Abstract
Telomere shortening is a hallmark of aging and is counteracted by
telomerase. As in humans, the zebrafish gut is one of the organs with
the fastest rate of telomere decline, triggering early tissue
dysfunction during normal zebrafish aging and in prematurely aged
telomerase mutants. However, whether telomere-dependent aging of an
individual organ, the gut, causes systemic aging is unknown. Here we
show that tissue-specific telomerase expression in the gut can prevent
telomere shortening and rescues premature aging of tert^−/−. Induction
of telomerase rescues gut senescence and low cell proliferation, while
restoring tissue integrity, inflammation and age-dependent microbiota
dysbiosis. Averting gut aging causes systemic beneficial impacts,
rescuing aging of distant organs such as reproductive and hematopoietic
systems. Conclusively, we show that gut-specific telomerase expression
extends the lifespan of tert^−/− by 40%, while ameliorating natural
aging. Our work demonstrates that gut-specific rescue of telomerase
expression leading to telomere elongation is sufficient to systemically
counteract aging in zebrafish.
Subject terms: Telomeres, Ageing, Senescence
__________________________________________________________________
Telomere shortening is a hallmark of aging and genetic telomerase
deficiency causes premature aging phenotypes and reduces lifespan. Here
the authors show that gut-specific telomerase expression is sufficient
to rescue aging phenotypes and extend the lifespan of
telomerase-deficient zebrafish, as well as ameliorate signs of aging in
wild-type animals.
Main
The discovery that the lifespan can be genetically extended in
Caenorhabditis elegans initiated a new era of research aiming to define
interventions to promote the lifespan and healthspan extension^[44]1.
Since then, improvements achieved by modulating the hallmarks of aging
have provided specific therapeutic targets for healthy aging^[45]2. For
example, reverting age-related deregulation of nutrient-sensing
mechanisms by interventions such as caloric restriction or rapamycin
(mammalian target of rapamycin (mTOR) inhibitor) treatment increases
the lifespan in several species^[46]3,[47]4. Similarly, genetic and
pharmacological removal of senescent cells can delay age-associated
defects resulting in lifespan extension in mice^[48]5,[49]6.
Telomere shortening and dysfunction are major determinants of
aging^[50]2. Telomeres protect chromosome ends from degradation and
recognition by DNA damage response pathways^[51]7. Due to the
‘end-replication problem’, telomeres gradually shorten with each round
of cell division. When telomeres are critically short, DNA damage
responses are triggered that culminate in cell cycle arrest,
replicative senescence^[52]8,[53]9 and loss of tissue integrity^[54]2.
Telomere shortening is counteracted by a specific reverse transcriptase
termed telomerase. TERT expression, the catalytic component of
telomerase, is restricted to stem or progenitor cells^[55]10,[56]11.
However, telomerase expression is insufficient to fully restore
telomere erosion throughout the lifespan of vertebrates; consequently,
aging organisms show signs of telomere dysfunction^[57]11.
Patients that carry mutations in telomerase or telomere maintenance
protein genes show premature shortening of telomeres, short life
expectancy and a set of pathologies known as telomere biology disorders
(TBDs)^[58]12,[59]13. Similarly, depletion of telomerase in zebrafish
accelerates telomere shortening, causing premature aging phenotypes and
reduced lifespan in tert^−/− animals^[60]14–[61]16. tert^−/− zebrafish
present the same dysfunction events observed during natural zebrafish
aging at an anticipated rate^[62]14–[63]16. DNA damage associated with
short telomeres is first observed in the gut; it is concomitant with
reduced cell proliferation, accumulation of senescent cells and
functional defects both in naturally aging and tert^−/−
zebrafish^[64]14,[65]17. Importantly, telomere shortening results in
cellular and functional defects in the gut at a time when other organs
are clear of tissue dysfunction^[66]14. As in zebrafish, the human
gastrointestinal system is one of the organs with the fastest rate of
telomere shortening^[67]18. Severe TBDs are often associated with
gastrointestinal syndromes^[68]19,[69]20; increased telomere shortening
was observed in the intestinal epithelium of patients with inflammatory
bowel disease^[70]21,[71]22. Therefore, gut homeostasis is heavily
dependent on telomere integrity.
Over a century ago, Metchnikov proposed that loss of tissue integrity
and aging derives from chronic systemic inflammation promoted by
increased intestine permeability and infiltration of microorganisms and
their products into the bloodstream. Even though weakening of the
intestinal barrier is a major feature of gut aging^[72]4, it is unclear
whether organ-specific decline influences overall organismal aging. In
this Article, we show that gut-specific telomerase expression in
tert^−/− zebrafish is sufficient to delay gut aging. Counteracting gut
aging improves health of the entire organism, reverting gut microbiota
dysbiosis and aging phenotypes in distant organs of tert^−/− zebrafish.
Finally, we show that the most relevant systemic effect of gut-specific
telomerase expression is lifespan extension, while improving natural
aging. Thus, gut telomere-dependent aging controls aging of the entire
organism.
Results
Tissue-specific telomerase expression rescues gut aging
To investigate how telomere-dependent gut aging impacts the organism,
we generated a zebrafish transgenic line harboring a Cre-inducible
zebrafish tert transgene driven by an enterocyte-specific fabp2
promoter^[73]23 in a tert^+/− genetic background (Fig. [74]1a). After
crossing this line with tert^+/− fish, we induced the tert transgene
expression by microinjection of Cre mRNA in one-cell-stage embryos,
creating the following sibling fish: (1) tert^−/− containing the full
construct (tert^−/− no Cre); (2) tert^−/−-expressing tert transgene
(tert^−/− + Cre); and (3) tert^+/+ containing the full construct
(wild-type (WT)).
Fig. 1. Gut-specific and Cre-mediated tert expression rescues gut aging
phenotypes.
[75]Fig. 1
[76]Open in a new tab
a, Schematic representation of the transgene for Cre-inducible and
tissue-specific expression of tert mRNA. b, RT–qPCR analysis of tert
transgene mRNA and total tert mRNA (endogenous + transgene) expression
in 9-month-old gut extracts (n[WT] = 5 and 6;
[MATH:
nter
t−/−noCre :MATH]
= 7 and 8 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 and 5 fish, respectively; levels were normalized by rps11 gene
expression levels). c, Quantification of mean telomere length by TRF
analysis (n[WT] = 7;
[MATH:
nter
t−/−noCre :MATH]
= 7 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish). d, Representative immunofluorescence images of DNA damage
staining (γH2AX; left) and quantification (right; n[WT] = 6;
[MATH:
Nter
t−/−noCre :MATH]
= 6 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish). e, Quantification of p53 protein levels (normalized by
β-actin) analyzed by western blot (n[WT] = 6;
[MATH:
Nter
t−/−noCre :MATH]
= 7 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish). f, Representative immunofluorescence images of
proliferation staining (left, proliferation cell nuclear antigen
(PCNA)) and quantification (right, n[WT] = 6;
[MATH:
nter
t−/−noCre :MATH]
= 6 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish). g, Representative image of SA-β-Gal staining. h,i, RT–qPCR
analysis of the senescence-associated genes ink4a/b (p15/16) (h) and
cdkn1a (p21) (i) expression (n[WT] = 6;
[MATH:
nter
t−/−noCre :MATH]
= 7 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish). j,k, Representative hematoxylin and eosin (H&E)-stained
sections of the gut (j). The yellow arrows delineate the lamina propria
width quantified in k (n[WT] = 7;
[MATH:
nter
t−/−noCre :MATH]
= 8 and
[MATH:
nter
t−/−+Cre
:MATH]
= 7 fish). l,m, RT–qPCR analysis of the YAP target genes cyr61 (l) and
ctgf expression (m) (n[WT] = 6;
[MATH:
nter
t−/−noCre :MATH]
= 8 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish). n, RT–qPCR analysis of the junction protein-associated gene
claudin-2 expression (n[WT] = 5;
[MATH:
nter
t−/−noCre :MATH]
= 7 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish). o, Representative immunofluorescence images of immune cell
staining (left, L-plastin) and quantification (right, n[WT] = 6 fish;
[MATH:
nter
t−/−noCre :MATH]
= 6 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 7 fish). p, Representative immunofluorescence images of neutrophil
staining (left, myeloperoxidase (MPX)) and quantification (right,
n[WT] = 5 fish;
[MATH:
nter
t−/−noCre :MATH]
= 5 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish). All analyses are based on 9-month-old fish gut sections or
extracts. Scale bar, 20 µm. The dashed lines delineate the gut villi.
All data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01,
***P < 0.001, using a one-way ANOVA and post hoc Tukey test; *P < 0.05,
**P < 0.01, using a Kruskal–Wallis and post hoc Dunn test.
[77]Source data
As expected, we did not detect expression of the tert transgene in
mock-injected fish, while Cre microinjection resulted in the excision
of the STOP cassette and tert transgene expression (Fig. [78]1b, left).
This led to an approximate fivefold enrichment of total tert mRNA
(endogenous and transgene tert mRNA) in the gut of tert^−/− + Cre fish
when compared to mock-injected control tissues (tert^−/− no Cre and WT;
Fig. [79]1b, right). Consequently, we observed a higher telomerase
activity in tert^−/− + Cre compared to both WT and tert^−/− no Cre
(Extended Data Fig. [80]1f). To test whether expression of the tert
transgene is sufficient to prevent telomere shortening, we performed
telomere restriction fragment (TRF) analysis on gut samples of
9-month-old fish. As described previously^[81]14,[82]15, we noted that
the range of telomere length in the gut of WT fish exhibited a bimodal
pattern (Extended Data Fig. [83]1c,d). This pattern reflects the
differences in telomere length between cell types. The telomere length
of WT blood cells was longer (approximately 19 kb) than other tissues
(approximately 9 kb) leading to a two-peak densitometry
pattern^[84]14,[85]15,[86]24. Reflecting the requirement of telomerase
to sustain long telomeres in blood cells, the telomere length of
tert^−/− blood cells was drastically reduced compared to WT (as seen by
the loss of the longer peak; Extended Data Fig. [87]1d)^[88]14,[89]15.
Even though expression of tert complementary DNA (cDNA) driven by the
fabp2 promoter did not restore telomere length to WT levels, induction
of the tert transgene was sufficient to elongate telomeres in the
whole-gut tissues of tert^−/^− + Cre fish (7.9–8.4 kb, n = 6–7,
P < 0.05; Fig. [90]1c and Extended Data Fig. [91]1c,e). Like tert^−/−
no Cre fish, tert^−/− + Cre fish lacked the longer telomere peak,
indicating that the tert transgene is not expressed in blood cells. As
described previously^[92]14,[93]15, telomere shortening in the gut of
tert^−/− no Cre fish leads to an increase in DNA damage, as observed by
γH2AX immunofluorescence and p53 protein levels, when compared to WT
fish (Fig. [94]1d,e). Consistent with telomere elongation, these
markers are reverted by telomerase expression in the gut of
tert^−/− + Cre fish. Thus, tert transgene expression is sufficient to
counteract telomere dysfunction in the gut of tert^−/− fish by
extending telomere length.
Extended Data Fig. 1. The tert transgene is specifically expressed in the gut
resulting in telomerase activity and telomere elongation in the gut of
tert^−/− zebrafish.
[95]Extended Data Fig. 1
[96]Open in a new tab
a, b. RT-qPCR analysis of tert transgene mRNA (A.) and total tert mRNA
(B.) expression in gut (N[tert−/− No Cre]=7 and 8 and N[tert−/− +Cre]=6
and 5 fish respectively), testes (N[tert−/− No Cre]=6 and 7 and
N[tert−/− +Cre]=5 and 6 fish respectively) and kidney marrow (N[tert−/−
No Cre]=9 and 8 and N[tert−/− +Cre]=7 and 4 fish respectively) extracts
derived from 9-month-old fish. c–e. Telomere length analyses of genomic
DNA extracted from 9-month-old gut samples (N[WT]=7, N[tert−/− No
Cre]=7 and N[tert−/− +Cre]=6 fish). Representative images of TRF
analysis (blue bars represents mean telomere length) (C.), mean TRF
densitometry curves (D.), and quantification of median TRF of the
longest (90^th percentile; left panel) and the shortest (10^th
percentile; right panel) telomeres (E.). f. Quantification of
telomerase activity in gut of 12-month-old zebrafish using quantitative
Telomerase Repeated Amplification Protocol (qTRAP) assay (N=3 fish per
condition). Hela cell extracts were used as positive control for
telomerase activity. g–j. Telomere length analyses of genomic DNA
extracted from 9-month-old testes samples (N = 7 fish per condition).
Representative images of TRF analysis (blue bars represents mean
telomere length). Dashed lines delineate cropped parts of the same
Southern blot image (G.), mean TRF densitometry curves (H.),
quantification of median TRF of the longest (90^th percentile; left
panel) and the shortest (10^th percentile; right panel) telomeres (I.)
and quantification of mean telomere length (J.). k. Quantification of
telomerase activity in testes of 12-month-old zebrafish by qTRAP (N = 3
fish per condition except N[WT] = 6). l–o. Telomere length analyses of
genomic DNA extracted from 9-month-old kidney marrow samples (N = 7
fish per condition). Representative images of TRF analysis (blue bars
represents mean telomere length)(L.), mean TRF densitometry curves
(M.), quantification of median TRF of the longest (90^th percentile;
left panel) and the shortest (10^th percentile; right panel) telomeres
(N.) and quantification of mean telomere length (O.). p. Quantification
of telomerase activity in kidney marrow of 12-month-old zebrafish using
qTRAP assay (N = 3 fish per condition except N[WT] = 6). Data are
represented as mean +/−SEM (***p-value<0.001, using one-way ANOVA and
post-hoc Tukey tests). Boxes of Tukey boxplots represent the median and
interquartile range.
[97]Source data
To test whether tert transgene expression can rescue the aging defects
of telomerase-deficient animals, we analyzed the gut of 9-month-old
fish. As observed previously^[98]14,[99]15, the gut of tert^−/− no Cre
fish showed reduced cell proliferation compared to WT fish.
Enterocyte-specific telomerase expression rescued the proliferative
capacity of this organ to WT levels (Fig. [100]1f).
Senescence-associated β-galactosidase (SA-β-gal) assays and
transcription levels of the senescence-associated genes ink4a/b
(p15/16) and cdkn1a (p21) revealed that telomerase expression reduced
cell senescence to WT levels (Fig. [101]1g–i). Consistent with our
previous work^[102]17, we detected no differences in apoptosis in the
gut of WT, tert^−/− no Cre and tert^−/− + Cre of 9-month-old fish
(Extended Data Fig. [103]2a).
Extended Data Fig. 2. 9-month-old fish do not exhibit differences in
apoptosis between groups.
[104]Extended Data Fig. 2
[105]Open in a new tab
At 9-months of age, no differences in apoptosis were detected in gut,
testes and kidney marrow comparing tert^−/− No Cre, tert^−/− +Cre and
WT fish. a–c. Representative immunofluorescence images of apoptotic
cell staining (TUNEL assay; left panel) and quantification (right
panel) in gut (A.; N[WT] = 5, N[tert−/− No Cre] = 6 and N[tert−/− +Cre]
= 7 fish), testes (B.; N[WT] = 6, N[tert−/− No Cre] = 7 and N[tert−/−
+Cre] = 7 fish) and kidney marrow (C.; N[WT] = 6, N[tert−/− No Cre] = 7
and N[tert−/− +Cre] = 6 fish) tissues of 9-month-old zebrafish. Scale
bar: 20µm. Dashed lines delineate gut villi (A.), mature spermatid area
(B.), or kidney tubules (C.). All data are represented as mean +/- SEM
(no significance was detected comparing all conditions and using
one-way ANOVA and post-hoc Tukey tests).
[106]Source data
These cellular defects observed in tert^−/− fish impact tissue
integrity^[107]14,[108]15,[109]17. We observed that tert^−/− no Cre
fish exhibited morphological tissue defects with thickening of the
lamina propria (Fig. [110]1j,k). Loss of intestinal barrier integrity
led to activation of the Yes-associated protein (YAP) transcription
factor responsible for tissue regeneration^[111]25,[112]26. Consistent
with loss of gut integrity, expression of the YAP target genes cyr61
and ctgf was increased in tert^−/− no Cre fish (Fig. [113]1l,m).
Likewise, claudin-2 mRNA levels were higher in tert^−/− no Cre fish
(Fig. [114]1n). Increased gene expression of the tight junction protein
claudin-2 occurs during primate aging and enhances in vivo intestinal
permeability^[115]27,[116]28. Strikingly, all these phenotypes were
rescued in tert^−/^− + Cre fish (Fig. [117]1j–n).
We observed that the number of proliferative cells in individual
intervilli was negatively correlated with the thickness of the lamina
propria (Extended Data Fig. [118]3). Plotting either individual
intervilli (Extended Data Fig. [119]3a) or individual fish (Extended
Data Fig. [120]3b), we noticed that WT and tert^−/− + Cre clustered
separately from tert^−/− no Cre samples. In addition, we observed
higher infiltration of total immune cells and neutrophils in the
intestinal epithelium of the tert^−/− no Cre fish compared to WT (Fig.
[121]1o,p). In line with a rescue of intestinal integrity, the number
of immune cells was reverted to WT levels in tert^−/− + Cre fish.
Considering that thickening of the gut lamina propria results from
immune cell infiltrates, these results suggest that cell proliferation
is locally affected by inflammation. Thus, rescuing tissue integrity
promotes the proliferative capacity of the gut in part by reducing
tissue inflammation.
Extended Data Fig. 3. Proliferation in individual intervilli negatively
correlates with local inflammation.
[122]Extended Data Fig. 3
[123]Open in a new tab
Intervillus-based correlation plot between cell proliferation and gut
lamina propria width in 9-month-old fish. Plots illustrate the
correlation between the number of PCNA positive cells within each
intervillus and lamina propria width below each respective intervillus
(analyzed based on the immunofluorescence staining experiment of Fig.
[124]1f). a. Each point represents a single intervillus analyzed from
either WT, tert^−/− No Cre or tert^−/− +Cre. b. Each point represents a
single zebrafish analyzed from either WT, tert^−/− No Cre or tert^−/−
+Cre (N = 6 fish per condition).
[125]Source data
Local effects
Gut tert rescues gene expression and metabolism
By comparing the expression profiles of whole-gut tissues using RNA
sequencing (RNA-seq), we observed a distinguishable transcriptomics
signature in tert^−/− no Cre, while WT and tert^−/− + Cre clustered
together (Fig. [126]2a and Supplementary [127]Data). Gene set
enrichment analysis (GSEA) showed that most of the hallmarks were
similarly deregulated in tert^−/− no Cre than either WT or
tert^−/− + Cre. The transcriptomics profiles of the tert^−/− no Cre gut
are enriched in gene expression related to senescence, inflammation and
morphogenesis (Fig. [128]2b), while the hallmarks of proliferation or
oxidative phosphorylation are downregulated (Fig. [129]2c). We further
validated this transcriptomics recovery of senescence-associated
secretory phenotype (SASP)/inflammation-related genes by analyzing the
transcription levels of the il6, tnfa, cxcl12a, tgfb1b, tgfb5 and mmp2
genes (Fig. [130]2d). In line with the previous results, these
transcription profiles confirmed that telomerase expression rescued
cell proliferation, loss of tissue integrity, senescence and
inflammation seen in the gut of tert^−/− no Cre fish.
Fig. 2. Gut-specific tert expression rescues gut transcriptomics and
metabolomics profiles.
[131]Fig. 2
[132]Open in a new tab
a, Principal component analysis (PCA)-based on untargeted
transcriptomics data of 9-month-old gut samples. A clustering between
tert^−/− + Cre and WT was observed while the tert^−/− no Cre group was
clearly distinguishable from tert^−/− no Cre fish (n = 3 per group).
b,c, Identification of upregulated (b) or downregulated (c) hallmarks
in tert^−/− no Cre compared to either WT or tert^−/− + Cre, based on
GSEA. The normalized enrichment scores (NES) depict to what degree the
pathway genes are overrepresented in WT or tert^−/− + Cre, compared to
tert^−/− no Cre. Gene sets related to senescence, inflammation and
morphogenesis were enriched while the hallmarks of proliferation and
oxidative phosphorylation were downregulated in the gut of tert^−/− no
Cre fish compared to the other two groups. d, RT–qPCR analysis of
inflammation-related gene expression (il6, tnfa, tgfb1b and tgfb5) and
SASP-related gene expression (il6, tnfa, cxcl12a, tgfb1b, tgfb5 and
mmp2) in 9-month-old gut samples (n[WT] = 8 fish,
[MATH:
nter
t−/−noCre :MATH]
= 10 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 8 fish for il6; n[WT] = 8 fish,
[MATH:
nter
t−/−noCre :MATH]
= 9 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 8 fish for tnfa; n[WT] = 8 fish,
[MATH:
nter
t−/−noCre :MATH]
= 11 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 8 fish for cxcl12; n[WT] = 7 fish,
[MATH:
nter
t−/−noCre :MATH]
= 9 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 7 fish for tgfb1b; n[WT] = 7 fish,
[MATH:
nter
t−/−noCre :MATH]
= 11 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish for tgfb5; and n[WT] = 8 fish,
[MATH:
nter
t−/−noCre :MATH]
= 10 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 7 fish for mmp2). e,f, PCA (e) and partial least squares
discriminant analysis (PLS-DA) (f) clustering analysis based on
untargeted metabolomics data of 9-month-old gut samples. A clustering
between tert^−/− + Cre and WT was observed while the tert^−/− no Cre
group was clearly distinguishable from the other (n[WT] = 8 fish,
[MATH:
nter
t−/−noCre :MATH]
= 8 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 9 fish). The score plot is presented with a confidence ellipse of
95%. g–i, Metabolomics analysis of energy metabolites (g), inflammatory
metabolites (h) and methionine cycle pathway (i) in 9-month-old gut
samples (n[WT] = 8 fish,
[MATH:
nter
t−/−noCre :MATH]
= 8 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 9 fish). All data are presented as the mean ± s.e.m.; *P < 0.05,
**P < 0.01, ***P < 0.001, using a one-way ANOVA and post hoc Tukey
test; *P < 0.05; *P < 0.01, ***P < 0.001, using a Kruskal–Wallis and
post hoc Dunn test).
[133]Source data
Changes in metabolism have been associated with aging and might reflect
cellular defects, such as gradual mitochondrial dysfunction with
age^[134]29,[135]30. Consistently, we previously showed that
9-month-old tert^−/− gut exhibits mitochondrial dysfunction accompanied
by low ATP and high reactive oxygen species (ROS) levels^[136]17.
Unsupervised and supervised clustering analyses of metabolomics
profiles revealed that both WT and tert^−/− + Cre samples clustered
tightly while tert^−/− no Cre samples differed from the other groups
(Fig. [137]2e,f and Extended Data Fig. [138]5a). Most metabolites were
reduced (621) or enriched (141) in both WT and tert^−/− + Cre when
compared to tert^−/− no Cre fish (Extended Data Fig. [139]5b).
Consistent with our previous work^[140]17, we observed a drastic
reduction of energetic metabolites, such as ATP, ADP, nicotinamide
adenine dinucleotide (NAD), NAD phosphate (NADP) and coenzyme A (CoA),
in tert^−/− no Cre fish (Fig. [141]2g). Following the anaerobic
glycolysis pathway, we noticed lower levels of glucose-6-phosphate and
fructose 1,6-bisphosphate and higher amounts of pyruvate and lactate
(Extended Data Fig. [142]5c). Considering that glucose did not vary
between groups, our results suggest that the gut of tert^−/− no Cre
fish acquired higher levels of anaerobic glycolysis. We also detected
higher pentose shunt activity in tert^−/− no Cre gut, evidenced by
increased amounts of ribose-5-phosphate and erythrose 4-phosphate
(Extended Data Fig. [143]5d). Except for citrate levels, all the
detected metabolites of the citric acid cycle were elevated in tert^−/−
no Cre fish (Extended Data Fig. [144]6a). Altogether, the gut energetic
metabolism of tert^−/− no Cre fish were engaged in uncoupled oxidative
phosphorylation, consistent with damaged mitochondria, low ATP levels
and higher production of ROS. By expressing tert transgene in the gut,
tert^−/− no Cre metabolic alterations were prevented in the entire
tissue.
Extended Data Fig. 5. Gut-specific telomerase activity rescues gut
metabolomic profile.
[145]Extended Data Fig. 5
[146]Open in a new tab
a. Heatmap clustering analysis based on untargeted metabolomic data of
9-month-old gut samples. A clustering between tert^−/− +Cre and WT
while tert^−/− No Cre group was clearly distinguishable from others. b.
Venn diagram representing downregulated (left panel) or upregulated
(right panel) gut metabolites comparing the three conditions. Most
metabolites detected in the gut of 9 months-old fish are concomitantly
down or up-regulated in tert^−/− +Cre and WT groups compared to
tert^−/− No Cre fish. c, d. Anaerobic glycolysis and pentose shunt
metabolic profiles are rescued to WT levels in the gut of tert^−/− +Cre
compared tert^−/− No Cre fish. Metabolomic analysis of the anaerobic
glycolysis (C.) and pentose shunt pathways (D.) in gut of 9-month-old
fish. All data are represented as mean +/− SEM (N[WT] = 8 fish,
N[tert−/− No Cre] = 8 fish and N[tert−/− +Cre] = 9 fish; *
p-value<0.05; ** p-value<0.01, *** p-value<0.001, using one-way ANOVA
and post-hoc Tukey tests). Red squares: detected metabolites; blue
squares: undetected metabolites.
[147]Source data
Extended Data Fig. 6. Gut-specific telomerase activity rescues citric acid
cycle and steroid metabolism alterations in the gut of tert^−/− fish.
[148]Extended Data Fig. 6
[149]Open in a new tab
a, b. Metabolomic analysis of the citric acid cycle (A.) and steroid
metabolism (B.) in gut of 9-month-old fish. Citric cycle and steroid
metabolic profiles in the gut of tert^−/− +Cre is similar to WT when
compared tert^−/− No Cre fish. All data are represented as mean +/- SEM
(N[WT] = 8 fish, N[tert−/− No Cre] = 8 fish and N[tert−/− +Cre] = 9
fish; * p-value<0.05; ** p-value<0.01, using one-way ANOVA and post-hoc
Tukey tests; ## p-value<0.01, using Kruskal-Wallis and post-hoc Dunn’s
tests). Red squares: detected metabolites; blue squares: undetected
metabolites.
[150]Source data
In line with our previous results depicting higher inflammation of
tert^−/− no Cre fish, we observed an overall increase in arachidonic
metabolism with higher levels of pro-inflammatory molecules, such as
prostaglandins and leukotrienes (Fig. [151]2h). Consistently, we
detected lower amounts of anti-inflammatory resolvin D2 in tert^−/− no
Cre fish when compared to the other groups. Among the detected amino
acids, methionine was significantly enriched in tert^−/− no Cre gut
compared to the other genotypes (Fig. [152]2i). We also observed an
overall increase in methionine metabolites in the mutant gut that might
be allowed by higher levels of nicotinamides. The steroid pathway was
also enriched in tert^−/− no Cre fish. Not only the stress hormone
cortisol but also female hormones (such as 16-oxoestrone or estradiol)
were elevated in tert^−/− no Cre male fish (Extended Data Fig.
[153]6b). Overall, our unbiased metabolomics analysis described an
altered metabolism profile in tert^−/− no Cre that was recovered by
gut-specific telomerase expression.
Local effects
Gut tert rescues gut microbiota dysbiosis
Gut microbiota dysbiosis is associated with a dysfunctional intestinal
barrier and is suggested to generate a feed-forward loop involving gut
permeability, inflammation and dysbiosis in aging^[154]31,[155]32.
However, it was unclear whether delaying gut aging would counteract gut
microbiota dysbiosis. To investigate if telomerase expression in the
gut of tert^−/− fish ameliorates gut dysbiosis, we performed
high-throughput sequencing of the V3 and V4 regions of 16S ribosomal
DNA of 9-month-old zebrafish gut. As described for human
aging^[156]33,[157]34, we observed diminished microbial diversity in
tert^−/− no Cre when compared to WT controls. Both α (within samples)
and β (within groups) analyses showed lower diversity in tert^−/− no
Cre individuals compared to other groups (Fig. [158]3a,b). According to
a reduced β diversity, using principal coordinates analysis (PCoA), we
observed a clustering of tert^−/− no Cre samples while WT and
tert^−/− + Cre samples were more dispersed (Fig. [159]3c).
Fig. 3. Gut-specific tert expression rescues gut microbiota dysbiosis.
[160]Fig. 3
[161]Open in a new tab
Telomere elongation in the gut of tert^−/− + Cre fish rescued gut
microbiota composition and diversity to WT levels compared to tert^−/−
no Cre fish, which exhibited gut microbiota dysbiosis. a,
Quantification of microbiome α diversity (within samples) using the
Shannon index (P values were determined using a two-sided Wilcoxon
signed-rank test) in the gut of 9-month-old fish. b, Quantification of
microbiome β diversity using weighted UniFrac distance (within groups;
***P < 0.001 using a two-sided Tukey test) in the gut of 9-month-old
fish. c, PCoA of the β diversity distance (weighted UniFrac) in the gut
of 9-month-old fish. d, Relative abundance of top 10 bacterial classes
in the microbiome of the three different groups in the gut of
9-month-old fish. e, Relative abundance of top 10 bacterial genera in
the microbiome of the three different groups in the gut of 9-month-old
fish. For all the figures, n[WT] = 15 fish,
[MATH:
nter
t−/−noCre :MATH]
= 15 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 14 fish; α and β diversity data are shown as Tukey boxplots, where
the boxes represent the median and interquartile range and the bars
represent the minimum and maximum values.
[162]Source data
The relative abundance of bacterial taxonomic units at the class level
revealed an overall alteration of gut microbiota composition in
tert^−/− no Cre fish that was recovered by tert expression (Fig.
[163]3d). At the class level, we observed in the tert^−/− no Cre group
a decreased abundance of Alphaproteobacteria and Planctomycetes along
with an enrichment in Gammaproteobacteria, Bacteroidia and
Fibrobacteria (Fig. [164]3d and Extended Data Fig. [165]7a). While
Alphaproteobacteria inhibit host cell death and promote
proliferation^[166]35, Gammaproteobacteria expansion is associated with
early age-dependent loss of intestinal barrier integrity in
flies^[167]32. Similarly, at the genus level, the Alphaproteobacteria
Reyranella and Defluviimonas were reduced while the Gammaproteobacteria
Aeromonas and Shewanella along with Bacteroides, a Bacteroidia-related
genus, were enriched in tert^−/− no Cre fish (Fig. [168]3e and Extended
Data Fig. [169]7b). Both Shewanella and Aeromonas have been described
as deleterious in humans, with Shewanella causing intra-abdominal
infections^[170]36 and Aeromonas being associated with inflammatory
bowel disease and inflammation^[171]37,[172]38. Within the Aeromonas
genus, Aeromonas veronii was strikingly overrepresented in tert^−/− no
Cre fish (Extended Data Fig. [173]7c). From the Bacteroidia class,
Bacteroides uniformis, Parabacteroides merdae and Bacteroides ovatus
were similarly enriched in tert^−/− no Cre and are considered
‘pathobionts’ that profit from a dysregulated environment to overtake
commensal symbionts and become pathogenic^[174]39–[175]41. Overall, our
analysis of gut microbiota composition revealed a wrongly balanced gut
microbiome in tert^−/− no Cre fish, containing a less diverse bacterial
community with increased representation of otherwise pathogenic taxa
microbiota being more pathogenic. These features were reverted by
gut-specific telomerase expression.
Extended Data Fig. 7. Gut-specific tert expression rescues alterations of gut
microbiota composition.
[176]Extended Data Fig. 7
[177]Open in a new tab
a–c. tert mRNA expression in gut of tert^−/− fish (tert^−/− +Cre)
recapitulates bacteria abundance at the class and species levels to WT
profile compared to tert^−/− No Cre in which pathogenic bacteria are
enriched. Relative abundance analysis of bacteria at the level of class
(A.); genus (B.) and species (C.) N[WT] = 15 fish, N[tert−/− No Cre] =
15 fish and N[tert−/− +Cre] = 14 fish; p values were determined using
two-sided Multiple hypothesis-test for sparsely sampled features and
false discovery rate (FDR).
[178]Source data
Systemic effects
Gut tert rescues tissue degeneration
Intestinal dysfunction is a major feature of aging^[179]4. To
investigate whether gut aging influences overall organismal aging, we
explored the systemic impact of gut-specific telomerase expression
using histological analyses of a broad spectrum of tissues. As reported
previously^[180]14,[181]16,[182]17, we observed a reduction in mature
spermatids area (with severe testes atrophy), in adipocyte size in
visceral adipose tissues, in muscle fiber thickness and in retinal
pigmented epithelium width in the tert^−/− no Cre fish compared to WT
(Fig. [183]4a,b). Strikingly, gut-specific telomerase expression
recovered all these morphological defects. Of note, no histological
differences were detected in kidney marrow between each condition.
Moreover, unlike previous observations^[184]14,[185]16, the adipocyte
size of subcutaneous adipose tissue and the photoreceptor layer did not
differ between the three genotypes. Therefore, our results indicate
that counteracting telomere shortening in the gut systemically
ameliorates age-dependant tissue degeneration.
Fig. 4. Gut-specific tert expression rescues systemic tissue degeneration.
[186]Fig. 4
[187]Open in a new tab
Expression of telomerase in the gut of tert mutant fish rescued tissue
degeneration in the testes, visceral adipose tissue, muscle and eye. a,
Representative image of a longitudinal section of a zebrafish stained
with H&E. The locations of each tissue analyzed in the study are
indicated by arrows. b, Representative images of testes, kidney,
visceral adipose tissue, subcutaneous adipose tissue, muscle and eye
from 9-month-old fish stained with H&E (right). Except for the kidney,
histological quantifications were performed for each tissue (left),
namely the mature spermatids area (n[WT] = 10 fish,
[MATH:
nter
t−/−noCre :MATH]
= 8 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 9 fish), adipocyte area (n[WT] = 9 fish,
[MATH:
nter
t−/−noCre :MATH]
= 9 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 9 fish), muscle fiber thickness (n = 8 fish per group), retinal
pigmented epithelium (RPE) and photoreceptor layer (PRL) (n[WT] = 7
fish,
[MATH:
nter
t−/−noCre :MATH]
= 8 fish and
[MATH:
nter
t−/−+Cre
:MATH]
= 8 fish), respectively. Scale bar, 20 µm. All data are presented as
the mean ± s.e.m.; *P < 0.05; **P < 0.01, ***P < 0.001, using a one-way
ANOVA and post hoc Tukey test; *P < 0.05, using a Kruskal–Wallis test
and post hoc Dunn test.
[188]Source data
Gut short telomeres drive systemic DNA damage and inflammation
We decided to study the extent of systemic aging recovery of specific
distant organs. Given the importance of anemia in patients with
TBD^[189]42,[190]43, and the drastic histological phenotype seen in the
testes of tert^−/− no Cre fish, we further detailed the rescue in the
kidney marrow (the adult hematopoietic organ in zebrafish) and testes
(the reproductive system). As described previously^[191]14,[192]17, we
observed increased γH2AX-positive cells and high levels of p53 in both
the testes and kidney marrow of tert^−/− no Cre fish (Figs. [193]5a,b
and [194]6a,b). These organs were affected by reduced cell
proliferation and high senescence (Figs. [195]5c–f and [196]6c–f). In
the kidney, even though most cells affected by DNA damage and low
proliferation reside in the hematopoietic compartment, we also observed
SA-β-Gal-positive cells in kidney tubules, suggesting that both
hematopoietic and nephrotic functions were affected in tert^−/− no Cre
fish.
Fig. 5. Gut-specific tert expression rescues the aging phenotypes of testes.
[197]Fig. 5
[198]Open in a new tab
a–e, Delaying gut aging in tert^−/− + Cre fish rescues DNA damage,
proliferation and senescence in the testes compared to tert^−/^− no Cre
fish. a, Representative immunofluorescence images of DNA damage
staining (γH2AX, left) and quantification (right, n[WT] = 6,
[MATH:
nter
t−/−noCre :MATH]
= 5 and
[MATH:
nter
t−/−+Cre
:MATH]
= 5 fish) in the tissue of testes. b, Quantification of p53 protein
levels (normalized by β-actin) in 9-month-old testes extracts analyzed
by western blot (n[WT] = 6,
[MATH:
nter
t−/−noCre :MATH]
= 8 and
[MATH:
nter
t−/−+Cre
:MATH]
= 8 fish). c, Representative immunofluorescence images of
proliferation staining (left, PCNA) and quantification (right, n = 6
fish per group) in the tissue of testes. d, Representative image of
SA-β-Gal staining of 9-month-old testes cryosections. e,f, RT–qPCR
analysis of the senescence-associated genes ink4a/b (p15/16) (e) and
cdkn1a (p21) (f) expression in testes samples (n[WT] = 6 and 5,
[MATH:
nter
t−/−noCre :MATH]
= 7 and 6 and
[MATH:
nter
t−/−+Cre
:MATH]
= 5 and 5 fish, respectively). g, Representative immunofluorescence
images of immune cell staining (left, L-plastin) and quantification
(right, n[WT] = 6,
[MATH:
nter
t−/−noCre :MATH]
= 6 and
[MATH:
nter
t−/−+Cre
:MATH]
= 7 fish) in testes tissues. h, Representative immunofluorescence
images of neutrophil staining (left, MPX) and quantification (right,
n[WT] = 6,
[MATH:
nter
t−/−noCre :MATH]
= 5 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish) in the tissue of testes. i, Identification of upregulated
(left) or downregulated (right) hallmarks in the testes of tert^−/− no
Cre fish compared to either WT or tert^−/− + Cre, based on GSEA. The
NES depict to what degree the pathway’s genes are overrepresented in WT
or tert^−/− + Cre, compared to tert^−/− no Cre fish. j, Quantification
of male fertility of fish determined by counting the percentage of
fertilized eggs (detected by successful embryogenesis events) after
individually crossing 9-month-old males with a young (3–6-month-old) WT
female (n[WT] = 19,
[MATH:
nter
t−/−noCre :MATH]
= 16 and
[MATH:
nter
t−/−+Cre
:MATH]
= 13 fish). All analyses were done on sections of 9-month-old fish
testes or extracts. Scale bar, 20 µm. The dashed lines delineate the
area of mature spermatids. All data are presented as the mean ± s.e.m.
*P < 0.05, **P < 0.01, ***P < 0.001, using a one-way ANOVA and post hoc
Tukey test; and *P < 0.05, **P < 0.01 using a Kruskal–Wallis and post
hoc Dunn test. The RT–qPCR graphs represent the mean ± s.e.m. Note the
mRNA fold increase after normalization by rps11 gene expression levels.
[199]Source data
Fig. 6. Gut-specific tert expression rescues aging of the hematopoietic
system (kidney marrow).
[200]Fig. 6
[201]Open in a new tab
a–f, Delaying gut aging in tert^−/− + Cre fish rescues DNA damage,
proliferation and senescence in the kidney marrow when compared to
tert^−/− no Cre fish. a, Representative immunofluorescence images of
DNA damage staining (left, γH2AX) and quantification (right, n[WT] = 5,
[MATH:
nter
t−/−noCre :MATH]
= 6 and
[MATH:
nter
t−/−+Cre
:MATH]
= 5 fish) in 9-month-old kidney marrow tissues. b, Quantification of
p53 protein levels in 9-month-old kidney extracts analyzed by western
blot (n[WT] = 6,
[MATH:
nter
t−/−noCre :MATH]
= 8 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish). c, Representative immunofluorescence images of
proliferation staining (left, PCNA) and quantification (right, n = 6
fish per group) in 9-month-old kidney marrow tissues. d, Representative
images of SA-β-Gal staining of 9-month-old kidney marrow cryosections.
e,f, RT–qPCR analysis of senescence-associated genes ink4a/b (p15/16)
(n[WT] = 5,
[MATH:
nter
t−/−noCre :MATH]
= 6 and
[MATH:
nter
t−/−+Cre
:MATH]
= 5 fish) (e) and cdkn1a (p21) (n[WT] = 6,
[MATH:
nter
t−/−noCre :MATH]
= 7 and
[MATH:
nter
t−/−+Cre
:MATH]
= 4 fish) (f) expression in 9-month-old kidney marrow samples. g–i,
tert mRNA expression in the gut of tert^−/− fish (tert^−/− + Cre fish)
have beneficial hematopoietic effects by reducing kidney marrow
inflammation and increasing immune compartment compared to tert^−/− no
Cre fish. g, Representative immunofluorescence images of immune cell
staining (left, L-plastin) and quantification (right, n[WT] = 6,
[MATH:
nter
t−/−noCre :MATH]
= 6 and
[MATH:
nter
t−/−+Cre
:MATH]
= 7 fish) in the tissue of 9-month-old testes. h, Representative
immunofluorescence images of neutrophil staining (left, MPX) and
quantification (right, n[WT] = 6,
[MATH:
nter
t−/−noCre :MATH]
= 5 and
[MATH:
nter
t−/−+Cre
:MATH]
= 6 fish) in the tissue of 9-month-old kidney marrow. i,
Identification of upregulated (left) or downregulated (right) hallmarks
in the kidney marrow of tert^−/− no Cre compared to either WT or
tert^−/− + Cre fish based on GSEA. The NES depicts to what degree the
pathway genes are overrepresented in WT or tert^−/− + Cre, compared to
tert^−/− no Cre fish. Scale bar, 20 µm. The dashed lines delineate the
kidney tubules. All data are presented as the mean ± s.e.m. (*P < 0.05;
**P < 0.01, ***P < 0.001, using a one-way ANOVA and post hoc Tukey
test). The western blot graphs represent the mean ± s.e.m. of p53
normalized by β-actin band intensities. All RT–qPCR graphs represent
the mean ± s.e.m. mRNA fold increase after normalization by rps11 gene
expression levels.
[202]Source data
Surprisingly, gut-specific telomerase expression in tert^−/− mutants
resulted in a reduction in DNA damage, p53 levels and recovery of cell
proliferation in both testes and kidney marrow (Figs. [203]5a–c and
[204]6a–c). Moreover, SA-β-Gal and ink4a/b (p15/16) mRNA levels were
reduced to WT levels in tert^−/− + Cre testes and kidney marrow (Figs.
[205]5d,e and [206]6d,e). While cdkn1a (p21) mRNA levels were
maintained in the testes of tert^−/− no Cre fish, these were rescued in
kidney marrow of tert^−/− + Cre fish (Figs. [207]5f and [208]6f).
Consistent with what we observed in the gut, apoptosis did not vary in
either testes or kidney marrow (Extended Data Fig. [209]2b,c).
Therefore, gut-specific telomerase expression unexpectedly rescues DNA
damage, proliferation and senescence in both the reproductive and
hematopoietic systems.
The increased immune infiltrates present in the testes of tert^−/− no
Cre fish were also reverted in the tert^−/− + Cre fish (Fig.
[210]5g,h). However, in contrast to the gut and testes, we observed a
considerable reduction of immune cells in the kidney marrow of tert^−/−
no Cre fish (Fig. [211]6g,h). These numbers were reverted to WT levels
in tert^−/− + Cre fish. Thus, our results provide evidence for a
decreased reserve pool of immune cells in tert^−/− no Cre fish that is
rescued by gut-specific telomerase expression. Decline of immune cells
in the kidney marrow constitutes an early sign of hematopoietic
dysfunction, which is comparable to the bone marrow failure described
in patients with TBD^[212]42,[213]43.
To ensure that these effects were not due to leaky fabp2 enterocyte
promoter expression in other tissues, we performed quantitative PCR
with reverse transcription (RT–qPCR) experiments on testes and kidney
marrow. While a clear induction of the tert transgene and total tert
mRNA was observed in the gut of tert^−/− + Cre fish, no expression of
the transgene was detected in either distant organ (Extended Data Fig.
[214]1a,b). Consistently, the DsRed reporter for transgene expression
showed that the fabp2 promoter was solely expressed in gut
differentiated cells but not the testes or kidney marrow (Extended Data
Fig. [215]4). As expected, we observed neither telomerase activity nor
telomere elongation in distant organs (Extended Data Fig. [216]1g–p).
In contrast, telomere shortening was observed in tert^−/− + Cre kidney
marrow and testes, similar to the telomere length of tert^−/− no Cre
fish. These experiments support a systemic role of gut-specific
telomerase expression.
Extended Data Fig. 4. The fabp2 promoter regulates gut specific transgene
expression.
[217]Extended Data Fig. 4
[218]Open in a new tab
Representative images of DsRed immunofluorescence staining of
cryosection from gut, testes and kidney marrow cryosections from
zebrafish containing no transgene, No Cre fabp2:
loxp-dsred-loxp-tert-t2a-cfp transgene or Cre-induced fabp2:
loxp-dsred-loxp-tert-t2a-cfp transgene.
Fertility decreases during natural aging of zebrafish and most mammals.
Loss of male fertility is accelerated in murine and fish premature
tert^−/− aging models^[219]14,[220]44. To test the male reproductive
function, we crossed 9-month-old males of the three groups with young
WT females. The percentages of fertilized eggs spawned by young females
were scored as a male fertility index. Consistent with a reduction of
mature spermatid content, tert^−/− no Cre male fish exhibited a drastic
reduction of fertility (Fig. [221]5j). In contrast, we observed a full
recovery of male fertility in tert^−/− + Cre fish. Therefore,
gut-specific telomerase expression not only improves cellular and
morphological defects of the male reproductive system, but also rescues
age-dependent loss of fertility.
Finally, to understand the mechanism through which gut decline
influences aging of distant organs, we analyzed the transcriptomics
profile of testes and kidney marrow. As in the gut, we observed similar
GSEA hallmark profiles when comparing tert^−/− no Cre to either WT or
tert^−/− + Cre (Figs. [222]5i and [223]6i), indicating that telomerase
expression in the gut rescues the transcriptomics profile of tert
mutant testes and kidney marrow. As in the gut, we observed a marked
enrichment of hallmarks of inflammation and a reduction of
proliferation-related genes in tert^−/− no Cre. Hallmarks of metabolic
pathways were also upregulated in tert^−/− no Cre indicating a
metabolic shift in this condition. Unexpectedly, even though senescence
was higher in all organs of tert^−/− no Cre fish (Figs. [224]5d–f and
[225]6d–f), in contrast to the gut, we observed a downregulation of the
SASP hallmark in both the testes and kidney marrow. This result
suggests that paracrine senescence of distant organs initiated by the
gut may have limited expression of SASP molecules, as previously
observed in secondary senescent cells^[226]45.
Gut tert extends tert^−/− lifespan and improves WT healthspan
We next tested whether telomerase expression in the gut would influence
the lifespan of zebrafish. As described previously^[227]14–[228]16,
telomere shortening in tert^−/− no Cre fish reduces the average
lifespan to 12–18 months compared to more than 42 months in WT fish
(Fig. [229]7). Strikingly, delaying gut aging was sufficient to extend
the average lifespan of tert^−/− fish by approximately 40%. The average
lifespan of tert^−/− no Cre fish was extended from 17 months to 24
months in tert^−/− + Cre fish (Fig. [230]7). Nevertheless, telomerase
expression in the gut was not sufficient to fully rescue life
expectancy to WT levels, suggesting that telomere shortening in other
organs may be limiting in later stages.
Fig. 7. Gut-specific tert expression extends the lifespan of tert^−/−
zebrafish.
[231]Fig. 7
[232]Open in a new tab
Gut-specific telomerase activity extends the lifespan, increasing
median life from 17 months in tert^−/− no Cre fish to 24 months in
tert^−/− + Cre fish. The survival curve of WT (n = 42 fish), tert^−/−
no Cre (n = 38 fish) and tert^−/− + Cre (n = 26 fish) zebrafish
(**P < 0.01 using the log-rank test) is shown.
[233]Source data
Finally, to extend our discovery to the natural aging of zebrafish, we
studied the recovery of 24–27-month-old WT zebrafish expressing
(WT + Cre) or not expressing (WT no Cre) the tert transgene in the gut.
At that age, we did not yet distinguish differences in survival between
the two groups (Fig. [234]8d). However, we observed that gut-specific
telomerase expression in WT fish increased cell proliferation, reduced
gut lamina propria width and counteracted cell senescence in the gut
compared to WT no Cre (Fig. [235]8a–c). As observed in tert^−/− fish,
expressing telomerase in the gut of WT fish is sufficient to improve
the proliferative capacity of distant organs such as the testes and
kidney marrow (Fig. [236]8a). Except for a partial rescue of ink4a/b
(p15/16) mRNA levels in kidney marrow, we did not observed signs of
senescence in either distant organ using SA-β-Gal assays or assessing
for transcription levels of ink4a/b (p15/16) and cdkn1a (p21) (Fig.
[237]8b). Consistently, we did not observe histological defects in
distant organs (Fig. [238]8c). Therefore, while 24–27-month-old WT fish
do not fully exhibit natural aging phenotypes, our data revealed that
delaying gut aging by gut-specific tert overexpression is sufficient to
counteract the early signs of aging, such as loss of proliferative
capacity. It also confirms that the gut is one of the earliest organs
affected in natural aging.
Fig. 8. Gut-specific tert expression extends the healthspan of naturally aged
zebrafish.
[239]Fig. 8
[240]Open in a new tab
Expression of tert transgene in the gut of WT fish delays local aging
phenotypes such as proliferation, senescence and tissue degeneration.
This leads to beneficial systemic impact improving early aging
phenotypes such as proliferation capacity. a, Representative
immunofluorescence images of proliferation staining (left, PCNA) and
quantification (right) in the gut, testes and kidney marrow of
27-month-old WT zebrafish expressing (WT + Cre; n = 7 fish for the gut
and kidney marrow and n = 6 fish for the testes) or not expressing (WT
no Cre; n = 8 fish for the gut and kidney marrow and n = 6 fish for the
testes) tert transgene in the gut. b, Representative images of SA-β-Gal
staining of gut, testes and kidney marrow sections of 24-month-old WT
zebrafish expressing (WT + Cre) or not expressing (WT no Cre) the tert
transgene in the gut (left). RT–qPCR analysis of senescence-associated
genes ink4a/b (p15/16) and cdkn1a (p21) in the gut, testes and kidney
marrow of either 9- (n = 6 fish) or 27-month-old WT zebrafish
expressing (WT + Cre; n = 8 fish) or not expressing (WT no Cre; n = 8
fish) tert mRNA in the gut (right). c, Representative H&E-stained
sections of the gut, testes and kidney marrow of 27-month-old WT
zebrafish expressing (WT + Cre) or not expressing (WT no Cre) the tert
transgene in the gut (left) and respective quantifications of the width
of the gut lamina propria (right; n = 7 fish for WT no Cre and n = 6
fish for WT + Cre) and mature spermatid area (right; n = 7 fish for WT
no Cre and WT + Cre). The yellow arrows indicate the width of the
lamina propria quantified on the left. The dashed lines delineate the
mature area of the spermatids. d, Survival curve of WT no Cre (n = 42
fish; similar to the WT curve in Fig. [241]7) and WT + Cre (n = 36
fish) zebrafish. All data are presented as the mean ± s.e.m. *P < 0.05,
**P < 0.01, ***P < 0.001, using a two-tailed unpaired t-test for a,c or
a one-way ANOVA and post hoc Tukey tests for b; **P < 0.01, a using
two-tailed Mann–Whitney U-test). All RT–qPCR graphs represent the
mean ± s.e.m. mRNA fold increase after normalization by rps11 gene
expression levels. Scale bar, 20 µm.
[242]Source data
Discussion
The gut is a central organ in aging and it constitutes the most
extensive and selective living barrier to the external environment.
Besides its function in nutrient uptake, it has an important role in
immune modulation and supports a complex interaction with the gut
microbiota^[243]4.
Broader keratinocyte promoter-driven telomerase expression was shown to
counteract degenerative phenotypes of late-generation tert^−/−
mice^[244]46,[245]47. Aging phenotypes were ameliorated, not only in
the gut, but also in other organs such as the testes, kidney and skin.
However, in these studies, tert expression was not targeted to a
specific organ. The dePinho laboratory recently showed that telomere
shortening in mice triggers gut inflammation through the YAP
pathway^[246]48. Mosaic expression of tert in the LGR5 cells of
tert^−/− mice improved intestinal function and inflammation. However,
no significant systemic effects were reported apart from body weight
gain and a modest increase in survival. Consistently, we showed that
YAP target genes were likewise induced in tert^−/− no Cre fish. These
were rescued in tert^−/− + Cre fish that not only reverted the YAP
pathway, but also rescued local inflammation. Moreover, we showed that
counteracting gut telomere dysfunction also delays remote organ
dysfunction and overall organismal aging.
In our study, we showed that enterocyte-specific telomerase expression
in zebrafish is sufficient to prolong maintenance of gut homeostasis
with age. Rescue of gut aging was observed in the context of a minor,
but significant, telomere elongation in the gut of tert^−/− fish.
Consistent with an increase in telomere length of the shortest telomere
population (tenth percentile), this was sufficient to abrogate DNA
damage, higher p53 levels and cell senescence in this organ.
Nevertheless, considering the potential noncanonical roles of
telomerase in proliferation and resistance to oxidative stress^[247]49,
we cannot exclude these effects in our work. Similarly, trace amounts
of fabp2 transcripts were previously reported in zebrafish in the
liver, brain and kidney marrow, but not in the testes^[248]50. While we
did not measure any fabp2-dependent transgene mRNA in other tissues, we
cannot exclude that undetected spurious expression may participate in
the systemic effects.
Common laboratory mice possess long telomeres (ranging from 40 to
150 kb) compared to humans and zebrafish (5–15 kb). Therefore,
producing telomerase-deficient mice requires several generations of
in-breeding (G3–G4) before mice show premature aging
phenotypes^[249]44,[250]51. We designed a new vertebrate model to study
the systemic effects of delaying aging of an individual organ, the gut,
by maintaining telomere length through enforced telomerase expression.
We report that delaying telomere-dependent gut aging has beneficial
systemic effects not only in a premature aging model, that is, tert^−/−
mutants, but also in a context of natural aging of zebrafish. Notably,
our study indicates that proliferative organs, such as the reproductive
or hematopoietic systems, can conserve their regenerative capacity even
in a context of shorter telomeres. This was observed in the rescue of
telomerase deficiency by tp53 mutations in mice and
zebrafish^[251]15,[252]52. Thus, maintenance of proliferative capacity
and tissue integrity in these organs relies on external cues from an
aging gut. We propose that the intestine is at the top of a cascade of
events that initiate systemic aging; thus, restoring intestine
integrity can result in organismal rejuvenation.
How would gut aging influence the entire organism? Aging is associated
with persistent DNA damage and inflammation^[253]2. In recent years, we
have seen a flurry of studies supporting the role of inflammation and
SASP in inducing paracrine senescence in remote
tissues^[254]53,[255]54. Senescent cells accumulate with age in tissues
and promote aging by secreting molecules such as inflammatory
cytokines, chemokines and other molecules, also known as SASP^[256]54.
Clearance of these cells delays age-associated defects and leads to
lifespan extension^[257]5,[258]6. We previously reported that some
organs in zebrafish, such as the kidney marrow, exhibit cellular
senescence before exhibiting critically short
telomeres^[259]14,[260]15. We now show that gut of tert^−/− no Cre fish
accumulates senescent cells and expresses SASP and inflammatory
molecules, such as interleukin-6 (IL-6) or transforming growth factor-β
(TGFβ). Senescent cells can induce ROS-mediated DNA damage in distant
tissues by secreting TGFβ and IL-1β^[261]55,[262]56. Therefore, we
anticipate that inflammation and SASP factors secreted by an aging gut
trigger DNA damage in distant organs. This mechanism results in
secondary senescence, affecting cell proliferation systemically and
leading to loss of tissue homeostasis and aging in the entire organism.
Alterations in gut microbiota have been linked to aging^[263]34,[264]57
and are involved in age-related systemic inflammation^[265]31. Specific
bacterial taxa are capable of inducing gut cell senescence but also in
distant organs (for example, the liver)^[266]58,[267]59. We show that
delaying gut aging counteracted gut microbiota dysbiosis. We anticipate
that, due to limiting gut telomere length, increasingly dysbiotic
microbiota will cause systemic aging, either directly through microbial
components or by triggering systemic inflammation or senescence. This
idea is supported by work showing that stool transfers from young to
middle-aged individuals is sufficient to extend the lifespan of
short-lived killifish^[268]60.
We noticed an accumulation of methionine and its metabolites
(S-adenosylmethionine (SAM), S-adenosylhomocysteine and homocysteine)
in the gut of tert^−/− no Cre fish. Similar enrichment with age has
been reported in humans and mice^[269]29,[270]61. Dietary methionine
restriction or impeding SAM accumulation extends the lifespan in
different animal models^[271]4,[272]62–[273]66. Hyperhomocysteinemia
has also been implicated in several age-related disorders^[274]61.
Mechanistically, deleterious effects of methionine and its metabolites
involves DNA methylation drift, mTOR activation, inflammation and
oxidative stress^[275]4,[276]30,[277]63. We suggest that propagation of
these molecules throughout the zebrafish organism contributes to
systemic aging.
Overall, the present work describes a central role of telomere
shortening in the gut during the aging of a vertebrate organism. We
provide several mechanistic clues on how this organ influences aging of
the entire organism, namely through microbiota dysbiosis, inflammation
and SASP, and dysregulation of methionine metabolism. Our future work
will disentangle these mechanisms by targeting them independently in a
unique organ, the gut, as an exciting strategy to extend the healthspan
and lifespan.
Methods
Plasmid construct
Zebrafish tert cDNA was obtained using the TertFL-pCR-II-Topo plasmid
provided by the Kishi laboratory^[278]58. Using Gibson assembly
recombination methods, tert cDNA and enhanced constitutively
fluorescent protein (eCFP) cDNA were linked by the T2A sequence and
inserted into the Ubi: loxP-dsRed-loxP-EGFP vector plasmid (a gift from
the Zon laboratory derived from Ubi: Switch and lmo2: Switch
contructs^[279]59). The enterocyte-specific intestinal fatty acid
binding protein promoter (−2.3 kb fabp2, also called i-fabp) was
amplified using high-fidelity PCR (iProof High-Fidelity DNA Polymerase;
Bio-Rad Laboratories) from the p5E–2.3ifabp plasmid (gifted by the
Rawls laboratory). The −2.3-kb fabp2 PCR product was then cloned into
the Ubi: loxP-dsRed-loxP-tert-T2A-CFP using sfI/FseI digestion to
provide the final construct: fabp2: loxP-dsRed-loxP-tert-T2A-CFP.
Generation of transgenic fish
Tol2 mRNA was synthesized with SP6 RNA polymerase from the
pCS2FA-transposase plasmid (Tol2Kit) using the mMESSAGE mMACHINE SP6
transcription kit (Invitrogen). One-cell-stage zebrafish embryos were
microinjected with 1.4 nl of a mixture containing 25 ng µl^−1 of
linearized plasmid and 100 ng µl^−1 of Tol2 mRNA, diluted with
RNase-free water. Injected fish were raised to adulthood and
germline-transmitting fish were selected and outcrossed to WT AB until
a single-copy transgenic line Tg was obtained (fabp2:
loxP-dsRed-loxP-tert-T2A-CFP).
Zebrafish lines and maintenance
Zebrafish were maintained in accordance with institutional and national
animal care protocols. Generation and maintenance of the telomerase
mutant line tert AB/hu3430 (referred in this work as tert^+/−) were
described previously^[280]14,[281]15,[282]17. This line was outcrossed
with Tg(fabp2: loxP-dsRed-loxP-tert-T2A-CFP) line to obtain a stock
that combined both transgenics. All stocks were kept in heterozygous
form for the tert mutation and were strictly maintained by outcrossing
to AB strains to avoid haploinsufficiency effects in the progeny.
Experimental fish were obtained by crossing tert^+/− fish with tert
^+/−; fabp2: loxP-dsRed-loxP-tert-T2A-CFP. Their embryos were
microinjected with 1.4 nl of either 25 ng µl^−1 Cre mRNA diluted in
RNase-free water (Cre-induced fish) or RNase-free water alone
(mock-injected fish). This experimental setup provided sibling fish
that were either tert^−/−; fabp2: loxP-dsRed-loxP-tert-T2A-CFP
(mock-injected tert^−/−, referred to as tert^−/− no Cre), tert^−/−;
fabp2: tert-T2A-CFP (Cre-induced tert^−/−, referred to as
tert^−/− + Cre) or tert^+/+; fabp2: loxP-dsRed-loxP-tert-T2A-CFP
(mock-injected WT, referred to as WT). Overall characterization of
these three genotypes was performed in F1 siblings at 9 months of age.
Due to a male sex bias in our crosses, primarily observed in the
tert^−/− progeny, we were unable to obtain significant numbers of
females for analysis; thus, all but our survival data are restricted to
males.
Fertility assays
To assess male fertility, 9-month-old males from the three different
genotypes were housed separately overnight in external breeding tanks
with a young 3–6-month-old WT female. Breeding pairs were left to cross
and lay eggs the following morning. Embryos were collected
approximately 2 h after fertilization and allowed to develop at 28 °C.
Assessment of egg fertilization and embryo viability was conducted
between 2 and 4 h after fertilization. At least 14 independent crosses
were conducted for each genotype to evaluate male fertility. Only
successful breeding trials were scored. Events where females laid a
normal clutch of eggs were scored.
Histology
Zebrafish were killed by lethal dose of 1 g l^−1 of MS-222
(Sigma-Aldrich), fixed for 72 h in 10% neutral buffered formalin and
decalcified in 0.5 M EDTA for 48 h at room temperature. Whole fish were
paraffin-embedded to create 5-µm sagittal section slides. Slides were
stained with H&E for histopathological analysis. Microphotographs
(n ≥ 6 fish per genotype) were acquired with a Leica DM4000 B
microscope coupled to a Leica DFC425 C camera (Leica Microsystems).
Senescence-associated β-galactosidase staining
Tissues were fixed with 4% paraformaldehyde for 3 h at 4 °C. After
washing with PBS, they were incubated in 30% sucrose (Sigma-Aldrich) at
4 °C until sinking (24–48 h). Fixed tissues were then embedded in
optimal cutting temperature medium (MM France) and kept at −80 °C.
Senescence-associated β-galactosidase staining was performed on slides
of 5-µm cryosections using the Senescence β-Galactosidase Staining Kit
(catalog no. 9860, Cell Signaling Technology) following manufacturer’s
instructions. After 16-h (testes, kidney marrow) or 3-h (gut)
incubations with the X-Gal staining solution at 37 °C, slides were
washed with PBS and counterstained for 1 min with Nuclear Fast Red
solution (Sigma-Aldrich) before being dehydrated and mounted.
Immunofluorescence
Deparaffinized and rehydrated slides were microwaved for 20 min at
550 W in citrate buffer (10 mM sodium citrate, pH 6) to allow for
antigen retrieval. Slides were washed twice in PBS for 5 min each and
blocked for 1 h at room temperature in 0.5% Triton X-100 and 5% normal
goat serum in PBS (blocking solution). Subsequently, slides were
incubated overnight at 4 °C with 1:50 dilution of primary antibody in
the blocking solution. The following primary antibodies were used:
rabbit polyclonal anti-histone H2A.XS139ph (γH2AX, phospho Ser139, 1:50
dilution, catalog no. GTX127342, GeneTex); rabbit polyclonal
anti-L-plastin (1:100 dilution, catalog no. GTX124420, GeneTex); mouse
monoclonal antibody anti-PCNA (1:50 dilution, catalog no. sc56, Santa
Cruz Biotechnology); and rabbit polyclonal anti-MPX (1:50 dilution,
catalog no. GTX128379, GeneTex). After two PBS washes, overnight
incubation at 4 °C was performed with 1:500 dilution of the Alexa Fluor
488 goat anti-rabbit or anti-mouse secondary antibody (Invitrogen).
Finally, after 4,6-diamidino-2-phenylindole staining (DAPI)
(Sigma-Aldrich), slides were mounted in DAKO Fluorescence Mounting
Medium (Sigma-Aldrich).
Apoptosis was detected using the In Situ Cell Death Detection Kit
(Roche) as described previously^[283]14,[284]17. Briefly,
deparaffinized sections were permeabilized by 1-h incubation at 37 °C
with 40 μg ml^−1 proteinase K (Sigma-Aldrich) in 10 mM Tris-HCl, pH
7.4. After washing with PBS, slides were incubated for 1 h at 37 °C
with TUNEL Label Mix (according to the manufacturer’s instructions)
before DAPI staining and mounting.
Immunofluorescence images were acquired on the Delta Vision Elite
microscope (GE Healthcare) using an OLYMPUS ×20/0.75 objective. For
quantitative and comparative imaging, equivalent image acquisition
parameters were used. The percentage of positive nuclei was determined
by counting a total of 500–1,000 cells per slide (n ≥ 6 zebrafish per
genotype).
Western blot
Proteins for the western blot were extracted according to the
manufacturer’s protocol with TRIzol (Invitrogen) and protein in
microliters was quantified with QuantiPro BCA Assay Kit
(Sigma-Aldrich). A total of 30 μg of protein was loaded per lane and
resolved in 10% resolution gel at 120 V for 2 h and transferred to a
nitrocellulose membrane (LI-COR) at 20 V for 70 min with the Trans-Blot
SD Semi-Dry Electrophoretic Transfer Cell system (Bio-Rad
Laboratories). Transfer and quality were checked with Ponceau staining
(VWR) and washed thoroughly with 1× PBS with 0.05% Tween 20 (PBST).
Membranes were incubated with primary antibody (anti-p53, 1:1,000
dilution, catalog no. 55342, AnaSpec and anti-actin 1:1,000 dilution,
catalog no. A2066, Sigma-Aldrich) overnight at 4 °C with gentle shaking
after 1-h blocking in 5% skimmed milk (Sigma-Aldrich) in 0.05% PBST at
room temperature with gentle shaking. After three washes with 0.05%
PBST, membranes were incubated with secondary antibody (anti-rabbit,
1:10,000, catalog no. sc-2357, Santa Cruz Biotechnology) for 1 h at
room temperature with gentle shaking. This was followed by three washes
with 0.05% PBST and membranes were then revealed with Amersham ECL
Select (Cytiva) using the Fusion Solo system (Vilber Lourmat).
TRF analysis by Southern blot
Isolated tissues were lysed in lysis buffer at 50 °C overnight (catalog
no. K0512, Thermo Fisher Scientific) supplemented with 1 mg ml^−1
proteinase K and RNase A (1:100 dilution, Sigma-Aldrich). Genomic DNA
(gDNA) was extracted using equilibrated phenol-chloroform
(Sigma-Aldrich) and chloroform-isoamyl alcohol extraction
(Sigma-Aldrich). Equal amounts of gDNA were digested with the Rsal and
HinFl enzymes (New England Biolabs) for 12 h at 37 °C. After digestion,
samples were loaded on a 0.6% agarose gel, in 0.5% Tris/Borate/EDTA
buffer and run on a CHEF-DRII pulse field electrophoresis apparatus
(Bio-Rad Laboratories). The electrophoresis conditions were as follows:
initial switch 1 s, final switch 6 s; voltage 4 V cm^−2; at 4 °C for
20 h. Gels were then processed for Southern blotting using a 1.6-kb
telomere probe, (TTAGGG)n, labeled with [α-32P]-dCTP.
Telomerase activity assay
A real-time quantitative TRAP (Q-TRAP) assay was performed as described
previously^[285]67. Protein extracts were obtained by adding 0.5% CHAPS
to dissociate the tissue followed by 30 min of incubation on ice.
Samples were centrifuged (16,000g for 20 min at 4 °C) and the
supernatant was collected. Protein concentration was assessed with a
Bradford assay, according to the manufacturer´s instructions. Then,
0.5 µg protein was added to the TRAP master mix (1× ABI SYBR Green,
10 mM EGTA, 100 ng ACX primer (5′-GCGCGGCTTACCCTTACCCTTACCCTAACC-3′),
100 ng TS (5′-AATCCGTCGAGCAGAGTT-3′), primer and RNase-free water up to
25 µl in a 96-well plate and incubated for 30 min at 28 °C in the dark.
Real-time PCR was performed with a StepOnePlus Real-Time PCR System
(Thermo Fisher Scientific): 95 °C for 10 min; 40 cycles at 95 °C for
15 s; and at 60 °C for 60 s. Each sample was performed in triplicate.
As a negative control, samples were incubated with 1 mg RNase for
20 min at 37 °C. A standard curve for telomerase activity was obtained
using 1:5 serial dilutions of HeLa extract. Data are presented as
relative telomerase activity units, which was calculated according to
the following formula: 10^((Ct sample]−Y[int])/slope).
Real-time qPCR and RNA-seq
Zebrafish were killed by lethal dose of 1 g l^−1 of MS-222 and each
tissue (gut, testes and kidney marrow) were dissected and immediately
snap-frozen in liquid nitrogen. RNA extraction was performed by
disrupting individual tissues with a pestle in TRIzol followed by
chloroform extraction. The quality of RNA samples was assessed with a
BioAnalyzer (Agilent Technologies). Retrotranscription into cDNA was
performed using the QuantiTect Reverse Transcription Kit (QIAGEN).
qPCR was performed using the FastStart Universal SYBR Green Master mix
(Roche) and a 7900HT Fast Real-Time PCR Detection System (Thermo Fisher
Scientific). qPCR was carried out in triplicate for each cDNA sample.
Relative mRNA expression was normalized against rps11 mRNA expression
using the 2^−ΔΔCT method as compared to the control condition. Primer
sequences are listed in the [286]Supplementary Information.
RNA-seq was performed by the Beijing Genomics Institute using, for each
condition, biological triplicates, each consisting of a pool of two
individual tissues. DNase-treated total RNA samples were enriched for
mRNAs using oligo(dT) magnetic beads. In turn, mRNAs were fragmented
into 200-bp fragments and the first strands of cDNAs were synthesized
using random hexamers. To generate the library products,
double-stranded cDNA from the second strand synthesis was purified
using magnetic beads followed by A-tailing and RNA adapter ligation.
The library was amplified with phi29 to make a DNA nanoball (DNB) that
had more than 300 copies of each molecule. Paired-end, 150-bp reads
were sequenced via combinatorial Probe-Anchor Synthesis on the DNBseq
platform; 100 M clean reads per sample were generated. Raw data with
adapter sequences or low-quality sequences were filtered using the
SOAPnuke software developed by the Beijing Genomics Institute.
RNA-seq reads were analyzed via an internal pipeline for transcript
quantification, normalization and comparison. Briefly, the human
reference genome assembly vGRCh38 (retrieved from
[287]http://www.ensembl.org) and gencode annotation v.37 (retrieved
from [288]https://www.gencodegenes.org/) were processed with gffread
v.0.12.2 to extract the human reference transcriptome. Based on this
extracted reference transcriptome, Salmon v.1.4 was used to perform
transcript quantification via quasi-mapping. RUVSeq v.1.20.0 was used
for data transformation by rlog and data normalization by replicates.
DESeq2 v.1.26.0 was used for differentially expressed gene (DEG)
analysis. A false discovery rate (FDR) cutoff of 0.1 was explored for
the DEG analysis (Supplementary [289]Data).
The pathway enrichment analysis was performed using a GSEA approach,
implemented using the Broad Institute’s GSEA
software^[290]68^,^[291]69. The enrichment was run using the hallmark
geneset, retrieved from the Molecular Signature
Database^[292]68^,^[293]70, as well as the Fridman senescence UP
geneset^[294]71 and Reactome’s SASP geneset
([295]https://reactome.org/PathwayBrowser/#/R-HSA-2559582). Before
enrichment, all geneset genes were mapped to zebrafish orthologs using
the Ensembl’s BioMart database (Ensembl release 107, 2022). The GSEA
parameters were set as follows: permutations = 1,000, permutation
type = gene_set. Significant enrichment results were genesets with
nominal P values and an FDR <0.05. Borderline significance (to indicate
the directionality of a pathway) was set at nominal P values <0.05 and
an FDR <0.25.
Metagenomics
gDNA was extracted from the gut of sibling fish as described for the
TRF analysis. The V3–V4 hypervariable regions of bacterial 16S rRNA
genes were amplified by PCR with the Phusion High-Fidelity PCR
mastermix (New England Biolabs) using the primer described
previously^[296]60. PCR products were mixed at equal density ratios and
purified with the Gel Extraction Kit (QIAGEN). Sequencing libraries
were generated using the NEBNext Ultra DNA Library Prep Kit and
sequenced on an Illumina NovaSeq 6000 paired-end platform to generate
250 bp paired-end raw reads. Sequence analysis was performed using the
UPARSE software with all effective tags. Sequences with 97% or more
similarity were assigned to the same operational taxonomic units
(OTUs). Representative sequences for each OTU were screened for further
annotation. For each representative sequence, the Mothur software was
applied against the SILVA Small Subunit rRNA database for species
annotation at each taxonomic rank (threshold: 0.8–1). QIIME and R were
used to calculate α and β diversity metrics and generate plots. PCoA
was performed to get principal coordinates and visualize complex,
multidimensional data.
Metabolomics
Each frozen gut sample was homogenized in 600 µl of methanol (HPLC
grade, Merck Millipore) and incubated overnight at −20 °C. Tubes were
vortexed and incubated overnight at −20 °C for protein precipitation.
After centrifugation, the supernatants were removed, dried using a
SpeedVac concentrator (Savant SVC100H, Thermo Fisher Scientific),
resuspended in 80 µl of a 20:80 acetonitrile-H[2]O mixture (HPLC grade,
Merck Millipore) and stored at −20 °C until use for the metabolomics
analysis.
Chromatographic analysis was performed using a Dionex UltiMate 3000
HPLC system coupled to a chromatographic column (Phenomenex Synergi
4 µm Hydro-RP 80 Å 250 × 3.0 mm) set at 40 °C and a flow rate of
0.9 ml min^−1. The gradients of mobile phases (mobile phase A: 0.1%
formic acid in water and mobile phase B: 0.1% formic acid in
acetonitrile) were performed for a total of 25 min. Mass spectrometry
analysis was carried out on an Exactive Plus Benchtop Orbitrap mass
spectrometer (Thermo Fisher Scientific). The heated electrospray
ionization source (HESI II) was used in positive and negative ion
modes. The instrument was operated in full-scan mode from 67 to
1,000 m/z. The post-treatment of data was performed using MZmine2
v.2.39 ([297]http://mzmine.github.io/). Metabolites were identified
using the Human Metabolome Database v.5.0 ([298]http://www.hmdb.ca). We
only used ions identified as (M + H)^+ adducts in the positive mode and
(M-H)^− adducts in the negative mode and ions found in all the samples
after gap filling. For dataset denoising, only ions with average peak
intensities greater than 10 × 10^5 were considered.
Statistics and reproducibility
Graphs and statistical analyses were performed in Prism 8 (GraphPad
Software). For multiple comparisons, a one-way analysis of variance
(ANOVA) with Tukey’s post hoc correction was used for normally
distributed data and a Kruskal–Wallis test with a Dunn’s post hoc test
was used for data that did not meet normality. A critical value for
significance of P < 0.05 was used throughout the study. For the
survival analysis, log-rank tests were performed with Prism8 to
determine statistical differences of the survival curves.
Untargeted metabolomics analysis of gut samples was processed using
statistical analysis (one-factor) modules proposed by MetaboAnalyst
v.5.0 ([299]https://www.metaboanalyst.ca). For each comparison, peak
intensities were log-transformed. Clustering analysis was performed
using PCA, PLS-DA and heatmap tools provided by MetaboAnalyst.
Statistical parameters and methods are reported in the figure legends.
No statistical method was used to predetermine sample size. Sample
sizes were chosen according to professional standards of the field for
individual assays. Outlier identification was pre-established and
performed using the Tukey’s method. Reported results were acquired
using independent fish that were randomly collected for each group.
(The number of fish used for each experiment is specified in each
figure legend.) Except for the lifespan experiments, the investigators
were not blinded to allocation during the experiments or data
collection and outcome assessment or analysis.
Ethics statement
The zebrafish work was conducted according to local and international
institutional guidelines and was approved in France by the Animal Care
Committee of the Institute for Research on Cancer and Aging, Nice, the
regional (CIEPAL Côte d’Azur no. 697) and national (French Ministry of
Research no. 27673-2020092817202619) authorities and in Portugal by the
Ethics Committee of the Instituto Gulbenkian de Ciência and approved by
the competent Portuguese authority (Direcção Geral de Alimentação e
Veterinária; approval no. 0421/000/000/2015).
Reporting summary
Further information on research design is available in the [300]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[301]Supplementary Information^ (305.5KB, pdf)
Supplementary information list of primers used in RT–qPCR expression
analysis.
[302]Reporting Summary^ (2MB, pdf)
[303]Supplementary Data Supplementary Tables 1–6^ (4.6MB, xlsx)
Differentially expressed genes.
Acknowledgements