Supplementary Material
Antidepressants increase human hippocampal neurogenesis
by activating the glucocorticoid receptor
Christoph Anacker, MSc1,2,3, Patricia A. Zunszain, PhD1, Annamaria Cattaneo,
PhD1,4, Livia A. Carvalho, PhD1, Michael J. Garabedian, PhD5,
Sandrine Thuret, PhD3,*, Jack Price, PhD3, and Carmine M. Pariante, MD, PhD1,2,*
1
King’s College London, Institute of Psychiatry, Section of Perinatal Psychiatry and
Stress, Psychiatry and Immunology (SPI-lab), Department of Psychological
Medicine, 125 Coldharbour Lane, SE5 9NU, London, UK
2
National Institute for Health Research “Biomedical Research Centre for Mental
Health”, Institute of Psychiatry and South London and Maudsley NHS Foundation
Trust, London, UK
3
King’s College London, Institute of Psychiatry, Centre for the Cellular Basis of
Behaviour (CCBB), 125 Coldharbour Lane, SE5 9NU, London, UK
4
Genetics Unit, IRCCS San Giovanni di Dio, Fatebenefratelli, Brescia, Italy
5
Department of Microbiology, NYU School of Medicine, 550 First Avenue, New York, NY 10016
Supplementary Materials and Methods
Cell Culture
HPC03A/07 cells were originally obtained from a 12-week old male fetus and
conditionally immortalized with the c-myc-ERTM transgene.1,2,3 This construct is
exclusively responsive to the synthetic steroid 4-hydroxytamoxifen (4-OHT).1
HPC03A/07 cells proliferate indefinitely in the presence of epidermal growth factor
(EGF), fibroblast growth factor (bFGF) and 4-OHT, whereas proliferation is ceased
upon their removal.2,3 During normal expansion, HPC03A/07 cells proliferate with a
doubling time of 72 hours (80% confluence).4 We thus cultured cells for 72 hours in
1
proliferation media containing EGF, bFGF and 4-OHT, and subsequently removed
growth factors and 4-OHT for 7 days to induce differentiation.
HPC03A/07 cells were grown in reduced modified media (RMM) consisting of
Dulbecco’s Modified Eagle’s Media/ F12 (DMEM:F12, Invitrogen, Paisley, UK)
supplemented with 0.03% human albumin (Baxter Healthcare, Compton, UK), 100
µg/ml human apo-transferrin, 16.2 µg/ml human putrescine DiHCl, 5 µg/ml human
rec. insulin, 60ng/ml progesterone, 2 mM L-glutamine and 40 ng/ml sodium selenite.
To maintain proliferation, 10 ng/ml human bFGF, 20 ng/ml human EGF and 100 nM
4-OHT were added. The cell culture media is free of any glucocorticoids unless
dexamethasone or cortisol is used as a treatment.
Immunocytochemistry
Differentiation was assessed by S100ß, O1 and glial-fibrillary-acidic protein (GFAP)
immunocytochemistry (see Supplementary Fig. 11a-d). Briefly, PFA-fixed cells were
incubated in blocking solution (5% normal goat serum (NGS), Alpha Diagnostics,
San Antonio, Texas) in PBS containing 0.3% Triton-X for 2 hours at room
temperature, and with primary antibodies (rabbit anti-S100ß, 1:500, DAKO, and
rabbit anti-GFAP, 1:500, Sigma-Aldrich) at 4°C over night. For O1
immunocytochemistry, cells were incubated in blocking solution without Triton-X for
2 hours at room temperature, and subsequently incubated with primary antibody
(mouse anti-O1, 1:1000, Millipore). Cells were incubated sequentially in blocking
solution for 30min, secondary antibodies (Alexa 594 goat anti-rabbit; 1:1000; Alexa
488 goat anti-mouse, 1:500, Invitrogen) for 1 hour, and Hoechst 33342 dye (0.01
mg/ml, Invitrogen) for 5 min at room temperature. Co-localization studies were
2
conducted with an inverted microscope (IX70, Olympus, Hamburg, Germany) and
ImageJ 1.41 software.
Proliferation assay using lower BrdU concentrations and using Ki67
immunocytochemistry
To assess progenitor cell proliferation with a lower concentration of BrdU (1 µM
instead of 10 µM), HPC03A/07 cells were plated on 96-well plates (Nunclon) at a
density of 1.1 x 104 cell per well, and cultured for 72 hours in the presence of growth
factors and 4-OHT. BrdU was added at a concentration of 1 µM to the culture media 4
hours before the end of the incubation and cells were fixed with 4% PFA for 20 min.
BrdU immunocytochemistry was conducted as described in ‘Materials and Methods’.
Proliferation was further investigated using the proliferation marker Ki67. Cells were
plated as described above and fixed with 4% PFA for 20min after a proliferation
period of 72 hours. Cells were incubated in blocking solution (5% NGS, Alpha
Diagnostics, San Antonio, Texas, USA) in PBS containing 0.1% Triton-X for 1h at
room temperature and subsequently incubated with primary antibody (rabbit antiKi67, 1:500, abcam) at 4° over night. Cells were then incubated in blocking solution
for 30min, secondary antibody (Alexa 594 goat anti-rabbit; 1:1000, Invitrogen) for 1
hour, and Hoechst 33342 dye (0.01 mg/ml, Invitrogen) for 5 min at room temperature.
The number of Ki67-positive cells over total Hoechst 33342 positive cells was
counted in an unbiased setup with an inverted microscope (IX70, Olympus, Hamburg,
Germany) and ImageJ 1.41 software.
3
Cell proliferation after dexamethasone pre-treatment
To investigate changes in cell proliferation after a period of glucocorticoid pretreatment, HPC03A/07 cells were plated on tissue culture flasks at a density of 1.2 x
106 cells/ flask and allowed to adhere to the flask for 24 hours. Cells were then treated
with dexamethasone (1 µM) or the respective vehicle control for 72 hours, and
subsequently plated on 96-well plates at a density of 1.1 x 104 cells/ well. Incubation
was continued on the 96-well plate with dexamethasone (1 µM), sertraline (1 µM) and
with dexamethasone and sertraline co-treatment for another 72 hours. BrdU was
incorporated 4 hours before cessation of treatment and proliferation was assessed as
described above.
Cell death assay
To determine changes in cell death after treatment with dexamethasone (100 nM, 1
µM, 10 µM), sertraline (100 nM, 1 µM), dexamethasone and sertraline co-treatment
(each at 1 µM) and cortisol (10 µM, 100 µM), we selectively labelled dead cells with
propidium iodide. Briefly, propidium iodide was added to the cell culture media at a
concentration of 1 µg/ml 30min before the end of the 72 hours incubation period.
Cells were fixed in 4% PFA for 20min at room temperature, and rinsed with Hoechst
33342 dye (0.01 mg/ml, Invitrogen) for 5 min. The number of propidium iodide
positive cells over total Hoechst 33342 positive cells was counted in an unbiased
setup with an inverted microscope (IX70, Olympus, Hamburg, Germany) and ImageJ
1.41 software.
4
Gene expression analysis
RNA was isolated using RNeasy mini kit (Qiagen, Crawley, UK) according to the
manufacturer’s instructions. Samples were treated with DNase (Ambion, Warrington,
UK) and RNA quantity was assessed by evaluation of the A260/280 and A260/230
ratios using a Nanodrop spectrometer (NanoDrop Technologies, Wilmington, USA).
Superscript III enzyme (Invitrogen) was used to reverse transcribe 1 µg total RNA.
Quantitative Real-Time PCR was performed using HOT FIREPol® EvaGreen® qPCR
Mix (Solis BioDyne, Tartu, Estonia) according to the SYBR Green method. PCR
cycles consisted of an initial heating step at 95 °C for 15 min to activate the
polymerase, 45 PCR cycles were performed. Each cycle consisted of a denaturation
step at 95 °C for 30 s, an annealing step at 60 °C for 30 s and an elongation step at 72
°C for 30 s. For each target primer set, a validation experiment was performed to
demonstrate that PCR efficiencies were within the range of 90-100% and
approximately equal to the efficiencies of the reference genes.
Protein Quantification
Protein concentrations were quantified using a bicinchoninic acid (BCA) colorimetric
assay system (Merck, UK). Protein samples (whole cell lysates or nuclear fractions)
were incubated with the kit reaction mixture in a ratio 1:8 for 30min at 37°C,
absorbance was measured with a microplate reader (DTX 880 Multimode Detector,
Beckman Coulter, Brea, USA) at 562nm. The protein concentration per sample was
determined based on a bovine serum albumin (BSA) standard curve (0µg/ml,
1.25µg/ml, 5µg/ml, 10µg/ml, 25µg/ml, 75µg/ml, 125µg/ml, 250µg/ml, 500µg/ml,
750µg/ml).
5
GR-transactivation assay
Nuclear extracts were obtained after treatment with sertraline for 1 hour, 6 hours, 12
hours, 24 hours and 72 hours using the a commercially available nuclear extraction kit
(Active Motif, Rixensart, Belgium) according to the manufacturers instructions. GR
binding to the GRE consensus sequence (5’-GGTACAnnnTGTTCT-3’) was analyzed
using the TransAM GRTM assay (Active Motif). Briefly, 25 µg nuclear protein
extracts were incubated in binding buffer for 1 hour with the immobilized GRE
oligonucleotide and sequentially incubated with GR-antibody (1:1000, Active Motif)
and with HRP-conjugated antibody (1:1000, Active Motif) for 1 hour at room
temperature. Plates were developed for 6 min before absorbance was read with a
spectrophotometer (DTX 880 Multimode Detector, Beckman Coulter, Brea, USA) at
450 nm with a reference wavelength of 655 nm. Specificity of the assay was
confirmed using wild-type and mutated oligonucleotide sequences.
Western Blot for MAP2 a,b,c isoforms
To investigate the specificity of the MAP2 [HM] antibody (abcam) for mature
neurons (MAP2 isoforms a and b), HPC03A/07 cells were cultured for 72 hours in
proliferation conditions, washed twice for 15min in differentiation media without
EGF, bFGF and 4-hydroxytamoxifen (4-OHT), and cultured under differentiation
conditions for another 7 days. Cells were then lysed in standard RIPA buffer
containing protease and phosphatase inhibitors (Pierce, Rockford, IL) for 20min on a
rotator at 4°C. Protein samples containing 20µg, 30µg and 50µg of total protein were
used for Western Blot analysis (see detailed information on Western Blot analysis in
‘Materials and Methods’) and immunoprobed with the monoclonoal mouse antiMAP2 [HM] antibody (abcam) at a concentration of 2 µg/ml in 5% non-fat dry milk
6
at 4°C over night. Membranes were washed and incubated with a HRP-conjugated
goat anti-mouse secondary antibody (1:2000, Serotec, Kidlington, UK) in 5% non-fat
dry milk in TBST for 1h at room temperature. Membranes were washed and proteins
were visualized with enhanced chemiluminescence (ECL) detection system (GE
Healthcare, UK).
Supplementary Results
HPC03A/07 cultures
The differentiation model implemented in this study was chosen to differentially
investigate the effect of glucocorticoid hormones and antidepressants on early
development of hippocampal progenitor cells. This model does not require the
addition of neurotrophic factors to artificially promote neuronal differentiation, and
therefore excludes such confounding factors. Using this model, we obtained 52% of
TuJ1-positive cells (of which 35% were Dcx-positive neuroblasts, 25% were MAP2positive mature neurons, and 8% labeled positive for both, Dcx and MAP2).
Furthermore, we obtained 27% of S100ß-positive astrocytes, 2% of O1-positive
oligodendrocytes and 19% of GFAP-positive immature progenitor cells in the control
condition. Temporal expression of Dcx in proliferation conditions was 4% of total
cells compared to 35% after 10 days of culture (72 hours of proliferation and 7 days
of differentiation).
BrdU incorporation for 4 hours at the end of the 72 hours proliferation period resulted
in 35% of BrdU-positive cells in the control condition.
7
Sertraline increases the number of TuJ1-positive neurons
To further confirm that sertraline increases neuronal differentiation, HPC03A/07 cells
were immunostained for the pan-neuronal marker TuJ1. Sertraline significantly
increased the number of TuJ1-positive neurons when cells were treated during both,
the proliferation and the differentiation phase (by 18%; Supplementary Fig. 2b) or
only during the proliferation phase (by 17%; Supplementary Fig. 2c). As for the
effects on Dcx and MAP2, no effect of sertraline on the number of TuJ1-positive
neurons was observed when cells were treated only during the differentiation phase
but not during the preceding proliferation phase (Supplementary Fig. 2d).
Dexamethasone dose dependently decreases cell proliferation
In order to choose the appropriate concentration of dexamethasone to be used in
proliferation and differentiation experiments, we treated HPC03A/07 cells with
concentrations ranging from 10 nM to 5 µM dexamethasone. We assessed changes in
cell proliferation using BrdU incorporation and immunocytochemistry for the
endogenous cell proliferation marker Ki67. Dexamethasone dose-dependently
decreased hippocampal progenitor cell proliferation with increasing concentrations
(Supplementary Fig. 12). The number of BrdU positive cells decreased from 35% in
control to 32% (for DEX 10 nM), 31% (for DEX 100 nM), 27% (for DEX 1 µM) and
23% (for DEX 5 µM) (Supplementary Figure 12b). The number of Ki67 positive cells
was decreased from 69% in the vehicle treated control condition, to 63% (DEX 10
nM), 60% (DEX 100 nM), 51% (DEX 1 µM) and 42% (DEX 5 µM) (Supplementary
Figure 12c). Notably, even though the total number of cells, which stop to proliferate,
is higher in the Ki67 condition because all proliferating cells are being labelled, the
8
effect sizes are similar for both BrdU and Ki67 staining (for comparison see
Supplementary Fig. 12b,c).
Different classes of antidepressants have the same effect on cell proliferation
To additionally investigate whether chemically different antidepressants (selective
serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants) have similar
effects on progenitor cell proliferation, we treated HPC03A/07 cells with different
concentrations of sertraline, amitriptyline and clomipramine (100 nM and1 µM), with
or without two different concentrations of dexamethasone (100nM and 1µM). Indeed,
we observed a dose-dependent reduction in cell proliferation for all three
antidepressants tested (Supplementary Fig. 5a-c). If co-treated with dexamethasone,
all three antidepressants increased cell proliferation (Supplementary Fig. 5d-f; squared
columns).
Effects of glucocorticoids and sertraline on cell death
Considering that glucocorticoids have been described to induce cell death in rodent
neurons and neural stem cell cultures,5,6,7 we have conducted propidium iodide live
staining to selectively label dead cells in our dexamethasone and cortisol treated cell
cultures. Using this cell death assay, we have found no significant effects of
dexamethasone on cell death for concentrations up to 1 µM (Supplementary Fig. 10b).
Cell death was only induced by higher concentrations of dexamethasone (10 µM).
Sertraline, and co-treatment of sertraline (1 µM) with dexamethasone (1 µM), did also
not affect cell death at the concentrations used (Supplementary Fig. 10c). Treatment
with the endogenous glucocorticoid cortisol did also not induce cell death at the
concentrations used in this study (10 µM and 100 µM) (Supplementary Fig. 10d).
9
MAP2 specifically labels mature neurons
In our experiments, we investigated neuronal maturation by immunocytochemistry for
MAP2, a marker which is expressed only by mature neurons. To confirm that the
MAP2 [HM] antibody used in this study specifically detects mature neurons in our
HPC03A/07 cultures, we conducted co-labelling experiments of MAP2 and S100ß (a
marker for astrocytes), GFAP (a marker for immature progenitor cells) and TuJ1 (a
marker for both, immature and mature neurons). We did not observe any co-staining
for MAP2 and S100ß or GFAP, confirming that the MAP2 antibody does not detect
astrocytes or immature progenitor cells in our culture (Supplementary Fig. 11a,b).
Furthermore, a subpopulation of TuJ-1-positive cells stained also positive for MAP2,
further supporting the notion that MAP2 is expressed only by mature neurons
(Supplementary Fig. 11c). Co-labelling experiments for MAP2 and O1 (a marker for
oligodendrocytes) could not be performed, because the available antibodies were the
same isotope of the same species. However, the low number of O1-positive
oligodendrocytes in our cultures (~2% of total cells) exhibit a characteristic highly
branched and multipolar morphology which was distinctly different from the
morphology of MAP2 positive neurons (Supplementary Fig. 11d). It can thus also be
excluded that MAP2 co-labels with oligodendrocytes in our cell culture preparations.
Furthermore, we conducted Western Blot analyses on protein lysates of HPC03A/07
cells after 7 days of differentiation. We detected a protein band at 280kDa which
corresponds to the neuronal isoforms MAP2ab. Even for high concentrations of total
protein or longer exposure times, no band was detected at 70kDa, the molecular
weight of the MAP2c isoform (Supplementary Fig. 11e). These data further confirm
that the MAP2 antibody selectively labels mature neurons in differentiated
HPC03A/07 cells.
10
Differential changes in gene expression upon treatment with sertraline and
dexamethasone
In order to explore how sertraline, dexamethasone and dexamethasone and sertraline
co-treatment exert their differential effects on neurogenesis, we investigated changes
in gene expression in the different treatment paradigms at 6 hours, 12 hours, 24 hours,
48 hours and 72 hours during progenitor cell proliferation, a phase which is critical
for the antidepressant-induced increase in neuronal differentiation (as shown in Fig.
1). In addition to the gene expression changes presented in ‘Results’ (GR, p27Kip1 and
p57Kip2, p11, CCND1, HDM2, FKBP5, SGK1, FOXO1, GADD45B), we specifically
investigated changes in the cell cycle related genes p21Cip1, CDK2, CCNA2, p53, and
BDNF, which have been implicated in depression and antidepressant-related changes
in neurogenesis8,9,10,11,12-14,15,16,17 (Supplementary Fig. 8a-d, k, m). No changes in gene
expression were observed for the cell cycle genes p21Cip1, CDK2, CCNA2 and p53
upon any of the treatments used (Supplementary Fig. 8a-d). Furthermore, we did not
observe any changes in BDNF gene expression for sertraline, dexamethasone or
sertraline and dexamethasone co-treatment (Supplementary Fig. 8k).
Rolipram decreases cell proliferation by activating the GR
To explore the effect of the PDE4-inhibitor rolipram on neurogenesis, we have treated
proliferating HPC03A/07 cells with concentrations of 10 nM to 10 µM rolipram.
Rolipram did not alter cell proliferation at a concentration of 100 nM, the
concentration which we used for our co-treatment experiments (Supplementary Fig.
6). At a higher concentration of 10 µM, however, rolipram significantly decreased cell
proliferation, similar to the effect of sertraline (Supplementary Fig. 6). To test
whether this effect, like the effect of sertraline, is dependent on the GR, we co-treated
11
cells with rolipram (10 µM) and the GR-antagonist RU486 (50 nM). RU486
counteracted the effect of rolipram on cell proliferation, confirming that cAMP/PKA
signaling decreases progenitor cell proliferation at least partially by an effect on the
GR, just like sertraline does. It is reasonable to assume that alternative cAMPdependent signaling mechanisms may be activated by rolipram at such high
concentrations. The cAMP response element binding protein (CREB), for example,
has been implicated in neuronal differentiation and may thus represent an additional,
alternative pathway.18,19
Cell proliferation after dexamethasone pre-treatment and cortisol
In order to mimic the effect of glucocorticoids and antidepressants in an in vitro
model similar to the clinical condition, we pre-treated HPC03A/07 hippocampal
progenitor cells for 72 hours with the glucocorticoid dexamethasone (1 µM) before
the commencement of the 72 hours co-treatment period of dexamethasone (1 µM)
with the antidepressant sertraline (1 µM) (for experimental timeline see
Supplementary Fig. 3b). BrdU was incorporated 4 hours before the cessation of the 6
days total treatment period, and cell proliferation was assessed by BrdU
immunocytochemistry. In this condition, in which cells were pre-treated with
dexamethasone before the subsequent co-treatment with sertraline, proliferation was
decreased by dexamethasone and increased after dexamethasone and sertraline cotreatment (Supplementary Fig. 3b). Notably, this is the same effect which we
observed after co-treatment with dexamethasone and sertraline without any preceding
period of dexamethasone treatment (see Fig. 4a and Supplementary Fig. 3b for
comparison).
12
To further model the effect on cell proliferation with an endogenous glucocorticoid,
cells were treated for 72 hours with cortisol (10 µM, 100 µM), sertraline (1 µM) and
cortisol (100 µM) and sertraline (1 µM) together. Cortisol significantly decreased cell
proliferation at a concentration of 100 µM, whereas cell proliferation was increased
when cells were co-treated with cortisol and sertraline (Supplementary Fig. 3a).
Furthermore, cortisol decreased neuronal differentiation into Dcx-positive neuroblasts
and MAP2-positive neurons (Supplementary Fig. 1). This effect is again similar to the
effect of dexamethasone (as described in Fig. 1). The high concentrations of cortisol
used in this study were required to provide specific maximal GR occupancy over MR
occupancy (Anacker et al., unpublished data) and did not induce cell death in our
cultures (Supplementary Fig. 10d).
Supplementary Discussion
Gene interactions
In our study, we found differential effects of antidepressants, glucocorticoids and
antidepressant and glucocorticoid co-treatment on human hippocampal neurogenesis.
Importantly, these effects are dependent on the GR, because co-treatment with the
GR-antagonist RU486 abolishes these effects. Our data demonstrate that the
antidepressant, sertraline, increases neuronal differentiation while concomitantly
decreasing cell proliferation by activating the GR via PKA signaling. In contrast, the
glucocorticoids, dexamethasone and cortisol, decrease both, neuronal differentiation
and progenitor cell proliferation, again by an effect on the GR. Co-treatment of
antidepressants and glucocorticoids, however, increases progenitor cell proliferation
without any changes in neuronal differentiation. These effects are also dependent on
13
the GR. Our gene expression analysis provides a possible answer for these different
GR-dependent effects. Specifically, our data demonstrate that the three different
treatment conditions induce distinct GR phosphorylation patterns and subsequently
activate different sets of genes which have been implicated in neurogenesis, stress,
depression and cell cycle regulation.
Effects of sertraline
Sertraline increases expression of the CDK2-inhibitors p27Kip1 and p57Kip2, as well as
of the serotonin receptor related genes p11 and ß-arrestin-2, while expression of the
growth arrest and DNA damage-inducible gene GADD45B was decreased.
Importantly, p27Kip1 and p57Kip2 are GR-target genes which promote cell cycle exit
and increase neuronal differentiation in the developing rat brain.20, 21,22,23,24 These
findings are thus consistent with the effect of sertraline, because the increased
expression of p27Kip1 and p57Kip2 may account for the decreased cell proliferation and
the increased neuronal differentiation in our model (as shown in Fig. 1 and Fig. 4).
The sertraline-induced expression of the genes, p27Kip1 and p57Kip2, was reduced in
the presence of the GR-antagonist RU486, further confirming their modulation by the
GR. We observed a partial reduction in p57Kip2 expression upon sertraline and RU486
co-treatment. This may be explained by alternative antidepressant-signaling pathways,
such as e.g. CREB activation, which may further regulate p57Kip2 expression. Also,
higher concentrations of RU486 may be required to completely block GR-dependent
p57Kip2 expression. Nevertheless, considering that the concentration of RU486 that we
used (50 nM) is sufficient to abolish the antidepressant-induced effects on
neurogenesis, this partial reduction in p57Kip2 expression by RU486 may already be
sufficient to abolish its biological activity.
14
Furthermore, p11 has recently been shown to be increased upon antidepressant
treatment,25,26 and p11 is also necessary for antidepressants to exert an effect on
neurogenesis in rodents.27,28 Our findings in the cell culture model are in line with this
data from rodents, as we observe an increase in p11 expression after 12 hours of
treatment, the time point of GR-transactivation (Supplementary Fig. 8m and Figure
5g). It is noteworthy that the p11 promoter contains GR-response elements (GREs),29
supporting the notion that p11 expression upon sertraline treatment is indeed mediated
by the GR.
We have also investigated changes in expression of ß-arrestin-2, which has recently
been shown to be increased upon antidepressant treatment in cellular models30 and in
fluoxetine-treated rodents, in which it contributes to antidepressant-induced changes
in neurogenesis.31 We observed a 1.5 fold increase in ß-arrestin-2 expression after 6
hours of treatment with sertraline, which is in line with the studies indicated above.
Effects of dexamethasone
Interestingly, dexamethasone treatment decreased p27Kip1 and p57Kip2 expression and
did not affect p11 and ß-arrestin-2 expression. However, dexamethasone increased the
expression of the stress-responsive genes FKBP5, SGK1, FOXO1 and GADD45B.
These findings demonstrate that sertraline and dexamethasone both act on the GR but
activate different sets of genes (for summary, see Supplementary Fig. 13a and b).
Importantly, FOXO1 has been demonstrated to inhibit cell cycle progression
independent of the CDK2 inhibitors p27Kip1 and p57Kip2, which we find upregulated
only by sertraline treatment, but downregulated by dexamethasone.32 Interestingly,
SGK1 is consistently upregulated upon exposure to stress and glucocorticoids,33,34 and
15
SGK1 is also strongly increased after dexamethasone treatment in our study, which
further supports the use of this glucocorticoid to model stressful conditions. It will be
interesting for future studies to explore the specific role of SGK1 in the
glucocorticoid-dependent decrease in hippocampal neurogenesis.
Effects of sertraline and dexamethasone co-treatment
Co-treatment with sertraline and dexamethasone also reduced expression of p27Kip1
and p57Kip2, did not affect p11, ß-arrestin-2 or GADD45B expression, but increased
cell cycle promoting genes, such as CCND1 and HDM2 (Supplementary Fig. 8).
Again, these data demonstrate that in this co-treatment condition, a third set of genes
is activated which differs from the gene expression changes upon treatment with
either sertraline or dexamethasone alone. Indeed, CCND1 is known to enhance
progenitor cell proliferation but not neuronal differentiation,35,36 and could therefore
significantly contribute to increased cell proliferation without concomitant changes in
neuronal differentiation (as we observe it in the sertraline and dexamethasone cotreatment condition in our study). Furthermore, increased expression of CCND1 has
been shown to prevail over the anti-proliferative effect of FOXO1.32 This mechanistic
link between CCND1 and FOXO1 is important with regards to the changes in
neurogenesis observed in our experiments: Indeed, FOXO1 is upregulated by
dexamethasone, but also by dexamethasone and sertraline co-treatment (most likely
because sertraline alone does not exert any effect on FOXO1 expression as shown in
Supplementary Fig. 8i). However, CCND1 expression is only increased by the cotreatment, which increases cell proliferation, but not by dexamethasone alone
(Supplementary Fig. 8f,i). This increase in CCND1 may therefore overcome the effect
16
of increased FOXO1 expression, and thus ultimately enhance cell proliferation as a
net effect of this co-treatment condition.
Negative findings
No changes in gene expression of the GR-responsive cell cycle gene p53 were
observed upon any of the treatments used (Supplementary Fig. 7d). However, HDM2
has been reported to increase cell proliferation by inhibiting p53 activity,10,37 and the
increase in HDM2 expression may therefore indeed represent a possible mechanism
which contributes to the increased cell proliferation upon dexamethasone and
sertraline co-treatment, possibly by inhibiting p53 activity.
Additionally, we have investigated changes in expression of the neurotrophic factor
BDNF. We did not find changes in BDNF expression for any of the treatment
conditions (Supplementary Fig. 8k), which is consistent with previous studies that
have suggested a role for BDNF in non-hippocampal cell populations, or in neuronal
survival, but not in progenitor cell proliferation.38,12,13
17
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Supplementary Figure Legends
Supplementary Figure 1
Cortisol reduces neuronal differentiation and maturation by activating the GR.
Immunocytochemistry (ICC) for doublecortin (Dcx) and microtubulin-associated
protein-2 (MAP2) was used to assess neuronal differentiation and maturation,
respectively. When HPC03A/07 cells were treated during the proliferation phase (72
hours) and the subsequent differentiation phase (7 days), cortisol (100 µM)
significantly decreased the number of MAP2-positive neurons and of Dcx-positive
neuroblasts. These effects were abolished by the GR-antagonist RU486 (50 nM) (a).
When HPC03A/07 cells were treated only during the proliferation phase, cortisol also
decreased the number of MAP2 and Dcx-positive cells. These effects were again
abolished by RU486 (b). Cortisol treatment only during the differentiation phase did
not exert an effect on MAP2-positive or on Dcx-positive neuroblasts (c). Three
independent experiments were conducted on 3 independent cultures (n=3), 4 wells
were analyzed per treatment condition in each experiment and 3 random, nonoverlapping pictures were analyzed for each well. All data are mean ± s.e.m.
**P<0.01 compared with the corresponding vehicle treated control.
Supplementary Figure 2
Sertraline induces neuronal differentiation into TuJ1-positive neurons.
Immunocytochemistry (ICC) for the pan-neuronal marker TuJ1 was used to assess
neuronal differentiation upon treatment with sertraline (a). When HPC03A/07 cells
were treated during the proliferation phase (72 hours) and the subsequent
differentiation phase (7 days), Sertraline (SERT, 1µM) increased the number of TuJ1-
22
positive neurons (b). The same effect was observed when cells were treated only
during the proliferation phase (c). Treatment only during the differentiation phase did
not affect differentiation into TuJ1-positive neurons (d). Three independent
experiments were conducted on 3 independent cultures (n=3), 4 wells were analyzed
per treatment condition in each experiment and 3 random, non-overlapping pictures
were analyzed for each well. All data are mean ± s.e.m. *P<0.05 compared with the
corresponding vehicle treated control.
Supplementary Figure 3
Glucocorticoids and antidepressants interact to modulate HPC03A/07 proliferation.
Cortisol dose-dependently decreased cell proliferation (One-Way ANOVA, p=
0.0022, F1,4= 5.7, R2= 0.27, n=3). Co-treatment with cortisol (100 µM) and SERT (1
µM) increased cell proliferation (a). Pre-treatment with DEX (1 µM) for 72 hours
before commencement of the 72 hours co-treatment period of DEX+SERT (b, left
panel). Treatment with either DEX or SERT decreased HPC03A/07 proliferation
whereas cell proliferation was significantly increased after co-treatment (b, right
panel). Three independent experiments were conducted on 3 independent cultures
(n=3), 4 wells were analyzed per treatment condition in each experiment and 3
random, non-overlapping pictures were analyzed for each well. All data are mean ±
s.e.m. *P<0.05 and **P<0.01 compared with the corresponding vehicle treated
control.
Supplementary Figure 4
Effects of sertraline on progenitor cell proliferation using 1 µM BrdU.
SERT (1 µM) and DEX (1 µM) decreased the number of BrdU-positive cells (control:
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36%, SERT 1µM: 31%, DEX 1µM: 31% of total cells). These effects were abolished
by the GR-antagonist RU486 (50 nM). Co-treatment of DEX+SERT increased the
number of BrdU-positive cells (40% of total cells). This effect was also abolished by
RU486. RU486 treatment alone did not show any effect. Three independent
experiments were conducted on 3 independent cultures (n=3), 4 wells were analyzed
per treatment condition in each experiment and 3 random, non-overlapping pictures
were analyzed for each well. All data are mean ± s.e.m. *P<0.05 and **P<0.01
compared with the corresponding vehicle treated control.
Supplementary Figure 5
Chemically unrelated antidepressants have similar effects on HPC03A/07
proliferation. HPC03A/07 proliferation was dose-dependently decreased by 100 nM
and 1 µM of sertraline (SERT) (One-Way ANOVA, p= 0.005, F1,3= 10.3, R2= 0.7,
n=4) (a), amitriptyline (AMI) (One-Way ANOVA, p= 0.002, F1,3= 13.5, R2= 0.8,
n=4) (b), and clomipramine (CMI) (One-Way ANOVA, p= 0.046, F1,3= 4.95, R2=
0.6, n=3) (c). SERT dose-dependently increased cell proliferation if co-treated with
DEX 100 nM (One-Way ANOVA, p= 0.02, F1,3= 9.6, R2= 0.8, n=4) and DEX 1 µM
(One-Way ANOVA, p= 0.009, F1,3= 7.1, R2= 0.54, n=4) (d). AMI increased cell
proliferation if co-treated with DEX 100 nM (One-Way ANOVA, p= 0.16, F1,3= 3.6,
R2= 0.7, n=3) and DEX 1 µM (One-Way ANOVA, p= 0.0007, F1,3= 20.9, R2= 0.84,
n=3) (e). CMI also increased cell proliferation if co-treated with DEX 100 nM (OneWay ANOVA, p= 0.003, F1,3= 10.4, R2= 0.65, n=3) and DEX 1 µM (One-Way
ANOVA, p= 0.02, F1,3= 5.4, R2= 0.4, n=3) (f). Three to four independent experiments
were conducted on 3 to 4 independent cultures (n=3 and n=4, respectively), 4 wells
were analyzed per treatment condition in each experiment and 3 random, non-
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overlapping pictures were analyzed for each well. All data are mean ± s.e.m. *P<0.05,
**P<0.01, ***P<0.001 compared with the corresponding vehicle treated control.
Supplementary Figure 6
Rolipram decreases HPC03A/07 proliferation by activating the GR. Rolipram dose
dependently decreases HPC03A/07 proliferation (One-Way ANOVA, p= 0.01, F1,5=
9.6, R2= 0.5, n=3). Co-treatment with RU486 (50 nM) partially abolished the effect of
rolipram (10 µM). Three independent experiments were conducted on 3 independent
cultures (n=3), 4 wells were analyzed per treatment condition in each experiment and
3 random, non-overlapping pictures were analyzed for each well. All data are mean ±
s.e.m. **P<0.01 compared with the corresponding vehicle treated control, *P<0.05
for rolipram (10 µM) vs. rolipram 10µM + RU486.
Supplementary Figure 7
Sertraline and dexamethasone decrease GR mRNA and protein expression after 12
hours of treatment. SERT (1 µM) significantly decreased GR (a) and GRα (b) mRNA
after 12 hours of treatment. SERT, DEX and SERT+DEX co-treatment also
significantly decreased GR protein expression after 12 hours of treatment (c). The
decreased GR protein expression after 12 hours of SERT treatment was abolished by
H89 (d). Representative Western Blots are shown after 12 hours of treatment. All data
are mean ± s.e.m. *P<0.05, **P<0.01 and ***P<0.001 compared with the
corresponding vehicle treated control at the same time point.
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Supplementary Figure 8
Gene expression analysis. Gene expression was analyzed at 6h, 12h, 24h, 48h and 72h
during HPC03A/07 proliferation. Genes are grouped into cell cycle genes (a-f), stress
and glucocorticoid-responsive genes (g-j) and neurotrophic factors and 5-HT-receptor
related genes (k-m). No significant changes were observed for p21Cip1 (a), CDK2 (b),
CCNA2 (c) and p53 (d). The cell cycle promoting genes CCND1 and HDM2 were
significantly upregulated only by DEX+SERT co-treatment (e,f). Among the stress
and glucocorticoid-responsive genes, FKBP5, SGK1 and FOXO1 were significantly
increased by DEX treatment, without any effect of SERT (g-i). The growth arrest
gene GADD45B was upregulated only by DEX, but downregulated by SERT. No
effect on GADD45B expression was observed for DEX+SERT co-treatment (j).
Treatment did not regulate BDNF expression (k). The neurogenesis and 5-HTreceptor related genes p11 and ß-arrestin-2 were increased only by SERT treatment,
but not by DEX or by DEX+SERT co-treatment (l,m). Three independent
experiments were conducted on 3 independent cultures (n=3). All data are mean ±
s.e.m. *P<0.05, **P<0.01 and ***P<0.001 compared with the corresponding vehicle
treated control at the same time point.
Supplementary Figure 9
Treatment did not regulate phosphorylation of the GR at the S226 phosphosite at any
time point. Western Blots for the GR-phosphoisoform S226 is shown after 1 hour of
treatment. S226 phosphorylation is normalized to the expression of the
unphosphorylated, total GR protein.
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Supplementary Figure 10
Cell death analysis. Total cell death was determined by propidium iodide (PI) and
Hoechst 33342 co-staining (a). DEX induced cell death only at concentrations ≥10
µM (One-Way ANOVA, p= 0.006, F1,4= 5.3, R2= 0.4, n=3) (b). SERT, and
DEX+SERT co-treatment did not induce cell death at the concentrations used (OneWay ANOVA, p= 0.27, F1,4= 1.4, R2= 0.1, n=3) (c). CORT did also not induce cell
death at the concentrations used (One-Way ANOVA, p= 0.98, F1,3=1.7, R2= 0.4, n=3).
Three independent experiments were conducted on 3 independent cultures (n=3), 4
wells were analyzed per treatment condition in each experiment and 3 random, nonoverlapping pictures were analyzed for each well. All data are mean ± s.e.m.
**P<0.01 for Newman-Keuls post-hoc test.
Supplementary Figure 11
MAP2 specifically labels mature neurons in differentiated HPC03A/07 cells. MAP2
does not co-label with S100ß (a) or GFAP (b), but co-labels a subpopulation of TuJ1positive neurons (c). O1-positive oligodendrocytes exhibit a characteristic, highly
branched, multipolar morphology which is distinctly different from MAP2-positive
neurons (d). Western Blot analysis for MAP2 in differentiated cell cultures revealed a
protein band at 280kDa, corresponding to the neuronal isoforms MAP2ab. No band
was detected for the MAP2c isoform at 70kDa (e).
Supplementary Figure 12
Dexamethasone dose response. Dexamethasone dose-dependently decreased the
number of BrdU-positive cells (control: 35%, DEX 10nM: 32%, DEX 100nM: 31%,
DEX 1µM: 27%, DEX 5µM: 23% of total cells, One-Way ANOVA, p<0.0001,
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F1,5=11.53, R2= 0.4, n=6) (b) and of Ki67-positive cells (control: 70%, DEX 10nM:
63%, DEX 100nM: 60%, DEX 1µM: 51%, DEX 5µM: 42% of total cells, One-Way
ANOVA, p= 0.0003, F1,5=10.38, R2= 0.7, n=4) (c). Independent experiments were
conducted on independent cultures (n), 4 wells were analyzed per treatment condition
in each experiment and 3 random, non-overlapping pictures were analyzed for each
well. All data are mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001 for Newman-Keuls
post-hoc test.
Supplementary Figure 13
Proposed mechanism of antidepressant action. Sertraline induces GR-dependent gene
transcription by activating the GR via PKA-signaling. Sertraline initially
downregulates, and then continuously upregulates GR phosphorylation at the S203
phosphosite via PKA-signaling. This GR-phosphoisoform (S203) then ultimately
activates gene expression of a particular subset of genes which cause cell cycle exit
and neuronal differentiation (a). Dexamethasone binds directly to the GR and thereby
induces an initial increase in S203 phosphorylation, followed by a decrease in S203
phosphorylation which is independent of PKA-signaling. Dexamethasone strongly
induces phosphorylation at the S211 phosphosite, which is again independent of PKA.
This GR-phosphoisoform (S211) ultimately initiates transcription of a second set of
genes which causes decreased cell proliferation and decreased neuronal differentiation
(b). Co-treatment with sertraline and glucocorticoids results in yet a third pattern of
GR phosphorylation (increased phosphorylation at S203 and hyperphosphorylation at
S211). This third phosphoisoform then induces transcription of yet a third set of
genes, which leads to increased progenitor cell proliferation without concomitant
changes in neuronal differentiation (c).
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