The Wnt Antagonist, Dickkopf-1, as a Target for the Treatment of Neurodegenerative Disorders

The canonical Wnt pathway contributes to the regulation of neuronal survival and homeostasis in the CNS. Recent evidence suggests that an increased expression of Dickkopf-1 (Dkk-1), a secreted protein that negatively modulates the canonical Wnt pathway, is causally related to processes of neurodegeneration in a number of CNS disorders, including Alzheimer’s disease (AD), brain ischemia and temporal lobe epilepsy (TLE). Dkk-1 induction precedes neuronal death in cellular and animal models of excitotoxicity, β-amyloid toxicity, transient global ischemia, and kainate-induced epilepsy. In addition, Dkk-1, which is barely visible in the healthy brain, is strongly induced in brain tissue from AD patients or from patients with TLE associated with hippocampal sclerosis. These data raise the attractive possibility that Dkk-1 antagonists or neutralizing antibodies behave as neuroprotective agents by rescuing the activity of the canonical Wnt pathway. Special issue article in honor of Anna Maria Giuffrida-Stella. Agata Copani and Ferdinando Nicoletti—Co-senior authors. Filippo Caraci—PhD Program in Neuropharmacology.     

Wnt/β‐catenin signalling pathway mediated aberrant hippocampal neurogenesis in kainic acid‐induced epilepsy

Temporal lobe epilepsy is a chronic disorder of nerve system, mainly characterized by hippocampal sclerosis with massive neuronal loss and severe gliosis. Aberrant neurogenesis has been shown in the epileptogenesis process of temporal lobe epilepsy. However, the molecular mechanisms underlying aberrant neurogenesis remain unclear. The roles of Wnt signalling cascade have been well established in neurogenesis during multiple aspects. Here, we used kainic acid‐induced rat epilepsy model to investigate whether Wnt/β‐catenin signalling pathway is involved in the aberrant neurogenesis in temporal lobe epilepsy. Immunostaining and western blotting results showed that the expression levels of β‐catenin, Wnt3a, and cyclin D1, the key regulators in Wnt signalling pathway, were up‐regulated during acute epilepsy induced by the injection of kainic acids, indicating that Wnt signalling pathway was activated in kainic acid‐induced temporal lobe epilepsy. Moreover, BrdU labelling results showed that blockade of the Wnt signalling by knocking down β‐catenin attenuated aberrant neurogenesis induced by kainic acids injection. Altogether, Wnt/β‐catenin signalling pathway mediated hippocampal neurogenesis during epilepsy, which might provide new strategies for clinical treatment of temporal lobe epilepsy. Temporal lobe epilepsy is a chronic disorder of nerve system, mainly characterized by hippocampal sclerosis. Aberrant neurogenesis has been shown to involve in the epileptogenesis process of temporal lobe epilepsy. In the present study, we discovered that Wnt3a/β‐catenin signalling pathway serves as a link between aberrant neurogenesis and underlying remodelling in the hippocampus, leading to temporal lobe epilepsy, which might provide new strategies for clinical treatment of temporal lobe epilepsy.     

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co-receptor low-density lipoprotein receptor-related protein (LRP) type 5. A 4-day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk-1, and induced the synthesis of the Wnt/Dkk-1 co-receptor LRP6. Recombinant Dkk-1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal-less homeobox gene (Dlx-2). Recombinant Dkk-1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear beta-catenin levels. Remarkably, the antisense- or small interfering RNA-induced knockdown of Dkk-1 largely reduced the expression of Dlx-2, and the neuronal marker beta-III tubulin in EBs exposed to RA. These data suggest that induction of Dkk-1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.     

Sexual Experience Promotes Adult Neurogenesis in the Hippocampus Despite an Initial Elevation in Stress Hormones

Aversive stressful experiences are typically associated with increased anxiety and a predisposition to develop mood disorders. Negative stress also suppresses adult neurogenesis and restricts dendritic architecture in the hippocampus, a brain region associated with anxiety regulation. The effects of aversive stress on hippocampal structure and function have been linked to stress-induced elevations in glucocorticoids. Normalizing corticosterone levels prevents some of the deleterious consequences of stress, including increased anxiety and suppressed structural plasticity in the hippocampus. Here we examined whether a rewarding stressor, namely sexual experience, also adversely affects hippocampal structure and function in adult rats. Adult male rats were exposed to a sexually-receptive female once (acute) or once daily for 14 consecutive days (chronic) and levels of circulating glucocorticoids were measured. Separate cohorts of sexually experienced rats were injected with the thymidine analog bromodeoxyuridine in order to measure cell proliferation and neurogenesis in the hippocampus. In addition, brains were processed using Golgi impregnation to assess the effects of sexual experience on dendritic spines and dendritic complexity in the hippocampus. Finally, to evaluate whether sexual experience alters hippocampal function, rats were tested on two tests of anxiety-like behavior: novelty suppressed feeding and the elevated plus maze. We found that acute sexual experience increased circulating corticosterone levels and the number of new neurons in the hippocampus. Chronic sexual experience no longer produced an increase in corticosterone levels but continued to promote adult neurogenesis and stimulate the growth of dendritic spines and dendritic architecture. Chronic sexual experience also reduced anxiety-like behavior. These findings suggest that a rewarding experience not only buffers against the deleterious actions of early elevated glucocorticoids but actually promotes neuronal growth and reduces anxiety.     

En1 and Wnt7a interact with Dkk1 during limb development in the mouse

Wnt signaling plays an essential role in induction and development of the limb. Missing digits are one consequence of the reduced Wnt signaling in Wnt7a null mice, while extra digits result from excess Wnt signaling in mice null for the Wnt antagonist Dkk1. The extra digits and expanded apical ectodermal ridge (AER) of Dkk1-deficient mice closely resemble En1 null mice. To evaluate the in vivo interaction between En1 and the canonical Wnt signaling pathway, we generated double and triple mutants combining the hypomorphic doubleridge allele of Dkk1 with null alleles of En1 and Wnt7a. Reducing Dkk1 expression in Dkk1d/+Wnt7a-/- double mutants prevented digit loss, indicating that Wnt7a acts through the canonical pathway during limb development. Reducing Dkk1 levels in Dkk1d/dEn1-/- double mutants resulted in severe phenotypes not seen in either single mutant, including fused bones in the autopod, extensive defects of the zeugopod, and loss of the ischial bone. The subsequent elimination of Wnt7a in Dkk1d/dEn1-/-Wnt7a-/- triple mutants resulted in correction of most, but not all, of these defects. The failure of Wnt7a inactivation to completely correct the limb defects of Dkk1d/dEn1-/- double mutants indicates that Wnt7a is not the only gene regulated by En1 during development of the mouse limb.     

Dickkopf-1 antagonizes Wnt signaling independent of beta-catenin in human mesothelioma

Dickkopf-1 (Dkk-1) is a secreted protein that acts as a potent inhibitor of the Wnt signal transduction pathway. It is thought that the antagonistic effect of Dkk-1 is specific to the canonical (Wnt/beta-catenin) pathway. In this study, we demonstrate that restoration of Dkk-1 expression suppresses cell growth and induces apoptotic cell death in beta-catenin-deficient mesothelioma cell lines H28 and MS-1. Furthermore, we found that a small-molecule inhibitor of JNK inhibited the apoptosis induced by Dkk-1 overexpression in these cells. Together, our data suggest that Dkk-1 may be able to antagonize Wnt signaling and exert its tumor suppressive effects through beta-catenin-independent non-canonical pathways (i.e., the Wnt/JNK pathway).     

The Wnt signaling inhibitor dickkopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow

Adult human mesenchymal stem cells from bone marrow stroma (hMSCs) differentiate into numerous mesenchymal tissue lineages and are attractive candidates for cell and gene therapy. When early passage hMSCs are plated or replated at low density, the cultures display a lag phase of 3-5 days, a phase of rapid exponential growth, and then enter a stationary phase without the cultures reaching confluence. We found that as the cultures leave the lag phase, they secrete high levels of dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt signaling pathway. The addition of recombinant Dkk-1 toward the end of the lag period increased proliferation and decreased the cellular concentration of beta-catenin. The addition of antibodies to Dkk-1 in the early log phase decreased proliferation. Also, expression of Dkk-1 in hMSCs decreased during cell cycle arrest induced by serum starvation. The results indicated that high levels of Dkk-1 allow the cells to reenter the cell cycle by inhibiting the canonical Wnt/beta-catenin signaling pathway. Since antibodies to Dkk-1 also increased the lag phase of an osteosarcoma line that expressed the gene, Dkk-1 may have a similar role in some other cell systems.     

Aggravation of chronic stress effects on hippocampal neurogenesis and spatial memory in LPA₁ receptor knockout mice

Background

The lysophosphatidic acid LPA₁ receptor regulates plasticity and neurogenesis in the adult hippocampus. Here, we studied whether absence of the LPA₁ receptor modulated the detrimental effects of chronic stress on hippocampal neurogenesis and spatial memory.

Methodology/Principal Findings

Male LPA₁-null (NULL) and wild-type (WT) mice were assigned to control or chronic stress conditions (21 days of restraint, 3 h/day). Immunohistochemistry for bromodeoxyuridine and endogenous markers was performed to examine hippocampal cell proliferation, survival, number and maturation of young neurons, hippocampal structure and apoptosis in the hippocampus. Corticosterone levels were measured in another a separate cohort of mice. Finally, the hole-board test assessed spatial reference and working memory. Under control conditions, NULL mice showed reduced cell proliferation, a defective population of young neurons, reduced hippocampal volume and moderate spatial memory deficits. However, the primary result is that chronic stress impaired hippocampal neurogenesis in NULLs more severely than in WT mice in terms of cell proliferation; apoptosis; the number and maturation of young neurons; and both the volume and neuronal density in the granular zone. Only stressed NULLs presented hypocortisolemia. Moreover, a dramatic deficit in spatial reference memory consolidation was observed in chronically stressed NULL mice, which was in contrast to the minor effect observed in stressed WT mice.

Conclusions/Significance

These results reveal that the absence of the LPA₁ receptor aggravates the chronic stress-induced impairment to hippocampal neurogenesis and its dependent functions. Thus, modulation of the LPA₁ receptor pathway may be of interest with respect to the treatment of stress-induced hippocampal pathology.     

Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.

BACKGROUND: Dickkopf-1 (Dkk-1) is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. Although several families of secreted antagonists have been shown to inhibit Wnt signal transduction by binding to Wnt, the molecular mechanism of Dkk-1 action is unknown. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its candidate coreceptor, LDL receptor-related protein 6 (LRP6), presumably through Fz-LRP6 complex formation induced by Wnt. The significance of the Fz-LRP6 complex in signal transduction remains to be established. RESULTS: We report that Dkk-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. Dkk-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. Dkk-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LRP6 and to disrupt the Fz-LRP6 association. LRP6 function and Dkk-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. CONCLUSIONS: Our results demonstrate that Dkk-1 is an LRP6 ligand and inhibits Wnt signaling by blocking Wnt-induced Fz-LRP6 complex formation. Our findings thus reveal a novel mechanism for Wnt signal modulation. LRP6 is a Wnt coreceptor that appears to specify Wnt/Fz signaling to the beta-catenin pathway, and Dkk-1, distinct from Wnt binding antagonists, may be a specific inhibitor for Wnt/beta-catenin signaling. Our findings suggest that Wnt-Fz-LRP6 complex formation, but not Wnt-Fz interaction, triggers Wnt/beta-catenin signaling.     

Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis

Increasing evidence suggests that depression may be associated with a lack of hippocampal neurogenesis. It is well established that neuronal nitric oxide synthase (nNOS)-derived NO exerts a negative control on the hippocampal neurogenesis. Using genetic and pharmacological methods, we investigated the roles of nNOS in depression induced by chronic mild stress (CMS) in mice. Hippocampal nNOS over-expression was first observed 4 days and remained elevated 21 and 56 days after exposure to CMS. The mice exposed to CMS exhibited behavioral changes typical of depression, and impaired neurogenesis in the hippocampus. The CMS-induced behavioral despair and hippocampal neurogenesis impairment were prevented and reversed in the null mutant mice lacking nNOS gene (nNOS−/−) and in the mice receiving nNOS inhibitor. Disrupting hippocampal neurogenesis blocked the antidepressant effect of nNOS inhibition. Moreover, nNOS−/− mice exhibited antidepressant-like properties. Our findings suggest that nNOS over-expression in the hippocampus is essential for chronic stress-induced depression and inhibiting nNOS signaling in brain may represent a novel approach for the treatment of depressive disorders.     

Wnt7a Regulates Multiple Steps of Neurogenesis

Although Wnt7a has been implicated in axon guidance and synapse formation, investigations of its role in the early steps of neurogenesis have just begun. We show here that Wnt7a is essential for neural stem cell self-renewal and neural progenitor cell cycle progression in adult mouse brains. Loss of Wnt7a expression dramatically reduced the neural stem cell population and increased the rate of cell cycle exit in neural progenitors in the hippocampal dentate gyrus of adult mice. Furthermore, Wnt7a is important for neuronal differentiation and maturation. Loss of Wnt7a expression led to a substantial decrease in the number of newborn neurons in the hippocampal dentate gyrus. Wnt7a^−/− dentate granule neurons exhibited dramatically impaired dendritic development. Moreover, Wnt7a activated β-catenin and its downstream target genes to regulate neural stem cell proliferation and differentiation. Wnt7a stimulated neural stem cell proliferation by activating the β-catenin–cyclin D1 pathway and promoted neuronal differentiation and maturation by inducing the β-catenin–neurogenin 2 pathway. Thus, Wnt7a exercised critical control over multiple steps of neurogenesis by regulating genes involved in both cell cycle control and neuronal differentiation.     

Imbalance of Wnt/Dkk Negative Feedback Promotes Persistent Activation of Pancreatic Stellate Cells in Chronic Pancreatitis

The role of persistent activation of pancreatic stellate cells (PSCs) in the fibrosis associated with chronic pancreatitis (CP) is increasingly being recognized. Recent studies have shown that Wnt signaling is involved in the development of fibrosis in multiple organs, however, the role of specific Wnts in pancreatic fibrosis remains unknown. We investigated the role of Wnt signaling during PSC activation in CP and the effect of β-catenin inhibition and Dickkopf-related protein 1 (Dkk1) restoration on the phenotype of PSCs. CP was induced in mice by repetitive caerulein injection and mouse PSCs were isolated and activated in vitro. The expression of Wnts, β-catenin, secreted frizzled-related proteins (sFRPs) and Dkks was analyzed by quantitative RT-PCR and western blotting. The canonical Wnt signaling pathway was examined by immunofluorescence and western blot detection of nuclear β-catenin expression. The effect of recombinant mouse Dkk-1 (rmDkk-1) on cell proliferation and apoptosis was assessed by flow cytometry, immunofluorescence, immunocytochemistry and Cell Counting Kit-8 (CCK-8) analysis. The expression of β-catenin, collagen1α1, TGFβRII, PDGFRβ and α-SMA in PSCs treated with different concentrations of rmDkk-1 or siRNA against β-catenin was determined by quantitative RT-PCR and western blotting. Wnt2 was the only Wnt whose expression was significantly upregulated in response to PSC activation, and Wnt2 and β-catenin protein levels were significantly increased in the pancreas of CP mice, whereas Dkk-1 expression was evidently decreased. Nuclear β-catenin levels were markedly increased in activated PSCs, and rmDkk-1 suppressed the nuclear translocation of β-catenin and the proliferation and extracellular matrix production of PSCs through the downregulation of PDGFRβ and TGFβRII. Upregulation of Dkk-1 expression increased apoptosis in cultured PSCs. These results indicate that Wnt signaling may mediate the profibrotic effect of PSC activation, and Wnt2/Dkk-1 could be potential therapeutic targets for CP.     

Effects of parathyroid hormone on Wnt signaling pathway in bone

The Wnt signaling pathway has recently been demonstrated to play an important role in bone cell function. In previous studies using DNA microarray analyses, we observed a change in some of the molecular components of the canonical Wnt pathway namely, frizzled-1 (FZD-1) and axil, in response to continuous parathyroid hormone (PTH) treatment in rats. In the present study, we further explored other components of the Wnt signaling pathway in rat distal metaphyseal bone in vivo, and rat osteoblastic osteosarcoma cells (UMR 106) in culture. Several Wnt pathway components, including low-density lipoprotein-receptor-related protein 5 (LRP5), LRP6, FZD-1, Dickkopf-1 (Dkk-1), and Kremen-1 (KRM-1), were expressed in bone in vivo and in osteoblasts in vitro. Continuous exposure to PTH (1-38) both in vivo and in vitro upregulated the mRNA expression of LRP6 and FZD-1 and decreased LRP5 and Dkk-1. These effects in UMR 106 cells were associated with an increase in beta-catenin as measured by Western blots and resulted in functional activation (three to six-fold) of a downstream Wnt responsive TBE6-luciferase (TCF/LEF-binding element) reporter gene. Activation of the TBE6-luciferase reporter gene by PTH (1-38) in UMR 106 cells was inhibited by the protein kinase A (PKA) inhibitor, H89. Activation was mimicked by PTH (1-31), PTH-related protein (1-34), and forskolin, but both PTH (3-34) and (7-34) had no effect. These findings suggest that the effect of PTH on the canonical Wnt signaling pathway occurs at least in part via the cAMP-PKA pathway through the differential regulation of the receptor complex proteins (FZD-1/LRP5 or LRP6) and the antagonist (Dkk-1). Taken together, these results reveal a possible role for the Wnt signaling pathway in PTH actions in bone.     

Second cysteine-rich domain of Dickkopf-2 activates canonical Wnt signaling pathway via LRP-6 independently of dishevelled.

Recent evidence suggests that members of the Dickkopf (Dkk) family can directly bind to LDL-related protein (LRP)-6, resulting in inhibition of Wnt-activated signaling. To further characterize the interactions between Dkk and LRP proteins, conditioned media containing individually conserved cysteine-rich domains of Dkk-1 and Dkk-2 were prepared. Although full-length Dkk-1 and Dkk-2 and the second cysteine-rich domains of both Dkk molecules inhibited Wnt-3a-induced activation of lymphoid enhancing factor (LEF)-1, a downstream target of the canonical pathway, we found that the second cysteine-rich domain of Dkk-2 (Dkk-2C2) was able to stimulate the canonical pathway when LRP-6 was ectopically expressed in NIH3T3 cells. This effect of Dkk-2C2 could be blocked by a monoclonal antibody specific to the second YWTD repeat domain of LRP-5/6, suggesting that Dkk-2C2 acts via LRP-6. We also showed that while both Axin and the DIX domain of Dishevelled (Dvl) could inhibit Dkk-2C2-induced activation of LEF-1, the DEP domain of Dvl, which inhibited Wnt-induced activation of LEF-1, failed to inhibit the activation of LEF-1 by Dkk-2C2 or by an activated form of LRP-5, LRPC2. In addition, glycogen synthase kinase-3 beta, a potent inhibitor for both Dvl and Wnt, also failed to inhibit LRPC2 or Dkk-2C2. Furthermore, knocking-down the expression of Dvl molecules by short interfering RNAs specific to Dvl inhibited Wnt-induced, but not LRPC2-induced, activation of LEF-1. All the evidence indicates that Dkk-2C2 signals through LRP proteins, which does not require Dvl, while Wnt protein may employ both Dvl, presumably through Fz, and LRP to achieve more efficient signal transduction.     

Oxysterol-induced osteogenic differentiation of marrow stromal cells is regulated by Dkk-1 inhibitable and PI3-kinase mediated signaling

Osteoporosis and its complications cause morbidity and mortality in the aging population, and result from increased bone resorption by osteoclasts in parallel with decreased bone formation by osteoblasts. A widely accepted strategy for improving bone health is targeting osteoprogenitor cells in order to stimulate their osteogenic differentiation and bone forming properties through the use of osteoinductive/anabolic factors. We previously reported that specific naturally occurring oxysterols have potent osteoinductive properties, mediated in part through activation of hedgehog signaling in osteoprogenitor cells. In the present report, we further demonstrate the molecular mechanism(s) by which oxysterols induce osteogenesis. In addition to activating the hedgehog signaling pathway, oxysterol-induced osteogenic differentiation is mediated through a Wnt signaling-related, Dkk-1-inhibitable mechanism. Bone marrow stromal cells (MSC) treated with oxysterols demonstrated increased expression of osteogenic differentiation markers, along with selective induced expression of Wnt target genes. These oxysterol effects, which occurred in the absence of beta-catenin accumulation or TCF/Lef activation, were inhibited by the hedgehog pathway inhibitor, cyclopamine, and/or by the Wnt pathway inhibitor, Dkk-1. Furthermore, the inhibitors of PI3-Kinase signaling, LY 294002 and wortmanin, inhibited oxysterol-induced osteogenic differentiation and induction of Wnt signaling target genes. Finally, activators of canonical Wnt signaling, Wnt3a and Wnt1, inhibited spontaneous, oxysterol-, and Shh-induced osteogenic differentiation of bone marrow stromal cells, suggesting the involvement of a non-canonical Wnt pathway in pro-osteogenic differentiation events. Osteogenic oxysterols are, therefore, important small molecule modulators of critical signaling pathways in pluripotent mesenchymal cells that regulate numerous developmental and post-developmental processes.     

Differential effects of corticosterone and dexamethasone on hippocampal neurogenesis in vitro

Prenatal stress during fetal development results in the blockade of neurogenesis in the dentate gyrus in adulthood. Present study was undertaken to investigate the dominant role of the glucocorticoid receptors in corticosterone actions on the neurogenesis of fetal hippocampal progenitor cells. For that purpose, expressions of key molecules affected by corticosterone and dexamethasone were compared during proliferation and differentiation of the hippocampal progenitor cells. Corticosterone (2 microM) significantly decreased the number of bromodeoxyuridine-labeled cells (about 50%) and caused the dendritic atrophy in microtubule-associated protein 2-labeled cells. The expressions of NeuroD, BDNF, and NR1 mRNA levels and protein levels of p-ERK and p-CREB were remarkably decreased by corticosterone in a dose-dependent manner. In contrast, dexamethasone, a glucocorticoid receptor (GR) specific agonist, had an inhibitory effect on proliferation, but not differentiation. It is concluded that corticosterone elicits its effects on neurogenesis including proliferation and differentiation whereas stimulation of the glucocorticoid receptor is sufficient to decrease only proliferation.     

Hippocampal neurogenesis and dendritic plasticity support running-improved spatial learning and depression-like behaviour in stressed rats

Exercise promotes hippocampal neurogenesis and dendritic plasticity while stress shows the opposite effects, suggesting a possible mechanism for exercise to counteract stress. Changes in hippocampal neurogenesis and dendritic modification occur simultaneously in rats with stress or exercise; however, it is unclear whether neurogenesis or dendritic remodeling has a greater impact on mediating the effect of exercise on stress since they have been separately examined. Here we examined hippocampal cell proliferation in runners treated with different doses (low: 30 mg/kg; moderate: 40 mg/kg; high: 50 mg/kg) of corticosterone (CORT) for 14 days. Water maze task and forced swim tests were applied to assess hippocampal-dependent learning and depression-like behaviour respectively the day after the treatment. Repeated CORT treatment resulted in a graded increase in depression-like behaviour and impaired spatial learning that is associated with decreased hippocampal cell proliferation and BDNF levels. Running reversed these effects in rats treated with low or moderate, but not high doses of CORT. Using 40 mg/kg CORT-treated rats, we further studied the role of neurogenesis and dendritic remodeling in mediating the effects of exercise on stress. Co-labelling with BrdU (thymidine analog) /doublecortin (immature neuronal marker) showed that running increased neuronal differentiation in vehicle- and CORT-treated rats. Running also increased dendritic length and spine density in CA3 pyramidal neurons in 40 mg/kg CORT-treated rats. Ablation of neurogenesis with Ara-c infusion diminished the effect of running on restoring spatial learning and decreasing depression-like behaviour in 40 mg/kg CORT-treated animals in spite of dendritic and spine enhancement. but not normal runners with enhanced dendritic length. The results indicate that both restored hippocampal neurogenesis and dendritic remodelling within the hippocampus are essential for running to counteract stress.