Neuroscience

Elevated DNA Damage without signs of aging in the short-sleeping Mexican Cavefish

Evan Lloyd
Fanning Xia
Kinsley Moore
Carolina Zertuche
Aakriti Rastogi
Rob Kozol
Olga Kenzior
Wesley Warren
Lior Appelbaum
Rachel L Moran
Chongbei Zhao
Erik Duboue
Nicolas Rohner author has email address
Alex C Keene author has email address

Department of Biology, Texas A&M University, College Station, TX, United States
Stowers Institute for Medical Research, Kansas City, MO, United States
Harriet Wilkes Honors College, Florida Atlantic University, Jupiter, FL, United States
Department of Genomics, University of Missouri, Columbia, MO, United States
Faculty of Life Science and the Multidisciplinary Brain Research Center, Bar Illan University, Ramat Gan, Israel

https://doi.org/10.7554/eLife.99191.1

Open access
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Figures and data

Cavefish harbor increased neuronal DNA damage and gut ROS.
(A,B) Representative images of cells stained with DAPI and γH2AX in the rhombencephalon of surface fish (A) and cavefish (B). Scale bar = 5 μm. (C) Mean γH2AX fluorescence across three regions of surface fish and cavefish brains. (rhomb: rhombencephalon; mes: mesencephalon; tele: telencephalon) (Mixed-effects analysis: F_{1, 68} = 32.08, p < 0.0001). (D) Representative image of larval gut showing regions in false color (re: rectum). (E,F) Representative images of surface fish and cavefish guts stained with DHE marking ROS. Scale bar = 500 μm. (G) Mean DHE fluorescence across four regions of surface fish and cavefish guts (two-way repeated measures ANOVA: F_{1, 35} = 48.36, p < 0.0001).

Cavefish lack a sleep response to UV-B-induced DNA damage.
(A) The 24-hour sleep profiles of surface fish exposed to 30 or 60 seconds of UV-B light compared to controls. (B) Average sleep amount in surface fish in the 3 hours following UV-B exposure (one-way ANOVA: F_{2, 202} = 18.75, p < 0.0001). (C) Average bout length in surface fish in the 3 hours following UV-B exposure (one-way ANOVA: F₂_{, 201} = 8.301, p = 0.0003). (D) Bout number in the 3 hours following UV-B exposure (one-way ANOVA: F₂_{, 201} = 11.5, p < 0.0001). (E) The 24-hour sleep profiles of cavefish exposed to 30 or 60 seconds of UV-B light compared to controls. (F) Average sleep amount in cavefish in the 3 hours following UV-B exposure. (G) Average bout length in cavefish in the 3 hours following UV-B exposure. (H) Bout number in cavefish in the 3 hours following UV-B exposure. (ZT=Zeitgeber time). All treatments performed at ZT0.

Response in sleep pressure to UV-B-induced DNA damage.
(A) The 24-hour P(wake) profiles of surface fish exposed to 30 or 60 seconds of UV-B light compared to controls. (B) P(wake) in surface fish in the 3 hours following UV-B exposure (one-way ANOVA: F_{2, 202} = 21.08, p < 0.0001). (C) The 24-hour P(doze) profiles of surface fish exposed to 30 or 60 seconds of UV-B light compared to controls. (D) P(doze) in surface fish in the 3 hours following UV-B exposure (one-way ANOVA: F₂_{, 202} = 14.09, p < 0.0001). (E) The 24-hour P(wake) profiles of cavefish exposed to 30 or 60 seconds of UV-B light compared to controls. (F) P(wake) in cavefish in the 3 hours following UV-B exposure. (G) The 24-hour P(doze) profiles of cavefish exposed to 30 or 60 seconds of UV-B light compared to controls. (H) P(doze) in cavefish in the 3 hours following UV-B exposure. (ZT=Zeitgeber time). All treatments performed at ZT0.

Transcriptional responses to UV-B-induced DNA damage in surface fish and cavefish.
(A) Schematic of experimental design. (B) Multidimensional scaling plot depicting the variances in principal component space between the processed sequencing samples. PC1 (two-way ANOVA: (Treatment) F_1,10=6.388, p=0.03, (Population) F_1,10=4970, p<0.0001, (Interaction) F_1,10=17.56, p=0.0019. PC2 (two-way ANOVA: (Treatment) F_1,10=465.0, p<0.0001, (Population) F_1,10=0.4969, p=0.497, (Interaction) F_1,10=18.56, p=0.0015 (C) Bi-directional volcano plot of changes in gene expression in surface and cave larvae after exposure to DNA damaging UV-B radiation. (D) Heat map of gene expression in DNA repair genes which responded significantly in UV-B-exposed surface fish.

Transcriptional responses to UV-B-induced DNA damage in surface fish and cavefish.
(A) Volcano plot of gene expression in surface fish in response to UV-B radiation, with significant genes highlighted in red (adjusted p < 0.05). (B) Volcano plot of gene expression in cavefish in response to UV-B radiation, with significant genes highlighted in red (adjusted p < 0.05). (C) Most highly enriched GO terms in a gene set enrichment analysis of surface fish exposed to UV-B radiation. (D) Most highly enriched GO terms in a gene set enrichment analysis of cavefish exposed to UV-B radiation.

DNA repair gene expression in surface fish and cavefish.
(A,D) Adult fins were collected at Zeitgber or Circadian time 8. cpdp (A) and ddb2 (D) mRNA levels were determined by qPCR. Expression of cpdp (one-way ANOVA: F = 11.99, p = 0.0039) and ddb2 (one-way ANOVA: F = 0.7576, p = 0.4997) was compared among all populations. ns p ≥ 0.05, ** p < 0.01. (B,E) RNA sequencing data obtained from cpdp (B) and ddb2 (E) gene expression level in 30 dpf juvenile fish. (TPM: transcript per million). (C,F) Fish larvae aged 5 dpf were collected at different Zeitgber or Circadian times. cpdp (C) and ddb2 (F) mRNA levels were determined by qPCR.

Pachón cavefish-derived cells exhibit a lower UV-induced DNA damage response and repair compared to surface fish.
(A,B) Immunostaining of CPD shows a similar DNA damage level induced by UV in surface fish and Pachón cavefish embryonic fibroblasts. White circles indicate the nuclear area by DAPI staining. Orange indicates CPD. Scale bar, 40 μm. P-values were determined by two-way ANOVA: F = 0.09703, p =0.7586. ns p = 0.6404, **** p < 0.0001. (C,D) Western blot of γH2AX indicates a diminished DNA damage response in Pachón cavefish embryonic fibroblasts compared to surface fish cells. (E,F) Flow cytometry images and quantification for host cell reactivation assays in surface fish and Pachón cavefish embryonic fibroblasts. Red line sets the GFP positive signal threshold. P-values were determined by unpaired t-test. (G) Representative GFP images for host cell reactivation assays in surface fish and Pachón cavefish embryonic fibroblasts. Scale bars, 500 μm.

Pachón cavefish-derived cells exhibit a lower UV-induced DNA damage response and repair compared to surface fish.
(A) Bright field images of surface fish and Pachón cavefish embryonic fibroblasts. Scale bar, 200 μm. (B) Immunostaining of vimentin in surface fish and Pachón cavefish embryonic fibroblasts. Yellow and blue show vimentin and DAPI staining, respectively. Scale bar, 100 μm. (C) Heat map of differentially expressed genes among different cell types. The number below the heat map indicates independent biological replicates (n = 13). Red and blue indicate upregulated and downregulated genes, respectively. (SEF: surface fish embryonic fibroblast; PEF: Pachón cavefish embryonic fibroblast; MEF: mouse embryonic fibroblast; iPSC: induced pluripotent stem cell; mESC: mouse embryonic fibroblast; SFL: surface fish liver-derived cell; CFL: Pachón cavefish liver-derived cell). (D) Western blot of γH2AX indicates a diminished DNA damage response in Pachón cavefish liver-derived cells compared to surface fish liver-derived cells.

Transcriptional response to aging is diminished in cavefish across tissues.
(A) Multidimensional scaling plot plotting the distances in principal component space between the brain samples. PC1 (two-way ANOVA: (Treatment) F_1,11=4.209, p=0.0648, (Population) F_1,11=2133, p<0.0001, (Interaction) F_1,11=0.029, p=0.867 PC2 (two-way ANOVA: (Treatment) F_1,11=16.83, p=0.0018, (Population) F_1,11=0.0002, p<0.99, (Interaction) F_1,11=19.37, p=0.0011 (B) Number of differentially expressed genes in the aged condition across tissues. (C) Bi-directional volcano plot depicting differences in gene expression between young and aged brains. (D) Dot plot visualizing the top 8 activated and suppressed gene ontology terms in aged cavefish brains resulting from GSE analysis.

Aging-induced changes in gene expression.
Multi-dimensional scaling plots of gene expression in young and aged surface fish and cavefish samples, in the gut (A) PC1 (two-way ANOVA: (Treatment) F_1,11=10.41, p=0.0081, (Population) F_1,11=264, p<0.0001, (Interaction) F_1,11=3.135, p=0.1043. PC2 (two-way ANOVA: (Treatment) F_1,11=8.257, p=0.0151, (Population) F_1,11=0.594, p=0.4571, (Interaction) F_1,11=7.113, p=0.0219. Heart (B) PC1 (two-way ANOVA: (Treatment) F_1,12=0.0302, p=0.8649, (Population) F_1,12=0.8455, p=0.376, (Interaction) F_1,12=0.9535, p=0.3481. PC2 (two-way ANOVA: (Treatment) F_1,12=7.354, p=0.0189, (Population) F_1,12=0.7.584, p=0.0175, (Interaction) F_1,12=0.3345, p=0.5737. Liver (C) PC1 (two-way ANOVA: (Treatment) F_1,12=1.067, p=0.3219, (Population) F_1,12=4.521, p=0.0549, (Interaction) F_1,12=0.1329, p=0.7217. PC2 (two-way ANOVA: (Treatment) F_1,12=2.039, p=0.1788, (Population) F_1,12=6.986, p=0.0214, (Interaction) F_1,12=0.0419, p=0.8413. Muscle (D) PC1 (two-way ANOVA: (Treatment) F_1,12=0.5895, p=0.4574, (Population) F_1,12=0.7313, p=0.4092, (Interaction) F_1,12=0.01396, p=0.9079. PC2 (two-way ANOVA: (Treatment) F_1,12=0.15, p=0.7053, (Population) F_1,12=89.42, p<0.0001, (Interaction) F_1,12=0.009, p=0.9251.

GSEA analyses of aging surface fish and cavefish tissues.
Top GO terms resulting from GSEA analysis in aged surface fish (left) and cavefish (right) tissues. Gut (A,B), heart (C,D), liver (E,F), and muscle (G,H). Top 8 results in each direction are shown; if less than 8 terms were enriched, all results are shown.

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