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. Author manuscript; available in PMC: 2011 Jun 29.
Published in final edited form as: Int J Neuropsychopharmacol. 2010 Jul 7;14(5):711–717. doi: 10.1017/S1461145710000726

Lentivirally mediated GSK-3β silencing in the hippocampal dentate gyrus induces antidepressant-like effects in stressed mice

Naoto Omata 1,3,*, Chi-Tso Chiu 1,*, Pablo R Moya 2, Yan Leng 1, Zhifei Wang 1, Joshua G Hunsberger 1, Peter Leeds 1, De-Maw Chuang 1
PMCID: PMC3125712  NIHMSID: NIHMS305733  PMID: 20604988

Abstract

Inhibition of glycogen synthase kinase-3 (GSK-3) by pharmacological tools can produce antidepressant-like effects in rodents. However, the GSK-3 isoform(s) and brain region(s) involved in regulating these behavioral effects remain elusive. We studied the effects of bilateral intra-hippocampal injections of lentivirus-expressing shRNA targeting GSK-3β on behavioral performance in mice subjected to chronic stress. Pre-injection of lentivirus-expressing GSK-3β shRNA into the hippocampal dentate gyrus significantly decreased immobility time in both forced swim and tail suspension tests, while the locomotor activity of these mice was unchanged. These results suggest that lentiviral GSK-3β shRNA injection induces antidepressant-like effects in chronically stressed mice. Under these conditions, the expression levels of GSK-3β were persistently and markedly reduced in the hippocampus following GSK-3β shRNA injection. To our knowledge, this is the first demonstration that a single injection of lentivirus-expressing GSK-3β shRNA in the hippocampal dentate gyrus of chronically stressed mice has antidepressant-like effects elicited by gene silencing.

Keywords: GSK-3 shRNA, lentivirus, hippocampal dentate gyrus, antidepressant-like effects, forced swim test

Introduction

Glycogen synthase kinase-3 (GSK-3) has emerged as an important target for lithium, a major mood stabilizer used to treat bipolar disorder. The α and β isoforms of GSK-3 regulate a large number of proteins through phosphorylation mechanisms, and affect pathways involved in neuroplasticity, neurotrophicity, cell survival, and neurotransmission. Lithium appears to inhibit GSK-3 activity directly by competitive inhibition of Mg2+ binding to the active site of the enzyme, and indirectly by enhancing serine phosphorylation levels through multiple signaling pathways including Akt (reviewed in Jope, 2003; Rowe et al., 2007). Other compounds used to treat bipolar disorder, such as some antipsychotic medications and the mood stabilizers valproate and lamotrigine, also enhance GSK-3β serine 9 phosphorylation (reviewed in Jope, 2003; Rowe et al., 2007). Therefore, investigating treatments that target GSK-3-linked pathways is a rational strategy for developing novel therapeutics to treat this disorder.

GSK-3 abnormalities have been implicated in the pathophysiology of various mood disorders. For example, decreased Akt activity and increased GSK-3 activity were noted in the postmortem prefrontal cortex of depressed individuals who committed suicide (Karege et al., 2007). Using the forced swim test, chronic treatment with lithium produces antidepressant-like effects in mice, reminiscent of its clinical efficacy in patients with bipolar disorder (O’Brien et al., 2004). Other studies have noted that GSK-3 peptide inhibitors (Kaidanovich-Beilin et al., 2004) and novel GSK-3 inhibitors (Gould et al., 2004) also induce rapid antidepressant-like and/or antimanic-like effects in mice, suggesting that GSK-3 is a potential mood-stabilizing target of lithium. In addition, target deletion of GSK-3β gene to produce heterozygous GSK-3β+/− mice produces behavioral effects similar to the antidepressant-like effects of lithium (O’Brien et al., 2004). To date, however, the brain region(s) responsible for these drug-induced antidepressant-like effects remains elusive. In this context, chronic lithium treatment has been shown to have brain region-selective neuroprotective effects in rodents (Omata et al., 2008), and these neuroprotective effects may contribute to its clinical efficacy (van der Schot et al., 2009). Thus, it is unlikely that the antidepressant-like effects of GSK-3 inhibitors are mediated by actions occurring throughout the whole brain.

RNA interference (RNAi) has become an effective tool for selectively silencing the expression of GSK-3 isoforms and for elucidating GSK-3 isoform-associated neurobiological functions (Liang and Chuang, 2006; Liang and Chuang, 2007). We elucidated differences in GSK-3 isoforms where GSK-3β depletion is more effective than GSK-3α depletion in suppressing spontaneous cell death in extended culture (Liang and Chuang, 2007). While depletion of both isoforms was able to block glutamate-induced-excitotoxity and warrant further investigation, we focused on GSK-3β in the present study. Lentiviral vectors have been developed to effectively and safely transfer genes into dividing and non-dividing cells such as postmitotoic neurons. For example, the lentiviral vector that expresses a short-hairpin RNA (shRNA) inhibits the expression of target protein specifically and persistently in vitro and in the rat brain (Sapru et al., 2006). Furthermore, the hippocampal dentate gyrus has been implicated as one of the brain regions involved in modulating the efficacy of antidepressants via mechanisms involving brain-derived neurotrophic factor (BDNF) (Adachi et al., 2008; Shirayama et al., 2002). In this study, we used chronic restraint stress as a mouse model of depression. We examined the antidepressant-like effects of persistent GSK-3β inhibition induced by injection of lentiviral vectors expressing GSK-3β shRNA into the hippocampal dentate gyrus of these mice.

Materials and Methods

Small hairpin RNA design

GSK3β Mission® shRNA Plasmid (PLK 0.1-puro-GSK-3β-shRNA) and non-targeting shRNA control vector (PLK 0.1-puro-control shRNA) were purchased from SIGMA (St. Louis, MO). shRNA was designed against GSK3β mRNA, and the sequence was 5′-CCGGCCACTCAAGAACTGTCAAGTACTCGAGTACTTGACAGTTCTTGAGTGGTTTTT-3′. The control vector produced a corresponding scrambled shRNA, with a sequence of 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT-3′.

Lentiviral vector production and concentration

HEK 293T/17 cells were grown in DMEM/10% FBS and plated in 10 cm dishes at a density of 1 × 106 cells/dish. The day after plating, GSK-3β Mission® shRNA Plasmids were transfected as follows: 2.6 μg GSK-3β plasmid DNA, 26 μl lentiviral packaging mix (SIGMA), and 16 μl FuGENE transfection reagent (Roche, Nutley, NJ, USA) per dish in 10 ml DMEM. The day after transfection, culture medium was collected and immediately frozen for preparation of GSK-3β-containing particles. For the second harvest of viral particles, DMEM was replaced with fresh medium for another collection on the next day. Collected medium was filtered (0.45 μm pore) and ultracentrifuged at 4°C for 90 minutes at 25,000 rpm. The pellet was resuspended with 200 μl PBS and left standing 30 minutes at 4°C. Viral particles from three tubes were collected, and concentrated by ultracentrifugation. Viral titer was detected using an HIV-1 p24 Antigen Elisa kit (ZeptoMetrix Corporation, Buffalo, NY), and the final injection titer was 2.5 × 108 TU/ml.

Animals, stereotaxic surgery, and antidepressant treatment

Animal procedures were approved by the Animal Care and Use Committee of the National Institute of Mental Health, National Institutes of Health (Bethesda, MD, USA). Male eight-week-old CD-1 mice were bred and housed at 24 ± 1°C, light-dark cycle of 12 hours/12 hours, and free access to food and water. Animals were anesthetized by i.p. with ketamine (80 mg/kg) and xylazine (8 mg/kg), and then mounted onto a stereotaxic apparatus (Stoelting Instruments, Wood Dale, IL, USA). One μl of lentivirus was injected targeting both sides of the hippocampal dentate gyrus at a rate of 0.25 μl/min using a 25-gauge Hamilton syringe. Coordinates of injection site were anteroposterior −2.0, mediolateral ± 1.6 relative to the bregma, and dorsoventral −2.0 relative to the skull surface (Paxinos and Franklin, 2001). The needle was left in place an additional four minutes and then withdrawn. For the measurement of the behavioral effects of a reference antidepressant, desipramine hydrochloride (SIGMA) was freshly dissolved in deionized water before use and injected i.p. 30 min prior to behavioral testing in a volume of 10 ml/kg of body weight. Previous studies have shown that CD-1 mice are sensitive to the behavioral effects of desipramine, and 20 mg/kg of this drug markedly reduces the immobility time in the forced swim test (Lucki et al., 2001). Control animals received injection of deionized water as the vehicle.

Chronic restraint stress

Animals recovered for seven days after surgery before initiation of chronic restraint stress, which was performed as previously described (Pawlak et al., 2003) with minor modifications. Briefly, each mouse was placed into a Plexiglas tube (diameter, 2.5 cm) without access to either food or water for two hours a day for 14 consecutive days. Open field testing (see below) was conducted one day after chronic restraint stress ended. Two days after the last chronic restraint stress, mice were given either the forced swim test or the tail suspension test. Following evaluation of behavioral performance, animals were sacrificed for GSK-3β analysis.

Open field test

Briefly, as previously described (Fukui et al., 2007), each mouse was placed in the center of an open field (27 × 27 × 20 cm) with a white floor and clear plastic walls, and equipped with infrared sensors. Testing lasted for 30 minutes. Distance traveled and average velocity were measured using Activity Monitor software version 5 (MED Associates, Inc., St. Albans, VT).

Forced swim test

Briefly, as previously described (Porsolt et al., 1977), each mouse was placed for six minutes in a Plexiglas cylinder (height, 25 cm; diameter, 15 cm) containing 20 cm of water maintained at 25 ± 1°C. Sessions were digitally recorded for later analysis. Duration of immobility, defined as lack of activity, except movements made by mice to keep their heads above water, was scored during the last four minutes.

Tail suspension test

Briefly, as previously described (Steru et al., 1985), each mouse was suspended 30 cm from the floor by the distal portion of its tail with adhesive tape for six minutes. Sessions were digitally recorded and later scored for duration of immobility—defined as absence of active attempts to escape—from the beginning to the end of the test.

Western blotting

Both hippocampi were isolated and homogenized in lysis buffer. Protein concentrations were determined and 10 μg aliquots were separated by electrophoresis on 4–12% Nupage Bis-Tris gel. Proteins were subsequently transferred to a polyvinylidene difluoride membrane, incubated with primary antibody against GSK-3β (1:5000; BD Biosciences, San Jose, CA, USA), phospho-Ser9-GSK-3β (1:1000; Cell Signaling, Danvers, MA, USA), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000; Advanced Immunochemical, Long Beach, CA, USA). The membrane was washed with 0.1% Tween PBS, and then incubated with horseradish peroxidase-labeled secondary antibody (1:2000; GE Healthcare Bioscience Corp., Piscataway, NJ, USA). Reactive bands were visualized by detecting chemiluminescence and quantified by using an FLA-7000 imaging system and Multi Gauge software version 3.0 (Fuji Photo Film Co. Ltd., Tokyo, Japan).

Immunohistochemistry

Immunohistochemical analysis was performed using a R.T.U. VECTASTAIN Kit (Vector Laboratories, Burlingame, CA, USA). Mice were anesthetized and perfused through the left cardiac ventricle with normal saline followed by 4% paraformaldehyde. Brains were removed and cut using a cryostat (Leica Microsystems Inc., Bannockburn, IL, USA) at a thickness of 30 μm, and incubated with primary antibody against GSK-3β (1:1000; BD Biosciences). Brain slices were washed with TBS, and incubated with horseradish peroxidase-combined secondary antibody. Immunostained brain slices were further washed with TBS, and then developed with ImmPACT DAB substrate (Vector Laboratories).

Statistical analysis

All data are presented as mean ± SEM. For comparison between two groups, Student’s t-test was used. For comparison of more than three groups, statistical analysis was done by one-way analysis of variance (ANOVA) followed by post-hoc Student–Newman–Keuls multiple comparison tests. A p value of less than 0.05 was considered statistically significant.

Results

Effects of intra-hippocampal injection of lentiviral-based GSK-3β shRNA on behavioural performance in chronically stressed mice

In mice undergoing a model of stress-induced depression, lentiviral vector expressing shRNA of GSK-3β was injected bilaterally into the hippocampal dentate gyrus seven days prior to beginning the stress procedure in order to induce a persistent reduction of GSK-3β expression levels. Mice subsequently underwent the forced swim and tail suspension tests to assess any associated antidepressant-like effects. Compared with drug-naïve, non-stressed mice, chronic restraint stress markedly increased the immobility time in the forced swim test (from 39.81 ± 1.54% to 47.13 ± 1.03%. n = 8). This depressive-like behavior was suppressed by 30-min pretreatment with 20 mg/kg of an antidepressant, desipramine. Similar to the effect produced by this antidepressant, injection of the lentivirus expressing GSK-3β shRNA significantly reduced immobility time by approximately 35% compared to the group injected with the control lentiviral vector after chronic restraint stress (from 46.61 ± 3.39% to 30.47 ± 2.39%, n = 8) (Fig. 1a). Similarly, pretreatment of stressed mice with desipramine decreased the immobility time in the tail suspension test. A significant decrease in immobility time was also detected in mice injected with the lentivirus expressing GSK-3β shRNA compared with those injected with the control lentiviral vector (from 42.83 ± 3.68% to 31.48 ± 3.34%, n = 6) (Fig. 1b). In contrast, locomotor activity (expressed as distance traveled and average velocity) assessed by the open field test after chronic stress was unchanged by either desipramine or lentiviral GSK-3β shRNA injection (Fig. 1c and d). No difference in the temporal profiles of locomotor activity throughout the 30-min open field test was observed between different experimental groups (Supplemental Fig. S1). These results suggest that intra-hippocampal injection of lentivirus expressing GSK-3β shRNA affected neither basal locomotion nor the habituation of mice to a novel environment.

Fig. 1.

Fig. 1

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Intra-hippocampal injection of lentivirus expressing GSK-3β shRNA produced antidepressant-like effect, but did not affect locomotor activity, in chronically stressed mice. Intra-hippocampal injection of lentivirus expressing GSK-3β shRNA or i.p. pretreatment with desipramine decreased immobility time in the forced swim (a) and tail suspension tests (b). Data are mean ± SEM of immobility time expressed as percentage of the test session (%, n = 6–10). *p<0.05, **p < 0.01, by Student–Newman–Keuls multiple comparison test after a one-way ANOVA. Locomotor activity was measured by the open field test and expressed as the distance traveled (c) and average velocity (d).

Effects of intra-hippocampal injection of lentiviral-based GSK-3β shRNA on GSK-3β expression levels in the hippocampus

Animals were sacrificed after assessment of behavioral performance, in order to perform western blotting for GSK-3β protein in the hippocampus. We found that total hippocampal GSK-3β protein levels were decreased by about 30% following injection of the lentiviral vector expressing GSK-3β shRNA, compared with the group injected with the control vector (Fig. 2a and b). Furthermore, immunostaining results showed that GSK-3β was clearly detected in the hippocampal dentate gyrus, particularly in the subgranular zone in the control group (Fig. 2c). However, GSK-3β immunostaining in the dentate gyrus was robustly reduced by injection with the lentivirus expressing GSK-3β shRNA (Fig. 2d).

Fig. 2.

Fig. 2

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Protein levels and immunohistochemical staining of GSK-3β in the hippocampus were decreased by injection of lentiviral GSK-3β shRNA into the dentate gyrus. (a) Western blots of GSK-3β and GAPDH (used as a loading control) in the entire hippocampus from three mice randomly selected from each group. (b) Quantified results of the blots of GSK-3β shown in (a) (n = 6). *p < 0.05 compared to the control. Typical results of GSK-3β immunostaining in the hippocampal dentate gyrus of a mouse injected with a control vector (c) or lentiviral GSK-3β shRNA (d). Arrows indicate positive staining of GSK-3β-expressing cells. Scale bar = 50 μm.

Discussion

To the best of our knowledge, this study is the first to use local injections of lentivirus expressing shRNA to specifically knock down GSK-3β in the hippocampal dentate gyrus and to assess any associated behavioral changes. Notably, we found that a single injection of lentivirus-expressing GSK-3β shRNA in the hippocampal dentate gyrus elicited significant antidepressant-like effects in mice undergoing a chronic stress-induced animal model of depression.

In this study, total hippocampal GSK-3β protein levels were reduced to about 70% compared to control animals. This modest but significant knockdown of hippocampal GSK-3β may be explained by the use of the whole hippocampus for western blotting, where the inclusion of non-transduced tissue dilutes the knockdown effect of the procedure. Nevertheless, the GSK-3β knockdown was sufficient to elicit changes in behaviors revealed in our tests. A previous report indicates that chronic stress elevates GSK-3β mRNA in the hippocampus (Silva et al., 2008). We found that hippocampal GSK-3β protein levels were not significantly increased by stress; however, its GSK-3β Ser9 phosphorylation was significantly reduced, thus indicating enhanced GSK-3β activity (Supplemental Fig. S2). These data are reminiscent of the recent report that brain GSK-3β Ser9 phosphorylation is decreased in the learned helpless state after inescapable foot shock (Polter et al., 2010). We also showed that GSK-3β immunostaining was robustly reduced in the dentate gyrus of stressed mice injected with lentivirus expressing GSK-3β shRNA. Our results support the notion that lentiviral-mediated RNAi effectively disrupts region-selective gene expression for rapid functional analysis, thus circumventing the need to generate gene knockout animals.

Notably, treatment with lentivirus expressing GSK-3β shRNA significantly reduced the immobility time of the mice in the forced swim and tail suspension tests. These two tests are widely used for screening antidepressant activity, and reduced immobility time is considered a well-validated antidepressant-like effect (Porsolt et al., 1977; Steru et al., 1985). Because false-positive results can be obtained in these tests due to stimulation of locomotor activity (Bourin et al., 2001), we also assessed this measure in our study and found that lentiviral GSK-3β shRNA had no effect on locomotor activity. Thus, our results suggest that knockdown of GSK-3β produces antidepressant-like effects.

Previous studies have noted that GSK-3 inhibitors have acute antidepressant-like and/or antimanic-like effects in naïve rodents (Gould et al., 2004; Kaidanovich-Beilin et al., 2004). However, the lack of clear selectivity of these inhibitors for GSK-3 among various kinases has made it difficult to reliably assess the link between GSK-3 inhibition and mood-stabilizing effects (Hongisto et al., 2008). In addition, these inhibitors likely block the activity of both GSK-3α and GSK-3β; thus, the role that each individual GSK-3 isoform plays in mediating behavioral effects has to date remained unclear. Furthermore, neither pharmacological studies nor experiments using GSK-3β+/− knockout mice (O’Brien et al., 2004) provide information regarding which brain region is critically involved in GSK-3’s antidepressant-like effects. In contrast, lentiviral-mediated RNAi provides an alternative approach where expression of a particular GSK-3 isoform can be silenced selectively and persistently in a specific brain region for behavioral evaluations. To our awareness, our results are the first to demonstrate that administration of lentiviral-mediated GSK-3β shRNA into the dentate gyrus causes antidepressant-like effects in mice subjected to chronic stress.

While numerous brain regions are supported to contribute to the pathophysiology of mood disorders, previous studies have strongly implicated the hippocampal dentate gyrus and this brain region has been the focus of our investigation. Patients with major depressive disorder had reduced hippocampal volume (McKinnon et al., 2009). Infusion of BDNF into the dentate gyrus has been found to induce antidepressant-like effects in rodents (Shirayama et al., 2002), and selective loss of BDNF in the dentate gyrus, but not the CA1 region, attenuated the actions of antidepressant drugs in the forced swim test (Adachi et al., 2008). In addition, adult neurogenesis occurs in the hippocampal dentate gyrus; this event is facilitated by hippocampal BDNF infusion (Scharfman et al., 2005) which is also crucial for the sensitivity to antidepressant treatment (Li et al., 2008). Interestingly, recent work from our laboratory showed that selective knockdown of GSK-3β using specific siRNA induced BDNF expression via transcriptional activation of promoter IV in rat cortical neurons (Yasuda et al., 2009). Therefore, it is conceivable that the antidepressant-like effects elicited in this study by intra-hippocampal injection of lentiviral GSK-3β shRNA involve BDNF induction and subsequent enhanced neurogenesis in this brain region. Further studies are needed to address whether injection of lentiviral GSK-3β shRNA into the dentate gyrus or other brain region(s) has anti-manic effects similar to those seen in individuals with bipolar disorder who receive chronic lithium therapy. The role of GSK-3α isoforms in mediating mood states also remains to be investigated. Nevertheless, our findings provide evidence that persistent silencing of GSK-3β expression in the hippocampal dentate gyrus is sufficient to induce antidepressant-like behaviors in stressed mice.

Supplementary Material

1

Acknowledgements

This study was supported by the IRP of the NIMH, NIH. The editorial assistance of Ms. Ioline Henter is greatly appreciated.

Footnotes

Conflict of interest

The authors have no conflict of interest to disclose, financial or otherwise.

For submission to International Journal of Neuropsychopharmacology as a Brief Report

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Supplementary Materials

1
NIHMS305733-supplement-01.pdf (424.2KB, pdf)

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