letterS

A negative regulator of MAP kinase causes
depressive behavior© 2010 Nature America, Inc. All rights reserved.

Vanja Duric1, Mounira Banasr1, Pawel Licznerski1, Heath D Schmidt1, Craig A Stockmeier2,3, Arthur A Simen1,
Samuel S Newton1 & Ronald S Duman1
The lifetime prevalence (~16%)1 and the economic
burden ($100 billion annually)2,3 associated with major
depressive disorder (MDD) make it one of the most commonand debilitating neurobiological illnesses. To date, theexact cellular and molecular mechanisms underlying thepathophysiology of MDD have not been identified. Here weuse whole-genome expression profiling of postmortem tissue
and show significantly increased expression of mitogenactivated protein kinase (MAPK) phosphatase-1 (MKP-1,
encoded by DUSP1, but hereafter called MKP-1) in the
hippocampal subfields of subjects with MDD compared tomatched controls. MKP-1, also known as dual-specificity
phosphatase-1 (DUSP1), is a member of a family of proteinsthat dephosphorylate both threonine and tyrosine residues
and thereby serves as a key negative regulator of the MAPK
cascade4, a major signaling pathway involved in neuronalplasticity, function and survival5,6. We tested the role of altered
MKP-1 expression in rat and mouse models of depression
and found that increased hippocampal MKP-1 expression,
as a result of stress or viral-mediated gene transfer, causes
depressive behaviors. Conversely, chronic antidepressanttreatment normalizes stress-induced MKP-1 expression andbehavior, and mice lacking MKP-1 are resilient to stress.
These postmortem and preclinical studies identify MKP-1 asa key factor in MDD pathophysiology and as a new target fortherapeutic interventions.
Brain imaging and postmortem studies have provided evidence ofchanges in the cellular architecture of several limbic brain regions, mostnotably atrophy of hippocampal pyramidal neurons and a correspond-­
ing reduction in the volume of this region, in individuals with MDD7–10.
Preclinical studies also show that stress causes atrophy of the apical den-­drites of pyramidal neurons and decreases neurogenesis in the dentate
gyrus of the adult hippocampus11–13. Such alterations of the structureand function of the hippocampus could contribute to certain aspects of
MDD, including disruption of cognition, depressed mood, helplessness,anhedonia and control of the hypothalamic-­pituitary-adrenal axis14–18.
To characterize the molecular changes underlying the pathophysio­
logy of MDD, we conducted a whole-genome expression analysis of

postmortem hippocampal tissues from 21 individuals with depres-­sion and 18 healthy controls that were matched for age, gender,­tissue pH and postmortem interval (Supplementary Tables 1 and 2).
To decrease tissue heterogeneity, we conducted the analysis on
two microdissected hippocampal subfields, the dentate gyrus
granule cell layer and CA1 pyramidal cell layer (Supplementary
Data 1 and 2 contain complete lists of regulated genes). Rodent
studies have shown that stress causes atrophy of CA3 pyramidalcells11,12, but we could not reliably dissect this cell layer from humansections. We extracted total RNA and used the resulting cDNA forwhole-genome microarray analysis (48,958 probes). We identi-­
fied MKP-1 as significantly dysregulated in both the dentate gyrus(2.3-fold increase, P = 0.038) and CA1 (2.4-fold increase, P = 0.004)
of MDD subjects (Fig. 1a). Of the subjects with depression, 12 hada prescription for an antidepressant drug filled in the last monthof life, but only one depressed subject had measurable amounts ofan antidepressant in the bloodstream (Supplementary Table 2).
We also examined the expression of other members of the DUSP
family. mRNA levels of DUSP2 and DUSP19 were increased in thedentate gyrus, whereas DUSP9 and DUSP12 were significantly
upregulated and DUSP24 was significantly downregulated in theCA1 (Fig. 1a and Supplementary Table 3 contains a completelist). MKP-1 was the only DUSP whose expression was significantly
increased in both hippocampal subregions. Secondary validationof the microarray results by quantitative PCR (qPCR) confirmedthat MKP-1 mRNA expression was higher by over twofold in thedentate gyrus and CA1 of subjects with MDD compared to controls(Fig. 1b). We also assessed MKP-1 expression by in situ hybridiza-­tion in a separate cohort of subjects with MDD and matched healthy
controls (Supplementary Table 4). Levels of MKP-1 mRNA in thedentate gyrus and CA1 of subjects with MDD were increased by 31%(P = 0.016) and 16% (P = 0.128), respectively (Fig. 1c).
Because MKP-1 is a major negative regulator of the neurotrophic
factor–MAPK cascade, we also examined other components of thispathway. We observed significant downregulation of MEK2 (encoding
MAPK kinase-2) in the CA1 of subjects with MDD, whereas expres-­sion of ERK2, which encodes a MAPK directly regulated by MKP-1,
was decreased in the dentate gyrus (Fig. 1d–f). Sustained induction of
MKP-1 would lead to inhibition of ERK signaling (that is, decreased

1Department of Psychiatry, Yale University, New Haven, Connecticut, USA. 2Department of Psychiatry and Human Behavior, University of Mississippi Medical Center,

Jackson, Mississippi, USA. 3Department of Psychiatry, Case Western Reserve University, Cleveland, Ohio, USA. Correspondence should be addressed to
R.S.D. (ronald.duman@yale.edu).
Received 9 March; accepted 24 August; published online 17 October 2010; doi:10.1038/nm.2219

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levels of phospho-ERK), which has been previously demonstrated in
the postmortem hippocampus of depressed individuals who commit-­
ted suicide19–21. Moreover, we observed reduced gene expression ofother known MAPK signaling target proteins (RPS6KA5, encoding
mitogen- and stress-activated protein kinase-1 (here called MSK1)),
downstream transcription factors (CREB1, encoding cAMP response
element–binding protein, and ATF6B, encoding cAMP responsive
element–binding protein–like-1 (here called CREBL1)) and growth
factors (BDNF, encoding brain-derived neurotrophic factor, VGF,
encoding VGF nerve growth factor inducible, and VEGFA, encoding
vascular endothelial growth factor) in depressed subjects (Fig. 1d,e),
which is consistent with previous reports22–25 and could subsequently
contribute to the functional consequences of reduced ERK signaling.
All of the downregulated genes contain a cAMP response element(CRE) (Supplementary Table 5), suggesting that decreased MAPKCREB signaling could account for decreased expression of these genes
in MDD. However, the expression of most of the genes is decreased
in only one hippocampal subfield, and other genes containing a CRE
are not regulated in either subfield (Fig. 1d,e), indicating that themechanisms underlying altered gene expression are more complex
than a simple reduction in CRE-CREB activity. Together, the results
indicate a disruption of MAPK signaling at multiple levels, including
the expression of certain target genes.
To further examine the regulation and function of MKP-1, we con-­ducted studies in a rat chronic unpredictable stress (CUS) model ­(Fig. 2a),

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© 2010 Nature America, Inc. All rights reserved.

letters

CBP

CREB

BDNF, TRKB, NGF,
VEGF, VGF, NPY,...
Cell proliferation
Growth and maturation
Neural plasticity
Survival and resilience

VOLUME 16 | NUMBER 11 | NOVEMBER 2010

one of the most valid and relevant rodent models of depression26–28.
CUS results in depressive-like behaviors, notably anhedonia andhelplessness, core symptoms of MDD that are reversed by chronic,
but not acute, antidepressant administration26–28. Exposure to CUS(35 d) decreased sucrose preference and increased escape failures inan active avoidance test, measures of anhedonia and helplessness,
respectively (Fig. 2b, P < 0.05). Analysis of the hippocampal subfields
showed that CUS exposure caused a significant increase in levels of
Mkp-1 mRNA in the dentate gyrus (47%, F1,14 = 22.48, P = 0.0003),
CA1 (71%, F1,14 = 27.29, P = 0.0001) and CA3 (62%, F1,14 = 17.30,
P = 0.001) (Fig. 2c). Administration of fluoxetine, which blocks the
CUS-induced anhedonia and helpless behavior (Fig. 2b), also reversed
the CUS-induced upregulation of Mkp-1 mRNA in the dentate gyrus
and partially normalized the increase in CA1, but not CA3, cell layers(Fig. 2c). Two-way analysis of variance (ANOVA) showed a significant
interaction between CUS and fluoxetine in the dentate gyrus and CA1(F1,14 = 22.40, P = 0.0003; F1,14 = 8.65, P = 0.0107, respectively) and atrend in CA3 (F1,14 = 3.52, P = 0.081). CUS-exposed rats treated withfluoxetine showed ~30% reductions in Mkp-1 mRNA levels withinboth the dentate gyrus and CA1 compared to CUS alone (P = 0.0016
and P = 0.023, respectively). Neither stress nor fluoxetine had an effecton Mkp-1 gene expression in the cortex, indicating that this is not a
global effect (Fig. 2c). We generated a parallel set of CUS-exposed
rats with similar depressive-like behavioral deficits (data not shown)and used them for assessing Mkp-1 protein abundance. Western blot
analysis of whole-hippocampal homogenates (Fig. 2d) showed that
CUS induced a significant 30% increase in Mkp-1 protein abundance(F1,16 = 9.36, P = 0.003). Further ANOVA analysis showed a probabilityof P = 0.077 (F1,16 = 3.56) toward an interaction between CUS andfluoxetine, indicating that administration of fluoxetine only partiallyattenuated CUS-mediated induction of Mkp-1 protein expression.
CUS-mediated increases in hippocampal Mkp-1 mRNA and protein
levels suggest a potential role for stress- and depression-induced ele-­vation of adrenal glucocorticoids, which is often observed in people
with MDD29,30 and is consistent with reports that MKP-1 is a stressand glucocorticoid-responsive immediate-early gene31–33.
Despite the evidence that MKP-1 is dysregulated in MDD and
CUS, there are no data linking altered MKP-1 or other DUSP
Figure 1 MKP-1 is dysregulated in major depressive disorder (MDD).
(a) Microarray analysis of MDD postmortem brain samples showing alterationsin the expression of DUSP genes in hippocampal subfields. (b) qPCR
analysis of MKP-1 gene expression in samples from the same cohort asin a. Data are expressed as mean fold change ± s.e.m. (n = 6); *P ≤ 0.05
compared to the healthy controls (Student’s t test). (c) Representative
autoradiographs and quantitative analysis of hippocampal MKP-1 mRNAlevels by in situ hybridization in a separate cohort of subjects with MDD
and matched controls (scale bar, 5 mm). Results are shown as percentage
increase for each control and MDD-affected subject. *P < 0.02
compared to the healthy controls (Student’s t test). DG, dentate gyrus.
(d,e) Microarray-based expression levels of MAPKs (d) and downstream
transcription factors and target genes (e). CREBBP, CREB binding
protein (CBP); NPY, neuropeptide Y; RAF1, v-raf-1 murine leukemia viraloncogene homolog-1;RPS6KA2, ribosomal S6 kinase (RSK). (f) Model forneurotrophic and growth factor receptor activation of MAPK, downstream
transcription factors, and target genes. Microarray results (a,d and e) are
shown as an average fold change (dentate gyrus, n = 14; CA1, n = 15);
*P < 0.05, †P < 0.06 compared to the healthy controls (permutation tests,
P value adjusted to false discovery rate at 0.05). Fold change for specificsplice variants is reported for DUSP19.2, DUSP24.2, RPS6KA5.2 and
VEGFa.2. FLK-1, fetal liver kinase-1; TRKA–C, tyrosine kinase receptors
A,B and C; IGF-1R, insulin-like growth factor-1 receptor; FGFR1-4,
fibroblast growth factor receptors 1, 2, 3 and 4.

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lettersacSaline or fluoxetine i.p. injections (21 d)

d
CA1

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Figure 2 Influence of CUS and antidepressant treatment on behavior and MKP-1 expression. (a) Schematic of the rat CUS paradigm. Rats were exposed
to CUS or control conditions and were then given either saline or fluoxetine (FLX) for 21 d. Locomotor activity (LA), active avoidance test (AAT) andsucrose preference test (SPT) behaviors were determined. (b) Behavioral results for AAT and SPT, expressed as mean ± s.e.m. (n = 8). (c) Representative
autoradiographs (top) and quantitative analysis (bottom) of Mkp-1 mRNA levels by in situ hybridization on coronal sections of rat hippocampus (scale bar,
1.0 mm). Results are expressed as mean ± s.e.m. (n = 4 or 5). (d) Western blot analysis showing the effects of CUS and FLX treatments on hippocampal
Mkp-1 protein abundance. Tissue amounts of β-actin were used as loading controls. Data are expressed as mean ± s.e.m. percentage change overnonstressed control group (n = 5); *P < 0.05 compared to the nonstressed control group, #P < 0.05 compared to CUS group (two-way ANOVA andFisher’s protected least significant difference post hoc analysis).

subtypes with depressive behaviors. To directly address this issue, weused both viral vector and mutant mouse approaches to determine theinfluence of increased expression or deletion of MKP-1 on depression
behaviors in rat and mouse models. We used a viral vector (recom-­binant adeno-associated virus (rAAV)) to locally express Mkp-1
in the hippocampal subfields of rat brain (Fig. 3a). We targeted
infusions of rAAV–Mkp-1 to the dentate gyrus cell layer because of
the opposing regulation of Mkp-1 by CUS and antidepressant treat-­ment (Fig. 2). Viral infusions increased Mkp-1 expression ­primarilyarAAV-control
ITR

CMV

GFP

ITR

CMV

Mkp-1

CMV

rAAV-Mkp-1
ITR

GFP

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ITR

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rAAV-control
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Anti-GFP
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Anti-GFP*in the dentate gyrus, although we also observed increases in the
CA1, probably as a result of the virus traveling up the cannula track(Fig. 3b,c). Behavioral analysis showed that rAAV–Mkp-1 infu-­
sion into unstressed rats produced anhedonic responses, evident
from significantly decreased sucrose preference (Fig. 3d, P = 0.002)
and increased escape failures in the active avoidance test (Fig. 3e,
P = 0.021), behaviors similar to those observed in rats exposed to CUS(Fig. 2b). Infusion of rAAV–Mkp-1 also increased the latency to feedin the novelty-suppressed feeding test (Fig. 3f, P = 0.093) and signifi-­
cantly increased immobility in the forced swim test (Supplementary
Fig. 1, P = 0.021). There were no significant effects in the elevated plus
maze or on locomotor activity, indicating no change in overall ambu-­
latory behavior (Supplementary Fig. 1). Subsequent studies showedthat infusions of rAAV–Mkp-1 into the CA1 subfield also decreased
sucrose preference (data not shown). These results demonstrate thattargeted viral expression of Mkp-1 in the dentate gyrus subfield ofnonstressed rats produces profound depressive-like responses similar
to the effect of CUS.
We examined the influence of MKP-1 deletion on behavior in Mkp1–null (Mkp-1−/−) mice. Previous studies have reported that Mkp-1−/−
mice have no obvious behavioral or histological abnormalities34 andrAAV–Mkp-1

f

900
Latency to feed (s)dNumber of escape failures

rAAV–Mkp-1
Anti-GFP

Percentage sucrose
consumed in total fluid© 2010 Nature America, Inc. All rights reserved.

#

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consumed in total fluid100

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Figure 3 Influence of MKP-1 overexpression on behavior in rodent
models of depression. (a) Depiction of the rAAV engineered to locallyoverexpress MKP-1 (rAAV–Mkp-1), and compared to a control vector that
expresses green fluorescent protein (rAAV-GFP). ITR, inverted terminal
repeats; CMV, cytomegalovirus promoter. (b,c) Rats received bilateral
intrahippocampal infusions of rAAV-Mkp-1 or rAAV-GFP. Expression levelsof GFP protein (b) and Mkp-1 mRNA (c) are shown (representative images;
scale bars in b, 100 μm; scale bar in c, 500 μm). (d–f) Effects of rAAVMkp-1 infusions on rat behavior, compared to rAAV-GFP controls, for thepercentage sucrose consumed compared to total fluid consumption (waterand sucrose) in the SPT (d), the number of escape failures in the AAT (e)and latency to feed in the novelty suppressed feeding test (f). Behavioral
data are expressed as mean ± s.e.m. (n = 6–9); *P < 0.02, †P < 0.10
compared to the rAAV-GFP control group (Student’s t test).

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letters
Figure 4 Influence of MKP-1 deletion
CUS
Nonstressedon behavioral models of depression.
D0
D17
D20
D30
D32
D33 D35
Mkp-1+/++/+–/–(a) Experimental paradigm for behavioral testing
pErk1
CUS
pErk2
and CUS exposure of Mkp-1–knockout mice
Erk1
(Mkp-1−/−; n = 9 or 10) and WT littermates
SCT
SCT
WCT
SCT OFT EPM
Erk2(Mkp-1+/+; n = 8). SCT, sucrose consumption
β-actintest; WCT, water consumption test; OFT,
open field test; EPM, elevated plus maze.
pErk1
pErk2
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16
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#
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#
(b) Baseline sucrose consumption on day 0,#
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14followed by post-stress measurements
100
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*
conducted on days 17 and 30. Water
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consumption was also measured throughout the
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stress paradigm (data shown for water test on
20
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15
1.5
day 20). (c) The number of entries into the open
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or closed arms, and the total time spent in the
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open arms during the elevated plus maze test.
0
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0
Results are expressed as mean ± s.e.m.; *P < 0.05#
compared to the WT control group and P < 0.05
compared to the WT stress group (sucrose
Mkp-1+/+
Mkp-1–/–
CUS
and water consumption: repeated-measures
ANOVA and Dunnett’s test; elevated plus maze:
Student’s t test). (d) Representative blots (top) and quantitative results (bottom) of western blot analysis showing the effects of CUS on hippocampal
phospho-Erk (pErk) abundance in WT and Mkp-1−/− mice compared to nonstressed WT control mice. Tissue amounts of total Erk and β-actin wereused as loading controls. Optical density values are expressed as a ratio of phospho-Erk to total Erk. Data are expressed as mean ± s.e.m. percentagechange over nonstressed WT control (n = 3); *P < 0.05 compared to WT control group and #P < 0.05 compared to the WT stress group (ANOVA andStudent-Newman-Keuls’ post hoc analysis).

adnature medicine

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pErk (% control)

+

M
kp
-1 +
/+
M
kp
1 –/–
N
on
s
M treskp s- 1 + ed
/

C
lo
ar se
m d

Number of entriesOar penmDay

20

Water (ml)

(b Da
as y
el 0
in
e)
D
ay
17
D
ay
30

© 2010 Nature America, Inc. All rights reserved.

have normal locomotor activity and food intake, although there is areduction in weight with age due to an increase in fat metabolism35.
Our preliminary baseline behavioral analysis (before initiation of
CUS) was consistent with these reports (that is, there were no grossdifferences between Mkp-1−/− and wild-type (WT or Mkp-1+/+) lit-­
termate controls in the open field, forced swim or elevated plus maze
tests; Supplementary Fig. 2). We conducted experiments to determine
whether deletion of Mkp-1 influences the response to CUS (Fig. 4a).
Prior to stress, we observed no difference between Mkp-1−/− and WT
mice in sucrose consumption, but exposure to CUS resulted in a pro-­
gressive, significant reduction in sucrose consumption in WT mice,indicative of stress-induced anhedonia (Fig. 4b, P < 0.05). However,Mkp-1−/− mice exposed to CUS consumed sucrose volumes that weresimilar to the baseline levels (measured before exposure to stress) andwere significantly higher than the sucrose volumes consumed by theWT group exposed to stress (Fig. 4b, P < 0.05). We saw similar results
on days 17 and 30 of CUS, indicating that the effects were persistent.
In contrast, there was no significant effect on water consumption(Fig. 4b) or time spent in an open field (center versus peripheral
zones; Supplementary Fig. 3). In the elevated plus maze, Mkp-1−/−
mice exposed to CUS spent significantly more time in the open arms
compared to CUS-exposed WT mice (P = 0.050), but there was noeffect on the number of entries into the open arms (Fig. 4c). These
results demonstrate that Mkp-1−/− mice are normal in the absence ofstress and are resistant to CUS-induced behavioral deficits.
We assessed the function of MAPK signaling by analysis of phosphoErk in the hippocampus. Exposure to CUS decreased the amount ofboth phospho-Erk1 and phospho-Erk2 (Fig. 4d). The CUS effect on
phospho-Erk2 amounts in WT mice was more robust and statisticallysignificant compared to nonstressed controls (P = 0.049) or to Mkp-1−/−
mice (P = 0.014). There was no significant effect of CUS on phosphoErk1 and phospho-Erk2 in Mkp-1−/− mice compared to nonstressed
controls, and there were no significant effects on total Erk under anyof the conditions tested. These results are consistent with the hypo­thesis that decreased ERK signaling, as well as sucrose consumption, in
response to CUS requires MKP-1. The results are also consistent withprevious studies demonstrating that pharmacological blockade or nullTime in open arm (s)cSucrose (ml)b­ utation of MEK-ERK signaling prevents antidepressant responses36–38,malthough these studies have been confounded by the locomotor activat-­
ing effects of chemical inhibitors as well as deletion of ERK38–40.
Our results indicate that induction of MKP-1 is not only a directconsequence of stress but also a key negative regulator of MAPK that
contributes to the expression of depressive symptoms. ERK signaling
and function have been linked with synaptic plasticity and survival
of neurons5,6, and sustained disruption of this pathway via MKP-1
would be expected to have negative consequences on the function ofpyramidal and granule cells in the hippocampus. The stress resistanceobserved in Mkp-1−/− mice also indicates that pharmacological block-­ade of MKP-1 would produce a resilient or antidepressant response tostress, or possibly an enhanced response to other classes of antidepres-­sants. Although kinases have received more attention in the controlof biological processes, the enzymatic power of phosphatases is muchgreater (100 to 1,000 times), because dephosphorylation is a direct
and more efficient process than phosphorylation4. Phosphatases suchas MKP-1 are powerful negative regulators of intracellular signalingthat represent promising new drug targets for treating depression andpossibly other mood disorders.
Methods
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/naturemedicine/.
Accession codes. Microarray data have been deposited in the Gene
Expression Omnibus with accession code GSE24095.
Note: Supplementary information is available on the Nature Medicine website.
Acknowledgments
This work is supported by US Public Health Service grants MH45481 (R.S.D.),
2 P01 MH25642 (R.S.D.), MH67996 (C.A.S.) and P20 RR17701 (C.A.S.) andthe Connecticut Mental Health Center (R.S.D.). We acknowledge the invaluable
contributions made by the families consenting to donate brain tissue and beinterviewed. We also thank the Cuyahoga County Coroner and staff for their
willing assistance. We are grateful for the contributions of J. Overholser, G. Jurjus,
H. Meltzer, L. Konick, L. Dieter, N. Herbst, G. Mahajan, H. Kooiman and J. Cobb in
the psychiatric assessment and human tissue dissection and preparation. We also1331

letters
thank R. Terwilliger and H.J. Kang for technical assistance with gene expression
analysis, A. Bennett (Yale University School of Medicine) for Mkp-1−/− mice,
X.Y. Li for assistance in breeding and genotyping Mkp-1−/− mice and R. DiLeone(Yale University School of Medicine) for generously donating AAV-CMV-GFP
plasmid used to generate recombinant AAVs.

© 2010 Nature America, Inc. All rights reserved.

AUTHOR CONTRIBUTIONS
V.D. prepared the original draft of the manuscript and was involved in all aspectsof the experimental design and research, including execution of all microarray andmolecular experiments, as well as behavioral tests in rats and mice. M.B. conducted
the behavioral aspects of the rat CUS study, assisted with rat surgeries and was
involved in analysis and interpretation of behavioral tests. P.L. was responsible
for optimization, construction and preparation of recombinant AAVs. H.D.S.
conducted baseline behavior tests in Mkp-1−/− mice. C.A.S. was responsible forhuman tissue generation and preparation of relevant human subjects’ informationtables and methodology. A.A.S. conducted statistical analysis of microarrayexperiments. S.S.N. assisted in the development and optimization of microarrayexperiments. R.S.D. was involved in all aspects of study design, data analysis,
interpretation of results and preparation of the manuscript and figures. All authors
discussed the results presented in the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturemedicine/.
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.
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VOLUME 16 | NUMBER 11 | NOVEMBER 2010

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ONLINE METHODS© 2010 Nature America, Inc. All rights reserved.

Human subjects and tissue preparation. We obtained tissues from 28
depressed subjects and 25 age-matched psychiatrically healthy control sub-­jects at autopsy from the Coroner’s Office of Cuyahoga County, Cleveland,Ohio, USA. An ethical protocol approved by the Institutional Review Boardof the University Hospitals of Cleveland was used, and informed written con-­sent was obtained from the next-of-kin for all subjects. All depressed subjects(12 women and 16 men) met diagnostic criteria for MDD according to the
Diagnostic and Statistical Manual of Mental Disorders IV. The control subjects(12 women and 13 men) never met criteria for an axis I disorder at any timein their lives. For the microarray analysis and qPCR, samples from 15 pairs ofsubjects for the dentate gyrus and CA1 were matched for age, gender, tissue
pH and postmortem interval (Supplementary Tables 1 and 2). For the micro­
array and qPCR studies, we collected punches from the granule cell layer ofthe dentate gyrus and the CA1 pyramidal cell layers. For in situ hybridization,
we used 20-μm-thick frozen hippocampal sections from a separate cohortof seven pairs of subjects matched for age, gender, tissue pH and postmor-­
tem interval (Supplementary Table 3). For full description of human subject
selection process, MDD diagnosis criteria and tissue collection, please see theSupplementary Methods.
Microarray analysis. We used human whole-genome expression MI Readymicroarrays (Microarray, Inc.) to analyze changes in gene expression. Thirty
two-channel arrays were used to hybridize all dentate gyrus and CA1 samples.
Data were analyzed with R programming language and Bioconductor
software (http://www.r-project.org/ and http://www.bioconductor.org); statis-­tical analysis was executed by 1,000 permutation tests with a high-performancecomputing cluster. We then adjusted P values to control false discovery rate at
0.05 with the Q-value package. We calculated P values with distributions gene­
rated by permutation methods, and standard error estimates were therefore
not used in the P value calculations (see the Supplementary Methods for fulldetails of microarray experimental conditions and analysis).
Quantitative real-time PCR and in situ hybridization analysis. qPCR was
performed with a hot-start SYBR Green (Qiagen) method. MKP-1 gene foldchanges in subjects with MDD versus controls were determined by using
ΔΔCt, an analytical method that includes normalization against housekeeping
genes PPID (encoding cyclophilin D) and GAPDH (encoding glyceraldehyde
3-phosphate dehydrogenase). For a detailed description of primer design andsequences, in situ hybridization procedures, test conditions and analysis, please
see the Supplementary Methods.
Western blot analysis and immunohistochemistry. Proteins from fresh rat
and mouse hippocampal tissue were electrophoretically separated on an SDSPAGE gel (10% Tris-HCl; Bio-Rad) and transferred to polyvinylidene difluo-­
ride membranes (0.2 μm pores; Millipore) for western blot analysis.

doi:10.1038/nm.2219

Coronal rat brain sections (60 µm) were immunohistochemically stained for
GFP. Images were captured using Olympus Fluoview FV1000 confocal micro-­scope (Olympus Corporation) and Zeiss Axioskop 2 fluorescent microscopewith AxioVision 3.1 software (Carl Zeiss Imaging Solutions GmbH).
For complete technical details of experimental conditions, antibodies used
and analysis, see the Supplementary Methods.
Construction, preparation and infusion of recombinant adeno-associatedvirus. The rat Mkp-1 cDNA was amplified from a rat hippocampal cDNA
library and subcloned into an AAV2 backbone, containing two CMV promot-­ers to independently drive the expression of target protein (Mkp-1) and EGFP.
The same backbone carrying no Mkp-1 cDNA was used as a control (rAAVcontrol). For a full description of recombinant AAV 2/1 pseudotyped virus
preparation and aseptic infusion surgeries, see the Supplementary Methods.
Chronic unpredictable stress and behavioral testing. Male Sprague-Dawley rats
(Charles River), wild-type (Mkp-1+/+) and homozygotic null (Mkp-1−/−) mice34,35(129J/C57BL/6 strain, kindly provided by A. Bennett) were housed in groups oftwo to four per cage under a 12-h light-dark cycle at constant temperature (25 °C)
and humidity with ad libitum access to food and water (except where indicated).
Prior to any treatments or experiments, animals were allowed at least 1 week ofhabituation to the housing conditions. All animals were age and weight matched(rats: 250–300 g, mice: 29–33 g) at the time of the first stressor. The maintenance of
rat and mouse colonies and all animal treatments and procedures were in accord-­ance with US National Institutes of Health laboratory care standards and approvedby the Yale University Care and Use of Laboratory Animals guidelines.
CUS is a rodent model of depression where animals are exposed to a sequenceof mild and unpredictable stressors designed to prevent habituation26,28. Animals
were subjected to a sequence of 12 different stressors (rats: two per day; mice:
three per day) for 35 d as previously described28 (Supplementary Table 6).
For a full description of rodent stress models and behavioral tests used in thisstudy (rats: sucrose preference test, active avoidance test and novelty suppressed
feeding test; mice: sucrose and water consumption tests, elevated plus maze,
open field test and forced swim test), see the Supplementary Methods.
Statistical analyses. Data from molecular and behavioral experiments were ana-­lyzed with Student’s t test for two-group comparisons. Two-way ANOVA with
Fisher’s protected least significant difference post hoc comparison tests was usedin experiments with four groups, and one-way ANOVA followed by StudentNewman-Keuls’ post hoc analysis was performed in experiments with three treat-­ment groups. In behavioral experiments where multiple tests were conducted on
the same sets of animals, repeated-measures ANOVA with Dunnett’s post hoc test
was used. Significance was set at P ≤ 0.05 (StatView 5.0.1, SAS Institute).
Additional methods. Detailed methodology is described in the Supplementary
Methods.

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