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Aging and Lifespan
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Drosophila genes associated with
Aging and Lifespan
anterior open
azot
flower
Indy
lamin-B
methuselah
NMNAT
Ras oncogene at 85D
Rpd3
rolled
Sir-2
Sod
Tequila
Related terms


Circadian clock
Corpora allata
Insulin signaling
Memory
Mushroom body
Relevant studies of Aging and Lifespan

Obata, F. and Miura, M. (2015). Enhancing S-adenosyl-methionine catabolism extends Drosophila lifespan. Nat Commun 6: 8332. PubMed ID: 26383889

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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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

ronmental stresses experienced during development exert many long-term effects upon health and disease. For example, chemical oxidants or genetic perturbations that induce low levels of reactive oxygen species can extend lifespan in several species. In some cases, the beneficial effects of low-dose oxidants are attributed to adaptive protective mechanisms such as mitohormesis, which involve long-term increases in the expression of stress response genes. This study shows in Drosophila that transient exposure to low concentrations of oxidants during development leads to an extension of adult lifespan. Surprisingly, this depends upon oxidants acting in an antibiotic-like manner to selectively deplete the microbiome of Acetobacter proteobacteria. The presence of Acetobacter species, such as A. aceti, in the indigenous microbiota increases age-related gut dysfunction and shortens lifespan. This study demonstrates that low-dose oxidant exposure during early life can extend lifespan via microbiome remodelling rather than mitohormesis (Obata, 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 (CG5147EY22749, henceforth called dC53EY, was backcrossed into a healthy, outbred population of flies. Homozygous dC53EY/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-tRNAHis, pre-tRNAAla and pre-tRNALeu, 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).

Avanesov AS, Ma S, Pierce KA, Yim SH, Lee BC, Clish CB, Gladyshev VN. 2014. Age- and diet-associated metabolome remodeling characterizes the aging process driven by damage accumulation. eLife 3: e02077. PubMed ID: 24843015

Hoffman JM, Soltow QA, Li S, Sidik A, Jones DP, Promislow DE. 2014. Effects of age, sex, and genotype on high-sensitivity metabolomic profiles in the fruit fly, Drosophila melanogaster. Aging Cell 13: 596-604. PubMed ID: 24636523

Laye MJ, Tran V, Jones DP, Kapahi P, Promislow DE. 2015. The effects of age and dietary restriction on the tissue-specific metabolome of Drosophila. Aging Cell 14: 797-808. PubMed ID: 26085309

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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

He, Y. and Jasper, H. (2014). Studying aging in Drosophila. Methods 68: 129-133. PubMed ID: 24751824 

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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Date revised: 25 April 2018

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