Systemic GDF11 attenuates depression-like phenotype in aged mice via stimulation of neuronal autophagy

Abstract

Cognitive decline and mood disorders increase in frequency with age. Many efforts are focused on the identification of molecules and pathways to treat these conditions. Here, we demonstrate that systemic administration of growth differentiation factor 11 (GDF11) in aged mice improves memory and alleviates senescence and depression-like symptoms in a neurogenesis-independent manner. Mechanistically, GDF11 acts directly on hippocampal neurons to enhance neuronal activity via stimulation of autophagy. Transcriptomic and biochemical analyses of these neurons reveal that GDF11 reduces the activity of mammalian target of rapamycin (mTOR), a master regulator of autophagy. Using a murine model of corticosterone-induced depression-like phenotype, we also show that GDF11 attenuates the depressive-like behavior of young mice. Analysis of sera from young adults with major depressive disorder (MDD) reveals reduced GDF11 levels. These findings identify mechanistic pathways related to GDF11 action in the brain and uncover an unknown role for GDF11 as an antidepressant candidate and biomarker.

Full text

Nature Aging | Volume 3 | February 2023 | 213–228 213
nature aging
https://doi.org/10.1038/s43587-022-00352-3
Article
Systemic GDF11 attenuates depression-like
phenotype in aged mice via stimulation of
neuronal autophagy
Carine Moigneu1,9, Soumia Abdellaoui1,8,9, Mariana Ramos-Brossier 2,
Bianca Pfaffenseller3, Bianca Wollenhaupt-Aguiar3,
Taiane de Azevedo Cardoso3, Aurélie Chiche4, Nicolas Kuperwasser2,
Ricardo Azevedo da Silva5, Fernanda Pedrotti Moreira 5, Han Li4,
Franck Oury 2, Flávio Kapczinski3,6,7, Pierre-Marie Lledo 1,10 &
Lida Katsimpardi 1,8,10
Cognitive decline and mood disorders increase in frequency with age. Many
eorts are focused on the identication of molecules and pathways to treat
these conditions. Here, we demonstrate that systemic administration of
growth dierentiation factor 11 (GDF11) in aged mice improves memory
and alleviates senescence and depression-like symptoms in a neurogenesis-
independent manner. Mechanistically, GDF11 acts directly on hippocampal
neurons to enhance neuronal activity via stimulation of autophagy.
Transcriptomic and biochemical analyses of these neurons reveal that GDF11
reduces the activity of mammalian target of rapamycin (mTOR), a master
regulator of autophagy. Using a murine model of corticosterone-induced
depression-like phenotype, we also show that GDF11 attenuates the depressive-
like behavior of young mice. Analysis of sera from young adults with major
depressive disorder (MDD) reveals reduced GDF11 levels. These ndings
identify mechanistic pathways related to GDF11 action in the brain and uncover
an unknown role for GDF11 as an antidepressant candidate and biomarker.
Aging is often accompanied by severe cognitive impairments, memory
loss and age-related depression1. Independently of age, MDD affects
around 20% of the population and correlates with short-term and work-
ing memory deficits, which exacerbate the effects of aging in some
older adults2. Depression can resemble a state of accelerated aging as
depressed individuals often exhibit a higher incidence of age-associated
diseases, including Alzheimer’s and other neurodegenerative diseases
3
.
The hippocampus plays a key role in the regulation of memory and
depression. Small hippocampal volumes in humans have been associ-
ated with MDD, other psychiatric disorders and memory impairments,
establishing the hippocampus as a structural link among aging, mood
disorders and memory decline
4
. In animal models, depression-like
behavior is also associated with structural and functional alterations of
the hippocampus and is most often related to impaired neurogenesis5.
Received: 5 February 2021
Accepted: 19 December 2022
Published online: 2 February 2023
Check for updates
1Perception and Memory Lab, Institut Pasteur, Université Paris Cité, CNRS UMR3571, Paris, France. 2Institut Necker Enfants Malades, INSERM UMR-S1151,
Université Paris Cité, Paris, France. 3Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, ON, Canada.
4Cellular Plasticity in Age-Related Pathologies Laboratory, Institut Pasteur, Université Paris Cité, CNRS UMR3738, Paris, France. 5Department of Health and
Behavior, Catholic University of Pelotas, Pelotas, Brazil. 6Instituto Nacional de Ciência e Tecnologia Translacional em Medicina (INCT-TM), Porto Alegre,
Brazil. 7Department of Psychiatry, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil. 8Present address: Institut Necker Enfants
Malades, INSERM UMR-S1151, Université Paris Cité, Paris, France. 9These authors contributed equally: Carine Moigneu, Soumia Abdellaoui.
10These authors jointly supervised this work: Pierre-Marie Lledo, Lida Katsimpardi. e-mail: pmlledo@pasteur.fr; lida.katsimpardi@inserm.fr
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Article https://doi.org/10.1038/s43587-022-00352-3
Here, we report that systemic GDF11 treatment in aged mice was
sufficient to prevent memory decline and depression-like behavior,
enhance hippocampal neurogenesis and autophagy and reduce hip-
pocampal senescence. Mechanistically, GDF11 stimulated neuronal
autophagy and inhibited the mTOR pathway in a neurogenesis-inde-
pendent manner. Using the corticosterone (CORT)-induced murine
model of depression-like behavior, we demonstrated that GDF11 inhib-
ited a depression-like phenotype in young mice. Finally, we revealed
that GDF11 levels were decreased in young adults with MDD or present-
ing a current depressive episode, making it a potential biomarker of
depression and a possible agent for therapeutic interventions.
Results
Systemic GDF11 administration restores memory decline and
attenuates the depression-like phenotype
We treated 22-month-old C57BL/6JRj male mice with rGDF11 via daily
intraperitoneal (i.p.) injections at a dose of 1 mg kg
−1
, as previously
described
24
. Aged-matched controls and young (3-month-old) mice
were injected with saline. Mice were injected over the course of 3
weeks, and behavioral tests were performed on the third week (Fig. 1a).
Assessing memory using the novel object recognition test (NORT)
At the cellular level, hippocampal neurogenesis is crucial for mem-
ory formation and cognitive function, whereas impaired adult neu-
rogenesis has been linked to memory decline and depression-like
phenotypes6,7.
Infusion of youthful blood factors or blocking proaging factors
has been shown to attenuate age-related impairments in neurogenesis,
memory decline and olfactory perception8–13. One of these blood fac-
tors is growth differentiation factor 11 (GDF11), a member of the trans-
forming growth factor beta (TGF-β) superfamily, which has a critical
role in embryonic development as a key regulator of patterning and
formation of several tissues
14–19
. The role of GDF11 in the developing
central nervous system regulates the progression of neurogenesis
as well as differentiation of neural subtypes
19–22
. In adults, GDF11 was
recently shown to negatively regulate neurogenesis
23
. Because cir-
culating levels of GDF11 decline with age
24,25
, supplementation with
recombinant GDF11 (rGDF11) in aged mice was previously shown to
rejuvenate neurogenesis in both the subventricular zone and hip-
pocampal dentate gyrus (DG) and improve cerebral vasculature8,26.
Despite increasing reports on the various effects of GDF11, it remains
unknown whether GDF11 improves cognitive impairments, as well as
its precise mechanism of action in the brain.
- Memory
- Depression and anxiety
- Physical performance
- General well-being
a
Aged mice
Week 1
GDF11 or vehicle
daily i.p. injections
bNORT
Week 2 Week 3
NOLT
Novel location
investigation time, %
Y maze
c d
0
20
40
60
80
100
Young
Aged
Aged +
GDF11
Young
Aged
Aged +
GDF11
Young
Aged
Aged +
GDF11
Discrimination index, %
P = 0.0014
P = 0.0012
0
20
40
60
80
P = 0.0370
P = 0.0216
0
20
40
60
80
100 P = 0.0202
P = 0.0177
0
5
10
15
Grooming frequency
P = 0.0002
P = 0.0005
Week 4
e f
Splash test TST Sucrose preference Active avoidance
Time spent avoidinng intruder (s)
Sucrose preference index
Immobility (s)
g h
Behavioral tests
0
50
100
150
200
250
P < 0.0001
P < 0.0001
P = 0.0021
0
20
40
60
80
100
P = 0.0046
P = 0.0056
0
2
4
6
8
10 P < 0.0001
P < 0.0001
Novel arm
investigation time, %
Young
Aged
Aged +
GDF11
Young
Aged
Aged +
GDF11
Young
Aged
Aged +
GDF11
Young
Aged
Aged +
GDF11
Fig. 1 | Systemic GDF11 administration restores memory decline and
depression-like phenotype in aged mice. a, Schematic representation of the
experimental procedure. b, Measurement of discrimination index during the
NORT (percentage of time spent to observe the novel object divided by the total
investigation time for both objects) (nYoung = 10 mice, nAged = 9 mice, nAged+GDF11 = 8
mice; F (2, 24) = 10.9). c, Measurement of percent investigation time of the novel
location during the novel object location test (NOLT) (percentage of time spent
to observe the novel location of the object divided by the total investigation
time for both objects) (nYoung = 10 mice, nAged = 11 mice, nAged+GDF11 = 12 mice;
F (2, 30) = 5.5). d, Measurement of percent investigation time of the novel arm
during the Y-maze test (percentage of time spent to observe the novel arm
divided by the total investigation time for both arms) (nYoung = 10 mice, nAged = 12
mice, nAged+GDF11 = 11 mice; F (2, 30) = 5). e, Measurement of grooming frequency
during the Splash test (nYoung = 7 mice, nAged = 7 mice, nAged+GDF11 = 8 mice;
F (2, 19) = 15.3). f, Measurement of immobility time during the TST (nYoung = 8
mice, nAged = 8 mice, nAged+GDF11 = 7 mice; F (2, 20) = 45.5). g, Measurement of the
sucrose preference index over two days (volume consumed from the sucrose
bottle divided by the total volume consumed) (nYoung = 10 mice, nAged = 12 mice,
nAged+GDF11 = 12 mice; F (2, 31) = 7.8). h, Measurement of the time spent avoiding
the intruder during the social interaction test (nYoung = 6 mice, nAged = 6 mice,
nAged+GDF11 = 6 mice; F (2, 15) = 33.7). One-way analysis of variance (ANOVA) and
Tukey’s post hoc test for multiple comparisons; F (DFn, DFd) values presented
for each ANOVA statistical analysis; P values <0.05 are represented on the graph;
mean ± standard error of the mean (s.e.m.).
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Article https://doi.org/10.1038/s43587-022-00352-3
AgedAged + GDF11Young
Aged + GDF11
Aged
Young
Aged + GDF11
Aged control
Sox2/DAPI
a
d e
f g
h
Number of SA-βgal+
cells in SGZ/field
SA-βGal
Sox2+ cells/SGL field
b
0
50
100
150
200
250
P
< 0.0001
P = 0.0015
P = 0.0498
Young
Aged
Aged +
GDF11
c
DCX+ cells/SGL field
0
2
4
6
8
10
30
40
50
60
70
80
P < 0.0001
#P = 0.0084
0
10
20
30 P = 0.0025
P = 0.0155
p19 fold change
relative to aged
0
0.5
1.0
1.5 P < 0.0001
P < 0.0001
P = 0.0071
p16 fold change
relative to aged
0
0.5
1.0
1.5
2.0
P < 0.0001
P = 0.0061
Aged Aged + GDF11
Young Aged Aged + GDF11
i
FoxO3a fold change
relative to aged
0
2
4
6
P = 0.0009
P = 0.0043
P < 0.0001
j
Beclin1 fold change
relative to aged
0
1
2
3
4
P < 0.0001
P < 0.0001
k
LC3 II/I fold change
relative to aged
0
0.5
1.0
1.5
2.0 P = 0.0243
45 kDa
60 kDa
97 kDa
45 kDa
18 kDa
16 kDa
45 kDa
Young
Aged
Aged +
GDF11
Young
Aged
Aged +
GDF11
Young
Aged
Aged +
GDF11
Young
Aged
Aged +
GDF11
FoxO3a
Actin
Beclin1
Actin
LC3 I/II
Actin
Young
Aged
Aged +
GDF11
Young
Aged
Aged +
GDF11
Young
Aged
Aged +
GDF11
Fig. 2 | Cellular changes induced by GDF11 treatment in the brains of aged
mice. a, Representative confocal images of the DG of the hippocampus of young,
aged and aged-GDF11 mice immunostained for Sox2 (red) and DAPI (blue). Scale
bar: 100 μm. b, Quantification of Sox2+ NSCs in the SGL of the DG (nYoung =
6 mice, nAged = 7 mice, nAged+GDF11 = 8 mice; F (2, 18) = 21). c, Quantification of DCX+
neuroblasts in the SGL of the DG (nYoung = 8 mice, nAged = 9 mice, nAged+GDF11 =
9 mice; F (2, 23) = 30.3; #P value by Mann–Whitney test between aged and aged
+ GDF11). d, Representative images of SA-βGal staining in the DG of young, aged
and aged-GDF11 mice. Scale bar: 100 μm. e, Quantification of SA-βGal+ cells
in the SGZ of the DG (n = 5 mice per group; F (2, 12) = 10.3). f,g, Real-time qPCR
for hallmarks of senescence showing fold changes in mRNA levels of p16 (f)
(nYoung = 8 mice, nAged = 9 mice, nAged+GDF11 = 4 mice; F (2, 18) = 22.2) and p19 (g)
(nYoung = 8 mice, nAged = 9 mice, nAged+GDF11 = 4 mice; F (2, 18) = 68) in hippocampi
of young, aged and aged-GDF11 mice, relative to aged mice. h, Representative
Western blot images of hippocampal lysates from young, aged and GDF11-treated
aged mice after 9 days of treatment. i–k, Quantification of western blots by
optical intensity for Foxo3a (i) (nYoung = 3 mice, nAged = 3 mice, nAged+GDF11 = 5 mice;
F (2, 8) = 63), Beclin 1 (j) (nYoung = 3 mice, nAged = 6 mice, nAged+GDF11 = 10 mice;
F (2, 16) = 34.9), and LC3 (k) (nYoung = 3 mice, nAged = 3 mice, nAged+GDF11 = 5 mice;
F (2, 8) = 5.7). One-way ANOVA and Tukey’s post hoc test for multiple
comparisons; F (DFn, DFd) values presented for each ANOVA statistical analysis;
P values < 0.05 are represented on the graph; mean ± s.e.m.
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Article https://doi.org/10.1038/s43587-022-00352-3
showed that aged mice were unable to discriminate the novel object and
scored significantly lower than young mice (aged, 48.2% ± 5.5; young,
68.2 ± 1.7; Fig. 1b). On the contrary, GDF11-treated aged mice presented
a significant 44.2% increase in the novel object discrimination index
(GDF11 = 69.6 ± 3%; aged, 48.2% ± 5.5; Fig. 1b). Hippocampus-dependent
spatial memory was specifically assessed with the novel object location
test, where aged mice spent significantly less time investigating the
novel location compared to both young and aged GDF11-treated mice
(aged, 42 ± 3.3%; young = 58.2 ± 2.2%; GDF11 = 57.8 ± 5%; Fig. 1c). In the
Y-maze test (used to assess recognition memory), young and GDF11-
treated aged mice spent significantly more time investigating the novel
arm of the maze than aged mice (aged, 24.4 ± 2.7%; young, 36.1 ± 2.9%;
GDF11 = 36.9 ± 3.7%), confirming the effect of GDF11 in improving age-
related memory impairments (Fig. 1d).
Subsequently, young and aged mice were subjected to a series
of behavioral tests aimed at assessing the level of a depression-like
phenotype, often associated with advanced aging. During the splash
test, aged mice exhibited a significant 2.8-fold decrease in grooming
frequency compared to young mice (aged, 3 ± 0.7; young, 8.4 ± 0.8),
confirming the known effect of aging on depression27 (Fig. 1e). On
the contrary, aged GDF11-treated mice showed a significant 2.5-fold
increase in grooming frequency compared to aged mice (Fig. 1e).
The tail suspension test (TST), which measures despair-like behavior,
showed that aged mice remained immobile twice as long as young
mice (aged, 180 ± 7.3 s; young, 90 ± 7.6 s) and 1.5-times longer than
aged GDF11-treated mice (aged, 180 ± 7.3 s; GDF11,142 ± 4.7 s; Fig. 1f).
Overall, the score of GDF11-treated mice suggests improved symptoms
of depression-like phenotype. Next, we tested anhedonia, another
trait of depression-like phenotype, with the sucrose preference test
and found that aged mice showed significantly decreased preference
for the sucrose solution compared to young (aged, 73.2 ± 4.5; young,
87.9 ± 1.2), but GDF11 treatment in aged mice restored this preference
(GDF11, 86.9 ± 1.2; aged, 73.2 ± 4.5; Fig. 1g). Finally, the measurement
of intruder avoidance during the social interaction test revealed that
both young and aged GDF11-treated mice spent less time avoiding the
intruder than aged mice (aged, 5.6 ± 0.9 s; GDF11, 0.2 ± 0.2 s; young,
0.1 ± 0.1 s; Fig. 1h).
Given the above results, we next asked whether GDF11 would also
influence anxiety-like state. Using the light/dark box (LDB) test, we
found that young mice covered a significantly longer distance (young,
726 ± 27 cm; aged, 532 ± 38 cm) and spent more time (young, 252 ± 11.4 s;
aged, 191.9 ± 14.5 s) in the light box than aged mice (Extended Data
Fig. 1a,b). However, GDF11 treatment had no effect compared to con-
trol in aged-matched mice (distance: GDF11, 445.7 ± 46.2 cm; aged,
531.9 ± 37.9 cm; time: GDF11,158.6 ± 19.3 s; aged, 191.9 ± 14.5 s; Extended
Data Fig. 1a,b). Next, the elevated plus maze (EPM) test, for anxiety-like
phenotype, revealed no difference between aged control and aged
GDF11-treated mice in the duration/time spent in open arms (GDF11,
185.5 ± 34.6 s; aged, 139.4 ± 25 s), despite the trend of a subgroup of
GDF11-treated mice exceeding the mean duration (Extended Data Fig.
1c). Collectively, these results show that GDF11 has a distinct effect on
the depression-like phenotype.
To examine whether GDF11 has a broader impact on behavior, we
performed additional assays targeting general well-being and physical
performance. First, we measured nest building as a general index of
well-being by assessing nest height and building scores. Overall, the
mice showed the same capacities for nest building (Extended Data Fig.
1d,e). Next, we evaluated burrowing behavior and found that all mice
behaved similarly (Extended Data Fig. 1f). Using the open field test to
assess physical performance, we measured the total distance traveled
and found no difference between GDF11 or vehicle treatment in aged
mice (Extended Data Fig. 1g). We assessed strength and endurance with
the hanging wire test and saw no difference regardless of age or treat-
ment (Extended Data Fig. 1h). Then, we performed the gait test to evalu-
ate possible locomotion problems. Once again, we found no significant
difference between the groups. These results demonstrate that GDF11
in aged mice has no effect on physical performance. Therefore, the
vast panel of behavioral tests demonstrates that GDF11 administration
specifically improves memory and depression-like phenotype.
Cellular changes induced by GDF11 treatment
We next sought to identify the cellular mechanisms underlying GDF11
treatment by analyzing the brains of young and aged mice at 9 days of
treatment (the earliest time point where we previously saw increased
olfactory neurogenesis), for hallmarks of aging, including neurogenesis
decline, increased senescence and autophagy impairments.
We measured levels of neurogenesis by assessing the levels of
Sox2 and doublecortin (DCX). We found a 35% significant increase in
Sox2+ NSCs in the subgranular layer (SGL) of the aged GDF11-treated
DG compared to aged mice (Fig. 2a,b), and analysis of DCX revealed a
53% increase in the number of immature neuroblasts in the neurogenic
SGL of GDF11-treated aged mice compared to aged controls (Fig. 2c and
Extended Data Fig. 2a).
Next, we examined hippocampal senescence by quantifying
SA-βGal
+
cells in the SGZ of the DG. We found a significant twofold
increase in the aged SGZ compared to the young (aged, 19.2 ± 1.7; young,
9.6 ± 1.2), but GDF11 treatment resulted in a decrease in the number
of SA-βGal+ cells (GDF11, 11.8 ± 1.6) (Fig. 2d,e). Moreover, we quanti-
fied the expression of Cdkn2a (cyclin-dependent kinase inhibitor 2 A)
locus transcripts encoding p16INK4A and p19ARF, critical senescence
mediators. Consistent with previous reports28, we observed a tenfold
increase in p16INK4A (Fig. 2f) and a fivefold increase in p19ARF expression
(Fig. 2g) with aging. Remarkably, p16
INK4A
and p19
ARF
expression were
significantly reduced by 70% (Fig. 2f) and 30% (Fig. 2g), respectively,
in GDF11-treated aged mice.
Subsequently, we analyzed the expression of key proteins involved
in autophagy, the impairment of which has been linked to cognitive
decline29. Western blot analyses of hippocampal tissues revealed sig-
nificant decreases with aging in the levels of the following proteins
(71% for FoxO3a, 70% for Beclin 1 and 23% for LC3 lipidation (Fig. 2i–k)
respectively). Interestingly, aged mice treated systemically with GDF11
showed a significant upregulation of all the above proteins, including a
significant 5.2-fold increase in FoxO3a (Fig. 2h,i), a significant 2.5-fold
increase in Beclin 1 (Fig. 2h,j) and a significant 1.4-fold increase in LC3
lipidation (Fig. 2h,k).
Together, these analyses demonstrate that systemic GDF11 treat-
ment profoundly affects cellular events in the aged brain.
Brain infusion of GDF11 recapitulates the effects of systemic
administration in a neurogenesis-independent fashion
To examine the direct effect of GDF11 on NSCs and neurons, we per-
formed brain infusions using intracerebroventricularly (ICV) implanted
cannulas connected to miniosmotic pumps into the lateral brain ven-
tricle of 21-month-old C57BL/6JRj male mice. The pumps continuously
delivered rGDF11 (0.3 mg kg
−1
) or vehicle for 2 weeks (Extended Data
Fig. 3a), after which memory, anxiety-like and depression-like pheno-
types were analyzed by behavioral tests. In the NORT, GDF11-infused
aged mice showed a significant 1.8-fold increase in the discrimina-
tion index compared to vehicle-infused mice (GDF11, 89.4 ± 1.9%;
vehicle, 49.3 ± 18.6%; Extended Data Fig. 3b). In the splash test, ICV
GDF11 induced a significant 2.4-fold increase in grooming frequency
compared to control (GDF11, 9.6 ± 1.4; vehicle, 4 ± 1.1; Extended Data
Fig. 3c). Moreover, ICV GDF11-infused mice exhibited a 1.4-fold sig
-
nificant increase in the time spent in the light box (GDF11, 380 ± 22.5 s;
vehicle, 272 ± 29.3; Extended Data Fig. 3d). During the novelty sup-
pressed feeding test, GDF11-infused mice exhibited shorter latency to
eat compared to vehicle-infused mice (Extended Data Fig. 3e). These
findings demonstrate that direct GDF11 infusion into the brain results
in the same behavioral outcomes as systemic injections. Next, we exam-
ined neurogenesis and autophagy. Quantification of Sox2
+
NSCs and
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Nature Aging | Volume 3 | February 2023 | 213–228 217
Article https://doi.org/10.1038/s43587-022-00352-3
DCX
+
neuroblasts in the DG of GDF11- or vehicle-infused aged mice
revealed that direct ICV infusion of GDF11 had no effect on hippocampal
neurogenesis (Extended Data Fig. 3f,g, respectively). Conversely, west-
ern blot analysis of hippocampi showed a significant 1.4-fold increase
in Beclin 1, a significant 2.3-fold increase in Atg5 and a significant 2-fold
increase in Lamp1 (Extended Data Fig. 3h–k, respectively). Thus, direct
infusion of GDF11 in the brain has distinct effects compared to systemic
administration and does not affect neurogenesis.
Hippocampal
neurons
Ctrl cLTP GDF11
GDF11/cLTP
stimulation
a
b
d
cFos
+
neurons/field, %
Number of spines/10 µm
cFos/Hoechst
MAP2/GFAP
e
Beclin1
Actin
p62
Actin
LC3 I/II
f
Actin
pSMAD2/3
GDF11 Control
c
Ctrl cLTP GDF11 Ctrl cLTP GDF11
0
2
4
6
8
10
12
14
P = 0.0010
P = 0.0385
0
2
4
6
8P = 0.0430
P < 0.0001
P = 0.0093
g h
pSMAD2/3
Fold change relative to control
Beclin1
i
LC3 I/II p62
j
0
0.5
1.0
1.5
2.0 P = 0.0286
Ctrl GDF11
0
0.5
1.0
1.5
2.0
2.5 P = 0.0379
Ctrl GDF11
Fold change relative to control
0
0.5
1.0
1.5
2.0
2.5
P = 0.0286
Fold change relative to control
Ctrl GDF11
0
0.5
1.0
1.5
2.0
2.5
P = 0.0286
Ctrl GDF11
Fold change relative to control
GFP
Actin
DIV11
GFP transfection
DIV18
62 kDa
60 kDa
45 kDa
45 kDa
18 kDa
16 kDa
45 kDa
60 kDa
52 kDa
45 kDa
Fig. 3 | GDF11 modulates neuronal activity and enhances autophagy in
hippocampal neurons in vitro. a, Schematic representation of the primary
hippocampal neuronal cultures in vitro. Neurons were transfected with a
GFP plasmid on DIV11 (only for the spine density experiment) and treated
on DIV18. Scale bar: 20 μm. b, Representative confocal images of primary
hippocampal neurons in vitro treated with vehicle (Ctrl), cLTP (positive control)
or rGDF11 (40 ng ml−1) for 2 h on DIV18 and immunostained with cFos (red)
and Hoechst (blue). Scale bar: 100 μm. c, Representative confocal images of
GFP+ hippocampal neurons transfected with GFP (green) on DIV11 and treated
with either vehicle (Ctrl) or cLTP (positive control) or rGDF11 (40 ng ml−1) on
DIV18. Scale bar: 4 μm. d, Quantification of % cFos+ neurons per field (nCtrl = 12
fields, ncLTP = 10 fields, nGDF11 = 13 fields; F (2, 32) = 8.3). e, Quantification of
dendritic spine density after a 2-h stimulation with either GDF11 or cLTP or
vehicle (nCtrl = 18, ncLTP = 19, nGDF11 = 15 neurons examined over three independent
experiments; F (2, 49) = 14.4). Numbers represent the number of spines for every
10 μm of primary dendrite. f, Western blots images of lysates from hippocampal
neurons treated with GDF11 or control (vehicle). g–j, Quantification of western
blots by optical density for phospho-SMAD2/3 (g) (n = 4 biologically independent
samples per condition), Beclin 1 (h) (n = 8 biologically independent samples
per condition), LC3 (i) (n = 4 biologically independent samples per condition)
and p62 (j) (n = 4 biologically independent samples per condition). One-way
one-sided ANOVA and Tukey’s post hoc test for multiple comparisons; two-sided
Mann–Whitney test for two-group comparisons; F (DFn, DFd) values presented
for each ANOVA statistical analysis; P values <0.05 are represented on the graph;
mean ± s.e.m.
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Treatment of NSCs with rGDF11 does not promote neuronal
differentiation
To further examine the direct effect of GDF11 on NSCs and progenitor
cells, we used primary cultures of neurospheres derived from 2-month-
old C57BL/6JRj male mice
8,30
. Adherent neurospheres were incubated
with rGDF11 (40 ng ml−1), and serum isolated from either young or old
mice was used as a positive or negative control, respectively, as previ-
ously shown
31
. After 4 days of incubation, we measured total neurite
outgrowth, a crucial process of neural stem cell differentiation and
subsequent neuroblast maturation, by analyzing total neuronal pro-
cess extensions. Young serum induced a 1.4-fold significant increase
in neurite length compared to old serum, 1.6-fold compared to rGDF11
and 2.1-fold compared to the serum-free/growth factor-free condition
(young serum, 154.5 ± 10.1; aged serum, 112.2 ± 6.6, GDF11, 96.7 ± 4.6,
control:71.5 ± 3.8; Extended Data Fig. 4a,b). GDF11 treatment had the
same effect as the serum-free/growth factor-free control (Extended
Data Fig. 4a,b), suggesting that GDF11 does not enhance neurogenesis,
in agreement with in vivo results.
Together, the above results indicate a neurogenesis-independent
pathway accounting for the effects of GDF11 on both memory and
depression-like behavior.
GDF11 modulates neuronal activity and enhances autophagy
in hippocampal neurons in vitro
Next, we sought to investigate the direct effect of GDF11 on hippocam-
pal neurons. We established in vitro cultures of primary hippocampal
neurons derived from C57BL/6JRj mouse embryonic day 15.5 (E15.5)
hippocampi. As depicted in Fig. 3a, hippocampal neurons were plated
on day 0 and were allowed to mature for the next 18 days in vitro (DIV).
On DIV18, neurons were stimulated with either rGDF11 (40 ng ml−1),
chemical LTP (cLTP; positive control) or vehicle for 30 min and changes
in neuronal activity were assessed by immunostaining for the expres-
sion of cFos. We found that the number of neurons expressing cFos was
significantly enhanced 11-fold by cLTP and 7.5-fold by GDF11, compared
to control (cLTP, 5.1 ± 1.3%; GDF11, 3.2 ± 0.6%; control, 0.44 ± 0.08%;
Fig. 3b,d). To assess dendritic spine density, another measure of neuronal
activity, the neurons were first transfected with a GFP plasmid on DIV11
to allow subsequent visualization of spines (Fig. 1a). On DIV18, neurons
were stimulated with either GDF11 (40 ng ml
−1
), cLTP or vehicle for 2 h.
cLTP induced a significant increase of spine density by 23% compared
to control, whereas GDF11 induced a 48% significant increase compared
to control and 21% compared to cLTP (cLTP, 4.3 ± 0.2; GDF11, 5.2 ± 0.2;
control, 3.6 ± 0.1; Fig. 3c,e). These results suggest that GDF11 can directly
stimulate neuronal activity in hippocampal neurons, at least in vitro.
Next, we examined whether GDF11 signals via the canonical
SMAD2/3 pathway. On DIV18, the neurons were treated with rGDF11
(40 ng ml−1) or vehicle. Western blot analysis showed enhanced
SMAD2/3 phosphorylation in GDF11-treated neurons (Fig. 3f,g). Fur-
thermore, we measured the levels of Beclin 1, which is activated at
the initiation of the autophagy process; LC3I/II, which allows for the
evaluation of autophagosome formation; and p62. Western blot analy-
sis revealed that a 30-min GDF11 stimulation significantly increased the
expression of Beclin 1 by 1.4-fold (Fig. 3f,h), LC3I/II by 1.7-fold (Fig. 3f,i)
and p62 by 1.9-fold (Fig. 3f,j).
Taken together, our results showed that GDF11 regulates neuronal
activity and its canonical SMAD2/3 signaling pathway and enhances
autophagy in primary hippocampal neurons.
GDF11 stimulation of neuronal activity is mediated by
autophagy
To investigate whether GDF11-induced stimulation of neuronal activity
was mediated through autophagy, we knocked down Beclin 1, which
has been shown to attenuate neuronal function
29
. Beclin 1 was silenced
by transfection with an isopropyl β-D-1-thiogalactopyranoside (IPTG)-
inducible short hairpin RNA (shBec) on DIV11. A GFP plasmid was used
as a marker of co-transfection, and neurons were stained with anti-LacI
to confirm co-transfection with the GFP. On DIV18, transfected neurons
were treated with either rGDF11 (40 ng ml
−1
) or vehicle for 2 h. GDF11
treatment significantly increased spine density by 22.5% compared to
shBec-silenced neurons without treatment (GDF11, 6.0 ± 0.1%; shBec,
4.9 ± 0.2%; Fig. 4a,b). However, GDF11 treatment on shBec-silenced
neurons significantly reduced spine density by 10% compared to non-
silenced neurons (GDF11, 6.0 ± 0.1%; shBec + GDF11, 5.4 ± 0.1%; Fig. 4a,b).
We also perturbed the autophagic process by blocking autophago-
some–lysosome fusion using Bafilomycin A1 (Baf). DIV18 neurons were
treated with Baf (100 nM), GDF11 or both (Baf, 100 nM + GDF11 (40 ng
ml
−1
)) for 2 h. cFos activation was used to evaluate neuronal activity. The
decrease in neuronal autophagy by Baf resulted in a significant reduc-
tion in the number of cFos
+
neurons (control, 5.6 ± 0.4%; Baf, 4 ± 0.5%;),
whereas treatment with GDF11 increased the number of cFos
+
neurons
compared to control (Ctrl, 5.6 ± 0.4%; GDF11, 8 ± 1%; Fig. 4c,d). However,
Baf treatment resulted in an attenuation of GDF11’s effect, as shown by
the decrease in the number of cFos+ neurons in the Baf + GDF11 condi-
tion compared to the GDF11-only condition (GDF11, 8 ± 1%; Baf + GDF11,
4.4 ± 0.3%; Fig. 4c,d). These results indicate that blocking autophagy
interferes with GDF11 action on hippocampal neuron activation.
Because autophagy is tightly linked to the bioenergetic function of
the cell, we examined the energy phenotypes of primary hippocampal
neurons treated with Baf (100 nM) and/or GDF11 (40 ng ml
−1
) using the
Seahorse analyzer. Interestingly, GDF11 treatment induced an increase
in the basal OCR of neurons compared to control (Fig. 4e,f). However,
neurons treated with either Baf alone or in combination with GDF11
showed decreased levels of basal OCR (Fig. 4e,f). These results show
that the metabolic effect of GDF11 on neuron metabolism is abrogated
when autophagy and catabolic functions of lysosomes are blocked
by Baf.
To gain a deeper mechanistic understanding of the signaling path-
ways engaged by GDF11 treatment, we performed bulk RNA sequencing
(RNA-seq) on primary neurons treated with GDF11 or control. Gene set
enrichment analysis (GSEA) revealed that pathways related to mTOR
Fig. 4 | GDF11 stimulation of neuronal activity is mediated by autophagy.
a, Representative confocal images of GFP+ dendrites and spines from
hippocampal neurons in culture transfected with either shBeclin 1 or GFP
(green) and treated with either GDF11 or control (vehicle). Scale bar: 4 μm.
b, Quantification of dendritic spine density after a 2-h stimulation with either
GDF11 or control (number represents number of spines for every 10 μm of
dendrite; nCtrl = 31, nshBec = 18, nGDF11 = 36, nshBec+GDF11 = 35 neurons examined over
three independent experiments; F (3, 116) = 8.2). c, Representative confocal
images of hippocampal neurons in vitro treated with vehicle, Baf or GDF11
or both and immunostained with cFos (green) and Hoechst (blue). Scale
bar: 100 μm. d, Quantification of % cFos+ neurons per field (nCtrl = 31 fields,
nBaf = 28 fields, nGDF11 = 28 fields, nBaf+GDF11 = 32 fields; F (3, 115) = 7.2). e, Oxygen
consumption rate (OCR) in primary hippocampal neurons in vitro treated for
2 h with either GDF11 or Baf or both and measured with the Seahorse analyzer
(nCtrl = 20, nBaf = 8, nGDF11 = 8, nBaf+GDF11 = 8 biologically independent samples).
f, Measurement of basal OCR before oligomycin/FCCP addition (for each
condition the three measurements were pooled; nCtrl = 24, nBaf = 21, nGDF11 = 21,
nBaf+GDF11 = 24 biologically independent samples; F (3, 86) = 5.5). g, GSEA from
RNA-seq on neurons treated with GDF11 or vehicle for 2 h. h, Western blots
images of lysates from hippocampal neurons treated with GDF11 or control
(vehicle). i–k, Quantification of western blots by optical density for Deptor (i)
(n = 4 biologically independent samples), phospho-S6K1 (j) (n = 8 biologically
independent samples) and 4E-BP1 (k) (n = 4 biologically independent
samples). Baf refers to Baf. One-way one-sided ANOVA and Tukey’s post hoc
test for multiple comparisons; F (DFn, DFd) values presented for each ANOVA
statistical analysis; two-sided Mann–Whitney test for two-group comparisons;
P values < 0.05 are represented on the graph; mean ± s.e.m.
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activity were enriched with GDF11 treatment (Fig. 4g), which is highly
relevant, as the mTOR pathway is a major regulator of autophagy32. In
particular, we identified Deptor, an inhibitor of both mTOR complexes33
(Fig. 4g). To confirm the RNA-seq results, we analyzed Deptor protein
levels in primary hippocampal neurons treated with GDF11 (40 ng ml
−1
)
or vehicle, for 2 h. Western blot analysis revealed that Deptor levels were
significantly increased by 2.8-fold in GDF11-treated neurons (Fig. 4h,i).
We next examined two downstream targets of mTOR, S6 Kinase 1 (S6K1)
and 4E-BP1, which are positively and negatively regulated, respectively,
and showed that S6K1 phosphorylation was significantly decreased by
shBec GDF11Ctrl medium shBec + GDF11 b
Baf GDF11 Baf + GDF11
GFP
a
cFos/Hoechst
cCtrl
Ctrl
shBec
GDF11
shBec
+ GDF11
Dendritic spine density
g h
d e
0 20 40 60
0
100
200
300
400
Time (minutes)
OCR (pmol/min)
Oligomycin
FCCP
Basal OCR (pmol/min)
f
Ctrl
GDF11
Baf
Baf + GDF11
cFos+ neurons, %
0
4
8
12
16
P = 0.0003
P = 0.0009
Ctrl
Baf + GDF11
GDF11
Baf
0
100
200
300
400
500 P = 0.0127
P = 0.0015
GDF11 Control
p-S6K1
Fold change relative to control
k4E-BP1
j
0
0.5
1.0
1.5
2.0 P = 0.0002
0
1
2
3
4
5P = 0.0286
Fold change relative to control
iDeptor
Ctrl
GDF11
0
1
2
3
4P = 0.0286
Fold change relative to control
0
2
4
6
8
P = 0.0127
P < 0.0001
P = 0.0071
70 kDa
48 kDa
45 kDa
45 kDa
20 kDa
RNA-seq on neurons
GSEA mTOR pathways
Ctrl
GDF11
Baf
Baf + GDF11
p-S6K1
Deptor
Actin
4E-BP1
Actin
Ctrl
GDF11
Ctrl
GDF11
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70% (Fig. 4h,j) and that 4E-BP1 levels were significantly increased by
2.4-fold by GDF11 treatment (Fig. 4h,k). These findings indicate that
GDF11 treatment reduces mTOR activity in hippocampal neurons,
which could explain the increase in neuronal autophagy.
Systemic GDF11 administration alleviates depressive-like
symptoms in a preclinical murine model of depression
To investigate whether GDF11 could have an antidepressant effect
in mice, we used a well-established preclinical model of depression-
like phenotype based on chronic treatment with CORT
34
. This model
produces behavioral and other alterations that resemble a limited set
of features of human anxiety and depression and has previously been
validated
5
. Young (2-month-old) male C57BL/6NTac male mice were
given CORT in their drinking water (5 mg kg
−1
per day), whereas con-
trol mice received vehicle (10% (2-hydroxypropyl)-beta-cyclodextrin
(β-CD)). After 4 weeks of CORT or vehicle treatment, we divided these
populations into two subgroups. Half of each group received daily
i.p. injections of rGDF11 (1 mg kg−1), and the other half received saline
injections (Fig. 5a), for another 3 weeks. All mice were weighed twice
a week during the 7 weeks of the experiment.
After the second week of treatment, we observed a consistent
weight gain for CORT mice compared to control (Extended Data Fig. 5a),
which has been previously reported as a side effect of water retention
due to CORT treatment34. Interestingly, 1 week after GDF11 injections
in CORT mice, we observed a significant 11% decrease in body weight
(CORT, 33.2 ± 0.5 g; CORT-GDF11, 29.5 ± 0.7 g; Extended Data Fig. 5b,c).
This observation is in accordance with the previously reported role for
GDF11 in inducing weight loss in mice25,35.
During the last 2 weeks of treatment, mice were subjected to
behavioral tests to assess the effect of GDF11 on depression-like and
anxiety-like phenotypes. First, we evaluated time spent in the center
of the open field, which is inversely associated to anxiety-like and
depression-like states (Fig. 5b). CORT mice spent 51% significantly less
time in the center than control mice (Ctrl, 178 ± 22 s; CORT, 87 ± 15 s;
Fig. 5c). Remarkably, CORT-GDF11 mice exhibited a significant 89%
increase in time spent in the center compared to CORT mice (CORT,
87 ± 15 s; CORT-GDF11, 164 ± 18 s; Fig. 5c). No significant change was
observed in the total distance traveled (Extended Data Fig. 5d), suggest-
ing that the alleviation of the anxious-like/depressive-like phenotype
was not due to possible locomotor changes. Next, we analyzed the
anxiety-like state using the LDB test. CORT mice exhibited a signifi-
cant 39% decrease in the distance moved in the light box compared to
control mice (Ctrl, 1015 ± 113 cm; CORT, 614 ± 61 cm; Fig. 5d). On the
contrary, CORT-GDF11 mice exhibited a significant 44% increase com-
pared to CORT mice (CORT, 614 ± 61 cm; CORT-GDF11, 882 ± 72 cm;
Fig. 5d), suggesting a dampening of the anxious-like phenotype. Finally,
we assessed the coat state, a well-validated index of the depressive-like
state
36
. A deterioration of the coat state was observed in CORT mice
compared to control mice as shown by a significant increase in coat
scoring, which was significantly attenuated in CORT-GDF11 mice (Ctrl,
0, CORT: 3.3 ± 0.3; GDF11, 0.1 ± 0.1; CORT-GDF11, 2.1 ± 0.3; Fig. 5e). Our
findings reveal that GDF11 administration in a preclinical model of
depression-like behavior was able to attenuate the depressive-like phe-
notype in young mice, consistent with the above results in aged mice.
Blood levels of GDF11 inversely correlate with MDD in human
subjects
These results prompted us to explore whether blood levels of GDF11
correlate with a related pathology in humans. To address this ques-
tion, we recruited participants with MDD and healthy controls of the
same age and education levels. All participants were subjected to a
psychiatric evaluation using the Mini International Neuropsychiatric
Interview, based on the Diagnostic and Statistical Manual of Mental
Disorders IV diagnostic criteria, and the Montgomery–Åsberg Depres-
sion Rating Scale (MADRS) was used to assess the severity of depres-
sive symptoms (Fig. 6a). Blood was collected from MDD subjects and
healthy age-matched controls. Serum was analyzed by enzyme-linked
immunosorbent assay (ELISA) for the detection of GDF11, as previ-
ously described35. We first confirmed that we could exclusively detect
GDF11 and not GDF8. We included young (mean age of 26) adults with
MDD and healthy controls matched by sex, age, and years of education
(Fig. 6a). Interestingly, serum GDF11 levels were significantly decreased
in the blood of MDD subjects (n = 57), compared to healthy age-matched
controls (n = 51) (median
CONTROL
= 22.69 pg ml
−1
, interquartile range
(IQR): 9.63–76.40), median
MDD
= 11.66 pg ml
−1
(IQR, 3.89–42.10); Fig. 6b).
Light/dark box
Coat score
Coat test
Time spent in center (s)
Open ield test
Distance moved in light box (cm)
Ctrl
GDF11
CORT
CORT-GDF11
a
CORT
Ctrl
CORT-GDF11
GDF11
b
c d e
0
100
200
300
400
P = 0.0270
P = 0.0035
P = 0.0221
0
1
2
3
4
5
P < 0.0001
P = 0.0022
P = 0.0307
0
400
800
1,200
1,600
P = 0.0084
P = 0.0463
1 2 3 4 5 6 7
CORT/Veh in drinking water
GDF11/saline
daily i.p. injections
Young mice
Weeks of treatment
Ctrl
GDF11
CORT
CORT-GDF11
Ctrl
GDF11
CORT
CORT-GDF11
Fig. 5 | Behavioral symptoms of the depressive-like phenotype are alleviated
after treatment with GDF11 in young CORT mice. a, Schematic representation
of the experimental timeline and conditions. Veh, vehicle. b, Representative
traces of the open field test. c, Measurement of the time spent in the center
during the open field test (nCtrl = 8 mice, nCORT = 15 mice, nGDF11 = 8 mice, nCOR T-
GDF11 = 16 mice; F (3, 43) = 5.9). d, Measurement of the distance moved in the light
box during the LDB test (nCtrl = 8 mice, nCORT = 15 mice, nGDF11 = 8 mice, nCORT-GDF11 = 15
mice; F (3, 42) = 4.6). e, Scoring of the coat state on five different areas of the
mouse fur (back, abdomen, tail, forepaws and hindpaws) (nCtrl = 8 mice, nCORT = 14
mice, nGDF11 = 8 mice, nCORT-GDF11 = 16 mice; F (3, 42) = 19.8). One-way ANOVA and
Tukey’s post hoc test for multiple comparisons; F (DFn, DFd) values presented
for each ANOVA statistical analysis; P values < 0.05 are represented on the graph;
mean ± s.e.m.
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Next, we evaluated levels of GDF11 in a larger cohort of young adults
who were presenting a depressive episode. This analysis included 759
young adults (103 in a current depressive episode and 656 controls)
aged between 21 and 32 years old. Sample characteristics and their asso-
ciation with a current major depressive episode are described in Fig. 6c.
Females presented a higher prevalence of a current major depres-
sive episode (P < 0.001), and individuals with a current major depres-
sive episode had less years of education as compared to controls
(P < 0.001). There was no significant difference between the groups
regarding age.
Individuals with a current depressive episode had lower serum
GDF11 levels (18.53 (IQR: 7.37–47.85)) than controls (26.97 (IQR: 11.79–
100.03), P = 0.001). These findings reveal that blood GDF11 levels are
associated with MDD as well as with a current depressive episode in
humans and suggest that GDF11 could be considered as a reliable bio-
marker for MDD in humans.
Discussion
We sought to determine the role of GDF11 in age-related depression-
like phenotype in mice and explore the underlying mechanism. Daily
systemic administration of rGDF11 resulted in an improvement of the
depression-like phenotype associated with aging, as well as reversed
memory decline. Likewise, direct infusion of GDF11 in the brain resulted
in the same behavioral outcome. These results come in accordance with
a positive effect in memory decline related to a pathological phenotype
of Alzheimer’s disease, as reported in AβPP/PS1 double transgenic
mice
37
. It is worth pointing out that the depressive-like phenotype and
memory decline observed in aged mice is not normative. As such, aging
is a risk factor for cognitive decline and depression, but only a subpopu-
lation of individuals will present these symptoms.
We further confirmed the antidepressant role of GDF11 in a pre-
clinical model of induced depressive-like phenotype due to chronic
CORT administration and found that depressive-like symptoms were
alleviated upon systemic GDF11 administration. This antidepressant
effect of GDF11 in both young and old mice highlights an unknown
role for GDF11 in the brain. It will be interesting to further compare
the effects of GDF11 to a known antidepressant, such as fluoxetine, on
possible restoration of neurogenesis and other parameters.
Regarding human pathology, we measured the levels of circulat-
ing GDF11 in the blood of young adults with MDD and found a decrease
compared to healthy controls. It is crucial to note that all persons
included in this study were young (average age of 26 years) and were
a b
Controls MDD
0
50
100
150
200
Human serum
Variables Controls
n = 51
MDD
n = 57 P value
Sex
Male
Female
Age (y)
Years of education
MADRS score
41 (80.4%) 46 (80.7%)
10 (19.6%) 11 (19.3%)
25.94 ± 2.20 26.14 ± 2.34
10.98 ± 3.08 10.35 ± 3.65
0.00 (0.00–2.00) 14.00 (10.00–21.00)
0.968
0.651
0.338
<0.001
MDD
GDF11 levels (pg ml−1)
c
Variables No
n = 656
Yes
n = 103 P-value
Sex
Male
Female
Age (y)
Years of education
MADRS score
331 (50.5%) 87 (84.5%)
325 (49.5%) 16 (15.5%)
25.76 ± 2.17 25.92 ± 2.23
11.32 ± 3.55 9.35 ± 3.29
0.00 (0.00–4.00) 20.00 (13.50–24.00)
<0.001
0.493
<0.001
<0.001
Individuals with current depressive episode d
Current depressive episode
Human serum
GDF11 levels (pg ml−1)
No (n = 656) Yes (n = 103)
0
50
100
150
200
250
300 P = 0.001
P = 0.035
Fig. 6 | Levels of GDF11 in the blood correlate with MDD in human subjects.
a, Table describing the clinical characteristics of the human young adults’ samples.
MDD and healthy control participants were matched by sex (Pearson chi-square
value = 0.002; df = 1; two-sided P value = 0.968), age (Student t test = −0.454;
df = 106; two-sided P value = 0.651) and years of education (Student t test = 0.963;
df = 106; two-sided P value = 0.338). There was a significant difference between
groups in MADRS scores (Mann–Whitney U = 0.000; two-sided P value <0.001).
b, Measurement of GDF11 by ELISA immunoassay in the serum of human healthy
controls or young adults with MDD. There was a significant difference between
groups in GDF11 levels (Mann–Whitney U = 1111.000; two-sided P value = 0.035).
c, Table describing the clinical characteristics of the young adult individuals with
a current depressive episode and controls. There was a significant difference
between groups in sex (Pearson chi-square value = 41.613; df = 1; two-sided
P value < 0.001), years of education (Student t test = 5.294; df = 752; two-sided
P value < 0.001) and MADRS scores (Mann–Whitney U = 2961.500; two-sided
P value < 0.001). There was no significant difference between groups in age
(Student t test = −0.686; df = 757; two-sided P value = 0.493). d, Measurement of
GDF11 by ELISA immunoassay in the serum of human controls or individuals with
a current depressive episode. There was a significant difference between groups
in GDF11 levels (Mann–Whitney U = 26894.000; two-sided P value = 0.001). The
statistical analysis was conducted using the chi-squared test (sex), Student t test
(age and years of education), and Mann–Whitney U test (MADRS and GDF11).
Age and years of education are presented as mean values ± standard deviation
(s.d.). MADRS are presented as the median and interquartile range. GDF11
levels are shown as Tukey boxplots, where the boxes represent the median and
interquartile range. *P < 0.05.
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Article https://doi.org/10.1038/s43587-022-00352-3
recruited from the community to have a population-based sample. Col-
lectively, our findings raise the possibility that serum GDF11 levels could
be used as a potential biomarker to uphold the presence of depressive
episodes. As these results demonstrate correlation and not causation,
further clinical studies should be conducted to conclude specificity in
humans. Also, it would be interesting to examine whether serum GDF11
levels are restored in MDD patients under antidepressant medication.
Given the tight links among hippocampal neurogenesis, depres-
sion and memory, we examined how GDF11 could be involved. We
discovered that the increase in neurogenesis does not require longer
than 9 days of GDF11 treatment and found a significant increase in both
Sox2+ NSC populations and DCX+ neuroblasts in the aged hippocampus.
Similar increases were reported in the hippocampus after 4 weeks of
GDF11 administration
26
, suggesting that NSC activation and produc-
tion of new neuroblasts is maintained at the same rate throughout
the 4-week treatment. It would be interesting to examine how long
this effect lasts, whether new neurons keep integrating in the DG and
if there is a point in which the NSC pool would start being depleted.
Examination of the NSC pool using neurospheres showed that GDF11
had no effect on neurogenesis. Moreover, direct brain infusion of GDF11
showed no increase in NSC or DCX+ populations in the hippocampus.
These results agree with initial reports that demonstrate GDF11 tak-
ing part in a feedback inhibitory signal that limits neurogenesis in
the embryonic olfactory epithelium
18,20,38
. Moreover, it was recently
reported that GDF11 is a negative regulator of adult hippocampal neu-
rogenesis by depletion of endogenous GDF11 in a tamoxifen-inducible
mouse model
23
. Therefore, the observed effects on neurogenesis after
systemic GDF11 administration seem to be indirect, potentially through
activation of other factors in the periphery. For example, GDF11 stimu-
lates the secretion of adiponectin from white adipose tissue
35
, and it
could stimulate other peripheral molecules, such as osteocalcin. Both
molecules are known to cross the blood–brain barrier and enhance
neurogenesis and cognition39–42.
Both systemic administration and ICV infusion of GDF11 resulted
in the upregulation of hippocampal autophagy. Further examination
showed that GDF11 acts directly on neurons via phosphorylation of
SMAD2/3 and that the autophagic process is necessary for the enhance-
ment of neuronal activity mediated by GDF11. This comes in accordance
with previous reports for GDF11 in stimulating autophagy in carotid arte-
rial smooth muscle cells
43
and skeletal muscle fibers
44,45
. In addition to its
well-known role in lifespan extension46, autophagy is crucial for proper
neuronal function and synaptic plasticity47. The fact that autophagy
dysfunction leads to neurodegeneration, memory impairments and
changes in mood states47,48, as a consequence of advanced aging, supports
this idea. Likewise, the effect of GDF11 on ameliorating memory in aged
mice might result from GDF11 acting as an autophagy inducer. Indeed, it
was recently reported that autophagy is required for memory formation
and that induction of autophagy in the brains of aged mice is sufficient
to repair age-related memory decline
29
. There is some evidence that
enhancement of the autophagic pathway can alleviate the depressive-like
features in mice, while inhibition of Beclin 1 blocks the effects of anti-
depressants
49
. Moreover, in human pathology, anti-depressants might
involve modulation of autophagic pathways50. Mechanistically, we pro-
pose that GDF11 stimulates autophagy via inhibition of mTOR. Recently,
the mTOR pathway has been shown to be compromised in the prefrontal
cortex of MDD patients
51
, as well as in the murine hippocampus
52
. We
provide evidence that GDF11 upregulates Deptor, an inhibitor of mTOR,
and downregulates S6K1 activity, a downstream target of mTOR. Inter-
estingly, S6K1-deficient mice exhibit a calorie restriction phenotype
53
,
which resembles the effect of GDF11 in aged mice24. Taken together, our
findings point to the conclusion that GDF11 acts as an antidepressant
through modulation of autophagic pathways and mTOR.
These results reveal a connection among GDF11, mTOR, autophagy
and depression and indicate that GDF11 could be considered as a reli-
able biomarker for MDD in humans. This role for GDF11 sheds light on
its mechanism of action in the brain and allows for future therapeutic
interventions in the context of depression associated with aging.
Methods
Human study design and participants
This paper reports the second wave of a prospective cohort study,
including a population-based sample of young adults. In the first wave
(2007–2009), sampling was performed by clusters, considering a
population of 39 667 people in the target age range (18–24 years old),
according to the current census of 448 sectors in the city Pelotas/
Brazil. From these sectors, 89 census-based sectors were randomly
selected. The home selection in the sectors was performed according
to a systematic sampling, the first one being the house at the corner
pre-established as the beginning of the sector, and the interval of selec-
tion was determined by skipping two houses. Therefore, the sample
is representative of the target population due to the probabilistic
sampling adopted. The first wave included 1560 young adults aged
between 18 and 24 years. The second wave took place a mean of five
years later (2012–2014), and all young adults included in the first wave
were invited for a reassessment. Only data from the second wave are
described in this current study. All participants agreed to participate
in the study by providing their free and written informed consent.
Compensation for travel expenses was provided for the participants.
This study was approved by the Research Ethics Committee of the
Universidade Catolica de Pelotas under protocol number 2008/118.
The full description of the study design is published elsewhere54.
The psychiatric diagnosis was assessed using the Mini Interna-
tional Neuropsychiatric Interview–Plus by trained psychologists. In
addition, the severity of depressive symptoms was assessed using
the MADRS.
For the first analysis using human serum in the present study, we
selected 57 young adults with MDD and 51 healthy controls without
mood disorders (MDD or bipolar disorder), anxiety disorders (panic
disorder, agoraphobia, social phobia, generalized anxiety disorder),
obsessive-compulsive disorder, post-traumatic stress disorder, or
attention deficit hyperactivity disorder. The groups were matched by
sex, age and years of education.
In the second analysis using human serum, we included 103 young
adults presenting a current depressive episode and 656 controls with-
out a current depressive episode.
For the GDF11 measurement, 10 ml of blood was withdrawn from
each subject by venipuncture into a free-anticoagulant vacuum tube.
The blood was immediately centrifuged at 3,500 g for 15 min, and the
serum was kept frozen at −80 °C until analysis.
GDF11 serum levels were determined by sandwich-ELISA using the
Human GDF-11/BMP-11 DuoSet ELISA kit according to the manufac-
turer’s instructions (R&D Systems) and as in Katsimpardi et al.
35
Briefly,
microtiter plates (96-well flat bottom) were coated overnight at room
temperature with the anti-human GDF11 capture antibody at 4 μg ml
−1
in PBS. Thereafter, the plates were washed three times with wash buffer
and blocked with 1% BSA solution for 2 h at room temperature. After
washing, the plates were incubated overnight at 4 °C with the samples
and the standard curve ranged from 7.82 to 2,000 pg ml
−1
GDF11. Plates
were washed and biotinylated anti-human GDF11 detection antibody at
400 ng ml−1 was added, which was incubated for 2 h at room tempera-
ture. After washing, incubation with streptavidin-peroxidase conjugate
(diluted 1:40 in 1% BSA solution) for 20 min at room temperature was
performed and subsequently plates were washed again and incubated
with the substrate solution for 20 min at room temperature. Finally, the
stop solution (2 N sulfuric acid) was added and the amount of GDF11
was determined by measuring absorbance at 450 nm with correction
at 540 nm. The standard curve demonstrates a direct relation between
optical density and GDF11 concentration.
It is important to note that we used the GDF8 (also called myostatin)
as a control for the specificity of the GDF11 kit
48
. The GDF8 (Peprotech)
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Article https://doi.org/10.1038/s43587-022-00352-3
was tested at the same concentration as the highest GDF11 standard
(2,000 pg ml−1) as well as at a middle value of the GDF11 standard curve
(500 pg ml−1). GDF8 was not detected at any concentration. The statisti-
cal analysis was performed in the SPSS 21.
The distribution of numeric variables (age, years of education,
MADRS and GDF11 levels) was evaluated through histograms. Numer-
ical variables that presented normal distribution were compared
between groups using Student t test, and numerical variables that did
not present a normal distribution were compared between groups
using Mann–Whitney U test. The dichotomous variable (sex) was com-
pared between groups using the chi-squared test.
Statistics and reproducibility. No statistical method was used to
predetermine sample size, because this analysis is part of a larger
study; however, the sample size for these analyses is in agreement with
previous literature in the field55. For the second analysis using human
serum, we excluded from the control group (a) individuals who fulfilled
criteria for a past depressive episode and were not in a current depres-
sive episode, and (b) individuals with a history of hypomanic or manic
episodes who were not in a current depressive episode. Importantly,
individuals in the control group could have other psychological con-
ditions, such as anxiety disorders. The investigators were blinded to
allocation during experiments and outcome assessment.
Animals
Young (3-month-old) and aged (22-month-old) C57BL/6JRj male mice
were obtained from Janvier Labs. For the CORT experiments, 2-month-
old C57BL6NTac male mice were obtained from Taconic Biosciences.
All animals were group housed and provided free access to food and
water. Housing conditions were as follows: dark/light cycle from 7 am
to 9 pm, controlled temperature 20–24 °C, ambient humidity 60%
minimum to 70% maximum. All animal procedures were performed in
accordance with French legislation and in compliance with the Euro-
pean Communities Council Directives (2010/63/UE), according to the
regulations of Institut Pasteur Animal Care Committees.
GDF11 administration in mice
GDF11 (Peprotech, 120-11) was dissolved in water, further diluted accord-
ing to the manufacturer’s instructions and injected at a concentration
of 1 mg kg
−1
, as previously described in
24
. Control mice (young or aged)
were injected with equivalent volumes of saline. All mice were injected
daily at 7 pm for the duration of the experiment. All experimental pro-
cedures were performed in accordance to the Institut Pasteur ethical
committee and the French Ministry of Research (APAFiS; 16380).
Miniosmotic pump implantation and ICV GDF11 infusion in mice
Twenty-four hours before implantation, programmable and refill-
able miniosmotic pumps (iPrecio, SMP-300) were filled with saline,
under sterile conditions, and programmed to deliver the infusion at a
constant rate of 1 μl h−1. Twenty-four hours later, miniosmotic pumps
were implanted into the right lateral ventricle of 21-month-old mice
(antero-posterior −0.5; medial-lateral + 1.0; dorsal-ventralDV −2.0).
The pumps were refilled with GDF11 solution (0.3 mg kg
−1
rGDF11 in
saline) or saline every 4 days for a total volume of 120 μl.
Behavioral tests in mice
Open ield test. Four gray arenas (45 × 45 cm) with high walls were used
and luminosity was set at 30 lux throughout the habituation and the test
phases. Mouse cages were placed in the behavior room for habituation
30 min before the test. Each mouse was placed in the arena for 10 min
and then returned to their home cage.
NORT. Four gray arenas (45 × 45 cm) with high walls were used and
luminosity was set at 30 lux throughout the habituation and the test
phases. Mouse cages were placed in the behavior room for habituation
30 min before the test. Mice were habituated to the arenas for 3 con-
secutive days as follows. On habituation day 1, mice housed in the
same cage were placed in one arena for 30 min. Then each mouse was
individually placed in one arena for 10 min each. On days 2 and 3, mice
were individually placed in an arena for 10 min each. On day 4 (NORT),
mice were individually placed in the arena where two identical objects
were placed at equal distances from the walls and from each other, and
the mice were left to explore for 10 min and put back in their home cage.
Two hours later, mice were placed back in the arena where one of the
two objects was replaced with a novel object, and the mice were left to
explore for 10 min. A camera above the arena automatically captured
the locomotor activity of each mouse, and its behavioral pattern was
measured and analyzed using EthoVision XT software (Noldus) and
confirmed by a blind manual measurement of the time spent sniffing
the objects. To exclude a spontaneous preference towards one of
the objects, several pairs of objects had previously been tested with
different sets of naive young and aged animals, and objects with no
preference were chosen for the experiment.
Novel object location test. Same arenas and procedure was used as in
the NORT. The test took place after the NORT, so no additional habitua-
tion was performed. The objects used were different from those in the
NORT, and spatial cues were provided on the walls of the arenas (sun,
moon, square and triangle shaped). On the day of the test, each mouse
was individually placed in the arena where two identical objects were
placed at equal distances from the walls and from each other, and the
mice were left to explore for 10 min and put back in their home cage.
Two hours later, mice were placed back in the arena where one of the
two objects was placed in a diagonal compared to its previous position,
and the mice were left to explore for 10 min. A camera above the arena
automatically captured the locomotor activity of each mouse, and its
behavioral pattern was measured and analyzed using EthoVision XT
software (Noldus) and confirmed by a blind manual measurement of
the time spent sniffing the objects. To exclude a spontaneous prefer-
ence towards one of the objects, several pairs of objects had previously
been tested with different sets of naive young and aged animals, and
objects with no preference were chosen for the experiment.
Y-maze test. Mouse cages were placed in the behavior room for habitu-
ation 30 min before the experiment. A gray Y-maze arena (24.6 cm
length and 7.8 cm wide) was used and luminosity was set at 30 lux
throughout the training and the test phases. For the training phase,
one of the arms was blocked and the mouse was placed in the center of
the Y-maze to start exploring for 15 min. Then, the mouse was placed
back to the home cage for 1 h, and the maze was cleaned before the next
trial. For the test phase, the divider was removed and all three arms
were open. The mouse was placed in the center and was left to explore
the maze for 5 min. A camera above the arena automatically captured
the locomotor activity of each mouse, and its behavioral pattern was
measured and analyzed using EthoVision XT software (Noldus).
Splash test. Mouse cages were placed in the behavior room for habitua-
tion 30 min before the experiment. Then, all mice, except for the experi-
mental mouse, were transferred to a different cage. The experimental
mouse was given a spray shot of a 10% sucrose solution on the lower
back and then put back in the home cage. The viscosity of the sucrose
solution dirties the fur, inducing a grooming behavior. The mouse
was observed for 6 min, during which the frequency of grooming, the
latency to first groom and the total grooming times were measured.
After the test, the experimental mouse was placed in a different cage
until all the mice of the cage had been subjected to the test.
TST. Mouse cages were placed in the behavior room for habituation
30 min before the experiment. One mouse per condition was placed in
the TST apparatus by applying tape on the mouse’s tail and using it to
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hang the mouse upside down. The test lasted for 6 min, during which
immobility time was measured by a blind observer.
Sucrose preference test. Each mouse was housed individually start-
ing in the evening of day 0. Two bottles with water were placed in each
cage to habituate the mouse to the new drinking scheme. The next
evening (day 1), one of the two bottles was replaced with 1% (wt/vol)
solution and the other one with water. The next morning (day 2), water
and sucrose-water consumption was measured. Then, the position of
the bottles was changed to avoid place preference. The next morning
(day 3), water and sucrose-water consumption was measured again and
mice were returned to their home cages. The sucrose index we report
was the average of the two measurements.
Social interaction test. The same arenas as for the open field test,
NORT and novel object location test were used. Mouse cages were
placed in the behavior room for habituation 1 h before the experiment.
Each test mouse was placed in the center of the arena. Immediately
after, a stimulus mouse (intruder) was also placed in the center of the
arena. Mice were left to explore the arena and interact for 5 min. A cam-
era above the arena automatically captured the locomotor activity of
the mice, and their behavioral patterns were measured and analyzed
using EthoVision XT software (Noldus). Active avoidance was measured
manually by a blind investigator by considering a movement of the test
mouse further than 3 cm away when the intruder approaches.
Light/Dark box test. The light box (16.5 × 22 cm) was separated from
the dark box (16.5 × 22 cm) with a dark divider with a small opening
allowing the mice to go through. Time spent and frequency of entry in
the light box (178 lux) were measured during 10 min. A camera above
the arena automatically captured the locomotor activity of the mice,
and their behavioral patterns were measured and analyzed using Etho
Vision XT software (Noldus).
Coat state test. The total coat state score resulted from the sum of
the score of five different body parts: neck, dorsal/ventral coat, tail,
forepaws and hindpaws. For each body area, a score of 0 was given for
a well-groomed coat and 1 for an unkempt coat. The measurements
of the coat state were done by an experimenter blind to treatments.
Novelty suppressed feeding. Mice were put in new cages without any
food 12 h before testing. A large white arena with high walls was covered
with litter, and a food pellet was placed in the center under high bright-
ness (100 lux). The mouse was placed in the arena, and the latency to
first bite in the food pellet placed was measured (maximum of 5 min).
As soon as the mouse bit the food pellet, it was removed from the arena
and placed alone in its home cage for 5 min with one food pellet, which
was weighed beforehand. The food pellet was weighed again after the
5-min trial, and the mouse was put in a new cage with food ad libitum.
The amount of food consumed during these 5 min was measured.
EPM. Mouse cages were placed in the behavior room for habituation
1 h before the experiment. The apparatus consists of an elevated cross
with two closed arms, two open arms and a center area (intersection
between closed and open arms). The length of the open and closed arms
was of 37.5 cm and the height 53 cm. To measure stress and anxiety,
each mouse was placed in the center of the EPM and left to explore the
arms for 6 min. A camera above the apparatus automatically captured
the activity of the mouse and its behavioral pattern, as well as the time
spent in the open and closed arms. Measurements and analysis were
done using EthoVision XT software (Noldus).
Nest building test. Each mouse was housed individually starting in the
evening of the testing day. Two intact cotton pellets (2 × 2 cm; 2.5 g)
were placed in the cage and mice were left undisturbed overnight.
The next morning each nest was assessed for two parameters: the
height of the nest and the nest quality. The latter was given a score based
on percentage of the nest height compared to mouse size and shape
(0: material untouched; 5: cloud-like shape, material finely shredded).
Burrowing behavior test. Each mouse was individually housed during
the experiment. Burrowing tubes (17 × 5 cm) were placed in the cage
together with food pellets, all preweighed. Mice were left for 30 min
to burrow by removing the pellets. The weight of the removed pellets
was weighed and the burrowing behavior index was calculated as the
percentage of the weight of the removed pellets divided by the initial
weight.
Gait test. Mouse paws were painted with non-toxic, water-soluble paint
using a different color for forepaws and hindpaws. Immediately after,
mice were left to walk/run on a 60-cm paper-corridor. Stride length
was calculated as the distance between the center of the hind paw to
the center of the forepaw. Base length was calculated as the distance
between left and right paw, for both forepaws and hindpaws.
Chronic CORT administration depression model
CORT, purchased from Sigma-Aldrich, was sonicated for 2 h in a vehi-
cle made of 10% (2-hydroxypropyl)-beta-cyclodextrin (β-CD; Sigma-
Aldrich, H107) in water. After complete dissolution, the solution was
added to the appropriate amount of water to reach the final concentra-
tion of 35 μg ml−1 CORT and 0.45% β-CD. CORT (35 μg ml−1, equivalent
to about 5 mg kg
−1
per day) or vehicle (0.45% β-CD) was available ad
libitum in the drinking water in aluminum foil- wrapped bottles to
protect it from light. All the bottles were changed every 3–4 days to
prevent any possible degradation, as previously described34. Animals
were weighed twice a week to verify the increase in body mass already
described in this model34.
Neural stem cell cultures
Neural stem cell cultures (neurospheres) were performed as described
previously30. For proliferation, neurospheres were maintained in
serum-free medium containing EGF and bFGF (both 20 ng ml
−1
) (Gibco
human recombinant bFGF, 10612074 (Thermo Fisher Scientific); Gibco
human recombinant EGF, 10628523 (Thermo Fisher Scientific)].
For the differentiation assay, neurospheres were plated on poly-
lysine/laminin coated coverslips in serum-free, growth-factor-free
media as described previously. To assess the effect of serum on differ-
entiated neurospheres, we incubated the following sera for 4 days (20%
of serum in culture medium, replaced every 2 days): serum from young
mice (young serum), serum from 23-month-old mice (old serum). To
assess the effect of the recombinant protein alone, we incubated rGDF11
(40 ng ml−1) for 4 days, in the same experiment as the serum assay. All
incubations took place in 24-well plates. At the end of the experiment,
coverslips were fixed with 4% paraformaldehyde for 30 min and then
stained as described below.
Cultures of primary hippocampal neurons
Hippocampal neurons were isolated from mouse embryos (embryonic
day 16.5), as in Glatigny et al.29 Briefly, after dissection, hippocampi were
digested with trypsin 0.05% and EDTA 0.02% for 15 min at 37 °C. After
three washes with DMEM (Thermo Fisher Scientific, 61965059) sup-
plemented with 10% FBS, 100 U ml
−1
penicillin/streptomycin and 1x Glu-
taMAX (Thermo Fisher Scientific), cells were dissociated by pipetting
up and down, and then plated. The dissociated cells were plated onto
poly-L-lysine-coated plates or glass coverslips for microscopic exami-
nation. Twenty-four hours after plating, the media was replaced with
Neurobasal medium (Thermo Fisher Scientific) containing B27 sup-
plement (Thermo Fisher Scientific), GlutaMAX and Mycozap (Lonza).
Media was changed two times per week and neurons were maintained
in 5% CO2 at 37 °C. Experiments were performed on cells after 18 days
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Article https://doi.org/10.1038/s43587-022-00352-3
of culture. Plasmid transfection was performed with Lipofectamine
2000 (Invitrogen, 11668019), according to the manufacturer’s instruc-
tions. For Beclin1 silencing experiments, neurons were transfected
with shRNA targeting Beclin 1 lentivirus plasmid (pLKO-IPTG-3XLacO)
expressing an IPTG-inducible shRNA targeting mouse Beclin-1 (Becn 1)
(Sigma-Aldrich). Transfected neurons were treated with 5 mM IPTG on
DIV15 and for 72 h to induce shRNA-Beclin-1 expression. Neurons were
stained with anti-LacI antibody (05-503I, Sigma-Aldrich) to confirm
co-transfection with the GFP plasmid.
For neurons treated with Bafilomycin, neurons were incubated
with 100 nM of Baf (B1793, Sigma-Aldrich) for 2 h before fixation. The
same concentration was used for the Seahorse analysis.
RNA-seq of primary hippocampal neurons
RNA isolation. Total RNA was isolated from samples using Trizol (Life
Technologies) according to the manufacturer’s instructions. After
adding chloroform, the samples were centrifuged at maximum speed,
and the upper phase was then used for RNA cleanup using RNAeasy
(Qiagen) minicolumns following the manufacturer’s instructions.
Briefly, an equal volume of 70% ethanol was added to the upper phase
and processed through the columns. After two washes, the RNA was
eluted with water.
RNA-seq. RNA-seq and bioinformatic data analysis were performed by
NovoGene. Briefly, messenger RNA was purified from total RNA using
poly-T oligo-attached magnetic beads. After fragmentation, the first
strand cDNA was synthesized using random hexamer primers, followed
by second strand cDNA synthesis. The libraries were checked with
Qubit and real-time PCR for quantification and bioanalyzer for size
distribution detection. Quantified libraries were pooled and sequenced
on an Illumina platform, and paired-end reads were generated. Raw
reads were filtered to remove those containing adapters, poly-N and
low-quality reads from raw data, and Q20, Q30 and guanine-cytosine
(GC) were calculated. All downstream analyses were performed on
clean data with high quality. Paired-end reads were aligned to the
GRCm38/mm10 reference genome using Hisat2 v2.0.5, and feature-
Counts v1.5.0-p3 was used to count the read numbers mapped to each
gene, normalized by FPKM.
Differential expression analysis. Differential expression analysis of
two conditions/groups (two biological replicates per condition) was
performed using the DESeq2 R package (1.20.0). The resulting P values
were adjusted using the Benjamini and Hochberg’s approach, and
genes with an adjusted P value ≤ 0.05 found by DESeq2 were assigned
as differentially expressed.
Enrichment analysis of differentially expressed genes. Gene Ontol-
ogy enrichment analysis of differentially expressed genes was done
using the clusterProfiler R package for which gene length bias was
corrected. Genes were ranked according to the degree of differential
expression, and then predefined gene sets were tested to using a local
version of the GSEA analysis tool (http://www.broadinstitute.org/
gsea/index.jsp).
Seahorse metabolic analysis
For metabolic analyses, the Seahorse XF Cell Energy Phenotype test
kit (Agilent) was used. Primary hippocampal neurons were isolated as
described above and plated directly on poly-L-lysine-coated 96-well
plates provided in the kit by the manufacturer. Cells were treated for
2 h before measurement of their basal OCR, and then oligomycin and
FCCP were injected to induce maximal respiration in the neurons.
Immunohistochemistry
Mice were anesthetized with a mix of ketamine (80–100 mg kg−1) and
xylazine (10–12.5 mg kg−1), and their brains were removed and fixed
overnight in 4% PFA. Each brain was embedded in 4% agarose, and
40-μm-thick coronal sections were cut using a vibrating microtome
(VT1000S, Leica). For neurosphere cultures and primary neurons, cells
were fixed on poly-L-lysine-coated coverslips. Tissue sections or cells
were pre-incubated in 10% normal goat or donkey serum, 0.1% Triton-X
100 in PBS for 1 h and were incubated overnight at 4 °C with the follow-
ing antibodies: rabbit polyclonal anti-Sox2 (1:100, Cell Signaling Tech-
nology, #2748), chicken polyclonal anti-DCX (1:400, abcam, ab153668),
cFos (1:2000, ABE457, Millipore). Alexa Fluor (Life Technologies) sec-
ondary antibodies were used for detection of the primary antibody at a
dilution of 1:1,000 (Thermo Fisher Scientific, A-21244; goat polyclonal
anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa
Fluor 647, lot 2086678), Thermo Fisher, A-11039, Goat polyclonal anti-
Chicken IgY (H + L) Secondary Antibody, Alexa Fluor 488, lot 2180688;
Merck Millipore, AP180SA6, Donkey polyclonal anti-Goat IgG (H + L)
secondary antibody, Alexa Fluor 647, lot 3743391; Jackson Immunore-
search, 703-545-155, Donkey polyclonal Anti-Chicken IgY (IgG) (H + L)
secondary antibody Alexa Fluor 488 AffiniPure, lot 151980; Thermo
Fisher Scientific, A-21235, Goat polyclonal anti-Mouse IgG (H + L),
cross-adsorbed secondary antibody, Alexa Fluor 647.
SA-βGal assay
Mice were anesthetized with a mix of ketamine (80–100 mg kg−1)
and xylazine (10–12.5 mg kg−1), and brains were perfused with 4% PFA
and subsequently post-fixed in 4% PFA overnight. The following day,
brains were washed three times for 15 min with PBS. Each brain was
embedded in 4% agarose, and 40-μm-thick coronal sections were cut
using a vibrating microtome (VT1000S, Leica). Then, the SGZ sections
were selected and incubated in X-Gal solution containing 40 mM cit-
rate buffer (pH=6) (C7129, Sigma-Aldrich), 5 mM K3Fe (CN)6 (P8131,
Sigma-Aldrich), 5 mM K4Fe (CN)6 (P9387, Sigma-Aldrich), 2 mM MgCl
2
(208337, Sigma-Aldrich), 150 mM NaCl (31434, Sigma-Aldrich) and 1 mg
ml
−1
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal)
(10703729001, Sigma-Aldrich) in PBS (pH 6)] at 37 °C in the dark for
48 h. After incubation, the SGZ sections were post-fixed with 4% PFA
for 15 min and then washed three times for 15 min with PBS. Finally, the
sections were mounted on slides using Fluoromount-G (00-4958-02,
Thermo Fisher Scientific) and observed under an optical microscope
for the development of blue color referring to senescent cells. The
images were taken with a scanner (Olympus VS120) and quantification
of senescent cells was performed manually by a blinded investigator.
Western blots
Hippocampi were dissected and snap frozen in liquid nitrogen. Cell
cultures were spun down, the medium was removed and the pellet
was snap frozen in liquid nitrogen. Tissues and cell pellets were lysed
in RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40,
1% sodium deoxycholate, 0.1% SDS) (Pierce, Thermo Fisher Scien-
tific) and protease (cOmplete, Sigma-Aldrich) and phosphatase (phos
STOP, Sigma-Aldrich) inhibitors. Protein concentration was measured
with the Pierce BCA protein Assay Kit. Tissue lysates were mixed with
4 × NuPage LDS loading buffer (Invitrogen), and proteins were sepa-
rated on a 4–12% SDS-polyacrylamide gradient gel (Invitrogen) and
subsequently transferred by semi-dry or liquid transfer onto a polyvi-
nylidene fluoride membrane (Trans-blot Turbo Mini PVDF, Bio-Rad).
The blots were blocked in 5% BSA in Tris-buffered saline with Tween
(TBS-T) and incubated with: anti-Lamp1 (1:1,000, ab24170 abcam),
rabbit anti-LC3 (1:1,000, L7543, Sigma-Aldrich), rabbit anti-Beclin1
(1:1,000, 3495, Cell Signaling), rabbit anti-Atg5 (1:1,000, 12994, Cell
Signaling), rabbit polyclonal anti-FOXO3a (1:1,000, 2497, Cell Signaling
Technology), rabbit polyclonal anti-total-Smad2/3 (1:1000, #13820,
Cell Signaling), rabbit polyclonal anti-phospho-Smad2/3 (1:1,000,
8685, Cell Signaling), rabbit polyclonal anti-phospho-S6K1 (1:1,000,
9205, Cell Signaling), rabbit polyclonal anti-Deptor (1:1,000, Cat#
ABS222, Sigma-Aldrich), rabbit polyclonal anti-phospho-4E-BP1
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(1:1,000, 2855, Cell Signaling) and mouse anti-actin (1:6,000, A5441,
Sigma-Aldrich). To detect protein signals, the following horseradish
peroxidase (HRP)-conjugated secondary antibodies were used: Goat
Anti-Rabbit IgG (H + L)-HRP conjugate (1:6,000, #1706515, Bio-Rad) and
Goat Anti-Mouse IgG1 heavy chain (HRP) (1:6000, ab97240, Abcam).
Chemiluminescence detection of proteins was performed with Lumi-
nata Crescendo Western HRP Substrate (Merck Millipore) and imaged
with the Chemidoc Imaging System (Bio-Rad). Bands were quantified
using Fiji (ImageJ) software.
Real-time qPCR
Total RNA was extracted from tissue samples using the NucleoSpin
RNA/Protein kit (NucleoSpin
®
RNA/Protein, Macherey-Nagel, 740933)
following the manufacturer’s recommendations. Samples were treated
with DNase I before reverse transcription into cDNA. One microgram
of RNA was reverse-transcribed with the High-Capacity cDNA Reverse
Transcription Kit (Thermo Fisher Scientific, 4368813) following the
provider’s recommendations and diluted fivefold with water before
using for quantitative PCR. Quantitative PCR was performed using
of the SYBR Green Master Mix (Roche Applied Science) on a LightCy-
cler 480 Real-Time PCR System (Roche Applied Science, 4368708).
Expression values were obtained using the 2
−ΔΔCt
method, as previously
described
56
. All assay were performed in triplicate and were normalized
to Gapdh levels. Primers used for qPCR analysis as follows: mp16 for-
ward, 5′-CGTACCCCGATTCAGGTGAT-3′; mp16 reverse, 5′-TTGAGCAGA
AGAGCTGCTACGT-3′; mARF forward (p19), 5′-GCCGCACCGGAATC
CT-3′; mARF reverse (p19), 5′-TTGAGCAGAAGAGCTGCTACGT-3′.
Image acquisition and analysis/quantification
Imaging was performed using a Zeiss LSM 510 inverted confocal
microscope, and a Zeiss Apotome microscope. Sox2 quantifica-
tion was performed by batch analysis using Icy software (http://icy.
bioimageanalysis.org/). Numbers of DCX
+
cells in the DG were blindly
quantified by hand, using every other section and the same area for
each condition. Neurite length of neurospheres was measured with
IMARIS software (Bitplane).
Statistics and reproducibility
No statistical methods were used to predetermine sample sizes. Sample
size was determined in accordance with standard practices in the field
and based on our previous analyses and experience in these experi-
mental paradigms
8,24,31
. No data were excluded from the analysis. Sam-
ples, mice and mouse cages were randomly allocated to experimental
groups. Experiments were carried out in a blinded fashion; investiga-
tors were blinded during experimental procedures, data collection
and data analysis by assigning codes (prepared by other investigators
irrelevant to this study) to mice, mouse cages, cell samples and images
before processing, to ensure unbiased analysis. Statistical tests for
each experiment are mentioned in the corresponding figure legends.
All statistical analyses (except for RNA-seq) were performed using
GraphPad Prism (version 9), with one-way ANOVA and Tukey’s post hoc
test for multiple group comparisons and two-sided Mann–Whitney test
for two-group comparisons, assuming a two-tailed distribution. Statis-
tical significance was assigned for P < 0.05; results are shown as s.e.m.
Data distribution was assumed to be normal, but this was not
formally tested.
Reporting summary
Further information on research design is available in the Nature Port-
folio Reporting Summary linked to this article.
Data availability
The primary neuron RNA-seq datasets are publicly available at the
Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/PRJNA
913014; accession number PRJNA913014). Source files are available
online, and all data are available from the corresponding authors upon
reasonable request.
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Acknowledgements
We thank G. Lepousez, C. Mazo and F. Sonthonnax for advice on the
miniosmotic pumps; M. Pende for discussions and sharing mTOR-
pathway antibodies; S. Pons and N. Lemière for sharing equipment;
J. Carbonne for technical help with the in vitro experiments on neurons;
and B. Charbit for advice and assistance with the Seahorse apparatus.
L.K. acknowledges support by the AGEMED INSERM program on
Aging (Programme Transversal sur le Vieillissement 2016–2022) and
ADPS Recherche sur la Longévité. L.K. and A. C. received a fellowship
from LabEx/Revive. The Lledo lab was supported by Institut Pasteur,
Centre National pour la Recherche Scientiique, the life insurance
company ‘AG2R-La-Mondiale’, Agence Nationale de la Recherche
(ANR-15-CE37-0004-01), National Institutes of Health US-French
Research Proposal Grants 1R01DC015137-01 and ANR-15-NEUC-0004
(Circuit-OPL) and the Laboratory for Excellence Programme ‘Revive’
(Investissement d’Avenir, ANR-10-LABX-73). The Oury lab was
funded by Agence Nationale de la Recherche (ANR-17-CE14-0030-
Memorautophagy), Fondation pour la recherche médicale (FRM-
EQU201903007775), Emergence de la ville de Paris and AGEMED
INSERM program. The Li lab was funded by Institut Pasteur, Centre
National pour la Recherche Scientiique, and the Agence Nationale
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Article https://doi.org/10.1038/s43587-022-00352-3
de la Recherche (Laboratoire d’Excellence Revive, Investissement
d’Avenir; ANR-10-LABX- 73), the Agence Nationale de la Recherche
(ANR-16-CE13-0017-01, ANR-21-CE13-0006-01) and Fondation ARC
(PJA 20161205028, 20181208231). The Kapczinski lab was supported
by the Fundação de Amparo à Pesquisa do Estado do Rio Grande do
Sul (FAPERGS), Conselho Nacional de Desenvolvimento Cientíico
e Tecnológico (CNPq), Instituto Nacional de Ciência e Tecnologia
Translacional em Medicina (Porto Alegre, Brazil), the Strategic
Alignment Fund from McMaster University and the St. Joseph’s
Healthcare Foundation, Hamilton.
The funders had no role in study design, data collection and
analysis, decision to publish or preparation of the manuscript.
Author contributions
C.M. and S.A. performed mouse experiments and analyzed data;
M.R.B. gave advice and performed experiments on hippocampal
neuron cultures in vitro; B.P., B.W.A. and F.P.M. performed and
analyzed the immunoassays for GDF11 in the human samples and
interpreted the clinical data; T.A.C. collected, interpreted and
performed statistical analysis on the clinical data; A.C. performed
the senescence qPCR experiments, N.K. cloned plasmids, prepared
the samples for RNA-Sequencing, gave advice on experiments and
helped edit the inal version of the manuscript; R.A.S. conceptualized
and designed the clinical study; F.P.M. collected and organized
the biological samples for the clinical study; H.L. gave advice
analyzed and interpreted the senescence data; F.O. gave advice on
the autophagy experiments and shared plasmids; F.K. interpreted
the clinical data; P.M.L. secured funding and supervised the study;
L.K. conceptualized the project, performed in vivo and in vitro
experiments, analyzed data, secured funding, supervised the study
and wrote and edited all versions of the manuscript. All authors have
read and approved the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/
s43587-022-00352-3.
Supplementary information The online version contains supplementary
material available at https://doi.org/10.1038/s43587-022-00352-3.
Correspondence and requests for materials should be addressed to
Pierre-Marie Lledo or Lida Katsimpardi.
Peer review information Nature Aging thanks Evandro Fang and the
other, anonymous, reviewer(s) for their contribution to the peer review
of this work.
Reprints and permissions information is available at
www.nature.com/reprints.
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© The Author(s) 2023
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Extended Data Fig. 1 | See next page for caption.
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Nature Aging
Article https://doi.org/10.1038/s43587-022-00352-3
Extended Data Fig. 1 | GDF11 treatment does not affect anxiety-like state,
general well-being or physical performance. (a–c) Behavioral tests related
to anxiety: (a) Measurement of the distance moved in the Light Box, during the
Light/Dark Box test (nYoung = 10 mice, nAged = 10 mice, nAged+GDF11 = 12 mice; F (2,
31) = 12.8), (b) Measurement of the time spent in the Light Box, during the Light/
Dark Box test (nYoung = 10 mice, nAged = 10 mice, nAged+GDF11 = 12 mice; F (2, 31) = 8.5),
(c) Measurement of the time spent in the open arms, during the Elevated Plus
Maze test (nYoung = 10 mice, nAged = 10 mice, nAged+GDF11 = 12 mice; F (2, 31) = 1.0). (d–f)
Behavioral tests related to general well-being: (d) Measurement of the height
of the nest (nYoung = 10 mice, nAged = 10 mice, nAged+GDF11 = 12 mice; F (2, 31) = 0.8),
(e) Scoring of the nest quality based on five criteria (nYoung = 10 mice, nAged = 10
mice, nAged+GDF11 = 12 mice; F (2, 31) = 1.2), (f) measurement of the burrowing index
(percentage of weight of pellets removed from the burrow divided by total pellet
weight), during the Burrowing test (nYoung = 6 mice, nAged = 9 mice, nAged+GDF11 = 9
mice; F (2, 21) = 0.5). (g–j) Behavioral tests related to physical performance:
(g) measurement of the distance moved during the Open Field test (nYoung = 11
mice, nAged = 11 mice, nAged+GDF11 = 12 mice; F (2, 31) = 2.7), (h) measurement of the
time mice spent hanging on the wire during the Hanging Wire test (nYoung = 10
mice, nAged = 10 mice, nAged+GDF11 = 10 mice; F (2, 27) = 3.6), (i) measurement of
the hind paw and fore paw stride length during the Gait test (nYoung = 10 mice,
nAged = 13 mice, nAged+GDF11 = 11 mice; F (2, 62) = 0.03), (j) measurement of the hind
base and front base length during the Gait test (nYoung = 10 mice, nAged = 13 mice,
nAged+GDF11 = 11 mice; F (2, 62) = 7.4). One-way and two-way ANOVA and Tukey’s post
hoc test for multiple comparisons; F (DFn, DFd) value presented for each ANOVA
statistical analysis; P values < 0.05 are represented on the graph; mean ± s.e.m.
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Nature Aging
Article https://doi.org/10.1038/s43587-022-00352-3
Extended Data Fig. 2 | DCX expression in the DG after GDF11 treatment. (a) Representative confocal images of the dentate gyrus of the hippocampus
immunostained for DCX (doublecortin, a marker of immature neuroblasts) in green (nYoung = 8 mice, nAged = 9 mice, nAged+GDF11 = 9 mice) relative to quantification Fig. 2c.
Scale bar: 100 μm.
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Nature Aging
Article https://doi.org/10.1038/s43587-022-00352-3
Extended Data Fig. 3 | Intracerebroventricular GDF11 infusion recapitulates
all aspects of systemic treatment except neurogenesis. (a) Schematic
representation of the experimental procedure: miniosmotic pumps were
implanted in the lateral ventricle and rGDF11 or vehicle was infused at a
constant rate over 2 weeks. (b) Measurement of discrimination index during
the NORT (percentage of time spent to observe the novel object divided by the
percentage of total investigation time for both objects) (n = 4 mice per group).
(c) Measurement of grooming frequency during the Splash test (n = 5 mice
per group). (d) Measurement of time spent in the Light box during the Light/
Dark Box Test (n = 6 mice per group). (e) Representation of latency to eat for
mice during the Novelty Suppressed feeding test (n = 5 mice per group). (f)
Quantification of Sox2+ NSCs in the SGL of the dentate gyrus (nVeh = 4 mice,
nGDF11 = 5 mice). (g) Quantification of DCX+ neuroblasts in the SGL of the dentate
gyrus (nVeh = 4 mice, nGDF11 = 5 mice). (h–k) Quantification of Western blots by
optical density for Beclin 1 (i) (nVeh = 4 mice, nGDF11 = 5 mice), Atg5 (j) (nVeh = 4 mice,
nGDF11 = 5 mice), and Lamp1 (k) (nVeh = 4 mice, nGDF11 = 5 mice). Two-sided Mann–
Whitney test for two-group comparisons; P values < 0.05 are represented on the
graph; mean ± s.e.m.
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Nature Aging
Article https://doi.org/10.1038/s43587-022-00352-3
Extended Data Fig. 4 | GDF11 has no effect on neurogenesis in vitro. (a)
Representative confocal images of neural stem cell differentiation into
neuroblasts upon serum treatment (4 μl), rGDF11 protein (40 ng/ml) or no serum/
no growth factors in the culture medium, immunostained for DCX (green) and
Hoechst (blue). (b) Quantification of total neurite length per neuroblast (n = 5
wells per condition; F (3, 16) = 26.4). Scale bar, 20 μm. One-way ANOVA and
Tukey’s post hoc test for multiple comparisons; F (DFn, DFd) value presented for
each ANOVA statistical analysis; P values < 0.05 are represented on the graph;
mean ± s.e.m.
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Nature Aging
Article https://doi.org/10.1038/s43587-022-00352-3
Extended Data Fig. 5 | GDF11 treatment reduces weight of CORT mice. (a)
Measurement of weight over the weeks of CORT/veh treatment and GDF11/saline
injections (nCtrl = 8 mice, nCORT = 16 mice, nGDF11 = 8 mice, nCORT-GDF11 = 16 mice). (b)
Same measurement as (a), but only the CORT conditions are represented. (c)
Measurement of the weight for CORT and CORT-GDF11 mice (n = 16 mice per
group). (d) Measurement of the distance traveled during the Open Field test
(nCtrl = 8 mice, nCORT = 15 mice, nGDF11 = 8 mice, nCORT-GDF11 = 16 mice; F (3, 43) = 2).
One-way one-sided ANOVA and Tukey’s post hoc test for multiple comparisons;
F (DFn, DFd) value shown for each statistical analysis; two-sided Mann–Whitney
test for two-group comparisons; P values < 0.05 are represented on the graph;
mean ± s.e.m.
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nature portfolio | reporting summary March 2021
Corresponding author(s): Dr. Lida Katsimpardi
Last updated by author(s): Dec 3, 2022
Reporting Summary
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The statistical test(s) used AND whether they are one- or two-sided
Only common tests should be described solely by name; describe more complex techniques in the Methods section.
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AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)
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Give P values as exact values whenever suitable.
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Our web collection on statistics for biologists contains articles on many of the points above.
Software and code
Policy information about availability of computer code
Data collection This study did not generate new codes. The following commercial software packages were used for data collection:
Behavioral experiments: Ethovision Noldus (https://www.noldus.com/ethovision-xt)
Confocal microscopy: Zeiss 980 Airyscan2 (https://www.zeiss.com/microscopy/en/products/light-microscopes/confocal-microscopes/
lsm-980-with-airyscan-2.html)
Microscopy: Olympus VS120 (https://www.olympus-lifescience.com/en/microscopes/virtual/vs120/)
Microscopy: Zeiss Apotome 3 (https://www.zeiss.com/microscopy/en/products/light-microscopes/widefield-microscopes/apotome-3.html)
qPCR data acquisition: LightCycler 480 Real-Time PCR System, Roche (https://diagnostics.roche.com/global/en/products/instruments/
lightcycler-480-ins-445.html)
Western blots: Chemidoc, Biorad (https://www.bio-rad.com/fr-fr/category/chemidoc-imaging-systems?ID=NINJ0Z15)
RNA-Sequencing performed by Novogene (https://en.novogene.com/): Illumina platform,Hisat2 v2.0.5 and featureCounts v1.5.0-p3
Seahorse metabolic phenotyping: Seahorse XF Pro Analyzer (https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-
analysis/xf-analyzers/seahorse-xf-pro-analyzer-1980223)
ELISA immunoassay: iMark microplate reader, Biorad (https://www.bio-rad.com/fr-fr/product/imark-microplate-absorbance-reader?
ID=ada40399-b5fe-4917-8423-377a3e0c3b44)
Data analysis
1
This study did not generate new codes. The following commercial software packages were used for data analysis:
Statistical analysis and generation of graphs: GraphPad Prism 9.4.1 (https://www.graphpad.com/scientific-software/prism/) or R software,
with one-way ANOVA and Tukey's post hoc test for multiple group comparisons and Mann-Whitney test for two-group comparisons, assuming
a two-tailed distribution. Statistical significance was assigned for p<0.05; results are shown as standard error of the mean (S.E.M.).
Behavioral ex
periments: Ethovision XT software Noldus (https://www.noldus.com/ethovision-xt)
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Quantification and image analysis: Fiji (https://imagej.net/software/fiji/), Imaris (https://imaris.oxinst.com/imaris-viewer) and Icy (https://
icy.bioimageanalysis.org/) softwares
Seahorse metabolic phenotyping: Seahorse XF Pro Analyzer (https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-
analysis/xf-analyzers/seahorse-xf-pro-analyzer-1980223)
RNA-Sequencing performed by Novogene (https://en.novogene.com/): R package DESeq2 R package (1.20.0) and GSEA analysis tool (http://
www.broadinstitute.org/gsea/index.jsp).
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and
reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.
Data
Policy information about availability of data
All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:
-Accession codes, unique identifiers, or web links for publicly available datasets
- A description of any restrictions on data availability
-For clinical datasets or third party data, please ensure that the statement adheres to our policy
The primary neuron RNA-sequencing datasets are publicly available at Zenodo (DOI: 10.5281/zenodo.7389272). Source files are available online and all data are
available from the corresponding authors upon reasonable request.
Human research participants
Policy information about studies involving human research participants and Sex and Gender in Research.
Reporting on sex and gender In this population-based sample of young adults sex was considered and we have presented the analysis based on sex
differences.
Population characteristics In this population-based sample of young adults (18-24 years-old), the psychiatric diagnosis was assessed using the Mini
International Neuropsychiatric Interview – Plus (MINI-Plus) by trained psychologists. In addition, the severity of depressive
symptoms was assessed using the Montgomery–Åsberg Depression Rating Scale (MADRS).
For the first analysis using human serum in the present study, we selected 57 young adults with Major Depressive Disorder
(MDD) and 51 healthy controls without mood disorders (MDD or bipolar disorder), anxiety disorders (panic disorder,
agoraphobia, social phobia, generalized anxiety disorder), Obsessive-Compulsive Disorder, Post-traumatic Stress Disorder, or
Attention deficit hyperactivity disorder. The groups were matched by sex, age, and years of education.
In the second analysis using human serum, we included 103 young adults presenting a current depressive episode and 656
controls without a current depressive episode. We excluded from the control group (a) individuals who fulfilled criteria for a
past depressive episode and were not in a current depressive episode, and (b) individuals with a history of hypomanic or
manic episodes who were not in a current depressive episode. Importantly, individuals in the control group could have other
psychological conditions, such as anxiety disorders.
Recruitment This is the second wave of a prospective cohort study, including a population-based sample of young adults. In the first wave
(2007-2009), sampling was performed by clusters, considering a population of 39 667 people in the target age range (18-24
years-old), according to the current census of 448 sectors in the city Pelotas/Brazil. From these sectors, 89 census-based
sectors were randomly selected. The home selection in the sectors was performed according to a systematic sampling, the
first one being the house at the corner pre-established as the beginning of the sector, and the interval of selection was
determined by skipping two houses. Therefore, the sample is representative of the target population due to the probabilistic
sampling adopted. Although this sample is representative of the young adult population from the city of Pelotas in Brazil, our
results may not be generalizable for other age range groups (i.e. older populations). The first wave included 1560 young
adults aged between 18-24. The second wave took place a mean of five years later (2012-2014), and all young adults included
in the first wave were invited for a reassessment. Only data from the second wave are described in this current study. All
participants agreed to participate in the study by providing their free and informed consent.
Ethics oversight This study was approved by the Research Ethics Committee of the Universidade Católica de Pelotas (UCPel) under protocol
number 2008/118.
Note that full information on the approval of the study protocol must also be provided in the manuscript.
Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.
Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size Sample size was determined in accordance with standard practices in the field and based on our previous analyses and experience in these
experimental paradigms (PMID: 32187541, 31637864, 24797482).
Data exclusions No data were excluded from the analysis.
Replication Reproducibility of experimental findings was assured by repeating three times independent experiments, as explained in the text.
Randomization Samples, mice and mouse cages were randomly allocated to experimental groups.
Blinding Investigators were blinded during experimental procedures, data collection and data analysis by assigning codes (prepared by investigators
irrelevant to this study) to mice, mouse cages, cell samples and images before processing, to ensure unbiased analysis.
n/a Involved in the study
Antibodies
Eukaryotic cell lines
Palaeontology and archaeology
Animals and other organisms
Clinical data
Dual use research of concern
Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,
system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
Materials & experimental systems Methods
n/a Involved in the study
ChIP-seq
Flow cytometry
MRI-based neuroimaging
Antibodies
Antibodies used
3
Immunofluorescence
Primary antibodies
Cell signaling technology, Cat#2748S, Rabbit polyclonal anti-Sox2, lot: 2
Abcam, Cat#ab153668, Chicken polyclonal anti-Doublecortin, lot: GR3334644-1
Merckmillipore, Cat#ABE457, Rabbit polyclonal anti-cFos, lot: 3168266
Abcam, Cat#ab4674, Chicken polyclonal anti-GFAP, lot: GR3424848-1
Santa Cruz Biotechnology, Cat#sc-5359, Goat polyclonal anti- MAP-2 (D-19), lot: D152
LacI: Sigma-Aldrich, Cat#05-503I, Mouse Monoclonal anti-LacI, Clone 9A5
Secondary antibodies
ThermoFisher, Cat#A-21244, Goat polyclonal anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647, Lot:
2086678
ThermoFisher, Cat#A-11039, Goat polyclonal anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor™ 488, Lot: 2180688
Merckmillipore, Cat#AP180SA6, Donkey polyclonal anti-Goat IgG (H+L) Secondary Antibody, Alexa Fluor® 647, Lot: 3743391
Jacksonimmuno, Cat#703-545-155, Donkey polyclonal Anti-Chicken IgY (IgG) (H+L) Secondary Antibody Alexa Fluor® 488 AffiniPure,
Lot: 151980
ThermoFisher, Cat#A-21235, Goat polyclonal anti-Mouse IgG (H+L), Cross-Adsorbed Secondary Antibody, Alexa Fluor, 647
Western blot
Primary antibodies
LAMP1: Abcam, Cat#ab24170S, Rabbit polyclonal anti-LAMP1
LC3B: Sigmaaldrich, Cat#L7543S, Rabbit polyclonal anti-LC3B
Beclin-1 (D40C5): Cell signaling technology, Cat#3495S, Rabbit polyclonal anti- Beclin-1 (D40C5), lot: 6
Atg5 : Cell signaling technology, Cat#12994S,Rabbit polyclonal anti- Atg5, lot: 4
FoxO3a (75D8): Cell signaling technology, Cat#2497S, Rabbit polyclonal anti- FoxO3a (75D8), lot : 6
Smad2/3 (D7G7): Cell signaling technology, Cat#8685S, Rabbit polyclonal anti- Smad2/3 (D7G7), lot : 6
Phospho-p70 S6 Kinase (Thr389) : Cell signaling technology, Cat#9205S, Rabbit polyclonal anti- Phospho-p70 S6 Kinase (Thr389)
DEPTOR : Merckmillipore, Cat#3495S, Rabbit polyclonal anti-DEPTOR
Phospho-4E-BP1 (Thr37/46) (236B4): Cell signaling technology, Cat#2855, Rabbit polyclonal anti-Phospho-4E-BP1 (Thr37/46) (236B4)
p62/SQSTM1 : Abnova, Cat#H00008878-M01, Mouse Monoclonal anti- p62/SQSTM1, Clone 2C11, lot : I2271-2C11
Actin: Sigma-Aldrich, Cat# A5441, Mouse Monoclonal Anti-b-Actin, Clone AC-15
Secondary antibodies
Bio-rad, Cat#1706515, Goat Polyclonal anti-Rabbit IgG-HRP secondary antibody conjugate,
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4
nature portfolio | reporting summary March 2021
Abcam, Cat#ab97240, Goat Polyclonal anti-Mouse IgG1 heavy chain-HRP secondary antibody,
Val idat ion Antibodies used for immunostaining of brain tissue or cells correctly stained the subcellular localization of the protein as in previous
reports (PMID: 31637864, 30341181, 24797482, 19911428, 18499894). Antibodies used for Western blots (phospho- or total
proteins) were validated using appropriate kinase inhibitors or knockout/overexpression experiments.
Additional information for each antibody can be found on the manufacturer's website as follows:
rabbit polyclonal anti-Sox2 (1:100, Cell Signaling Technology, #2748), https://www.cellsignal.com/products/primary-antibodies/sox2-
antibody/2748
chicken polyclonal anti-doublecortin (DCX) (1:400, abcam, ab153668), https://www.abcam.com/doublecortin-antibody-
ab153668.html
rabbit polyclonal anti-cFos (1:2000, ABE457, Millipore), https://www.merckmillipore.com/FR/fr/product/Anti-c-Fos-
Antibody,MM_NF-ABE457
rabbit polyclonalanti-Lamp1 (1:1000, ab24170 abcam), https://www.abcam.com/lamp1-antibody-lysosome-marker-ab24170.html
rabbit polyclonalanti-LC3 (1:1000, L7543, Sigma), https://www.sigmaaldrich.com/FR/fr/product/sigma/l7543
rabbit polyclonalanti-Beclin1 (1:1000, #3495, Cell Signaling), https://www.cellsignal.com/products/primary-antibodies/beclin-1-
d40c5-rabbit-mab/3495?site-search-type=Products&N=4294956287&Ntt=3495%2C&fromPage=plp&_requestid=2730050
rabbit polyclonalanti-Atg5 (1:1000, #12994, Cell Signaling), https://www.cellsignal.com/products/primary-antibodies/atg5-d5f5u-
rabbit-mab/12994
rabbit polyclonal anti-FOXO3a (1:1000, #2497, Cell Signaling Technology), https://www.cellsignal.com/products/primary-antibodies/
foxo3a-75d8-rabbit-mab/2497
rabbit polyclonal anti-total-Smad2/3 (1:1000, #13820, Cell Signaling), https://www.cellsignal.com/products/primary-antibodies/
phospho-smad1-ser463-465-smad5-ser463-465-smad9-ser465-467-d5b10-rabbit-mab/13820
rabbit polyclonal anti-phospho-Smad2/3 (1:1000, #8828, Cell Signaling), https://www.cellsignal.com/products/primary-antibodies/
phospho-smad2-ser465-467-smad3-ser423-425-d27f4-rabbit-mab/8828
rabbit polyclonal anti-phospho-S6K1 (1:1000, #9205, Cell Signaling), https://www.cellsignal.com/products/primary-antibodies/
phospho-p70-s6-kinase-thr389-antibody/9205
rabbit polyclonal anti-Deptor (1:1000, Cat# ABS222, Sigma-Aldrich), https://www.sigmaaldrich.com/FR/fr/product/mm/abs222
rabbit polyclonal anti-phospho-4E-BP1 (1:1000, #2855, Cell Signaling) https://www.cellsignal.com/products/primary-antibodies/
phospho-4e-bp1-thr37-46-236b4-rabbit-mab/2855
mouse monoclonalanti-actin (1:6000, A5441, Sigma), https://www.sigmaaldrich.com/FR/fr/product/sigma/a5441
mouse monoclonal anti-LacI antibody (05-503I, Sigma-Aldrich), https://www.sigmaaldrich.com/FR/fr/product/mm/05503i
Animals and other research organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in
Research
Laboratory animals You n g (2-3-month-old) and aged (18-22-month-old) C57BL/6JRj male mice were obtained from Janvier Labs (France). Eight-week old
C57BL6NTac male mice were obtained from Taconic Biosciences only for depression experiments.Housing conditions were as follows:
dark/light cycle from 7am to 9pm, controlled temperature 20-24C, humidity 60% minimum to 70% maximum.
Wild animals This study did not involve wild animals.
Reporting on sex Findings involving in vivo experiments apply only to male mice.
Field-collected samples The study did not involve samples collected from the field.
Ethics oversight All animal procedures were performed in accordance with French legislation and in compliance with the European Communities
Council Directives (2010/63/UE), according to the regulations of Institut Pasteur Animal Care Committees.
Note that full information on the approval of the study protocol must also be provided in the manuscript.
Clinical data
Policy information about clinical studies
All manuscripts should comply with the ICMJE guidelines for publication of clinical research and a completed CONSORT checklist must be included with all submissions.
Clinical trial registration Provide the trial registration number from ClinicalTrials.gov or an equivalent agency.
Study protocol Note where the full trial protocol can be accessed OR if not available, explain why.
Data collection Describe the settings and locales of data collection, noting the time periods of recruitment and data collection.
Outcomes Describe how you pre-defined primary and secondary outcome measures and how you assessed these measures.
This checklist template is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
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