Blue light and aging

To investigate whether light affects Drosophila longevity, we first compared the lifespan of white (w1118, hereafter w) adult flies kept in daily cycles of 12-h white fluorescent light alternating with 12 h of darkness (L:D) or in constant darkness (D:D). Survival of flies in D:D was significantly extended compared with those in L:D (Log-rank test, p < 0.0001) and their median lifespan was extended by 42% (Fig. 1a). The difference in mortality could be caused by delayed aging or by other factors. Aging in flies is associated with slower climbing up the vial walls, which can be measured by using the rapid iterative negative geotaxis (RING) assay.13 To determine whether the lifespan extension of D:D flies was associated with delayed aging, we measured vertical locomotion by RING. Middle-aged (30-day-old) males kept in D:D had significantly better average climbing ability than flies kept in L:D (Fig. 1b), suggesting that reduced lifespan of L:D flies may be due to accelerated aging. The lack of pigment granules in the retina makes w flies sensitized to light;14 therefore, we also tested whether the longevity of wild-type Canton S (CS) flies was affected by light. Indeed, the lifespan of CS males and females was significantly reduced in L:D compared with D:D (Log-rank test, p < 0.0001), albeit not as strongly as in w flies (Fig. 1c). Consistent with these results, 30-day-old CS flies in L:D showed a trend toward reduced average climbing ability, which became statistically significant at a later age of 50 days (Fig. 1d).

Fig. 1 White fluorescent light shortens fly lifespan and decreases mobility. a Adult white (w) flies aged in constant darkness (D:D) have a significantly extended lifespan compared with those aged in white fluorescent light (L:D) (Log-rank test, p < 0.0001). b Average climbing ability was significantly lower in 30-day-old w males kept in L:D versus those kept in D:D (unpaired t test, p = 0.0066). c Canton S (CS) adult flies aged in D:D have a significantly extended lifespan compared with those aged in L:D (Log-rank test, p < 0.0001). d Average climbing ability was lower but not significant in 30-day-old CS males kept in L:D versus those kept in D:D, and significantly lower in 50-day-old CS males kept in L:D versus those kept in D:D (unpaired t test, p = 0.0434). For longevity experiments in (a, c), N = 100 for each genotype and light condition. Numbers above bars indicate the sample size in each light condition. Error bars show standard error of the mean (SEM) Full size image

The spectral composition of light used in the above experiments showed a substantial blue component (Supplementary Fig. 1a); therefore, we tested the contribution of blue wavelengths commonly used in human environments (LED with peak wavelength at ~460 nm) to the lifespan reduction. Lifespan was measured in flies kept in daily cycles of 12-h blue LED light and 12 h of darkness (B:D), or white LED light with blue wavelengths blocked by a yellow filter (W–B:D) (Supplementary Fig. 1b). To equalize the amount of exposure across light sources, all light sources hereafter were adjusted to emit similar photon flux density (PFD) as L:D, ranging from 20 to 30 µmol m−2 s−1, at the level where flies were kept. Compared with flies aged in D:D, the median lifespan of w flies was reduced by ~50% in B:D but only by 4% in W–B:D light (Fig. 2a). Likewise, blue light caused a more dramatic (~30%) reduction in the median lifespan of CS flies compared with W–B light, which shortened median lifespan by ~10% (Fig. 2b). We also determined that the lifespan reduction of both w and CS flies corresponded to increased intensity of blue light (Fig. 2c, d). Pairwise comparisons of mortality curves showed a dose-dependent effect, namely, increasing PFD from 4 to 11, from 11 to 17, and from 17 to 24 µmol each caused a significant increase in mortality (Log-rank tests with Bonferroni multiple correction, p < 0.0001). Taken together, these results suggest that irradiation by blue wavelengths is mainly responsible for the reduced longevity of flies exposed to light.

Fig. 2 Light in the blue spectrum is responsible for the decrease in fly lifespan. Lifespan of w a and CS b flies is dramatically reduced in B:D compared with D:D (Log-rank test, p < 0.0001), but minimally reduced in flies aged under white LEDs lacking blue wavelengths by means of a yellow filter (W–B:D). Median lifespan of w c and CS d flies in B:D is reduced with increasing photon flux density (PFD). Statistics shown are from pairwise comparisons of the corresponding mortality curves that showed a dose-dependent effect, increasing PFD from 4 to 11, from 11 to 17, and from 17 to 24 µmol each, causing a significant increase in mortality (Log-rank tests with Bonferroni multiple correction, p < 0.0001). Note that the y axis does not start at 0 to highlight these differences in median lifespan. e Survival of w and CS males kept in B:D or in B:D with added orange light (B + O:D). f Mortality curves of the white-eyed ninaE8 and red-eyed ninaE7 mutants in B:D and D:D. In all of the above experiments, N = 100 for each genotype and light condition Full size image

Blue light activates Rhodopsin 1, the prevalent opsin in the fly retina, which then requires exposure to orange light in order to regenerate.15 To test whether lack of orange light may contribute to the reduced lifespan, we kept flies under B:D alone or B:D of similar intensity with the addition of orange LED light (peak at 600 nm, 1.5 µmol m−2 s−1) to allow for Rhodopsin regeneration. Median lifespan of both w and CS flies was not extended by the addition of orange light (Fig. 2e), suggesting that defects in rhodopsin processing are not responsible for the reduced lifespan of flies maintained in blue light. It was reported that blue-light-induced photoreceptor death is ameliorated by mutations in the gene encoding Rhodopsin 1 (ninaE), which disrupt phototransduction;9 therefore, we tested the effects of blue light on the lifespan of ninaE7 and ninaE8 mutants, both with reduced rhodopsin levels.9,16 The lifespan of white-eyed ninaE8 flies was shortened significantly in B:D compared with D:D (Log-rank test, p < 0.0001) with median lifespan reduced by 21% (Fig. 2f). The lifespan of red-eyed ninaE7 flies was also shortened significantly in B:D compared with D:D (Log-rank test, p < 0.0001), with median lifespan reduced by 9% (Fig. 2f). The fact that the magnitude of lifespan reduction was smaller in mutants with impaired phototransduction than in w or CS flies suggests that phototransduction may partially contribute to the detrimental effects of blue light.

Blue light acts in the entrainment of the circadian clock even at low intensities;17 however, we reasoned that levels of blue light that negatively affect longevity could have damaging effects on the clock. To test this, we recorded locomotor activity of flies held in L:D or B:D cycles for 5 days and then transferred to D:D for 5 days. Flies in both L:D and B:D showed prominent morning and evening activity peaks; however, B:D flies were more active throughout the entire light phase, especially at younger ages (Supplementary Fig. 2). Upon transfer to D:D, young flies from both regimes showed strong free-running circadian rhythms (Supplementary Fig. 2), suggesting that light used in this study is not damaging to the clock. Given these results, we then tested whether disruption of the circadian clock increases the susceptibility to blue light, as it is known that an intact clock confers resistance to many stresses.18,19 We determined that the lifespan of flies with disrupted clocks due to a mutation in the core clock gene period (per01) was not reduced in B:D compared with w control flies with an intact clock (Supplementary Fig. 3), suggesting that a functional clock is not protective against the blue-light exposure used in our experiments.

It has been reported that mammalian and fly retinal photoreceptor cells subjected to acute strong blue light become damaged;9,20 therefore, we asked whether photoreceptor cells are affected by daily 12-h exposure to moderate blue light. The fly retina consists of ~800 identical units called ommatidia, containing 6 outer and 2 inner photoreceptor cells (PR), each possessing a rhabdomere consisting of tightly packed microvilli where the phototransduction occurs. We examined histologically the health of the PRs in w and CS flies kept in D:D or B:D by counting the number of identifiable rhabdomeres (arrows, Fig. 3) on the same area of retinal cross sections in different conditions. At the age of 35 days, w and CS flies in D:D showed the regular arrangement of PRs with the dark rhabdomeres clearly distinguishable (Fig. 3a, b). In contrast, retinal degeneration and disorganized rhabdomeres were evident at this age in flies kept in B:D (Fig. 3a, b). A quantification confirmed a significant reduction in the average number of distinct rhabdomeres in both w and CS flies in B:D relative to D:D (Fig. 3c, d). By comparing rhabdomere loss between genotypes in B:D, we determined that it was more significant (unpaired t test, p = 0.0018) in w flies than in CS flies with normal eye pigmentation. This is consistent with higher PR degeneration reported previously in w flies in unspecified light conditions.21

Fig. 3 Retinal photoreceptors degenerate under blue light in flies with white or red eyes. Representative retinal cross sections of 35-day-old w a and CS b males in D:D and B:D. Red arrows point to identifiable rhabdomeres. c, d The average number of rhabdomeres is significantly reduced in 35-day-old w and CS males in B:D compared with D:D (unpaired t test, ****p < 0.0001). Numbers above bars indicate the sample size in each light condition. Error bars show SEM Full size image

Since PR damage occurred even in wild-type flies with normal eye pigmentation, we next asked whether deeper brain tissues are affected by blue-light exposure. To examine the central brain, heads of CS flies aged in D:D or B:D for 52 days were sectioned to measure the size of vacuoles indicative of neuronal loss. A significant increase in the average area of brain vacuolization was detected in CS flies in B:D compared with age-matched flies in D:D (Fig. 4).

Fig. 4 Blue-light exposure leads to neurodegeneration in the aging fly brain. a Representative brain sections showing brain vacuoles (red arrows) in 52-day-old CS males in D:D compared with B:D. b Average area of vacuoles is significantly higher in B:D (unpaired t test, p = 0.0165). Numbers above bars indicate the sample size in each light condition. Error bars show SEM Full size image

The observation that blue-light exposure leads to damage in both PR and the brain raised the question of whether PR degeneration is causally involved in brain neurodegeneration, or alternatively, whether blue light affects the brain independent of the retinal status. To address this, we used eyes-absent (eya2) mutants,22 which do not develop compound eyes and thus lack PRs. The lifespan of eya2 flies was significantly shortened in B:D compared with D:D (Log-rank test, p < 0.0001), with median lifespan reduced by 37% for males and 42% for females (Fig. 5a). In contrast, median lifespan was reduced by only 6% and 4%, respectively, in males and females kept in white light with blue wavelengths blocked (W–B:D) compared with flies kept in D:D (Fig. 5a). Climbing ability was also severely compromised in eya2 flies in B:D compared with D:D (Fig. 5b). As in flies with normal eyes, this behavioral deficit was associated with a significant degree of brain degeneration, measured as an increased area of vacuoles in B:D eya2 flies (Fig. 5c, d). In an additional experiment, we measured the lifespan of another mutant lacking PRs, sine oculis (so1), and found that their lifespan was also significantly shortened by blue light; the median lifespan of so1 in B:D was reduced by 19% compared with D:D (Supplementary Fig. 4). Together, these data suggest that accelerated mortality and locomotor impairments of flies maintained in B:D may occur independently of retinal damage. We hypothesize that brain neurodegeneration is a culprit in accelerating aging; however, other organs not studied here may be also involved.

Fig. 5 Flies lacking retina show reduced lifespan and brain neurodegeneration in blue light. a Lifespan of eyes-absent mutant (eya2) flies is significantly reduced in B:D compared with D:D (Log-rank test, p < 0.0001 for males and females), but is similar in W–B:D conditions (N = 100 for each light condition). b Aged eya2 males show a significant reduction in the average vertical climbing ability in B:D compared with D:D (unpaired t test, p = 0.0009). c Representative brain sections showing brain vacuoles (red arrowheads) in 52-day-old eya2 males in D:D and B:D. d The average area of brain vacuolization of 52-day-old eya2 males was significantly increased in B:D compared with D:D (unpaired t test, p = 0.0352). Numbers above bars indicate the sample size in each light condition. Error bars show SEM Full size image

To begin investigating molecular pathways mediating the damaging action of blue light on the brain, we first considered cryptochrome, the blue-light-sensitive photoreceptor protein encoded by the gene cry. In flies, the CRY protein is the major light sensor for the entrainment of the circadian clock,23,24 and it is involved in modulation of neuronal activity and behavior by blue light.25,26 To test whether CRY could mediate the phototoxicity of blue light, we measured the lifespan of flies with genetically manipulated cry expression held in B:D or D:D. We found that neither a null mutation in the cry gene nor overexpression of cry affected survival in B:D conditions (relative to D:D) compared with their respective controls (Supplementary Fig. 5a), suggesting that CRY is not involved in the lifespan alterations caused by blue light. In addition to cry, we tested whether the recently identified Rhodopsin 7 (Rh7) plays a role in inducing the aging phenotypes. RH7 protein is sensitive to blue light and its mRNA is weakly expressed in both the brain and the retina.27 We determined that median lifespan was similarly shortened in B:D relative to D:D, both in Rh71 mutants and in flies overexpressing Rh7, compared with their respective controls (Supplementary Fig. 5b), suggesting that this chromoprotein is not involved in mediating the effects of daily blue-light exposure on longevity. We note that it is still possible that removing all photoreceptive pathways (i.e., cry and rhodopsins together) could reduce blue-light-induced damage.

What are the proximate causes of premature aging of flies in B:D? Our recent RNA-seq study comparing the diurnal transcriptome in heads of young and old flies demonstrated that several stress-response genes are upregulated in heads of 55-day-old w flies kept in L:D 12:12 cycles, and their maximal expression over a 24-h period occurred after 12 h of light exposure.28 These genes also become induced in young flies kept in L:D but subjected to oxidative stress by treatment with 100% oxygen.28 Given our observation that L:D shortens the lifespan in flies (Fig. 1), and reports that blue light induces oxidative stress in retinal cells9,20 and in the nematode Caenorhabditis elegans,29 we tested whether blue light increased the expression of genes known to be induced by oxidative stress. The expression of selected stress-response genes was measured in heads of day 5 or day 35 w flies maintained in B:D and collected at the end of their daily 12 h of blue-light exposure. To discern the effects of light, we collected simultaneously 5- or 35-day-old w flies maintained in D:D; these flies are expected to show average expression of diurnal genes due to the absence of clock entrainment by light. Some of the known oxidative stress-response genes (Gclc, GstO1) were not upregulated in B:D; however, expression of several other genes was significantly increased in 35-day-old flies in B:D compared with age-matched D:D controls (Fig. 6a). These included cnc (the fly homolog of the transcription factor Nrf2), thioredoxin reductase Trxr-1, glutathione S transferases GstD1 and GstD2, and several heat-shock proteins: Hsp23, Hsp68, and Hsp70. Most of the examined genes (with the exception of Gclc, Trxr-1, and GstD2) did not increase expression in 35-day-old D:D flies compared with 5-day-old D:D flies, suggesting that blue light plays a much bigger role in upregulation of stress-response genes than aging by itself. We also observed strong upregulation of the metabolic gene, lactate dehydrogenase (Ldh), which is known to increase with aging and stress.28 Importantly, none of the examined genes showed an increase in 5-day-old flies kept in B:D compared with D:D, suggesting that the cumulative action of blue light over many days is needed to induce stress-response genes, or that response to blue light is age-dependent (Fig. 6a). To explore these possibilities further, we tested survival of flies exposed to B:D or D:D for a set number of days and then switched to the opposite conditions. We kept w flies in B:D throughout their life, or for the first 25 or 30 days of adult life followed by a transfer to D:D, and compared their lifespan. As shown in Fig. 6b, exposure for the first 25 days of adulthood caused some flies to die within a few days, but most of the remaining flies survived nearly as long as flies that were always kept in D:D. However, exposing flies to B:D for the first 30 days of adulthood (only 5 days longer than in the previous experiment) followed by a transfer to D:D resulted in the majority of flies dying shortly after the switch to D:D. These flies had a median lifespan of 34 days, similar to the 33 days of controls kept continually in B:D. In a reverse experiment, we kept flies in D:D for 30 days and then exposed them to B:D for the rest of their lives. The median survival of these 30-day-old flies was 21 days after the switch to B:D, while the median lifespan of young flies exposed to B:D was 34 days. These results suggest two conclusions. First, blue light has cumulative damaging effects, but the damage can be halted upon removal of this type of stress, provided that it does not accumulate beyond a certain irreversible threshold that causes death. Second, blue-light damage affects flies differently across their lifespan with vulnerability to this part of the visible spectrum increasing with age. In other words, blue-light-induced damage seems to accumulate faster with advancing age.