Abstract
Given the growing interest in human exploration of space, it is crucial
to identify the effects of space conditions on biological processes.
Here, we analyze the transcriptomic response of Caenorhabditis elegans
to simulated microgravity and observe the maintained transcriptomic
response after returning to ground conditions for four, eight, and
twelve days. We show that 75% of the simulated microgravity-induced
changes on gene expression persist after returning to ground conditions
for four days while most of these changes are reverted after twelve
days. Our results from integrative RNA-seq and mass spectrometry
analyses suggest that simulated microgravity affects
longevity-regulating insulin/IGF-1 and sphingolipid signaling pathways.
Finally, we identified 118 genes that are commonly differentially
expressed in simulated microgravity- and space-exposed worms. Overall,
this work provides insight into the effect of microgravity on
biological systems during and after exposure.
Keywords: ceramide, sphinogolipid signaling, longevity, transcriptome,
space, intergenerational, microgravity
1. Introduction
Significant efforts are being made for the human exploration of space.
It is known that space conditions can negatively affect biological
processes by causing bone loss, muscle atrophy, and immune system
impairment, to name a few examples (reviewed in [[30]1,[31]2]).
However, the underlying molecular mechanisms that contribute to these
adverse effects is largely unknown.
Microgravity is one of the major stress factors causing detrimental
health effects on humans (reviewed in [[32]3]). Simulation of
microgravity is a cost-effective way to study the impact of
microgravity on biological systems. For this purpose, various platforms
including drop towers, parabolic flights and space flights are
available. Because of their advantages to enable long term simulations
in an effective and economical manner, clinostats are widely preferred
to study gravitational response in biological systems, mainly plants,
for decades [[33]4,[34]5,[35]6,[36]7,[37]8]. Clinostats rotate a sample
around a horizontal axis, thereby exposing the sample to a rotating
gravitational vector. This can reduce and even possibly remove
gravitational bias in the development of an organism
[[38]6,[39]9,[40]10,[41]11,[42]12,[43]13].
C. elegans is an exceptional model organism for space biology studies
[[44]14] with its completely documented cell lineage, high reproduction
rate, short lifespan, and high similarity to the human genome (reviewed
in [[45]15]). The usage of this organism in previous space missions
(such as International C. elegans Experiment in Space (ICE-First), C.
elegans RNAi space experiment (CERISE), Shenzhou-8, and Commercial
Generic Bioprocessing Apparatus Science Insert-01 (CSI-01)) have
provided valuable insights into the biological response of C. elegans
to space conditions
[[46]16,[47]17,[48]18,[49]19,[50]20,[51]21,[52]22,[53]23,[54]24].
However, these studies generally have utilized different strains and
liquid cultures instead of commonly used OP50-seeded agar plates to
reduce the impact of surface tension [[55]25]. We previously have shown
that the usage of different liquid cultures and strains cause highly
distinct intergenerational transcriptomic and phenotypical responses
[[56]26]. Similarly, a recent study reported dramatical
intergenerational changes in the physiology of liquid-grown worms
[[57]27]. Thus, complementary to the spaceflight studies, separate
investigations can provide insight into the impact of individual space
conditions on biological processes.
This study investigates the effect of simulated microgravity and the
sustained impacts after return to ground conditions on C. elegans
transcriptome. Through the RNA-sequencing (RNA-seq) and mass
spectrometry analyses, we reveal the downregulation of the sphingolipid
signaling pathway under simulated microgravity. In addition, we
identify a putative microgravity-responsive transcriptomic signature by
comparing our results with previous studies.
2. Materials and Methods
2.1. C. elegans Strain and Growth Conditions
Wild-type N2 strain was obtained from the Caenorhabditis Genetics
Center (CGC). The worms were grown at 21 °C. Stocks of C. elegans were
acclimated to CeHR medium for three weeks prior to microgravity
experiments. The ground control animals were maintained in CeHR medium
in 20 mL scintillation vials as described [[58]26]. Approximately
100,000 live worms (P[0] generation) were cleaned by sucrose
floatation, their embryos (F[1] generation) were harvested by bleaching
and placed in ~40 mL of CeHR culture in sterile VueLife culture bags
(“AC” Series 32-C, Saint-Gobain). [59]Supplementary Videos S1 and S2
show some of the worms in the VueLife culture bags. Culture bags were
mounted in the clinostat and rotated at 1 rad/s (~10 RPM) for four
days, which is the measured time to reach L4/adulthood [[60]28]. The
“F[2]” and subsequent generations were approximated by using four-day
intervals. Prior to RNA extraction, only live worms are retrieved using
the sucrose floatation method. We note that “F2” generation and beyond
will not maintain synchronicity, and all will contain a mixture of
generations, but recovering only the live animals enriches the RNA pool
for the expected (majority) generation as each adult will produce ~30
offspring. The reason for the usage of mixed stage worms were the
difficulties experienced (e.g., contamination of the liquid medium)
during the synchronizing of the animals.
2.2. RNA Isolation, Illumina Sequencing
Wild-type N2 strain was obtained from the Caenorhabditis Genetics
Center (University of Minnesota, Twin Cities, MN, USA). The worms were
grown at 21 °C. Stocks of C. elegans were acclimated to CeHR medium for
three weeks prior to microgravity experiments. Approximately 100,000
live worms (P0 generation) were cleaned by sucrose floatation, their
embryos (F1 generation) were harvested by bleaching and placed in ~40
mL of CeHR medium in sterile VueLife culture bags (“AC” Series 32-C,
Saint-Gobain, La Défense Cedex, France). Four culture bags were mounted
in a lab-built clinostat inside a laminar flow biosafety hood, and one
was kept aside as a ground control. The clinostat rotated the culture
bags at 1 rad/s (~10 RPM) for four days, which is the measured time to
reach L4/adulthood at 21 °C in CeHR medium [[61]28]. Worms from one bag
were immediately harvested to represent simulated microgravity
conditions. The remaining three culture bags were processed in four-day
intervals to represent the return to ground conditions. We note that F2
generation and beyond will not maintain synchronicity, and all will
contain a mixture of generations, but recovering only the live animals
enriches the RNA pool for the expected (majority) generation as each
adult will produce ~30 offspring in CeHR culture. The experimental
scheme is limited to using mixed stage worms because of the technical
difficulties associated with manually picking the desired age
(swimming) worms from liquid cultures. Live worms were selected for
harvesting by sucrose floatation and total RNA was isolated with TRIzol
treatment and recovered by alcohol precipitation. Total RNA was further
purified by PureLink ™ RNA Mini Kit (Life Technologies, Carlsbad, CA,
USA). Two micrograms of total RNA were used for library preparations
using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina). Libraries
were sequenced on an Illumina HiSeq 2500 instrument set to the rapid
run mode using single-end, 1 × 51 cycle sequencing reads as per
manufacturer’s instructions. RNA-seq was performed for each condition
with three replicates.
2.3. Gene Expression Analysis
Quality control on the RNA-seq data was done with FASTQC (version
0.11.2) [[62]29]. All the reads were in the “very good” quality range.
The DEGs were identified by using two separate approaches. First, the
Tuxedo pipeline [[63]30] were used with default parameters. Second, the
DESeq2 software (version 1.16.1) [[64]31] was utilized after
quantifying the expression of transcripts with Salmon (version 0.8.2)
[[65]32]. The reference genome (WBCel235) along with the annotation
file were retrieved from Ensembl [[66]33]. We considered genes as
differentially expressed if FDR adjusted p-values < 0.05, and log2 fold
change > 2 unless otherwise stated. The results from the Tuxedo
pipeline were used throughout the paper as explained in the results and
the results from DESeq2 were reported in the [67]supplementary data
([68]Supplementary Figure S4; [69]Supplementary Table S5). We discarded
miRNA, piRNA, and rRNA molecules from the analysis. The ncRNA molecules
(WS250) and unconfirmed genes were obtained from WormBase [[70]34].
Dendrogram was generated with Ward’s clustering criterion by using R
software (version 3.5.1).
2.4. Functional Analysis of the Genes
Gene ontology (GO) enrichment for biological processes and protein
domains were assessed with Database for Annotation, Visualization and
Integrated Discovery (DAVID) (v6.8) [[71]35]. The GO network for the
common DEGs between our simulated microgravity experiment and four-day
CERISE was created in Cytoscape (version 3.4.0) [[72]36] by using the
BINGO plugin [[73]37]. Pathway analysis was done using KEGG Mapper
([74]https://www.genome.jp/kegg/tool/map_pathway1.html (accessed on 1
January 2019)). Tissue enrichment analysis was performed by using
WormBase Gene Set Enrichment Analysis tool [[75]38], and FDR adj.
p-value < 0.05 considered significant and top ten tissues were
reported.
2.5. Comparison of DEGs to the CERISE Four Days
NASA’s GeneLab ([76]https://genelab.nasa.gov/ (accessed on 1 January
2019)) platform was used to acquire the Gene Expression Omnibus (GEO)
accession number for the previous studies on microgravity related gene
expressions in C. elegans. We identified four such studies with the
following GEO accession numbers: [77]GSE71771 (Expression Data from
International C. elegans Experiment 1st), [78]GSE71770 (four-day
CERISE), [79]GSE27338 (eight-day CERISE), and [80]GSE32949 by UKM
(PRJNA146465) from the query made on 30 June 2016 with “C. elegans” and
“microgravity” terms on GeneLab. We used data from only four-day CERISE
as the duration of the study is the same with ours. We analyzed the
publicly available microarray datasets with GEO2R
([81]https://www.ncbi.nlm.nih.gov/geo/geo2r/ (accessed on 1 January
2019)) for gene expression under microgravity at the ISS versus 1G
control in four day CERISE. The genes with logFC > 2 and Benjamini and
Hochberg corrected p-value < 0.05 were considered as differentially
expressed.
STRING database (version 10.5) [[82]39] was used to determine whether
the common DEGs from our simulated microgravity experiment and four-day
CERISE show enrichment for protein–protein interactions (PPI). We only
considered the interactions only from experiments, gene-fusion,
databases, and co-expression and selected “high-confidence (0.7)”
results. We excluded the interactions based on text-mining, fusion,
neighborhood, and co-occurrence as we were only interested in PPI with
higher experimental and/or computational confidence. PPI enrichment
showed significance at p-value < 1.0 × 10^−16 with 58 interacting nodes
and 155 edges. The PPI network was visualized with Cytoscape (version
3.4.0) [[83]36]. Edge width was determined based on the “combined
score” for the interactions obtained from the STRING database. We
identified the human orthologs of the common DEGs in our experiment and
four-day CERISE with OrthoList 2 with default settings [[84]40].
2.6. Mass Spectrometry for Ceramides
The worms were chopped manually with razor blades on ice-cold glass.
Three replicates from mixed stage worms grown in ground control and
simulated microgravity conditions were used simultaneously. Each sample
was sent to Avanti Polar Lipids Analytical Services as a frozen extract
in glass tubes for liquid chromatography with tandem mass spectrometry
(LC/MS/MS) experiment for detection of Ceramide levels. Each sample was
extracted by modified Bligh and Dyer extraction [[85]41]. The samples
were dried and resuspended in 50:50 Chloroform:Methanol and diluted
prior to analysis. The resolved sample was used for analyses and stored
at −20 °C until assayed. Samples were diluted as needed for analysis
with internal standards for Ceramides for quantization by injection on
LC/MS/MS. The individual molecular species for each sphingolipid group
were measured by reversed phase liquid chromatography tandem mass
spectrometry methods which separate the compounds by multiple reaction
monitoring m/z to fragments and retention time. Quantification was
performed by ratio of analyte to internal standard response multiplied
by ISTD concentration and m/z response correction factor. The results
are expressed as ng/rnl. The consistency among the replicates were
examined with Pearson correlation. All but one replicates in both the
experiments showed high correlation (R^2 > 0.9). The ground control
replicate which did not show a strong correlation (around 60%) with the
other replicates was excluded from the analysis. The data were analyzed
with t-test (two-tailed), and p-value < 0.05 was considered
significant. Equal variances were confirmed with Levene’s test at α =
0.05 for all the ceramides tested.
2.7. Microgravity Simulation with Clinorotation
We used a laboratory-built clinostat with an integrated microscope to
observe the motion of small objects including microspheres, yeast
cells, and C. elegans embryos during clinorotation. The trajectory of a
spherical particle in a rotating vessel has been described previously
in literature [[86]5,[87]11,[88]42,[89]43]. Dedolph and Dipert (1971)
separated the motion of a spherical particle into two parts: a circular
motion under the influence of gravity, the radius of which is inversely
proportional to the angular velocity (ω, rad/s), and a radial motion
due to centrifugal forces, the velocity of which is proportional to ω^2
[[90]42]. The equation of motion that describes the orbital
trajectories is given by:
[MATH:
md2qdx2<
/mfrac>+(2iωm+λ)d<
/mi>qdt−ω2(m−
mw)q=−ig(m−mw)e−iωt. :MATH]
In the above equation, λ is the viscous drag coefficient, m is the mass
of the object, m[w] is the mass of the displaced water, t is time, g is
the acceleration due to gravity, and q(x,y,z,t) is the rotating
reference frame defined as:
[MATH:
q≡xrot+iyrot=ze−iωt
. :MATH]
As a test of the performance of the clinostat we recorded the motion of
fluorescent melamine-formaldehyde microspheres (5.6 µm diameter, 1.51
g/cm^3 density; Corpuscular Inc., Cold Harbor, NY, USA) suspended in a
3% bovine serum albumin (BSA) solution ([91]Supplementary Video S3).
This solution was injected into an 8 mm diameter, 100-µm deep,
coverglass topped chamber mounted on the clinostat microscope. Some of
the melamine microspheres adhere to coverglass of the chamber,
conveniently providing fiducial markers which we were able to use to
compensate for the mechanical noise introduced into the micrographs by
the rotation of the clinostat. We measured the terminal velocity of the
suspended microspheres to be 3.2 ± 0.2 µm/s (mean ± SEM, n = 8).
[92]Figure S6 shows a plot of the measured radius of circular motion of
suspended microspheres as a function of inverse angular velocity (black
dots), and the calculated radius [[93]5] using the measured mean
terminal velocity (blue line). The shaded area encompasses calculated
radii for terminal velocities within one standard deviation of the
mean.
3. Results
To dissect the biological processes affected under simulated
microgravity and sustained after the exposure, we first cultured C.
elegans in CeHR for three weeks on ground control condition and allowed
the worms to acclimate to the liquid culture. Then, we exposed the
worms to clinostat-simulated microgravity for four days ([94]Figure 1a;
[95]Supplementary Video S1) and observed the maintained impacts at
four, eight, and twelve days after placing the worms back to ground
conditions ([96]Figure 1b). RNA-seq was performed for each condition
with three replicates ([97]Supplementary Table S1). Because RNA-seq can
detect low abundance transcripts and achieve less noise in the data,
unlike previous space biology studies on C. elegans, we preferred
RNA-seq over microarray [[98]44].
Figure 1.
[99]Figure 1
[100]Open in a new tab
Transcriptomic response of C. elegans to simulated microgravity and
return to ground conditions. (a) Clinostat used for simulating
microgravity. (b) Experimental design for the effect of simulated
microgravity on gene expressions during the exposure and after the
return. (c) Dendrogram of the gene expression profiles for ground
condition (GC), simulated microgravity (MG), four days after return to
ground conditions (R1_GC), eight days after return to ground conditions
(R2_GC), and twelve days after return to ground conditions (R3_GC). (d)
Log2 fold change of the gene expressions (FPKM) between the conditions.
We generated a dendrogram for the transcriptomic profile in each
condition tested and found significant differences during and after
exposure to simulate microgravity compared to the ground control
conditions ([101]Figure 1c). Thus, this result suggests that exposure
to simulated gravity induces highly distinct gene expression patterns
which are maintained even after 12 days return to ground conditions
(approximately three generations of C. elegans in axenic medium
[[102]28]).
3.1. Simulated Microgravity Triggers Differential Expression of Hundreds of
Genes
To identify the genes with the most distinctive expression levels after
the exposure, we determined the differentially expressed genes (DEGs)
in the simulated microgravity-exposed and returned worms against the
ground control. The genes with over two-fold log2 expression difference
with the FDR-adjusted p-value ≤ 0.05 were considered as DEGs. Hundreds
of genes demonstrated differential expression during exposure to
simulated microgravity and up to eight days after return to ground
conditions ([103]Figure 1d). Twelve days after the return, the gene
expression levels started to resemble the ones in the ground control
with only 91 upregulated and 13 downregulated genes.
The spatial expression of the DEGs is of great importance to identify
the potentially affected tissues. Thus, we performed tissue enrichment
analysis for the DEGs in simulated microgravity ([104]Figure 2a). The
downregulated genes were overrepresented in neuronal and epithelial
tissues, and intestine while the upregulated genes were enriched in the
reproductive system-related tissues.
Figure 2.
[105]Figure 2
[106]Open in a new tab
Differentially expressed gene profiles under simulated microgravity and
after return to ground conditions. (a) Tissue enrichment of the
upregulated (green) and downregulated (red) genes under simulated
microgravity. (b) Pathway enrichment of the upregulated (green) and
downregulated (red) genes during the exposure to simulated microgravity
(MG), and four and eight days after return to ground conditions (R1 GC
and R2 GC, respectively). (c) The number of differentially expressed
genes under simulated microgravity and the transmission of the
differential expression after return to ground conditions. (d)
Categorization of the upregulated genes in comparison to the ground
control animals, and the enriched gene ontology terms assigned to them.
(e) Categorization of the downregulated genes in comparison to the
ground control animals, and the enriched gene ontology terms assigned
to them.
Next, we conducted pathway enrichment analysis for the DEGs in
simulated microgravity and return to ground conditions to identify the
altered pathways and whether they remained altered after return to
ground conditions ([107]Figure 2b). Our results suggested an
upregulation of dorso–ventral axis formation and downregulation of
lysosome during the exposure, and these expression patterns were
maintained for four and eight days after the return, respectively.
3.2. Simulated Microgravity-Induced Gene Expression Differences Are Highly
Maintained for Eight Days after Return to Ground Conditions
We identified the number of simulated microgravity-induced DEGs that
preserved their expression patterns after the return to ground control
([108]Figure 2c). The majority of the DEGs (approximately 75%) from the
exposed animals maintained their expression patterns after the return
for four days. The shared number of DEGs decreased drastically at 12
days after the return to ground conditions: 16% for commonly
upregulated and <1% downregulated genes with the exposed animals.
To elucidate the genes showing altered expression under simulated
microgravity and maintaining these expression patterns after return to
ground conditions for short (four days) and long-term (12 days), we
categorized the DEGs in Venn diagrams ([109]Figure 2d,e;
[110]Supplementary Table S2). The genes solely upregulated in the
simulated microgravity exposed animals did not exhibit enrichment for
any gene ontology (GO) term. The genes upregulated during the exposure
and four days after the return, however, showed an overrepresentation
for reproduction-related processes ([111]Figure 2d). Chitin metabolic
process-related genes were induced at four days after return to ground
conditions, and the expression profiles were conserved for eight days
after the return.
We found that the downregulated genes were enriched for the biological
processes which are affected in space conditions. In particular, genes
functioning in body morphology, collagen and cuticulin-based cuticle
development, defense response, and locomotion were downregulated under
simulated microgravity and up to eight days after return to ground
conditions ([112]Figure 2e). In simulated microgravity and spaceflight
studies, both small movement defects [[113]18] and no difference in
locomotory behavior [[114]19,[115]45] have been noted. The reason
behind these discrepancies is unclear, but our results indicated
differences in the expression of the locomotion genes in close to
weightless environment. Similarly, neuropeptide signaling pathway genes
(flp-22, flp-8, flp-26, nlp-10, nlp-12, nlp-17, nlp-20, nlp-24, nlp-25,
nlp-26, nlp-39, nlp-33, nlp-28, nlp-29, nlp-30, and flp-24) were
downregulated during the exposure, and this expression profile was lost
after return to ground conditions ([116]Figure 2e). These neuropeptide
signaling pathway genes also exhibit enrichment for movement variant
phenotypes ([117]Supplementary Figure S1) indicating altered locomotion
in response to simulated microgravity.
During spaceflight, the neuromuscular system and collagen are
negatively affected [[118]18]. In agreement with these findings,
collagen genes were downregulated in our experiment during the
exposure, and the downregulation was sustained for eight days after
return to ground conditions. Previously, Higashibata et al. (2006)
reported decreased expression for the myogenic transcription factors
and myosin heavy chains in space-flown worms [[119]18]. We did not
observe differential expression for these muscle-related genes. Since
their reported flight to ground gene expression ratio for the
downregulated genes was less than one, and we only consider the genes
with the log2 ratio greater than two as DEG, we did not expect to
observe those genes in our group of DEGs. To test whether other
muscle-related genes are differentially expressed under simulated
microgravity, we compared our DEGs to 287 genes reported in WormBase
for involvement in muscle system morphology variant or any of its
transitive descendant terms via RNAi or variation. Only T14A8.2,
col-103, and sqt-3 among the 287 genes showed downregulation under
simulated microgravity (hypergeometric test, p-value = 0.99). Thus, our
results did not suggest a significant change in the expression of the
genes known to function in muscle morphology.
In our previous work, we revealed that the expression of many ncRNA
molecules is triggered in response to environmental changes [[120]26].
To identify whether a similar pattern occurs under simulated
microgravity, we determined the expressed ncRNA molecules in ground
control, simulated microgravity, and return. Since the housekeeping
gene pmp-3 presents consistent expression patterns both in our study
and previous studies [[121]26], we considered its minimum expression
level (FPKM = 24.7) in our experiment as the cutoff for the expression
of ncRNA molecules. The number of expressed ncRNA molecules slightly
decreased in response to simulated microgravity ([122]Supplementary
Figure S2A). Interestingly, this number increased on the eighth day of
return. We found that the expression of 126 ncRNA molecules is induced
during simulated microgravity exposure and for 12 days after the return
([123]Supplementary Figure S2B) while the expression of 16 ncRNA
molecules is lost during and after simulated microgravity
([124]Supplementary Figure S2C). Among the classified ncRNA molecules,
mostly small nucleolar RNA (snoRNA) and long noncoding RNA (lincRNA)
molecules were induced while other sets of snoRNA and antisense RNA
(asRNA) molecules lost expression in simulated microgravity. For
instance, asRNA molecules anr-33, K12G11.14, and ZK822.8 are induced
whereas anr-2, anr-9, and Y49A3A.6 are silenced during and 12 days
after the exposure.
We next sought to identify the putative transcriptional regulators
(i.e., transcription factors) of the simulated microgravity-induced
genes and whether these regulators maintain their impact after return
to ground conditions. That is, we determined the putative transcription
factor (TF) genes [[125]46] that are upregulated under simulated
microgravity and after the return to ground conditions. Our results
have revealed that 20 TF genes are upregulated during the exposure and
90%, 75%, and 15% of these genes maintained their upregulation after
the return for four, eight, and twelve days, respectively
([126]Supplementary Figure S3). These TFs play a role in a variety of
mechanisms such as double strain break repair or sex determination. For
example, tbx-43 is upregulated during the exposure and for at least 12
days after the return to ground. The best human BLASTP-match of tbx-43
in WormBase, TBR, functions in developmental processes and is required
for normal brain development (E-value = 3 × 10^−33; identity = 68.5%).
However, many of the upregulated TFs do not have a known function.
Hence, our list of simulated microgravity-induced TF gene expressions
can be a rich source for the discovery of the microgravity-related
transcriptional regulators.
3.3. Longevity Regulating Pathways Are Affected under Simulated Microgravity
Spaceflight affects the mechanisms involved in delayed aging in worms.
For instance, Honda et al., found that age-dependent increase in
35-glutamine repeat aggregation is suppressed and seven
longevity-controlling genes are differentially expressed in spaceflight
[[127]20,[128]24]. To examine whether simulated microgravity induces
such changes, we mapped the DEGs (simulated microgravity versus ground
control) to longevity regulating pathways in the Kyoto Encyclopedia of
Genes and Genomes (KEGG). We used a less stringent criterion for
differential expression by including the genes with log[2](FPKM) > 1.5
in this analysis. We determined 11 DEGs in the longevity regulating
pathway genes most of which are involved in the insulin/insulin-like
growth factor signaling pathway (insulin/IGF-1) ([129]Figure 3).
Interestingly, the transcription factor DAF-16 gene did not exhibit
differential expression unlike its targets sod-3, lips-17, and ctl-1.
Because the translocation of DAF-16 into the nucleus activates or
represses target genes functioning in longevity, metabolism, and stress
response, this finding was unexpected
[[130]47,[131]48,[132]49,[133]50]. The nuclear localization of DAF-16
is antagonized by transcription factor PQM-1 [[134]51], and thus an
upregulation in pqm-1 may indicate inhibition of DAF-16 translocation.
Our data, however, did not present a differential expression for pqm-1
and thus it does not suggest inhibition of the DAF-16 translocation. It
is possible that the DAF-16 targets are differentially expressed due to
the translocation state of DAF-16 or involvement of other factors
(e.g., other transcriptional regulators).
Figure 3.
[135]Figure 3
[136]Open in a new tab
Longevity regulating pathway genes are differentially expressed under
simulated microgravity. Adopted from the KEGG longevity regulating
pathway—worm (cel04212).
The upregulation of the longevity regulating pathway transcriptional
targets generally contributes to increased lifespan. Since many of
these targets were downregulated under simulated microgravity, we
sought to identify the potential impact of their downregulation by
acquiring their RNAi phenotype from WormBase [[137]34]. Lifespan
variants (WBRNAi00063155 and WBRNAi00063156) for hsp-12.6 and extended
lifespan (WBRNAi00064044) for sod-3 have been reported. Follow-up
experiments are needed to fully understand the role of these genes in
the longevity regulation under simulated microgravity.
To further investigate the involvement of the longevity genes in
simulated microgravity, we compared the DEGs to the DAF-16-responsive
genes [[138]51]. We found 144 DAF-16-induced genes were downregulated
and this overlap was statistically significant (hypergeometric test,
p-value < 0.0001). The number of shared genes between the simulated
microgravity-induced genes and the genes induced or repressed by DAF-16
are 11 and 35, respectively (hypergeometric test, p-value > 0.05 for
both). Similarly, the number of downregulated DAF-16-repressed genes
were insignificant with a total of 57 shared genes (hypergeometric
test, p-value > 0.05) ([139]Supplementary Table S3). Together, our
results suggest that the longevity regulation genes are affected under
simulated microgravity and that along with the DAF-16-regulation, other
mechanisms have an important function in this process.
3.4. Sphingolipid Signaling Pathway Is Suppressed in Response to Simulated
Microgravity
Previous studies have suggested that the sphingolipid signaling pathway
plays a role in the expression of DAF-16/FOXO-regulated genes
[[140]52,[141]53]. In line with this, our results showed that the
sphingolipid signaling pathway is downregulated under simulated
microgravity. That is, putative glucosylceramidase 4 gene (gba-4) and
putative sphingomyelin phosphodiesterase asm-3 are downregulated and
these downregulation patterns were sustained for eight days after
return to ground conditions ([142]Figure 4a). This downregulation
pattern indicates an attenuation in the ceramide levels through a
potential decrease in the degradation of sphingomyelin and
glucosylceramide to ceramide. Along with the other biological functions
such as autophagy, senescence, and apoptosis, the sphingolipid
signaling pathway has critical functions in aging and longevity
regulation [[143]52,[144]54,[145]55]. The inhibition of this conserved
pathway results in an extension of lifespan in animals from worms to
humans [[146]54,[147]55]. For example, the inactivation of asm-3 in C.
elegans causes translocation of DAF-16 into the nucleus, promotion of
DAF-16 target gene expression, and extension of lifespan by 14–19%
[[148]53].
Figure 4.
[149]Figure 4
[150]Open in a new tab
Sphingolipid signaling pathway is downregulated under simulated
microgravity. (a) The sphingolipid signaling pathway genes asah-1,
asm-3, and gba-4 are downregulated under simulated microgravity. The
downregulation pattern of asm-3 and gba-4 is maintained for eight days
after return to ground conditions. (b) The levels of d18:1 sphingosine,
total ceramide, hexosylceramide, and sphingoid base reduced while total
lactosylceramide level increased under simulated microgravity. (c) The
levels of different acyl-chain ceramides show alterations under
simulated microgravity. The error bars represent the standard error of
the mean (* p < 0.05, ** p < 0.01, *** p < 0.001).
To validate that the ceramide and sphingosine levels are indeed
decreased in response to the aforementioned gene downregulation, we
performed mass spectrometry analysis in ground control and simulated
microgravity-exposed worms concurrently ([151]Supplementary Table S4;
[152]Figure 4b,c). Our results revealed that total ceramide, total
hexosylceramide (HexCer), and d18:1 sphingosine are lower (1.5-, 3.6-,
and 1.4-fold, respectively) under simulated microgravity (t-test,
p-value < 0.05). Similarly, sphingoid base (SB) levels decreased
1.9-fold under simulated microgravity, but this decrease was not
significant (t-test, p-value = 0.055) ([153]Figure 4b). Interestingly,
the levels of total lactosylceramide (LacCer) increased 27-fold
(t-test, p-value < 0.05). Increase in the LacCer and HexCer levels have
been determined as biomarkers of aging in humans and murine [[154]56]
while LacCer C18:1 was unaffected during aging in C. elegans [[155]54].
It is unclear why LacCer levels are elevated when the HexCer levels are
decreased, but the overall pattern indicates a downregulation in the
sphingolipid signaling pathway under simulated microgravity.
The levels of long acyl-chain ceramides (≥C24) are elevated with
advancing age and in age-related diseases such as diabetes and
cardiovascular disease while C20 and C26 ceramides are unaffected with
aging or developmental stage in C. elegans [[156]54,[157]57,[158]58].
To further decipher the potential effect of simulated microgravity on
aging-related mechanisms, we quantified the levels of different
acyl-chain ceramides ([159]Supplementary Table S4; [160]Figure 4c). We
observed a decrease in d18:1-C24, and d18:1-C24:1 ceramides (1.2-, and
3.2-fold, respectively) under simulated microgravity (t-test, p-value <
0.05). It has been found that the inhibition of C24–C26
ceramide-inducing ceramide synthase HYL-1 causes improvements in
neuromuscular function and age-dependent hypoxia and stress response
[[161]59,[162]60]. Hence, the lower levels of C24 and C24:1 ceramides
in the simulated microgravity-exposed worms may indicate their positive
impact on the stress response and aging of the worms.
Along with their roles in aging and longevity, different acyl-chain
ceramides have other distinctive functions. For example, C16-ceramide
induces germ cell apoptosis [[163]61] and C20–C22 ceramide functions in
resistance to hypoxia [[164]60]. We determined that ceramides with
different acyl-chain showed altered levels in response to simulated
microgravity. While d18:1-C16 and d18:1-C18 exhibited a decrease (2.8-
and 1.6-fold, respectively), d18:1-C20 and d18:1-C22 ceramides
exhibited an increase (3.7- and 1.5-fold, respectively) under simulated
microgravity (t-test, p-value < 0.05). Collectively, our findings
suggest that sphingolipid and insulin/IGF-1 pathways play a role in
simulated microgravity response.
3.5. Identification of the Common Microgravity-Responsive Genes between
Four-Day CERISE and Simulated Microgravity
To investigate the microgravity-responsive genes, we compared the
results from our simulated microgravity experiment to those from
four-day CERISE spaceflight experiment on C. elegans reported in NASA’s
GeneLab database ([165]https://genelab.nasa.gov/ (accessed on 1 January
2019)). For this analysis, we used GEO2R to determine the microarray
DEGs (|log2 fold change (logFC)| > 2 and FDR < 0.05). We reasoned that
the genes that are commonly differentially expressed in all the studies
should be the core genes responding to microgravity.
We analyzed our RNA-seq data both with the Tuxedo pipeline [[166]30]
and DEseq2 [[167]31] to confirm that the results are not dependent upon
the analysis method. The results from both the methods were clustered
together indicating their similarity ([168]Figure 5b). The highest
number of DEG overlap was between our experiment and four-day CERISE,
and the overlap was significant for both the methods (hypergeometric
test, p-value < 0.05) ([169]Figure 5c). Since the overlap between the
four-day CERISE and our results from the Tuxedo pipeline is higher, we
decided to use the results from this pipeline throughout the
manuscript. The results from DEseq2 are reported in the
[170]supplementary data ([171]Supplementary Figure S4;
[172]Supplementary Table S5).
Figure 5.
[173]Figure 5
[174]Open in a new tab
Microgravity-responsive genes are consistently differentially expressed
in the ISS and under simulated microgravity. (a) Shared DEGs among the
space-flown worms and our simulated microgravity experiment. The
highest number of common DEGs are between our experiment and four-day
CERISE. These genes are named as the “microgravity-responsive genes”.
(b) The hierarchical clustering of the overall gene expressions among
the space-flown worms and our simulated microgravity experiment. Our
results analyzed with two different data analysis pipelines (*,**)
demonstrated highly similar patterns. (c) The number of common DEGs
were high for our results analyzed with two different pipelines (*,**).
Both of the pipelines showed that the common number of DEGs are higher
than the values expected by chance (hypergeometric text, p < 0.05). (d)
Protein–protein interaction of the microgravity-responsive genes from
STRING database. (e) Gene ontology enrichment of the
microgravity-responsive genes. (f) Protein domain enrichment of the
microgravity-responsive genes.
We determined a total of 134 common DEGs between our experiment and
four-day CERISE and categorized these 134 genes as the putative
microgravity-responsive genes ([175]Figure 5a,c; [176]Supplementary
Table S6). We discarded 16 DEGs showing conflicting patterns between
the experiments (i.e., upregulated in one experiment and downregulated
in the other) for further analyses. We reasoned that if the remaining
genes are collaboratively involved in a biologically relevant process,
they might have protein–protein interactions (PPIs) with each other. To
test this hypothesis, we examined the enrichment for PPI of these genes
by using the STRING database [[177]39]. Among 118 DEGs, 58 had known
PPIs with each other (FDR < 1.0 × 10^−16) indicating their
collaborative involvement in a biological process ([178]Figure 5d). The
GO analysis revealed that the microgravity-responsive genes play a
functional role in locomotion, body morphogenesis, and collagen and
cuticulin-based cuticle development ([179]Figure 5e). Similarly, the
products of microgravity-responsive genes exhibit enrichment for
nematode cuticle collagen N-terminal and collagen triple helix repeat
domains ([180]Figure 5f). Together, our findings suggest that mainly
the collagen genes are affected under microgravity and this effect is
reproducible between the studies.
Next, we asked whether the human orthologs of the
microgravity-responsive genes have relevant functions which might be
affected in astronauts. We identified 64 human orthologs to 44
microgravity-responsive genes in worms ([181]Supplementary Table S7).
The human orthologs exhibited overrepresentation for protein domains
including calpain large subunit domain III and calpain family cysteine
protease ([182]Supplementary Figure S5). Given the reported
upregulation of calpain under microgravity and its function in muscle
atrophy [[183]62,[184]63], these results provide an additional support
for the conserved microgravity-responsive genes in worms and humans.
Our analyses revealed that 20 putative TF genes are upregulated under
simulated microgravity conditions and compared these to the four-day
CERISE mission. Among the 20 upregulated putative TF genes, Y56A3A.28
and T24C4.2 showed upregulation during the four-day CERISE, indicating
a potential transcriptional regulation role for these two in response
to microgravity. Our results additionally suggested that the
upregulation pattern of these TFs is maintained for eight and twelve
days after the return, respectively. We could not find a human ortholog
for T24C4.2 while the WormBase-suggested human ortholog of Y56A3A.28 is
PRDM16. PRDM16 regulates the switch between skeletal muscle and brown
fat cells by inhibiting the skeletal muscle development and gene
expression and stimulating brown adipogenesis [[185]64]. Therefore,
Y56A3A.28 can be a strong candidate as a negative regulator of muscle
development under microgravity conditions.
4. Discussion
In this study, we examined the simulated microgravity responsive gene
expression patterns and their maintained levels for four, eight, and
twelve days after return to ground conditions. Longevity regulating
pathways such as insulin/IGF-1 and sphingolipid signaling were affected
under simulated microgravity. We identified the putative
microgravity-responsive genes by determining the common microgravity
responsive transcriptomic signatures by incorporating our study to a
previous one with the same exposure time. Moreover, we revealed that
the microgravity-responsive genes are potentially conserved between
worms and humans by performing function analysis for the human
orthologs of the worm microgravity-responsive genes and identifying
some of their potential transcriptional regulators.
Sphingolipid signaling pathway plays crucial biological roles such as
lifespan regulation, apoptosis, and oxidative-stress response
[[186]52,[187]54,[188]65]. Our results revealed an overall
downregulation of the sphingolipid pathway which is generally related
to an increase in longevity [[189]54]. A recent study identified
decreased ASM-1 and ASM-2 levels in the ISS-housed worms indicating a
similar downregulation in response to microgravity [[190]66]. Mutant
strains or RNAi knockdown of acid sphingomyelinase ASM-1, ASM-2, and
ASM-3 genes increase the lifespan, ASM-3 being the most prominent one
[[191]53]. Overall, both simulated and the ISS-introduced microgravity
seem to contribute to a potential increase in longevity through the
downregulation of the sphingolipid signaling pathway. Follow-up
experiments are needed to delimitate the contribution of sphingolipids
on longevity under microgravity.
In the Twins Study of NASA, Scott Kelly was sent to the ISS for 340
days while his identical twin Mark Kelly remained on Earth. The results
from this investigation showed that Scott Kelly’s telomere length
significantly increased (14.5%) during the mission. In addition, the
sphingolipid signaling pathway was differentially expressed in Scott
Kelly. The telomere length of Mark Kelly, however, remained relatively
stable [[192]67]. Telomere length and lifespan have a strong
relationship. That is, longer telomeres are linked to longer lifespan
and increased resistance to environmental stress while shorter
telomeres are linked to accelerated aging and reduced longevity
[[193]68,[194]69,[195]70,[196]71]. Ceramide functions in the regulation
of telomere length [[197]55,[198]72]. Considering our findings on
decreased ceramide levels and the observations on increased telomere in
the ISS, investigation of the ceramide and telomere length relationship
might prove important to uncover the mechanisms affecting longevity in
space.
Most of the space flight-triggered physiological changes are reverted
after return to Earth [[199]24,[200]67]. Similarly, we observed that
the majority of the simulated microgravity-induced DEGs (over 84%) are
reverted 12 days after return to ground conditions. Our results have
revealed that while some biological processes are only affected during
the exposure, others can be affected for at least eight days
(approximately two generations) after the return. For example,
neuropeptide signaling pathway genes were differentially expressed only
during the exposure whereas defense response genes remain downregulated
for eight days after the return. Approximately 50% of the astronauts
from Apollo mission experienced minor bacterial or viral infections at
the first week of their return. Later studies reported that space
flight-induced reactivation of latent herpes viruses which lasted for a
week after the return to Earth (reviewed in [[201]73]). These
consistent observations indicate that the detrimental effects of space
conditions (i.e., microgravity) on the immune system carry over even
after the return. Given the longevity differences in worms and humans,
future investigations could provide insight into whether the duration
of the exposure has an impact on the duration of the lasting effects.
The limitation of our study is the inclusion of mixed stage worms and
the collection of samples from whole animals. This limitation is common
among the worm experiments conducted in the ISS
[[202]23,[203]74,[204]75]. Considering the significantly different gene
expression patterns among developmental stages and across cell types
[[205]76], it is possible to retrieve biased results from the averaged
gene expression levels. It is essential to address these issues in
future studies.
The comparison of spaceflight responsive genes in C. elegans and
Drosophila melanogaster demonstrated only six common genes from the
European Soyuz flights to the ISS [[206]17]. Similarly, the overlap
between our results and four-day CERISE were relatively small. One of
the reasons for these discrepancies can be the batch effects introduced
due to the differences in the experimental designs. For instance,
different exposure times to the space conditions or the usage of
different food source or strain may induce distinct responses in the
worms. It is crucial to examine the effects of individual factors
(e.g., diet and gravitational force) along with the combinatory ones.
Towards that end, we previously reported the impact of liquid
cultivation of two C. elegans strains on gene expression and phenotype
[[207]26,[208]77] and here, studied the impact of simulated
microgravity.
5. Conclusions
Given the elevated interest in the human exploration of space, it is
crucial to determine the detrimental effects of the space conditions on
biological systems. We believe that our results will be a valuable
reference for the studies on transcriptomic response to microgravity by
controlling for the renowned batch effects from space conditions,
allowing the worms to acclimate to liquid cultivation before the
experiment, presenting the sustained transcriptomic responses after
return to ground along with their putative regulators, integrating the
results from a previous study conducted at the ISS, and providing the
response of different ceramide profiles to simulated microgravity.
Acknowledgments