Near-infrared light increases ATP, extends lifespan and improves mobility in aged Drosophila melanogaster Rana Begum 1 , Karin Calaza 2 , Jaimie Hoh Kam 1 , Thomas E. Salt 1 , Chris Hogg 3 and Glen Jeffery Institute of Ophthalmology, University College London, London EC1V 9EL, UK 2 Program of Neuroscience, Institute de Biologia, Universidade Federal Fluminense, Rio de Janeiro 24210130, Brazil 3 Moorfields Eye Hospital, London EC1V 2PD, UK Ageing is an irreversible cellular decline partly driven by failing mitochondrial integrity. Mitochondria accumulate DNA mutations and reduce ATP production necessary for cellular metabolism. This is associated with inflammation. Near-infrared exposure increases retinal ATP in old mice via cytochrome c oxidase absorption and reduces inflammation. Here, we expose fruitflies daily to 670 nm radiation, revealing elevated ATP and reduced inflam- mation with age. Critically, there was a significant increase in average lifespan: 100–175% more flies survived into old age following 670 nm exposure and these had significantly improved mobility. This may be a simple route to extending lifespan and improving function in old age. 1. Introduction Mitochondria provide cellular energy via adenosine triphosphate (ATP). But, their DNA (mtDNA) suffers from progressive mutations resulting in reduced ATP production, which is thought to run concomitantly with an increase in pro-inflammatory reactive oxygen species (ROS) [1,2]. Hence, hallmarks of ageing are reduced cellular energy and progressive systemic inflammation. Meta- bolic demand also plays a role as tissues and organisms with high metabolic rates generally suffer from rapid ageing [3,4]. The retina has the greatest metabolic demand in the body [5], but ATP decline in the central nervous system can be significantly improved by near-infrared/infrared light (NIR/IR, [6]). Specific wavelengths in this range are absorbed by cytochrome c oxidase in mitochondrial respiration, improving its efficiency [7–10]. These wavelengths improve mito- chondrial membrane potentials, significantly reduce inflammation and reduce macrophage numbers with brief exposures of around 60–90 s repeated over approximately a week [11,12]. NIR/IR also reduces experimental pathology when insult impacts on mitochondrial function, as in experimental Parkinson’s disease, where NIR significantly reduces cell death in the substantia nigra [13]. However, NIR/IR studies have largely used light for short periods and their impact on lifespan has not been assessed [7,11,12]. If NIR improves mitochondrial function we predict it may extend life. The fly has been used here because of its relatively short life [14]. Hence, we ask if long-term exposure to 670 nm in Drosophila melanogaster can increase lifespan and improve function in old age. 2. Material and methods Drosophila melanogaster were used. Hatched male flies were housed on 12/12 light cycle at 258C within a season. Half were exposed to 670 nm for 20 min per day at & 2015 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. on March 18, 2015http://rsbl.royalsocietypublishing.org/Downloaded from 40 mW cm 22 in clear plastic 50 cm 3 (28 mm wide) containers, illuminating flies from either side, which were counted weekly. Room illumination was 2 mW cm 22 . 670 nm energies were approximately 100 times lower than indirect sunlight, consistent with earlier studies [7]. Light devices were built by C. H. Elec- tronics UK and contained 50 670 nm LEDS over 20 cm 2 . Six independent replicates were used in lifespan experiments (n ¼ 620 flies). ATP, inflammation and mobility were assessed at seven weeks, when ATP and mobility are known to decline [15]. ATP was measured by luciferin–luciferase assay (Enliten w ATP Assay System, Promega). Flies were killed with liquid nitrogen, transferred to 2.5% trichoroacetic acid (TCA), then homogenized at 48C. Supernatant was collected and the TCA was neutralized with 1 M Tris–acetate buffer (pH 7.75, final TCA concentration 0.0625%); 10 ml of neutralized solution was added to 100 ml of luci- ferin–luciferase in fresh buffer. ATP was measured using an Orion microplate luminometer (Berthold Detection Systems GmbH) and data normalized to fly numbers. Tissues were homogenized in 2% sodium dodecyl sulfate (SDS) with protease inhibitor cocktail for Western blot (Roche Diagnos- tics), and centrifuged; the supernatant was pipetted out, separated with 10% SDS–PAGE and electrophoretically transferred onto nylon membranes. Immunoblotting was undertaken for complement component C3 (Cappel, MP Biomedicals), which is highly conserved [16]. Protein was quantified by densitometric X-ray scanning and values were normalized to a-tubulin. Fly mobility assessment was as Bjedov et al. [14]. Flies were placed in 100 ml clear cylinders (seven flies per trial), tapped to the bottom and then videoed, the last two steps repeated three times. Using the videos, the number of flies above the 50 ml mark (9 cm from the bottom) was counted after 1 min. Individual flies were traced, with absolute distance travelled measured. Data w er e analysed with GraphPad P RISM v. 5 and sta t is ti cal analysis was undertaken using Mann –Whitne y U non-parametric and log-rank tests. 3. Results (a) ATP levels are elevated and systemic inflammation reduced Whole body ATP declines with age only after appr o xima tely seven weeks [14], when ATP was measured here. A TP concen- trations wer e significantly grea t er, by approxima tely 80%, in 670 nm exposed animals compared with unexposed (figure 1a, Mann–Whitney test p ¼ 0.028). At seven weeks, W estern blots wer e undertaken for inflammatory marker complement com- ponent C3. This was reduced in 670 nm exposed flies compared with controls (figure 1b). Hence, 670 nm radiation elevates ATP and reduces inflammation. (b) Lifespan increases Fly numbers in experimental and control groups were similar in the two weeks post-hatching. From week 3, fly deaths were greater in controls than 670 nm exposed flies and they remained so at each time point until week 11–12, when all flies were dead in both groups. This difference was significant (figure 2, log-rank test p ¼ 0.008). The progressive mean percentage increase in 670 nm flies alive over controls is given in figure 2b. Group differences accelerated from week 4, when 10% extra 670 nm treated flies were alive compared with controls, to approximately 50% extra when the control population had halved. By the time the control population was reduced by 80%, at week 8, more than 100% extra 670 nm treated flies remained alive. Subsequently, group differences reached almost 180% before declining to zero in both groups at week 11–12. Hence, 670 nm did not extend absolute lifespan. (c) Aged mobility increases Mobility of 670 nm treated and control flies was measured at seven weeks. Significantly more 670 nm treated flies climbed above the 50 ml level (9 cm) and significantly more travelled a greater distance than controls (Mann–Whitney test p ¼ 0.028, p ¼ 0.014, respectively). Twice as many 670 nm flies climbed above 50 ml (9 cm) compared with controls and these travelled twice the distance in 1 min compared with controls (figure 2c,d). Hence, 670 nm exposure significantly improves both lifespan and mobility. 4. Discussion Drosophila melanogaster has been widely used in lifespan studies as they are short lived and their genomic sequence is relatively well understood [14,17], hence their adoption experimentally here to extend lifespan. Our results reveal [ATP] (nM)/fly ATP level(a) C3 expression (Western blot)(b) control absolute intensity 670 control 670 control C3 ~ 110 kDa a-tubulin 55 kDa 670 * 0 0 500 1000 200 400 600 Figure 1. Exposur e to 670 nm radiation increases ATP in aged flies and reduces inflammati on. (a) Sev en week old flies exposed to 670 nm had a significant incr ease in whole body ATP compared with controls, p ¼ 0.028. n ¼ 25 flies per group. (b) Whole body inflammation (C3) was measured in seven week flies using W estern blot. This was reduced in 670 nm exposed flies by approxima tely 15%. Here, flies were pooled within groups as C3 protein levels were lo w in individuals. Hence there are no error bars. n ¼ 15 flies per group. (Online version in colour.) rsbl.royalsocietypublishing.org Biol. Lett. 11: 20150073 2 on March 18, 2015http://rsbl.royalsocietypublishing.org/Downloaded from that when flies are exposed to 670 nm radiation they have reduced inflammation, improved ATP, improved mobility and extended average lifespans. These data are consistent with the majority of studies undertaken using 670 nm on mammals, showing reduced inflammation in experimental models and in ageing, and improved ATP levels [6,7]. How- ever, it would be difficult to undertake lifespan experiments in mice as the light would not penetrate the entire body as it does in flies and hence its influence would not be systemic. There are many factors and pathways in ageing, and nine candidate hallmarks have been suggested, which may be separate, but also are likely to have interactions [1]. Mito- chondrial function is one. Previously, mitochondrial function and ageing were viewed within a framework of pro- gressive mtDNA mutations/deletions resulting in reduced ATP and increased ROS. The balance of these factors was seen as a driver in the mitochondrial theory of ageing [18]. However, evidence has undermined the role of ROS in ageing [19,20]. Hence, some mutant mice have reduced life- span as a result of mtDNA mutations/deletions not associated with increased ROS [21,22]. Further, increased ROS can prolong lifespan in yeast and Caenorhabditis elegans [22,23], and in mammals it does not accelerate ageing [20]. These data are reviewed by Lopez-Otin et al. [1], who argue that low ROS may activate compensatory mechanisms and not directly contribute to ageing. Such data may undermine the ROS element in Harman’s mitochondrial theory [18]. If correct, it places greater potential emphasis on ATP in ageing. NIR has been successful in treating induced pathology [7] and ageing, particularly in the retina, where progressive age- related inflammation is marked owing to high metabolic rate [11,12]. These wavelengths penetrate deeply and 670 nm trans-illuminated our flies at 40 mW cm 22 . In relation to this, it may be significant that, while old domestic incandescent light- ing contained significant NIR elements, none is present in modern strip lighting or energy-saving domestic lighting [12]. The absence of these wavelengths from artificial lighting may have long-term consequences. As longer wavelengths penetrate deeply, this may be of significance not only for the ageing eye, but also potentially for other tissues. Ethics statement. Fly research is free of legal ethical constraint. Data accessibility. All data are presented in the manuscript. Acknowledgement. We thank Iris Salecker, Giovanna Vinti and Tobi Weinrrich for technical assistance. Author contributions. G.J. designed experiments and wrote the manu- script. All authors undertook the experiments and approved the final version of the manuscript. R.B. analysed the data. Funding statement. Supported by the Rosetrees Trust UK. K.C. was a research fellow from CAPES Brazil (proc. 18134/12-2). Competing interests. We have no competing interests. References 1. Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. 2013 The hallmarks of aging. Cell 153, 1194–1217. (doi:10.1016/ j.cell.2013.05.039) 2. Balaban RS, Nemoto S, Finkel T. 2005 Mitochondrial, oxidants and aging. Cell 120, 483–495. (doi:10.1016/j.cell.2005.02.001) 3. Speakman JR. 2005 Body size, energy, metabolism and lifespan. J. Exp. Biol. 208, 1717–1730. (doi:10. 1242/jeb.01556) 4. Wang Z, Ying Z, Bosy-Westphal A, Zhang J, Schautz B, Later W, Heymsfield SB, Mu¨ller MJ. 2010 Specific metabolic rates of major organs and tissues across adulthood: evolution by mechanistic model of resting expenditure. Am. J. Clin. Nutr. 92, 1369– 1377. (doi:10.3945/ajcn.2010.29885) 5. Yu DY, Cringle SJ. 2001 Oxygen distribution and consumption within the retina in vascularized and avascular retinas and in animal models of disease. Prog. Retin. Eye Res. 20, 175–208. (doi:10.1016/ S1350-9462(00)00027-6) 0 0 10 20 30 40 50 60 70 80 100 (a) (c) (d ) (b) % survival over controls n = 620 flies fly survival curves for 670 nm (–) and control (–) 90 123456 time (weeks) % survival 78910 0 0 100 200 300 123456 no. weeks % increase over control control distance travelled in 1 min 670 control 670 * 0 20 60 40 80 100 distance (mm) fly climbing (>90 mm) * 0 20 60 40 80 % >90 mm in 1 min 78910 11 12 Figure 2. Lifespan and mobility. (a) Fly numbers at progressive weeks in groups exposed to 670 nm supplemented light each day (red line) and controls (black line). Curves are averages for six independent experiments with a minimum of 40 flies per group in each experiment. Fly death rates separated between three and six weeks with fewer flies dying in 670 nm exposed animals. Reduction in the two population followed similar patterns from six weeks but with the 670 nm exposed group having greater numbers at any point until week 12. In all replicates, there was no indication that 670 nm increased absolute lifespan beyond weeks 11 – 12. Differences between the two groups were statistically significant ( p ¼ 0.008). (b) Inset: percentage increase of 670 nm exposed flies alive at pro- gressive weeks. (c) Seven week old 670 nm exposed flies were more active than controls. (d) Mobility measures the percentage of flies that climbed above 90 mm in a clear 100 ml cylinder. (d) This was filmed and then the distance travelled by each fly was measured in each group. In both cases, the 670 nm exposed flies where significantly more mobile. There were 21 flies in each group in each condition. (Online verion in colour.) rsbl.royalsocietypublishing.org Biol. Lett. 11: 20150073 3 on March 18, 2015http://rsbl.royalsocietypublishing.org/Downloaded from 6. Gkotsi D, Begum R, Salt T, Lascaratos G, Hogg C, Chau KY, Schapira AH, Jeffery G. 2014 Recharging mitochondrial batteries in old eyes. Near infra-red increases ATP. Exp. Eye Res. 122, 50 –53. (doi:10. 1016/j.exer.2014.02.023) 7. Fitzgerald M et al . 2013 Red/near-infrared irradiation therapy for treatment of central nervous system injuries and disorders. Rev. Neurosci. 24, 205–226. (doi:10.1515/revneuro-2012-0086) 8. Wilson M, Greenwood C. 1970 The long-wavelength absortion band of cytochrome c oxidase. Biochem. J. 116, 17 –18. 9. Karu TI, Pyatibrat LV, Kolyakov SF, Afanasyeva NI. 2005 Absorption measurements of cell monolayer relevant to phototherapy: reduction of cytochrome c oxidase under near IR radiation. J. Photochem. Photobiol. 81, 98– 106. (doi:10.1016/j.jphotobiol.2005.07.002) 10. Cooper CE, Springett R. 1997 Measurement of cytochrome oxidase and mitochondrial energetics by near-infrared spectroscopy. Phil. Trans. R. Soc. Lond. B 352, 669– 676. (doi:10.1098/rstb.1997.0048) 11. Kokkinopoulos I, Colman A, Hogg C, Heckenlively J, Jeffery G. 2013 Age-related inflammation is reduced by 670 nm light via increased mitochondrial membrane potential. Neurobiol. Aging 34, 602– 609. (doi:10.1016/j.neurobiolaging.2012.04.014) 12. Begum R, Powner MB, Hudson N, Hogg C, Jeffery G. 2013 Treatment with 670 nm up regulates cytochrome C oxidase expression and reduces inflammation in an age-related macular degeneration model. PLoS ONE 8, e57828. (doi:10. 1371/journal.pone.0057828) 13. Purushothuman S, Nandasena C, Johnstone DM, Stone J, Mitrofanis J. 2013 The impact of near- infrared light on dopaminergic cell survival in a transgenic mouse model of parkinsonism. Brain Res. 1535, 61 –70. (doi:10.1016/j.brainres.2013.08.047) 14. Bjedov I, Toivonen JM, Kerr F, Slack C, Foley A, Partridge L. 2010 Mechanisms of life span extension by rampamycin in the fruit fly Drosophila melanogaster. Cell Metab. 11, 35–46. (doi:10.1016/ j.cmet.2009.11.010) 15. Vernace VA, Arnaud L, Schmidt-Glenewinkel T, Figueiredo-Pereira ME. 2007 Aging perturbs 26S proteasome assembly in Drosophila melanogaster . FASEB J. 21, 2672–2682. (doi:10.1096/fj.06- 6751com) 16. Nonaka M, Kimura A. 2006 Genomic view of the evolution of the complement system. Immunogenetics 58, 701– 713. (doi:10.1007/ s00251-006-0142-1) 17. Celniker SE, Rubin GM. 2003 The Drosophila melanogaster genome. Annu. Rev. Genomics Hum. Genet. 4, 89–117. (doi:10.1146/annurev.genom.4. 070802.110323) 18. Harman D. 1981 The ageing process. Proc. Natl Acad. Sci. USA 78, 7124 –7128. (doi:10.1073/pnas. 78.11.7124) 19. Edgar D et al. 2009 Random point mutations with major effects on protein coding genes are the driving force behind premature aging in mtDNA mutator mice. Cell Metab. 10, 131–138. (doi:10. 1016/j.cmet.2009.06.010) 20. Hiona A et al. 2010 Mitcohondrial DNA mutations induce mitochondrial dysfunction, apoptosis and sarcopenia in skeletal muscle of mitochondrial mutator mice. PLoS ONE 5, e11468. (doi:10.1371/ journal.pone.0011468) 21. Doonan R, McElwee JJ, Matthijssens F, Walker GA, Houthoofd K, Back P, Matscheski A, Vanfleteren JR, Gems D. 2008 Against the oxidative damage theory of aging: superoxide disumatases protect against oxidative stress but have little or no effect on life span in Caenorhabditis elegans. Genes Dev. 22, 3236–3241. (doi:10.1101/gad. 504808) 22. Mesquita A et al. 2010 Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H 2 O 2 and superoxide dismutase activity. Proc. Natl Acad. Sci. USA 107, 15 123–15 128. (doi:10.1073/pnas.1004432107) 23. Zhang Y et al. 2009 Mice deficient in both Mn superoxide dismutase and glutathione peroxidase-1 have increased oxidative damage and a greater incidence of pathology but no reduction in longevity. J. Gerontol. A Biol. Sci. Med. Sci. 64, 1212– 1220. (doi:10.1093/gerona/glp132) rsbl.royalsocietypublishing.org Biol. Lett. 11: 20150073 4 on March 18, 2015http://rsbl.royalsocietypublishing.org/Downloaded from

Original Source: https://www.researchgate.net/publication/273781783_Near-infrared_light_increases_ATP_extends_lifespan_and_improves_mobility_in_aged_Drosophila_melanogaster