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iScience
Article
Spaceflight affects neuronal morphology and
alters transcellular degradation of neuronal debris
in adult Caenorhabditis elegans
Ricardo
Laranjeiro, Girish
Harinath, Amelia
K. Pollard, ...,
Timothy
Etheridge,
Nathaniel J.
Szewczyk, Monica
Driscoll
ricardo_laranjeiro@hotmail.
com
HIGHLIGHTS
Spaceflight induces
morphological
remodeling of adult
neurons in C. elegans
Hyperbranching is a
common response of
adult neurons to
spaceflight
Neuronal debris
accumulates in the
hypodermis of proteo-
stressed space-flown
animals
Laranjeiro et al., iScience 24,
102105
February 19, 2021 ª2021 The
Author(s).
https://doi.org/10.1016/
j.isci.2021.102105
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iScience
Article
Spaceflight affects neuronal morphology
and alters transcellular degradation
of neuronal debris in adult Caenorhabditis elegans
Ricardo Laranjeiro,
1,7,
*Girish Harinath,
1
Amelia K. Pollard,
2
Christopher J. Gaffney,
3,4
Colleen S. Deane,
3
Siva A. Vanapalli,
5
Timothy Etheridge,
3
Nathaniel J. Szewczyk,
2,6
and Monica Driscoll
1
SUMMARY
Extended space travel is a goal of government space agencies and private com-
panies. However, spaceflight poses risks to human health, and the effects on
the nervous system have to be better characterized. Here, we exploited the
unique experimental advantages of the nematode Caenorhabditis elegans to
explore how spaceflight affects adult neurons in vivo. We found that animals
that lived 5 days of adulthood on the International Space Station exhibited hyper-
branching in PVD and touch receptor neurons. We also found that, in the presence
of a neuronal proteotoxic stress, spaceflight promotes a remarkable accumula-
tion of neuronal-derived waste in the surrounding tissues, suggesting an impaired
transcellular degradation of debris released from neurons. Our data reveal that
spaceflight can significantly affect adult neuronal morphology and clearance of
neuronal trash, highlighting the need to carefully assess the risks of long-duration
spaceflight on the nervous system and to develop adequate countermeasures for
safe space exploration.
INTRODUCTION
Humankind as long been fascinated by space exploration. Yuri Gagarin first showed in 1961 that humans
could survive in space, and Neil Armstrong and Edwin ‘Buzz’ Aldrin became the first to walk on the
moon in 1969. Now, the International Space Station (ISS), a multinational collaborative station that orbits
Earth, has been continuously occupied with crew since November 2000. Into the future, both governmental
space agencies and private companies plan to send crewed missions to Mars and beyond. Research to
date, however, makes evident that spaceflight poses health risks to the human body, including detrimental
effects to musculoskeletal, cardiovascular, and immune systems (Crucian et al., 2018;Fitts et al., 2001;Shen
and Frishman, 2019;Sibonga, 2013;Williams et al., 2009). Particularly striking is the loss of up to 20% of mus-
cle mass in short-duration spaceflights (Adams et al., 2003;Edgerton et al., 1995), which can be ameliorated
by regular physical exercise (Loehr et al., 2015). Much less is known about the effects of spaceflight on
neuronal morphology and function, especially in vivo and at the single-neuron level. Given the impracti-
cality of conducting such studies in humans, the use of animal models in which detailed high-resolution
neuronal analyses can be performed is invaluable for assessing conserved neuronal responses to space-
flight and developing effective countermeasures to mitigate the consequences of long-duration missions.
The nematode Caenorhabditis elegans is a genetic model widely used in neurobiology, aging, and stress
research that is amenable for assessing the effects of spaceflight on neuronal biology given its unique ad-
vantages: ease of culture in large numbers due to its microscopic size; a transparent body that permits
in vivo imaging of multiple tissues, single cells, and evencellular organelles; and a short lifespan (2–3 weeks)
that allows for even a short-duration spaceflight mission to correspond to a large portion of the C. elegans
lifetime. Importantly, the spaceflight-induced muscle atrophy observed in humans is conserved in
C. elegans, as several studies reported a downregulation of muscular components in space-flown nema-
todes (Adenle et al., 2009;Higashibata et al., 2006,2016;Selch et al., 2008). The aging C. elegans muscu-
lature also displays the fundamental characteristics of human sarcopenia (the pervasive age-associated
loss of muscle mass and strength that contributes to human frailty) (Herndon et al., 2002), and physical ex-
ercise in C. elegans mimics human exercise by inducing multi-systemic health benefits (Hartman et al.,
2018;Laranjeiro et al., 2017,2019). In addition to muscle changes, space-flown C. elegans have altered
1
Department of Molecular
Biology and Biochemistry,
Rutgers, The State University
of New Jersey, Piscataway,
NJ 08854, USA
2
MRC Versus Arthritis Centre
for Musculoskeletal Ageing
Research and NIHR
Nottingham BRC, University
of Nottingham, Medical
School Royal Derby Hospital,
Derby, DE22 3DT, UK
3
Sport and Health Sciences,
University of Exeter, Exeter,
EX1 2LU, UK
4
Lancaster Medical School,
Health Innovation One,
Lancaster University,
Lancaster, LA1 4AT, UK
5
Department of Chemical
Engineering, Texas Tech
University, Lubbock, TX
79409, USA
6
Ohio Musculoskeletal and
Neurologic Institute and
Department of Biomedical
Sciences, Heritage College of
Osteopathic Medicine, Ohio
University, Athens, OH 45701,
USA
7
Lead contact
*Correspondence:
ricardo_laranjeiro@hotmail.
com
https://doi.org/10.1016/j.isci.
2021.102105
iScience 24, 102105, February 19, 2021 ª2021 The Author(s).
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
1
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metabolism and differential expression of longevity-related genes (Adenle et al., 2009;Higashibata et al.,
2016;Honda et al., 2012;Selch et al., 2008). The C. elegans nervous system is composed of exactly 302 neu-
rons, each one with a well-described morphology. Thus, transgenic nematodes in which neuronal subpop-
ulations are fluorescently labeled allow for in vivo single-neuron assessments that are virtually impossible in
the highly complex mammalian brain.
Here, we show that adult C. elegans that lived 5 days on the ISS exhibit morphological changes in two
distinct types of sensory neurons when compared to ground control animals. We identify hyperbranching
as a common response of adult neurons to spaceflight. We also studied proteostressed touch neurons with
a focus on a formerly unrecognized mechanism by which C. elegans neurons can extrude large membrane-
surrounded vesicles that contain neuronal waste (e.g. protein aggregates and damaged organelles) (Ar-
nold et al., 2020;Melentijevic et al., 2017). On Earth, the extruded neurotoxic components are, in most
cases, efficiently degraded by the surrounding tissues, which in the case studied is the nematode hypoder-
mis (Arnold et al., 2020;Melentijevic et al., 2017). We find that, under spaceflight conditions, proteos-
tressed neurons are associated with a striking accumulation of neuronal debris in the surrounding tissues
not apparent in ground controls, indicating a severe dysregulation in the ability to clear neuronal waste in
space-flown, middle-aged animals. Our results reveal spaceflight-associated challenges to systemic pro-
teostasis and underscore the need to carefully assess the cellular consequences of long-duration space-
flight as strategies to maintain health are developed.
RESULTS
A spaceflight protocol that features adult-only C. elegans culture and enables analysis of
microgravity impact on a cohort that spans early adult life to middle age
For all experiments presented in this study, we grew C. elegans in a liquid culture of S-Basal with freeze-dried
E. coli OP50 as a food source. Prior to launch, we cultured synchronized populations of young adult nematodes
that we loaded intopolyethylene (PE) bags with6.5 mL of liquid culture (300 animals/bag) on December 2, 2018
(see detailedoptimization of culture conditions (Pollardet al., 2020)). Importantly,given our goal of assessing the
effects of spaceflight during adult life rather than during animal development, we added 5-fluoro-20-deoxyuri-
dine (FUdR) to the culture bagsto suppress production of progeny and to guarantee that the animals we loaded
into the bags would be the same ones we scored upon their return to Earth, as opposed to their progeny. Our
samples were loaded into SpaceX CRS-16, the 16
th
Commercial Resupply Service mission to the ISS, and
launched on December 5, 2018 aboard a Falcon 9 rocket. SpaceX CRS-16 docked to the ISS on December 8,
2018, and the C. elegans samples, which were kept in cold stowage (8–13C) once introduced into their flight
bags, were transferred to 20C for five days, beginning on December9, 2018. After fivedays, the C. elegans cul-
ture bags were transferred to 80C and kept frozen until return to Earth (Figure 1). For ground control samples,
we loaded young adultnematodes into PE bags on December5, 2018 and exposed C. elegans to the same time
Figure 1. A spaceflight protocol to assess microgravity impact on C. elegans adult life
Diagram showing the timeline of C. elegans life at 20C, with approximately 2.5 days of embryonic and larval
development and approximately three weeks (21 days) of adulthood. We indicate in red the crucial events in our
experimental design by which we obtained middle-aged animals that experienced most of their physiological aging on
the International Space Station (ISS). Note that C. elegans were kept in cold stowage (8–13C) for approximately 7 days,
relatively low temperatures that significantly delayed progression through adult stages. We estimated the cold stowage
period to correspond to approximately 3 days at 20C. After 5 days on the ISS at 20C, samples were frozen and returned
to Earth for analysis.
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A
B
C
D
Figure 2. The PVD dendritic tree is affected by spaceflight
(A) Diagram showing the morphology of adult PVD sensory neurons. The characteristic PVD branches that form during
larval development (1–4branches) to produce the repetitive structural units called menorahs are indicated in red. We
performed all quantifications in the 100 mm anterior to the PVD cell body as depicted by the blue dashed box. A, anterior;
P, posterior.
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frame/temperaturesas space samples but maintainedcultures on Earth. A key aspectof our experimental design
is thus that we generated middle-aged nematodes that experienced most of their physiological aging in micro-
gravity (ISS) vs. normal gravity (Earth).
Spaceflight induces morphological remodeling of adult PVD sensory neurons
We started by studying the effects of spaceflight in the morphology of PVD sensory neurons, polymodal
nociceptors with spectacular morphologies that sense harsh touch (Way and Chalfie, 1989), cold temper-
ature (Chatzigeorgiou et al., 2010), and posture (Albeg et al., 2011). The two PVD neurons (one on the left
side and one on the right side of the body) extend processes that branch to cover most of the body and,
together with FLP neurons in the head, possess the most complex dendritic arborization structure of all
C. elegans neurons (Figure 2A). The highly branched, yet stereotyped, morphology of PVD dendrites arises
sequentially during larval development, with primary (1) branches forming during the L2 stage, secondary
(2) and tertiary (3) branches forming during the L3 stage, and quaternary (4) branches forming during the
early L4 stage (Oren-Suissa et al., 2010). By the late L4 stage, PVD patterning is completed, and the repet-
itive structural units resembling candelabras or menorahs become apparent (Figure 2A).
We used a C. elegans strain in which PVD neurons express a translational fusion of transmembrane protein
DES-2 and green fluorescent protein (GFP) (DES-2::GFP) (Oren-Suissa et al., 2010). All tiers of the PVD den-
drites are well visualized by GFP in this strain, and approaches toward quantitative imaging of branching
have been published (Kravtsov et al., 2017;Oren-Suissa et al., 2010,2017). We compared PVD neurons
from middle-aged space-flown vs. ground control animals by scoring 1–4branch structures. We found
that the space-flown PVD neurons did not degenerate en masse nor did they exhibit major dendritic
gaps relative to ground control PVD neurons (n= 51–56 PVD neurons/condition) (Figures 2Band2C).How-
ever, when we carefully quantified the number of branches and other menorah-related phenotypes in a
100-mm domain anterior to the PVD soma (Figure 2A), we registered con sistent differences between control
and spaceflight animals. More specifically, space-flown animals exhibited increased numbers of 2and 3
branches, whereas the number of 4branches remained constant between control and spaceflight animals
(Figure 2D), indicating a plastic morphological restructuring during adult life with outcome that differs
consequent to microgravity experience.
On Earth, the PVD dendritic pattern is established during larval stages, but adult neurons still exhibit a
limited plasticity of the dendritic trees, with dynamic growth and retraction events occurring during lifetime
(Kravtsov et al., 2017). Moreover, the PVD dendritic trees show age-dependent hyperbranching, disorga-
nization, and loss of self-avoidance within tree branches (Kravtsov et al., 2017). Whether these changes
reflect deleterious aspects of aging or adaptive processes as the animal grows remains unclear. With re-
gard to spaceflight samples, our quantification of the number of extra branches derived from 4branches
(5,6
,and7
branches) and the number of ectopic branches (ectopic 2,3
,and4
) found that space-flown
animals displayed an increased number of 5branches (Figure 3A) and ectopic 3branches (Figure 3B) rela-
tive to ground control animals. Retrograde branches (4or higher-order branches that migrate toward the
3branch by forming a hook) were also present at significantly higher numbers in spaceflight PVD neurons
when compared to control counterparts (Figure 3C). We observed that spaceflight increased the propor-
tion of disorganized menorahs (menorahs with extra/ectopic branches) (Figure 3D) and the proportion of
self-avoidance defects (no gap in 3branch between adjacent menorahs) (Figure 3E). PVD dendrites also
exhibit age-associated degeneration characterized by the appearance of bead- or bubble-like structures
along the dendrites, which are enriched in autophagosomes and fragmented microtubules (Eetal.,
2018). We focused on this morphological feature but found no difference in the number of beads/bubbles
present in PVD dendrites of space-flown animals relative to ground control animals (Figure 3F).
Overall, our data reveal that spaceflight induces significant remodeling of the PVD dendritic tree in adult
C. elegans without promoting neurodegeneration. Importantly, the morphological changes we report here
Figure 2. Continued
(B and C) Representative maximum intensity projection confocal images of ground control (B) and spaceflight (C) PVD
neurons from DES-2::GFP animals. Scale bars, 10 mm.
(D) Number of 2,3
,and4
branches in ground control and spaceflight PVD neurons. Number of animals used for
analysis: n
Control
=51,n
Space
= 56. We determined statistical significance by unpaired two-tailed Student’s ttest. *p %
0.05, **p %0.01. Data are presented as mean Gstandard error of the mean.
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A
B
C
D
E
F
Figure 3. Spaceflight promotes hyperbranching, disorganization, and self-avoidance defects in PVD sensory
neurons
(A–F) Quantifications in ground control and spaceflight PVD neurons of 5,6
,and7
branches (A), ectopic 2,ectopic3
,
and ectopic 4branches (B), retrograde branches (C), disorganized menorahs (D), self-avoidance defects (E), and beads/
bubbles (F). We highlight each PVD remodeling phenotype with a diagram (left panel) and a representative confocal
image from DES-2::GFP animals (center panel). Number of animals used for analysis in A–E: n
Control
=51,n
Space
=56.
Number of animals used for analysis in F: n
Control
= 12, n
Space
= 12 (we randomly selected 12 ground control and
spaceflight animals for scoring; each point represents a single animal). We determined statistical significance by unpaired
two-tailed Student’s ttest (A–C, F) and by Fisher’s exact test (D, E). *p %0.05, **p %0.01, ***p %0.001. Data are
presented as mean Gstandard error of the mean.
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for sensory neurons occurred during adult life, indicating that microgravity can influence the morphologyof
adult neurons.
Spaceflight promotes modest morphological changes in adult touch receptor neurons
The complex dendritic tree of PVD neurons contrasts with the simple morphology of a single unbranched
process adopted by most C. elegans neurons (White et al., 1986). The six touch receptor neurons (AVM,
ALML, ALMR, PVM, PLML, and PLMR) mediate the response to gentle touch, with each extending a single
major dendritic process anteriorly (Chalfie and Sulston, 1981)(Figure 4A). We sought to investigate the ef-
fects of spaceflight on touch receptor neuron morphology by studying a C. elegans strain in which touch
receptor neurons are specifically labeled with soluble GFP (P
mec-4
GFP).
We found that middle-aged nematodes that lived five days of adulthood on the ISS exhibited touch recep-
tor neurons with an overall morphology similar to ground control animals, with no obvious degeneration,
dendritic gaps, or soma/process mislocalization (n= 54–64 of each touch receptor neuron/condition) (Fig-
ures 4B and 4C). For a detailed morphological characterization of each neuron, we quantified age-depen-
dent morphological changes previously reported for touch receptor neurons (Toth et al., 2012), including
new branches that extend from the main process (‘‘branches’’) and new processes emerging from the soma
(‘‘outgrowths’’) (Figure 4D). We observed an overall trend in space-flown animals for increased number of
branches in all six touch receptor neurons (statistically significant for ALMR and PLML) relative to ground
control animals (Figure 4E), whereas we found the number of outgrowths was generally unchanged by
spaceflight (except for PVM) (Figure 4F). Thus, C. elegans adult touch receptor neurons undergo minor
structural changes consequent to space travel. Together, our data reveal that spaceflight during adult
life does not promote major morphological changes in touch receptor neurons. Still, the hyperbranching
phenotype detected in specific adult touch receptor neurons and the morphological changes observed in
adult PVD neurons suggest that modest neuronal restructuring might constitute a general neuronal
response to microgravity and/or other stresses associated with spaceflight.
Spaceflight induces a distinctive response to neuronal mCherry extrusion
Spaceflight includes novel stresses experienced by organisms during lift-off/reentry and extended micro-
gravity periods, and some of these stresses have been suggested to contribute to accelerated aging (De-
montis et al., 2017). Among the many stress response pathways activated and compromised by aging are
proteostasis pathways such as autophagy and protein degradation processes (Kaushik and Cuervo, 2015).
Although fluxes through such pathways are difficult to measure in spaceflight samples, we considered
some physical readouts thought to reflect proteostasis status.
Expression of GFP in touch receptor neurons can induce high-intensity fluorescent puncta in the soma (Fig-
ure 4D). We found that the absolute numbers of GFP puncta in touch neurons did not differ between mid-
dle-aged nematodes on Earth and the ones that spent the corresponding five days on the ISS (Figure S1A).
However, we did observe a trend in space-flown animals for an increased proportion of large GFP puncta
(diameter R0.9 mm) in the soma of touch receptor neurons as compared to ground control animals (Fig-
ure S1B). Although we cannot distinguish whether the touch neuron puncta correspond to aggregates,
developing aggresomes, or liquid phase droplets, the differential handling of introduced GFP suggests
physiological differences between Earth and spaceflight neurons.
We also reasoned that we might readily evaluate one proteostress response using fluorescence imaging of
marked neurons – the extrusion of neuronal garbage. Touch receptor neurons can extrude large mem-
brane-surrounded vesicles called exophers that contain protein aggregates and organelles (Melentijevic
et al., 2017)(Figure 4D). Exopher production increases under enhanced proteostress, as well as under envi-
ronmental stresses such as elevated superoxide production (Arnold et al., 2020;Melentijevic et al., 2017),
possibly as a conserved protective mechanism of proteostasis. Under standard conditions, exopher pro-
duction in GFP-labeled neurons is a relatively rare event (Melentijevic et al., 2017). We found that space-
flight did not significantly increase overall exopher detection in touch receptor neurons of P
mec-4
GFP an-
imals (Figure 4G). We did, however, note an interesting switch in the apparent rates of exopher
production in individual touch receptor neurons. On Earth, adult ALMR neurons reproducibly produce
more baseline exophers than other neurons, including the left side counterpart ALML (Melentijevic
et al., 2017),butinspaceflightanimals,leftsideALML produced more baseline exophers (Figure 4G).
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A
BC
D
EF
G
Figure 4. Spaceflight promotes modest morphological changes in adult touch receptor neurons
(A) GFP-labeled touch receptor neurons in a young adult P
mec-4
GFP animal. Image adapted from www.wormatlas.org.
NR, nerve ring; VNC, ventral nerve cord.
(B and C) Representative confocal images of ground control (B) and spaceflight (C) P
mec-4
GFP animals. Scale bars, 10 mm.
(D) Diagram showing a touch receptor neuron with a branch, an outgrowth, high-intensity fluorescent puncta in the cell
body, and an exopher.
(E–G) Quantifications in ground control and spaceflight P
mec-4
GFP animals of the number of branches (E), outgrowths (F),
and exophers (G) in each of the six touch receptor neurons. We highlight each touch receptor neuron phenotype with a
representative confocal image from P
mec-4
GFP animals. Number of touch receptor neurons used for analysis: n
Control
=
54–61, n
Space
= 60–64. Note that the different number of neurons used for analysis in each condition derives from the fact
that, depending on the animal position during imaging, not all touch receptor neurons are visible in certain animals. We
determined statistical significance by unpaired two-tailed Student’s ttest. *p %0.05, ***p %0.001. Data are presented as
mean Gstandard error of the mean.
See also Figure S1.
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Our observations raise the possibility that maintained gravitational forces may influence left/right asymme-
tries associated with stresses or their management in particular neurons.
GFP-expressing touch receptor neurons have been studied extensively and are not thought to experience
any particular stress as a consequence of the GFP expression (Chalfie et al., 1994;Herndon et al., 2002).
Other transgenically supplied reporters such as mCherry can appear disruptive to normal physiology
and are associated with an enhanced level of selective extrusion in exophers (Melentijevic et al., 2017).
We wondered whether ‘‘at risk’’ neurons, pre-sensitized by the genetic introduction of a noxious
mCherry-induced proteostress, might be impacted by spaceflight. Thus, we examined space-flown animals
of a strain that over-produces an mCherry reporter well characterized to elevate the production of exo-
phers in touch receptor neurons (P
mec-4
mCherry1)(Melentijevic et al., 2017). We examined the neuronal
morphology, apparent protein aggregation, and exopher-genesis in the presence of the mCherry stressor.
We found that touch receptor neurons of space-flown P
mec-4
mCherry1 animals exhibited a generally
normal neuronal morphology (n= 42–66 of each touch receptor neuron/condition). We observed a trend
in spaceflight animals for increased number of mCherry foci in the soma of specific touch receptor neurons
(AVM, ALMR, PLML, and statistically significant in PLMR) relative to ground control animals (Figure S2A),
whereas we detected no significant differences in the proportion of large internal mCherry foci (diameter
R0.9 mm) between both conditions (Figure S2B). We found no significant differences in the absolute num-
ber of exophers present in middle-aged control vs. spaceflight touch neurons (Figure S2C).
We did, however, observe a striking difference in mCherry reporter signal present outside the touch recep-
tor neurons: a remarkably large proportion of P
mec-4
mCherry1 animals that spent 5 days of adulthood on
the ISS contained numerous mCherry-fluorescent structures throughout the body, a phenotype we never
observed in ground control animals (Figures 5A, 5B, and S3). While exophers produced by ground control
animals are found almost exclusively near touch receptor neuron cell bodies, in limited numbers (one or
two exophers produced at the most per neuron), and with characteristic sizes (Arnold et al., 2020;Melenti-
jevic et al., 2017), fluorescent structures in space-flown animals ranged from small particles to large
rounded structures that could appear as a continuous layer covering multiple regions of the body (Figures
5BandS3).
C. elegans touch receptor neurons run through and are fully surrounded by hypodermal tissue. When exo-
phers are extruded, they must therefore enter the hypodermis, which attempts digestion of exopher con-
tents via its extensive lysosomal network. mCherry exopher contents that cannot be digested in the sur-
rounding hypodermis are eventually extruded from the hypodermis into the C. elegans pseudocoelom.
AB
Figure 5. Spaceflight leads to accumulation of neuronal-derived mCherry throughout the body of middle-aged
nematodes
(A and B) Representative confocal images of ground control (A) and spaceflight (B) P
mec-4
mCherry1 animals. Scale bars,
10 mm.
See also Figures S2 and S3.
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Materials floating through the pseudocoelom can be taken up by scavenger cells called coelomocytes (Ar-
nold et al., 2020;Melentijevic et al., 2017). During the process of transit through the hypodermis, the
mCherry exopher contents often become dispersed in the hypodermal lysosomal network, appearing as
small fluorescent particles (we refer to this as a ‘‘Starry Night’’ phenotype) (Arnold et al., 2020;Melentijevic
et al., 2017). The unexpected distribution of fluorescence that we observed in spaceflight P
mec-4
mCherry1
animals appears comprised of two main components (Figure 6A): small fluorescent particles resembling
the Starry Night phenotype and large rounded fluorescent structures (‘‘spaceflight vesicles’’ or ‘‘sVesicles’’)
that we have not previously found in this C. elegans strain under standard culture conditions (even in much
older adults) (Arnold et al., 2020;Melentijevic et al., 2017). These large rounded structures have the appear-
ance of the enlarged hypodermal lysosomes that can be found in C. elegans mutants defective in lysosomal
function and assembly (Li et al., 2016;Liu et al., 2018;Wang et al., 2019).
We characterized the presence and location of both Starry Night and sVesicles in our samples. We found a
striking increase in the proportion of animals with Starry Night in control vs. spaceflight (7% vs. 67%) (Fig-
ure 6B). In ground control animals, we always found Starry Night in the mid-body and in the hypodermis
(peripheral layer surrounding the body) (Figures 6C and 6D). In space-flown animals, we still found Starry
Night predominantly in the mid-body, but in approximately half of the cases, the Starry Night dispersed
fluorescence expanded into the anterior and/or the posterior region of the body (Figure 6C). Regarding
tissue location, we detected that Starry Night after spaceflight was always present in the hypodermis
(Videos S1 and S2) but could also spread into the intestine (15% of animals) or other tissues (48% of animals)
(Figure 6D). We did not observe sVesicles in any of the 72 ground control animals analyzed, whereas 52 out
of the 83 space-flown animals (63%) exhibited sVesicles (Figure 6E). The location distribution of sVesicles in
spaceflight animals was similar to Starry Night location, with sVesicles found predominantly in the mid-
body but also in the anterior and posterior regions of the body (Figure 6F), and the primarily hypodermal
location (Videos S1 and S2) extended into the intestine (7% of animals) or other tissues (56% of animals)
(Figure 6G). Despite the extensive mCherry fluorescence found outside the touch receptor neurons in
spaceflight animals, we rarely observed mCherry fluorescence in coelomocytes in this sample (only 1 out
of 56 animals). The similar distribution patterns of Starry Night and sVesicles, together with the fact that
96% of the animals that exhibited sVesicles also had Starry Night, strongly support a link between both
phenomena.
We tried to identify the sVesicles found in spaceflight nematodes by performing antibody staining for the
lysosomal marker LMP-1 but were unable to get specific staining, possibly due to the paraformaldehyde
fixation that we had to perform when thawing the frozen nematodes (essential to maintain a strong fluores-
cence from reporters). Moreover, it appeared that the sVesicles could be readily disrupted as we found that
alternative fixation methods (e.g. methanol and acetic acid) led to the disappearance of most mCherry fluo-
rescence outside the touch receptor neurons. The fragility of the sVesicle structures limited our ability to
definitively confirm the nature of the hypodermal structures we observed. We did find that the fluorescence
intensity of the sVesicles varied greatly, ranging from a bright to a diffuse, dim fluorescence (Figure 6H),
which is consistent with sVesicles being at different stages of lysosomal degradation.
We were fortunate that an unanticipated feature of the flight population enabled us to compare mCherry
distribution in some escaper progeny in the culture bag from the ISS. We found that despite our use of
FUdR to limit reproduction, some progeny were able to develop up to young adulthood. We could easily
identify these animals from older adults by their reduced size. Young adult animals present in the same cul-
ture bag as the middle-aged adults that were our major focus exhibited the fluorescent structures at
extremely low levels (Figure S4), suggesting that the dramatic sVesicle profiles in the hypodermis could
be age dependent. We cannot rule out, however, that a variable introduced during development in space-
flight could be responsible for the lack of the large sVesicle pattern in younger animals.
Overall, our data reveal that, in the presence of an internal cell-specific proteotoxic stress (i.e., mCherry
overexpression in touch receptor neurons), spaceflight is associated with significant extrusion of the offen-
sive protein in middle-aged touch receptor neurons. Unexpectedly, the extruded material appears to
become stuck in the hypodermal lysosomal network, and the lysosomal network adopts a morphology
similar to that observed when lysosomes are unable to degrade their substrates. Our data suggest that
in microgravity, extruded neuronal garbage is not handled as efficiently by the neighboring cell that col-
lects neuronal debris and attempts degradation.
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A
B
CD
EF
GH
Figure 6. Spaceflight induces a distinctive hypodermal response to neuronal mCherry extrusion
(A) Confocal image of a spaceflight P
mec-4
mCherry1 animal in which we identified Starry Night and spaceflight vesicles
(sVesicles) as two distinct fluorescent components. Scale bar, 10 mm.
(B) Proportion of ground control and spaceflight P
mec-4
mCherry1 animals with/without Starry Night. Number of animals
used for analysis: n
Control
= 72, n
Space
= 83. We determined statistical significance by Fisher’s exact test. ****p %0.0001.
(C and D) Location of Starry Night in ground control and spaceflight P
mec-4
mCherry1 animals in the anterior-posterior
body axis (C) and in different tissues (D). Number of animals used for analysis: n
Control
=5,n
Space
=33.
(E) Proportion of ground control and spaceflight P
mec-4
mCherry1 animals with/without sVesicles. Number of animals used
for analysis: n
Control
= 72, n
Space
= 83. We determined statistical significance by Fisher’s exact test. ****p %0.0001.
(F and G) Location of sVesicles in spaceflight P
mec-4
mCherry1 animals in the anterior-posterior body axis (F) and in
different tissues (G). Number of animals used for analysis: n
Space
= 27. We scored Starry Night and sVesicles as ‘‘anterior’’ if
location was anterior to the AVM neuron, ‘‘mid’’ if location was in between the AVM and the PVM neurons, and ‘‘posterior’’
if location was posterior to the PVM neuron. We scored tissue location by imaging different planes on each animal;
‘‘hypodermal’’ corresponded to the peripheral layer surrounding the body, ‘‘intestinal’’ corresponded to the cells
surrounding the intestinal lumen, and ‘‘other’’ corresponded to locations in the body not easily identifiable. We randomly
selected a subgroup of spaceflight P
mec-4
mCherry1 animals to score the location of Starry Night and sVesicles in the
anterior-posterior body axis and in different tissues.
(H) Confocal image of a spaceflight P
mec-4
mCherry1 animal showing the variation in fluorescence intensity of sVesicles.
Scale bar, 10 mm.
See also Figure S4.
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Future experiments will be necessary to determine the exact mechanism causing mCherry accumulation in
large quantities in multiple tissues of C. elegans consequent to spaceflight. Still, it is interesting that our
results suggest that spaceflight can increase mCherry spread from touch receptor neurons to neighboring
tissues (via exophers and/or unknown mechanisms) and that these significant extrusions are not processed
in neighboring tissues as occurs on Earth. The abnormal fluorescent structures observed primarily in the
hypodermis suggest that the degradative pathways of middle-aged animals may become overwhelmed
in response to spaceflight with the resulting inability to quickly clear neuronal trash. Thus, in upcoming
spaceflight missions, it will be fascinating to specifically track the hypodermal lysosomal network and to
determine whether other proteostressors such as aggregation-prone human neurodegenerative disease
proteins tau, amyloid beta, huntingtin, or alpha-synuclein promote similar distinctive outcomes.
DISCUSSION
Spaceflight has been shown to induce adverse effects on the human body at multiple levels, including in
musculoskeletal, cardiovascular, and immune systems (Crucian et al., 2018;Fitts et al., 2001;Shen and Frish-
man, 2019;Sibonga, 2013;Williams et al., 2009). However, the effect of spaceflight on neuronal
morphology and function, especially in vivo, is largely unknown. In this study, we took advantage of the
unique characteristics of the nematode C. elegans (e.g. ease of culture in large numbers and transparent
body allowing for in vivo imaging of fluorescently labeled transgenic lines) to address how spaceflight af-
fects the morphology of different adult neurons and the response to a neuronal proteotoxic stress. We
show that spaceflight induces extensive remodeling of the PVD dendritic tree in adult C. elegans relative
to ground control animals, whereas touch receptor neurons exhibit modest morphological changes in
response to spaceflight. Importantly, hyperbranching is a consistent response to spaceflight between
different adult neuron types. We also report on a striking difference in the ability to respond to a neuronal
proteostress (i.e., mCherry overexpression) in spaceflight vs. Earth samples, in which adults that aged on
the ISS exhibited accumulated extruded neuron al garbage outside the neurons, distributed in neighboring
tissues. Our observations raise the intriguing possibility that spaceflight may impair neuronal proteostasis/
clearance pathways, which may hold significant health implications for long-duration spaceflights.
Spaceflight promotes remodeling of complex adult dendritic trees
We found that spaceflight promoted hyperbranching, disorganization, and loss of self-avoidance in mid-
dle-aged PVD neurons. These phenotypes have been shown to increase during aging in C. elegans (Kravt-
sov et al., 2017), raising the possibility that space-flown animals experienced accelerated aging compared
to ground control counterparts. However, we observed no increase in space-flown animals of age-associ-
ated PVD degeneration (i.e. number of beads/bubbles), which is tightly linked to the functional aging of
PVD neurons (Eetal.,2018). Adult animals with disorganized PVD dendritic trees rarely show defects in
response to harsh touch (E et al., 2018;Kravtsov et al., 2017), suggesting that changes in dendritic architec-
ture are not necessarily a deleterious aspect of aging but rather might be part of normal neuronal mainte-
nance and an adaptive response to intrinsic/extrinsic cues. Furthermore, in other models, most physiolog-
ical changes induced by spaceflight are reversed upon return to Earth and therefore can be considered
physiological adaptations to the spaceflight environment (Garrett-Bakelman et al., 2019;Honda et al.,
2014;Williams et al., 2009). Thus, we hypothesize that the PVD remodeling we describe here is more likely
an adaptive change to spaceflight conditions rather than an overall increase in aging rate. Testing this hy-
pothesis is a challenge for spaceflight experiments as the C. elegans lifespan is shorter than typical
missions.
A complex cross talk between muscle, skin (hypodermis), and PVD neurons defines the pattern of higher-
order branches in the PVD dendritic tree (Diaz-Balzac et al., 2016;Liang et al., 2015;Salzberg et al., 2013;
Yang and Chien, 2019;Zhu et al., 2017). Briefly, the extracellular matrix protein UNC-52/Perlecan forms reg-
ular stripes by following the location of the dense bodies of sarcomeres and is essential for the formation of
regular hypodermal hemidesmosomes, which are the attachments that mechanically link thehypodermis to
the muscle. The intermediate filament MUA-6, which is a component of the hemidesmosome, patterns the
transmembrane cell adhesion molecule SAX-7/L1CAM in hypodermal stripes by locally excluding SAX-7
from the regions where MUA-6 is present. Then, SAX-7 physically interacts with the transmembrane recep-
tor DMA-1 in PVD neurons to instruct the growth of 4branches. Given the well-established and conserved
role of spaceflight in muscle degeneration (Adenle et al., 2009;Fitts et al., 2001;Higashibata et al., 2016;
Williams et al., 2009), we propose that PVD dendritic morphology in space-flown C. elegans may be indi-
rectly affected by changes at the body wall muscle level. In fact, microgravity in C. elegans has been shown
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to downregulate the expression of multiple muscle dense body genes and intermediate filament genes,
including mua-6 (Higashibata et al., 2016). Areas where MUA-6 is absent have abnormally shaped PVD den-
drites (Liang et al., 2015), suggesting that the downregulation of muscle structural genes together with
mua-6 by microgravity could contribute to the changes we observed in space-flown PVD neurons, partic-
ularly the hyperbranching, menorah disorganization, and increase in retrograde branches.
Spaceflight has also been suggested to reduce insulin signaling in C. elegans (Honda et al., 2012;Selch
et al., 2008), whereas the insulin/IGF-1 receptor daf-2 mutant exhibits an increase in PVD 2branches
(Gioran et al., 2014) and self-avoidance defects (Kravtsov et al., 2017). Thus, a putative modulation in insulin
signaling in space-flown C. elegans might contribute to some of the PVD remodeling phenotypes that we
describe in this study.
Hyperbranching may be a conserved response of adult neurons to spaceflight
We found that space-flown C. elegans exhibited touch receptor neurons with modest morphological
changes (i.e., hyperbranching) when compared to ground control animals. Interestingly, impaired mito-
chondrial respiration increases branching in both touch receptor neurons and PVD neurons (Gioran
et al., 2014), akin to our observations after spaceflight. Moreover, spaceflight in C. elegans has been shown
to induce a metabolic shift (Adenle et al., 2009;Selch et al., 2008) and decrease the expression of numerous
enzymes involved in the tricarboxylic acid cycle and of all electron transport chain complexes (Higashibata
et al., 2016), strongly indicating that mitochondrial respiration is reduced during spaceflight. Therefore,
changes in mitochondrial function during spaceflight may contribute to the hyperbranching phenotype
that we described in different adult neuronal cell types.
The subtle morphological changes induced by spaceflight in touch receptor neurons contrasted with an
extensive remodeling of the PVD dendritic tree. This difference in response to spaceflight can be explain
bytheinnatemorphologyofeachneuron(i.e.,thehighlybrancheddendritictreeofPVDneuronsallowsfor
the detection of multiple morphological abnormalities vs. a single unbranched process of touch receptor
neurons), the stimuli sensed by each neuron (i.e., PVD neurons may sense microgravity more directly than
touch receptor neurons given their role in proprioception (Albeg et al., 2011)), and/or the fact that PVD
morphology is indirectly affected by changes at the muscle level (as discussed previously). Despite the dif-
ferences between neuron types, our results suggest hyperbranching as a common neuronal adaptive
response to spaceflight. This response may in fact be conserved from C. elegans to mammals given that
Septin 7, a GTP-binding protein critical for dendrite branching (Xie et al., 2007), has been shown to be
significantly upregulated in the brain of mice exposed to the ISS environment for 3 months (Santucci
et al., 2012).
Clearance of neuronal waste is not affected by spaceflight in normal conditions
In addition to neuronal morphological changes, we also assessed how spaceflight modulated the extrusion
of neuronal garbage by touch receptor neurons, with a focus on the potential extrusion of large membrane-
surrounded vesicles called exophers (Melentijevic et al., 2017). We found that GFP-labeled touch receptor
neurons, which are not thought to be stressed by transgene expression, exhibited an overall similar number
of detectable exophers in middle-aged animals exposed to the ISS vs. Earth environment. It is interesting
that for GFP-labeled anterior touch neurons, we measured a difference in the left-right asymmetry in exo-
pher production levels. On Earth, the ALM neuron on the right side of the body (ALMR) consistently pro-
duces more exophers than the left side ALM neuron (ALML) (Melentijevic et al., 2017). However, in space-
flown animals, we observed a reversal in exopher-genesis rate between ALMR and ALML neurons.
Although the reason underlying this reversal is unclear, a recent study of reproducible transcriptomic
changes in space-flown C. elegans (Willis et al., 2020) identified downregulated genes predicted to be un-
der the control of the transcription factor NSY-7, which is involved in determination of neuronal left/right
asymmetry (Lesch et al., 2009). Together, these results suggest that spaceflight may influence the left/right
asymmetries in the C. elegans nervous system and potentially affect side-specific neuronal functions.
Exopher production is a relatively rare event under normal conditions (i.e., without a proteotoxic stress),
and the majority of exophers are produced on adult days 2 and 3, followed by a reduction in abundance
during midlife (Arnold et al., 2020;Melentijevic et al., 2017). In this study, we quantified the number of exo-
phers present in middle-aged animals, a time window several days past the peak in exopher-genesis, and
therefore, we cannot definitively draw comparisons to Earth samples regarding the effect of spaceflight on
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exopher production. We can, however, conclude that spaceflight did not induce any exopher-clearance
problems in animals with GFP-labeled touch receptor neurons since we observed no abnormal accumula-
tion of GFP fluorescence in middle-aged C. elegans.
Debris originating in neurons accumulates in surrounding tissues of space-flown animals in
the presence of a proteotoxic stress
In the presence of a neuronal proteotoxic stress (i.e., in a line overexpressing mCherry in touch receptor
neurons), we observed a surprising accumulation of mCherry fluorescence in the hypodermis (and other
tissues) of space-flown animals. Given that mCherry is expressed exclusively in the touch receptor neurons,
this fluorescence accumulation in the hypodermis strongly supports that mCherry extruded from touch re-
ceptor neurons by exophers and/or other processes is not efficiently cleared in the surrounding hypoder-
maltissueinspace-flownanimals.
A major question that arises from our results is whether spaceflight increases the extrusion of neuronal trash
in proteotoxic conditions or decreases the degradative/clearance ability of non-neuronal neighboring tis-
sues (or a combination of both). We found a modest increase in the number of mCherry foci in the soma of
certain touch receptor neurons exposed to the ISS environment, suggesting that neuronal protein aggre-
gation may be enhanced by spaceflight. This, in turn, could increase the extrusion of neuronal trash (e.g.
aggregates) through exophers. We did not identify more exophers in space-flown, mCherry-expressing
touch receptor neurons when compared to ground control neurons, but, as discussed above, our quanti-
fication occurred in middle-aged animals rather than at the peak of exopher-genesis (adult days 2 and 3).
Additionally, given that exopher identification is based on the presence of large fluorescent vesicles nearby
the neuron cell body, we likely missed several exophers in space-flown P
mec-4
mCherry1 animals due to
masking by all the extra fluorescence present. Regardless of a putative increase in neuronal trash extrusion
by spaceflight, the remarkable mCherry accumulation throughout the body of over 60% of the space-flown
animals indicates to us that the transcellular degradation/management of neuronal trash is severely
compromised in the ISS environment.
In mammals, glial cells play a vital role in the central nervous system in the elimination of waste proteins and
metabolites produced by neurons (Jessen et al., 2015;Lim and Yue, 2015;Weber and Barros, 2015). These
non-neuronal cells maintain homeostasis in the brain, and their dysregulation can contribute to the devel-
opment of neurodegenerative diseases, including Alzheimer disease. In fact, microglia and astrocytes play
critical roles in the regulation of amyloid-beta(Ab) clearance and degradation (Ries and Sastre, 2016). In the
case of C. elegans, touch receptor neurons are fully surrounded by hypodermal tissue, and therefore, the
hypodermis assumes an analogous function to mammalian glia regarding the clearance of neuronal waste.
Even though we were unable to definitively identify the hypodermal structures in which mCherry fluores-
cence accumulates in response to spaceflight, it is striking that the large rounded fluorescent structures
(sVesicles) resemble enlarged hypodermal lysosomes found in animals defective in lysosomal function
and assembly (Li et al., 2016;Liu et al., 2018;Wang et al., 2019). Moreover, the small fluorescent particles
(Starry Night) have been previously described as neuron-derived mCherry that becomes dispersed in the
hypodermal lysosomal network (Arnold et al., 2020;Melentijevic et al., 2017). Thus, our results suggest that
transcellular management of neuronal waste by the hypodermis is defective in space-flown animals, lead-
ing to accumulation of impaired degradative organelles such as lysosomes. Our observations also suggest
that the inability to efficiently clear neuronal trash is associated with the aging process given the absence of
fluorescence accumulation in space-flown young adult animals that were escapers in our study.
On Earth, mCherry extruded from neurons that cannot be digested by the hypodermis is re-released by the
hypodermis into the C. elegans pseudocoelom and taken up for degradation by scavenger cells called coe-
lomocytes (Arnold et al., 2020;Melentijevic et al., 2017). Interestingly, despite the accumulation of mCherry
throughout the body of space-flown animals, we rarely found mCherry fluorescence in coelomocytes, sug-
gesting a defect in hypodermal re-release or an uptake problem in coelomocytes. Alternatively, a distinct
debris uptake pathway in other tissues like intestine might be induced in response to spaceflight, so that
extruded material is handled differently from neuronal waste on Earth.
Autophagy is a conserved lysosomal-dependent degradative pathway that is critical for cellular homeosta-
sis and has been shown to increase in response to spaceflight or simulated microgravity in multiple cell
types, such as muscle (Ryu et al., 2014), bone (Sambandam et al., 2014), liver (Blaber et al., 2017), kidney
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(Ryu et al., 2014), endothelial (Locatelli et al., 2020), and cancer (Ferranti et al., 2014;Fukazawa et al., 2019;
Jeong et al., 2018) cells. However, to the best of our knowledge, nothing is known about the effect of space-
flight on autophagy or other degradative pathways in glial cells. Our results may even be consistent with an
increase in autophagy markers in the hypodermis of space-flown animals, but the final steps of degradation
seem to stall at some point leading to the accumulation of waste-filled vesicles. Future missions should
address the impact of spaceflight on glial clearance pathways, particularly in the presence of proteostres-
sors such as neurodegenerative disease proteins tau, Ab, huntingtin, or alpha-synuclein.
What are the consequences of spaceflight to neuronal function?
In this study, we show that spaceflight can promote significant morphologi cal changes in adult neurons and
affect the clearance of neuronal trash from the surrounding tissues. Despite microgravity being the major
difference experienced by space-flown animals compared to ground control animals, we cannot exclude
other environmental factors (e.g. radiation) as partially responsible for the observed phenotypes. The
impact of these changes to neuronal function remains in question; however, our results raise the intriguing
possibility that long-duration spaceflights may affect brain function due to neuronal morphological remod-
eling and deficits in waste degradation. Interestingly, the recent National Aeronautics and Space Admin-
istration (NASA) twins study found that the twin astronaut that spent 1 year onboard the ISS exhibited post-
flight decline in cognitive performance when compared to his identical brother who remained on Earth
(Garrett-Bakelman et al., 2019). Future missions will need to carefully address the impact of spaceflight
on overall brain structure and function to properly assess the risks of long-duration missions planned for
the medium future to take humans to Mars and beyond.
Limitations of the study
Our study reveals how spaceflight canaffectadultneuronalmorphologyandtheresponsetoextruded
neuronal waste. However, we were unable to mechanistically dissect these phenotypes given the inability
to send new samples to the ISS on a regular basis. Future missions will be essential to clarify how consistent
these adaptations to spaceflight are and to further explore the molecular, cellular, and tissue responses to
spaceflight. In addition, even though C. elegans provides unique opportunities to identify in vivo single-
neuron responses to spaceflight, future studies in mammalian models and ultimately in humans will be
required to assess the risks of long-duration spaceflight and to develop countermeasures for safe space
exploration.
Resource availability
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by
the lead contact, Ricardo Laranjeiro (ricardo_laranjeiro@hotmail.com).
Material availability
All unique strains generated in this study are available from the lead contact without restriction.
Data and code availability
No large-scale data sets or codes were generated or analyzed in this study. We deposited detailed tem-
perature recordings from spaceflight and control samples in Harvard Dataverse: https://doi.org/10.
7910/DVN/ATOJCJ. The raw data supporting the current study are available from the lead contact upon
request.
METHODS
AllmethodscanbefoundintheaccompanyingTransparent methods supplemental file.
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102105.
ACKNOWLEDGMENTS
We thank NASA’s Cold Stowage team (payload loading), the Biotechnology Space Support Center
(BIOTESC; control of payload operations), and the crew of Expedition 57 (Alexander Gerst, Serena
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Aun
˜o
´n-Chancellor, and Sergey Prokopyev; payload handling). R.L., G.H., and M.D. thank Cousin Tina Lara
for accommodations. We thank Sander van den Heuvel (Utrecht University) for permission to use the
C. elegans microscopy image in the graphical abstract and Beata Edyta Mierzwa (www.BeataScienceArt.
com) for permission to use the Molecular Muscle Experiment mission logo in the Graphical Abstract. We
thank the UK Space Agency, the European Space Agency (ESA), the Japan Aerospace Exploration Agency
(JAXA), and the National Aeronautics and Space Administration (NASA) for their support of the Molecular
Muscle Experiment. This work was supported by the Biotechnology and Biological Sciences Research
Council (BBSRC United Kingdom) (Grant Nos. BB/N015894/1) and the European Space Agency (ESA) (des-
ignations Molecular Muscle Experiment and ESA-14-ISLRA_Prop-0029). S.A.V. acknowledges support from
the National Aeronautics and Space Administration (NASA) (Grant No. NNX15AL16G).
AUTHOR CONTRIBUTIONS
S.A.V., T.E., and N.J.S. collaborated on the Molecular Muscle Experiment team who organized the mission
project. R.L., T.E., N.J.S., and M.D. conceived and designed the experiments. R.L., G.H., A.K.P., C.J.G.,
C.S.D., T.E., and N.J.S. prepared samples for spaceflight and ground control experiments. R.L. and G.H.
scored samples and analyzed data. R.L. and M.D. wrote the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: October 29, 2020
Revised: December 17, 2020
Accepted: January 21, 2021
Published: February 19, 2021
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16 iScience 24, 102105, February 19, 2021
iScienc
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Article
iScience, Volume 24
Supplemental Information
Spaceflight affects neuronal morphology
and alters transcellular degradation
of neuronal debris in adult Caenorhabditis elegans
Ricardo Laranjeiro, Girish Harinath, Amelia K. Pollard, Christopher J. Gaffney, Colleen S.
Deane, Siva A. Vanapalli, Timothy Etheridge, Nathaniel J. Szewczyk, and Monica Driscoll
Figure S1. Spaceflight modulates the size of high-intensity GFP fluorescent puncta in touch
receptor neurons. Related to Figure 4. (A) Number of GFP puncta per cell body in each of the six touch
receptor neurons in ground control and spaceflight Pmec-4GFP animals. We include a confocal image from
a Pmec-4GFP animal showing GFP puncta in an ALML cell body. Number of touch receptor neurons used
for analysis: nControl = 54-61, nSpace = 60-64. We determined statistical significance by unpaired two-tailed
Student’s t test. (B) Proportion of large GFP puncta per cell body in each of the six touch receptor
neurons in ground control and spaceflight Pmec-4GFP animals. We scored GFP puncta as ‘large’ when
diameter ≥ 0.9 µm. Number of touch receptor neurons used for analysis: nControl = 54-61, nSpace = 60-64.
We determined statistical significance by Fisher’s exact test. *P ≤ 0.05.
Figure S2. Spaceflight modulates the number of mCherry foci in touch receptor neurons. Related
to Figure 5. (A) Number of mCherry foci per cell body in each of the six touch receptor neurons in ground
control and spaceflight Pmec-4mCherry1 animals. We include a confocal image from a Pmec-4mCherry1
animal showing mCherry foci in a PLMR cell body. Number of touch receptor neurons used for analysis:
nControl = 42-61, nSpace = 42-66. We determined statistical significance by unpaired two-tailed Student’s t
test. (B) Proportion of large mCherry foci per cell body in each of the six touch receptor neurons in ground
control and spaceflight Pmec-4mCherry1 animals. We scored mCherry foci as ‘large’ when diameter ≥ 0.9
µm. Number of touch receptor neurons used for analysis: nControl = 42-61, nSpace = 42-66. We determined
statistical significance by Fisher’s exact test. (C) Number of exophers per each of the six touch receptor
neurons in ground control and spaceflight Pmec-4mCherry1 animals. We include a confocal image from a
Pmec-4mCherry1 animal showing an ALML cell body extruding an exopher. *P ≤ 0.05.
Figure S3. Spaceflight leads to accumulation of neuronal-derived mCherry throughout the body of
middle-aged nematodes. Related to Figure 5. Additional representative confocal images of spaceflight
Pmec-4mCherry1 animals. Scale bars, 10 µm.
Figure S4. Accumulation of neuronal-derived mCherry throughout the body of space-flown
animals appears to be age-dependent. Related to Figure 6. (A, B) Proportion of ground control,
spaceflight, and young spaceflight Pmec-4mCherry1 animals with/without Starry Night (A) and with/without
sVesicles (B). Note that we obtained young adults from the same spaceflight bag as the middle-aged
adults given that some progeny were able to escape FUdR inhibition to develop in the presence of FUdR.
We easily identified the spaceflight young adults from the middle-aged adults by their reduced size and
we randomly selected 50 young adults for scoring. Number of animals used for analysis: nControl = 72,
nSpace = 83, nSpace (young animals) = 50. We determined statistical significance by Fisher’s exact test.
****P ≤ 0.0001.
Transparent Methods
C. elegans strains and maintenance
The C. elegans strains used in this study were: MF190 hmIs4[des-2::GFP + rol-6(su1006)]
(Oren-Suissa et al., 2010), ZB4510 zdIs5[Pmec-4GFP + lin-15(+)] I, and ZB4065 bzIs166[Pmec-
4mCherry1] (Melentijevic et al., 2017). ZB4510 was generated by outcrossing SK4005
zdIs5[Pmec-4GFP + lin-15(+)] I (Clark and Chiu, 2003) to N2 five times. Approximately two weeks
prior to the launch procedure, we transferred animals from NGM agar plates seeded with live
Escherichia coli OP50-1 to a liquid culture of S-Basal with freeze-dried Escherichia coli OP50
(LabTIE, 1 vial per 250 mL of S-Basal). After this point, we maintained all C. elegans strains at
20°C in the liquid culture in 75 cm2 cell culture flasks (Pollard et al., 2020).
Sample preparation for spaceflight
We synchronized C. elegans populations by bleaching (20% bleach in 2N NaOH) to obtain eggs
or by gravity settling (Gaffney et al., 2014) to obtain L1 larvae. When the synchronized animals
reached L4/young adult stage, we moved approximately 300 animals per strain to 6-well tissue
culture dishes in 1.2 mL of S-Basal with freeze-dried OP50 and 400 µM FUdR. After an
overnight incubation at 20°C, we added 5.3 mL of S-Basal with freeze-dried OP50 and 400 µM
FUdR to each well and loaded the entire volume (6.5 mL) into PE flight culture bags (Pollard et
al., 2020) on December 2, 2018. Culture bags were then placed into experiment cassettes
(ECs, Kayser Italia) (Pollard et al., 2020) to provide an additional level of containment and to
protect the C. elegans cultures from launch and/or in-flight damage. Our samples were then
kept in cold stowage (8-13°C) until they reached the ISS. Launch of SpaceX CRS-16 occurred
on December 5, 2018 and docking to the ISS occurred on December 8, 2018. On December 9,
2018, our C. elegans samples were transferred to 20°C for five days on the ISS. After the five
days, samples were transferred to −80°C and kept frozen until return to Earth.
For ground control samples, we performed the same synchronization protocol as for spaceflight
samples and we loaded C. elegans into the PE flight culture bags on December 5, 2018. We
also placed ground control bags into ECs and exposed them to the same time
frame/temperatures as spaceflight samples but we maintained cultures always on Earth.
Temperature conditions experienced by spaceflight samples were recorded by iButton digital
thermometers (iButtonLink) placed inside ECs and allowed us to expose ground control
samples to similar conditions. We deposited detailed temperature recordings from spaceflight
and control samples in the Harvard Dataverse repository
(https://doi.org/10.7910/DVN/ATOJCJ).
Confocal microscopy
We stored the frozen PE flight culture bags at −80°C until we were ready to start the confocal
imaging. We thawed small fragments of each culture bag in 2% PFA/M9 buffer for
approximately 30 min at room temperature. Based on previous testing, we determined that
thawing C. elegans while simultaneously fixing them in PFA was essential to maintain a strong
fluorescence signal in the reporter strains used in this study. After thaw/fixation, we carefully
selected, under a stereo microscope, the middle-aged adults present in the sample, which we
could easily identify from young animals by their large size. We stored animals in M9 buffer at
4°C until imaging. We performed confocal imaging with an X-Light V2 TP spinning disk unit
(CrestOptics) mounted to an Axio Observer.Z1 microscope (ZEISS) using MetaMorph Premier
software (Molecular Devices). We scored all middle-aged adults that we recovered from the
frozen culture bags, except to score the number of beads/bubbles in PVD dendrites and the
location of Starry Night and sVesicles in the anterior-posterior body axis and in different tissues,
for which we randomly selected a subgroup of middle-aged animals. For PVD neurons, we first
acquired a series of z-stack images from the region anterior to the PVD soma and then obtained
the maximum intensity projection image using ImageJ, in which we scored PVD dendritic
morphologies. For touch receptor neurons, we scored phenotypes directly on the confocal
microscope and we took representative images and z-stack series.
Whole-mount LMP-1 antibody staining
After thawing/fixing Pmec-4mCherry1 animals in 2% PFA/M9 buffer for 30 min at room
temperature, we tried different permeabilization methods prior to anti-LMP-1 antibody
incubation. We permeabilized animals in 1% Triton X-100/M9 buffer for 45 min, in 1% glacial
acetic acid/ethanol for 30 min, or at −80°C in methanol for 1 hour then in acetone for 30 min
followed by a serial rehydration at room temperature in 75%, 50%, 25%, and 0% methanol in
TBS (100mM NaCl, 50mM Tris-HCl, pH7.5) (Djeddi et al., 2015). We washed samples with
0.1% Tween-20/M9 buffer followed by blocking in 2% BSA/0.1% Tween-20/M9 buffer for 1 hour
at room temperature. We incubated samples with anti-LMP-1 antibody (1D4B, Developmental
Studies Hybridoma Bank, 1:5 or 1:10 dilution of supernatant) (Hadwiger et al., 2010) in blocking
solution overnight at 4°C. After several washes with 0.1% Tween-20/M9 buffer, we incubated
samples with 1:1000 or 1:2000 secondary antibody Goat anti-Mouse, Alexa Fluor 488
(Invitrogen) in blocking solution at room temperature. After washing thoroughly with 0.1%
Tween-20/M9 buffer, we imaged samples in the confocal microscope. Despite the different
permeabilization methods and antibody concentrations tested, we were unable to observe
specific LMP-1 staining in Pmec-4mCherry1 animals.
Statistical analyses
The data in this study are presented as the mean ± standard error of the mean (SEM). The
specific number of data points and the test used for each statistical analysis are presented in
the figure legends.
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