A negative regulator of MAP kinase causes depressive behavior

Abstract

The lifetime prevalence (∼16%) and the economic burden ($100 billion annually) associated with major depressive disorder (MDD) make it one of the most common and debilitating neurobiological illnesses. To date, the exact cellular and molecular mechanisms underlying the pathophysiology of MDD have not been identified. Here we use whole-genome expression profiling of postmortem tissue and show significantly increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1, encoded by DUSP1, but hereafter called MKP-1) in the hippocampal subfields of subjects with MDD compared to matched controls. MKP-1, also known as dual-specificity phosphatase-1 (DUSP1), is a member of a family of proteins that dephosphorylate both threonine and tyrosine residues and thereby serves as a key negative regulator of the MAPK cascade, a major signaling pathway involved in neuronal plasticity, function and survival. 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 antidepressant treatment normalizes stress-induced MKP-1 expression and behavior, and mice lacking MKP-1 are resilient to stress. These postmortem and preclinical studies identify MKP-1 as a key factor in MDD pathophysiology and as a new target for therapeutic interventions.

Figure 1

MKP-1 is dysregulated in major depressive disorder (MDD). a) Microarray analysis of MDD postmortem brain samples demonstrates significant alterations in the expression of DUSP genes in hippocampal subfields. b) Microarray findings for MKP-1 gene expression were validated by qRT-PCR of samples from the same cohort. 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 mRNA levels by in situ hybridization in a separate cohort of MDD subjects and matched controls (scale bar = 5 mm). Results are shown as percent increase for each control and MDD subject. *P < 0.02 compared to the healthy controls (Student’s t-test). Microarray-based expression levels of (d) MAP kinases and (e) downstream transcription factors and target genes. (f) Model for neurotrophic/growth factor receptor activation of MAPK, down-stream transcription factors, and target genes that play a key role in neuronal proliferation, survival and plasticity. 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 FDR at 0.05). Fold change for specific splice variants is reported for DUSP19.2, DUSP24.2, RPS6KA5.2 (MSK1) and VEGFa.2. BDNF, brain-derived neurotrophic factor; CREB, cyclic-AMP response element binding protein; CBP, CREB binding protein; CREBL, CREB-like; ERK, extracellular signal-regulated kinase; MAPK, mitogen activated protein kinase; MEK, MAPK kinase; MSK, mitogen and stress-activated protein kinase; NPY, neuropeptide Y; RAF, v-raf-1 murine leukemia viral oncogene homolog 1; RSK, ribosomal S6 kinase; VEGF, vascular endothelial growth factor; VGF, VGF nerve growth factor inducible.

Figure 2

Influence of chronic unpredictable stress (CUS) and antidepressant treatment on behavior and MKP-1 expression. a) Rats were exposed to CUS or control conditions and then were administered either saline or fluoxetine (FLX) for 21 d. Locomotor activity (LA), active avoidance test (AAT), and sucrose preference test (SPT) behaviors were determined. b) Behavioral results for AAT and SPT, expressed as mean ± S.E.M. (n = 8). c) Representative autoradiographs and quantitative analysis 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 levels. Tissue levels of β-actin were used as loading controls. Data are expressed as mean ± S.E.M. percent change over non-stressed control group (n = 5); *P < 0.05 compared to the non-stressed control group, ^#P < 0.05 compared to CUS group (two-way ANOVA and Fisher’s PLSD post hoc analysis).

Figure 3

Influence of Mkp-1 over-expression on behavior in rodent models of depression. a) Recombinant adeno-associated virus was engineered to locally over-express MKP-1 (rAAV-Mkp-1), and compared to a control vector that expresses green fluorescent protein (rAAV-GFP). Rats received bilateral intrahippocampal infusions of rAAV-Mkp-1 or rAAV-GFP. Expression levels of GFP protein (b) and Mkp-1 mRNA (c) are shown [representative figures; b) scale bars = 100 μm, c) scale bar = 500 μm]. Effects of rAAV-Mkp-1 infusions on animal behavior, compared to rAAV-GFP controls, was evaluated for (d) the percent sucrose consumed compared to total fluid consumption (water and sucrose) in the SPT, (e) number of escape failures in the AAT, and (f) latency to feed in the novelty supressed feeding (NSF) test. 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). ITR, inverted terminal repeats; CMV, cytomegalovirus promoter.

Figure 4

Influence of MKP-1 deletion on behavioral models of depression. a) Experimental paradigm for behavioral testing and CUS exposure of Mkp-1 knockout mice (Mkp-1^−/−; n = 9–10) and wildtype (WT) littermates (Mkp-1^+/+; n = 8). b) Baseline sucrose consumption was tested on day 0, followed by post-stress measurements conducted on days 17 and 30. Water consumption was also measured throughout the stress paradigm (data shown for water test on day 20). c) Both genotypes were further tested in the elevated plus maze test; the number of entries into the open or closed arms, and the total time spent in the open arms are shown. 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 and water consumption: repeated-measures ANOVA and Dunnett’s; elevated plus maze: Student’s t-test). d) Representative images and quantitative results of western blot analysis showing the effects of CUS on hippocampal phospho-Erk levels in WT and Mkp-1^−/−, compared to non-stressed WT control mice. Tissue levels of total Erk and beta-actin were used as loading controls. Optical density values are expressed as a ratio of the pErk and total Erk. Data are expressed as mean ± S.E.M. percent change over non-stressed wildtype control (n = 3); *P < 0.05 compared to WT control group and ^#P < 0.05 compared to the WT stress group (ANOVA and Student-Newman-Keuls’ post hoc analysis). SCT, sucrose consumption test; WCT, water consumption test; OFT, open field test; EPM, elevated plus maze.