changes in the Drosophila melanogaster whole-brain transcriptome by comparing 5-, 20-, 30- and 40-day-old flies of both sexes. RNA-sequencing of dissected brain samples followed by differential expression, temporal clustering, co-expression network and gene ontology enrichment analyses were performed. An overall decline was observed in expression of genes from the mitochondrial oxidative phosphorylation pathway that occurred as part of aging. In females, a pattern of continuously declining expression was detected for many neuronal function genes, which was unexpectedly reversed later in life. This group of genes was highly enriched in memory-impairing genes previously identified through an RNAi screen. Deficits in short-term olfactory memory performance was observed in older flies of both sexes, some of which matched the timing of certain changes in the brain transcriptome. This study provides the first transcriptome profile of aging brains from fruit flies of both sexes, and it will serve as an important resource for those who study aging and cognitive decline in this model (Pacifico, 2018).

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Nguyen, N. N., Rana, A., Goldman, C., Moore, R., Tai, J., Hong, Y., Shen, J., Walker, D. W. and Hur, J. H.(2019). Proteasome beta5 subunit overexpression improves proteostasis during aging and extends lifespan in Drosophila melanogaster. Sci Rep 9(1): 3170. PubMed ID: 30816680

The beta5 subunit of the proteasome has been shown in worms and in human cell lines to be regulatory. In these models, beta5 overexpression results in upregulation of the entire proteasome complex which is sufficient to increase proteotoxic stress resistance, improve metabolic parameters, and increase longevity. However, fundamental questions remain unanswered, including the temporal requirements for beta5 overexpression and whether beta5 overexpression can extend lifespan in other species. To determine if adult-only overexpression of the beta5 subunit can increase proteasome activity in a different model, this study characterized phenotypes associated with beta5 overexpression in Drosophila melanogaster adults. Adult-only overexpression of the beta5 subunit does not result in transcriptional upregulation of the other subunits of the proteasome as they do in nematodes and human cell culture. Despite this lack of a regulatory role, boosting beta5 expression increases the chymotrypsin-like activity associated with the proteasome, reduces both the size and number of ubiquitinated protein aggregates in aged flies, and increases longevity. Surprisingly, these phenotypes were not associated with increased resistance to acute proteotoxic insults or improved metabolic parameters (Nguyen, 2019).

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Bhukel, A., Beuschel, C. B., Maglione, M., Lehmann, M., Juhasz, G., Madeo, F. and Sigrist, S. J.(2019). Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner. Nat Commun 10(1): 1318. PubMed ID: 30899013

Macroautophagy is an evolutionarily conserved cellular maintenance program, meant to protect the brain from premature aging and neurodegeneration. How neuronal autophagy, usually loosing efficacy with age, intersects with neuronal processes mediating brain maintenance remains to be explored. This study shows that impairing autophagy in the Drosophila learning center (mushroom body, MB) but not in other brain regions triggered changes normally restricted to aged brains: impaired associative olfactory memory as well as a brain-wide ultrastructural increase of presynaptic active zones (metaplasticity), a state non-compatible with memory formation. Mechanistically, decreasing autophagy within the MBs reduced expression of an NPY-family neuropeptide, and interfering with autocrine NPY signaling of the MBs provoked similar brain-wide metaplastic changes. The results in an exemplary fashion show that autophagy-regulated signaling emanating from a higher brain integration center can execute high-level control over other brain regions to steer life-strategy decisions such as whether or not to form memories (Bhukel, 2019).

The maintenance of neuronal homeostasis is severely threatened by aging. The strictly postnatal character of deficits observed after KD of core autophagy machinery triggered the hope that autophagy might have a specific relation to the aging process. The last few years have indeed seen an accumulation of evidences that the efficiency of autophagic clearance in neurons declines with age on organismal level. Hence, rejuvenating autophagy in aging neurons is considered a promising strategy to restore cognitive performance. Successfully exploring this direction will, however, depend on deepening insights at the intersection of autophagy, the relevant neuronal sub-cellular compartments, importantly synaptic specializations, and relevant neuron populations/brain regions (Bhukel, 2019).

The endogenous polyamine spermidine has prominent cardio-protective and neuro-protective effects and recent work finds spermidine restoration to counteract otherwise deteriorating health in aging mice in an autophagy-dependent manner. In Drosophila, restoring spermidine specifically suppressed age-induced decay in their ability to form olfactory memories, again in an autophagy-dependent manner. Concomitantly, in the aged Drosophila brain, previous work found a brain-wide, age-induced upshift in the ultrastructural size (EM: larger T-bars; STED: increased diameter of BRP scaffold) of presynaptic AZs (metaplasticity). Two findings causally linked this upshift to decreased olfactory memory performance. First, when continuously fed with spermidine, flies of 30 days of age (normally suffering from a complete loss of age-sensitive component of memory) were largely protected from these changes. Secondly, genetically provoking this up-shift eliminated the normally age-sensitive memory component in young animals already. An upshift in the AZ size should increase synaptic strength, evident in increased SV release in response to natural odors observed in aged but not aged-spermidine-fed flies. Presynaptic plasticity is crucial for forming memory traces in Drosophila. Previous work thus suggests that this presynaptic metaplasticity shifts the operational range of synapses in a way that they become unable to execute the plastic changes faithfully in response to conditioning stimuli (Bhukel, 2019).

This study further addressed the relation between defective autophagy, presynaptic ultrastructure and plasticity and olfactory memory formation. Autophagosome biogenesis is very dominant close to presynaptic specializations in distal axons in compartmentalized fashion and efficient macro-autophagy is essential for neuronal homeostasis and survival. Retrograde transport of autophagosomes might play a role in broader neuronal signaling processes, promoting neuronal complexity and preventing neurodegeneration. Surprisingly, however, the data do not favor a direct substrate relationship between AZ proteins and autophagy. Instead, evidence was found for a seemingly non-cell autonomous relation between brain-wide synapse organization and the autophagic status of the mere MB. After genetic impairment of autophagy (via atg5 or atg9 KD) using two different MB-specific Gal4-driver lines, the presynaptic metaplasticity was observed across the Drosophila olfactory system and beyond. While the autophagic arrest (p62 staining) was largely limited to the expression domain of these drivers, the synapses were pushed towards a state of metaplasticity. Since the ultrastructural size of AZs and the per AZ BRP levels increased equally in aged and MB-autophagy-challenged animals, it is concluded that the autophagic status of the MB neuron population executes a signaling process, which can control the per AZ amounts of BRP and other AZ proteins. Further studies are warranted to dissect the nature of these signaling processes (Bhukel, 2019).

Notably, accumulating evidences support the important role of neuropeptide Y (NPY) in aging and lifespan determination. NPY levels decrease with age in mice and re-substituting NPY is able to counteract age-induced changes of the brain at several levels. A cross-talk between autophagy and NPY in regulating the feeding behavior has been demonstrated in mice (Bhukel, 2019).

This study found that transcript expression level of an NPY family member (sNPF) are controlled by autophagy within the MBs. snpf hypomorph allele mimicking the MB reduction of sNPF of the MB-specific autophagy KD situations as well as the sNPF expression in aged animals. In this hypomorph allele a similar up regulation was observed in BRP Nc82 signal. KD of the snpfr using an MB-specific driver drove the brain-wide metaplastic change even stronger than the sNPF hypomorph (obviously only partially affecting the sNPF-specific signaling). This scenario in ultrastructural detail resembled both the age-induced and MB-specific autophagy-KD-induced metaplasticity phenotypes. These results, therefore, support the essential role of MB in integrating the metabolic state of Drosophila in an autocrine fashion to modulate the presynaptic release scaffold state throughout the fly brain. The mechanistic basis of this exciting regulation warrants further investigation. Interestingly, elevated cAMP signaling is generally driving plasticity in Drosophila neurons, while sNPF signaling is meant to reduce cAMP and thus potentially might be able to reset plastic changes such as increased BRP levels. In apparent contradiction to sNPF signaling directly widely controlling metaplasticity is the finding that MB-specific KD of the sNPFR sufficed to increase BRP levels. At this moment, it can only be speculated as to why KD of sNPF-receptor also results in extended metaplastic changes. Potentially, sNPF-receptor signaling within the MB might be important to control sNPF secretion in a physiological manner via a quasi-autocrine mechanism (Bhukel, 2019).

Intriguingly, the metaplastic state characterized both aged and MB-specific autophagy KD animals, and in both cases provoked a specific loss of the ASM component of memory. Notably, olfactory MTM measured in this study, are considered to be the direct precursor of olfactory LTM, which in turn have been shown to be energetically costly. Notably, autophagy and NPY signaling are prime candidate mechanisms for the therapy of age-induced cognitive processes (Bhukel, 2019).

Recent research has uncovered several examples connecting autophagy and hormonal-type regulations interacting between organ systems in non-cell autonomous regimes. For instance, Atg18 acts non-cell autonomously both in neurons and in intestines to firstly, maintain the wild-type lifespan of C.elegans and secondly, to respond to the dietary restriction and DAF-2 longevity signals. Atg18 in chemosensory neurons and intestines acts in parallel and converges on unidentified neurons that secrete neuropeptides to mediate the influence of Daf-2 on C.elegans lifespan through the transcription factor DAF-16/FOXO in response to reduced IGF signaling. In Drosophila, neuronal up-regulation of AMPK induces autophagy, via up-regulation of Atg1 non-cell autonomously in intestines and slows intestinal aging and vice versa. Moreover, up-regulation of Atg1 in neurons extends lifespan and maintains intestinal homeostasis during aging and these inter-tissue effects of AMPK/Atg1 were linked to altered insulin-like signaling. On the contrary, this study found the insulin producing cells (IPCs) themselves to not mediate the observed metaplastic state, as neither the KD of atg9 nor the KD of snpfr in Pars intercerebralis had any impact on the synaptic status of these flies (Bhukel, 2019).

Autophagy regulation is tightly connected to cellular energetics, nutrient recycling, and the maintenance of cellular energy status. The fruit fly can evaluate its metabolic state by integrating hunger and satiety signals at the very KC-to-MBON synapses in MB under control of dopaminergic neurons to control hunger-driven food-seeking behavior. At the same time, long-term memory encoding necessitates an increase in MB energy flux with dopamine signaling mediating this energy switch in the MB. In line with these findings, this study now provides a modeling basis to study these delicate relations in an exemplary fashion. Taken together, these data suggest that MB integrates the metabolic state of the flies via cross talk between autophagy and sNPF signaling with the decision whether to form memories or not and a block in this cross talk with aging gives rise to synaptic metaplasticity which initiates the age-induced memory impairment in Drosophila. It is tempting to speculate that the MB executes hierarchically, a high-level control integrating the metabolic and caloric situation with a life-strategy decision of whether or not to form mid-term memories (Bhukel, 2019).

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Wang, M. and Lemos, B. (2019). Ribosomal DNA harbors an evolutionarily conserved clock of biological aging. Genome Res. PubMed ID: 30765617

The ribosomal DNA (rDNA) is the most evolutionarily conserved segment of the genome and gives origin to the nucleolus, an energy intensive nuclear organelle and major hub influencing myriad molecular processes from cellular metabolism to epigenetic states of the genome. The rDNA/nucleolus has been directly and mechanistically implicated in aging and longevity in organisms as diverse as yeasts, Drosophila, and humans. The rDNA is also a significant target of DNA methylation that silences supernumerary rDNA units and regulates nucleolar activity. This study introduced an age clock built exclusively with CpG methylation within the rDNA. The ribosomal clock is sufficient to accurately estimate individual age within species, is responsive to genetic and environmental interventions that modulate life-span, and operates across species as distant as humans, mice, and dogs. Further analyses revealed a significant excess of age-associated hypermethylation in the rDNA relative to other segments of the genome, and which forms the basis of the rDNA clock. These observations identified an evolutionarily conserved marker of aging that is easily ascertained, grounded on nucleolar biology, and could serve as a universal marker to gauge individual age and response to interventions in humans as well as laboratory and wild organisms across a wide diversity of species (Wang, 2019).

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Kennerdell, J. R., Liu, N. and Bonini, N. M. (2018). MiR-34 inhibits polycomb repressive complex 2 to modulate chaperone expression and promote healthy brain aging. Nat Commun 9(1): 4188. PubMed ID: 30305625

Aging is a prominent risk factor for neurodegenerative disease. Defining gene expression mechanisms affecting healthy brain aging should lead to insight into genes that modulate susceptibility to disease. To define such mechanisms, analysis of miR-34 mutants have been pursued in Drosophila. The miR-34 mutant brain displays a gene expression profile of accelerated aging, and miR-34 upregulation is a potent suppressor of polyglutamine-induced neurodegeneration. Pcl and Su(z)12, two components of polycomb repressive complex 2, (PRC2), are targets of miR-34, with implications for age-associated processes. Because PRC2 confers the repressive H3K27me3 mark, it is hypothesized that miR-34 modulates PRC2 activity to relieve silencing of genes promoting healthful aging. Gene expression profiling of the brains of hypomorphic mutants in Enhancer of zeste (E(z)), the enzymatic methyltransferase component of PRC2, revealed a younger brain transcriptome profile and identified the small heat shock proteins as key genes reduced in expression with age (Kennerdell, 2018).

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Joseph, N. M., Elphick, N. Y., Mohammad, S. and Bauer, J. H. (2018). Altered pheromone biosynthesis is associated with sex-specific changes in life span and behavior in Drosophila melanogaster. Mech Ageing Dev. PubMed ID: 30312624

Many insect behaviors, including foraging, aggression, mating or group behavior, are tightly regulated by pheromones. Recently, it has been shown that pheromones may influence extreme longevity in the honeybee Apis mellifera, while changes in pheromone profile have been observed during ageing in Drosophila melanogaster. These data suggest a potential link between the pheromone system, behavior and longevity in insects. This study investigated this potential link by examining changes in behavior and longevity in fruit flies with altered pheromone profiles. Oenocyte-specific reduction of desaturase activity was shown to be sufficient to dramatically alter the composition of the hydrocarbon mix displayed by the flies. In addition, flies with altered desaturase activity display changes in fecundity and stereotypical mating behavior, and, importantly, extended longevity. These data provide evidence for a potential link between hydrocarbon synthesis and life span, and suggest that longevity may be influenced by behavior (Joseph, 2018).

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Westfall, S., Lomis, N. and Prakash, S. (2018). Longevity extension in Drosophila through gut-brain communication. Sci Rep 8(1): 8362. Pubmed ID: 29849035

Aging and chronic disease development are multifactorial processes involving the cumulative effects of metabolic distress, inflammation, oxidative stress and mitochondrial dynamics. Recently, variations in the gut microbiota have been associated with age-related phenotypes and probiotics have shown promise in managing chronic disease progression. In this study, novel probiotic and synbiotic formulations are shown to combinatorially extend longevity in male Drosophila melanogaster through mechanisms of gut-brain-axis communication with implications in chronic disease management. Both the probiotic and synbiotic formulations rescued markers of metabolic stress by managing insulin resistance and energy regulatory pathways. Both formulations also ameliorated elevations in inflammation, oxidative stress and the loss of mitochondrial complex integrity. In almost all the measured pathways, the synbiotic formulation has a more robust impact than its individual components insinuating its combinatorial effect. The concomitant action of the gut microbiota on each of the key risk factors of aging and makes it a powerful therapeutic tool against neurodegeneration, diabetes, obesity, cardiovascular disease and other age-related chronic diseases (Westfall, 2018).

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Davie, K., Janssens, J., Koldere, D., De Waegeneer, M., Pech, U., Kreft, L., Aibar, S., Makhzami, S., Christiaens, V., Bravo Gonzalez-Blas, C., Poovathingal, S., Hulselmans, G., Spanier, K. I., Moerman, T., Vanspauwen, B., Geurs, S., Voet, T., Lammertyn, J., Thienpont, B., Liu, S., Konstantinides, N., Fiers, M., Verstreken, P. and Aerts, S. (2018). A single-cell transcriptome atlas of the aging Drosophila brain. Cell. PubMed ID: 29909982

The diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. This paper presents a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. The data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in this unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain (Davie, 2018).

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Catterson, J. H., Khericha, M., Dyson, M. C., Vincent, A. J., Callard, R., Haveron, S. M., Rajasingam, A., Ahmad, M. and Partridge, L. (2018). Short-term, intermittent fasting induces long-lasting gut health and TOR-independent lifespan extension. Curr Biol. PubMed ID: 29779873

Intermittent fasting (IF) can improve function and health during aging in laboratory model organisms, but the mechanisms at work await elucidation. This study subjected fruit flies (Drosophila melanogaster) to varying degrees of IF and found that just one month of a 2-day fed:5-day fasted IF regime at the beginning of adulthood was sufficient to extend lifespan. This long-lasting, beneficial effect of early IF was not due to reduced fecundity. Starvation resistance and resistance to oxidative and xenobiotic stress were increased after IF. Early-life IF also led to higher lipid content in 60-day-old flies, a potential explanation for increased longevity. Guts of flies 40 days post-IF showed a significant reduction in age-related pathologies and improved gut barrier function. Improved gut health was also associated with reduced relative bacterial abundance. Early IF thus induced profound long-term changes. Pharmacological and genetic epistasis analysis showed that IF acted independently of the TOR pathway because rapamycin and IF acted additively to extend lifespan, and global expression of a constitutively active S6K did not attenuate the IF-induced lifespan extension. It is concluded that short-term IF during early life can induce long-lasting beneficial effects, with robust increase in lifespan in a TOR-independent manner, probably at least in part by preserving gut health (Catterson, 2018).

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Liang, Y., Liu, C., Lu, M., Dong, Q., Wang, Z., Wang, Z., Xiong, W., Zhang, N., Zhou, J., Liu, Q., Wang, X. and Wang, Z. (2018). Calorie restriction is the most reasonable anti-ageing intervention: a meta-analysis of survival curves. Sci Rep 8(1): 5779. PubMed ID: 29636552

Despite technological advances, the survival records from longevity experiments remain the most indispensable tool in ageing-related research. A variety of interventions, including medications, genetic manipulations and calorie restriction (CR), have been demonstrated to extend the lifespan of several species. Surprisingly, few systematic studies have investigated the differences among these anti-ageing strategies using survival data. This study conductd a comprehensive and comparative meta-analysis of numerous published studies on Caenorhabditis elegans and Drosophila. CR and genetic manipulations were found to be generally more effective than medications at extending the total lifespan in both models, and CR can improve the ageing pattern of C. elegans. The survival variation for different anti-ageing medications was examined and determined that hypoglycaemic agents and antioxidants are advantageous despite only moderately increasing the overall lifespan; therefore, these two types of medications are promising CR mimetics. Analysis of genetic manipulations also indicated that the genes or pathways that extend lifespan in a healthier pattern are associated with CR. These results suggest that CR or CR mimetics may be the most reasonable and potentially beneficial anti-ageing strategy (Liang, 2018).

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Yan, Y., Wang, H., Hu, M., Jiang, L., Wang, Y., Liu, P., Liang, X., Liu, J., Li, C., Lindstrom-Battle, A., Lam, S. M., Shui, G., Deng, W. M. and Jiao, R. (2017). HDAC6 suppresses age-dependent ectopic fat accumulation by maintaining the proteostasis of PLIN2 in Drosophila. Dev Cell 43(1): 99-111.e115. PubMed ID: 28966044

Age-dependent ectopic fat accumulation (EFA) in animals contributes to the progression of tissue aging and diseases such as obesity, diabetes, and cancer. However, the primary causes of age-dependent EFA remain largely elusive. This study characterized the occurrence of age-dependent EFA in Drosophila and identified HDAC6, a cytosolic histone deacetylase, as a suppressor of EFA. Loss of HDAC6 leads to significant age-dependent EFA, lipid composition imbalance, and reduced animal longevity on a high-fat diet. The EFA and longevity phenotypes are ameliorated by a reduction of the lipid-droplet-resident protein PLIN2. HDAC6 was found to be associated physically with the chaperone protein dHsc4/Hsc70 to maintain the proteostasis of PLIN2. These findings indicate that proteostasis collapse serves as an intrinsic cue to cause age-dependent EFA. This study suggests that manipulation of proteostasis could be an alternative approach to the treatment of age-related metabolic diseases such as obesity and diabetes (Yan, 2017).

Age-dependent EFA occurs in mammals as a hallmark of aging and contributes to age-related tissue deterioration and dysfunction. This study used a Drosophila model to assess the molecular basis of age-dependent EFA formation. Age-dependent EFA appears mainly in the thoracic jump muscles of adult flies in an age-dependent manner. Further, proteostatic regulators, dHDAC6 and dHs4, are identified to suppress age-dependent EFA. The genetic and biochemical data indicate that dHDAC6 maintains the proteostasis of lipid droplet protein PLIN2 by modulating the acetylation level of dHsc4. The dHDAC6-dHsc4-PLIN2 axis links proteostasis to fat metabolism during aging. These results also highlight that it is the protein quality rather than the protein quantity of PLIN2 that controls age-dependent EFA (Yan, 2017).

PLIN2, belonging to the PAT family, is an lipid droplet (LD) coating protein that has been shown to play important roles in the formation and turnover of LDs in non-adipose tissues such as the skeletal muscle, pancreas, gonads, and gut. PLIN2 accumulates in human muscle with age and is associated with muscle weakness, obesity, and diabetes. Since the activity of both ubiquitin-proteasome and lysosome weakens during aging, it is plausible to infer that the increase in PLIN2 protein levels in aged individuals are caused by lowered activity of either ubiquitin-proteasome or lysosome. The results demonstrate that the degradation of PLIN2 is mediated by dHDAC6 through chaperone dHsc4-assisted autophagy but not macro-autophagy, and that the quality but not the quantity of PLIN2 plays an important role in EFA formation and tissue dysfunction during aging. The substrates of chaperone Hsc70/dHsc4 exhibit a consensus pentapeptide KFERQ motif, and Hsc70 has been reported to mediate the degradation of PAT family proteins, PLIN2 and PLIN3, in mouse. This study excluded the possibility that dHsc4/Hsc70 mediates the degradation of PLIN2 through the CMA machinery based on the following evidence: First, no conserved KREFQ motif specific for CMA degradation was found in Drosophila PLIN2; second, a mutant form of Drosophila PLIN2 was made in the non-canonical KREFQ motif region, which did not show any decreased degradation rate; Third, CMA degradation in mammals requires the lysosome receptor LAMP2A, however, Lamp1, the Drosophila homolog of LAMP2A, is not involved in age-dependent EFA. Therefore, it is speculated that dHsc4/Hsc70 may mediate the degradation of PLIN2 through chaperone-assisted selective autophagy, which involves co-chaperones and ubiquitination to degrade mainly insoluble proteins. Supporting this hypothesis, a significant amount of ubiquitinated aggregates were detected to accumulate on the surface of LDs in the jump muscles of dHDAC6 mutants, which colocalize with PLIN2. On the other hand, several co-chaperones (Dnaj-1, HspB8, Dnaj-2, mrj, and CG5001) and the E3 ligase CHIP were tested, but none of them were required for the chaperone-assisted selective autophagy process to lead to EFA. It is speculated that there may be another unknown co-chaperone(s) that functions with dHDAC6/dHsc4 in Drosophila (Yan, 2017).

Recently, studies show that PLIN2 is associated with the progression of age-related diseases, such as insulin resistance, fatty liver, type 2 diabetes, sarcopenia, and cancer. All the diseases reported thus far that are associated with PLIN2 are linked to aging, implying that the changes in PLIN2 during aging might have a pivotal contribution to the severity of these age-related diseases. This study assessed the changes in soluble and insoluble PLIN2 protein levels during aging and showed that only the insoluble PLIN2 protein level was increased and associated with the increase in age-dependent EFA in the jump muscle. The results suggest a possibility of improving the proteostasis of PLIN2 as an efficient way to ameliorate the progressive defects of age-related metabolic diseases (Yan, 2017).

Another question is how increased insoluble PLIN2 can cause increased EFA in aging muscle. Insoluble proteins exhibit hydrophobic aggregation properties and LDs containing a hydrophobic core are prone to act as an anchoring site for hydrophobic proteins. Thus, it is proposed that insoluble PLIN2 is prone to be anchored on the LDs to sequester more hydrophobic lipases. Anchored insoluble PLIN2 or insoluble PLIN2 aggregates prevent triglyceride lipases from reaching the LD surface to mediate lipid breakdown. In support of this hypothesis, the data show that increased LD accumulation in the jump muscle of the dHDAC6 mutant could not be reverted by overexpression of lipases such as Bmm or dHSL. However, more investigations are needed to explore precisely how the proteostasis of PLIN2 affects LD turnover in the aging process and whether the proteostasis of PLIN2 may also be involved in other physiological processes (Yan, 2017).

The maintenance of proteostasis in organelles, such as the endoplasmic reticulum, mitochondrion, and the nucleus, involves specialized cellular compartments. Mitochondrial proteostasis requires mitochondrial chaperones, ATFS-1 signaling, and GCN2 signaling to activate mitochondrial unfolded protein response (UPRmt); whereas nuclear proteostasis requires nuclear envelope, nuclear pore complexes, and transport pathways. In Drosophila, proteostasis of the muscles controls systemic aging and requires Foxo/4E-BP signaling and Activin signaling. As the primary site of lipid metabolism, LDs are considered as dynamic organelles, but little is known about how proteostasis of LDs is maintained. This study identified that the chaperone dHsc4 and the deacetylase dHDAC6 play key roles in maintaining LD proteostasis. More importantly, proteostasis of the LD protein PLIN2 links the proteostasis network to age-dependent EFA (Yan, 2017).

Age-dependent EFA appears mainly in the tubular jump muscle, but not in the fibrous indirect flight muscle, which is a large part of adult thoracic muscles, indicating LD accumulation is more sensitive to aging in jump muscle than in indirect flight muscle. Age-dependent EFA was also detected in other regions of an aging fly particularly in the posterior midgut and the tip region of testis. However, EFA in these tissues appears to be regulated in a non-tissue autonomous manner, since muscle-specific expression of dHDAC6 in the dHDAC6 mutants reverted the EFA-increase phenotype not only in the jump muscle but also in the posterior midgut and the testis. Recently, fat accumulation has also been shown to occur in other conditions, such as in the niche glia of stem cells of larval CNS under oxidant/oxidative stress and in the adult pigment cells of mitochondrial mutants. LD formation in the niche glia functions as a protective organelle to sequester polyunsaturated fatty acids and to reduce the levels of reactive oxygen species, whereas LD accumulation in adult pigment cells appears to increase lipid peroxidation and to promote neurodegenerative disease. LD accumulation in either the niche glia cells or the pigment cells can be reverted by overexpression of triglyceride lipases, indicating that the LDs formed in such conditions are an 'active' organelle. However, the LD accumulation in the aging jump muscle described in this study could not be reverted by overexpression of lipases, implying that the LDs in aging jump muscle seem to be a 'steady' organelle. This 'steady' LD formation can only be ameliorated by improving the proteostasis of LD-resident protein PLIN2. LD accumulation could also be induced by altering some of the fat-metabolism-related genes, but not in an age-dependent manner; thus, age-dependent EFA occurs in a distinct way contributing to the dysfunction of muscle aging. This study suggests that improvement of proteostasis of PLIN2 may be a new approach to ameliorate age-related metabolic diseases such as obesity (Yan, 2017).

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Lee, B. C., Lee, H. M., Kim, S., Avanesov, A. S., Lee, A., Chun, B. H., Vorbruggen, G. and Gladyshev, V. N. (2018). Expression of the methionine sulfoxide reductase lost during evolution extends Drosophila lifespan in a methionine-dependent manner. Sci Rep 8(1): 1010. PubMed ID: 29343716

Accumulation of oxidized amino acids, including methionine, has been implicated in aging. The ability to reduce one of the products of methionine oxidation, free methionine-R-sulfoxide (Met-R-SO), is widespread in microorganisms, but during evolution this function, conferred by the enzyme fRMsr, was lost in metazoa. This study examined whether restoration of the fRMsr function in an animal can alleviate the consequences of methionine oxidation. Ectopic expression of yeast fRMsr supported the ability of Drosophila to catalyze free Met-R-SO reduction without affecting fecundity, food consumption, and response to starvation. fRMsr expression also increased resistance to oxidative stress. Moreover, it extended lifespan of flies in a methionine-dependent manner. Thus, expression of an oxidoreductase lost during evolution can enhance metabolic and redox functions and lead to an increase in lifespan in an animal model. More broadly, this study exposes the potential of a combination of genetic and nutritional strategies in lifespan control (Lee, 2018).

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Pyo, J. H., Jeon, H. J., Park, J. S., Lee, J. S., Chung, H. Y. and Yoo, M. A. (2018). Drosophila PEBP1 inhibits intestinal stem cell aging via suppression of ERK pathway. Oncotarget 9(26): 17980-17993. PubMed ID: 29719584

The intestine is a high cellular turnover tissue largely dependent on the regenerative function of stem cell throughout life, and a signaling center for the health and viability of organisms. Therefore, better understanding of the mechanisms underlying the regulation of intestinal stem cell (ISC) regenerative potential is essential for the possible intervention of aging process and age-related diseases. Drosophila midgut is a well-established model system for studying the mechanisms underlying ISC regenerative potential during aging. This study reporta the requirement of Drosophila phosphatidylethanolamine binding protein 1 (PEBP1) in ISC regenerative potential. PEBP1 was strongly expressed in enterocytes (ECs) of guts and its decrease with age and oxidative stress. Furthermore, the downregulation of PEBP1 in ECs accelerates ISC aging, as evidenced by ISC hyper-proliferation, gammaH2AX accumulation, and centrosome amplification, and intestinal hyperplasia. The decrease in PEBP1 expression was associated with increased extracellular signal-regulated kinase (ERK) activity in ECs. All these phenotypes by EC-specific depletion of PEBP1 were rescued by the concomitant inhibition of ERK signaling. These findings evidence that the age-related downregulation of PEBP1 in ECs is a novel cause accelerating ISC aging and that PEBP1 is an EC-intrinsic suppressor of epidermal growth factor receptor (EGFR)/ERK signaling. This study provides molecular insights into the tight regulation of EGFR/ERK signaling in niches for stem cell regenerative potential (Pyo, 2018).

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Brenman-Suttner, D. B., Long, S. Q., Kamesan, V., de Belle, J. N., Yost, R. T., Kanippayoor, R. L. and Simon, A. F. (2018). Progeny of old parents have increased social space in Drosophila melanogaster. Sci Rep 8(1): 3673. PubMed ID: 29487349

This study report the effects of aging and parental age in Drosophila melanogaster on two types of responses to social cues: the choice of preferred social spacing in an undisturbed group and the response to the Drosophila stress odorant (dSO) emitted by stressed flies. The patterns of changes during aging were notably different for these two social responses. Flies were initially closer in space and then became further apart. However, the pattern of change in response to dSO followed a more typical decline in performance, similarly to changes in locomotion. Interestingly, the increased social space of old parents, as well as their reduced performance in avoiding dSO, was passed on to their progeny, such that young adults adopted the behavioural characteristic of their old parents. While the response to social cues was inherited, the changes in locomotion were not. It was possible to scale the changes in the social space of parents and their progeny by accelerating or decelerating the physiological process of aging by increasing temperatures and exposure to oxidative stress, or via caloric restriction, respectively. Finally, when only one parent was aged, only the male progeny of old fathers and the progeny of very old mothers were more distant (Brenman-Suttner, 2018).

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Becker, L., Nogueira, M. S., Klima, C., de Angelis, M. H. and Peleg, S. (2018). Rapid and transient oxygen consumption increase following acute HDAC/KDAC inhibition in Drosophila tissue. Sci Rep 8(1): 4199. PubMed ID: 29520020

Epigenetic deregulation, such as the reduction of histone acetylation levels, is thought to be causally linked to various maladies associated with aging. Consequently, histone deacetylase inhibitors are suggested to serve as epigenetic therapy by increasing histone acetylation. However, previous work suggests that many non-histone proteins, including metabolic enzymes, are also acetylated and that post transitional modifications may impact their activity. Furthermore, deacetylase inhibitors were recently shown to impact the acetylation of a variety of proteins. By utilizing a novel technique to measure oxygen consumption rate from whole living tissue, this study demonstrated that treatment of whole living fly heads by the HDAC/KDAC inhibitors sodium butyrate and Trichostatin A, induces a rapid and transient increase of oxygen consumption rate. In addition, this study indicates that the rate increase is markedly attenuated in midlife fly head tissue. Overall, this data suggest that HDAC/KDAC inhibitors may induce enhanced mitochondrial activity in a rapid manner. This observed metabolic boost provides further, but novel evidence, that treating various maladies with deacetylase inhibitors may be beneficial (Becker, 2018).

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Wen, D. T., Zheng, L., Yang, F., Li, H. Z. and Chen, J. (2018). Endurance exercise prevents high-fat-diet induced locomotor impairment, cardiac dysfunction, lifespan shortening, and dSir2 expression decline in aging Drosophila. Exp Gerontol [Epub ahead of print]. PubMed ID: 29355704

High-Fat-Diet (HFD)-induced obesity is a major contributor to premature senescence in both Drosophila and humans, which includes locomotor impairment, cardiac dysfunction, and decrease in lifespan. While, few studies have shown that HFD could affect the expression dSir2 genes since the dSir2 genes are closely related to aging. Endurance exercise can efficiently prevent obesity and delay age-related functional decline in Drosophila and humans. However, few directed reports showing that exercise can resist HFD-induced locomotor impairment, cardiac dysfunction and decrease in lifespan. It is also unclear whether exercise training can relieve the harmful HFD-induced influence on the dSir2 gene and prevent premature senescence in flies. In this study, flies were fed a HFD and trained from when they were one week old until they were five weeks old. Then, TAG levels, climbing index, cardiac function, lifespan, and dSir2 mRNA expressions were measured. It was found that endurance exercise could protect Drosophila from HFD-induced and age-related body lipid accumulation, locomotor impairment, cardiac contraction dysfunction, heart fibrillation rise, lifespan shortening, and dSir2 expression decline in aging flies. Therefore, this study confirmed that exercise effectively prevented flies from HFD-induced premature aging (Wen, 2018).

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Romey-Glusing, R., Li, Y., Hoffmann, J., von Frieling, J., Knop, M., Pfefferkorn, R., Bruchhaus, I., Fink, C. and Roeder, T. (2017). Nutritional regimens with periodically recurring phases of dietary restriction extend lifespan in Drosophila. Faseb J. PubMed ID: 29196499

Nutritional interventions such as caloric and dietary restriction increase lifespan in various animal models. To identify alternative and less demanding nutritional interventions that extend lifespan, fruit flies (Drosophila melanogaster) were subjected to weekly nutritional regimens that involved alternating a conventional diet with dietary restriction. Short periods of dietary restriction (up to 2 d) followed by longer periods of a conventional diet yielded minimal increases in lifespan. Three or more days of contiguous dietary restriction (DR) was necessary to yield a lifespan extension similar to that observed with persistent DR. Female flies were more responsive to these interventions than males. Physiologic changes known to be associated with prolonged DR, such as reduced metabolic rates, showed the same time course as lifespan extension. Moreover, concurrent transcriptional changes indicative of reduced insulin signaling were identified with DR. These physiologic and transcriptional changes were sustained, as they were detectable several days after switching to conventional diets. Taken together, diets with longer periods of DR extended lifespan concurrently with physiologic and transcriptional changes that may underlie this increase in lifespan (Romey-Glusing, 2017).

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Filer, D., Thompson, M. A., Takhaveev, V., Dobson, A. J., Kotronaki, I., Green, J. W. M., Heinemann, M., Tullet, J. M. A. and Alic, N. (2017). RNA polymerase III limits longevity downstream of TORC1. Nature 552(7684): 263-267. PubMed ID: 29186112

Abstract

Three distinct RNA polymerases transcribe different classes of genes in the eukaryotic nucleus. RNA polymerase (Pol) III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including tRNAs and 5S rRNA. The historical focus on transcription of protein-coding genes has left the roles of Pol III in organismal physiology relatively unexplored. Target of rapamycin kinase complex 1 (TORC1) regulates Pol III activity, and is also an important determinant of longevity. This raises the possibility that Pol III is involved in ageing. This study shows that Pol III limits lifespan downstream of TORC1. A reduction in Pol III extends chronological lifespan in yeast and organismal lifespan in worms and flies. Inhibiting the activity of Pol III in the gut of adult worms or flies is sufficient to extend lifespan; in flies, longevity can be achieved by Pol III inhibition specifically in intestinal stem cells. The longevity phenotype is associated with amelioration of age-related gut pathology and functional decline, dampened protein synthesis and increased tolerance of proteostatic stress. Pol III acts on lifespan downstream of TORC1, and limiting Pol III activity in the adult gut achieves the full longevity benefit of systemic TORC1 inhibition. Hence, Pol III is a pivotal mediator of this key nutrient-signalling network for longevity; the growth-promoting anabolic activity of Pol III mediates the acceleration of ageing by TORC1. The evolutionary conservation of Pol III affirms its potential as a therapeutic target (Filer, 2017).

The task of carrying out transcription in the eukaryotic nucleus is divided among RNA Pol I, II and III. This specialization is evident in the biogenesis of the translation machinery, a task that requires the co-ordinated activity of all three polymerases: Pol I generates the 45S pre-rRNA that is subsequently processed into mature rRNAs, Pol II transcribes various RNAs including mRNAs encoding ribosomal proteins, while Pol III provides the tRNAs and 5S rRNA. This costly process of generating protein synthetic capacity is tightly regulated to match the extrinsic conditions and the intrinsic need for protein synthesis by the key driver of cellular anabolism, TORC1. The central position of TORC1 in the control of fundamental cellular processes is mirrored by the notable effect of its activity on organismal physiology: following its initial discovery in worms, inhibition of TORC1 has been demonstrated to extend lifespan in all tested organisms, from yeast to mice, with beneficial effects on a range of age-related diseases and dysfunctions. TORC1 strongly activates Pol III transcription and this relationship suggests the possibility that inhibition of Pol III promotes longevity (Filer, 2017).

In Saccharomyces cerevisiae, each of the 17 Pol III subunits is encoded by an essential gene. This study generated a yeast strain in which the largest Pol III subunit (C160, encoded by RPC160, also known as RPO31) is fused to the auxin-inducible degron (AID). The fusion protein can be targeted for degradation by the ectopically expressed E3 ubiquitin ligase (OsTir) in the presence of indole-3-acetic acid (IAA) to achieve conditional inhibition of Pol III. It was confirmed that IAA treatment triggered degradation of the fusion protein, and it was observed that IAA treatment also improved the survival of the RPC160-AID strain upon prolonged culture. In addition, IAA treatment of the control strain lacking the AID fusion reduced its survival relative to both the same strain in the absence of IAA and to the RPC160-AID strain in the presence of IAA. Hence, Pol III depletion appears to extend the chronological lifespan in yeast. While IAA had no substantial effect on the survival of a strain carrying the AID domain fused to the largest subunit of Pol II (RPB220, also known as RP021), this strain appeared to survive better than the control strain did in the presence of IAA, indicating that inhibition of Pol II may also extend chronological lifespan. Chronological lifespan of yeast is a measure of survival in a nutritionally limited, quiescent population, whereas replicative lifespan measures the number of daughters produced by a single mother cell in its lifetime. No evidence was found that inhibition of Pol III causes an increase in the replicative lifespan in yeast (Filer, 2017).

The observed increase in chronological lifespan may simply indicate increased stress resistance and hence be of limited relevance to organismal ageing. To examine the role of Pol III in organismal ageing directly, animal models were examined. RNA-mediated interference (RNAi) was initiated against rpc-1, the Caenorhabditis elegans orthologue of RPC160, in worms from the L4 stage, causing a partial knockdown of rpc-1 mRNA. This consistently extended the lifespan of worms at both 20°C and 25°C. To reduce Pol III activity in Drosophila melanogaster, a P-element insertion that deletes the transcriptional start site of the gene encoding the Pol III-specific subunit C53 (CG5147^EY22749, henceforth called dC53^EY, was backcrossed into a healthy, outbred population of flies. Homozygous dC53^EY/EY mutants were not viable, but heterozygous females had reduced dC53 mRNA levels and lived longer than controls. Taken together, these data strongly indicate that Pol III limits lifespan in multiple model organisms and conversely, that partial inhibition of its activity is an intervention that increases longevity in multiple species (Filer, 2017).

The longevity of an animal can be governed from a single organ. In the worm, this role is often played by the gut. To restrict the rpc-1 knockdown to the gut, worms were used that were deficient in rde-1, in which the RNAi machinery deficiency is restored in the gut by gut-specific rde-1 rescue. rpc-1 RNAi extended the lifespan of this strain, both at 20°C and 25°C. Similarly, in the adult fly, driving an RNAi construct targeting the RPC160 orthologue (CG17209, henceforth called dC160,with the mid-gut-specific, RU486-inducible driver TIGS extended the lifespan of females, while the presence of the inducer (RU486) did not affect survival of the control strains. The longevity phenotype could also be recapitulated with RNAi against dC53, another Pol III subunit, indicating that the phenotype was not subunit-specific or due to off-target effects. As well as the gut, longevity can also be associated with the fat body and neurons in flies. However, the longevity phenotype caused by dC160 RNAi appears to be specific to the gut, since no significant lifespan extension was observed upon induction of dC160 RNAi in the fat body of the adult fly, and only a modest, albeit significant, extension resulted from neuronal induction of dC160 RNAi (Filer, 2017).

The worm gut is composed of only post-mitotic cells. In flies, as in mammals, the adult gut epithelium contains mitotically active intestinal stem cells, and the mid-gut-specific driver TIGS appears to be active in at least some ISCs, prompting restricting of dC160 RNAi induction to this cell type. ISC-specific dC160 RNAi, achieved with the GS5961 driver, was sufficient to promote longevity. In summary, Pol III activity in the gut limits survival in worms and flies, and in the fly, Pol III can drive ageing specifically from the gut stem-cell compartment (Filer, 2017).

The consequences of Pol III inhibition in the fly gut was assessed. Pol III acts to generate precursor tRNAs (pre-tRNAs) that are processed rapidly to mature tRNAs. Owing to their short half-lives, pre-tRNAs are useful as readouts of in vivo Pol III activity. Profiling the levels of specific pre-tRNAs, pre-tRNA^His, pre-tRNA^Ala and pre-tRNA^Leu, relative to the levels of U3 (a small nucleolar RNA transcribed by Pol II) revealed a moderate but significant reduction in Pol III activity upon gut-specific induction of dC160 RNAi. The three polymerases can be directly coordinated to generate the translation machinery. Indeed, Pol III inhibition had knock-on effects on Pol I- but not Pol II-generated transcripts, revealing partial cross-talk. dC160 RNAi also reduced protein synthesis in the gut, consistent with reduced Pol III activity. These effects (reduction in pre-tRNAs or protein synthesis) were not observed after feeding RU486 to the driver-only control. The reduction in protein synthesis was not pathological: total protein content of the gut was unaltered; fecundity, a sensitive readout of a female's nutritional status, was unaffected; and the flies' weight, triacylglycerol and protein levels remained unchanged. Reduced protein synthesis can liberate protein-folding machinery from protein production and increase homeostatic capacity. Indeed, induction of dC160 RNAi in the gut increased the resistance of adult flies to proteostatic challenge with tunicamycin for TIGS-only control. Hence, Pol III can fine-tune the rate of protein synthesis in the adult fly gut without obvious detrimental outcomes, while increasing resistance to proteotoxic stress (Filer, 2017).

Having demonstrated the relevance of Pol III for ageing, whether it acts on lifespan downstream of TORC1 was investigated in Drosophila. Numerous observations in several organisms support the model in which TORC1 localizes on Pol III-transcribed loci and promotes phosphorylation of the components of the Pol III transcriptional machinery to activate transcription, in part by inhibition of the Pol III repressor, Maf1. Using chromatin immunoprecipitation (ChIP) with two independently generated antibodies against Drosophila TOR (target of rapamycin), TOR enrichment was observed on Pol III-target genes in the adult fly, relative to Pol II targets. Inhibition of TORC1 by feeding rapamycin to flies reduced the levels of pre-tRNAs in whole flies. Rapamycin also reduced pre-tRNA levels specifically in the gut relative to U3. Since rapamycin results in re-scaling of the gut, evidenced by the reduction in the total RNA content of the organ, it was also confirmed that the drug reduced pre-tRNA levels relative to total RNA. Interestingly, rapamycin did not cause a decrease in 45S pre-rRNA in the gut, suggesting a lack of sustained Pol I inhibition. Additionally, gut-specific overexpression of Maf1 reduced the levels of pre-tRNAs and extended lifespan, confirming that Maf1 acts on Pol III in the adult gut. These data are consistent with TORC1 driving systemic and gut-specific Pol III activity in the adult fruitfly (Filer, 2017).

To examine whether the lifespan effects of Pol III are downstream of TORC1, adult-onset Pol III inhibition was combined with rapamycin treatment. Rapamycin feeding or gut-specific dC160 RNAi resulted in the same magnitude of lifespan extension. The two treatments were not additive, consistent with their acting on the same longevity pathway. The same effect was observed with RNAi against dC53 in the gut, as well as when dC160 RNAi was restricted to the ISCs. Importantly, rapamycin feeding also inhibited phosphorylation of the TORC1 substrate, S6 kinase (S6K), in both the gut and the whole fly, and decreased fecundity, while gut-specific C160 RNAi did not have these effects. This confirms that Pol III inhibition does not impact TORC1 activity locally or systemically, and therefore, Pol III acts downstream of TORC1 in ageing (Filer, 2017).

TORC1 inhibition is known to ameliorate age-related pathology and functional decline of the gut. Whether inhibition of Pol III was sufficient to block the dysplasia resulting from hyperproliferation and aberrant differentiation of ISCs was examined by assessing the characteristic, age-dependent increase in dividing phospho-histone H3 (pH3)-positive cells. Inducing dC160 RNAi in the fly gut or solely in the ISCs ameliorated this pathology. These treatments also counteracted the age-related loss of gut barrier function, decreasing the number of flies displaying extra-intestinal accumulation of a blue food dye (the 'Smurf' phenotype). It as also found that rpc-1 RNAi reduced the severity of age-related loss of gut-barrier function in worms. In Drosophila, gut health and TORC1 inhibition are specifically linked to female survival. Indeed, induction of dC160 RNAi in the gut had a sexually dimorphic effect on lifespan, as the effect on males, although significant, was lower in magnitude relative to the effect on females. Overall, the data show that gut- or ISC-specific inhibition of Pol III, which extends lifespan, is sufficient to ameliorate age-related impairments in gut health, which may be causative of or correlate with this longevity (Filer, 2017).

This study demonstrates that the adult-onset decrease in the growth-promoting anabolic function mediated by Pol III in the gut, and specifically in the intestinal stem-cell compartment, is sufficient to recapitulate the longevity benefits of rapamycin treatment. Pol III activity is essential for growth; its detrimental effects on ageing suggest an antagonistic pleiotropy in which wild-type levels of Pol III activity are optimised for growth and reproductive fitness in early life but prove detrimental for later health. This study reveals a fundamental role for Pol III in adult physiology, implicating wild-type Pol III activity in age-related stem-cell dysfunction, declining gut health and organismal survival downstream of nutrient signalling pathways. The longevity resulting from partial Pol III inhibition in adulthood is likely to result from the reduced provision of protein synthetic machinery; however, differential regulation of tRNA genes or Pol III-mediated changes to chromatin organization may also be involved, as has been suggested in other contexts (Arimbasseri, 2016). The strong structural and functional conservation of Pol III in eukaryotes suggests that studies of its influence on mammalian ageing are warranted and could lead to important therapies (Filer, 2017).

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Kayashima, Y., Katayanagi, Y., Tanaka, K., Fukutomi, R., Hiramoto, S. and Imai, S. (2017). Alkylresorcinols activate SIRT1 and delay ageing in Drosophila melanogaster. Sci Rep 7: 43679. PubMed ID: 28252007

Abstract

Sirtuins are enzymes that catalyze NAD+ dependent protein deacetylation. The natural polyphenolic compound resveratrol received renewed interest when recent findings implicated resveratrol as a potent SIRT1 activator capable of mimicking the effects of calorie restriction. However, resveratrol directly interacts with fluorophore-containing peptide substrates. It was demonstrated that the SIRT1 activation of resveratrol is affected by the amino acid composition of the substrate. Resveratrol did increase the enzyme activity in cases in which hydrophobic amino acids are at the +1 position to the acetylated lysine in the substrate. Alkylresorcinols (ARs) are compounds that belong to the family of phenolic lipids, and they are found in numerous biological species. This study shows that the natural activators ARs increased the Vmax of recombinant SIRT1 for NAD+ and peptide substrate, and that ARs decreased acetylated histone in human monocyte cells by stimulating SIRT1-dependent deacetylation of substrates. ARs also extended the lifespan of Drosophila melanogaster, which was shown to be dependent on functional Sir2. These results demonstrated that ARs are natural catalytic activators for sirtuin (Kayashima, 2017).

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Klassen, M. P., Peters, C. J., Zhou, S., Williams, H. H., Jan, L. Y. and Jan, Y. N. (2017). Age-dependent diastolic heart failure in an in vivo Drosophila model. Elife 6 [Epub ahead of print]. PubMed ID: 28328397

Abstract

While the signals and complexes that coordinate the heartbeat are well established, how the heart maintains its electromechanical rhythm over a lifetime remains an open question with significant implications to human health. Reasoning that this homeostatic challenge confronts all pulsatile organs, this study developed a high resolution imaging and analysis toolset for measuring cardiac function in intact, unanesthetized Drosophila melanogaster. As in humans, normal aging primarily manifests as defects in relaxation (diastole) while preserving contractile performance. Using this approach, it was discovered that a pair of two-pore potassium channel (K2P) subunits (FlyBase gene: sandman), largely dispensable early in life, are necessary for terminating contraction (systole) in aged animals, where their loss culminates in fibrillatory cardiac arrest. As the pumping function of its heart is acutely dispensable for survival, Drosophila represents a uniquely accessible model for understanding the signaling networks maintaining cardiac performance during normal aging (Klassen, 2017).

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Stefana, M. I., Driscoll, P. C., Obata, F., Pengelly, A. R., Newell, C. L., MacRae, J. I. and Gould, A. P. (2017). Developmental diet regulates Drosophila lifespan via lipid autotoxins. Nat Commun 8(1): 1384. PubMed ID: 29123106

Abstract

Early-life nourishment exerts long-term influences upon adult physiology and disease risk. These lasting effects of diet are well established but the underlying mechanisms are only partially understood. This study shows that restricting dietary yeast during Drosophila development can, depending upon the subsequent adult environment, more than double median lifespan. Developmental diet acts via a long-term influence upon the adult production of toxic molecules, which are termed autotoxins, that are shed into the environment and shorten the lifespan of both sexes. Autotoxins are synthesised by oenocytes and some of them correspond to alkene hydrocarbons that also act as pheromones. This study identifies a mechanism by which the developmental dietary history of an animal regulates its own longevity and that of its conspecific neighbours. It also has important implications for the design of lifespan experiments as autotoxins can influence the regulation of longevity by other factors including diet, sex, insulin signalling and population density (Stefana, 2017).

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Garschall, K., Dellago, H., Galikova, M., Schosserer, M., Flatt, T. and Grillari, J. (2017). Ubiquitous overexpression of the DNA repair factor dPrp19 reduces DNA damage and extends Drosophila life span. NPJ Aging Mech Dis 3: 5. PubMed ID: 28649423

Abstract

Mechanisms that ensure and maintain the stability of genetic information are fundamentally important for organismal function and can have a large impact on disease, aging, and life span. While a multi-layered cellular apparatus exists to detect and respond to DNA damage, various insults from environmental and endogenous sources continuously affect DNA integrity. Over time this can lead to the accumulation of somatic mutations, which is thought to be one of the major causes of aging. Previous work has found that overexpression of the essential human DNA repair and splicing factor SNEV, also called PRP19 or hPso4, extends replicative life span of cultured human endothelial cells and impedes accumulation of DNA damage. This study show that adult-specific overexpression of dPrp19, the D. melanogaster ortholog of human SNEV/PRP19/hPso4, robustly extends life span in female fruit flies. This increase in life span is accompanied by reduced levels of DNA damage and improved resistance to oxidative and genotoxic stress. These findings suggest that dPrp19 plays an evolutionarily conserved role in aging, life span modulation and stress resistance, and support the notion that superior DNA maintenance is key to longevity (Garschall, 2017).

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Ruth Archer, C., Basellini, U., Hunt, J., Simpson, S. J., Lee, K. P. and Baudisch, A. (2017). iet has independent effects on the pace and shape of aging in Drosophila melanogaster. Biogerontology [Epub ahead of print]. PubMed ID: 28914388

Abstract

Studies examining how diet affects mortality risk over age typically characterise mortality using parameters such as aging rates, which condense how much and how quickly the risk of dying changes over time into a single measure. Demographers have suggested that decoupling the tempo and the magnitude of changing mortality risk may facilitate comparative analyses of mortality trajectories, but it is unclear what biologically meaningful information this approach offers. This study has determined how the amount and ratio of protein and carbohydrate ingested by female Drosophila melanogaster affects how much mortality risk increases over a time-standardised life-course (the shape of aging) and the tempo at which animals live and die (the pace of aging). Pace values increased as flies consumed more carbohydrate but declined with increasing protein consumption. Shape values were independent of protein intake but were lowest in flies consuming ~90 mug of carbohydrate daily. As protein intake only affected the pace of aging, varying protein intake rescaled mortality trajectories (i.e. stretched or compressed survival curves), while varying carbohydrate consumption caused deviation from temporal rescaling (i.e. changed the topography of time-standardised survival curves), by affecting pace and shape. Clearly, the pace and shape of aging may vary independently in response to dietary manipulation. This suggests that there is the potential for pace and shape to evolve independently of one another and respond to different physiological processes. Understanding the mechanisms responsible for independent variation in pace and shape, may offer insight into the factors underlying diverse mortality trajectories (Ruth Archer, 2017).

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Bloch Qazi, M. C., Miller, P. B., Poeschel, P. M., Phan, M. H., Thayer, J. L. and Medrano, C. L. (2017). Transgenerational effects of maternal and grandmaternal age on offspring viability and performance in Drosophila melanogaster. J Insect Physiol 100: 43-52. PubMed ID: 28529156

Abstract

In non-social insects, fitness is determined by relative lifetime fertility. Fertility generally declines with age as a part of senescence. For females, senescence has profound effects on fitness by decreasing viability and fertility as well as those of her offspring. However, important aspects of these maternal effects, including the cause(s) of reduced offspring performance and carry-over effects of maternal age, are poorly understood. Drosophila melanogaster is a useful system for examining potential transgenerational effects of increasing maternal age, because of their use as a model system for studying the physiology and genetic architecture of both reproduction and senescence. To test the hypothesis that female senescence has transgenerational effects on offspring viability and development, this study measured the effects of maternal age on offspring survival over two generations and under two larval densities in two laboratory strains of flies (Oregon-R and Canton-S). Transgenerational effects of maternal age influence embryonic viability and embryonic to adult viability in both strains. However, the generation causing the effects, and the magnitude and direction of those effects differed by genotype. The effects of maternal age on embryonic-to-adult viability when larvae are stressed was also genotype-specific. Maternal effects involve provisioning: older females produced smaller eggs and larger offspring. These results show that maternal age has profound, complex, and multigenerational consequences on several components of offspring fitness and traits. This study contributes to a body of work demonstrating that female age is an important condition affecting phenotypic variation and viability across multiple generations (Bloch Qazi, 2017).

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Lucchetta, E.M. and Ohlstein, B. (2017). Amitosis of polyploid cells regenerates functional stem cells in the Drosophila intestine. Cell Stem Cell [Epub ahead of print]. PubMed ID: 28343984

Abstract

Organ fitness depends on appropriate maintenance of stem cell populations, and aberrations in functional stem cell numbers are associated with malignancies and aging. Symmetrical division is the best characterized mechanism of stem cell replacement, but other mechanisms could also be deployed, particularly in situations of high stress. This study shows that after severe depletion, intestinal stem cells (ISCs) in the Drosophila midgut are replaced by spindle-independent ploidy reduction of cells in the enterocyte lineage through a process known as amitosis. Amitosis is also induced by the functional loss of ISCs coupled with tissue demand and in aging flies, underscoring the generality of this mechanism. However, random homologous chromosome segregation during ploidy reduction can expose deleterious mutations through loss of heterozygosity. Together, these data highlight amitosis as an unappreciated mechanism for restoring stem cell homeostasis, but one with some associated risk in animals carrying mutations (Lucchetta, 2017).

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Garcia, J. F., Carbone, M. A., Mackay, T. F. C. and Anholt, R. R. H. (2017). Regulation of Drosophila lifespan by bellwether promoter alleles. Sci Rep 7(1): 4109. PubMed ID: 28646164

Abstract

Longevity varies among individuals, but how natural genetic variation contributes to variation in lifespan is poorly understood. Drosophila melanogaster presents an advantageous model system to explore the genetic underpinnings of longevity, since its generation time is brief and both the genetic background and rearing environment can be precisely controlled. The bellwether (blw) gene encodes the alpha subunit of mitochondrial ATP synthase. Since metabolic rate may influence lifespan, this study investigated whether alternative haplotypes in the blw promoter affect lifespan when expressed in a co-isogenic background. 521 bp upstream promoter sequences containing the alternative SNP haplotypes (G/T and A/G) were amplified, and promoter activity was assessed both in vitro and in vivo using a luciferase reporter system. The AG haplotype showed significantly greater expression of luciferase than the GT haplotype. A blw cDNA construct driven by either the AG or GT haplotype promoter was driven in transgenic flies, and the AG haplotype was shown to results in greater blw cDNA expression and a significant decrease in lifespan relative to the GT promoter haplotype, in male flies only. Thus, the results show that naturally occurring regulatory variants of blw affect lifespan in a sex-specific manner (Garcia, 2017).

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Carazo, P., Green, J., Sepil, I., Pizzari, T. and Wigby, S. (2016). Inbreeding removes sex differences in lifespan in a population of Drosophila melanogaster Biol Lett 12. PubMed ID: 27354712

Abstract

Sex differences in ageing rates and lifespan are common in nature, and an enduring puzzle for evolutionary biology. One possibility is that sex-specific mortality rates may result from recessive deleterious alleles in 'unguarded' heterogametic X or Z sex chromosomes (the unguarded X hypothesis). Empirical evidence for this is, however, limited. This study tests a fundamental prediction of the unguarded X hypothesis in Drosophila melanogaster, namely that inbreeding shortens lifespan more in females (the homogametic sex in Drosophila) than in males. To test for additional sex-specific social effects, the lifespan of males and females kept in isolation was studied, in related same-sex groups, and in unrelated same-sex groups. As expected, outbred females outlive outbred males and inbreeding shortens lifespan. However, inbreeding-mediated reductions in lifespan are stronger for females, such that lifespan is similar in inbred females and males. It was also shown that the social environment, independent of inbreeding, affects male, but not female lifespan. In conjunction with recent studies, these data suggest that asymmetric inheritance mechanisms may play an important role in the evolution of sex-specific lifespan and that social effects must be considered explicitly when studying these fundamental patterns (Carazo, 2016). 

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Moskalev, A., Shaposhnikov, M., Proshkina, E., Belyi, A., Fedintsev, A., Zhikrivetskaya, S., Guvatova, Z., Sadritdinova, A., Snezhkina, A., Krasnov, G. and Kudryavtseva, A. (2016). The influence of pro-longevity gene Gclc overexpression on the age-dependent changes in Drosophila transcriptome and biological functions. BMC Genomics 17(Suppl 14): 1046. PubMed ID: 28105938

Abstract

Transcriptional changes that contribute to the organism's longevity and prevent the age-dependent decline of biological functions are not well understood. This study overexpressed pro-longevity gene encoding glutamate-cysteine ligase catalytic subunit (Gclc) and analyzed age-dependent changes in transcriptome that associated with the longevity, stress resistance, locomotor activity, circadian rhythmicity, and fertility. The life extension effect of neuronal overexpression of the Gclc gene were reproduced, and its influence on the age-depended dynamics of transcriptome and biological functions such as fecundity, spontaneous locomotor activity and circadian rhythmicity were investigated, as well as on the resistance to oxidative, proteotoxic and osmotic stresses. It was shown that Gclc overexpression reduces locomotor activity in the young and middle ages compared to control flies. Gclc overexpression slowed down the age-dependent decline of locomotor activity and circadian rhythmicity, and resistance to stress treatments. Gclc level demonstrated associations with the expression of genes involved in a variety of cellular processes including Jak-STAT, MAPK, FOXO, Notch, mTOR, TGF-beta signaling pathways, translation, protein processing in endoplasmic reticulum, proteasomal degradation, glycolysis, oxidative phosphorylation, apoptosis, regulation of circadian rhythms, differentiation of neurons, synaptic plasticity and transmission. This study revealed that Gclc overexpression induces transcriptional changes associated with the lifespan extension and uncovered pathways that may be associated with the age-dependent decline of biological functions.

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Vonk, J. J., et al. (2017). Drosophila Vps13 Is required for protein homeostasis in the brain. PLoS One 12(1): e0170106. PubMed ID: 28107480

Abstract

Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A) gene. This study characterized a Drosophila Vps13 mutant line. The data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2)P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. The results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis (Vonk, 2017).

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M'Angale, P. G. and Staveley, B. E. (2016). Inhibition of Atg6 and Pi3K59F autophagy genes in neurons decreases lifespan and locomotor ability in Drosophila melanogaster. Genet Mol Res 15. PubMed ID: 27813607

Abstract

Autophagy is a cellular mechanism implicated in the pathology of Parkinson's disease. The proteins Atg6 (Beclin 1) and Pi3K59F are involved in autophagosome formation, a key step in the initiation of autophagy. This study used the GMR-Gal4 driver to determine the effect of reducing the expression of the genes encoding these proteins on the developing Drosophila eye. Subsequently, their expression in D. melanogaster neurons was inhibited under the direction of a Dopa decarboxylase (Ddc) transgene, and the effects on longevity and motor function were examined. Decreased longevity coupled with an age-dependent loss of climbing ability was observed. In addition, the roles of these genes were investigated in the well-studied alpha-synuclein-induced Drosophila model of Parkinson's disease. In this context, lowered expression of Atg6 or Pi3K59F in Ddc-Gal4-expressing neurons results in decreased longevity and associated age-dependent loss of locomotor ability. Inhibition of Atg6 or Pi3K59F together with overexpression of the sole pro-survival Bcl-2 Drosophila homolog Buffy in Ddc-Gal4-expressing neurons resulted in further decrease in the survival and climbing ability of Atg6-RNAi flies, whereas these measures were ameliorated in Pi3K59F-RNAi flies (M'Angale, 2016).

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Delabaere, L., et al. (2016). Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells. Aging Cell [Epub ahead of print]. PubMed ID: 28000382

Abstract

Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability. This study shows that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, it is proposed that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals (Delabaere, 2016).

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Vienne, J., Spann, R., Guo, F. and Rosbash, M. (2016). Age-related reduction of recovery sleep and arousal threshold in Drosophila. Sleep [Epub ahead of print]. PubMed ID: 27306274

Abstract
Physiological studies show that aging affects both sleep quality and quantity in humans, and sleep complaints increase with age. Along with knowledge about the negative effects of poor sleep on health, understanding the enigmatic relationship between sleep and aging is important. Because human sleep is similar to Drosophila (fruit fly) sleep in many ways, this study addressed the effects of aging on sleep in this model organism. Baseline sleep was recorded in five different Drosophila genotypes raised at either 21 ° C or 25 ° C. The amount of sleep recovered was then investigated after a nighttime of sleep deprivation (12 h) and after chronic sleep deprivation (3 h every night for multiple nights). Finally, the effects of aging on arousal, namely, sensitivity to neuronal and mechanical stimuli, were studied. Fly sleep was shown to be affected by age in a manner similar to that of humans and other mammals. Not only do older flies of several genotypes have more fragmented sleep and reduced total sleep time compared to young flies, but older flies also fail to recover as much sleep after sleep deprivation. This suggests either lower sleep homeostasis and/or a failure to properly recover sleep. Older flies also show a decreased arousal threshold, i.e., an increased response to neuronal and mechanical wake-promoting stimuli. The reduced threshold may either reflect or cause the reduced recovery sleep of older flies compared to young flies after sleep deprivation. It is concluded that further studies are certainly needed, but it is suggested that the lower homeostatic sleep drive of older flies causes their decreased arousal threshold (Vienne, 2016).

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Wu, Q., Lian, T., Fan, X., Song, C., Gaur, U., Mao, X., Yang, D., Piper, M. D. and Yang, M. (2016). 2,5-Dimethyl-Celecoxib extends Drosophila life span via a mechanism that requires insulin and Target of rapamycin signaling. J Gerontol A Biol Sci Med Sci [Epub ahead of print]. PubMed ID: 28025308

Abstract

The search for antiaging drugs is a key component of gerontology research. A few drugs with positive effects on life span in model organisms have been found. This study reports that 2,5-dimethyl-celecoxib, a derivative of the anti-inflammatory drug celecoxib, can extend Drosophila life span and delay aging by a mechanism involving insulin signaling and target of rapamycin signaling. Importantly, its positive effects were apparent when the treatment window was restricted to the beginning of life or the later half. 2,5-Dimethyl-celecoxib-induced longevity was also associated with improvements in physical activity, intestinal integrity, and increased autophagy. In addition, 2,5-dimethyl-celecoxib exhibited protective effects against several kinds of stress such as starvation and heat. The generally positive effects of 2,5-dimethyl-celecoxib on both health and life span, combined with its mode of action via evolutionarily conserved signaling pathways, indicate that it has the potential to become an effective antiaging drug (Wu, 2016).

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Heintz, C., et al. (2016). Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans. Nature [Epub ahead of print]. PubMed ID: 27919065

Abstract

Ageing is driven by a loss of transcriptional and protein homeostasis and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses. However, the role of splicing homeostasis in healthy ageing remains unclear. This study demonstrates that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, this study found defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP; see Drosophila SF1). SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. It was also demonstrated that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing (Heintz, 2016).

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Johnson, J. L., Huang, W., Roman, G. and Costa-Mattioli, M. (2016). TORC2: a novel target for treating age-associated memory impairment. Sci Rep 5: 15193. PubMed ID: 26489398

Abstract

Memory decline is one of the greatest health threats of the twenty-first century. Because of the widespread increase in life expectancy, 20 percent of the global population will be over 60 in 2050 and the problems caused by age-related memory loss will be dramatically aggravated. However, the molecular mechanisms underlying this inevitable process are not well understood. This study shows that the activity of the recently discovered mechanistic target of rapamycin (mTOR) complex 2 (mTORC2, see Drosophila TOR and Rictor) declines with age in the brain of both fruit flies and rodents and that the loss of mTORC2-mediated actin polymerization contributes to age-associated memory loss. Intriguingly, treatment with a small molecule that activates mTORC2 (A-443654; a specific Akt inhibitor that activates mTORC2-mediated phosphorylation of Akt) reverses long-term memory (LTM) deficits in both aged mice and flies. In addition, pharmacologically boosting either mTORC2 or actin polymerization enhances LTM. In contrast to the current approaches to enhance memory that have primarily targeted the regulation of gene expression (epigenetic, transcriptional, and translational), the data points to a novel, evolutionarily conserved mechanism for restoring memory that is dependent on structural plasticity. These insights into the molecular basis of age-related memory loss may hold promise for new treatments for cognitive disorders (Johnson, 2016).

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Gupta, V. K., Pech, U., Bhukel, A., Fulterer, A., Ender, A., Mauermann, S. F., Andlauer, T. F., Antwi-Adjei, E., Beuschel, C., Thriene, K., Maglione, M., Quentin, C., Bushow, R., Schwarzel, M., Mielke, T., Madeo, F., Dengjel, J., Fiala, A. and Sigrist, S. J. (2016) PLoS Biol 14: e1002563. PubMed ID: 27684064

Abstract

Memories are assumed to be formed by sets of synapses changing their structural or functional performance. The efficacy of forming new memories declines with advancing age, but the synaptic changes underlying age-induced memory impairment remain poorly understood. Spermidine feeding has been found to specifically suppress age-dependent impairments in forming olfactory memories, providing a mean to search for synaptic changes involved in age-dependent memory impairment. This study shows that a specific synaptic compartment, the presynaptic active zone (AZ) of the adult brain, increases the size of its ultrastructural elaboration and releases significantly more synaptic vesicles with advancing age. These age-induced AZ changes, however, were fully suppressed by spermidine feeding. A genetically enforced enlargement of AZ scaffolds (four gene-copies of BRP) impaired memory formation in young animals. Thus, in the Drosophila nervous system, aging AZs seem to steer towards the upper limit of their operational range, limiting synaptic plasticity and contributing to impairment of memory formation. Spermidine feeding suppresses age-dependent memory impairment by counteracting these age-dependent changes directly at the synapse. The results suggest that the integrity of the autophagic system is crucial for the spermidine-mediated protection from age-associated increase in AZ scaffold components (Gupta, 2016).

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Wen, D. T., Zheng, L., Ni, L., Wang, H., Feng, Y. and Zhang, M. (2016). The expression of CG9940 affects the adaptation of cardiac function, mobility, and lifespan to exercise in aging Drosophila. Exp Gerontol 83: 6-14. PubMed ID: 27448710

Abstract

The CG9940 gene, which encodes the NAD+ synthase protein in Drosophila, is conserved in human, zebra fish, and mosquito. NAD+ synthase is a homodimer, which catalyzes the final step in de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis, an amide transfer from either ammonia or glutamine to nicotinic acid adenine dinucleotide (NaAD). Both the CG9940 and exercise are closely relative to NAD+ level, and NAD+ plays important roles not only in energy metabolism and mitochondrial functions but also in aging. This study changed expression of CG9940 by UAS/GAL4 system in Drosophila. Flies were trained by a training device. Cardiac function was analyzed by M-mode traces, climbing index was measured through negative geotaxis assay, and lifespan was measured via lifespan assays. The important new findings from this study included the following: (1) the expression of the CG9940 could affect cardiac function, mobility, and lifespan in Drosophila. Over-expression of the CG9940 gene had positive effects on Drosophila, such as enhanced aging cardiac output, reduced heart failure, delayed age-related mobility decline, and prolonged lifespan, but lower-expression of the CG9940 had negative effects on them. (2) Different expressions of the CG9940 resulted in different influences on the adaptation of cardiac function, mobility, and lifespan to exercise in aging Drosophila. Both normal-expression and over-expression of the CG9940 resulted in positive influences on the adaptation of cardiac functions, mobility, and lifespan to exercise in aging Drosophila such as exercise slowed age-related decline of cardiac function, mobility and extent of lifespan in these flies, while lower-expression of the CG9940 led to negative impacts on the adaptation of mobility and lifespan to exercise in Drosophila (Wen, 2016).

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Parkhitko, A.A., Binari, R., Zhang, N., Asara, J.M., Demontis, F. and Perrimon, N. (2016). Tissue-specific down-regulation of S-adenosyl-homocysteine via suppression of dAhcyL1/dAhcyL2 extends health span and life span in Drosophila. Genes Dev [Epub ahead of print]. PubMed ID: 27313316

Abstract
Aging is a risk factor for many human pathologies and is characterized by extensive metabolic changes. Using targeted high-throughput metabolite profiling in Drosophila melanogaster at different ages, this study demonstrates that methionine metabolism changes strikingly during aging. Methionine generates the methyl donor S-adenosyl-methionine (SAM), which is converted via methylation to S-adenosyl-homocysteine (SAH), which accumulates during aging. A targeted RNAi screen against methionine pathway components reveals significant life span extension in response to down-regulation of two noncanonical Drosophila homologs of the SAH hydrolase Ahcy (S-adenosyl-L-homocysteine hydrolase [SAHH]), CG9977/dAhcyL1 and Ahcy89E/CG8956/dAhcyL2, which act as dominant-negative regulators of canonical AHCY. Importantly, tissue-specific down-regulation of dAhcyL1/L2 in the brain and intestine extends health and life span. Furthermore, metabolomic analysis of dAhcyL1-deficient flies reveals its effect on age-dependent metabolic reprogramming and H3K4 methylation. Altogether, reprogramming of methionine metabolism in young flies and suppression of age-dependent SAH accumulation lead to increased life span. These studies highlight the role of noncanonical Ahcy enzymes as determinants of healthy aging and longevity (Parkhitko, 2006).

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Regan, J.C., Khericha, M., Dobson, A.J., Bolukbasi, E., Rattanavirotkul, N. and Partridge, L. (2016). Sex difference in pathology of the ageing gut mediates the greater response of female lifespan to dietary restriction. Elife 5:e10956. PubMed ID: 26878754

Abstract

Women live on average longer than men, but have greater levels of late-life morbidity. This study uncovers a substantial sex difference in the pathology of the ageing gut in Drosophila. The intestinal epithelium of the ageing female undergoes major deterioration, driven by intestinal stem cell (ISC) division, while lower ISC activity in males associates with delay or absence of pathology, and better barrier function, even at old ages. Males succumb to intestinal challenges to which females are resistant, associated with fewer proliferating ISCs, suggesting a trade-off between highly active repair mechanisms and late-life pathology in females. Dietary restriction reduces gut pathology in ageing females, and extends female lifespan more than male. By genetic sex reversal of a specific gut region, female-like ageing pathologies were induced in males, associated with decreased lifespan, but also with a greater increase in longevity in response to dietary restriction (Regan, 2016).

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Xia, B. and de Belle, S. (2016). Transgenerational programming of longevity and reproduction by post-eclosion dietary manipulation in Drosophila. Aging 8(5):1115-34. PubMed ID: 27025190

Abstract

Accumulating evidence suggests that early-life diet may program one's health status by causing permanent alternations in specific organs, tissues, or metabolic or homeostatic pathways, and such programming effects may propagate across generations through heritable epigenetic modifications. However, it remains uninvestigated whether postnatal dietary changes may program longevity across generations. To address this question of important biological and public health implications, newly-born flies (F0) were collected and subjected to various post-eclosion dietary manipulations (PDMs) with different protein-carbohydrate (i.e., LP, IP or HP for low-, intermediate- or high-protein) contents or a control diet (CD). Longevity and fecundity analyses were performed with these treated F0 flies and their F1, F2 and F3 offspring, while maintained on CD at all times. The LP and HP PDMs were found to shorten longevity, while the IP PDM extends longevity significantly up to the F3 generation. Furthermore, the LP reduces while the IP PDM increases lifetime fecundity across the F0-F2 generations. These observations establish the first animal model for studying transgenerational inheritance of nutritional programming of longevity, making it possible to investigate the underlying epigenetic mechanisms and identify gene targets for drug discovery in future studies (Xia, 2006).

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Slade, J. D. and Staveley, B. E.(2016). Enhanced survival of Drosophila Akt1 hypomorphs during amino-acid starvation requires foxo. Genome 59(2):87-93. PubMed ID: 26783834

Abstract

Disordered eating includes any pattern of irregular eating that may lead to either extreme weight loss or obesity. The conserved insulin receptor signalling pathway acts to regulate energy balance and nutrient intake, and its central component Akt1 and endpoint effector foxo are pivotal for survival during nutritional stress. Recently generated Akt1 hypomorphic mutant lines exhibit a moderate decrease in lifespan when aged upon standard media, yet show a considerable increase in survival upon amino-acid starvation media. While the loss of foxo function significantly reduces the survival response to amino-acid starvation, a combination of these Akt1 hypomorphs and a null foxo mutation reveal a synergystic and severe reduction in lifespan upon standard media, and an epistatic relationship when undergoing amino-acid starvation. Evaluation of survivorship upon amino-acid starvation media of these double mutants indicate a phenotype similar to the original foxo mutant demonstrating the role of foxo in this Akt1 phenotype. These results indicate that the subtle manipulation of foxo through Akt1 can enhance survival during adverse nutrient conditions to model the ability of individuals to tolerate nutrient deprivation. Ultimately, a Drosophila model of disordered eating could generate new avenues to develop potential therapies for related human conditions (Slade, 2016).

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Castillo-Quan, J.I., Li, L., Kinghorn, K.J., Ivanov, D.K., Tain, L.S., Slack, C., Kerr, F., Nespital, T., Thornton, J., Hardy, J., Bjedov, I. and Partridge, L. (2016). Lithium promotes longevity through GSK3/NRF2-dependent hormesis. Cell Rep [Epub ahead of print]. PubMed ID: 27068460

Abstract

The quest to extend healthspan via pharmacological means is becoming increasingly urgent, both from a health and economic perspective. This study shows that lithium, a drug approved for human use, promotes longevity and healthspan. Lithium was shown to extend lifespan in female and male Drosophila, when administered throughout adulthood or only later in life. The life-extending mechanism involves the inhibition of glycogen synthase kinase-3 (GSK-3) and activation of the transcription factor nuclear factor erythroid 2-related factor (NRF-2). Combining genetic loss of the NRF-2 repressor Kelch-like ECH-associated protein 1 (Keap1) with lithium treatment revealed that high levels of NRF-2 activation confer stress resistance, while low levels additionally promote longevity. The discovery of GSK-3 as a therapeutic target for aging will likely lead to more effective treatments that can modulate mammalian aging and further improve health in later life (Castillo-Quan, 2016).

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Parkhitko, A.A., Binari, R., Zhang, N., Asara, J.M., Demontis, F. and Perrimon, N. (2016). Tissue-specific down-regulation of S-adenosyl-homocysteine via suppression of dAhcyL1/dAhcyL2 extends health span and life span in Drosophila. Genes Dev 30(12):1409-22. PubMed ID: 27313316

Abstract

Aging is a risk factor for many human pathologies and is characterized by extensive metabolic changes. Using targeted high-throughput metabolite profiling in Drosophila melanogaster at different ages, this study demonstrates that methionine metabolism changes strikingly during aging. Methionine generates the methyl donor S-adenosyl-methionine (SAM), which is converted via methylation to S-adenosyl-homocysteine (SAH), which accumulates during aging. A targeted RNAi screen against methionine pathway components reveals significant life span extension in response to down-regulation of two noncanonical Drosophila homologs of the SAH hydrolase Ahcy (S-adenosyl-L-homocysteine hydrolase [SAHH]), CG9977/dAhcyL1 and Ahcy89E/CG8956/dAhcyL2, which act as dominant-negative regulators of canonical AHCY. Importantly, tissue-specific down-regulation of dAhcyL1/L2 in the brain and intestine extends health and life span. Furthermore, metabolomic analysis of dAhcyL1-deficient flies reveals its effect on age-dependent metabolic reprogramming and H3K4 methylation. Altogether, reprogramming of methionine metabolism in young flies and suppression of age-dependent SAH accumulation lead to increased life span. These studies highlight the role of noncanonical Ahcy enzymes as determinants of healthy aging and longevity (Parkhitko, 2016).

Discussion

Aging is the primary risk factor for many major human pathologies, including cancer, diabetes, cardiovascular disorders, and neurodegenerative diseases. Previous studies of the transcriptional changes that occur during Drosophila aging have revealed that genes encoding members of metabolic pathways are among the most affected. In addition, analyses of changes associated with dietary restriction (DR) that slows down the aging process have also demonstrated dramatic changes in the expression of different metabolic genes. Similarly, studies in worms, mice, and humans have documented changes in the metabolome during the aging process. Recently, untargeted metabolomics analysis in flies (Hoffman, 2014) has suggested that DR might reverse age-dependent metabolic reprogramming at the tissue (Laye, 2015) and whole-organism (Avanesov, 2014) levels. Despite these studies, the mechanisms underlying age-dependent metabolic reprogramming, the nature of the metabolites that change with time, and their effect on life span are still poorly characterized (Parkhitko, 2016).

A number of alterations in metabolic pathway activities are known to extend life span in flies and other organisms. Among them, perturbation of components of the mitochondrial respiratory complexes I, III, IV, and V; increased mitochondrial uncoupling via expression of human UCP2; heterozygous mutations of AMP biosynthetic enzymes; reduced levels of Enigma, the enzyme responsible for β-oxidation of fatty acids; and reduced levels of Indy, which functions as a cation-independent electroneutral transporter for a variety of tricarboxylic acid cycle intermediates extend life span in Drosophila. In addition, key longevity regulators such as insulin receptor substrate (IRS)/chico and JNK are known to reprogram whole-body metabolism, but it is unknown whether this reprogramming is responsible for life span extension (Parkhitko, 2016).

Another level of complexity between metabolism and aging arises from the observation that different tissues have different metabolic requirements and that alterations of different metabolic components or upstream regulators of metabolism in one tissue can affect aging of other tissues and life span. For example, muscle-specific FOXO/4E-BP signaling retards muscle aging in Drosophila in a cell-autonomous manner and nonautonomously extends life span and preserves proteostasis in other aging tissues such as the brain, the retina, and adipose tissue. Similarly, overexpression of AMPK in the adult Drosophila nervous system nonautonomously maintains proteostasis during muscle aging and extends organismal life span. In addition, muscle-specific mitochondrial injury promotes organismal life span via activation of mtUPR and increased production of ImpL2, an insulin growth factor-binding protein (IGFBP)-like protein. Moreover, muscle-specific expression of the transcription factor Mnt extends life span by reducing ribosome biogenesis and promoting the expression of the myokine Myoglianin (Parkhitko, 2016).

To expand knowledge of the regulation of life span by metabolism, high-throughput metabolite profiling of Drosophila melanogaster was performed to identify changes that may correlate with aging. Strikingly, methionine metabolism emerged as one of the most regulated metabolic pathways with age. To test the role of the methionine pathway in life span determination, a targeted RNAi screen against most of the methionine pathway components and related enzymes was performed. Unexpectedly, ubiquitous down-regulation of two Drosophila homologs of S-adenosyl-homocysteine (SAH) hydrolase-like proteins, CG9977/dAhcyL1 (S-adenosyl-L-homocysteine hydrolase [SAHH]) and CG8956/Ahcy89E/dAhcyL2, significantly extended life span. Moreover, brain-specific down-regulation of dAhcyL1 and intestine-specific down-regulation of both dAhcyL1 and dAhcyL2 increased life span. Importantly, down-regulation of dAhcyL1 extended not only life span but also health span. Finally, suppression of dAhcyL1 activities decreased the level of SAH, as determined by tandem mass spectrometry (MS/MS), and suppressed H3K4 trimethylation (H3K4me3), thus phenocopying methionine starvation. Altogether, these data demonstrate that dAhcyL1 and dAhcyL2 encode new key regulators of age-dependent metabolic reprogramming and control both health span and life span (Parkhitko, 2016).

By studying metabolic changes during fly aging, two potential targets for health and life span extension were identified, CG9977/dAhcyL1 and Ahcy89E/CG8956/dAhcyL2. Whole-body and tissue-specific down-regulation of these two noncanonical, dominant-negative Drosophila homologs of Ahcy (AHCY is the rate-limiting enzyme in methionine metabolism that hydrolyzes SAH to adenosine and homocysteine) significantly extended life span, decreased levels of SAH, and suppressed H3K4me3 (Parkhitko, 2016).

Fly metabolome changes caused by aging were sought and it was hypothesized that preventing some of these changes would increase life span and prevent age-dependent health deterioration. Metabolite profiling revealed striking changes in the metabolome of aged flies, including altered levels of multiple methionine metabolism intermediates as well as several other previously known pathways affected by aging, including glutamate metabolism, glutathione metabolism, and the mitochondrial electron transport chain (Parkhitko, 2016).

Restriction of a single amino acid, either methionine or tryptophan, extends life span in rodents. In addition, methionine restriction extends life span in yeast, flies, rodents, and human diploid fibroblasts. Methionine metabolism consists of three branches: salvage, de novo, and transsulfuration pathways. Methionine is converted into glutathione and taurine via the transsulfuration pathway supplying cells with antioxidant defense. In accordance, overexpression of CBS, the rate-limiting enzyme in the transsulfuration pathway, extends life span. Moreover, CBS is one of the primary sources of hydrogen sulfide production, which has been shown to function as an evolutionarily conserved mediator of DR-mediated longevity (Parkhitko, 2016).

This study identified two novel members of methionine metabolism (located upstream of the transsulfuration pathway) that can extend life span when down-regulated. Both dAhcyL1/CG9977 and dAhcyL2/Ahcy89E/CG8956 encode noncanonical AHCY/SAHH enzymes that most likely suppress the function of the canonical AHCY enzyme. A possible explanation for their effects on life span extension is that down-regulation of dAhcyL1 and dAhcyL2 would enhance flux into the transsulfuration pathway. However, cycloleucine, an upstream inhibitor of Sam-S, also increases life span, suggesting that life span extension is most likely due to the clearance of metabolites between Sam-S and Ahcy13 (SAM and SAH) (Parkhitko, 2016).

Gnmt catalyzes the conversion of glycine to sarcosine using SAM as a donor of the methyl group, and recent studies have shown that Gnmt overexpression decreases levels of SAM and extends life span in flies. Although Gnmt overexpression extends life span, its product, sarcosine, was identified as a metabolite contributing to prostate cancer progression, and high cytoplasmic GNMT expression in patient tumor samples correlated with more aggressive forms of prostate cancer. Based on these data, dAhcyL1 and dAhcyL2 could represent better targets for developing methionine restriction mimetics, as they affect methionine indirectly via regulation of AHCY activity (Parkhitko, 2016).

AHCY (Ahcy13 in flies) is a tetrameric enzyme that catalyzes the reversible hydrolysis of SAH to adenosine and L-homocysteine. SAH is formed as a by-product of SAM through methylation reactions, and hydrolysis of SAH is required to maintain proper concentrations of SAH, which serves as an inhibitor of SAM-dependent methylation reactions. Accordingly, inhibition of AHCY, which is associated with decreased life span, results in the intracellular accumulation of SAH (whole-body adult-onset Ahcy13 RNAi expression caused an ~15-fold increase in whole-body SAH level). In contrast, down-regulation of dAhcyL1 moderately suppressed levels of SAH (approximately twofold decrease) and increased life span. Interestingly, the level of SAH was increased with age in OreR flies and was significantly lower in naturally selected long-lived flies compared with control flies at 7 wk of age. The mechanisms underlying the age-dependent changes of SAH and which of them affect the age-dependent changes in Ahcy13 activity and methionine pathway activity are unknown. Possibly, age-dependent increased oxidative stress can redirect methionine flux into the transsulfuration pathway for glutathione production. It is also worth noting that alterations in SAH levels do not equally affect the activity of methyltransferases, an observation that warrants further investigation (Parkhitko, 2016).

dAHCYL1/dAHCYL2 proteins consist of a C-terminal AHCY domain and an N-terminal IRBIT domain. Due to the fact that dAHCYL1/dAHCYL2 proteins likely have lost their enzyme activity, they can suppress Ahcy13 function via heteromultimerization. As the presence of an N-terminal IRBIT domain gives dAHCYL1/dAHCYL2 proteins new functions in Ca²⁺ signaling, intracellular pH regulation, and production of deoxyribonucleotides (which are referred as noncanonical functions), tests were performed to see which downstream effectors of dAHCYL1/dAHCYL2 proteins are responsible for life span extension. Down-regulation of dAhcyL1 in the whole body did not affect the levels of deoxyribonucleotides, and the down-regulation of RnrL and Itp-r83A or overexpression of Itp-r83A had no effect on life span. Furthermore, dAhcyL1 down-regulation suppressed the levels of SAH and increased life span, contrary to Ahcy13 function. Altogether, these results suggest that down-regulation of dAhcyL1 promotes life span via modulating Ahcy13 function but not through noncanonical functions. The modular structure of AHCYL proteins suggests that they integrate different signals from Ca²⁺ signaling, pH regulation, production of deoxyribonucleotides, SAH clearance, and life span regulation (Parkhitko, 2016).

Several studies have shown that specific heterochromatin regions are remodeled during aging and that life span-extending interventions such as calorie restriction suppress age-dependent heterochromatin remodeling. Moreover, heterochromatin formation prolongs life span, and its status depends on the levels of HP1 and H3K9me. As the status of methionine metabolism is sufficient to determine the H3K4me3 (but not H3K9me3 or H3K27me3) levels in human cells, it was hypothesized that down-regulation of dAhcyL1 and dAhcyL2 would mimic methionine starvation and affect H3K4me3 levels and the level of heterochromatinization. Accordingly, it was found that down-regulation of dAhcyL1 suppressed H3K4me3 levels. In contrast, no effect of down-regulation of dAhcyL1 was observed on expression of a LacZ reporter gene located in the heterochromatin, which could be explained by differences in the effects of calorie restriction and methionine starvation (Parkhitko, 2016).

Interestingly, the mammalian homolog of Ahcy13 (SAHH) was recovered in a loss-of-function genetic screen as a putative tumor suppressor gene, and its mRNA was lost in 50% of tumor tissues studied in comparison with normal tissue. Moreover, elevated homocysteine levels have been reported as a risk factor for dementia and Alzheimer's disease. As it was proposed that down-regulation of dAhcyL1 and dAhcyL2 activates Ahcy13 (SAHH), it will be important to examine whether dAhcyL1/2 can affect carcinogenesis. In addition, methionine metabolism and SAM levels have been shown to be critical for the maintenance and differentiation of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Interestingly, wild-type flies exhibit age-dependent intestine stem cell (ISC) hyperproliferation and misdifferentiation, causing loss of intestinal integrity, whereas genetic manipulations that improve proliferative homeostasis extend life span. The current data suggest that down-regulation of dAhcyL1 and dAhcyL2 suppresses age-dependent SAH accumulation and prevents lossof intestinal integrity (as revealed by the 'Smurf' assay) and that their ubiquitous and tissue-specific down-regulation extends life span. The precise mechanisms of ISC regulation by methionine metabolism and SAH are interesting subjects for further studies (Parkhitko, 2016).

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Talbert, M. E., Barnett, B., Hoff, R., Amella, M., Kuczynski, K., Lavington, E., Koury, S., Brud, E. and Eanes, W. F. (2015). Genetic perturbation of key central metabolic genes extends lifespan in Drosophila and affects response to dietary restriction. Proc Biol Sci 282. PubMed ID: 26378219

Abstract

There is a connection between nutrient inputs, energy-sensing pathways, lifespan variation and aging. Despite the role of metabolic enzymes in energy homeostasis and their metabolites as nutrient signals, little is known about how their gene expression impacts lifespan. This report uses P-element mutagenesis in Drosophila to study the effect on lifespan of reductions in expression of seven central metabolic enzymes and contrasts the effects on normal diet and dietary restriction. The major observation is that for five of seven genes, the reduction of gene expression extends lifespan on one or both diets. Two genes are involved in redox balance, and it was observed that lower activity genotypes significantly extend lifespan. The hexokinases also show extension of lifespan with reduced gene activity. Since both affect the ATP/ADP ratio, this connects with the role of AMP-activated protein kinase as an energy sensor in regulating lifespan and mediating caloric restriction. These genes possess significant expression variation in natural populations, and the experimental genotypes span this level of natural activity variation. These studies link the readout of energy state with the perturbation of the genes of central metabolism and demonstrate their effect on lifespan (Talbert, 2015).

Discussion

In their lifetime, all organisms experience environments that change both temporally and spatially in their nutrient availability and energy content. The optimal utilization and storage of available energy is a physiological challenge that shapes variation in life-history phenotypes, and in principle sets the trade-off between lifespan and reproduction. Failing to allocate energy optimally to the changing availability of nutrients would be expected to have significant fitness costs. As a consequence, nutrient sensing and response networks are strongly conserved pathways. Since there are optimal physiological responses that reset internal energy balance for different environments, genetic variation associated with these responses would be expected (Talbert, 2015).

As potential modifiers of cell energy state through their action on metabolite levels, the genes of central metabolism are potential sources for this genetic variation. The response to changing nutrient input results in intercellular signals that drive a cascade of downstream gene transcription shifts that facilitate energy utilization and storage. The proximal signal is generally derived from specific metabolite levels as they change under shifting nutrient load. There is considerable precedent for this general mechanism; for example, the well-known action of glucose on insulin secretion in vertebrates, and the signals associated with the secretion and action of adipokinetic hormone (AKH) in insects. The effect of energy balance on longevity in yeast is well known. Since metabolite concentrations act as proximal signals and also appear to correlate with the associated gene expression levels of the component steps in metabolism, this relationship clearly implicates the natural genetic variation in expression (or activity) of the central metabolic genes as potential sources of genetic variation in setting metabolite levels, and thus play a role in sensing and setting responses. Moreover, specific enzymes are expected to emerge as the targets of natural selection where genetic expression or activity level will act as an 'energy-stat'. Different genotypes will bracket and set the nutrient levels involved in triggering downstream responses that affect traits associated with fitness, such as lifespan variation and fecundity (Talbert, 2015).

The well-established observation that nutritional or dietary restriction (DR) extends lifespan in many species is mechanistically related to energy-state signalling. In Drosophila, many studies have shown DR to extend lifespan, and using genetic manipulation, many of the signalling pathways have been identified that extend lifespan and thus effectively mimic DR. Experimental work has shown connections between energy-signalling steps and other energy correlated phenotypes such as starvation resistance, nutrient storage and stress resistance that have connections to DR (Talbert, 2015).

In both plants and animals, it is becoming apparent that many metabolic genes and their enzyme products are associated with roles other than simple processing of metabolites. Based on evidence from RNAi reduction screens of central metabolic genes in Caenorhabditis elegans, it is estimated that as much as 25% of the genes screened implicate metabolism in longevity extension. RNAi knockdown of expression of several genes in the mitochondrial respiratory chain has been shown to extend lifespan in Drosophila. In yeast, the overexpression of several of the genes involved in cofactor shuttles actually extends lifespan. Despite its importance as a model in lifespan studies, and the connection between metabolism and lifespan seen in other models, no studies in Drosophila have examined the mutational perturbation of central metabolic gene activity and their effects on longevity (Talbert, 2015).

Natural populations of Drosophila melanogaster vary in both average lifespan and response of lifespan to dietary challenge. In D. melanogaster, the genes of the central metabolic pathway harbour considerable sequence and expression variation, which often shows change with latitude and season. In this regard, unlike the other experimental models used in aging studies, Drosophila offers the unique opportunity to associate aging and signalling pathways with their population genetics (Talbert, 2015).

The long-term goal of these studies is to integrate geographical and seasonal variation in metabolic genes with life-history phenotypes, such as longevity and its fitness correlates. This report used matched sets of P-element excision alleles in D. melanogaster to create genotypes that possess modest reductions in gene expression and subsequently examined the impact of these perturbations on lifespan under normal and restricted diets. Seven metabolic genes were studied: Idh, Mdh2, Hex-C, Hex-A, Gpdh, Gdh and Men. These enzymes involve possible signalling via glucose, ATP/ADP, NAD/NADH and NADP/NADPH ratios, citrate, pyruvate, malate and glutamate, and they also involve genes with primary expression restricted to the cytoplasm or the mitochondria. A range of effects was observed on lifespan, from none at all to very significant increases in lifespan that can depend on diet (Talbert, 2015).

This study observed that the genetic perturbation of central metabolic genes has significant effects on lifespan. Moreover, the general observation is that low-activity genotypes show extension of lifespan. Perhaps not surprisingly, some genotype effects are also diet dependent. Some genes (Idh, Mdh2, Hex-C) show an increase in lifespan under DR, but no effect of genotype, while others show a strong (Gdh, Men) genotype dependence in their response to DR. Finally, Gpdh shows a strong dependence on genotype activity, yet no effect of DR. These differences are expected because the observations represent seven enzymes that act on different metabolites and cofactors, and are limited to mitochondrial or cytosolic function (Talbert, 2015).

In this work, experimental outcomes across genes are not strictly comparable. First, while single gene effects are being studied within identical genetic backgrounds, the backgrounds differ across the seven gene sets. The P-element progenitor lines differ in the type of element used in the excision series (e.g. KG versus EP), and while the replacement backgrounds possess some chromosomes in common (often the 6326 chromosomes), they generally differ in others. Second, while all of the genotype comparisons involve reductions of activity of 50% or less, the same level of flux control across enzymes cannot be expected. Thus, cytosolic IDH may possess little flux control over NADPH/NADP levels, while MEN may exercise greater control over these metabolites, especially at reduced nutrient levels. It should be pointed out that in the Raleigh population the cytosolic Idh gene bears little molecular polymorphism and the few SNPs seen show little cis-based expression effect or clinal change (Talbert, 2015).

It should also be emphasized that unlike many gene-targeted lifespan studies, this study was not using full knockout genotypes, or genotypes where the relative functional reduction is unknown, and precise estimates of genotype activity are available. Moreover, these activity differences are representative of the range of much of the cis-associated SNP expression variation seen in natural populations. The observation that metabolic genes, when perturbed modestly in activity, have an effect on lifespan is certainly relevant to discussions of the maintenance of genetic variation in these genes in natural populations, especially as nutrient levels vary geographically and seasonally (Talbert, 2015).

Gpdh, Gdh and Hex-A were highlighted in this study study of clinal SNP expression variation in the pathway. These genes, among others, showed significant changes in gene transcript expression with latitude. The observations in this study add a fitness component to the causes of genetic expression variation of metabolic genes in natural populations of Drosophila. Also, the expectation that the gene-specific extension of lifespan can depend on dietary level adds complexity, since nutritional background is expected to shift locally and seasonally in this species. The effect of reduced activity is incrementally small in terms of daily survival, but when integrated over the average lifespan of a fly this can be very significant. This relationship is in contrast to flight metabolism, where similar activity changes have no effects on flight performance (Talbert, 2015).

The enzymes were targeted because they act on different metabolites and cofactors, and are limited to either mitochondrial or cytosolic function. Both IDH and MEN are cytosolic enzymes and NADPH dependent, and, along with the pentose shunt enzymes, provide a significant contribution to the NADPH/NADP pool. Both glutamate (GDH) and malate dehydrogenase (MDH2) are limited to mitochondrial function and, like GPDH, are dependent on NAD/NADH, and will impact that redox balance and its effect on signalling and aging. The two hexokinases, HEX-A and HEX-C, potentially affect the ADP/ATP ratio and have different tissue expressions. They could vary the ADP/ATP content, and in that fashion set energy-state response via regulating AMPK signalling. This AMP-activated kinase is a sensor that has effects in Drosophila, which provides a link between lifespan and caloric restriction (Talbert, 2015).

The observations for the Gpdh and Gdh genes implicate mitochondrial function and the redox balance with lifespan extension. GPDH is part of the essential mitochondrial phosphoglycerol shuttle in insects and is often considered a point of ROS production. The NAD/NADH redox balance is emerging as an important element of lifespan extension in yeast and is often considered a direct readout of metabolic state. Moreover, the lifespan extension associated with these genes might also act through the Sir-like enzymes, which are NAD-dependent histone deacetylases that silence chromatin and thus control transcription in a fashion directly coupled to energy-state imbalance. This relationship is important because starvation in Drosophila has been clearly shown to significantly raise the NAD/NADH ratio, although the role of Sir2 in lifespan extension in Drosophila has been questioned. GDH is also limited to mitochondrial function, and potentially affects the redox balance and NAD/NADH ratio. It connects glutamate, a key energy-state signalling molecule, to metabolic control, and sits at the important crossroads of carbohydrate and amino acid metabolism. As well, glutamate is at the hub of connectivity in the large central metabolic network. Both of these enzymes show significant extension of lifespan with only 40% reductions in whole-body activity. However, all mitochondrial or NAD-dependent genes are not similar in affecting lifespan; comparable activity changes in mitochondrial and NAD-dependent MDH (Mdh2) have little effect on lifespan in either dietary condition (Talbert, 2015).

The dependence of the results on diet is important. Over the past two decades, DR has been shown to impose a trade-off where it extends lifespan and reduces reproduction fecundity. It has gained prominence because of its association with aging research in general, but the phenomenon of DR has obvious relevance to studies of life-history evolution because it will be associated with plastic responses to nutritional challenges in nature, and the potential maintenance of genetic variation. Studies on model organisms have led to the discovery of many genes where mutational perturbation extends lifespan and thus mimics DR restriction. This is most notable in the parallel effects of disruption of genes specifically associated with energy-sensing pathways and signalling of dietary state and DR. Despite its general occurrence in D. melanogaster, an effect of DR on lifespan is not seen in some of the experimental lines. Two (Gpdh, Hex-A) of the seven genes show no DR effect in general. For Gdh, DR is seen just for the low-activity genotype. This different response to DR is suggested by studies where line-by-diet interactions are noted, but is shown more definitively in studies in mice and yeast, where it becomes clear that genetic background affects the response to DR. In another study, 166 single, non-essential genes were made deficient in yeast, and a large proportion of genes showed loss of DR extension capability, as well as an enhanced DR response. Clearly, DR response can be modified genetically and it would not be surprising if genetic variation in natural populations were to reflect this observation (Talbert, 2015).

Does the failure of some expression modified genotypes to respond to DR imply a mechanistic connection to the signalling associated with DR? An interaction between genotype and diet would suggest that the lifespan responses to DR may be coupled to metabolic signals associated with these enzymes or pathways. For example, the full-activity Men genotype shows a significant DR lifespan response, yet the Men low-activity genotype appears resistant to lifespan extension under DR. This may suggest that the NADPH/NADP ratio in the case of MEN is a signal associated with DR. However, the NADPH-dependent IDH shows no genotypic effect on DR, which may contradict this suggestion or simply be because IDH possesses low control over cofactor pool levels. Conversely, the Gdh normal activity genotype shows no DR response, while a genotype reduction in Gdh activity strongly enhances a DR response. Perhaps the reduction in amino acids that is associated with DR in Drosophila is enhanced by reduction of Gdh activity, since glutamate and GDH sit at the crossover of carbohydrate and amino acid metabolism in the mitochondria. However, this interaction must be interpreted with caution, since tests of genotype dependence of DR should be tested by using a range of diet changes (Talbert, 2015).

Where might the mechanism of action reside that extends lifespan or is associated with a genotype-dependent response to DR for these metabolic genes? Discussions of energy-signalling pathways typically start with the statement that nutrient levels are first 'sensed' and then the pathway of interest is addressed (e.g. the insulin receptor insulin/TOR pathway in Drosophila). Presumably, this initial sensing must emanate from direct immediate readouts of the cell's metabolic state (i.e., metabolites). In Drosophila, dietary sugars induce significant metabolite changes. These metabolite levels changes trigger the secretion of neuropeptides from specialized neurosecretory cells. This model of sensing is similar to the regulation of glucagon in mammalian pancreatic cells, and a similar case has been made for the sensing and regulation of AKH by the corpora cardiac cells in Drosophila. It is possible that either these cell-specific or just systemic metabolite levels initiate the signalling process. Genetic variation will regulate these metabolite levels in conjunction with nutrient inputs. In this sense, lifespan extension by some metabolic genes is top-down (Talbert, 2015).

Over the past two decades, a large number of genes in several model species have been observed to extend lifespan when mutated. In Drosophila, most studies have emphasized the signalling cascades emanating from neurosecretory cells. However, the most proximal steps of central metabolism must set the signalling environments because their metabolite levels respond immediately to nutrient inputs. This study places the metabolic pathway in the discussion of energy signalling and looked at the impact of genetic perturbation of some key genes on lifespan. The outcome is that there are numerous examples where reductions in activity extend lifespan. Perhaps not surprisingly, this extension depends on the nutrient environment. It is proposed that this setting of lifespan response to gene expression variation also provides a selective context for naturally segregating metabolic gene variation, and moreover may contribute to unravelling the patterns of genetic variation observed in natural populations (Talbert, 2015).

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Kopp, Z. A., Hsieh, J. L., Li, A., Wang, W., Bhatt, D. T., Lee, A., Kim, S. Y., Fan, D., Shah, V., Siddiqui, E., Ragam, R., Park, K., Ardeshna, D., Park, K., Wu, R., Parikh, H., Parikh, A., Lin, Y. R. and Park, Y. (2015). Heart-specific Rpd3 downregulation enhances cardiac function and longevity. Aging (Albany NY) 7: 648-663. PubMed ID: 26399365

Abstract

Downregulation of Rpd3, a homologue of mammalian Histone Deacetylase 1 (HDAC1), extends lifespan in Drosophila melanogaster. Once revealed that long-lived fruit flies exhibit limited cardiac decline, this study investigated whether Rpd3 downregulation would improve stress resistance and/or lifespan when targeted in the heart. Contested against three different stressors (oxidation, starvation and heat), heart-specific Rpd3 downregulation significantly enhanced stress resistance in flies. However, these higher levels of resistance were not observed when Rpd3 downregulation was targeted in other tissues or when other long-lived flies were tested in the heart-specific manner. Interestingly, the expressions of anti-aging genes such as sod2, foxo and Thor, were systemically increased as a consequence of heart-specific Rpd3 downregulation. Showing higher resistance to oxidative stress, the heart-specific Rpd3 downregulation concurrently exhibited improved cardiac functions, demonstrating an increased heart rate, decreased heart failure and accelerated heart recovery. Conversely, Rpd3 upregulation in cardiac tissue reduced systemic resistance against heat stress with decreased heart function, also specifying phosphorylated Rpd3 levels as a significant modulator. Continual downregulation of Rpd3 throughout aging increased lifespan, implicating that Rpd3 deacetylase in the heart plays a significant role in cardiac function and longevity to systemically modulate the fly's response to the environment (Kopp, 2015).

Discussion

The data showed that decreased Rpd3 expression in Drosophila has a benefit for stress resistance against the environment. To downregulate the rpd3 gene in whole body, two ways were approached using the heterozygous rpd3 mutant (P{PZ}rpd3[04556]/+) and the UAS/Gal4 system to carry out RNAi (rpd3Ri/armG4). Both flies showed higher survivorship under oxidative stress compared to the control flies. However, the flies differed in increased survivorship percent (rpd3-/+: 31% and rpd3Ri/armG4: 22%). Considering that the downregulation yield of the rpd3 gene was different between the two approaches (rpd3-/+: 54% and rpd3Ri/armG4: 40%), it is possible that more downregulation of the rpd3 gene may induce higher resistance to stress. In the heart-specific Rpd3 downregulation, a similar pattern was observed between the rpd3Ri/tinG4 and rpd3RiS/tinG4 flies. The 21bp target sequence of rpd3RiS transgene was less effective at rpd3 downregulation compared to the 482bp sequences of rpd3Ri transgene when tested in the whole body. Thus, the rpd3RiS/tinG4 flies showed a 23% increase in survivorship compared to a 35% increase in rpd3Ri/tinG4 flies (Fig. 3A). Those data let to a speculation that the content of rpd3 downregulation determines the consequent stress-resistance enhancement (Kopp, 2015).

It was found that heart-specific Rpd3 downregulation systemically increases expression of anti-aging genes such as Sod2 and dFOXO. It was also shown that more downregulation of the rpd3 gene in a heart induces higher expression of anti-aging genes. This may provide an explanation of how Rpd3 downregulation in the heart enhances stress resistance mechanism, particularly since dFOXO is considered to activate sod2 gene. In response to cellular stresses, such as nutrient deprivation or increased levels of reactive oxygen species, dFOXO is activated and inhibits growth through acting on target genes such as Thor (d4E-BP). As a translational repressor, 4E-BP activity is shown to be critical for survival under dietary restriction and oxidative stress, and is linked to lifespan. This dFOXO/4E-BP signaling is also revealed to play a key role in the coordination of organismal and tissue aging through an organism-wide regulation of proteostasis in response to muscle aging. Interestingly, this Drosophila forkhead transcription factor (dFOXO) activates d4E-BP transcription, which is upregulated under stressed conditions. Consistent with increased foxo expression in flies with heart-specific Rpd3 downregulation, the data also showed that Thor was significantly upregulated with heart-specific Rpd3 downregulation. When induced by stress, fat body antimicrobial peptide (AMP) genes are activated in response to nuclear dFOXO activity. Upregulation of both foxo and DptB (one of target AMP) genes in heart-specific Rpd3 downregulation illustrates that Rpd3 downregulation in the heart modulates gene expression in other tissues such as fat body for stress adaption. One possible mechanism of this modulation is that heart-specific Rpd3 downregulation produces secreted proteins through Rpd3 deacetylase activity from heart, which thus regulates gene expression in other tissues (Kopp, 2015).

A positive correlation between stress resistance and lifespan extension was shown in several long-lived mutant flies. Previous findings have also suggested that enhanced stress resistance may extend lifespan in Drosophila. The data indicated that downregulating the rpd3 gene in the whole body or heart enhances both stress resistance and lifespan with improved cardiac function. However, insufficient heart-specific Rpd3 downregulation in older aged flies failed to prolong lifespan or improve cardiac condition, implying that throughout lifetime, Rpd3 in the heart influences both cardiac function and lifespan. Currently, although a conclusion of whether improved cardiac function from heart-specific Rpd3 modulation directly impacts longevity mechanism cannot yet be made, it is reported that enhanced cardiac capability could extend the lifespan of Drosophila (Kopp, 2015).

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Peleg, S., Feller, C., Forne, I., Schiller, E., Sévin, D.C., Schauer, T., Regnard, C., Straub, T., Prestel, M., Klima, C., Schmitt Nogueira, M., Becker, L., Klopstock, T., Sauer, U., Becker, P.B., Imhof, A. and Ladurner, A.G. (2016). Life span extension by targeting a link between metabolism and histone acetylation in Drosophila. EMBO Rep [Epub ahead of print]. PubMed ID: 26781291

Abstract

Old age is associated with a progressive decline of mitochondrial function and changes in nuclear chromatin. However, little is known about how metabolic activity and epigenetic modifications change as organisms reach their midlife. This study assessed how cellular metabolism and protein acetylation change during early aging in Drosophila melanogaster. Contrary to common assumptions, it was found that flies increase oxygen consumption and become less sensitive to histone deacetylase inhibitors as they reach midlife. Further, midlife flies show changes in the metabolome, elevated acetyl-CoA levels, alterations in protein-notably histone-acetylation, as well as associated transcriptome changes. Based on these observations, the activity of the acetyl-CoA-synthesizing enzyme ATP citrate lyase (ATPCL) or the levels of the histone H4 K12-specific acetyltransferase Chameau were decreased. It was found that these targeted interventions both alleviate the observed aging-associated changes and promote longevity. These findings reveal a pathway that couples changes of intermediate metabolism during aging with the chromatin-mediated regulation of transcription and changes in the activity of associated enzymes that modulate organismal life span (Peleg, 2016).

The process of aging is characterized by a deterioration of multiple interconnected cellular pathways, which makes the identification of molecular mechanisms of phenotypic aging and death particularly difficult. Many molecular analyses have focused on the comparison of young and old organisms, which resulted in the formulation of nine hallmarks of aging ranging from telomere shortening and epigenetic alterations, to differences in nutrient sensing and stem cell depletion. While many of these experiments have identified valuable paths toward life span extension, such studies face the complication that old individuals suffer from the progressive deterioration of multiple cellular systems, which can make it challenging to distinguish primary from secondary effects. To identify changes involved in the onset of aging, this study compared D. melanogaster flies at young age and during midlife at the onset of a premortality plateau, when most individuals of a population are still alive (Peleg, 2016).

Surprisingly, heads from midlife flies consume more oxygen than the young ones. This is in apparent contradiction to the general observation of a reduced metabolism when animals age, which this study also observe in old flies. There are several possible explanations for this unexpected finding. In many studies, the oxygen consumption rate was extrapolated from measurements of isolated mitochondria, which may lack crucial extra-mitochondrial signals when investigated in isolation, whereas this study has measured activity in isolated fly heads. Alternatively, flies may change their feeding behavior when reaching midlife, or switch from an anaerobic to a more aerobic metabolism due to their decreased activity, which is consistent with higher levels of metabolites generated by oxidative processes in midlife flies. Finally, the metabolic changes may be due to a feed-forward activation of metabolic enzymes that become stimulated by hyper-acetylation. The observation that the treatment of isolated fly heads with lysine deacetylase (KDAC) inhibitors increases oxygen consumption rate (OCR) within minutes suggests that such a direct feed-forward mechanism might indeed exist. The finding that midlife flies have a higher ground state of acetylation and are less susceptible to a stimulation by KDAC inhibitors argues for similar acetylation events triggered by KDAC inhibitor treatment and aging (Peleg, 2016).

The increased level of acetyl-CoA in midlife flies correlates with a very specific change in the histone modification pattern as flies reach midlife. As it is not possible to distinguish between mitochondrial and cytosolic acetyl-CoA, the substrate for acetyltransferases, the observed correlation may not be causal. However, an increased activity of the main enzyme was also observed for the synthesis of cytosolic acetyl-CoA, ATPCL, in midlife flies, and therefore it was assumed that the cytosolic acetyl-CoA level is indeed higher when flies reach midlife. Interestingly, this increased activity is not caused by increased protein synthesis of ATPCL, but potentially by posttranslational mechanisms such as a hyper-acetylation. This is also supported by the observation that a fly strain heterozygous for an atpcl mutation shows only a 15% reduction in ATPCL activity, suggesting that there is a substantial degree of posttranscriptional regulation of this enzymatic activity. Such a regulation of ATPCL has also been proposed to stimulate lipid synthesis and tumor growth in rats. The current findings that a fly strain carrying a mutation in the ATPCL gene has an extended life span and a delayed onset of aging further confirm the importance of extra-mitochondrial acetyl-CoA for the regulation of aging. Interestingly, the reduction in ATPCL has a much stronger effect on the metabolism of midlife animals when compared to young animals. The effects observed analyzing head tissue of Drosophila melanogaster are in line with earlier reports that the targeted depletion of an unrelated acetyl-CoA synthase in fly neurons extends life span. It will be interesting to resolve the physiological effects of ATPCL mutation on the metabolome, the histone acetylation, and the transcriptome in isolated neurons (Peleg, 2016).

The ATPCL mutation results in a rather specific change in histone acetylation and does not affect all acetylation sites to the same degree. In midlife animals, the ATPCL mutation has the strongest effect on H4K12ac-an acetylation site that had been implicated in age-dependent memory impairment and transcriptional elongation and which is increased when flies reach their premortality plateau phase. This may be due to modulation of the enzymatic properties of Chameau or of a corresponding deacetylase. An increased activity in several deacetylases has been shown to extend life span in various organisms and higher concentrations of the sirtuin cofactor NAD⁺ have been shown to be beneficial for life span extension. However, the effect of sirtuins on life span continues to be debated and their effect has so far not been associated with a particular histone modification pattern. The quantitative analysis of specific histone modifications in this study has allowed identification of Chameau as an enzyme responsible for the increased modification in midlife flies. It is worth mentioning that the chm mutant allele is homozygous lethal and the beneficial effect on life span is more pronounced in males than in females, suggesting that Chameau has additional function, which are not yet fully understood. However, the fact that a reduction in the activity of the acetyltransferase Chameau robustly promotes longevity in male flies supports the hypothesis that this enzyme has an active role in modulating life span at least in Drosophila males (Peleg, 2016).

Previous studies demonstrated that old flies show an impaired transcriptome surveillance, as manifested in increased transcriptional noise and expression of aberrant or immature mRNAs. This study found substantial changes in the transcriptional profile as flies reach midlife, suggesting that the differential regulation of gene expression is one of the early hallmarks of the aging process. It remains to be explored how specific changes in gene expression integrate with regulatory modifications and metabolic activity. Chameau appears to promote the expression of a large number of genes particularly during the midlife period genes. Conceivably, the enhanced H4K12 acetylation leads to widespread chromatin opening, with positive effects for the transcription of specific genes. A side effect of this loosening of chromatin structure may be the increased transcriptional noise, which might compromise a variety of physiological functions. Considering the localization of H4K12ac at the gene body of highly expressed genes, it will be interesting to investigate whether the increased transcription during midlife is due to a higher rate of transcript elongation or a higher activity of cryptic promoters. It is hypothesized that the attenuation of this effect in chm mutant flies is the cause for their extended life span. A similar effect is also seen in ATPCL mutant flies, and the observation that life span is not further extended if the ATPCL and chm alleles are combined suggests that the two enzymes may act in the same pathway (Peleg, 2016).

These data provide an overview of the metabolic, proteomic, and transcriptomic changes that occur as flies reach the premortality plateau phase. Conceivably, metabolic processes are linked to changes in gene expression through differential protein acetylation, in general, and histone acetylation, in particular. Currently it cannot be unambiguously distinguish whether the shift in metabolic activity upon fly aging precedes the increases in protein/histone acetylation, or whether increases in protein/histone acetylation result in specific metabolic changes. Most likely, both principles affect each other in a complex network of feedback and feed-forward loops. Indeed, many mitochondrial enzymes that have been shown to be acetylated in response to metabolic changes either gain or lose enzymatic activity (Peleg, 2016).

Considering the high conservation of central metabolism, metabolic regulation, and epigenetics between flies and humans, these data raise the possibility that small molecule regulators of acetyl-CoA production or consumption, or changes in the activity of selective acetyltransferase functions, could prolong a healthy midlife also in humans. These model organism data reveal a potential alternative strategy that could extend midlife and delay aging-associated homeostatic decline in humans (Peleg, 2016).

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Chen, N., Guo, A. and Li, Y. (2015). Aging accelerates memory extinction and impairs memory restoration in Drosophila. Biochem Biophys Res Commun 460: 944-948. PubMed ID: 25842205

Abstract
Age-related memory impairment (AMI) is a phenomenon observed from invertebrates to human. Memory extinction is proposed to be an active inhibitory modification of memory, however, whether extinction is affected in aging animals remains to be elucidated. Employing a modified paradigm for studying memory extinction in fruit flies, this study found that only the stable, but not the labile memory component was suppressed by extinction, thus effectively resulting in higher memory loss in aging flies. Strikingly, young flies were able to fully restore the stable memory component 3 h post extinction, while aging flies failed to do so. In conclusion, these findings reveal that both accelerated extinction and impaired restoration contribute to memory impairment in aging animals (Chen, 2015).

Highlights

• Higher proportion of memory loss in aging flies.
• Memory reduction was restored in young flies, while restoration was absent in aging flies.
• PCOP specifically suppressed ARM, but not ASM.
• Extinction-induced ARM impairment was not recovered in aging flies.

Discussion
Simultaneously exposing the flies with one odor (conditioned odor) and electric shock, then another odor (unconditioned odor) without electric shock sequentially make them learn to avoid the conditioned odor. Cycles of extinction procedures, which are performed as the presentation of conditioned odor without electric shock, impair aversive olfactory memory. In a previous report, memory was reduced about 10% following 10 cycles of odor presentation. To improve extinction efficiency, this study modified the original paradigm by performing the extinction procedures between the presentation of the conditioned odor and unconditioned odor, and named this treatment PCOP (Chen, 2015).

It was found that the performance index decreased gradually with the increase of extinction cycle numbers. Furthermore, when equal or more than four cycles of PCOP was performed, the ratio of memory reduction was more than 30%, which was a more significant decrease than previous paradigm. These findings suggested that the presenting time of PCOP and the unconditioned odor affected extinction efficiency. By adjusting the sequence of 4 cycles of PCOP and the unconditioned odor, it was found that the earlier presentation of PCOP, the more significant memory extinction induced. Therefore, four cycles of PCOP before the unconditioned odor were used in all subsequent experiments (Chen, 2015).

To investigate the effect of aging on extinction, the memory index upon extinction procedures in flies at 2, 10, 20, 30 and 50 days of age was measured. It was found that the aversive olfactory memory was reduced significantly by PCOP among these flies. Strikingly, the memory reduction ratio in flies at 20, 30 and 50 days of age was statistically higher than the younger flies. These results indicated that memory extinction in aging flies was more severe than in younger flies, in accordance with the faster extinction performance in aging rats (Chen, 2015).

Several earlier reports showed that extinguished memory can be restored in the presence of an unconditioned stimulus. To test whether the extinction effect changed over time in flies, the memory 3 h post conditioning was evaluated. It was found that PCOP-induced memory reduction was spontaneously recovered within 3 h in flies at 2 days or 10 days of age. Strikingly, this memory restoration was not observed in flies at 20, 30 and 50 days of age, suggesting more severe memory deficiencies in aging flies (Chen, 2015).

It has been reported that aging specifically impaired anesthesia-sensitive memory (ASM) while leaving anesthesia-resistant memory (ARM) intact. Given these findings that aging flies exhibited higher ratios of memory extinction, it was then examined whether extinction affected ARM and ASM differently, using amnX8 and rsh1 mutant flies. It was found that amnX8 mutant flies exhibited significant memory extinction, with a reduction comparable to that in wild-type flies. Unexpectedly, little memory extinction was observed in rsh1 mutant flies. These results suggested that PCOP specifically suppressed ARM, whereas ASM was unaffected (Chen, 2015).

Radish was reported to be strongly expressed in both the mushroom body (MB) and ellipsoid body in the adult fly brain. It was found that expression of radish with c739-Gal4 in the MB α/β lobes rescued the ARM formation, and re-established PCOP-induced memory extinction. In contrast, expressing Radish in the MB α′/β′ lobes and ellipsoid body with c305a-Gal4 failed to do so. Together, these findings suggested that Radish expression in MB α/β lobes was required for memory extinction (Chen, 2015).

Bruchpilot (Brp), a ubiquitous presynaptic active zone protein, has been reported to be specifically required in the MB for ARM formation. Similar to rsh1 mutant flies, MB-specific brp-knocking down flies exhibited no significant memory extinction in the PCOP assay. Taken together, these results suggested that prolonged odor presentation specifically impaired ARM, but not ASM (Chen, 2015).

To test if the impaired ARM was restored, or whether ASM was elevated after 3 h, a 2-min cold shock 2 h after the conditioning step was introduced to examine ARM. In young wild-type flies, the PCOP group showed comparable ARM to that in the air control group, indicating that the extinction-induced impairment of ARM was restored. In contrast, amnX8 mutant flies still exhibited significant PCOP-induced reduction of ARM, and showed almost no detectable ARM in the PCOP group. Since amnX8 mutant flies were deficient in ASM, the study proposed that recovery of the suppressed ARM required the presence of ASM. Overall, these findings reveal that upon aging, memory extinction is becoming more and more severe, and once in place, this reduction cannot be restored (Chen, 2015).

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Tonoki, A. and Davis, R.L. (2015). Aging impairs protein-synthesis-dependent long-term memory in Drosophila. J Neurosci 35: 1173-1180. PubMed ID: 25609631

Abstract
Although aging is known to impair intermediate-term memory in Drosophila, its effect on protein-synthesis-dependent long-term memory (LTM) is unknown. This study shows that LTM is impaired with age, not due to functional defects in synaptic output of mushroom body (MB) neurons, but due to connectivity defects of dorsal paired medial (DPM) neurons with their postsynaptic MB neurons. GFP reconstitution across synaptic partners (GRASP) experiments revealed structural connectivity defects in aged animals of DPM neurons with MB axons in the α lobe neuropil. As a consequence, a protein-synthesis-dependent LTM trace in the α/β MB neurons failed to form. Aging thus impairs protein-synthesis-dependent LTM along with the α/β MB neuron LTM trace by lessening the connectivity of DPM and α/β MB neurons (Tonoki, 2015).

Highlights

• Aging impairs protein-synthesis-dependent LTM.
• Aging disrupts the formation of a LTM trace.
• Aging fails to alter the synaptic transmission requirements of the α/β MB neurons for LTM.
• Aging occludes DPM synaptic transmission requirement for LTM.
• Aging alters the contacts between DPM and MB neurons specifically in the α tip as revealed by GRASP.

Discussion
The data presented in this study offer several important findings about the neural circuitry and the forms of memory disrupted by aging. First, it shows that aging impairs only one of the two mechanistically distinct forms of LTM generated by spaced, aversive classical conditioning in Drosophila. LTM that is independent of protein synthesis remains unaffected by age, whereas that form of LTM requiring protein synthesis becomes impaired. Therefore, there is mechanistic specificity in the effects of aging on LTM. Although aging, in principal, could disrupt processes like protein synthesis at the molecular level leading to a LTM deficit, these results indicate that the problem is traceable to the circuitry involved in generating protein-synthesis-dependent LTM (Tonoki, 2015).

The normal synaptic transmission from DPM neurons onto follower neurons during spaced training that is required for generating LTM is lost with age. This is attributable to the reduction of synaptic contacts between DPM neuron processes and MB axons specifically in the tip of the α lobe neuropil as revealed by GRASP signals. The loss of synaptic contacts between DPM and MB neurons in this region also may explain why synaptic blockade of DPM neurons during acquisition disrupts protein-synthesis-dependent LTM in young but not old flies. Therefore, a second major finding is that neural contacts and subsequent synaptic activity between DPM and α/β MB neurons are required for generating protein-synthesis-dependent LTM, and aging impairs this process. Consistent with this model, it is found that aging blocks the formation of a calcium-based, protein-synthesis-dependent memory trace in the α/β MB neurons (Tonoki, 2015).

It has been found previously that ITM is impaired in flies of 30 d of age along with the capacity to form an ITM trace in the DPM neurons. Nevertheless, aging does not compromise the capacity to form an STM trace in the α'/β' MB neurons. Therefore, aging disrupts specific temporal forms of memory, including ITM and protein synthesis LTM, but not STM and protein-synthesis-independent LTM. It is possible that the loss of connectivity of DPM neurons with the α tip neuropil is responsible for the loss of both ITM and protein-synthesis-dependent LTM, along with their respective memory traces. Previous and this study's data indicate that STM appears to bypass the DPM neurons, whereas the reciprocal activity between DPM and MB neurons is required for ITM and LTM. Aging puts a kink in this neural system by impairing connectivity (Tonoki, 2015).

The study offers a model to explain the neural circuitry involved in protein-synthesis-dependent LTM formation and how aging impairs this form of memory. Although DPM neurons make contacts widely throughout the MB lobe neuropil with processes of many cell types, the critical interaction for LTM formation occurs in the vertical lobes of the MB through contacts onto the axons of α/β MB neurons. DPM neuron synaptic activity during spaced training, which occurs due to their stimulation by MB neurons, promotes synaptic changes in the postsynaptic α/β MB neurons and leads to the formation of memory trace in the α/β MB neurons. Aging impairs protein-synthesis-dependent LTM along with a LTM trace that normally forms in the α/β MB neurons by lessening the connectivity of DPM and α/β MB neurons. Identifying the mechanisms by which the DPM neurons lose their connectivity with only the tips of α/β MB neurons might reveal how aging impairs protein-synthesis-dependent LTM (Tonoki, 2015).

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Slack, C., Alic, N., Foley, A., Cabecinha, M., Hoddinott, M.P. and Partridge, L. (2015). The Ras-Erk-ETS-signaling pathway is a drug target for longevity. Cell 162(1):72-83. PubMed ID: 26119340

Abstract

Identifying the molecular mechanisms that underlie aging and their pharmacological manipulation are key aims for improving lifelong human health. This study has identified a critical role for Ras-Erk-ETS signaling in aging in Drosophila. Inhibition of Ras was shown to be sufficient for lifespan extension downstream of reduced insulin/IGF-1 (IIS) signaling. Moreover, direct reduction of Ras or Erk activity leads to increased lifespan. ETS transcriptional repressor Anterior open (Aop) was identified as central to lifespan extension caused by reduced IIS or Ras attenuation. Importantly, it was demonstrates that adult-onset administration of the drug trametinib, a highly specific inhibitor of Ras-Erk-ETS signaling, can extend lifespan. This discovery of the Ras-Erk-ETS pathway as a pharmacological target for animal aging, together with the high degree of evolutionary conservation of the pathway, suggests that inhibition of Ras-Erk-ETS signaling may provide an effective target for anti-aging interventions in mammals (Slack, 2015).

Discussion

The key role of IIS in determining animal lifespan has been well appreciated for more than two decades and shows strong evolutionary conservation. Alleles of genes encoding components of this pathway have also been linked to longevity in humans. Multiple studies have demonstrated the importance of the PI3K-Akt-Foxo branch of IIS, while this study has identified an equally important role for Ras-Erk-ETS signaling in IIS-dependent lifespan extension (Slack, 2015).

Downstream of chico, preventing the activation of either Ras or PI3K is sufficient to extend lifespan. Ras can interact directly with the catalytic subunit of PI3K, which is required for maximal PI3K activation during growth. Thus, inhibition of Ras could increase lifespan via inactivation of PI3K. However, several lines of evidence indicate that the Erk-ETS pathway must also, if not solely, be involved. In this study and elsewhere, it has been demonstrated that direct inhibition of the Ras-dependent kinase, Erk, or activation of the Aop transcription factor, a negative effector of the Ras-Erk pathway, is sufficient to extend lifespan. Importantly, this study shows that Ras-Erk-ETS signaling is genetically linked to chico because activation of Aop is required for lifespan extension due to chico loss of function. Furthermore, altering the ability of Chico to activate Ras or PI3K does not result in equivalent phenotypes: it has been shown that mutation of the Grb2/Drk docking site in Chico is dispensable for multiple developmental phenotypes associated with chico mutation, while disruption of the Chico-PI3K interaction is not. Overall, the observations strongly suggest that lifespan extension downstream of chicomutation involves inhibition of the Ras-Erk-ETS-signaling pathway (Slack, 2015).

A simple model integrates the role of Ras-Erk-ETS signaling with the PI3K-Akt-Foxo branch in extension of lifespan by reduced IIS. It is proposed that, downstream of Chico, the IIS pathway bifurcates into branches delineated by Erk and Akt, with inhibition of either sufficient to extend lifespan, as is activation of either responsive TF, Aop or Foxo. The two branches are not redundant, because mutation of chico or the loss of its ability to activate either branch results in the same magnitude of lifespan extension. Furthermore, Aop and Foxo are each individually required downstream of chico mutation for lifespan extension. At the same time, the effects of the two branches are not additive, as simultaneous activation of Aop and Foxo does not extend lifespan more than activation of either TF alone. Taken together, these data suggest that the two pathways re-join for transcriptional regulation, where Aop and Foxo co-operatively regulate genes required for lifespan extension. The model is corroborated by a previous finding that, in the adult gut and fat body, some 60% of genomic locations bound by Foxo overlap with regions of activated-Aop binding (Slack, 2015).

It is proposed that functional interactions of Aop and Foxo at these sites may be such that each factor is both necessary and sufficient to achieve the beneficial changes in target gene expression upon reduced IIS. It remains to be determined how promoter-based Foxo and Aop interactions produce such physiologically relevant, transcriptional changes. It is, however, curious that activation of either TF alone promotes longevity when one is known as a transcriptional activator (Foxo) and the other as a transcriptional repressor (Aop). A subset of Foxo-bound genes, albeit a minority, has been consistently observed that are transcriptionally repressed when Foxo is activated. Furthermore, the Foxo target gene myc is downregulated in larval muscle when Foxo is active under low insulin conditions, while deletion of foxo or its binding site within the myc promoter results in de-repression of myc expression in adipose of fed larvae (Teleman, 2008). Thus, on some promoters under certain conditions, Drosophila Foxo appears to act as a transcriptional repressor. Mammalian Foxo3a may also directly repress some genes. It will therefore be important to test whether the lifespan-relevant interactions between Foxo and Aop occur on promoters where Foxo acts as a repressor with Foxo possibly acting as a cofactor for Aop or vice versa (Slack, 2015).

In mediating the effects of IIS on lifespan, the Ras-Erk-ETS- and PI3K-Akt-Foxo-signaling pathways both appear to inhibit Aop/Foxo. To understand why signaling might be so wired, it is important to consider that the two pathways are also regulated by other stimuli, such as other growth factors, stress signals, and nutritional cues. The re-joining of the two branches at the transcriptional level would therefore allow for their outputs to be integrated, producing a concerted transcriptional response, a feature that is also seen in other contexts. For example, stability of the Myc transcription factor is differentially regulated in response to Erk and PI3K signals, allowing it to integrate signals from the two kinases. Transcriptional integration in response to RTK signaling also confers specificity during cell differentiation, with combinatorial effects of multiple transcriptional modulators inducing tissue-specific responses to inductive Ras signals. Similar integrated responses of lifespan could be orchestrated by transcriptional coordination of Aop and Foxo (Slack, 2015).

Direct inhibition of Ras in Drosophila can extend lifespan, suggesting that the role of Ras in aging is evolutionarily conserved. In budding yeast, deletion of RAS1 extends replicative lifespan, and deletion of RAS2 increases chronological lifespan by altering signaling through cyclic-AMP/protein kinase A (cAMP/PKA), downregulation of which is sufficient to extend both replicative and chronological lifespan. This role of cAMP/PKA in aging may be conserved in mammals, as disruption of adenylyl cyclase 5' and PKA function extend murine lifespan. However, cAMP/PKA are not generally considered mediators of Ras function in metazoa. Instead, the data suggest that signaling through Erk and the ETS TFs mediates the longevity response to Ras. Interestingly, fibroblasts isolated from long-lived mutant strains of mice and long-lived species of mammals and birds show altered dynamics of Erk phosphorylation in response to stress, further suggesting a link between Erk activity and longevity. Importantly, the ETS TFs are conserved mediators of Ras-Erk signaling in mammals. Investigation of the effects of Ras inhibition on mammalian lifespan and the role of the mammalian Aop ortholog Etv6 are now warranted (Slack, 2015).

A role for Ras-Erk-ETS signaling in lifespan offers multiple potential targets for small-molecule inhibitors that could function as anti-aging interventions. Importantly, due to the key role of this pathway in cancer, multiple such inhibitors exist or are in development (Slack, 2015).

This study has shown that trametinib, a highly specific allosteric inhibitor of the Mek kinase, prolongs Drosophila lifespan, thus validating the Ras-Erk-ETS pathway as a pharmacological target for anti-aging therapeutics. Trametinib joins a very exclusive list of FDA-approved drugs that promote longevity in animals, the most convincing other example being rapamycin (Slack, 2015).

Rapamycin not only increases lifespan in multiple organisms, including mammals, but also improves several indices of function during aging. While rapamycin can protect against tumor growth, the effects on longevity appear to be independent of cancer prevention, as rapamcyin-treated animals still develop tumors and rapamycin can increase lifespan in tumor-free species. Furthermore, increased activity of certain tumor suppressors such as lnk4a/Arf and PTEN as well as the RasGrf1 deficiency all increase lifespan independently of anti-tumor activity. The findings that trametinib can increase lifespan inDrosophila, which are mainly post-mitotic in adulthood, and that doses of trametinib that increase lifespan do not alter proliferation rates of ISCs inDrosophila suggest that the anti-aging effects of trametinib are separable from its anti-cancer activity (Slack, 2015).

Finally, due to the high degree of evolutionary conservation in the Ras-Erk-ETS pathway, this study suggests the intriguing possibility that pharmacological inhibition of Ras-Erk-ETS may also increase lifespan in mammal (Slack, 2015).

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Yamamoto, R., Bai, H., Dolezal, A.G., Amdam, G. and Tatar, M. (2013). Juvenile hormone regulation of Drosophila aging. BMC Biol 11: 85. PubMed ID: 23866071

Abstract
Juvenile hormone (JH) has been demonstrated to control adult lifespan in a number of non-model insects where surgical removal of the corpora allata eliminates the hormone's source. In contrast, little is known about how juvenile hormone affects adult Drosophila melanogaster. Previous work suggests that insulin signaling may modulate Drosophila aging in part through its impact on juvenile hormone titer, but no data yet address whether reduction of juvenile hormone is sufficient to control Drosophila life span. This study adapts a genetic approach to knock out the corpora allata in adult Drosophila melanogaster and characterize adult life history phenotypes produced by reduction of juvenile hormone. With this system, potential explanations for how juvenile hormone modulates aging were tested. A tissue specific driver inducing an inhibitor of a protein phosphatase was used to ablate the corpora allata while permitting normal development of adult flies. Corpora allata knockout adults had greatly reduced fecundity, inhibited oogenesis, impaired adult fat body development and extended lifespan. Treating these adults with the juvenile hormone analog methoprene restored all traits toward wildtype. Knockout females remained relatively long-lived even when crossed into a genotype that blocked all egg production. Dietary restriction further extended the lifespan of knockout females. In an analysis of expression profiles of knockout females in fertile and sterile backgrounds, about 100 genes changed in response to loss of juvenile hormone independent of reproductive state. Reduced juvenile hormone alone was sufficient to extend the lifespan of Drosophila melanogaster. Reduced juvenile hormone limited reproduction by inhibiting the production of yolked eggs, and this might arise because juvenile hormone is required for the post-eclosion development of the vitellogenin-producing adult fat body. These data do not support a mechanism for juvenile hormone control of longevity simply based on reducing the physiological costs of egg production. Nor does the longevity benefit appear to function through mechanisms by which dietary restriction extends longevity. The study identifies transcripts that change in response to juvenile hormone independent of reproductive state and suggests these represent somatically expressed genes that could modulate how juvenile hormone controls persistence and longevity (Yamamoto, 2013).

Highlights

• Corpora allata knockout (CAKO) reduces fecundity and impedes adult fat body development.
• CAKO extends lifespan independent of egg production.
• CAKO independent of reproduction modulates somatically related genes.

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Whitaker, R., Faulkner, S. Miyokawa, R. Burhenn, L., Henriksen, M., Wood, J.G. and Helfand, S.L. (2013). Increased expression of Drosophila Sir2 extends life span in a dose-dependent manner. Aging (Albany NY). 5: 682-691. PubMed ID: 24036492 

Abstract
Sir2, a member of the sirtuin family of protein acylases, deacetylates lysine residues within many proteins and is associated with lifespan extension in a variety of model organisms. Recent studies have questioned the positive effects of Sir2 on lifespan in Drosophila. Several studies have shown that increased expression of the Drosophila Sir2 homolog (dSir2) extends life span while other studies have reported no effect on life span or suggested that increased dSir2 expression was cytotoxic. To attempt to reconcile the differences in these observed effects of dSir2 on Drosophila life span, this study hypothesized that a critical level of dSir2 may be necessary to mediate life span extension. Using approaches that allowed titration of dSir2 expression, a strong dose-dependent effect of dSir2 on life span was described. Using the two transgenic dSir2 lines that were reported not to extend life span, significant life span extension when dSir2 expression was induced between 2 and 5-fold was shown. However, higher levels decreased life span and could induce cellular toxicity, manifested by increased expression of the JNK-signaling molecule Puc phosphatase and induction of dnaJ-H. These results help to resolve the apparently conflicting reports by demonstrating that the effects of increased dSir2 expression on life span in Drosophila are dependent upon dSir2 dosage (Whitaker, 2013).

Highlights

• dSir2 expression from UAS-dSir2 transgenes can increase or decrease life span.
• Moderate increases in dSir2 expression from UAS-dSir2 transgenes extend life span.
• dSir2-mediated life span extension is dose-dependent.
• High levels of dSir2 expressed from transgenes induce cellular toxicity and dnaJ-H expression.

Discussion
This study shows that in Drosophila, increased expression of dSir2 extends life span in a dose-dependent manner, thereby resolving apparent controversies in the field about the role of sirtuins in fly aging. By measuring life span while directly titrating the increase in dSir2 expression through use of a series of new and available UAS-dSir2 transgenes, and by determining the level of dSir2 expression under conditions for previously published life span studies, it is shown that when dSir2 expression is increased to moderate levels (approximately 2-5 fold increased over normal), life span is consistently extended. Expression below this range (less than 2-fold increase), or slightly above it (between 5-10 fold increase) inconsistently extends life span, while higher levels of expression are detrimental to life span and can induce JNK signaling and dnaJ-H expression (Whitaker, 2013).

The study notes that induction of dSir2 from its native locus using the dSir2EP2300 insertion allele extends life span more reliably and to a greater extent than increased expression from the dSir2 transgenes at other genomic locations. This may be due to the lower level of dSir2 induction seen with dSir2EP2300, but it could also be due to the presence of favorable endogenous regulatory elements that are maintained when dSir2 is expressed from its native genomic location (Whitaker, 2013).

In a previous study, an increase in dnaJ-H was observed when dSir2EP2300 was used to increase expression of dSir2 in the eye using a GMR-Gal4 driver, indicating that Gal4 induction of dSir2EP2300 may increase expression of both dnaJ-H and dSir2 due to their overlapping genetic locus and that increased co-expression may account for the observed life span extension. However, it has subsequently been shown that dnaJ-H is not induced when dSir2EP2300 is used to increase expression of dSir2 in life span-extending conditions in adult neurons or in adult fat body cells. Furthermore, this study found that dnaJ-H is induced when dSir2 or GFP is highly expressed from transgenic lines that are not located near the native dSir2 / dnaJ-H genomic locus. The dSir2 expression conditions that exhibited an increase in dnaJ-H levels also showed elevated transcripts of puc phosphatase, a target of JNK signaling. Taken together, these results show that increased expression of dSir2 can induce JNK signaling/puc phosphatase as previously reported, but only when dSir2 is expressed at high levels (Whitaker, 2013).

This study's observation that high levels of dSir2 over expression is toxic fits with previously published results showing that expression of dSir2 from a transgene induced lethality and activated JNK signaling. This finding that high levels of dSir2 expression can lead to cytotoxicity is perhaps not surprising given the many known interacting partners of dSir2, including proteins central to metabolism (FOXO), mitochondrial biogenesis (PGC-1α), and genomic defense (p53, Ku70) (Whitaker, 2013).

The study concludes that increasing dSir2 expression in Drosophila can extend life span, but cautions that experiments testing the overexpression of dSir2 should ensure that dSir2 levels are increased to a sufficient extent to induce a positive effect on life span, but not high enough to induce cytotoxicity. The conflicting reports in the literature over whether increased dSir2 expression extends life span are resolved when the dosage of dSir2 is taken into consideration (Whitaker, 2013).

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Klichko, V.I., Chow, E.S., Kotwica-Rolinska, J., Orr, W.C., Giebultowicz, J.M. and Radyuk, S.N. (2015). Aging alters circadian regulation of redox in Drosophila. Front Genet 6: 83. PubMed ID: 25806044 

Abstract
Circadian coordination of metabolism, physiology, and neural functions contributes to healthy aging and disease prevention. Clock genes govern the daily rhythmic expression of target genes whose activities underlie such broad physiological parameters as maintenance of redox homeostasis. Previously, it was reported that glutathione (GSH) biosynthesis is controlled by the circadian system via effects of the clock genes on expression of the catalytic (Gclc) and modulatory (Gclm) subunits comprising the glutamate cysteine ligase (GCL) holoenzyme. The objective of this study was to determine whether and how aging, which leads to weakened circadian oscillations, affects the daily profiles of redox-active biomolecules. It was found that fly aging is associated with altered profiles of Gclc and Gclm expression at both the mRNA and protein levels. Analysis of free aminothiols and GCL activity revealed that aging abolished daily oscillations in GSH levels and altered the activity of glutathione biosynthetic pathways. Unlike GSH, its precursors and products of catabolism, methionine, cysteine and cysteinyl-glycine, were not rhythmic in young or old flies, while rhythms of the glutathione oxidation product, GSSG, were detectable. The study concludes that the temporal regulation of GSH biosynthesis is altered in the aging organism and that age-related loss of circadian modulation of pathways involved in glutathione production is likely to impair temporal redox homeostasis (Klichko, 2015).

Highlights

• Age-related changes in GCLc and GCLm profiles.
• Circadian characteristics of free aminothiols determined in heads of young and old flies.

Discussion
This study investigated a role for the circadian system in regulating redox in the context of organismal aging. Cellular redox homeostasis largely relies on the redox-active compound, glutathione, which is present at concentrations many fold higher compared to concentrations of other redox-active molecules. It was previously shown that the circadian clocks modulate the de novo synthesis of GSH via transcriptional control of GCLc and GCLm, the subunits that comprise the GCL holoenzyme. This study broadened the investigation of the relationship between clock and redox to determine the levels of other redox-active molecules, involved in glutathione synthesis and metabolism. It investigated around the clock expression profiles of both cysteine and its precursor methionine (Klichko, 2015).

As neither cysteine nor methionine exhibited evidence for diurnal rhythms in either young or old flies, it appears that the contribution of the trans-sulfuration pathway to glutathione homeostasis is not regulated by circadian clocks. Consistent with these findings, methionine was found to be arrhythmic in the study of the human metabolome of blood plasma and saliva although the analyses were performed with healthy but older (57–61 years) males, where age-dependent effects might have influenced the oscillations. In contrast, mouse hepatic metabolome and transcriptome studies revealed rhythmicity in metabolic sub-pathways, where oscillations in glutathione were ascertained by oscillations in its precursors, cysteine and methionine, albeit with a lower amplitude for the latter (Klichko, 2015).

Analysis of the Drosophila heads revealed no cycling in the concentrations of Cys-Gly, consistent with the arrhythmic behavior of cysteine and methionine. Given that Cys-Gly also serves as a signature of glutathione degradation, interpretation of these results are somewhat tentative. Nevertheless, these results revealed no rhythmicity in cysteine, methionine and Cys-Gly, and suggest that, at least in flies, the pathways responsible for the supply of sulfur-containing precursors for glutathione synthesis are not regulated by the circadian clocks. It should be noted that the mammalian liver is a homogenous tissue with a strong food-entrained clock mechanism, while fly heads are enriched in nervous tissues with clocks entrained by light-dark cycles. Moreover recent analysis of the circadian transcriptome shows that liver possesses the highest number of rhythmic genes, while brain has the lowest (Klichko, 2015).

Another important finding of this study is that the diurnal fluctuations in GSH levels were not followed by similar changes in the products of its degradation (Cys-Gly) and oxidation (GSSG). While Cys-Gly was completely arrhythmic, changes in GSSG profile did not mirror those observed for GSH. Even though both shared the same slow drop-off from ZT0 to ZT8 as well as the ZT8 trough, their peaks were quite distinct (ZT12 for GSSG and ZT20 for GSH). Also in old flies, a certain degree of rhythmicity is maintained for GSSG in contrast to the absence of any diurnal GSH patterns (Klichko, 2015).

An additional important finding of this study is that the rhythms in glutathione levels observed in young flies were lost in old flies, presumably due to the loss of diurnal fluctuations of GCLc, GCLm as well as GCL activity, in response to the weakening of the circadian clocks. In contrast, GSSG rhythms were largely preserved in older flies, suggesting that the daily changes in glutathione disulfide levels are supported by enzymatic reactions that are not under clock control (Klichko, 2015).

Despite loss of circadian regulation, average daily levels of GSH remained unchanged during aging, while the levels of GSSG were slightly higher, mainly due to lesser drop in the early morning. In Drosophila, it has been established that whole body GSH levels were either relatively constant or slightly decreased during aging while GSSG rose 2–3 fold. Similar age-related changes were documented in different mammalian tissues with the most significant reduction in GSH and accumulation of GSSG in the brain, indicative of a more pro-oxidative cellular environment. As such changes in GSH and GSSG were frequently associated with increases in enzyme activities related to GSH usage, the relatively steady glutathione concentrations observed in the heads of old flies could point to less efficient GSH utilization (Klichko, 2015).

The rather unexpected finding of this study is that the expression of Gclc at both mRNA and protein levels significantly increased in the heads of old flies, and this increase was associated with about 25% higher average daily GCL activity. Despite this increase, the average daily levels of GSH remained unchanged suggesting a loss in GCL catalytic efficiency or an age-related increase in GSH utilization. One possible scenario is that the efficiency of GSH synthesis can be induced by oxidative stress, in part through the well-documented increase in H2O2 signaling that accompanies aging. For instance, post-translational control of γ-glutamylcysteine (γ-Glu-Cys) synthesis is influenced by oxidative stress, which can dramatically affect formation of GCL holoenzyme and its stabilization (Klichko, 2015).

Consistent with induction of GCL by stress, it was previously reported that per-null mutants with disrupted clock displayed arrhythmic as well as elevated GCL activity, which was also reflected in arrhythmic and elevated ROS levels relative to the control. It should be noted that previous studies comparing GCL activity and GSH levels in young and old rats showed a decrease of both parameters in liver, while in aging brain and heart GSH decreased but GCL activity remained unchanged, pointing again to catalytic deficiency of the enzyme (Klichko, 2015).

Other aminothiols that did not show cycling in young flies also remained arrhythmic in old flies, but displayed changes in their steady state levels. Consistent with previous reports, the amounts of Cys-Gly were ∼50% higher in older flies. Cys-Gly, derived from the breakdown of glutathione, is required for GSH synthesis as a precursor of cysteine, but at the same time it is also a prooxidant generated during the catabolism of glutathione. The requirement of Cys-Gly for GSH synthesis justifies its increase with age, as the tissues require an increased supply of precursors for GSH biosynthesis in older flies. However, an increase in cysteine levels during aging was not observed. A more plausible explanation is that the increase in Cys-Gly is indicative of an increase in oxidative stress and GSH degradation. In agreement with this view, the average daily levels of methionine were about 35% lower in old flies suggesting the likelihood of an increase in oxidation of methionine to methionine sulfoxide by ROS rather than an increase in methionine consumption for cysteine biosynthesis. Together, these changes indicate a shift in redox homeostasis in the heads of older flies, consistent with the earlier reports in whole flies. Similar alterations in the redox components were also indicative of heightened oxidative stress in pathologies like systemic lupus erythematosus (Klichko, 2015).

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Blake, M.R., Holbrook, S.D., Kotwica-Rolinska, J., Chow, E.S., Kretzschmar, D. and Giebultowicz, J.M. (2015). Manipulations of amyloid precursor protein cleavage disrupt the circadian clock in aging Drosophila. Neurobiol Dis 77: 117-126. PubMed ID: 25766673

Abstract
Alzheimer's disease (AD) is a neurodegenerative disease characterized by severe cognitive deterioration. While causes of AD pathology are debated, a large body of evidence suggests that increased cleavage of Amyloid Precursor Protein (APP) producing the neurotoxic Amyloid-β (Aβ) peptide plays a fundamental role in AD pathogenesis. One of the detrimental behavioral symptoms commonly associated with AD is the fragmentation of sleep-activity cycles with increased nighttime activity and daytime naps in humans. Sleep-activity cycles, as well as physiological and cellular rhythms, which may be important for neuronal homeostasis, are generated by a molecular system known as the circadian clock. Links between AD and the circadian system are increasingly evident but not well understood. This study examined whether genetic manipulations of APP-like (APPL) protein cleavage in Drosophila melanogaster affect rest-activity rhythms and core circadian clock function in this model organism. It was shown that the increased β-cleavage of endogenous APPL by the β-secretase (dBACE) severely disrupts circadian behavior and leads to reduced expression of clock protein PER in central clock neurons of aging flies. The study's data suggest that behavioral rhythm disruption is not a product of APPL-derived Aβ production but rather may be caused by a mechanism common to both α and β-cleavage pathways. Specifically, it was shown that increased production of the endogenous Drosophila Amyloid Intracellular Domain (dAICD) causes disruption of circadian rest-activity rhythms, while flies overexpressing endogenous APPL maintain stronger circadian rhythms during aging. In summary, this study offers a novel entry point toward understanding the mechanism of circadian rhythm disruption in Alzheimer's disease (Blake, 2015).

Highlights

• Over-expression of dBACE in clock cells accelerates aging phenotypes and disrupts rest-activity rhythms.
• Neuronal over-expression of dBACE disrupts behavioral rest-activity rhythms.
• Over-expression of dBACE dampens the cycling of PER in central pacemaker neurons.
• Rest-activity rhythms are disrupted by KUZ over-expression.
• Expression of AICD disrupts rest-activity rhythms.
• dAICD is capable of entering the nucleus, but is not toxic to central pacemaker neurons.

Discussion
Loss of rest-activity rhythms is a well-established early symptom of AD in humans. Because disruption of circadian rhythms is detrimental to neuronal homeostasis, it is important to understand relationships between AD and circadian rhythms at the cellular and molecular levels. To address this question, this study examined how manipulations of the fly ortholog of APP and its cleaving enzymes affect endogenous rest-activity rhythms and clock mechanism in Drosophila. Over-expression of dBACE was found to disrupt behavioral rest-activity rhythms, and this effect is most severe in aged flies suggesting an age-dependent mechanism. Furthermore, dBACE expression resulted in dampened oscillation of the core clock protein PER in central pacemaker neurons, which are master regulators of rest activity rhythms. Significantly reduced PER levels are observed in the sLNv and lLNv neurons of age 50d flies expressing dBACE in all clock cells (including glia), all neurons, or only in PDF-positive sLNv and lLNv neurons. These data suggest that manipulation of APP-cleavage by dBACE over-expression directly affects the oscillation of PER protein in central pacemaker neurons in a cell-autonomous manner. Since a functional clock mechanism in sLNv is necessary and sufficient to maintain free running activity rhythms, reduced oscillations of PER in these neurons could be responsible for the loss of activity rhythms in age 50d flies. Importantly, the decline in PER levels occurrs only in flies with manipulated dBACE, not in old control flies. This is in agreement with earlier findings that aging does not dampen PER oscillations in pacemaker neurons of wild type flies, while it reduces clock oscillations in peripheral clocks  (Blake, 2015).

While this study reports that the loss of behavioral rhythms after manipulation of dBACE is associated with reduced expression of clock genes in the central pacemaker, other recent work shows that expression of human Aβ peptides leads to disruption of rest activity rhythms without interfering with PER oscillations in the central pacemaker. Even strongly neurotoxic Aβ peptides, such as Aβ42 arctic, do not cause rhythm disruption when expressed in central pacemaker neurons; rather, pan-neuronal expression is required. The fact that even the most neurotoxic Aβ peptides are not capable of dampening PER oscillation in pacemaker neurons suggests that Aβ production does not affect clock oscillations and that it is not Aβ production that causes the phenotype observed in this study upon over-expression of dBACE. This was confirmed by expression of KUZ, whose activity does not increase dAβ production; however, it also leads to disruption of rest-activity rhythms. Similar rhythm disruption by dBACE and KUZ suggests that an excess cleavage product of both pathways might be responsible for the disruption. Like in the mammalian APP cleavage pathway, in Drosophila cleavage of APPL by KUZ or dBACE results in a C-terminal fragment (CTF) that is subsequently cleaved by the ϒ-secretase resulting in the production of dAICD. Indeed, it was shown that expression of dAICD results in an age-dependent decline in rhythmic locomotor activity. As with dBACE and KUZ expression, dAICD expression causes weakening or complete loss of behavioral rhythms while age-matched control flies remain highly rhythmic. In this context, it is worth noting that α-secretase activators are considered for clinical trials to reduce Aβ production in AD patients. However, according to results in this study, this could lead to disruptions of circadian rhythms and sleep patterns thus negatively impacting the lives of patients and their caretakers (Blake, 2015).

This study's data suggest that increased dAICD may be the proximal cause of decay in rest-activity rhythms. The role of AICD in AD is increasingly evident but poorly understood. AICD is able to enter the nucleus and has been implicated in transcriptional regulation that may affect cell death, neurite outgrowth and neuronal excitability. Interestingly, transgenic mice expressing AICD have increased activity of GSK-3, which in flies affects the circadian clock. Over-expression of GSK-3 in Drosophila leads to altered circadian behavior by hyper-phosphorylation of TIMELESS (TIM), a key circadian protein which forms dimers with PER that enter the nucleus and regulate the clock mechanism. Of further interest, increased GSK-3 activity has been implicated in AD, and in Drosophila, increased GSK-3 activity mediates the toxicity of Aβ peptides (Blake, 2015).

Cleavage of APPL likely results in a significant decline in intact APPL, and this could be detrimental as APPL has neuroprotective effects. It was also recently shown that loss of full-length APPL induces cognitive deficits in memory. This study reports that flies over-expressing full-length APPL in central pacemaker neurons maintain stronger behavioral rest-activity rhythms during aging than control flies; however this effect is not observed when APPL is expressed pan-neuronally. This could be caused by negative effects of APPL when expressed in other unspecified neurons, or could be related to driver strength. Overall, the study suggests that the loss of full-length APPL might negatively affect circadian behavior by way of the central pacemaker neurons (Blake, 2015).

Over-expression of dAICD induces a severe phenotype, disrupting rest-activity rhythms as early as age 5d when expressed in central pacemaker neurons and by age 35d with pan-neuronal expression. Taken together these results suggest that while loss of full-length APPL by over-expression of its secretases might negatively impact circadian behavior, the cleavage product dAICD induces the most severe behavioral rest-activity disruption. Interestingly, the observed effect is not likely a product of neurodegeneration as it was previously shown that dAICD has no effect on neurodegeneration, and this study shows that the pacemaker cells appear intact in pdf > dAICD flies. In addition, it was shown that dAICD, like the vertebrate AICD, can be found in the nucleus. Therefore, this study suggests that dAICD may directly or indirectly affect the expression of clock genes. This offers a novel entry point toward understanding the mechanism of circadian rhythm disruption in Alzheimer's disease (Blake, 2015).

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Wang, L., Ryoo, H.D., Qi, Y. and Jasper, H. (2015). PERK limits Drosophila lifespan by promoting intestinal stem cell proliferation in response to ER stress. PLoS Genet 11: e1005220. PubMed ID: 25945494 

Abstract
Intestinal homeostasis requires precise control of intestinal stem cell (ISC) proliferation. In Drosophila, this control declines with age largely due to chronic activation of stress signaling and associated chronic inflammatory conditions. An important contributor to this condition is the age-associated increase in endoplasmic reticulum (ER) stress. This study shows that the PKR-like ER kinase (PERK) integrates both cell-autonomous and non-autonomous ER stress stimuli to induce ISC proliferation. In addition to responding to cell-intrinsic ER stress, PERK is also specifically activated in ISCs by JAK/Stat signaling in response to ER stress in neighboring cells. The activation of PERK is required for homeostatic regeneration, as well as for acute regenerative responses, yet the chronic engagement of this response becomes deleterious in aging flies. Accordingly, knocking down PERK in ISCs is sufficient to promote intestinal homeostasis and extend lifespan. These studies highlight the significance of the PERK branch of the unfolded protein response of the ER (UPRER) in intestinal homeostasis and provide a viable strategy to improve organismal health- and lifespan (Wang, 2015).

Highlights

• ROS-independent induction of ISC proliferation by ER stress.
• Non-autonomous activation of PERK in ISCs by JAK/Stat signaling.
• Knockdown of PERK in ISCs extends lifespan.

Discussion
This study identifies the PERK branch of the UPRER as a central node in the control of proliferative homeostasis in the intestinal epithelium, and establishes a previously unrecognized role for PERK in promoting regenerative responses to both tissue-wide and cell-autonomous ER stress. This critical function of PERK in tissue regeneration, however, also results in the aging-associated loss of proliferative homeostasis in the intestinal epithelium, limiting organismal lifespan. The unique and specific increase in eIF2α phosphorylation in ISCs in stressed and aging conditions suggests a differential activation of the PERK-eIF2α branch of the UPRER between ISCs and their daughter cells. It remains unclear whether this differential regulation reflects different strategies in combating ER stress between these cell populations, and additional studies are necessary to address this interesting question (Wang, 2015).

Drosophila ISCs, as many other stem cell types, are controlled extensively by redox signals. Previous work, as well as the results shown in this study, suggests that ER-induced oxidative stress plays a central role in the control of ISC proliferation after a proteostatic challenge. The results support the notion that ER-induced ROS is a consequence of the PDI/Ero1L system, as has been proposed in mammalian cells. However, Ero1L, as a thiol oxidase, may also affect the proper folding and maturation of Notch directly (as described previously), inhibiting ISC differentiation, and resulting in stem cell tumors. The phenotype of Ero1L-deficient ISC lineages supports a role for Ero1L in Notch signaling (tumors with elevated numbers of Dl+ cells). At the same time, this study's results also support a role for Ero1L in limiting ISC proliferation directly through the UPRER (and independently of Notch signaling or oxidative signals), as loss of Ero1L induces PERK activity without promoting ROS production in these cells. PERK itself is required for the induction of cell cycle and DNA replication genes in ISCs responding to TM treatment, yet it also induces antioxidant genes under these conditions, suggesting complex crosstalk between PERK-mediated control of mitotic activity of ISCs and the control of redox homeostasis in these cells (Wang, 2015).

The fact that loss of Ero1L activates PERK while not inducing Xbp1 in ISCs suggests selective activation mechanisms for these two branches of the UPRER. The study proposes that this selectivity is associated with the production of ROS and that ER protein stress activates the Xbp1 branch when associated with a ROS signal, while PERK can be activated by unfolded proteins independently of ROS production. Further studies are needed to dissect the relative contribution of ROS production, PERK activation and Notch perturbation in the control of ISC proliferation in Ero1L loss of function conditions (Wang, 2015).

This study highlights the interaction between cell-autonomous and non-autonomous events in the ER stress response of ISCs and supports the notion that improving proteostasis by boosting ER folding capacity in stem cells improves long-term tissue homeostasis and can impact lifespan. The regulation of PERK activity in ISCs by the JAK/Stat signaling pathway provides a tentative mechanism for the interaction between IECs experiencing ER stress and ISCs: the study proposes that JNK-mediated release of JAK/Stat ligands from stressed IECs results in JAK/Stat mediated activation of PERK in ISCs, and that this activation is required for the proliferative response of ISCs to epithelial dysfunction. The activation of JAK/Stat signaling in the intestinal epithelium of animals in which Xbp1 is knocked down in ECs, the requirement for JNK activation and Upd expression in ECs for ISC proliferation in response to stress, and the requirement for Stat (and Hop and Dome) in ISCs for the activation of eIF2α phosphorylation and stress-induced ISC proliferation, support this model. The mechanisms by which Stat mediates activation of PERK remain unclear, and will be interesting topics of further study (Wang, 2015).

Studies in worms have established the UPRER as a critical determinant of longevity, and Xbp1 extends lifespan by improving ER stress resistance. This study's data further support the notion that regulating ER stress response pathways is critical to increase health- and lifespan. Here, chronic PERK activation can be considered a downstream readout of the buildup of proteotoxic stress in the intestinal epithelium during aging, which then perturbs proliferative homeostasis by continuously providing pro-mitotic signals to ISCs. Knocking down PERK in ISCs limits these pro-mitotic signals, improving homeostasis and barrier function, and extending lifespan. Lifespan is generally extended when ISC proliferation is limited in older flies, but not when it is completely inhibited. Accordingly, lifespan extension is observed when PERK is knocked down using an RNAi approach that does not completely ablate PERK function (Wang, 2015).

ER stress has been documented as tightly associated with intestinal inflammation and the development of IBDs in mice and humans. Genetic variants in Xbp1 are associated with higher susceptibility to IBD and a recent study indicates that Xbp1 can act as a tumor suppressor in the intestinal epithelium, by limiting intestinal proliferative responses and tumor development through the control of local inflammation. In this context, the specific role of PERK in the control of ISC proliferation in the fly gut is consistent with the function of PERK in the intestinal epithelium of mice, where activation of PERK can promote transition of ISCs into the transient amplifying cell population. While the Drosophila midgut epithelium does not contain a transit amplifying cell population, this study's data suggest that a role for PERK in the proliferative response of the ISC lineage to ER stress is conserved (Wang, 2015).

Due to the importance of the UPRER in the maintenance of tissue homeostasis in aging organisms, therapies targeting the UPRER are promising strategies to delay the aging process. Accordingly, pharmaceuticals that can limit ER stress (such as Tauroursodeoxycholic acid, TUDCA and 4-phenylbutyrate, PBA) have had therapeutic success in various human disorders. Interestingly, flies fed PBA show increased lifespan, yet the effects of PBA on intestinal homeostasis have not yet been explored. Based on this work, it is likely that further characterization of the effects of UPRER-targeting drugs on ISC function and intestinal homeostasis will help develop clinically relevant strategies to limit human aging and extend healthspan (Wang, 2015).

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Kao, S.H., Tseng, C.Y., Wan, C.L., Su, Y.H., Hsieh, C.C., Pi, H. and Hsu, H.J. (2015). Aging and insulin signaling differentially control normal and tumorous germline stem cells. Aging Cell 14: 25-34. PubMed ID: 25470527

Abstract
Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. This study reports that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age-dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, it is shown that GSCs from mated and unmated females behave similarly, indicating that female GSC-male communication does not affect GSCs with age. These results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging (Kao, 2015).

Highlights

• GSC number is decreased with age, regardless of mating history.
• The Fucci Cdt1 probe is not a valid G1 marker for GSCs, as it is present throughout the cell cycle.
• GSCs exhibit an extremely short G1 phase.
• Aging delays S phase progression in GSCs.
• DNA damage accumulates in GSCs with age.
• Aging induces tumor-like GSCs and enlarged and mislocated niche cells in the ovary.
• Insulin signaling and GSC cell cycle progression contribute to GSC maintenance with age.
• Interactions with a normal niche are not required for the age-induced decline of GSC proliferation.
• FACS-based DNA histograms from GSCs exhibit an inverted pattern to those of other cell types.
• Tumor GSCs respond to aging in a similar manner to normal GSCs.

Discussion
Although aging results in a decline in stem cell proliferation, relatively few studies have addressed how stem cell cycle progression is altered by aging. DNA damage is mainly induced by by-products of cellular metabolism, such as reactive oxygen species (ROS) and environmentally induced lesions upon irradiation. Accumulation of irreversible genomic DNA damage has been implicated as a prominent cause of aging, both at the organismal and at the cellular levels. Cells respond to DNA damage by activating checkpoint pathways, which delay cell cycle progression and allow for repair of the defects. This study observed that aged GSCs exhibit accumulation of DNA damage and a prolonged S phase, suggesting that the former may be responsible for the latter in GSCs during aging (Kao, 2015).

DNA breaks result in activation of ATM/ATR kinases (ataxia-telangiectasia mutated and Rad3 related), which phosphorylate a variant of histone H2A (H2AX); this histone variant is a critical factor in facilitating the assembly of specific DNA-repair complexes on damaged DNA. ATM/ATR kinase-mediated signaling is part of the intra-S phase checkpoint pathway, and its activation is often associated with a delay in S phase progression. However, ATR heterozygous mutant (mei-41D3/+) GSCs still exhibited a similar degree of S phase delay compared to wild-type, suggesting that ATR may be dispensable for age-induced S phase delay, although it is possible that disruption of one copy of ATR may not be sufficient to block the intra-S check point pathway (Kao, 2015).

Surprisingly, it was observed that there was a 65% increase of aged tufeatm−8/+ GSCs in S phase (1.98-fold increase relative to young tufeatm−8/+ GSCs), as compared to its sibling controls at the same age (1.33-fold increase relative to young control GSCs). Coincidently, a recent publication on Drosophila reported that ATM functions in DNA damage repair and exerts negative feedback control over the level of programmed double strand breaks (DSBs) during meiosis, and thus the number of H2AX foci (a marker of DNA damage) is dramatically increased in tufeatm−8 mutant germ cells. This study speculates that tufeatm−8/+ GSCs may induce more DNA damage via feedback regulation, thereby causing more severe S phase delay. However, in mice, Atm−/− undifferentiated spermatogonia are not maintained in the testis due to DNA damage-induced cell cycle G1 arrest, suggesting that ATM may function in the G1 phase in response to DNA damage. Nevertheless, it remains to be elucidated whether ATM mediates different cell cycle regulators in different cell contexts or in response to different types of stress-induced DNA damage (Kao, 2015).

With age, cells may accumulate DNA mutations that allow them to escape normal regulatory processes and become tumor cells. Although tumorigenesis is harmful to health in the long term, it may also serve as a survival and protective mechanism when the body is highly threatened. While the germarium normally houses differentiating 8- or 16-germ cell cysts interconnected with branched fusomes, this study found that the middle portion of the aged germarium was occupied by tumor-like GSCs, which express pMad (a Dpp signaling effector) and possess rounded fusomes. This result recalls an earlier report that forced stemness Dpp signaling causes differentiating germ cell cysts to revert into functional stem cells in Drosophila ovaries, through the induction of ring canal closure and fusome scission (Kao, 2015).

It has also been reported that aged human epidermal cells can dedifferentiate into stem cell-like cells via Wnt/β-catenin signaling, and injury can drive the dedifferentiation of epidermal cells via the β-integrin-mediated signaling pathway; these findings suggest that dedifferentiation is a process by which organisms address aging or tissue damage. Given that GSCs play a fundamental role in producing the next generation, this study suspects that these tumor-like GSCs may be derived from germ cell cysts through a dedifferentiation process triggered by aging; however, they cannot rule out the possibility that these tumor-like GSCs are derived from the transformation of normal GSCs (Kao, 2015).

In invertebrates, including C. elegans and Drosophila, mating is detrimental to the lifespan of females, to increase progeny production. In Drosophila, mating females die earlier than unmated females, and sex peptides, produced from the male accessory gland, may be responsible for this effect. In C. elegans, females shrink and die after mating, and this is partially due to the stimulation of GSC proliferation by sperm. This study, however, did not observe differences in GSC proliferation rates between mated and unmated females at any age, suggesting that the promotion of GSC proliferation by mating may be specific to C. elegans. In addition, it is also found that sex peptides do not affect GSCs, at least at the level of proliferation. Moreover, similar rates of aging-induced GSC loss in mated and unmated females are observed, suggesting that mating does not affect the physiological status of GSCs (Kao, 2015).

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Li, Y., Hassinger, L., Thomson, T., Ding, B., Ashley, J., Hassinger, W. and Budnik, V. (2016). Lamin mutations accelerate aging via defective export of mitochondrial mRNAs through nuclear envelope budding. Curr Biol [Epub ahead of print]. PubMed ID: 27451905

Abstract

Defective RNA metabolism and transport are implicated in aging and degeneration, but the underlying mechanisms remain poorly understood. A prevalent feature of aging is mitochondrial deterioration. This study links a novel mechanism for RNA export through nuclear envelope (NE) budding that requires A-type lamin, an inner nuclear membrane-associated protein, to accelerated aging observed in Drosophila LaminC (LamC) mutations. These LamC mutations were modeled after A-lamin (LMNA) mutations causing progeroid syndromes (PSs) in humans. Mitochondrial assembly regulatory factor (Marf), a mitochondrial fusion factor (mitofusin) as well as other transcripts required for mitochondrial integrity and function, were identified in a screen for RNAs that exit the nucleus through NE budding. PS-modeled LamC mutations induced premature aging in adult flight muscles, including decreased levels of specific mitochondrial protein transcripts (RNA) and progressive mitochondrial degradation. PS-modeled LamC mutations also induced the accelerated appearance of other phenotypes associated with aging, including a progressive accumulation of polyubiquitin aggregates and myofibril disorganization. Consistent with these observations, the mutants had progressive jumping and flight defects. Downregulating marf alone induced the above aging defects. Nevertheless, restoring marf was insufficient for rescuing the aging phenotypes in PS-modeled LamC mutations, as other mitochondrial RNAs are affected by inhibition of NE budding. Analysis of NE budding in dominant and recessive PS-modeled LamC mutations suggests a mechanism by which abnormal lamina organization prevents the egress of these RNAs via NE budding. These studies connect defects in RNA export through NE budding to progressive loss of mitochondrial integrity and premature aging (Li, 2016).

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Niccoli, T., Cabecinha, M., Tillmann, A., Kerr, F., Wong, C.T., Cardenes, D., Vincent, A.J., Bettedi, L., Li, L., Grönke, S. Dols, J. and Partridge, L. (2016). Increased glucose transport into neurons rescues Aβ toxicity in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 27524482

Abstract

Glucose hypometabolism is a prominent feature of the brains of patients with Alzheimer's disease (AD). Disease progression is associated with a reduction in glucose transporters in both neurons and endothelial cells of the blood-brain barrier. However, whether increasing glucose transport into either of these cell types offers therapeutic potential remains unknown. Using an adult-onset Drosophila model of Aβ (amyloid beta) toxicity, this study shows that genetic overexpression of a glucose transporter specifically in neurons, rescues lifespan, behavioral phenotypes, and neuronal morphology. This amelioration of Aβ toxicity is associated with a reduction in the protein levels of the unfolded protein response (UPR) negative master regulator Grp78 and an increase in the UPR. Genetic downregulation of Grp78 activity also protects against Aβ toxicity, confirming a causal effect of its alteration on AD-related pathology. Metformin, a drug that stimulates glucose uptake in cells, mimics these effects, with a concomitant reduction in Grp78 levels and rescue of the shortened lifespan and climbing defects of Aβ-expressing flies. These findings demonstrate a protective effect of increased neuronal uptake of glucose against Aβ toxicity and highlight Grp78 as a novel therapeutic target for the treatment of AD (Niccoli, 2016).

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Branco, A.T., Schilling, L., Silkaitis, K., Dowling, D.K. and Lemos, B. (2016). Reproductive activity triggers accelerated male mortality and decreases lifespan: genetic and gene expression determinants in Drosophila. Heredity (Edinb) [Epub ahead of print]. PubMed ID: 27731328

Abstract

Reproduction and aging evolved to be intimately associated. Experimental selection for early-life reproduction drives the evolution of decreased longevity in Drosophila whereas experimental selection for increased longevity leads to changes in reproduction. Although life history theory offers hypotheses to explain these relationships, the genetic architecture and molecular mechanisms underlying reproduction-longevity associations remain a matter of debate. This study shows that mating triggers accelerated mortality in males and identifies hundreds of genes that are modulated upon mating in the fruit fly Drosophila melanogaster. Interrogation of genome-wide gene expression in virgin and recently mated males revealed coherent responses, with biological processes that are upregulated (testis-specific gene expression) or downregulated (metabolism and mitochondria-related functions) upon mating. Furthermore, using a panel of genotypes from the Drosophila Synthetic Population Resource (DSPR) as a source of naturally occurring genetic perturbation, abundant variation in longevity and reproduction-induced mortality among genotypes was uncovered. Genotypes display more than fourfold variation in longevity and reproduction-induced mortality that can be traced to variation in specific segments of the genome. The data reveal individual variation in sensitivity to reproduction and physiological processes that are enhanced and suppressed upon mating. These results raise the prospect that variation in longevity and age-related traits could be traced to processes that coordinate germline and somatic function (Branco, 2016).

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Wood, J.G., Jones, B.C., Jiang, N., Chang, C., Hosier, S., Wickremesinghe, P., Garcia, M., Hartnett, D.A., Burhenn, L., Neretti, N. and Helfand, S.L. (2016). (2016). Chromatin-modifying genetic interventions suppress age-associated transposable element activation and extend life span in Drosophila. Proc Natl Acad Sci U S A 113(40):11277-11282. PubMed ID: 27621458

Abstract

Transposable elements (TEs) are mobile genetic elements, highly enriched in heterochromatin, that constitute a large percentage of the DNA content of eukaryotic genomes. Aging in Drosophila melanogaster is characterized by loss of repressive heterochromatin structure and loss of silencing of reporter genes in constitutive heterochromatin regions. Using next-generation sequencing, this study found that transcripts of many genes native to heterochromatic regions and TEs increased with age in fly heads and fat bodies. A dietary restriction regimen, known to extend life span, represses the age-related increased expression of genes located in heterochromatin, as well as TEs. A corresponding age-associated increase in TE transposition in fly fat body cells was also observed that was delayed by dietary restriction. Furthermore, manipulating genes known to affect heterochromatin structure, including overexpression of Sir2, Su(var)3-9, and Dicer-2, as well as decreased expression of Adar, mitigate age-related increases in expression of TEs. Increasing expression of either Su(var)3-9 or Dicer-2 also leads to an increase in life span. Mutation of Dicer-2 leads to an increase in DNA double-strand breaks. Treatment with the reverse transcriptase inhibitor 3TC results in decreased TE transposition as well as increased life span in TE-sensitized Dicer-2 mutants. Together, these data support the retrotransposon theory of aging, which hypothesizes that epigenetically silenced TEs become deleteriously activated as cellular defense and surveillance mechanisms break down with age. Furthermore, interventions that maintain repressive heterochromatin and preserve TE silencing may prove key to preventing damage caused by TE activation and extending healthy life span (Wood, 2016).

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Yang, D., Lian, T., Tu, J., Gaur, U., Mao, X., Fan, X., Li, D., Li, Y. and Yang, M. (2016). LncRNA mediated regulation of aging pathways in Drosophila melanogaster during dietary restriction. Aging (Albany NY) 8: 2182-2203. PubMed ID: 27687893

Abstract

Dietary restriction (DR) extends lifespan in many species which is a well-known phenomenon. Long non-coding RNAs (lncRNAs) play an important role in regulation of cell senescence and important age-related signaling pathways. This study profiled the lncRNA and mRNA transcriptome of fruit flies at 7 day and 42 day during DR and fully-fed conditions, respectively. A large number of differentially expressed lncRNAs and their targets enriched in GO and KEGG analysis were found. Some new aging related signaling pathways during DR, such as hippo signaling pathway-fly, phototransduction-fly and protein processing in endoplasmic reticulum etc were also found. Novel lncRNAs XLOC_092363 and XLOC_166557 were found to be located in 10 kb upstream sequences of hairy and ems promoters, respectively. Furthermore, tissue specificity of some novel lncRNAs was analyzed at 7 day of DR in fly head, gut and fat body. Also the silencing of lncRNA XLOC_076307 resulted in altered expression level of its targets including Gadd45 (involved in FoxO signaling pathway). Together, these results implicate many lncRNAs closely associated with dietary restriction, which could provide a resource for lncRNA in aging and age-related disease field (Yang, 2016).

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Shahrestani, P., Wilson, J. B., Mueller, L. D. and Rose, M. R. (2016). Patterns of physiological decline due to age and selection in Drosophila melanogaster. Evolution [Epub ahead of print]. PubMed ID: 27624548

Abstract

In outbred sexually reproducing populations, age-specific mortality rates reach a plateau in late life following the exponential increase in mortality rates that marks aging. Little is known about what happens to physiology when cohorts transition from aging to late life. Age-specific values were measured for starvation resistance, desiccation resistance, time-in-motion and geotaxis in ten Drosophila melanogaster populations: five populations selected for rapid development and five control populations. Adulthood was divided into two stages, the aging phase and the late-life phase according to demographic data. Consistent with previous studies, populations selected for rapid development entered the late-life phase at an earlier age than the controls. Age-specific rates of change for all physiological phenotypes showed differences between the aging phase and the late-life phase. This result suggests that late life is physiologically distinct from aging. The ages of transitions in physiological characteristics from aging to late life statistically match the age at which the demographic transition from aging to late life occurs, in all cases but one. These experimental results support evolutionary theories of late life that depend on patterns of decline and stabilization in the forces of natural selection (Shahrestani, 2016).

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Rogers, R.P. and Rogina, B. (2014). Increased mitochondrial biogenesis preserves intestinal stem cell homeostasis and contributes to longevity in Indy mutant flies. Aging (Albany NY). 6: 335-350. PubMed ID: 24827528

Abstract
The Drosophila Indy (I'm Not Dead Yet) gene encodes a plasma membrane transporter of Krebs cycle intermediates, with robust expression in tissues associated with metabolism. Reduced INDY alters metabolism and extends longevity in a manner similar to caloric restriction (CR); however, little is known about the tissue specific physiological effects of INDY reduction. This study focused on the effects of INDY reduction in the Drosophila midgut due to the importance of intestinal tissue homeostasis in healthy aging and longevity. The expression of Indy mRNA in the midgut changes in response to aging and nutrition. Genetic reduction of Indy expression increases midgut expression of the mitochondrial regulator spargel/dPGC-1, which is accompanied by increased mitochondrial biogenesis and reduced reactive oxygen species (ROS). These physiological changes in the Indy mutant midgut preserve intestinal stem cell (ISC) homeostasis and are associated with healthy aging. Genetic studies confirm that dPGC-1 mediates the regulatory effects of INDY, as illustrated by lack of longevity extension and ISC homeostasis in flies with mutations in both Indy and dPGC1. These data suggest INDY may be a physiological regulator that modulates intermediary metabolism in response to changes in nutrient availability and organismal needs by modulating dPGC-1 (Rogers, 2014).

Highlights

• Aging increases Indy mRNA levels in the midgut of control flies.
• Indy reduction is associated with increased dPGC-1 levels in the midgut.
• Reduced Indy increases dPGC-1 medited mitochondrial biogenesis.
• Indy mutants have enhanced mitochondrial activity and reduced ROS levels in the midgut.
• Indy mutant flies have increased resistance to oxidative stress.
• Indy mutations preserve ISC homeostasis and intestinal integrity.
• Indy-longevity is mediated by dPGC-1.

Discussion
Reduction of Indy gene activity in fruit flies, and homologs in worms, extends lifespan by altering energy metabolism in a manner similar to caloric restriction (CR). Indy mutant flies on regular food share many characteristics with CR flies and do not have further longevity extension when aged on a CR diet. Furthermore, mINDY−/− mice on regular chow share 80% of the transcriptional changes observed in CR mice, supporting a conserved role for INDY in metabolic regulation that mimics CR and promotes healthy aging. This study shifted from systemic to the tissue specific effects of INDY reduction, focusing on the midgut due to the high levels of INDY protein expression in wild type flies and the importance of regulated intestinal homeostasis during aging. It provides evidence that supports a role for INDY as a physiological regulator that senses changes in nutrient availability and alters mitochondrial physiology to sustain tissue-specific energetic requirements (Rogers, 2014).

The study shows an age-associated increase in midgut Indy mRNA levels that can be replicated by manipulations that accelerate aging such as increasing the caloric content of food or exposing flies to paraquat. Conversely, it is also shown that CR decreases Indy mRNA in control midgut tissues, which is consistent with previous findings in fly muscle and mouse liver. Diet-induced variation in midgut Indy expression suggests that INDY regulates intermediary metabolism by modifying citrate transport to meet tissue or cell-specific bioenergetic needs. Specifically, as a plasma membrane transporter INDY can regulate cytoplasmic citrate, thereby affecting fat metabolism, respiration, and via conversion to malate, the TCA cycle. Reduced INDY-mediated transport activity in the midgut could prevent age-related ISC-hyperproliferation by decreasing the available energy needed to initiate proliferation, thereby preserving tissue function during aging. This is supported by findings that nutrient availability affects ISC proliferation in adult flies and that CR can affect stem cell quiescence and activation (Rogers, 2014).

One of the hallmarks of CR-mediated longevity extension is increased mitochondrial biogenesis mediated by dPGC-1. Increased dPGC-1 levels and mitochondrial biogenesis have been described in the muscle of Indy mutant flies, the liver of mIndy−/− mice, and this study describes it in the midgut of Indy mutant flies. One possible mechanism for these effects can be attributed to the physiological effects of reduced INDY transport activity. Reduced INDY-mediated transport activity could lead to reduced mitochondrial substrates, an increase in the ADP/ATP ratio, activation of AMPK, and dPGC-1 synthesis. This is consistent with findings in CR flies and the livers of mINDY−/− mice. This study's analysis of mitochondrial physiology in the Indy mutant midgut shows upregulation of respiratory proteins, maintenance of mitochondrial potential and increased mitochondrial biogenesis, all of which are signs of enhanced mitochondrial health. The observed increase in dPGC-1 levels in Indy mutant midgut therefore appears to promote mitochondrial biogenesis and functional efficiency, representing a protective mechanism activated in response to reduced energy availability (Rogers, 2014).

Genetic interventions that conserve mitochondrial energetic capacity have been shown to maintain a favorable redox state and regenerative tissue homeostasis. This is particularly beneficial in the fly midgut, which facilitates nutrient uptake, waste removal and response to bacterial infection. Indy mutant flies have striking increases in the steady-state expression of the GstE1 and GstD5 ROS detoxification genes. As a result, any increase in ROS levels, whether from mitochondrial demise or exposure to external ROS sources can be readily metabolized to prevent accumulation of oxidative damage. Such conditions not only promote oxidative stress resistance, but also preserve ISC homeostasis as demonstrated by consistent proliferation rates throughout Indy mutant lifespan and preserved intestinal architecture in aged Indy mutant midguts. Thus, enhanced ROS detoxification mechanisms induced by Indy reduction and subsequent elevation of dPGC-1 contributes to preservation of ISC functional efficiency, and may be a contributing factor to the long-lived phenotype of Indy mutant flies (Rogers, 2014).

Several lines of evidence indicate that INDY and dPGC-1 are part of the same regulatory network in the midgut, in which dPGC-1 functions as a downstream effector of INDY. The similarity between dPGC-1 mRNA levels and survivorship of flies overexpressing dPGC-1 in esg-positive cells and Indy mutant flies suggests that Indy and dPGC-1 interact to extend lifespan. This is further supported by the lack of additional longevity extension when dPGC-1 is overexpressed in esg-positive cells of Indy mutant flies. Moreover, hypomorphic dPGC-1 flies in an Indy mutant background are similar to controls with respect to life span, declines in mitochondrial activity and ROS-detoxification. Together, these data suggest that dPGC-1 must be present to mediate the downstream physiological benefits and lifespan extension of Indy mutant flies (Rogers, 2014).

There are some physiological differences between the effects of Indy mutation and dPGC-1 overexpression in esg-positive cells. While Indy mutant flies are less resistant to starvation and more resistant to paraquat, a recent report showed that overexpressing dPGC-1 in esg-positive cells has no effect on resistance to starvation or oxidative stress. Additionally, mice lacking skeletal muscle PGC-1α were found to lack mitochondrial changes associated with CR but still showed other CR-mediated metabolic changes. In the fly INDY is predominantly expressed in the midgut, fat body and oenocytes, though there is also low level expression in the malpighian tubules, salivary glands, antenae, heart and female follicle cell membranes. Thus, the effects of INDY on intermediary metabolism and longevity could be partially independent from dPGC-1 or related to changes in tissues other than the midgut (Rogers, 2014).

This study suggests that INDY may function as a physiological regulator of mitochondrial function and related metabolic pathways, by modulating nutrient flux in response to nutrient availability and energetic demands. Given the localization of INDY in metabolic tissues, and importance of regulated tissue homeostasis during aging, these studies highlight INDY as a potential target to improved health and longevity. Reduced Indy expression causes similar physiological changes in flies, worms and mice indicating its regulatory role would be conserved. Further work should examine the interplay between Indy mutation and metabolic pathways, such as insulin signaling, which have been shown to promote stem cell maintenance and healthy aging in flies and mice. In doing so, the molecular mechanisms, which underlie Indy mutant longevity may provide insight for anti-aging therapies (Rogers, 2014).

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Merino, M.M., Rhiner, C., Lopez-Gay, J.M., Buechel, D., Hauert, B. and Moreno, E. (2015). Elimination of unfit cells maintains tissue health and prolongs lifespan. Cell 160: 461-476. PubMed ID: 25601460

Abstract
Viable yet damaged cells can accumulate during development and aging. Although eliminating those cells may benefit organ function, identification of this less fit cell population remains challenging. Previously, a molecular mechanism based on "fitness fingerprints" displayed on cell membranes, which allowed direct fitness comparison among cells in Drosophila was identified. This study reports the physiological consequences of efficient cell selection for the whole organism. It is found that fitness-based cell culling is naturally used to maintain tissue health, delay aging, and extend lifespan in Drosophila. A gene, azot, which ensures the elimination of less fit cells, is identified. Lack of azot increases morphological malformations and susceptibility to random mutations and accelerates tissue degeneration. On the contrary, improving the efficiency of cell selection is beneficial for tissue health and extends lifespan (Merino, 2015).

Highlights

• Azot is expressed in cells undergoing negative selection.
• Azot is required to eliminate loser cells and unwanted neurons.
• Azot maintains tissue fitness during development.
• azot promoter computes relative FlowerLose and Sparc levels.
• Cell selection is active during adulthood.
• Death of unfit cells is sufficient and required for multicellular fitness maintenance.
• Death of unfit cells extends lifespan.

Discussion
This study shows that active elimination of unfit cells is required to maintain tissue health during development and adulthood. It identifies a gene (azot), whose expression is confined to suboptimal or misspecified but morphologically normal and viable cells. When tissues become scattered with suboptimal cells, lack of azot increases morphological malformations and susceptibility to random mutations and accelerates age-dependent tissue degeneration. On the contrary, experimental stimulation of azot function is beneficial for tissue health and extends lifespan. Therefore, elimination of less fit cells fulfils the criteria for a hallmark of aging (Merino, 2015).

Although cancer and aging can both be considered consequences of cellular damage, this study did not find evidence for fitness-based cell selection having a role as a tumor suppressor in Drosophila. Their results rather support that accumulation of unfit cells affect organ integrity and that, once organ function falls below a critical threshold, the individual dies (Merino, 2015).

The study finds Azot expression in a wide range of “less fit” cells, such as WT cells challenged by the presence of “supercompetitors,” slow proliferating cells confronted with normal proliferating cells, cells with mutations in several signaling pathways (i.e., Wingless, JAK/STAT, Dpp), or photoreceptor neurons forming incomplete ommatidia. In order to be expressed specifically in “less fit” cells, the transcriptional regulation of azot integrates fitness information from at least three levels: (1) the cell’s own levels of FlowerLose isoforms, (2) the levels of Sparc, and (3) the levels of Lose isoforms in neighboring cells. Therefore, Azot ON/OFF regulation acts as a cell-fitness checkpoint deciding which viable cells are eliminated (Merino, 2015).

The study proposes that by implementing a cell-fitness checkpoint, multicellular communities became more robust and less sensitive to several mutations that create viable but potentially harmful cells. Moreover, azot is not involved in other types of apoptosis, suggesting a dedicated function, and- given the evolutionary conservation of Azot- pointing to the existence of central cell selection pathways in multicellular animals (Merino, 2015).

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Ali, Y. O., Ruan, K. and Zhai, R. G. (2012). NMNAT suppresses tau-induced neurodegeneration by promoting clearance of hyperphosphorylated tau oligomers in a Drosophila model of tauopathy. Hum Mol Genet 21(2): 237-250. PubMed ID: 21965302

Abstract

Tauopathies, including Alzheimer's disease, are a group of neurodegenerative diseases characterized by abnormal tau hyperphosphorylation that leads to formation of neurofibrillary tangles. Drosophila models of tauopathy display prominent features of the human disease including compromised lifespan, impairments of learning, memory and locomotor functions and age-dependent neurodegeneration visible as vacuolization. This study used a Drosophila model of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), in order to study the neuroprotective capacity of a recently identified neuronal maintenance factor, nicotinamide mononucleotide (NAD) adenylyl transferase (NMNAT), a protein that has both NAD synthase and chaperone function. NMNAT is essential for maintaining neuronal integrity under normal conditions and has been shown to protect against several neurodegenerative conditions. However, its protective role in tauopathy has not been examined. This study shows that overexpression of NMNAT significantly suppresses both behavioral and morphological deficits associated with tauopathy by means of reducing the levels of hyperphosphorylated tau oligomers. Importantly, the protective activity of NMNAT protein is independent of its NAD synthesis activity, indicating a role for direct protein-protein interaction. Next, it was shown that NMNAT interacts with phosphorylated tau in vivo and promotes the ubiquitination and clearance of toxic tau species. Consequently, apoptosis activation was significantly reduced in brains overexpressing NMNAT, and neurodegeneration was suppressed. This report on the molecular basis of NMNAT-mediated neuroprotection in tauopathies opens future investigation of this factor in other protein foldopathies (Ali, 2012).

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Chen, H., Zheng, X. and Zheng, Y. (2014). Age-associated loss of Lamin-B leads to systemic inflammation and gut hyperplasia. Cell 159: 829-843. PubMed ID: 25417159

Abstract
Aging of immune organs, termed as immunosenescence, is suspected to promote systemic inflammation and age-associated disease. The cause of immunosenescence and how it promotes disease, however, has remained unclear. This study reports that the Drosophila fat body, a major immune organ, undergoes immunosenescence and mounts strong systemic inflammation that leads to deregulation of immune deficiency (IMD) signaling in the midgut of old animals. Inflamed old fat bodies secrete circulating peptidoglycan recognition proteins that repress IMD activity in the midgut, thereby promoting gut hyperplasia. Further, fat body immunosenecence is caused by age-associated lamin-B reduction specifically in fat body cells, which then contributes to heterochromatin loss and derepression of genes involved in immune responses. As lamin-associated heterochromatin domains are enriched for genes involved in immune response in both Drosophila and mammalian cells, these findings may provide insights into the cause and consequence of immunosenescence during mammalian aging (Chen, 2014).

Highlights

• Age-associated upregulation of systemic immune response by fat body may repress IMD signaling in the midgut.
• Systemic inflammation caused by old fat body leads to intestinal hyperplasia.
• Age-associated lamin-B loss in fat body cells correlates with enhanced IMD signaling.
• Lamin-B represses immune response in fat bodies.
• Lamin-B in fat body represses intestinal hyperplasia and promotes survival.
• Lamin-B maintains midgut IMD signaling and homeostasis by inhibiting systemic inflammation in fat bodies.
• Lamin-B inhibits systemic inflammation by maintaining heterochromatin in the fat body.

Discussion
Aging of immune organs, termed as immunosenescence, is suspected to promote systemic inflammation and age-associated disease. The cause of immunosenescence and how it promotes disease, however, has remained unclear. This study reports that the Drosophila fat body, a major immune organ, undergoes immunosenescence and mounts strong systemic inflammation that leads to deregulation of immune deficiency (IMD) signaling in the midgut of old animals. Inflamed old fat bodies secrete circulating peptidoglycan recognition proteins that repress IMD activity in the midgut, thereby promoting gut hyperplasia. Further, fat body immunosenecence is caused by age-associated lamin-B reduction specifically in fat body cells, which then contributes to heterochromatin loss and derepression of genes involved in immune responses. As lamin-associated heterochromatin domains are enriched for genes involved in immune response in both Drosophila and mammalian cells, these findings may provide insights into the cause and consequence of immunosenescence during mammalian aging. (Chen, 2014).

By analyzing gene expression changes upon aging in fat bodies and midguts, it was shown that an increase of immune response in the fat body is accompanied by a striking reduction in the midgut. Specifically, it was demonstrated that the age-associated increase in Immune deficiency (IMD) signaling in fat bodies leads to reduction of IMD activity in the midgut, which in turn contributes to midgut hyperplasia. This fat body to midgut effect requires peptidoglycan recognition proteins (PGRPs) secreted from fat body cells and is mediated by both bacteria dependent and independent pathways. Therefore, fat body aging contributes to systemic inflammation, which contributes to the disruption of gut homeostasis. Importantly, it was shown that the age-associated lamin-B loss in fat body cells causes the derepression of a large number of immune responsive genes, thereby resulting in fat body-based systemic inflammation (Chen, 2014).

B-type lamins have long been suggested to have a role in maintaining heterochromatin and gene repression. Consistently, this study's global analyses of fat body depleted of lamin-B revealed a loss of heterochromatin and derepression of a large number of immune responsive genes. This is further supported by ChIP-qPCR analyses of H3K9me3 on specific IMD regulators. Recent studies in different cell types show that tethering genes to nuclear lamins do not always lead to their repression. Deleting B-type lamins or all lamins in mouse ES cells or trophectdoderm cells does not result in derepression of all genes in LADs. In light of these studies, it is suggested that the transcriptional repression function of lamin-B could be gene and cell type dependent.

Interestingly, GO analyses revealed a significant enrichment of immune responsive genes in Lamin-associated domains (LADs) in four different mammalian cell types and Drosophila Kc cells. Since the large-scale pattern of LADs is conserved in different cell types in mammals, it is possible that the immune-responsive genes are also enriched in LADs in the fly fat body cells. Supporting this notion, the IKKγ, key, which is one of the two derepressed IMD regulators and was found to exhibit H3K9me3 reduction and gene activation, is localized to LADs in Kc cells. It is speculated that lamin-B might play an evolutionarily conserved role in repressing a subset of inflammatory genes in certain tissues, such as the immune organs, in the absence of infection or injury. Consistently, senescence-associated lamin-B1 loss in mammalian fibroblasts is correlated with senescence-associated secretory phenotype (SASP). Although the in vivo relevance of fibroblast SASP in chronic inflammation and aging-associated diseases in mammals remains to be established, the findings in Drosophila provide insights and impetus to investigate the role of lamins in immunosenescence and systemic inflammation in mammals (Chen, 2014).

Lamin-B gradually decreases in fat body cells of aging flies, whereas lamin-C amount remains the same. Since it has been recently shown that the assembly of an even and dense nuclear lamina is dependent on the total lamin concentration, the age-associated appearance of lamin-B and lamin-C gaps around the nuclear periphery of fat body cells is likely caused by the drop of the lamin-B level. How aging triggers lamin-B loss is unknown, but it appears to be posttranscriptional, because lamin-B transcripts in fat bodies remain unchanged upon aging. Interestingly, among the tissues examined, no changes of lamin-B and lamin-C proteins were found in cells in the heart tube, oenocytes, or gut epithelia in old flies. Therefore, the age-associated lamin-B loss does not occur in all cell types in vivo. A systematic survey to establish the cell/tissue types that undergo age-associated reduction of lamins in both flies and mammals should provide clues to the cause of loss. Deciphering how advanced age leads to lamin loss should open the door to further investigate the cellular mechanism that contributes to chronic systemic inflammation and how it in turn promotes age-associated diseases in humans (Chen, 2014).

Old Drosophila gut is known to exhibit increased microbial load, which would cause increased stress response and activation of tissue repair, thereby leading to midgut hyperplasia. Systemic inflammation caused by lamin-B loss in fat body leads to repression of local midgut IMD signaling. The upregulation of targets of IMD in the aged whole gut has been recently reported, while a downregulation of target genes was observed in the current analyses of the midgut. However, the previous study found a similar upregulation of the genes when performing RNA-seq of the whole gut (Chen, 2014).

These studies reveal an involvement of bacteria in the repression of midgut IMD signaling by the PGRPs secreted from the fat body. How PGRPs from the fat body repress midgut IMD is still unknown. One possibility is that the body cavity bacteria contribute to the maintenance of midgut IMD activity, and the increased circulating PGRPs limit these bacteria. The circulating PGRPs may also reduce midgut IMD activity indirectly by affecting other tissues. The evidence suggests that lamin-B loss could also contribute to midgut hyperplasia independent of the IMD pathway. While it will be important to further address these possibilities, the findings have revealed a fat body mediated inflammatory pathway that can lead to reduced midgut IMD, increased gut microbial accumulation, and midgut hyperplasia upon aging (Chen, 2014).

Interestingly, microbiota changes also occur in aging human intestine and have been linked to altered intestinal inflammatory states and diseases. Although, much effort has been devoted to understand how local changes in aging mammalian intestines affect gut microbial community, the cause remains unclear. The findings in Drosophila reveal the importance of understanding the impact of immunosenescence and systemic inflammation on gut microbial homeostasis. Indeed, if increased circulating inflammatory cytokines perturb the ability of local intestine epithelium and the gut-associated lymphoid tissue to maintain a balanced microbial community, the unfavorable microbiota in the old intestine would cause chronic stress response and tissue repair, thereby leading to uncontrolled cell growth as observed in age-associated cancers (Chen, 2014).

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Reviews

Tatar, M., Post, S. and Yu, K. (2014). Nutrient control of Drosophila longevity. Trends Endocrinol Metab 25: 509-517. PubMed ID: 24685228

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Stress and lifespan in Drosophila

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Date revised: 25 April 2018

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