Dietary Restriction and Clock Delay Eye Aging to Extend Lifespan in D

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Dietary restriction and clock delay eye aging to extend lifespan in D. melanogaster

2 Brian A. Hodge1*, Geoffrey T. Meyerhof1,4, Subhash D. Katewa1,2, Ting Lian1,3, Charles

3 Lau1, Sudipta Bar1, Nicole Leung4,5, Menglin Li4, David Li-Kroeger6, Simon Melov1, Birgit

4 Schilling1, Craig Montell4, Pankaj Kapahi1*

5 1Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA 94945, USA.

6 2NGM Biopharmaceuticals, 333 Oyster Point Blvd, South San Francisco, CA 94080, USA.

7 3Sichuan Agricultural University, 46 Xinkang Rd, Yucheng District, Ya'an, Sichuan, China.

8 4Neuroscience Research Institute and Department of Molecular, Cellular and

9 Developmental Biology, University of California, Santa Barbara, Santa Barbara, 93106,

10 California, USA.

11 5Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA

12 94305, USA; Department of Neurobiology, Stanford University, Stanford, CA 94305, USA.

13 6Department of Neurology, Baylor College of Medicine, Houston, TX 77096, USA.

15 *Correspondence: [email protected], [email protected]

1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

25 Abstract

26 Many vital processes in the eye are under circadian regulation, and circadian dysfunction

27 has emerged as a potential driver of eye aging. Dietary restriction is one of the most

28 robust lifespan-extending therapies and amplifies circadian rhythms with age. Herein, we

29 demonstrate that dietary restriction extends lifespan in D. melanogaster by promoting

30 circadian homoeostatic processes that protect the visual system from age- and light-

31 associated damage. Disrupting circadian rhythms in the eye by inhibiting the transcription

32 factor, Clock (CLK), or CLK-output genes, accelerated visual senescence, induced a

33 systemic immune response, and shortened lifespan. Flies subjected to dietary restriction

34 were protected from the lifespan-shortening effects of photoreceptor activation.

35 Inversely, photoreceptor inactivation, achieved via mutating rhodopsin or housing flies in

36 constant darkness, primarily extended lifespan in flies reared on a high-nutrient diet. Our

37 findings establish the eye as a diet-sensitive modulator of lifespan and indicate that vision

38 is an antagonistically pleiotropic process that contributes to organismal aging.

2 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

48 Introduction

49 Circadian rhythms are approximate 24-hour oscillations in behavior, cellular physiology,

50 and biochemistry, which evolved to anticipate and manage predictable changes

51 associated with the solar day (e.g., predator/prey interactions, nutrient availability,

52 phototoxicity, etc.) [1]. Circadian rhythms are generated by endogenous clocks that sense

53 time-cues (e.g., light and food) to govern rhythmic oscillations of gene transcriptional

54 programs, synchronizing cellular physiology with daily environmental stressors [2]. In

55 addition to keeping time, the molecular clock regulates the temporal expression of

56 downstream genes, known as clock-controlled genes, to promote tissue-specific rhythms

57 in physiology [3]. The Drosophila molecular clock is comprised of transcriptional-

58 translational feedback loops, where the transcription factors Clock (CLK) and Cycle (CYC)

59 rhythmically activate their own repressors, Period and Timeless [2]. This feedback loop not

3 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

60 only exists in central pacemaker neurons, where it sets rhythms in locomotor activity, it

61 also functions in peripheral tissues, such as the eye [4].

62 Aging is associated with a progressive decline in visual function and an increase in

63 the incidence of ocular disease. Drosophila photoreceptor cells serve as a powerful model

64 of both visual senescence and retinal degeneration [5, 6]. Drosophila and mammalian

65 photoreceptors possess a cell-intrinsic molecular clock mechanism that temporally

66 regulates a large number of physiological processes, including light-sensitivity,

67 metabolism, pigment production, and susceptibility to light-mediated damage [7]. Visual

68 senescence is accompanied by a reduced circadian amplitude in core-clock gene

69 expression within the retina [8]. This reduction in retinal circadian rhythms may be causal

70 in eye aging, as mice harboring mutations in their core-clock genes, either throughout

71 their entire body, or just in their photoreceptor cells, display several early-onset aging

72 phenotypes within the eye. These mice prematurely form cataracts and have reduced

73 photoreceptor cell light-sensitivity and viability [8]. However, the molecular mechanisms

74 by which the molecular clock influences eye aging are not fully understood.

75 Dietary restriction (DR), defined by reducing specific nutrients or total calories, is

76 the most robust mechanism for delaying disease and extending lifespan [9]. The

77 mechanisms by which DR promotes health and lifespan may be integrally linked with

78 circadian function, as DR enhances the circadian transcriptional output of the molecular

79 clock and preserves circadian function with age [10]. Inversely, high-nutrient diets (i.e.,

4 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

80 excess consumption of protein, fats, or total calories) repress circadian rhythms and

81 accelerate organismal aging [11, 12]. However, how DR modulates circadian rhythms

82 within the eye, and how these rhythms influence DR-mediated lifespan extension, had yet

83 to be examined.

84 Herein, we sought to elucidate the circadian processes that are activated by DR by

85 performing an unbiased, 24-hour time-course mRNA expression analysis in whole flies.

86 We found that circadian processes within the eye are highly elevated in expression in flies

87 reared on DR. In particular, DR enhanced the rhythmic expression of genes involved in the

88 adaptation to light (i.e., calcium handling and de-activation of rhodopsin-mediated

89 signaling). Building on this observation, we demonstrate that the majority of these

90 circadian phototransduction components were transcriptionally regulated by CLK.

91 Eliminating CLK function either pan-neuronally, or just in the photoreceptors, accelerated

92 visual decline with age. Furthermore, disrupting photoreceptor homeostasis increased

93 systemic immune responses and shortened lifespan. Several eye-specific CLK-output

94 genes that were upregulated in expression in response to DR, were also required for DR-

95 to slow visual senescence and extend lifespan.

98 Results

5 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

99 Dietary restriction amplifies circadian transcriptional output and delays visual senescence

100 in a CLK-dependent manner

101 To determine how DR changes circadian transcriptional output, we performed a series of

102 microarray experiments over the span of 24-hours in female Canton-S flies (whole body)

103 reared on either a high-yeast (5%; ad libitum, AL) diet or a low-yeast (0.5%; DR) diet

104 (Supplementary Fig. 1a). Flies maintained on DR displayed nearly twice the number

105 circadian transcripts compared to flies on AL (Fig. 1a, b and Supplementary Fig. 1b).

106 Circadian gene expression was also more robust on DR vs AL. DR-specific oscillators were

107 statistically more rhythmic (lower JTK_CYCLE circadian p-values) and displayed larger

108 circadian amplitudes than AL-specific oscillators (Supplementary Fig. 1c, d). Diet also

109 drastically altered the circadian transcriptional profile, as only 16% of DR oscillators were

110 also oscillating on AL (Supplementary Fig. 1b). Furthermore, the AL and DR circadian

111 transcriptomes were enriched for distinct processes (Supplementary Fig. 1e, f and

112 Supplementary Data 1).

113 Transcripts that oscillate on both AL and DR diets were highly enriched for genes

114 that comprise the canonical phototransduction signaling cascade (Fig. 1c-d), which is the

115 process by which Drosophila photoreceptor cells, the primary light-sensitive neurons,

116 transduce light information into a chemical signal [13]. Briefly, light-mediated conversion

117 of rhodopsin proteins to their meta-rhodopsin state stimulates heterotrimeric Gq proteins

118 that activate phospholipase C (norpA), which produces secondary messengers and

6 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which a Circadianwas on not DR certified byb peer review) Fold-changeis the author/funder, who hasFigure granted bioRxiv1. Dietary a license to restrictiondisplay the preprint amplifies in perpetuity. Itcircadian is made tran- available under aCC-BY-NC-ND 4.0 International license. -3 30 Circadian on AL scriptional output and rhythmicity of phototransduction

genes. (a-c) Circadian transcriptome heatmaps for Can-

ZT 0 4 8 12 16 20 0 4 8 12 16 20 ton-S flies representing 24-hour expression plots for tran- (hr) AL DR

c Circadian on AL and DR scripts that cycle only on DR (a, n=1609 transcripts), only on AL (b, n=568 transcripts), or on both diets (c, n=301

ZT 0 4 8 12 16 20 0 4 8 12 16 20 ZT 0 4 8 12 16 20 0 4 8 12 16 20 (hr) AL DR (hr) AL DR transcripts). Circadian transcripts (24h period, JTK_CY- d Circadian on AL & DR e Fold-change phototransduction Phototransduction −2 0 2 CLE pvalue<0.05) are plotted by phase. (d) Gene-ontology response to pheromone ninaA inaD long-chain fatty-acyl-CoA inaC metabolic process Arr1 trpl enrichment categories corresponding to transcripts that olfactory behavior norpA entrainment of ninaC circadian clock Rh6 Rh5 trp cycle on both AL and DR diets. (e) Heatmap of phototrans- 0 5 10 15 ZT 0 4 8 12 16 20 0 4 8 12 16 20 P value (-log10) (hr) f AL DR duction transcript expression on AL and DR. (f) Pho- Phototransduction cascade

Rh5/6 Meta-Rh totransduction cascade diagram with components colored

inaC β β norpA Gqα Gqα Trp according to their circadian phase on DR. Arr1 γ γ inaD Ca2+ Trpl

cry ninaA ninaC F-actin

Phase (ZT) 0-5 6-11 12-17 ER bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

119 promotes the opening of Transient Receptor Potential channels (TRP, TRPL), ultimately

120 allowing Ca2+ and Na+ to depolarize the photoreceptor cell [14]. Although the

121 phototransduction transcripts were cyclic on both diets, on DR their expression became

122 more rhythmic (lower JTK_CYCLE p-values & larger circadian amplitudes) and elevated

123 (~2-fold increase in expression across all timepoints) (Fig. 1e and Supplementary Fig. 1i).

124 Since our time-course analyses were perfromed in whole-fly, we queried publicly available

125 circadian transcriptomes from wild-type heads to further investigate the rhythmic

126 oscillations of eye-related transcripts [15]. The majority of the DR-sensitive

127 phototransduction genes also robustly cycled in wild-type heads (Supplementary Table

128 1). Furthermore, the GO-term “phototransduction” (GO:0007602) was amongst the most

129 enriched cyclic processes in the heads of wild-type flies, as ~70% of the genes that

130 comprise the category oscillate in a circadian fashion (Supplemental Data 2).

131 In Drosophila and mammals, visual function oscillates to align with daily changes

132 in ambient illuminance from the sun, which can be 106 to 108-fold brighter during the day

133 than at night [16]. Photoreceptors are unique in that they have evolved mechanisms

134 responsible for maintaining homeostasis in the presence of light-induced calcium ion

135 gradients that are magnitudes greater than what other neuronal populations experience

136 [17, 18]. Mechanisms of light adaptation within photoreceptors include the rapid

137 (millisecond) closure of TRP channels (facilitated via enzymes scaffolded by inaD),

138 rhodopsin internalization from the rhabdomere membrane (e.g., arr1, arr2), and calcium

7 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

139 efflux (e.g., calx) [19, 20]. Acrophase analyses (i.e., time of peak expression) revealed that

140 circadian transcripts that promote photoreceptor activation (Ca2+ influx) reach peak

141 expression during the dark-phase, while genes that terminate the phototransduction

142 response (i.e., deactivation of rhodopsin mediated signaling) peak in anticipation of the

143 light-phase (Fig. 1f and Supplementary Fig. 1j). These findings provide a potential

144 mechanistic explanation for the rhythmic response pattern in light-sensitivity observed in

145 Drosophila photoreceptors and suggests that DR’s ability to delay visual senescence is

146 mediated in part by amplifying circadian rhythms within photoceptors (See Supplemental

147 Discussion 1 for additional interpretations).

148 To determine if molecular clocks mediate the enhanced rhythmic expression of

149 phototransduction genes on DR, we measured the transcriptome of fly heads with pan-

150 neuronal over-expression of a dominant negative form of the core-clock factor, CLK (Elav-

151 GS-GAL4>UAS-CLK-1; denoted nCLK-1) (Supplementary Fig. 2a). To avoid potential

152 developmental defects related to Clk disruption, we used a drug-inducible (RU486) “gene-

153 switch” driver to express CLK-∆ in adult flies. Genes downregulated in nCLK-1 heads

154 were enriched for light-response pathways, including “response to light stimulus” and

155 “deactivation of rhodopsin signaling” (Fig. 2a, b and Supplemental Data 3). Additionally,

156 genes that were both circadian in wild-type heads and downregulated in nCLK-1 were

157 highly enriched for homeostatic processes related to eye function (Supplementary Fig. 2c

8 a DownregulatedbioRxiv preprint in nCLK- doi:Δ1 https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made response to light stimulus available under aCC-BY-NC-NDFigure 4.02. DietaryInternational restriction license. delays visual senescence in a

cellular calcium ion homeostasis CLK-dependent manner. (a) GO enrichment scores corre- phototransduction sponding to downregulated light-response genes in heads deactivation of rhodopsin mediated signaling from RNA-Seq of nCLK-Δ1 (Elav-GS-GAL4> AL circadian rhythm DR

0 5 10 15 UAS-CLK-Δ1) vs controls. (b) Heatmap of normalized P value (-log10) b Fold-change Deactivation of rhodopsin mediated signaling -2 0 2 RNA-Seq expression corresponding to the gene-ontology Arr2 Gβ76C inaD sunglasses category “Deactivation of rhodopsin mediated signaling” ninaC Gprk1 rtp Δ inaC (GO:0016059) in nCLK- 1 and controls at zeitgeber times 0 Arr1 Ekar norpA Camta and 12 (lights on and lights off, respectively). (c) Positive inaF−B rdgB AL cnt AL Δ1 DR cnt DR Δ1 AL cnt AL Δ1 DR cnt DR Δ1 phototaxis responses for nCLK-Δ1 flies. For each timepoint ZT 0 ZT 12 c AL cnt AL nCLK-Δ1 DR cnt DR nCLK-Δ1 0.061 2.72e-5 results are represented as average percent positive photo- 5.52e-10 8.40e-7 0.722 0.016 5.50e-8 0.459 0.030 8.05e-6 100 0.030 4.74e-9 3.40e-8 1.53e-4 taxis +/- SEM (n=24 biological replicates, N=480 flies per 80 2.00e-8 condition). (d) Boxplots of electroretinogram amplitudes 60

40 for nCLK-Δ1 flies and controls at day 14 and 21. (e) Posi-

Phototaxis index (% ) 20 tive phototaxis responses for prCLK-Δ1 flies (Trpl-GAL4;

0 ts 2 6 10 14 18 GAL80 > UAS-CLK-Δ1) and control flies (Trpl-GAL4; Time (days) d 30 0.030 AL cnt DR Cnt ts AL nCLK-Δ1 DR nCLK-Δ1 0.027 GAL80 >CantonS) reared at 30°C. For each timepoint 0.219

20 results are represented as average percent positive photo-

0.628 0.002 0.143 taxis +/- SEM (n=16 biological replicates, N=320 flies per 10 Amplitude (mV ) condition). (f) Boxplots of electroretinogram amplitudes

0 for prCLK-Δ1 and control flies reared at 30°C. Illuminance 14 21 Time (days) e AL cnt AL prCLK-Δ1 DR cnt DR prCLK-Δ1 was set at 150 Lux. (c-f) Pvalues were determined by 0.116 0.020 2.92e-4 2.85e-9 100 0.170 0.247 1.23e-7 3.87e-4 two-tailed Student’s ttest (unpaired), comparing responses

80 3.17e-7 3.49e-6 6.97e-5 between diet and/or genotype at each timepoint. 8.02e-9 60 8.61e-5

7.95e-4 40

1.16e-4

Phototaxis index (% ) 20

0 2 6 10 14 18 Time (days) f 0.161 0.538 AL cnt DR cnt 8 0.361 AL prCLK-Δ1 DR prCLK-Δ1 0.059 0.005

6 7.76e-5

0.006 4 0.559 1.26e-5 0.003 0.004 3.64e-4

Amplitude (mV ) 2

0 2 6 10 14 Time (days) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

158 and Supplemental Data 4). Together, this indicates that CLK governs the circadian

159 transcriptional regulation of many eye-related processes in Drosophila.

160 Given DR’s ability to improve homeostasis across an array of tissues [21], and its

161 ability to enhance the circadian rhythmicity of light-response genes, we examined how

162 diet and clocks influence visual function with age. We longitudinally quantified the

163 positive phototaxis response of wild-type flies (Canton-S and Oregon-R) reared on either

164 AL or DR diets (experimental setup in Supplementary Fig. 2d). Compared to AL-fed flies,

165 DR slowed the decline in positive phototaxis observed with age (Supplementary Fig. 2e,

166 f). Importantly, this effect cannot solely be attributed to diet-dependent changes in

167 locomotor activity, as climbing activity and phototaxis declined at different rates with age

168 (Supplementary Fig. 2g). Compared to wild-type flies, DR minimally protected Clkout (Clk-

169 null) flies from age-related declines in phototaxis (Supplementary Fig. 2h). nCLK-1 and

170 nCLK-2 (an additional dominant negative Clk mutant, Elav-GS-GAL4>UAS-CLK-2) flies

171 displayed accelerated declines in positive phototaxis with age compared to controls (Fig.

172 2c and Supplementary Fig. 2i). Since the positive phototaxis assay measures a behavioral

173 response to light, we next evaluated how diet and CLK directly influence photoreceptor

174 function with age by performing extracellular electrophysiological recordings of the eye

175 (electroretinograms, ERG [22]). We observed larger ERG amplitudes, i.e. the light-induced

176 summation of receptor potentials from the photoreceptors [23], in control flies reared on

9 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

177 DR vs AL at day 14 (Fig. 2d). Furthermore, the DR-mediated enhancements in the ERG

178 amplitudes were significantly reduced in nCLK-1 flies with age (Fig. 2d).

179 Since the Elav-GS-GAL4 driver is expressed in a pan-neuronal fashion (i.e.,

180 photoreceptors + extra-ocular neurons), we sought to examine how disrupting CLK

181 function solely within photoreceptors influences visual function with age. To this end, we

182 crossed UAS-CLK-1 flies with a photoreceptor-specific GAL4 driver line under the

183 temporal control of the temperature sensitive GAL80 protein (Trpl-GAL4; GAL80ts>UAS-

184 CLK-1, denoted prCLK-1). To avoid disrupting CLK function during development,

185 prCLK-1 flies were raised at 18C (GAL80 active, GAL4 repressed) and then transferred

186 to 30C (GAL80 repressed, GAL4 active) following eclosion. When compared to control

187 flies (Trpl-GAL4; GAL80ts>CantonS), prCLK-1 flies displayed accelerated declines in both

188 positive phototaxis and ERG amplitude with age, and in a similar fashion to the nCLK-1

189 flies (Fig. 2e-f). Together, our gene expression, phototaxis, and ERG data indicate that DR

190 functions in a CLK-dependent manner to delay photoreceptor aging in the fly.

191 nCLK- drives a systemic immune response and reduces longevity

192 Age-related declines in tissue homeostasis are accompanied by elevated immune

193 responses and inflammation [24, 25]. Interestingly, we found that genes upregulated in

194 nCLK-1 fly heads were significantly enriched for immune and antimicrobial humoral

195 responses (Fig. 3a, b). In Drosophila, damage-associated molecular patterns can induce a

196 sterile immune response that is characterized by the expression of anti-microbial peptides

10 a UpregulatedbioRxiv in preprint nCLK-Δ1 doi: https://doi.org/10.1101/2021.05.08.443272; this version posted MayΔ 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, whoFigure has granted 3. bioRxiv nCLK- a license1 flies to display display the preprint elevated in perpetuity. immune It is made respons- humoral immune response available under aCC-BY-NC-ND 4.0 International license. es and shortened lifespan. (a) GO enrichment scores corre- response to toxic substance sponding to upregulated inflammatory genes in heads from immune response

AL RNA-Seq of nCLK-Δ1 vs controls. (b) Heatmap of normal- response to wounding DR

0 5 10 15 20 P value (-log10) ized RNA-Seq expression corresponding to the gene-ontolo- Antimicrobial humoral response b PGRP−SD PGRP−LF gy category “Antimicrobial humoral responses” Mtk AttB par−1 Δ mask (GO:0019730) in nCLK- 1 and controls. (c) Relative expres- AttA cact AttD lola sion of AMP genes (AttA, DiptB, and Dro) calculated by CecC Dro PGRP−LB Def RT-qPCR with mRNA isolated from nCLK-Δ1 bodies. AttC DiptB AL cnt AL Δ1 DR cnt DR Δ1 AL cnt AL Δ1 DR cnt DR Δ1 Fold-change Results are plotted as average Log2 fold-change in expres- ZT 0 ZT 12 -2 0 2 c AL cnt AL nCLK-Δ1 ΔΔ 6 DR cnt DR nCLK-Δ1 sion calculated by the -Ct method, normalized to DR vehi- 8.48e-5 0.014 0.814 4 cle treated control samples, as well as the housekeeping gene 0.855 0.001 0.856

2 rp49 +/- SEM (n=3 biological replicates, N=30 flies per

0 biological replicate). (d) Relative mRNA expression of AMP

Expression (Log2 FC) -2 genes calculated by RT-qPCR with mRNA isolated from 0.028 1.45e-4 1.02e-4 bodies of eye-specific ATPα knockdown flies -4 AttA DiptB Dro d 6 (GMR-GAL4>UAS-ATPα-RNAi) vs RNAi control flies AL RNAi cnt AL ATPα-RNAi * DR RNAi cnt DR ATPα-RNAi (GMR-GAL4>UAS-mCherry-RNAi). Results are plotted as 4 0.016 0.085 0.046 0.231 0.058 average Log2 fold-change in expression calculated by the 2 0.445 ΔΔ-Ct method, normalized to DR RNAi control samples as

Expression (Log2 FC) 0 * * * well as housekeeping gene rp49 +/- SEM (n=3 biological replicates, N=30 flies per biological replicate). (e) Relative -2 AttA DiptB Dro 1118 1118 2 AttaA DiptB Dro e AL w w ;rh3 1118 G mRNA expression of immune genes ( , , and ) 1118 17 1118 1 w ;rh6 4 DR w ;ninaE w ;rh4 0.176 0.566 0.027 0.161 0.081 0.045 calculated by RT-qPCR with mRNA isolated from bodies of 0.271 0.280 2 0.509 0.769 w1118 and rhodopsin mutant flies housed in 12:12h LD.

0 0.159 Results are plotted as average Log2 fold-change in expres- 0.041 -2 0.037 sion calculated by the ΔΔ-Ct method normalized w1118 DR Expression (Log2 FC ) 0.232 0.006 0.040 0.008 0.080 -4 0.036 0.099 0.098 0.011 control samples as well as rp49 +/- SEM (n=3 biological 0.023 0.604 -6 AttA DiptB Dro replicates, N=30 flies per biological replicate). (f) f 100 Δ AL cnt Kaplan-Meyer survival analysis of nCLK- 1 flies 80 AL nCLK-Δ1 (Elav-GS-GAL4>UAS-CLK-Δ1). Survival data is plotted as DR cnt DR nCLK-Δ1 60 an average of three independent lifespan repeats. Control

40 flies (vehicle treated): AL N=575, DR N=526; nCLK-Δ1 flies Percent survival

20 (RU486 treated): AL N=570, DR N=565. (c-e) Pvalues were calculated with the pairwise Student’s ttest comparing Log2 0 0 20 40 60 80 100 Time (days) fold-changes in expression. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

197 (AMPs), similar to the effects from infections by pathogens [26]. We quantified the mRNA

198 expression of AMPs in the bodies of nCLK-1 and nCLK-2 flies to determine if neuronal

199 damage signals propagate throughout the body to drive systemic immune responses; the

200 Drosophila fat body generates high levels of AMPs in response to intrinsic damage signals

201 [26]. AMP expression (AttA, DiptB, and Dro) was reduced in control flies reared on DR

202 compared to AL, however nCLK-1 and nCLK-2 elevated AMP expression on DR (Fig. 3c

203 and Supplementary Fig. 3a). To further investigate this systemic inflammatory response,

204 we isolated and quantified hemolymph from nCLK-1 and control flies. In agreement with

205 the transcriptional activation of AMPs in both the heads and bodies of nCLK-1 flies, we

206 found the most highly upregulated protein in nCLK-1 hemolymph to be the

207 antimicrobial peptide, AttC (Supplementary Fig. 3b). Furthermore, we observed an

208 enrichment for proteins associated with translational activation (e.g., cytoplasmic

209 translation and ribosomal biogenesis) within the upregulated proteins in the nCLK-1

210 hemolymph, which may reflect the activation of hemocytes, the immune effector cells in

211 Drosophila (Supplemental Data 5) [27]. Taken together, these data demonstrate that

212 disrupting neuronal CLK function elevates systemic immune responses.

213 To determine if photoreceptor degeneration induces a systemic immune response

214 in Drosophila, we forced photoreceptor degeneration by knocking down ATP within the

215 eye (GMR-GAL4>UAS-ATP-RNAi), and quantified expression of AMPs within the bodies.

216 ATP encodes the catalytic alpha subunit of the Na+K+ATPase responsible for

11 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

217 reestablishing ion balance in the eye during light responses [28, 29]. Our decision to use

218 ATP knockdown as a model of photoreceptor degeneration was motivated by previous

219 reports indicating that its expression is under circadian regulation [30] and that its

220 knockdown in the eye results in aberrant ion homeostasis that drives age-dependent,

221 light-independent photoreceptor degeneration [31]. Ocular knockdown of ATP

222 rendered flies blind in both AL and DR conditions compared to controls (Supplementary

223 Fig. 3c). Knocking down ATP in the eye also drove the expression of AMPs within the

224 bodies of flies reared on either an AL or DR diet (Fig. 3d). Thus, DR fails to suppress

225 immune responses in the context of forced photoreceptor degeneration.

226 Since we found that photoreceptor degeneration induced systemic immune

227 responses, we postulated that reducing phototransduction should reduce inflammation.

228 To assess how stress from environmental lighting influences immune responses, we

229 analyzed a circadian microarray dataset comparing gene expression changes in wild-type

230 (y,w) heads in flies reared in 12hr light and 12hr darkness (12:12LD) or constant darkness

231 [32]. We found immune response genes to be among the most highly enriched processes

232 upregulated in the flies housed in 12:12LD vs constant darkness (Supplementary Fig. 3d,

233 e and Supplemental Data 8). We quantified AMPs within the bodies of flies harboring

234 rhodopsin null mutations to evaluate how the different photoreceptor subtypes influence

235 systemic immune responses. The Drosophila ommatidia consists of eight photoreceptors

236 (R1-8) that express different rhodopsins with varying sensitives to distinct wavelengths of

12 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

237 light [33]. The R1-6 photoreceptors express the major rhodopsin Rh1, encoded by ninaE,

238 while the R7 photoreceptor expresses either Rh3 or Rh4. The R8 photoreceptor expresses

239 either Rh5 or Rh6 [34]. The rhodopsin null mutants [ninaE [35], rh3 [36], rh4 [37] , or rh6

240 [38]] displayed reductions in immune marker expression in their bodies compared to w1118

241 outcrossed controls (Fig. 3e). Taken together, these findings indicate that suppression of

242 rhodopsin mediated signaling is sufficient to suppress systemic immune responses in

243 Drosophila.

244 Given the strong associations between chronic immune activation and accelerated

245 aging, we examined the lifespans of nCLK- flies [24]. Both nCLK-1 and nCLK-2 flies

246 displayed significantly shortened lifespans, with a proportionally greater loss in median

247 lifespan in flies reared on DR compared to AL (Fig. 3f and Supplementary Fig. 3f-h). nCLK-

248  flies have altered CLK function throughout all neurons, however, it is possible that the

249 lifespan-shortening effect observed in these lines was substantially driven by loss of CLK-

250 function within the eye; Others have demonstrated that CLK is highly enriched (>5-fold)

251 within photoreceptors compared to other neuronal cell types in Drosophila

252 (Supplementary Fig. 3i) [5]. Furthermore, over-expressing CLK-1 within just

253 photoreceptors (prCLK-1) also shortened lifespan (Supplementary Fig. 3j). These findings

254 argue that neuronal CLK function is required for the full lifespan extension mediated by

255 DR and indicate that photoreceptor clocks are essential for maintenance of visual function

256 with age and organismal survival.

13 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

257 DR protects against lifespan shortening from photoreceptor cell stress

258 Previous reports have demonstrated that exposure to light can decrease lifespan—

259 extending the daily photoperiod, or housing flies in blue light reduces longevity [39, 40].

260 Since DR delays visual senescence and promotes the rhythmic expression of genes

261 involved in photoreceptor homeostasis (i.e., light adaptation, calcium handling), we

262 investigated how diet influences survival in the context of light and/or phototransduction.

263 To test the interrelationship among diet, light, and survival, we housed w1118 (white-eyed)

264 in either a 12:12 LD cycle or constant darkness. Housing flies in constant darkness

265 extended the lifespan of flies reared on AL, while the lifespans of flies reared on DR were

266 unaffected (Fig. 4a). Constant darkness failed to extend the lifespan of red-eyed (w+)

267 Canton-S wild-type flies (Supplementary Fig. 4a), suggesting that the ATP-binding

268 cassette transporter encoded by w, and the red-pigment within the cone-cells, helps to

269 protect against lifespan shortening from diet- and light-mediated stress [41]. White-eyed,

270 photoreceptor null flies (homozygous for TRPP365 mutation [42]) reared on AL failed to

271 display lifespan extension in constant darkness (Supplementary Fig. 4b), indicating that

272 the lifespan shortening effects of light exposure are primarily mitigated by the

273 photoreceptors.

274 We performed survival analyses in rhodopsin null flies to examine how activation

275 of the different photoreceptor subtypes influence lifespan on AL and DR. In agreement

276 with the reduction in systemic immune responses observed in the rhodopsin null strains,

14 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made a 100 b 100 AL w1118 AL w1118 Figure 4. Photoreceptor activation modulates lifespan in a LD available under aCC-BY-NC-ND 4.0 International license. DR w1118 DR w1118 80 1118 80 1118 17 AL w DD AL w ; ninaE 1118 DR w1118 DR w1118; ninaE17 diet-dependent fashion. (a) Survival analysis of w flies 60 60 40 40 housed in 12:12h LD or constant darkness (DD). Survival Percent survival Percent survival 20 20 data is plotted as an average of three independent lifespan 0 0 0 20 40 60 80 0 20 40 60 80 Time (days) Time (days) repeats. LD housed flies: AL N=560, DR N=584; DD: AL 100 c 100 1118 d AL w 1118 1118 AL w 1118 17 DR w 1118 80 1118 2 80 DR w N=460, DR N=462. (b-f) Survival analysis of w ; ninaE , AL w ; rh3 1118 1 1118 2 AL w ; rh4 DR w ; rh3 1118 1 60 60 DR w ; rh4 w1118; rh32, w1118; rh41, w1118; rh6G, and w1118; Gqα1 mutants com- 40 40 1118 20 20 Percent survival Percent survival pared to w control flies housed in 12:12h LD. Survival data

0 0 0 20 40 60 80 100 0 20 40 60 80 100 is plotted as an average of three independent lifespan Time (days) Time (days) e 100 f 100 1118 AL w1118 AL w1118 repeats. *Survival curves for w are re-plotted (b-f) for DR w1118 80 1118 80 DR w 1118 α1 1118 G AL w ; Gq 1118 AL w ; rh6 DR w1118; Gqα1 60 DR w1118; rh6G 60 visual comparison, and the w and rhodopsin null lifespans 40 40 repeats were performed simultaneously. All mutant lines

Percent survival 20 Percent survival 20 1118 1118 17 0 0 were outcrossed to w . w ; ninaE flies: AL N=514, DR 0 20 40 60 80 100 0 20 40 60 80 Time (days) Time (days) N=511; w1118; rh32 flies: AL N=543, DR N=597; w1118; rh41 flies: g **** h ns w1118; ninaE17 100 AL RNAi cnt 1118 G **** DR RNAi cnt AL N=550, DR N=593; w ; rh6 flies: AL N=533, DR N=563; 1118 2 80 *** w ; rh3 AL Arr1-RNAi DR Arr1-RNAi **** 60 1118 1 **** w1118; rh41 w ; Gqα flies: AL N=403, DR N=400. (g) Hazard ratios for **** 40 **** w1118; rh6G rhodopsin and Gq mutant flies compared to w1118 control flies AL **** Percent survival 20 DR **** w1118; Gqα1 0 0.0 0.5 1.0 1.5 0 20 40 60 80 100 (ratios<1 indicate flies that are more likely to survive com- Hazard ratio (logrank) vs. w1118 Time (days) i j 1118 100 100 pared to w ). Error bars indicate the 95% confidence inter- AL RNAi cnt 80 80 DR RNAi cnt AL ATPα-RNAi val of the hazard ratios. (h) Survival analysis of eye-specific 60 60 DR ATPα-RNAi

40 40 arr1-RNAi knockdown flies vs RNAi control flies. Survival

20 AL csChrimson Percent survival Percent survival DR csChrimson 20 AL csChrimson+retinal data is plotted as an average of two independent lifespan DR csChrimson+retinal 0 0 0 20 40 60 80 0 20 40 60 80 100 Time (days) Time (days) repeats for arr1-RNAi and one independent lifespan replicate for RNAi-controls. RNAi control flies: AL N=177, DR N=161; arr1-RNAi flies: AL N=333, DR N=322. (i) Survival analysis of retinal inducible, photoreceptor-specific optogenetic flies (Trpl-GAL4>UAS-csChrimson[red-shifted]) supplemented with retinal or vehicle control and housed in 12:12h red-light:dark. Survival data is plotted as an average of two independent lifespan repeats. Retinal treated flies: AL N=289, DR N=236; Vehicle treated flies: AL N= 256, DR N=126. (j) Survival analysis of eye-specific ATPα RNAi knockdown flies vs RNAi control flies. Survival data is plotted as an average of three independent lifespan repeats. RNAi control flies: AL N=493, DR N= 490; ATPα RNAi flies: AL N=510, DR N=535. (g) Pvalues were determined by Log-rank (Man- tel-Cox) test, ns denotes a non-significant pvalues, **** indicates pvalues less than 0.0001. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

277 these flies were also longer lived in comparison to w1118 outcrossed controls (Fig. 4b-e).

278 Furthermore, rh6G mutants, which displayed the largest reductions in inflammation, also

279 displayed the greatest extension in lifespan compared to the other rhodopsin null lines.

280 Gq1 mutants [43], which harbor a mutation in the G-protein that mediates activation of

281 TRP channels downstream of rhodopsin, also displayed increased longevity compared to

282 control flies (Fig. 4f). Interestingly, with the exception of Rh4, rhodopsin null mutations

283 and Gq1 mutants primarily extended lifespan on AL, indicated by the hazard ratios in Fig.

284 4g. We next sought to investigate how increases in rhodopsin-mediated signaling

285 influence survival. To this end, we knocked down the major arrestin protein, arr1, within

286 the eyes of flies (GMR-GAL4>UAS-arr1-RNAi). Arr1 is required for light-mediated

287 rhodopsin internalization from the rhabdomere membrane into endocytic vesicles, thus

288 suppressing rhodopsin-mediated signaling and associated Ca2+-mediated

289 phototoxicity/cell death [19, 44, 45]. In agreement with its physiological role in light-

290 adaptation, we found that arr1-RNAi knockdown flies were hypersensitized to light

291 (Supplementary Fig. 4i). In contrast to the Rhodopsin null strains which displayed greater

292 proportional improvements in survival on AL vs DR, arr1-RNAi knockdown flies displayed

293 significantly lifespan shortening on DR, while the lifespan on AL was indistinguishable

294 from the control (Fig. 4h). Together, these data argue that DR-protects against lifespan

295 shortening downstream of light and/or rhodopsin-mediated signaling in a manner that

296 requires light-adaptation, and by extension, arr1-mediated rhodopsin endocytosis.

15 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

297 We utilized an optogenetics approach to examine how chronic photoreceptor

298 activation influences survival in flies reared on AL or DR. Optogenetics is a powerful tool

299 for examining how photoreceptor activation/suppression influences lifespan as it allows

300 for the ability to compare lifespans within flies reared under the same lighting conditions,

301 thus diminishing potential confounding variables present when comparing lifespan in

302 different lighting conditions (i.e., LD vs constant darkness), such as extra-ocular effects of

303 light on survival. To generate optogenetic flies we expressed the red-light-sensitive

304 csChrimson cation channel [46] within photoreceptors (Trpl-GAL4>UAS-csChrimson). To

305 activate the csChrimson channels, we housed the optogenetic flies in a 12:12 red-

306 light:dark cycle and supplemented their food with either all-trans retinal (a chromophore

307 required for full activation of csChrimson channels [47]) or a vehicle control

308 (Supplementary Fig. 4c). Optogenetic activation of the photoreceptors (retinal treated)

309 drastically reduced AL lifespan compared to vehicle treated controls, while the lifespan on

310 DR was unaffected (Fig. 4i). Retinal did not appear to be toxic to flies lacking csChrimson

311 channels, as the lifespan of Canton-S wild-type flies were indistinguishable between

312 vehicle and retinal treated groups (Supplementary Fig. 4d).

313 Although DR protected flies from lifespan shortening from the optogenetic

314 activation of photoreceptors, we found that forcing photoreceptor degeneration, by

315 knocking down ATP in the eye shortened lifespan on both AL and DR (Fig. 4j). Similarly,

316 eye-specific knockdown of nervana-2 and -3 (nrv2, GMR-GAL4>UAS-nrv2-RNAi and nrv3,

16 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

317 GMR-GAL4>UAS-nrv3-RNAi), which encode the Beta subunit of the Na+K+ATPase of the

318 eye [31] also reduced phototaxis responses and shortened lifespan (Supplementary Fig.

319 4e-h). Taken together, these data support a model where DR protects flies from lifespan

320 shortening caused by photoreceptor stress, as chronic photoreceptor activation reduces

321 survival in flies reared on AL while having minimal to no effect on flies reared on DR.

322 Inversely, photoreceptor deactivation primarily improves survival of flies reared on AL.

323 Eye-specific, CLK-output genes modulate lifespan

324 We next sought to determine if CLK-output genes in the eye influence age-related visual

325 declines and lifespan. We employed a bioinformatics approach to identify candidate eye-

326 specific circadian genes transcriptionally regulated by CLK (Supplementary Fig. 5a and

327 Supplemental Data 6). First, we compared age-associated changes in photoreceptor-

328 enriched gene expression [5] to genes that were differentially expressed on DR compared

329 to AL. More than half of the photoreceptor-enriched genes that were downregulated with

330 age were also upregulated on DR at ZT 0 and ZT 12 (upper left quadrant of Fig. 5a, b and

331 Supplementary Fig. 5a). We then subset this gene list, selecting just transcripts whose

332 expression was downregulated with age and upregulated on DR, and examined how their

333 expression changed in nCLK-1 fly heads (Supplementary Fig. 5a). From this analysis, we

334 identified G76c, retinin, and sunglasses as genes that were significantly downregulated

335 in nCLK-1 fly heads at ZT 0 and/or ZT 12 (Fig. 5c, d and Supplementary Fig. 5a, e-g).

336 G76c encodes the eye-specific G beta subunit that plays an essential role in terminating

17 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which a was not certified byb peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made 1.0 1.0 available under aCC-BY-NC-NDFigure 4.05. InternationalKnockdown license. of DR-sensitive, eye-specific retinin

0.5 0.5 CLK-output genes reduces survival. (a-b) Scatterplot of Gβ76c sunglasses

0 0 circadian, photoreceptor-enriched gene changes with age in

−0.5 −0.5 wild-type heads (x-axis: 5- vs 55-day old flies) vs diet-depen- Log2 fold-change on DR Log2 fold-change

Age pvalue on DR Log2 fold-change Age pvalue 0.005 0.005 Δ −1.0 0.050 −1.0 0.050 dent gene expression changes in heads from nCLK- 1 0.250 0.250 1.000 1.000 −2 −1 0 1 −2 −1 0 RNA-Seq control flies (y-axis: DR- vs AL-minus RU486) at Log2 Fold-change with age Log2 fold-change with age c 0.4 d 0.50 ZT 0 (a) and ZT 12 (b). (c-d) Boxplots of the expression 1 (DR) 0.2 1 (DR) Gene changes at ZT 12 Gene changes at ZT Gene changes at ZT 0 Gene changes at ZT 0.25 changes in nCLK-Δ1 heads (DR plus- vs DR minus-RU486)

0 0 at ZT 0 (c) and ZT 12 (d) of genes that were downregulated −0.2 pvalue pvalue 0.90 −0.25 0.90 with age and upregulated on DR (upper left quadrants of retinin 0.65 sunglasses 0.65 −0.4 0.30 0.30 Log2 fold-change with CLK- Δ β Log2 fold-change with CLK- Δ G 76c 0.05 0.05 Fig. 5a, b). (e) Positive phototaxis responses with eye-specif- e DR RNAi cnt DR retinin-RNAi ic knockdown of Gβ76c (GMR-GAL4> UAS-Gβ76c-RNAi), DR sunglasses-RNAi DR Gβ76c-RNAi 8.6e-18 4.4e-17 3.9e-17 1.0e-19 2.5e-14 1.5e-7 1.6e-18 1.7e-11 1.1e-7 3.8e-8 2.3e-8 4.0e-9 9.1e-11 6.5e-8 retinin (GMR-GAL4>UAS-retinin-RNAi), and sunglasses 100 3.4e-10 (GMR-GAL4>UAS-sunglasses-RNAi) compared to RNAi 80 control flies (GMR-GAL4>UAS-mCherry-RNAi) reared on 60 DR. For each timepoint results are represented as average 40

Phototaxis index (% ) phototaxis response +/-SEM (RNAi control n=24 biological 20 replicates, N=480 flies per condition; Gβ76c RNAi n=24 0 2 6 10 14 18 Time (days) biological replicates, N=480 flies per condition; retinin RNAi f DR RNAi cnt DR Gβ76c-RNAi 100 DR retinin-RNAi DR sunglasses-RNAi n=16 biological replicates, N=384 flies per condition; sun-

80 glasses RNAi n=24 biological replicates, N=480 flies per con- β 60 dition). (f) Survival analysis of eye-specific G 76c, retinin, sunglasses, and RNAi knockdown flies compared to RNAi 40 Percent survival control flies reared on DR. Survival data is plotted as an 20 average of three independent lifespan repeats for RNAi con- 0 0 20 40 60 80 100 Time (days) trol, sunglasses, and Gβ76c flies and two independent lifespan repeats for retinin RNAi knockdown flies. RNAi-cnt flies: N=490; retinin-RNAi flies: N=363; sunglasses-RNAi flies: N=468; Gβ76c-RNAi flies: N=509. (e) Pvalues were determined by two-tailed Student’s ttest (unpaired) at each timepoint comparing the phototaxis index of RNAi control flies to Gβ76c-, retinin-, and sunglasses-RNAi flies. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

337 phototransduction [13, 48]. Retinin encodes one of the four most highly expressed

338 proteins in the lens of the Drosophila compound eye [49]. Furthermore, retinin functions

339 in the formation of corneal nanocoatings, knockdown of which results in degraded

340 nanostructures and a reduction in their anti-reflective properties [50]. Sunglasses, also

341 called Tsp42Ej, encodes for a lysosomal tetraspanin concentrated in the retina that

342 protects against photoreceptor degeneration by degrading rhodopsin in response to light

343 [51]. We analyzed a published CLK Chromatin Immunoprecipitation (ChIP-chip) dataset in

344 flies and observed rhythmic CLK binding at the 5’-untranslated region of sunglasses in

345 Drosophila eye tissue [52] (Supplementary Fig. 5h and Supplementary Table 1), which

346 supports our bioinformatics approach and provides further evidence that sunglasses is an

347 eye-specific CLK-output gene. Eye-specific knockdown of G76c (GMR-GAL4>UAS-

348 G76c-RNAi), retinin (GMR-GAL4>UAS-retinin-RNAi), and sunglasses (GMR-GAL4>UAS-

349 sunglasses-RNAi) reduced phototaxis responses (Fig. 5e and Supplementary Fig. 5i), and

350 shortened lifespan in comparison to RNAi control flies (GMR-GAL4>UAS-mCherry-RNAi)

351 (Fig. 5f, Supplementary Fig. 5j). These findings indicate that DR and CLK function together

352 in the regulation of eye-specific circadian genes involved in the negative regulation of

353 rhodopsin signaling (i.e., phototransduction termination). Furthermore, these

354 observations support previous findings that lifespan extension upon DR requires

355 functional circadian clocks [10, 53], and establishes CLK-output genes as diet-dependent

356 regulators of eye aging and lifespan in Drosophila.

18 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

357 Discussion

358 Progressive declines in circadian rhythms are one of the most common hallmarks of aging

359 observed across most lifeforms [54]. Quantifying the strength, or amplitude, of circadian

360 rhythms is an accurate metric for predicting chronological age [55]. Many cellular

361 processes involved in aging (e.g., metabolism, cellular proliferation, DNA repair

362 mechanisms, etc.) display robust cyclic activities. Both genetic and environmental

363 disruptions to circadian rhythms are associated with accelerated aging and reduced

364 longevity [56, 57]. These observations suggest that circadian rhythms may not merely be

365 a biomarker of aging; rather, declines in circadian rhythms might play a causal role. The

366 observation that DR and DR-memetics, such as calorie restriction and time-restricted

367 feeding, improve biological rhythms suggests that clocks may play a fundamental role in

368 mediating their lifespan-extending benefits.

369 Herein, we identified circadian processes that are selectively amplified by DR. Our findings

370 demonstrate that DR amplifies circadian homeostatic processes in the eye, some of which

371 are required for DR to delay visual senescence and improve longevity in Drosophila. Taken

372 together, our data demonstrate that photoreceptor stress has deleterious effects on

373 organismal health; disrupting CLK function and/or overstimulation of the photoreceptors

374 induced a systemic immune response and reduced longevity. Our findings establish the

375 eye as a diet-sensitive regulator of lifespan. DR’s neuroprotective role in the

376 photoreceptors appears to be mediated via the molecular clock, which promotes the

19 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

377 rhythmic oscillation of genes involved in the suppression of phototoxic cell stress (Fig. 6

378 and Supplemental Discussion 1). Our data also support the idea that age-related declines

379 in the visual system impose a high cost on an organism. Perhaps this is why a number of

380 long-lived animals have visual systems that have undergone regressive evolution (e.g.,

381 cave-dwelling fish and naked-mole rats) [58]. Failing to develop a visual system may help

382 these organisms avoid age-related damage and inflammation caused by retinal

383 degeneration. Ultimately, developing a visual system, which is critical for reproduction

384 and survival, may be detrimental to an organism later in life. Thus, vision may be an

385 example of an antagonistically pleiotropic mechanism that shapes lifespan.

386

20 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Ad libitumavailable under aCC-BY-NC-ND 4.0 InternationalDietary license Restriction. hν hν Lens Rh Trp Rh

Ca2+ Phototoxicity Aging CLK CLK Photoreceptor dysfunction

Systemic Visual function inflamation Lifespan Fig. 6. Dietary restriction extends lifespan by promoting rhythmic homeostatic processes in the eye. DR promotes CLK-output processes in the eye that suppress light/Ca2+-mediated phototoxicity to suppress systemic inflammaion, delay visual senesence, and improve survival. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

387 Methods

388 Fly stocks: The genotypes of the Drosophila lines used in this study are listed in

389 Supplemental Table 2. The following lines were obtained from the Bloomington

390 Drosophila Stock Center: Oregon R. (25125), GMR-GAL4 (1104), Elav-GS-GAL4 (43642),

391 Trpl-GAL4 (52274), ClkOUT (56754), UAS-csChrimson (55134), UAS-CLK-∆1 (36318), UAS-

392 CLK-∆2 (36319), G76c-RNAi (28507), tsp42Ej/sunglasses-RNAi (29392), retinin-RNAi

393 (57389), ATP-RNAi (28073), nrv2-RNAi (28666), nrv3-RNAi (60367), and mCherry-RNAi

394 (Bloomington RNAi-cnt, 35785). The following lines were obtained from the Vienna

395 Drosophila Resource Center: arr1-RNAi (22196), RNAi-cnt (empty vector, 60100). The

396 following lines were received from the laboratory of Craig Montell: CantonS, w1118, w1118;

397 ninaE17, w1118; rh32, w1118; rh41, w1118; rh6G, w1118; Gq1, and TRP365. The following lines were

398 outcrossed to w1118 for this manuscript: UAS-CLK-∆1OC and CantonSOC. The Trpl-GAL4 line

399 was recombined with GAL80 for this manuscript: Trpl-GAL4; GAL80ts.

400

401 Fly husbandry and survival analyses: All flies were maintained at 25±1 °C, 60% humidity

402 under a 12h:12h LD cycle (~750lux, as measured with a Digital Lux Meter, Dr. Meter Model

403 LX1330B) unless otherwise indicated. Fly stocks and crosses were maintained on a

404 standard fly media as described previously [59]. Briefly, standard fly media consisted of

405 1.5% yeast extract, 5% sucrose, 0.46% agar, 8.5% of corn meal, and 1% acid mix (a 1:1 mix

406 of 10% propionic acid and 83.6% phosphoric acid). Fly bottles were seeded with live yeast

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407 prior to collecting virgins or setting up crosses. Mated adult progeny were then

408 transferred to ad libitum (AL) or dietary restriction (DR) media within three days of

409 eclosion. Adult flies used in experiments were transferred to fresh media every 48h at

410 which point deaths were recorded for survival analysis. AL and DR fly media differed only

411 in its percentage of yeast extract, respectively containing 5% or 0.5% (Yeast Extract, B.D.

412 Bacto, Thermo Scientific 212720, Cat no. 90000-722). Optogenetic experiments: For

413 experiments using the csChrimson channel rhodopsin [46], adult flies were transferred to

414 media supplemented with 50μM all-trans-retinal (Sigma Aldrich, R2500-1G) or drug

415 vehicle (100% ethanol), and maintained under a 12h:12h red light:dark cycle, with ~10lux

416 of red light (~590nm) during the light phase. Elav-GeneSwitch flies: GeneSwitch [60], adult

417 flies were transferred to media supplemented with 200μM RU486 (Mifepristone, United

418 States Biological), indicated as either AL+ or DR+, for post-developmental induction of

419 transgenic elements; isogenic control flies were transferred to food supplemented with a

420 corresponding concentration of drug vehicle (100% ethanol), indicated as either AL- or

421 DR-. prCLK-1 experiments: GAL80 temperature sensitive crosses were set in bottles at

422 25°C, 60% humidity under a 12h:12h LD cycle for four days. Parental flies were removed,

423 and the bottles were transferred to 18°C for approximately three-weeks to suppress GAL4

424 activity throughout development. After ecolsion, the F1 generations were sorted onto AL

425 or DR food the flies were maintained at 30°C to de-repress GAL80 and activate GAL4 (60%

426 humidity under a 12h:12h LD cycle) for the remainder of their lifespans. The F1

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427 generations for these experiments share the same genetic background, as both the UAS-

428 CLK-1 and the CantonS control lines were fully outcrossed to the same w1118 strain prior

429 to setting up the cross with Trpl-GAL4; GAL80ts.

430

431 Circadian time-course expression analysis: Mated Canton-S females were reared on AL or

432 DR diets for seven days at 25±1 °C, under a 12:12h light-dark (LD) regimen. Beginning on

433 the seventh day, four independent biological replicates (per diet/timepoint) of

434 approximately 35 flies were collected on dry ice every four-hours for 20-hours starting at

435 ZT 0 (six total timepoints, 48 total samples). RNA extraction, DNA amplification/labeling,

436 and gene expression arrays were performed following the same protocols as in Katewa et

437 al., 2012 [61]. In summary, RNA was isolated from whole fly lysates with Qiagen’s RNeasey

438 Lipid Tissue Mini Kit (74804) and RNA quantity and quality were accessed with a Nanodrop

439 and Agilent’s bioanalyzer (RNA 600 Nano Kit (5067-15811)). DNA amplification from total

440 RNA was performed using Sigma’s TransPlex Complete Whole Transcriptome

441 Amplification Kit (WTA2) and purified with Qiagen’s QIAquick PCR Purification Kit (28104).

442 Gene expression labeling was performed with NimbleGen One-Color DNA Labeling Kit

443 (05223555001) and hybridized to NimbleGen 12-Plex gene expression arrays. Arrays were

444 quantitated with NimbleGen’s NimbleScan2 software, and downstream expression

445 analyses were conducted in R (http://www.r-project.org). Transcript-level expression from

446 the four independent biological replicates were averaged for each time-point.

23 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

447

448 nCLK-1 RNA-Seq analyses: nCLK-1 (Elav-GS-GAL4>UAS-nCLK-1) adult flies were

449 developed on standard stock food (1.5% yeast-extract) for four days. Three independent

450 biological replicates of 100 mated female flies were then reared on AL or DR diets treated

451 with RU486 or vehicle control at 25±1 °C, under a 12:12h LD regimen. Diets were changed

452 approximately every 48-hours, until the seventh day at which point flies were flash frozen

453 on dry-ice at ZT 0 and ZT 12 (lights-on and -off, respectively). See Supplemental Fig. 2a

454 for RNA-Seq. experimental design. RNA-extraction: Frozen flies were vortexed to remove

455 heads and mRNA from each biological replicate of pooled heads was isolated with the

456 Quick-RNA MiniPrep Kit (Zymo Research #11-328), per manufactures’ instructions.

457 Fragment library preparation and deep sequencing: Library preparation was performed

458 by the Functional Genomics Laboratory (FGL), a QB3-Berkeley Core Research Facility at

459 University of California, Berkeley. cDNA libraries were produced from the low-input RNA

460 using the Takara SMART-Seq v4 Ultra-low input RNA kit. An S220 Focused-Ultrasonicator

461 (Covaris®) was used to fragment the DNA, and library preparation was performed using

462 the KAPA hyper prep kit for DNA (KK8504). Truncated universal stub adapters were used

463 for ligation, and indexed primers were used during PCR amplification to complete the

464 adapters and to enrich the libraries for adapter-ligated fragments. Samples were checked

465 for quality on an AATI (now Agilent) Fragment Analyzer. Samples were then transferred

466 to the Vincent J. Coates Genomics Sequencing Laboratory (GSL), another QB3-Berkeley

24 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

467 Core Research Facility at UC Berkeley, where Illumina sequencing library molarity was

468 measured with quantitative PCR with the Kapa Biosystems Illumina Quant qPCR Kits on a

469 BioRad CFX Connect thermal cycler. Libraries were then pooled evenly by molarity and

470 sequenced on an Illumina NovaSeq6000 150PE S4 flowcell, generating 25M read pairs per

471 sample. Raw sequencing data was converted into fastq format, sample specific files using

472 the Illumina bcl2fastq2 software on the sequencing centers local linux server system. Read

473 alignment and differential expression analyses: Raw fastq reads were filtered by the

474 Trimmomatic software [62] (Trimmomatic-0.36) to remove Illumina-specific adapter

475 sequences and the minimal length was set to 36 (MINLEN) for trimming sequences. The

476 paired end filtered reads were then aligned to the D. Melanogaster dm6 genome (BDGP

477 Release 6 + ISO1 MT/dm6) by HISAT2 [63] to generate BAM files with the specific strand

478 information set to “Reverse”. Count files were then generated by featureCounts [64] and

479 the D. Melanogaster reference genome was utilized as the gene annotation file with

480 specific strand information set to “stranded (Reverse)”. Resulting count files (tabular

481 format) were then analyzed with DEseq2 [65] with fit-type set to “local”, and pvalues of

482 less than 0.05 were considered differentially expressed between factor levels. Normalized

483 count reads were outputted for visualization of expression (heatmaps), and Supplemental

484 Data Files 3a contains normalized count reads across all experimental samples. UCSC

485 genome browser visualization: The makeUCSCfile software package from HOMER was

25 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

486 utilized to generate bedGraph files for visualizing changes in tag density at exon 2 of clk

487 comparing nCLK-1 and control samples (Supplementary Fig. 2B).

488

489 Heatmap visualizations: We employed the heatmap2 function from R gplots package to

490 visualize bioinformatics data. Data were not clustered, and data were scaled by row for

491 normalization across time-points.

492

493 Electroretinogram assays: ERGs were performed and analyzed in two independent

494 laboratories. ERGs were recorded for nCLK-1 flies reared on AL or DR diets supplemented

495 with vehicle or RU486 at day 14 at the Baylor College of Medicine (BCM), and at day 21

496 at the University of California, Santa Barbara (UCSB). ERGs were recorded for prCLK-1

497 flies at UCSB reared on AL or DR and maintained at either 18C or 30C. BCM: ERG

498 recordings were performed as in Wang et al., 2014 [66]. Flies were glued on a glass slide.

499 A recording electrode was placed on the eye and a reference electrode was inserted into

500 the back of the fly head. Electrodes were filled with 0.1 M NaCl. During the recording, a

501 1 s pulse of light stimulation was given. The ERG traces of at least eight flies per

502 genotype/diet were recorded and analyzed by LabChart8 software (AD Instruments).

503 UCSB: ERG recordings were performed as in Wes et al., 1999 [67]. Two glass electrodes

504 were filled with Ringer’s solution and electrode cream was applied to immobilized flies. A

505 reference electrode was placed on the thorax, while the recording electrode was placed

26 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

506 on the eyes. Flies were then exposed to a 10s pulse of ~200lux white light, a light intensity

507 that is comparable to the phototaxis assay. An EI-210 amplifier (Warner Instruments) was

508 used for amplifying the electrical signal from the eye after light stimulation, and the data

509 were recorded using a Powerlab 4/30 device along with the LabChart 6 software (AD

510 Instruments). Raw data were then uploaded into R-statistical software for plotting and

511 statistical analysis. All electroretinograms were performed between ZT4-8 or ZT12-14.

512

513 Positive phototaxis assay: Positive phototaxis was performed using an adapted protocol

514 from Vang et al., 2014 [68] (Fig S2D). Phototaxis measurements were recorded

515 longitudinally on populations of female flies aged on either AL or DR food (with or without

516 200μM RU486 when indicated) at a density of 10-25 flies per tube prior to and after

517 phototaxis measurements. On the day of phototaxis recording, eight groups of flies (four

518 AL and four DR groups) were placed in separate 2.5cm x 20cm tubes (created from three

519 enjoined narrow fly vials [Genesee Scientific]) and dark-adapted for 15-minutes prior to

520 light exposure (no food was available in the vials during phototaxis assays). Flies were

521 then gently tapped to the bottom of the tube, placed horizontally, and exposed to white

522 light from an LED strip (Ustellar, UT33301-DW-NF). A gradient of light intensity was

523 created, with 500lux at the nearest point in the fly tube to the light source and 150lux at

524 the furthest point. Phototaxis activity was recorded by video at 4K resolution (GoPro,

525 Hero5 black). Positive phototaxis was scored manually as the percentage of flies that had

27 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

526 traveled >19cm toward the light source in three 15s intervals (15s, 30s, and 45s).

527 “Phototaxis index” was calculated by averaging the percent of positive phototaxis for each

528 vial at the three 15s intervals. To control for light-independent wandering activity, a

529 phototaxis index was also calculated when the light source was placed in parallel to the

530 fly tube, such that all parts of the tube were equally illuminated with 500lux. *Normalizing

531 phototaxis responses to wandering activity failed to significantly affect phototaxis index,

532 data not shown.

533

534 RNA extraction and cDNA preparation: Flies were maintained on AL or DR for the

535 indicated amount of time, then flash frozen on dry ice. Heads were separated from bodies

536 (thorax and abdomen) by vigorous shaking. Flies were then ground using a hand-held

537 homogenizer at room temperature following MiniPrep instructions. Total RNA was

538 isolated using the Quick-RNA MiniPrep Kit (Zymo Research, 11-328). In brief, flies were

539 maintained on AL or DR for the indicated amount of time, then flash frozen on dry ice.

540 Heads were separated from bodies (thorax and abdomen) by vigorous shaking. Flies were

541 then ground using a hand-held homogenizer at room temperature following MiniPrep

542 instructions. RNA was collected into 30μl DNAse/RNAse-free water and quantified using

543 the NanoDrop 1000 Spectrophotometer (Thermo Scientific). For each experiment, 120-

544 180 age-, genotype-, and diet-matched flies were collected, and three independent RNA

545 extractions were performed. To extract RNA from heads, 40-60 flies were used; to extract

28 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

546 RNA from bodies, 20-30 flies were used. cDNA preparation: The iScript Reverse

547 Transcription Supermix for RT-qPCR (Bio-Rad, 1708841) was used to generate cDNA from

548 RNA extracted from heads and bodies. For each group, 1 μg of total RNA was placed in a

549 volume of 4μl iScript master mix, then brought to 20μl with DNAse/RNAse-free water. A

550 T1000 thermocycler (BioRad) was used for first-strand RT-PCR reaction following iScript

551 manufacturers’ instructions—priming step (5min at 25°C), reverse transcription (30min at

552 42°C), and inactivation of the reaction (5min at 85°C).

553 Real-time quantitative PCR: Reactions were performed in a 384-well plate. Each reaction

554 contained 2μl of 1:20 diluted cDNA, 1μl of primers (forward and reverse at 10μM), 5μl

555 SensiFAST SYBR Green No-ROX Kit (BIOLINE, BIO-98020), and 2μl of DNAse/RNAse-free

556 water. The qPCR reactions were performed with a Light Cycler 480 Real-Time PCR machine

557 (Roche Applied Science) with the following run protocol: pre-incubation (95°C for 2 min),

558 forty PCR cycles of denaturing (95°C for 5s, ramp rate 4.8°C/s), and annealing and

559 extension (60°C for 20 s, ramp rate 2.5°C/s).

560

561 Hemolymph Mass spectrometry: Proteomic sample preparation: nCLK-1 female flies

562 (Elav-GeneSwitch-GAL4>UAS-nCLK-1) were reared on AL diet plus RU486 or vehicle

563 control (N=300 flies per biological replicate, n=3 biological replicates). At day 14, flies

564 were snap frozen on dry ice and transferred to pre-chilled vials. The vials were vortexed

565 for 5-10s to remove heads and the frozen bodies were transferred to room temperature

29 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

566 vials fitted with 40µm filters. Headless bodies were thawed at room temperature for 5

567 minutes and spun at 5000 rpm for 10min at 4 °C. Following the spin, hemolymph collected

568 at the bottom of each vial and the bodies remained within the filters. Digestion: A

569 Bicinchoninic Acid protein assay (BCA) was performed for each of the hemolymph samples

570 and a 100µg aliquot was used for tryptic digestion for each of the 6 samples. Protein

571 samples were added to a lysis buffer containing a final concentration of 5% SDS and 50

572 mM triethylammonium bicarbonate (TEAB), pH ~7.55. The samples were reduced in 20

573 mM dithiothreitol (DTT) for 10 minutes at 50⁰ C, subsequently cooled at room temperature

574 for 10 minutes, and then alkylated with 40 mM iodoacetamide (IAA) for 30 minutes at

575 room temperature in the dark. Samples were acidified with a final concentration of 1.2%

576 phosphoric acid, resulting in a visible protein colloid. 90% methanol in 100 mM TEAB was

577 added at a volume of 7 times the acidified lysate volume. Samples were vortexed until the

578 protein colloid was thoroughly dissolved in the 90% methanol. The entire volume of the

579 samples was spun through the micro S-Trap columns (Protifi) in a flow-through Eppendorf

580 tube. Samples were spun through in 200 µL aliquots for 20 seconds at 4,000 x g.

581 Subsequently, the S-Trap columns were washed with 200 µL of 90% methanol in 100 mM

582 TEAB (pH ~7.1) twice for 20 seconds each at 4,000 x g. S-Trap columns were placed in a

583 clean elution tube and incubated for 1 hour at 47⁰ C with 125 µL of trypsin digestion buffer

584 (50 mM TEAB, pH ~8) at a 1:25 ratio (protease:protein, wt:wt). The same mixture of trypsin

585 digestion buffer was added again for an overnight incubation at 37⁰ C.

30 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

586 Peptides were eluted from the S-Trap column the following morning in the same elution

587 tube as follows: 80 µL of 50 mM TEAB was spun through for 1 minute at 1,000 x g. 80 µL

588 of 0.5% formic acid was spun through next for 1 minute at 1,000 x g. Finally, 80 µL of 50%

589 acetonitrile in 0.5% formic acid was spun through the S-Trap column for 1 minute at 4,000

590 x g. These pooled elution solutions were dried in a speed vac and then re-suspended in

591 0.2% formic acid. Desalting: The re-suspended peptide samples were desalted with stage

592 tips containing a C18 disk, concentrated and re-suspended in aqueous 0.2% formic acid

593 containing “Hyper Reaction Monitoring” indexed retention time peptide standards (iRT,

594 Biognosys). Mass spectrometry system: Briefly, samples were analyzed by reverse-phase

595 HPLC-ESI-MS/MS using an Eksigent Ultra Plus nano-LC 2D HPLC system (Dublin, CA) with

596 a cHiPLC system (Eksigent) which was directly connected to a quadrupole time-of-flight

597 (QqTOF) TripleTOF 6600 mass spectrometer (SCIEX, Concord, CAN). After injection,

598 peptide mixtures were loaded onto a C18 pre-column chip (200 µm x 0.4 mm ChromXP

599 C18-CL chip, 3 µm, 120 Å, SCIEX) and washed at 2 µl/min for 10 min with the loading

600 solvent (H2O/0.1% formic acid) for desalting. Subsequently, peptides were transferred to

601 the 75 µm x 15 cm ChromXP C18-CL chip, 3 µm, 120 Å, (SCIEX), and eluted at a flow rate

602 of 300 nL/min with a 3 h gradient using aqueous and acetonitrile solvent buffers. Data-

603 dependent acquisitions (for spectral library building): For peptide and protein

604 identifications the mass spectrometer was operated in data-dependent acquisition [51]

605 mode, where the 30 most abundant precursor ions from the survey MS1 scan (250 msec)

31 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

606 were isolated at 1 m/z resolution for collision induced dissociation tandem mass

607 spectrometry (CID-MS/MS, 100 msec per MS/MS, ‘high sensitivity’ product ion scan

608 mode) using the Analyst 1.7 (build 96) software with a total cycle time of 3.3 sec as

609 previously described [69]. Data-independent acquisitions: For quantification, all peptide

610 samples were analyzed by data-independent acquisition (DIA, e.g. SWATH), using 64

611 variable-width isolation windows [70, 71]. The variable window width is adjusted

612 according to the complexity of the typical MS1 ion current observed within a certain m/z

613 range using a DIA ‘variable window method’ algorithm (more narrow windows were

614 chosen in ‘busy’ m/z ranges, wide windows in m/z ranges with few eluting precursor ions).

615 DIA acquisitions produce complex MS/MS spectra, which are a composite of all the

616 analytes within each selected Q1 m/z window. The DIA cycle time of 3.2 sec included a

617 250 msec precursor ion scan followed by 45 msec accumulation time for each of the 64

618 variable SWATH segments.

619

620 Identification of photoreceptor enriched CLK-output genes: Diagram of bioinformatics

621 steps reported in Supplementary Fig. 5A. Gene-lists are reported in Supplemental Data 6.

622 We identified the top 1,000 photoreceptor-enriched genes from Charlton-Perkins et al.,

623 2017 [72] (GSE93782). We then filtered this list for genes that oscillate in a circadian

624 fashion, and that are downregulated with age from Kuintzle et al., 2017 [15] (GSE81100).

625 Approximately 1/3 of the photoreceptor enriched genes (366 genes) were expressed in a

32 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

626 circadian fashion in young wild-type heads and approximately one-half of these (172

627 genes) displayed a significant loss in expression with age (5- vs 55-day old heads). We

628 further analyzed the remaining gene lists to identify those that are significantly

629 upregulated on DR compared to AL at either ZT 0 or ZT 12 from control (vehicle treated)

630 samples from our nCLK-1 RNA-Seq analyses. For the final filtering step, we analyzed the

631 genes that were significantly downregulated in nCLK-1 on DR (RU486 vs vehicle treated

632 controls), resulting in the identification of G76c, retinin, and sunglasses.

633

634 Statistical analysis

635 The individual biological replicates “n” and the number of individual flies “N” is denoted

636 in each figure legend along with the particular statistical test utilized. The pvalue statistics

637 are included in each figure. All error bars are represented as standard error of the mean

638 (SEM), and all graphs were generated in PRISM 9 (GraphPad). The experiments in this

639 manuscript were performed with populations of female flies (i.e., typically greater than 20

640 flies per technical replicate).

641 Time-course microarray analyses: Four independent biological replicates (per

642 diet/timepoint) of approximately 35 CantonS female flies were collected on dry ice every

643 four-hours for 20-hours starting at ZT 0 (six total timepoints, 48 total samples). Differential

644 expression was determined by two-tailed Student’s ttest (paired) comparing the averaged

645 transcript-level expression values between AL and DR samples across all timepoints, and

33 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

646 pvalues less than 0.05 were considered significant. The JTK_CYCLE algorithm [73] (v3.0)

647 was utilized to identify circadian transcripts from the AL and DR time-course expression

648 arrays. Transcript level expression values for each of the four biological replicates (per

649 timepoint/diet) were used as input for JTK_CYCLE, and period length was set to 24-hours.

650 We defined circadian transcripts as those displaying a JTK_CYCLE pvalue of less than 0.05.

651 Subsequent analyses compared diet-dependent changes in JTK_CYCLE outputs (phase

652 and amplitude).

653 nCLK-1 RNA-Seq: Three independent biological replicates of 100 mated female adult

654 flies were utilized per genotype/diet/time-point. DEseq2 software [65] was utilized and

655 pvalues of less than 0.05 were considered differentially expressed between factor levels.

656 ERG responses: For ERG experiments we quantified responses from 6-15 individual flies

657 per standards in the field. Statistical significance was determined by two-tailed Student’s

658 ttest (unpaired), comparing ERG responses between diet and genotypes. Full ERG statistics

659 are reported in Supplemental Data 10.

660 Survival analyses: The Log-rank (Mantel-Cox) test was used to determine statistical

661 significance comparing average lifespan curves from a minimum of two independent

662 lifespan replicates. Hazard Ratios (logrank) were also utilized to determine the probability

663 of death across genotypes, lighting conditions, and diet. Detailed Log-rank and hazard

664 ratios for each lifespan are reported in Supplemental Data 7.

34 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

665 Positive phototaxis assay: Statistical significance for phototaxis index at each timepoint

666 were calculated with the Student’s ttest (two-tailed, un-paired). 2way ANOVA or mixed-

667 effects models were performed to determine statistical significance between diet,

668 genotype, or time interactions. Full statistical output (2way ANOVA and ttest) for all

669 phototaxis experiments is reported in Supplemental Data 9.

670 Real time quantitative PCR: Fold-change in gene expression was calculated using the ΔΔCt

671 method and the values were normalized using rp49 as an internal control. Pvalues were

672 calculated with the pairwise Student’s t-test comparing Log2 fold-changes in expression.

673 Mass-spectrometric data processing, quantification and bioinformatics: Mass

674 spectrometric data-dependent acquisitions [51] were analyzed using the database search

675 engine ProteinPilot (SCIEX 5.0 revision 4769) using the Paragon algorithm (5.0.0.0,4767).

676 Using these database search engine results a MS/MS spectral library was generated in

677 Spectronaut 14.2.200619.47784 (Biognosys). The DIA/SWATH data was processed for

678 relative quantification comparing peptide peak areas from various different time points

679 during the cell cycle. For the DIA/SWATH MS2 data sets quantification was based on XICs

680 of 6-10 MS/MS fragment ions, typically y- and b-ions, matching to specific peptides

681 present in the spectral libraries. Peptides were identified at Q< 0.01%, significantly

682 changed proteins were accepted at a 5% FDR (q-value < 0.01).

683 Gene-ontology enrichment analysis: To identify enriched gene-ontology (i.e., bioprocess)

684 categories with the resultant lists from bioinformatics approaches, we utilized the

35 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

685 “findGO.pl” package from HOMER. Full gene-ontology lists including enrichment statistics

686 and associated gene-lists are reported in supplemental data files. A maximal limit of 200

687 gene identifiers per GO category was implemented to reduce the occurrence of large,

688 over-represented terms that lack specificity (i.e., metabolism). Full gene-ontology lists are

689 reported in supplemental data files.

690

691

692 Data availability

693 Time-course microarray data and accompanied JTK_CYCLE statistics that support the

694 findings in this study have been deposited in the Gene Expression Omnibus [74] with the

695 GSE158286 accession code. The RNAseq data and accompanied differential expression

696 analyses that support the findings in this study have been deposited to GEO with the

697 GSE158905 accession code. The mass spectrometric raw data are deposited at

698 ftp://[email protected] (MassIVE user ID: MSV000086781, password:

699 winter; preferred engine: Firefox); it is also available at ProteomeXchange with the ID

700 PXD023896. Additional mass spectrometric details from DIA and DDA acquisitions, such

701 as protein identification and quantification details are available at the repositories

702 (including all generated Spectronaut and Protein Pilot search engine files).

703

704

36 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

705

706

707

708

709

710

711

712

37 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

713 References

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878

879

41 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

880

881 Acknowledgements

882 We would like to thank the QB3 Genomics Functional Genomics Lab at UC Berkeley and

883 the Vincent J. Coates Genomics Sequencing Lab for RNA-seq library preparation and deep

884 sequencing. We thank the Bloomington Drosophila Stock Center, the Vienna Drosophila

885 Resource Center, and Craig Montell for providing flies used in this study. We would like

886 to acknowledge Hugo Bellen and Zhongyuan Zuo for contributing resources and insight.

887 We thank Daron Yim and John McCarthy for their assistance. We would like to

888 acknowledge the work of Paolo Sassone-Corsi and his recent contributions to the fields

889 on nutrition, circadian rhythms, and aging that helped in the conception of this project.

890 This work is funded by grants awarded to P.K. from the American Federation of Aging

891 Research, NIH grants R01 R01AG038688 and AG045835 and the Larry L. Hillblom

892 Foundation. B.A.H. is supported by NIH/NIA T32 award AG000266 and C.M. is supported

893 by NIH/NEI awards EY008117 and EY010852. We acknowledge the Buck Institute

894 Proteomics Core and the support of instrumentation from the NCRR shared

895 instrumentation grant 1S10 OD016281.

896

897 Author Contributions

898 Conceptualization, B.A.H., G.T.M., and S.K.; Software, B.A.H., G.T.M., B.S., and S.K.; Formal

899 analysis, B.A.H., G.T.M., and B.S.; Investigation, B.A.H., G.T.M., C.L., T.L., N.L., D.L-K., and S.B.;

42 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

900 Visualization, B.A.H.; Writing-Original Draft, B.A.H., Writing-Review & Editing, B.A.H.,

901 G.T.M., and P.K.; Data Curation, B.A.H., G.T.M., S.M., and B.S.; Methodology, G.T.M. and

902 M.L., Resources, C.M. and P.K., Supervision, P.K., Funding acquisition, C.M. and P.K.

903

904

905 Additional information

906 The authors declare no competing interests.

907

908 Supplementary Information is available for this paper

909 Correspondence and requests for materials should be addressed to 910 P.K. ([email protected]) 911

43 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Discussion 1.

DR amplifies and modifies circadian transcriptional output

DR and DR-memetics have long been known to improve circadian behavioral rhythms in

old age [1]. Over the past decade, improvements in molecular genetic techniques and

next-generation sequencing have allowed investigators to examine how nutrient

composition and time of feeding influence circadian transcriptional rhythms. Reports in

mammals have demonstrated that calorie restriction, a reduction in total calorie intake

without malnutrition, enhances the number and amplitude of rhythmic transcripts [2].

Inversely, high-nutrient diets, such as high-fat/western diets suppress circadian

transcriptional rhythms [3]. The near doubling in the number of circadian transcripts we

quantified on DR vs AL in Drosophila is consistent with observations in mammals.

Additionally, transcripts oscillating on DR displayed an increased circadian amplitude. DR-

mediated increases in the number of circadian transcripts, and their amplitude, is likely

due to enhanced transcriptional output by CLK/CYC. Recent reports in both mice and flies

have demonstrated that nutrient-sensing mechanisms (i.e., AMPK/TOR and Sirtuin

signaling) signal directly to the core-clock transcription factors to activate transcription

[4-6]. For instance, the Drosophila AMPK, which is activated in response to cellular energy

depletion (e.g., elevated AMP concentrations), directly phosphorylates CLK, enhancing its

circadian transcriptional output [4]. Because we extracted mRNA from populations of flies,

the transcript expression values we report here are influenced by both individual and bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

population-wide transcript expression levels. Therefore, the DR-mediated improvements

to the circadian transcriptome we have observed may also reflect greater synchronicity

between individual flies.

Interestingly, we also observed relatively low overlap between the transcripts that

oscillate on AL compared to DR. We found that DR-oscillating genes are enriched for

processes related to homeostatic function, while circadian processes on AL-oscillating

genes are enriched for processes related to damage-response pathways. These findings

are similar to those reported in mouse liver tissue, comparing transcriptome of mice

reared on standard chow versus those on calorie restriction [2]. A combination of aging

and damage response signals (e.g., reactive oxygen species) also influence which

transcripts cycle in the fly [7]. This phenomenon, termed “circadian reprogramming,” is

also observed in response to nutrient cues, where differing nutrient signals direct which

specific transcripts are transcriptionally targeted downstream of the molecular clock. The

similarities between the diet-dependent changes we report here and those previously

reported in mice on calorie restriction indicates that the molecular clock’s response to

nutrient restriction is evolutionarily conserved.

Given DR’s ability to robustly extend lifespan while amplifying circadian

transcriptional output, we postulated that DR-sensitive circadian processes play an

important role in slowing aging and improving survival. Although highly informative, to

date, the diet-dependent circadian transcriptome studies have analyzed only a small bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

number of mammalian tissues and thus have provided only a limited description of how

diet influences circadian transcriptional output at the whole-organism level. Our ability to

analyze the AL/DR circadian transcriptomes in the whole fly allowed for an unbiased

approach for identifying the most DR-sensitive, cyclic processes throughout the body.

This approach led to the observation that phototransduction was among the top circadian

processes amplified by DR. The phototransduction genes we identified were also cyclic in

flies reared on AL, albeit at a lower expression and circadian amplitude, indicating that

their transcriptional regulation is likely not a result of circadian reprogramming. This,

however, highlights the biological importance of their circadian regulation. A limitation of

our AL/DR circadian transcriptome analyses is that they are likely under-powered to

identify the full spectrum of eye-specific circadian transcripts, because our mRNA samples

were pooled from whole-body lysates and were collected for only one circadian cycle

(24hr). Analyses of a more robust circadian transcriptome, performed from mRNA

collected from heads, over 2 circadian cycles (48hr), indicated that phototransduction

components were among the most rhythmic circadian processes, thus underscoring the

importance of circadian regulation within the eye [7].

DR delays visual senescence by amplifying circadian rhythms in the eye

Metabolic dysfunction is strongly correlated with accelerated aging and eye-disease (e.g.

diabetic retinopathy) [8, 9]. Declines in the circadian amplitude of clocks within the eye

have been reported in wild-type mice with age and in models of diabetic retinopathy, bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

which may further exacerbate disease pathology [10, 11]. Calorie restriction protects

against several age-related eye diseases—dry-eye disease, cataracts, and age-related

macular degeneration [12]. Calorie restriction also has a neuroprotective effect in

photoreceptors and retinal-ganglion cells with age [12]. To date, no studies have

investigated whether calorie restriction enhances circadian amplitude within the eye or

whether its benefits within the eye are dependent on the molecular clock. Our results in

flies demonstrate that DR amplifies circadian rhythms within the eye and delays visual

senescence in a CLK-dependent manner. Additionally, we identified the DR-sensitive CLK-

output genes G76c, retinin, and sunglasses and demonstrated that their knockdown in

the eye accelerated visual declines, thus indicating that DR’s neuroprotective role in the

eye functions mechanistically through the molecular clock.

Several age-associated morphological and physiological declines have been

reported in circadian mutant mouse models [13]. The positive-limb of the core molecular

clock in mice is comprised of the basic-helix-loop-helix transcription factors BMAL1 and

CLOCK [14]. Mice harboring whole-body genetic knockouts of either BMAL1 or CLOCK

develop cataracts and corneal inflammation with age [13]. Additionally, photoreceptor-

specific (cone-cell, HRGP-Cre x Bmal1 fl/fl) BMAL1 knockout mice display a significantly

altered circadian transcriptome, a shift in the distribution of short vs medium wavelength

opsins, and a reduction in photoreceptor cell viability with age [15, 16]. Consistently, our

data demonstrates that diminishing CLK function in adult animals (post-development) is bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

sufficient to drive eye aging in flies. However, there are important distinctions between

the mechanism of phototransduction used by mammalian rod and cone photoreceptors,

and what exists in the fly.

In mammals, light-activated rhodopsin in rod and cone photoreceptor neurons

couples to, and inactivates, cyclic nucleotide gated channels, hyperpolarizing the cell [17].

This is distinctly different from what occurs in the fly, where light-activated rhodopsin

couples to a TRP channel, which when activated depolarizes the cell [18]. However, in a

third class of mammalian photoreceptors, the intrinsically-photosensitive retinal ganglion

cells (ipRGCs), there is a nearly identical mechanism of phototransduction to Drosophila

[19]. The ipRGCs play a role in non-image forming light sensation, effecting pupillary

constriction and the entrainment of the central circadian clock to light. There is some

evidence that eliminating Bmal1 in mice (either specifically in their ipRGCs or throughout

their entire body) impairs the functionality of the ipRGCs [20]. This is consistent with what

we observed when we disrupted clk in the Drosophila photoreceptors. Together, this

suggests that there may be a conserved mechanism through which circadian clocks

mediate the health of photoreceptor cells.

An inability to adequately respond to light stress may underly the accelerated

photoreceptor aging we observe when CLK function is diminished in the eye of adult flies.

Chronic exposure to phototoxic wavelengths or strong ambient light intensities, as well

as mutations in light adaptation proteins, elevates intracellular calcium ion concentrations bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

that result in rapid photoreceptor degeneration [21, 22]. Pittendrigh’s “escape the light”

hypothesis posits that circadian rhythms evolved as a means for cells/organisms to

anticipate and manage the deleterious effects of daily light exposure [23, 24]. One of the

key neuroprotective functions of intrinsic clocks within photoreceptors is their ability to

modulate time-of-day sensitivities to light. Electroretinogram (ERG) recordings in both

flies and mammals have revealed a circadian response pattern that peaks at night when

luminescence is approximately one-billion-fold less than during the day, and this pattern

in light sensitivity is abolished in in circadian mutants [25, 26]. Interestingly, exposing rats

to a bout of intense light at night results in significantly greater photoreceptor damage

and degeneration than when the same treatment is performed during the day, thus

highlighting the physiologic importance of the clocks in suppressing light sensitivity

during the day [27]. Our acrophase analyses revealed that circadian transcripts that

promote photoreceptor activation (Ca2+ influx) reach peak expression during the dark-

phase, while genes that terminate the phototransduction response (i.e., reducing

rhodopsin mediated signaling) peak in anticipation of the light-phase (Fig. 1f and

Supplementary Fig. 1j).

The eye regulates longevity in Drosophila

With age, declines in tissue homeostasis and chronic activation of the immune system

increases local and systemic inflammation, termed “inflammaging.” The deleterious

effects from inflammaging exacerbate pre-existing aging phenotypes and reduce survival bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

[28, 29]. Interestingly, partial inhibition of the primary immune response regulator,

NFkappaB, extends lifespan in Drosophila [30]. Photoreceptor degeneration is a main

source of inflammation within the mouse retina [31]. Here, our results demonstrate that

diminishing neuronal CLK function and forcing photoreceptor degeneration significantly

elevates systemic immune responses. Furthermore, we report dampened AMP expression

in the bodies of Rh null lines, indicating that reductions in phototransduction coincide

with reduced systemic inflammation in the fly. We have also found that flies reared on DR,

which improves photoreceptor viability, displayed dampened immune responses in

comparison to flies reared on AL. Interestingly, photoreceptor degeneration caused by

light- or calcium-mediated excitotoxicity is primarily the result of necrotic cell death [21].

Forced photoreceptor necrosis also results in necrotic cell death of surrounding cells [32].

Given that cytosolic f-actin can drive the sterile immune response in the fly, it is possible

that the increased systemic inflammation we report is due in part to elevations in necrosis

[33]. However, future studies will be needed to elucidate how diet and circadian rhythms

influence necrotic cell death in photoceptors, and the effect this has on the local niche.

Circadian disruption, achieved either genetically or via chronic circadian

misalignment with the environment, is associated with reduced longevity [34, 35]. Long-

lived humans (i.e., centenarians) display significantly improved behavioral rhythms

compared to “normal” aging groups [36]. Inversely, studies in mice and flies have

demonstrated that organisms that display arrhythmic, or non-24h rhythms, are bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

significantly shorter-lived than those who display near 24h (wild-type) circadian rhythms

[37, 38]. Furthermore, chronic phase-adjustments, as is common in shift workers, is

associated with early aging phenotypes and reduced lifespan in both mice and flies [35,

39]. Interestingly, placing BMAL1 knockout mice on calorie restriction fails to extend their

lifespan [40]; although, these mice lack BMAL1 expression in all tissues and throughout

development. To our knowledge, our study is the first to demonstrate that disruptions to

the photoreceptor, and in particular to the photoreceptor clocks, is sufficient to shorten

lifespan.

DR extends lifespan in part by maintaining photoreceptor homeostasis

A number of studies have previously investigated the effects of light exposure on lifespan

in Drosophila [41]. These studies, however, have not simultaneously examined the

influence of diet and the influence of the photoreceptor cells. Exposure to short-

wavelength light (i.e., blue-light), which is especially phototoxic, reduces survival in worms

and flies [22, 42]. Interestingly, housing flies in a 12:12 blue-light/dark cycle significantly

shortens lifespan even when those flies lack photoreceptors [22]. This effect appears to

be directly related to blue-light mediated neuronal cell death (i.e., extraocular blue light

sensing). Our lab previously demonstrated that DR-mediated lifespan extension is

completely abolished in flies reared in constant lighting conditions (LL), while flies reared

on AL experienced only a minor decrease in lifespan in LL [43]. We previously attested

that LL blocked DR’s lost ability to extend lifespan because it induces arrhythmicity. A new, bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

alternative, hypothesis is that the LL-mediated lifespan shortening on DR was also a result

of the phototoxic effects of LL, which force photoreceptors to rapidly degenerate. The

observation that knocking down ATP in the eye (a model of forced photoreceptor

degeneration) also significantly reduced lifespan on DR, further supports the

photoreceptor hypothesis. Furthermore, as discussed above, photoreceptors regulate the

timing of their light-sensitivity through the molecular clock. Therefore, housing a fly in LL

would likely render their photoreceptor clocks arrhythmic, and increase the photoreceptor

cells’ susceptibility to phototoxic stress. Interestingly, chronic dim-light exposure at night

also shortens lifespan in Drosophila [44]. Our results here, indicate that DR protects flies

from the lifespan-shortening effects of photoreceptor activation.

Although, forced photoreceptor degeneration is sufficient to significantly reduce

longevity on DR, we also demonstrate that DR protects against the lifespan-shortening

effects of photoreceptor activation during a normal 12:12 LD cycle. Flies reared on AL,

which display dampened circadian rhythms within the eye, were selectively sensitive to

lethality from the optogenetic activation of the photoreceptors. Inversely, we report that

white-eyed flies, which are highly susceptible to light-mediated retinal degeneration, only

display lifespan extension from constant darkness when they are reared on AL. Consistent

with this observation, Rh null flies (which have reduced photoreceptor activity) display

proportionally larger increases in lifespan when maintained on AL vs. DR. Importantly,

although Rh expression is enriched in the photoreceptor cells, it is also expressed in other bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

populations of neurons. Therefore, we cannot be certain that the lifespan extensions we

observe in Rh null flies is solely the result of diminished Rh levels in the photoreceptor

cells.

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which a Circadian time-course array b was not certified by peer review) is theexperimental author/funder, design who has granted bioRxiv a license to display the preprint in perpetuity. It is made Canton-Savailable underCanton-S aCC-BY-NC-ND 4.0 InternationalDR license. AL (n=4) Reared on diet DR (n=4) for 7 days AL

RNA isolation (whole fly) @ ZT 0 4 8 12 16 12 Microarray and JTK_CYCLE analysis 567 301 1609 (24hr period, p-value<0.05)

AL DR circadian circadian transcriptome transcriptome c d 1000 1000 AL AL 800 DR 800 DR 600 600

400 400 # of transcript s # of transcript s 200 200

0 0 1 2 3 4 5 >6 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 >1.6 JTK_CYCLE pvalue (-log10) Amplitude e Circadian on AL f Circadian on DR neutral lipid metabolic process chitin-based cuticle development

G protein-coupled receptor amino sugar metabolic process signaling pathway unsaturated fatty acid drug metabolic process biosynthetic process regulation of developmental triglyceride metabolic process pigmentation regulation of eye photoreceptor cell maintenance pigmentation 0 10 20 30 40 50 0 2 4 6 8 10 P value (-log10) P value (-log10) g Circadian on AL and DR h Circadian on AL and DR 80 60 AL AL 60 DR DR 40 40 20 Percent tota l 20 Percent tota l

0 0 1 2 3 4 5 >6 0 0.2 0.4 0.6 0.8 >1.0 JTK_CYCLE pvalue (-log10) Amplitude i Gene AL pvalue DR pvalue AL amplitude DR amplitude DR fold change -t test arr1 0.0302 0.1066 0.092 0.083 2.18 3.4e-4 cry 0.1415 4.07e-7 0.190 0.443 1.43 0.002 inaC 0.0030 0.0019 0.281 0.380 2.62 0.002 inaD 0.0423 0.0178 0.214 0.132 2.92 0.001 ninaA 0.0068 0.0001 0.352 0.350 2.69 0.034 ninaC 0.0178 0.0037 0.223 0.279 2.58 0.002 norpA 1.0000 0.0024 0.038 0.144 1.25 0.002 rh5 0.0068 0.0029 0.223 0.249 2.18 0.014 rh6 0.0030 0.0148 0.174 0.281 2.08 0.004 trp 0.0497 0.0045 0.147 0.189 2.82 0.004 trpl 0.0084 0.0009 0.243 0.250 2.27 7.3e-4

j Termination Activation 10

Arr2 8 sunglasses inaD 6 ninaA

value (-log10 ) inaC p Gbeta76C 4 rdgB fbxl4 trpl rtp trp stops Circadian 2 norpA Calx Arr1 ninaC santa-maria Rh4 Rh3 Gprk1 0 0 4 8 12 16 20 24 Phase (hr) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Figure 1. Dietary restriction amplifies circadian transcriptional output. (a) Experimental design of the time-course microarray and generation of the AL/DR circadian transcriptomes. Canton-S females were reared on AL or DR diets for 7 days. Flies were then collected, and mRNA was isolated from whole-fly lysates at 4-hour intervals for 24 hours (n=4 pooled mRNA samples from 30 flies per condition/timepoint). Circadian transcripts were identified with the JTK_CYCLE algorithm [45]. (b) Venn-diagram displaying the number of circadian transcripts that oscillate in flies reared on AL, DR, or in both diets. (c-d) Histograms of JTK_CYCLE pvalue statistics and circadian amplitudes of transcripts that cycle only on AL or DR diets. The y-axis indicates the total number of transcripts. A rightward shift was observed in pvalue and amplitude for transcripts that are circadian on DR compared to AL. (e-f) Gene-ontology enrichment categories corresponding to transcripts that cycle on AL (e) or DR (f). (g-h) Histograms of JTK_CYCLE pvalue statistics (g) and circadian amplitudes (h) of transcripts that are circadian on both diets. The y-axis indicates percent of transcripts out of the 301 total transcripts that oscillate on both diets. Transcripts that are circadian on both diets display smaller circadian pvalues and larger circadian amplitudes on DR compared to AL. (i) Table of phototransduction genes that are circadian on AL and DR. *cry is only circadian in DR, and arr1 is only circadian on DR. Fold-changes and ttest statistics were calculated by averaging the individual fold-changes in expression for each timepoint. (j) Circadian acrophase chart of transcripts that oscillate on DR and AL plotted as number of transcripts that peak at different timepoints throughout the day (as calculated by JTK_CYCLE algorithm). (k) Differential expression heatmap and associated GO terms for transcripts that are significantly upregulated (n=524) or downregulated (n=543) across all timepoints on DR compared to AL. Data were analyzed by student’s ttest comparing expression values from AL and DR transcriptome from ZT0-20, and transcripts that display a pvalue less than 0.05 were considered differentially expressed.

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which a Elav-GS-GAL4>UAS-CLK-Δ1 d Positive phototaxis assay e AL Canton-S DR Canton-S was not certified by peer review) is the author/funder,Start who hasFinish granted bioRxiv a license to display the preprint in perpetuity. It is made RNA-Seq experimental design 2.34e-4 0.011 0.004 4.46e-4 available under aCC-BY-NC-ND 4.0 International 100license. 5.15e-5 AL CLK-Δ1 AL cnt DR cnt DR CLK-Δ1 AL (+ RU486) (-RU486) (-RU486) (+RU486) DR 80 Reared on diet AL for 7 days 60 DR RNA isolation from heads at ZT 0 & 12 AL 40 DR AL 20 Phototaxis index (% ) RNA-Seq, differential expression analyses DR (DEseq2) 0 2 6 10 14 18 500 lux LED Time (days) Differential expression Differential expression Light Source AL nCLK-Δ1 vs cnt DR nCLK-Δ1 vs cnt f g AL Oregon-R DR Oregon-R AL phototaxis AL climbing 1.6e-4 DR phototaxis DR climbing b 2kb 100 0.754 0.002 1.2 7,771,500 7,771,000 7,770,500 7,770,000 7,769,500 7,769,000 7,768,500 0.015 Clk 8.8e-5 Exon 2 _ 82.14 80 1.0 _ 0

_ 89.59 p AL 60 =0.0041

_ 0 0.8 p

Clk-Δ1 _ 93.18 <0.0001

_ 0 40 _ 34.68 0.6

_ 0 _ 37.01 20 Phototaxis index (% )

AL cnt Fold-change from day 2 0.4 _ 0 _ 32.53 0 2 6 10 14 18 2 6 10 14 18 _ 0 _ 124.31 Time (days) Time (days) ZT 0

_ 0 h out out i AL cnt AL nCLK-Δ2 DR cnt DR nCLK-Δ2 DR _ 105.5 AL CLK DR CLK 0.014 0.697 0.028 0.046 0.063 Clk-Δ1 _ 0 100 _ 81.52 0.568 0.816 0.055 0.001 1.9e-4 0.173 100 7.7e-4 3.7e-4 1.4e-5 _ 0 7.6e-6 _ 31.52 80 0.469 0.095 3.5e-6 _ 0 0.077 80 _ 33.24 60 DR cnt 6.4e-5 _ 0 _ 40.71 40 60 _ 0 _ 150.31

_ 0 20 40 _ 110.9 Phototaxis index (% ) AL _ 0 0 Clk-Δ1 _ 131.77 Phototaxis index (% ) 2 6 10 14 18 20 _ 0 _ 9.97 Time (days)

_ 0 0 _ 16.71 AL cnt 2 6 10 14 18 _ 0 Time (days) _ 11.75 j AL cnt DR cnt _ 0 AL prCLK-Δ1 DR prCLK-Δ1 _ 109.32 15 ZT 12 ZT 0.470 _ 0 0.031 _ 126.25 DR 0.005 7.76e-5 _ 0 Clk-Δ1 _ 84.13

_ 0 10 _ 17.21

_ 0 _ 13.56 DR cnt _ 0 _ 10.35

Amplitude (mV ) 5 _ 0

c Circadian & down-regulated in nClk-Δ1 0 response to light stimulus 18 30 Temperature (°C) deactivation of rhodopsin mediated signaling phototransduction

retina homeostasis cellular calcium ion homeostasis DR photoreceptor AL cell maintenance 0 5 10 15 P value (-log10) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Figure 2. Design and additional analyses of nCLK-1 RNA-Seq., positive phototaxis, and ERG experiments. (a) Design of nCLK-1 RNA-Seq. Mated females were reared on AL or DR with the addition of vehicle or RU486 to induce the expression of CLK- 1 pan-neuronally for 7 days. mRNA was isolated from heads (n=3 biological replicates, N=30 heads per replicates) at ZT0 and ZT12. RNA-sequencing was performed and differentially expressed genes were identified with the DEseq2 software [46] package. (b) UCSC genome browser visualization of the individual tracks for each nCLK-1 RNA-Seq sample zoomed into exon 2 of clk (chr3L:7,766,807-7,773,169). Exon 2 (highlighted in red) of clk encodes the basic helix-loop-helix domain (DNA binding) of CLK that is selectively ablated in CLK-1 flies. Overexpression of CLK-1 results in a relative decrease in the ratio of tags at exon 2 vs exon 3-4 (right), while exon 3-4 display elevated tag density compared to control samples. Track size is normalized for each sample and the total number of tags is indicated as the top number (color coded to match each track) on the far right. (c) Gene- ontology enrichment terms and pvalue statistics for genes that are circadian in young heads (GEO81100) [7] and significantly down-regulated in the nCLK1 RNA-Seq on AL or DR. (d) Diagram of positive-phototaxis setup. Flies are sorted in clear elongated fly vials, dark adapted for 15 minutes, knocked to the bottom of the vial, and then laid horizontally and perpendicular to an LED light source. Once the light is turned on flies that reach the green line are scored as “positive-phototaxis” and counted at 15, 30, 45 seconds (See methods for additional details). (e) Positive phototaxis responses for Canton-S females reared on AL or DR diets. See methods for calculation of phototaxis index. For each timepoint results are represented as average percent positive phototaxis +/- SEM (n=24 biological reps, N=480 flies per condition). (f) Phototaxis responses for Oregon-R females. For each timepoint results are represented as average percent phototaxis response +/- SEM (n=8 biological replicates, N=160 flies per condition). (g) Canton-S climbing activity and positive phototaxis plotted as fold-change from responses at day 2. (n=24 biological replicates, N=480 flies per condition). (h) Positive phototaxis responses for Clkout females reared on AL or DR diets. For each timepoint results are represented as average percent positive phototaxis +/- SEM (n=24 biological reps, N=480 flies per condition). (i) Positive phototaxis responses for nCLK-2 flies (Elav-GS-GAL4>UAS-CLK-2. For each timepoint results are represented as average percent positive phototaxis +/- SEM (n=24 biological replicates, N=480 flies per condition). (j) Box-plots of electroretinogram amplitudes for prCLK-1 (Trpl-GAL4;GAL80>UAS-CLK-∆1OC) and control flies (Trpl- GAL4;GAL80>CantonSOC) reared at 18C (GAL80 active, GAL4 inactive) and 30C (GAL80 inactive, GAL4 active) for 6 days. Illuminance was set at 15000 Lux. (e-f and h-j) Pvalues were determined by two-tailed Student’s ttest (unpaired) at each timepoint. (g) Pvalues were determined by two-tailed Student’s ttest (unpaired) across genotypes.

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272a ; this versionb posted May 10, 2021. The copyright holder for this preprint (which nCLK-Δ1 hemolymph (mass-spec) was not certified by peer review) is ALthe cnt author/funder,AL nCLK- whoΔ2 has granted bioRxiv a licensecandidate tonon-candidate display the preprint in perpetuity. It is made DR cntavailable DRunder nCLK- aΔCC-BY-NC-ND2 50 4.0 International license. * 0.205 6 0.022 0.051 0.088 0.089 40 0.135 4 30

2 20 alue (−log10) Q v 0 * ** 10 Expression (Log2 FC) 0.024 0.013 AttC 0.158 -2 0 AttA DiptB Dro −4 −2 0 2 Log2 fold-change (nCLK-Δ1/cnt)

c 3.24e-39 100 2.5e-43 AL RNAi cnt AL ATPα-RNAi 80 DR RNAi cnt DR ATPα-RNAi 60

20 Phototaxis index (% )

0 Day 2 d e Upregulated in LD vs DD 4 Log antibacterial humoral response fold-change AttC 1.1 chitin-based cuticle development 3 DptB AttB 0 response to light stimulus DptA Mtk locomotory behavior

alue (−log10) 2 Listericin −1.1 Dro P v cuticle development

1 0 5 10 15 Pvalue (-log10) −1.0 −0.5 0.0 0.5 1.0 Log2 fold-change (LD/DD) f g 80 AL cnt DR cnt Log-rank 827.2 AL nCLK-Δ2 DR nCLK-Δ2 p<0.0001 100

60 80

Log-rank 103.0 60 40 p<0.0001 40 20 Percent survival 20 Median lifespan (days)

0 0 AL AL DR DR 0 20 40 60 80 100 h cnt nCLK-Δ1 cnt nCLK-Δ1 i Time (days)

100 Log-rank 1123.0 Photoreceptor-enriched transcription factors p<0.0001 6 80

Log-rank 210.3 5 Pvalue 60 p<0.0001 0.029 4 CG18011 Clk gl 0.020 40 Pif1A peb alue (−log10) Crg−1 3 CG31612 gol 0.010 retn 20 P v rgr dve Lmpt 0.001

Median lifespan (days) esc 2 oc CG31642 0 AL AL DR DR cnt nCLK-Δ2 cnt nCLK-Δ2 2 4 6 8 Enrichment (fold-change) j AL cnt DR cnt AL prCLK-Δ1 DR prCLK-Δ1 100

Percent survival 20

0 0 20 40 60 Time (days) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Figure 3. nCLK elevates immune responses and shortens longevity in a diet- dependent fashion. (a) Relative expression of AMP genes (AttA, DiptB, and Dro) calculated by RT-qPCR with mRNA isolated from nCLK-2 bodies. Results are plotted as average Log2 fold-change in expression calculated by the -Ct method, normalized to DR vehicle treated control samples as well as rp49 +/- SEM (n=3 biological replicates, N=30 flies per biological replicate). (b) Volcano-plot of hemolymph proteins identified by tandem mass- spectrometry comparing nCLK-1 (RU486 treated, N=300) and control (vehicle treated, N=300) flies reared on AL at day 14. Each dot represents an individual protein with a statistical significance less than 0.0001 comparing nCLK-1 and control hemolymph samples. Black dots are considered to be differentially expressed protein candidates with a Log2 fold-change cutoff of  0.6. AttC was the most highly up-regulated protein in nCLK-1 hemolymph compared to control. (c) Volcano-plot of gene expression changes in heads of y.w. flies housed in 12:12 LD vs constant darkness (DD) from Wijnen et al, 2006 (GSE3842) [47]. Fold-changes in response to light were calculated by averaging the changes in expression at each timepoint from a circadian time-course microarray (ZT 2, 6, 10, 14, 18, 22) and comparing expression between flies housed in LD compared to DD. (d) The top-5 enriched gene-ontology categories corresponding to genes that are upregulated in heads of flies housed in LD vs DD. (e) Positive phototaxis responses with eye-specific knockdown of ATP (GMR-GAL4>UAS-ATP-RNAi) compared to RNAi control flies (GMR-GAL4>UAS-mCherry-RNAi). For each timepoint results are represented as average phototaxis response +/- SEM (n=24 biological replicates, N=480 flies per condition). (f) Median lifespan of nCLK-1 flies corresponding to lifespans in (Fig. 3D). Data are plotted as the average median lifespan of the 3 biological replicates and error bars indicate +/- SEM. (g) Survival analysis of nCLK-2 flies. Survival data is plotted as an average of three independent lifespan repeats. Control flies (vehicle treated): AL N=505, DR N=504; nCLK-1 flies (RU486 treated): AL N=497, DR N=508. (h) Median lifespan of nCLK-2 flies corresponding to lifespans in (Supplemental Fig. 3D). Data are plotted as the average median lifespan of the 3 biological replicates and error bars indicate +/- SEM. (i) Volcano-plot of photoreceptor-specific transcription factors [48]. (j) Survival analysis of prCLK-1 flies. Survival data is plotted from one independent lifespan. Control flies (Trpl- GAL4;GAL80>UAS-CantonSOC): AL N=206, DR N=172; prCLK-1 flies (Trpl-GAL4;GAL80> UAS-CLK-1OC): AL N=154, DR N=190. *The CantonS and UAS-CLK-1 parental lines were outcrossed to w1118 and the F1 generations share the same genetic background. (a) Pvalues were calculated with the pairwise Student’s ttest comparing Log2 fold-changes in expression. (e) Pvalues were determined by two-tailed Student’s ttest (unpaired) at each timepoint. (f, h) Pvalues were determined by Chi square from Log-rank (Mantel-Cox) test.

bioRxivA preprint doi: https://doi.org/10.1101/2021.05.08.443272; thisB version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made 100 available under aCC-BY-NC-ND100 4.0 International license. AL Canton-S AL w1118 ;TRPP365 LD LD DR Canton-S DR w1118 ;TRPP365 1118 P365 80 AL Canton-S 80 AL w ;TRP DD 1118 P365 DD DR Canton-S DR w ;TRP

60 60

40 40 Percent surviva l Percent surviva l 20 20

0 0 0 20 40 60 80 100 0 20 40 60 80 Time (days) Time (days)

C Photoreceptor optogenetic activation D (Trpl-GAL4>UAS-csChrimson) Canton-S untreated all-trans retinal 100 + + + + AL cnt + + AL + retinal + + + + 80 + + + + DR cnt + + + DR + retinal + + 60

cytosol 40 + + + Percent surviva l + + + + 20 + + + + + + + + + + 0 + + + 0 20 40 60 80 100 ALDR ALDR Time (days) + +--

E AL RNAi cnt DR RNAi cnt F AL RNAi cnt AL nrv2-RNAi DR RNAi cnt DR nrv2-RNAi AL nrv3-RNAi DR nrv3-RNAi 0.004 3.9e-9 6.6e-5 1.4e-10 4.1e-6 2.3e-10 5.93-11 1.8e-12 100 0.860 5.8e-8 100 6.2e-9 1.9e-7 0.006 8.0e-6 0.003 2.7e-14 8.7e-8 0.896 8.8e-8 3.7e-7 80 80

60 60

40 40 Phototaxis index (% ) Phototaxis index (% ) 20 20

0 0 2 6 10 14 18 2 6 10 14 18 Time (days) Time (days) G H AL RNAi cnt AL nrv2-RNAi AL RNAi cnt AL nrv3-RNAi 100 DR RNAi cnt DR nrv2-RNAi 100 DR RNAi cnt DR nrv3-RNAi

80 80

60 60

40 40 Percent survival Percent survival 20 20

0 0 0 20 40 60 80 100 0 20 40 60 80 100 Time (days) Time (days)

I AL RNAi cnt DR RNAi cnt AL Arr1-RNAi DR Arr1-RNAi 0.744 0.618 0.897 0.258 0.096 2.39e-6 0.277 0.329 0.002 6.15e-8 3.10e-4 0.014 0.897 100 0.230 0.748 0.191 2.39e-6 1.69e-8 0.104 1.25e-8 80

40 Phototaxis index (% ) 20

0 2 6 10 14 18 Time (days) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Figure 4. Lighting and retinal control lifespans, optogenetic activation diagram, and lifespans of RNAi-mediated knockdown of ATP-subunits in the eye. (a) Survival analysis of Canton-S wildtype flies housed in 12:12h LD and constant darkness (DD). Survival data is plotted as an average of three independent lifespan crosses. AL LD N=549, AL DD N=510, DR LD N=558, DR DD N=509. (b) Survival analysis of white-eyed, photoreceptor null flies (w1118; TRPP365) housed in 12:12h LD or DD. Survival data is plotted as an average of two independent lifespan repeats. LD housed flies: AL N=290, DR N=373; DD housed flies: AL N=301, DR N=357. (c) Diagram of optogentic activation of photoreceptors. The photoreceptor-specific driver, Trpl-GAL4, drives the expression of a red-shifted csChrimson channel in R1-R8 photoreceptors. Addition of all-trans retinal (50M) in the fly media promotes the opening of optogenetic channels in the presence of red-light, allowing the flow of positively charged ions into the cytosol to activate photoreceptors. (d) Survival analysis of Canton-S flies reared in 12:12 red-light:dark on AL and DR diets with the addition of all-trans retinal or vehicle (control). Survival data is plotted as an average of two independent lifespan repeats. All-trans retinal treated flies: AL N= 340, DR N=328; Vehicle treated flies: AL N=347, DR N=328. (e-f) Positive phototaxis responses with eye-specific knockdown nrv2 (e, GMR-GAL4>UAS-nrv2-RNAi), and nrv3 (f, GMR-GAL4>UAS-nrv3-RNAi) compared to RNAi control flies (GMR-GAL4>UAS-mCherry- RNAi). For each timepoint results are represented as average phototaxis response +/- SEM (RNAi control and nrv2: n=24 biological replicates, N=480 flies per condition; nrv3: n=16 biological replicates, N=320 flies per condition). (g-h) Survival analysis of eye- specific nrv2 (g) and nrv3 (h) RNAi knockdown flies compared to RNAi control flies. Survival data is plotted as an average of three independent lifespan repeats for RNAi controls and nrv2 RNAi flies, and two independent lifespan crosses for nrv3 RNAi knockdown flies. RNAi cnt flies: AL N=493, DR N=490; nrv2 RNAi flies: AL N=482, DR N=513; nrv3 RNAi flies: AL N=301, DR N=288. (e-f) Pvalues were determined by two- tailed Student’s ttest (unpaired) at each timepoint.

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272b ; thisGβ version76c posted May 10, 2021.c The copyright holderretini nfor this preprint (which A Photoreceptor enriched genes 300 1,200 was not certified byn=1,000 peer genes review) is the author/funder, who has granted bioRxivYoung a license to display the preprint in perpetuity. It Yisoun madeg Circadian in young heads available under aCC-BY-NC-ND 4.0Ol dInternational license1,000. Old p=3.15e-6 n=366 genes 200 p=0.00015 800 p=0.02 Downregulted with age n=172 genes 600 Upregulated on DR vs AL (head RNA-Seq.) p=1.37e-10 ZT 0 n=102 genes ZT 12 n=99 genes 100 400

Downregulated in nCLK-Δ1 vs cnt (on DR) Expression (FPKM ) p=1.39e-5 p=0.07 Expression (FPKM ) 200 ZT 0 n=19 genes ZT 12 n=19 genes retinin, Gβ76c sunglasses 0 0 0 4 8 12 16 20 24 28 32 36 40 44 48 0 4 8 12 16 20 24 28 32 36 40 44 Zeitgeber time (hr) Zeitgeber time (hr) d sunglasses 120 Gβ76c f retinin e AL cnt DR cnt AL cnt DR cnt AL nCLK-Δ1 AL nCLK-Δ1 DR nCLK-Δ1 DR nCLK-Δ1 100 p=6.3e-5 20 30 9.6e-6 7.0e-5 0.024 0.006 80 2.4e-6 15 p=4.19e-7 0.777 60 20 0.014 p=0.08 9.2e-8 3.6e-10 0.0004 40 10 1.4e-5 0.014 (1000x) (1000x) Expression (FPKM ) 20 Young 10 Old 5 0 0 4 8 12 16 20 24 28 32 36 40 44 DEseq2 normalized count s Zeitgeber time (hr) DEseq2 normalized count s 0 0 0 12 0 12 Zeitgeber time (hr) Zeitgeber time (hr) g sunglasses h AL cnt DR cnt AL nCLK-Δ1 DR nCLK-Δ1 0.076 4.5 0.307

4.0 1.9e-5 sunglasses promotor TF binding 4.7e-15 0.012 CLK fold-change 3.5 POL 0.152 -2 0 2 2 6 8 12 16 20 (1000x) 3.0 ZT (hr) 2.5

DEseq2 normalized count s 2.0 0 12 Zeitgeber time (hr) i j AL RNAi cnt AL Retinin-RNAi AL RNAi cnt AL Gβ76c-RNAi AL Sunglasses-RNAi AL Gβ76c-RNAi 1.4e-16 1.3e-31 6.7e-16 100 AL Retinin-RNAi AL Sunglasses-RNAi 5.8e-4 100 1.9e-9 0.003 5.0e-17 1.4e-9 1.5e-4 0.099 0.001 2.2e-20 0.028 0.002 0.035 80 80

60 60

40 40 Percent survival

Phototaxis index (% ) 20 20

0 0 0 20 40 60 80 2 6 10 14 18 Time (days) Time (days) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Figure 5. Identification of photoreceptor enriched CLK-output genes, and additional analyses with eye-specific knockdown of G76c, retinin, and sunglasses. (a) Bioinformatics pipeline for identifying photoreceptor enriched, CLK-output genes. (b-d) Circadian expression of G76c, retinin, and sunglasses and their corresponding circadian pvalue statistics for young (5-day old) and old (55-day old) wildtype heads from Kuintzle et al., 2017 [7]. (e-g) Normalized expression counts for G76c, retinin, and sunglasses from the nCLK-1 RNA-Seq. Results are represented as average expression counts calculated by DEseq2 +/- SEM. (h) Heatmap of CLK and POL (Drosophila polymerase) tag-densities at the 5’-untranslated region of the sunglasses promoter over a circadian time-course from ChIP-Chip analyses [49]. Consistent with other direct CLK target genes, Abruzzi et al., 2011 report maximal CLK binding at ZT 12, while POL displayed antiphasic binding to that of CLK and aligned with the phase of sunglasses mRNA expression (ZT 0-2). *CLK binding was not observed in GMR-HID heads suggesting sunglasses is under CLK transcriptional regulation specifically in the neurons of the eye. (i) Positive phototaxis responses with eye-specific knockdown of G76c (GMR-GAL4>UAS- G76c-RNAi), retinin (GMR-GAL4>UAS-retinin-RNAi), and sunglasses (GMR-GAL4>UAS-sunglasses-RNAi) compared to RNAi control flies (GMR-GAL4>UAS-mCherry-RNAi) reared on AL. For each timepoint results are represented as average phototaxis response +/- SEM (RNAi control n=24 biological replicates, N=480 flies per condition; G76c RNAi n=24 biological replicates, N=480 flies per condition, retinin RNAi n=16 biological replicates, N=384 flies per condition; sunglasses RNAi n=24 biological replicates, N=480 flies per condition). (j) Survival analysis of eye-specific G76c-RNAi, retinin-RNAi, sunglasses-RNAi, and RNAi control knockdown flies compared to RNAi control flies reared on AL. Survival data is plotted as an average of three independent lifespan repeats for RNAi control, G76c- RNAi, sunglasses-RNAi flies and two independent lifespan repeats for retinin-RNAi flies. RNAi cnt flies: N=493; G76c RNAi flies: N=543; retinin RNAi flies: N=353; sunglasses RNAi flies: N=503. (b-d) Circadian pvalues were determined by ARSER algorithm by Kuintzle et al., 2017 [7] (AL=red, DR=blue). To compare gene expression profiles with age we utilized the two-tailed Student’s ttest (paired) to determine Pvalues (black). (e-g) Pvalues were determined by DEseq2 differential expression analysis. (i) Pvalues were determined by two-tailed Student’s ttest (unpaired) at each timepoint comparing the phototaxis index of RNAi control flies to retinin- and sunglasses-RNAi flies.

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which Genewas symbol not certified by peer review) is the author/funder,Gene name who has granted bioRxiv younga license pvalue to displayold pvalue the preprintAL pvalue in perpetuity.DR pvalue It isCLK made binding available under aCC-BY-NC-ND 4.0 International license. Arr1 Arrestin 1 0.0084 0.0040 0.0302 0.1066 * † Arr2 Arrestin 2 6.89e-09 0.0096 1 1 Calx Na/Ca-exchange protein 0.0059 0.0001 1 0.1623 * † Camta Calmodulin-binding transcription factor 0.0158 0.0029 1 1 * CdsA CDP diglyceride synthetase 8.67e-06 0.0026 1 0.1856 * † Cib2 Calcium and integrin binding family member 2 0.4123 0.0301 1 0.7501 cl clot 0.0069 0.0406 1 1 * † cry cryptochrome 0.0006 0.0005 0.1415 4.07e-7 * Dmn Dynactin 2, p50 subunit 0.0084 0.0401 1 1 Ekar Eye-enriched kainate receptor 0.0170 0.0432 1 0.6100 Fbxl4 F box and leucine-rich-repeat gene 4 0.0008 0.5073 0.2729 0.0583 * † Galphaq G protein alpha q subunit 0.1135 0.0076 1 1 * † Gbeta76c G protein beta-subunit 76C 3.15e-06 1.39e-05 1 1 Gprk1 G protein-coupled receptor kinase 1 0.0378 0.0260 1 1 * Gycalpha99B Guanylyl cyclase alpha-subunit at 99B 0.0060 0.0208 1 1 Ih I[[h]] channel 0.0001 0.0002 1 0.3086 * † inaC inactivation no afterpotential C 5.92e-05 0.0001 0.0030 0.0012 inaD inactivation no afterpoteinal D 5.99e-07 2.48e-05 0.0423 0.0178 Inx3 Innexin 3 0.0498 0.0212 1 0.0213 Inx7 Innexin 7 0.0254 0.3187 1 0.0583 Itpr Inositol 1,4,5,-tris-phosphate receptor 0.0261 0.1080 1 1 laza lazaro 0.0049 0.0024 1 0.0148 Lrpprc Leucine-rich pentatricopeptide repeat containing 2 0.0136 0.0918 0.8288 0.3086 ninaA neither inactivation nor afterpotential A 8.46e-06 0.0025 0.0068 0.0001 ninaB neither inactivation nor afterpotential B 0.3094 0.0049 1 0.9134 ninaC neither inactivation nor afterpotential C 0.0251 0.0001 0.0178 0.0037 * † ninaD neither inactivation nor afterpotential D 0.0241 0.1043 1 1 ninaE neither inactivation nor afterpotential E 0.1575 0.0044 0.3480 1 * † norpA no receptor potential A 0.0057 0.0447 1 0.0024 * † PAPLA1 Phosphatidic Acid Phospholipase A1 0.0241 0.8591 1 1 * Pdh Photoreceptor dehydrogenase 0.0017 0.0044 0.0003 3.37e-05 pinta prolonged depolarization afterpotential (PDA) is not apparent 0.0346 0.3933 1 1 PIP5k59B Phosphatidylinositol 4-phosphate 5-kinase 59B 0.2862 0.0159 1 0.2729 Pld Phospholipase D 0.0290 0.0860 1 1 * † porin porin 0.0095 0.0017 0.8288 1 * rdgB retinal degeneration B 0.0004 0.3399 1 1 * † rdgC retinal degeneration C 0.0681 0.0766 1 0.3914 * † rdhB retinol dehydrogenase B 2.70e-05 0.0074 0.4911 0.0148 Rh3 Rhodopsin 3 0.0107 0.4194 1 1 Rh4 Rhodopsin 4 0.0169 0.2723 1 1 Rh5 Rhodopsin 5 0.0546 0.0029 0.0068 0.0030 * Rh6 Rhodopsin 6 0.1415 0.0097 0.0030 0.0148 rtp retinophilin 0.0004 0.0272 1 1 santa-maria scavenger receptor acting in neural tissue and majority of rh is absent 0.0284 0.0002 1 0.0123 shakB shaking B 0.0120 0.0304 0.0084 0.5481 * † stmA stambha A 0.0074 0.0045 1 0.4390 * † stops slow termination of phototransduction 0.0037 0.0008 1 1 * † trp transient receptor potential 0.0040 0.0121 0.0497 0.0045 * † TrpA1 Transient receptor potential cation channel A1 0.1622 0.0208 0.6773 0.3914 trpl transient receptor potential-like 0.0013 0.0002 0.0084 0.0009 Tsp42Ej/sunglasses Tetraspanin 42Ej (sunglasses) 6.29e-05 0.0774 1 1 * † Xport-A exit protein of rhodopsin and TRP A 0.0022 0.0002 1 1 * Clk binding † CLK binding is eye-specific Supplemental Table 1. Circadian statistics and CLK binding of light-response genes. Circadi- an pvalue statistics for light response genes in young and old wildtype heads (calculated by ARSER by Kuintzle et al., 2017 [7]) and whole flies reared on AL and DR (calculated by JTK_CY- CLE in this study). bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which wasDrosophila not certified strain by peer review) is the author/funder,Genotype who has granted bioRxiv a license to display the preprintSource in perpetuity. It isCatalog made # available under aCC-BY-NC-ND 4.0 International license. ninaE17 outcrossed to w1118 w1118; ninaE17 Laboratory of Craig Montell N/A rh32 outcrossed to w1118 w1118; rh32 Laboratory of Craig Montell N/A rh41 outcrossed to w1118 w1118; rh41 Laboratory of Craig Montell N/A rh6G outcrossed to w1118 w1118; rh6G Laboratory of Craig Montell N/A Gqα1 outcrossed to w1118 w1118; Gqα1 Laboratory of Craig Montell N/A CantonS CantonS (Janelia Farm) Laboratory of Craig Montell N/A CantonS outcrossed to w1118 CantonS This manuscript N/A OregonR OregonR Bloomington Drosophila Stock Center BL25125 TrpP365 w[*]; trp[P365] Bloomington Drosophila Stock Center BL9044 GMR-GAL4 w[*]; P{w[+mC]=GAL4-ninaE.GMR}12 Bloomington Drosophila Stock Center BL1104 Elav-GS-GAL4 y[1] w[*]; P{w[+mC]=elav-Switch.O}GSG301 Bloomington Drosophila Stock Center BL43642 Trpl-GAL4 w;trpl-GAL4/Tm6B,Tb Bloomington Drosophila Stock Center BL52274 Trpl-GAL4; GAL80ts w;trpl-GAL4/CyO;tub-GAL80ts This manuscript N/A CLKout w*;[ClkOUT] Bloomington Drosophila Stock Center BL56754 UAS-csChrimson (optogenetic) w[1118] P{y[+t7.7] w[+mC]=20XUAS-IVS-CsChrimson.mVenus}attP18 Bloomington Drosophila Stock Center BL55134 UAS-CLK∆1 w[*]; P{w[+mC]=UAS-Clk.Delta}1 Bloomington Drosophila Stock Center BL36318 UAS-CLK∆1 outcrossed to w1118 w[*]; P{w[+mC]=UAS-Clk.Delta}1 This manuscript N/A UAS-CLK∆2 w[*]; P{w[+mC]=UAS-Clk.Delta}865 Bloomington Drosophila Stock Center BL36319 Gβ76c-RNAi y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF03127}attP2 Bloomington Drosophila Stock Center BL28507 tsp42Ej-RNAi (sunglasses) y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF03325}attP2/TM3, Sb[1] Bloomington Drosophila Stock Center BL29392 retinin-RNAi y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMC04693}attP40 Bloomington Drosophila Stock Center BL57389 ATPα-RNAi y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS00703}attP2 Bloomington Drosophila Stock Center BL28073 nrv2-RNAi y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF03081}attP2 Bloomington Drosophila Stock Center BL28666 nrv3-RNAi y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMJ22547}attP40 Bloomington Drosophila Stock Center BL60367 RNAi-cnt (BDSC) y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=VALIUM20-mCherry}attP2 Bloomington Drosophila Stock Center BL35785 arr1-RNAi w1118; P{GD11744}v22196/TM3 Vienna Drosophila Resource Center v22196 RNAi-cnt (VDRC) y,w[1118];P{attP,y[+],w[3`] Vienna Drosophila Resource Center v60100 Supplemental Table 2. Drosophila strains used in this study. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Data 1. AL and DR circadian transcriptome analyses. These files contain JTK_CYCLE statistics accompanied gene-ontology enrichment terms/scores for AL and DR circadian transcriptomes, circadian acrophase analyses, and differential gene-expression analyses.

Supplemental Data 2. Gene-ontology enrichment analyses of genes that are circadian in young heads. These files contain the enriched biological processes in young wild-type heads from Kuintzle et al., 2017, highlighting circadian processes within the eye.

Supplemental Data 3. Additional nCLK-1 RNA-Seq analyses. Included in these files are normalized count reads generated by DEseq2 across all experimental groups and replicates from the nCLK-1 RNA-Seq. The normalized expression counts across all samples for the gene-ontology terms “Deactivation of rhodopsin mediated signaling” and “Antimicrobial humoral response” (corresponding to Fig. 2b and 3b) are also reported.

Supplemental Data 4. Cross-comparison of wild-type circadian transcriptome and nCLK- 1 RNA-Seq analyses. These files include gene-ontology enrichment terms/scores for genes that are circadian in wild-type heads (from Kuintzle et al., 2017, GSE81100) and downregulated in nCLK-1 heads.

Supplemental Data 5. nCLK-1 hemolymph mass-spec analysis. These files contain the proteins identified and quantification of differential expression comparing proteomic profiles between nCLK-1 and control hemolymph. Enriched bioprocesses are also included for significantly up- or downregulated proteins.

Supplemental Data 6. Bioinformatic pipeline for identification of eye-specific and DR- sensitive CLK-output genes Gbeta76c, retinin, and sunglasses. These files provide the filtered gene-lists that correspond to the bioinformatic filtering steps performed in Fig. 5a-d and Supplementary Fig. 5a.

Supplemental Data 7. Survival analyses. These files report lifespan statistics (Log-Rank and Hazard Ratios) and group sizes (n) for the survival analyses performed.

Supplemental Data 8. Analyses of transcriptional responses to light. These files report gene-ontology enrichment terms/scores for genes that are differentially expressed in wild-type fly heads in response to being housed in 12:12h LD vs constant dark (from Wijnen et al., 2006, GSE3842) and correspond to Supplementary Fig. 3D-E. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.08.443272; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Data 9. Positive phototaxis responses and statistics. These files report detailed statistics (ttest and 2way ANOVA) for the positive phototaxis experiments performed.

Supplemental Data 10. Electroretinogram analyses and statistics. These files include detailed ttest statistics for the ERG assays performed at day 14 and 21 in nCLK-1 flies.

Supplemental Data 11. Experimental materials.

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