Exp Neurobiol 2023; 32(2): 91-101
Published online April 30, 2023
© The Korean Society for Brain and Neural Sciences
Yong-Jae Jeon1†, Bo-Ryoung Choi1†, Min-Sun Park1, Yoon-Sun Jang1, Sujung Yoon2, In Kyoon Lyoo2,3 and Jung-Soo Han1*
1Department of Biological Sciences, Konkuk University, Seoul 05029,
2Ewha Brain Institute and Department of Brain and Cognitive Sciences, Ewha Womans University, Seoul 03760,
3Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-450-3292, FAX: 82-2-3436-5432
†These authors contributed equally to this article.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
The FK506 binding protein 5 (FKBP5) is a co-chaperone that regulates the activity of the glucocorticoid receptor (GR) and has been reported to mediate stress resilience. This study aimed to determine the effects of
The hippocampus is vulnerable to uncontrollable stress [1-3] and is enriched with glucocorticoid receptors (GR) [4-6]. Accordingly, animals that experience acute uncontrollable stress perform poorly in hippocampal-dependent memory tasks, such as the hidden platform water maze and novel object recognition [3, 7-9]. Furthermore, GR signaling and expression levels of FK506-binding protein 5 (FKBP5) are altered in the hippocampus during acute or chronic stress [10-13]. Recently, levels of GR phosphorylation at serine 203 and serine 211 were reported to increase in the hippocampus of rats with chronic subcutaneous corticosterone injections .
Several studies have reported that FKBP5 is a co-chaperone that regulates GR activity, which in turn mediates stress resilience [13, 15, 16]. For example,
This study aimed to determine the effects of
Tail sampling was performed in three-week-old mice and then stored in a microcentrifuge tube (see Fig. 1). A tail mix buffer (0.5% sodium dodecyl sulfate [SDS], 0.1 M NaCl, 50 mM Tris [pH 8.0], 2 mM EDTA), and proteinase K (10 mg/ml) were added to the microcentrifuge tube containing the tail and incubated overnight in a 56℃ water bath. Following overnight incubation, 8 M potassium acetate (75 µl) and chloroform (400 µl) were added to the microcentrifuge tube and centrifuged (Smart R17, Hanil Science, South Korea) for 15 min, at 4℃, 9358×g. After centrifugation, the supernatant was transferred to a new tube, and 100% ethanol (1 ml) was added. Another centrifugation was performed (4℃, 18,341×g, 5 min), the supernatant was discarded, and 70% ethanol (700 µl) was added to dissolve the pellet. One last centrifugation (4℃, 18,341×g, 5 min) was performed, the supernatant was discarded, and the resulting deoxyribonucleic acid (DNA) pellet was stored in Tris-acetate-EDTA (TAE) buffer (100 µl). Polymerase chain reaction (PCR) was performed using 2× Biomix (Meridian Bioscience, Cincinnati, OH, USA), DNA, and primers. A common forward primer (5’-AAA GGA CAA TGA CTA CTG ATG AGG-3’) and two reverse primers (5’-AAG GAG GGG TTC TTT TGA GG-3’ and 5’-GTT GCA CCA CAG ATG AAA CG-3’) were used for wild-type and
Blood and hippocampal tissues were collected from wild-type and
Before the stress procedure, all mice were familiarized with the object recognition box for five days. The stress procedure consisted of restraining the animals for 60-min in a cone-shaped plastic bag and administering 60 intermittent tail-shocks (0.45 mA intensity; 1 s duration; 30 to 90 s inter-shock interval), through copper electrodes attached to their tails. The control animals were left undisturbed in their cages. At the end of the stress procedure, the mice were placed back in their home cages for 1 h of recovery before beginning the object recognition task (Fig. 1).
This study employed a modified version of the spontaneous object recognition task [8, 21-23], initially developed by Ennaceur and Delacour , which exploits rodents’ natural tendency to explore novel stimuli. The task was conducted inside a black open-field square box (27×34×26.5 cm) with a constant masking white noise of 70 dB source. The box was wiped with 70% ethanol between animals. Mice were handled for 5-min daily and were placed inside the box for 10 min daily to familiarize themselves with transportation and the empty arena for five days. On the sixth day, 1 h after the stress procedure, the animals underwent successive sessions of familiarization, delay, and recognition memory tests (Fig. 1). The familiarization phase consisted of 1-minute of re-habituation in the open-field box and a brief transfer of the animals to their home cages, while two identical objects were placed at the two corners of the box. The animals were then placed back in the box where they remained until they cumulatively explored the objects for 20 s. Upon reaching the 20 s of object exploration criterion (variable time in the arena), animals were placed back in their home cages for 3 h, until the test phase. During the delay period, an identical object to that placed in the familiarization phase (but not scent-marked) and a novel object were placed in the same two corners (counterbalanced between animals) as in the familiarization phase. After the delay, animals were reintroduced to the box and remained there until they accumulated a total of 20 s of exploration of the two different objects. Exploration behavior was quantified using a computer-assisted scoring program (QBASIC), where manual keystrokes on a computer keyboard recorded the duration and frequency of object exploration . Exploration was only scored when the snout of the mouse was directly facing and sniffing the objects, and not when another body part contacted the objects, such as the forepaws on the object and rearing. During the familiarization and test phases, one mouse did not meet the 20-s exploration criterion within the 10-min allotted time in the open-field box and was excluded from the study. Preference for the novel object was computed as the time spent exploring the novel object rather than the familiar object. Computer scoring of the behavior was performed by Y.-J.J. and Y.-S.J., who matched 87% of the joint observations based on randomly selected familiarization and test sessions from ten mice.
All mice were deeply anesthetized with isoflurane and perfused with ice-cold 0.01 M phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.01 M PBS for histological analysis. Brains were removed immediately and placed in 4% paraformaldehyde in 0.01 M PBS at 4℃ for 48 h. After fixation, the brain samples were embedded in 30% sucrose in 0.01 M PBS until the brain sunk down to the bottom of the glass jar. Each brain sample was quickly frozen using dry ice and stored at -70℃. Before staining, brain samples were embedded with Tissue-Tekⓡ (Sakura, Torrance, CA, USA), sliced into 30-µm coronal sections, and stored in cryoprotectant (30% ethylene glycol, 25% glycerol, 25% 0.1 M phosphate buffer, and 20% distilled water). For total protein extracts, individual tissue samples were homogenized in ice-cold lysis buffer consisting of 20 mM Tris (pH 7.5), 5% glycerol, 1.5 mM EDTA, 40 mM KCl, 0.5 mM dithiothreitol, and protease inhibitors. The homogenates were then centrifuged at 18,341×g for 1 h at 4℃, and the supernatant was harvested and stored at -80℃ until further analysis.
Protein extraction was performed using a ReadyPrepTM Protein Extraction Kit (Bio-Rad, Hercules, CA, USA). Hippocampi were dissected and homogenized with cytoplasmic protein extraction buffer (CPEB) in a glass tissue grinder (Radnoti, Covina, CA, USA). After centrifugation at 1,000×g for 10 min, at 4℃, the resulting supernatant was used as the cytoplasmic protein fraction. CPEB was added to extract nuclei from the pellet, and the mixture was centrifuged at 1,000×g for 10 min, at 4℃. Protein solubilization buffer was added to the mixture, which was then centrifuged at 16,000×g for 20 min, at 24℃. The resulting supernatant was used as the nucleic protein fraction.
Protein concentrations in the total cell extracts and cytosolic and nuclear fractions were determined using a bicinchoninic acid assay. Proteins in the extracts were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane using a Mini Trans-Blot Cell (Bio-Rad). After blocking, the membranes were incubated with polyclonal anti-Fkbp5 (1:1,000; Origene, Rockville, MD, USA), polyclonal anti-GR (1:1,000; Santa Cruz, Dallas, TX, USA), polyclonal anti-pGR S211 (1:1,000; Cell Signaling, Danvers, MA, USA), anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000; Cell Signaling) or anti-actin antibody (1:5,000, Sigma, St. Louis, MO 68178), followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoreactive proteins were visualized using an enhanced chemiluminescence system (GE Healthcare) and images were captured using an ImageQuant LAS 500 CCD camera (GE Healthcare). The protein band intensity of GR and pGR was normalized to the intensity of the GAPDH band or actin band using Image J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997~2018).
Brain sections were washed in PBS containing 0.3% Triton X-100 (PBS-T) and then incubated in a blocking solution (10% fetal horse serum and 0.3% Triton X-100 in PBS) for 2 h at room temperature. Next, they were incubated overnight with a cocktail of primary antibody solution (guinea pig anti-NeuN antibody, 1:1,000, Merck Millipore, Burlington, MA, USA; rabbit anti-pGR antibody, 1:200, Cell Signaling), which contained 1.5% horse serum in PBS-T. The sections were then washed in PBS-T and incubated for 1 h at room temperature with a cocktail of secondary antibody solution (Alexa Fluor 488 conjugated donkey anti-rabbit antibody, 1:200, A21206, Invitrogen, Waltham, MA, USA; Alexa Fluor 633 conjugated donkey anti-guinea pig, 1:200, A21105, Invitrogen), which contained 1.5% horse serum in PBS-T. Subsequently, sections were mounted on resin-coated slides, dried for 1 h, and finally coverslips were mounted with ProLong Gold Antifade Mountant (Invitrogen). Images were obtained using a confocal microscope (LSM 800, Carl Zeiss, Oberkochen, Germany). At least six sections were selected per animal.
The time spent exploring the two objects during the familiarization and test phases of the NOR task was evaluated in the same animals. Thus, we utilized a one-sample t-test (two-tailed significance) to analyze the behavioral data [8, 22], where a test value setting of 10 s denoted no object preference. The preference in the NOR task, corticosterone levels, total GR, and pGR levels were analyzed using analysis of variance (ANOVA) and an independent t-test. Post hoc analyses were conducted using Fisher’s least significant difference test, if necessary. All data are expressed as boxplots or means±standard error of the mean. The alpha level was set to 0.05. SPSS Statistics 25 (IBM, Armonk, NY, USA) and Prism 9 software (GraphPad Software, San Diego, CA, USA) were used for statistical analyses and graphical figures, respectively.
Thirty-six mice from all groups equally explored the two identical objects placed at the left and right sides (L and R) in the open-field box during the familiarization phase (all p>0.05), indicating no left-right side bias against the object location (Fig. 2A). All groups required a similar amount of time to reach the 20-s exploration criterion (F3,32<0.7, p>0.77) in the familiarization phase, suggesting that the behavioral impairment observed in this experiment was not due to alterations in exploratory or locomotor activity. During the test phase (Fig. 2A), significantly more time was spent exploring the novel object than the familiar object in the non-stressed wild-type mice (t7=10.77, p<0.001), non-stressed
The absence of Fkbp5 in the hippocampus of the
A two-way ANOVA of the corticosterone levels (genotype, F1,36=9.49, p<0.01; stress F1,36=67.06, p<0.001; interaction, F1,36=7.74, p<0.01) and post hoc analyses revealed that the corticosterone levels of stressed
Representative western blots of GR and pGR S211 in cytosolic and nuclear fractions are shown in Fig. 5. We assessed stress-induced GR translocation in the hippocampi of wild-type and
In addition, we measured pGR S211 levels in the cytosolic and nuclear fractions of the non-stressed and stressed hippocampi. A two-way ANOVA of cytosolic pGR S211 levels (genotype, F1,40=3.35, p<0.05; stress, F1,40=7.89, p<0.001; interaction, F1,40=3.80, p<0.05) and post hoc analyses revealed that cytosolic pGR S211 levels in stressed wild-type mice were higher than those in other groups (Fig. 5E). A two-way ANOVA of nuclear pGR S211 levels (genotype, F1,40=0.02, p=0.90; stress, F1,40=89.98, p<0.001; interaction, F1,40=2.34, p=0.13) revealed that nuclear pGR S211 levels increased in the hippocampus of wild-type and
The hippocampus, which is part of a system necessary for memory formation , is enriched with GR and terminates the stress response via glucocorticoid-mediated negative feedback of the HPA axis [4, 28]. Therefore, memory formation is susceptible to stress . Stress impairs hippocampal-dependent spatial memory in rodents [1, 3, 7]. Furthermore, recognition memory has been shown to be impaired in rats with hippocampal damage and stressed rats [8, 9, 21], similar to the stressed wild-type mice used in our experiment. However, recognition memory was intact in the
FKBP5, an inhibitor of GR activity, determines the binding affinity of GR to glucocorticoids, and thus regulates the negative feedback sensitivity of the HPA axis to stress [29, 30]. Moreover, stress-induced translocation of GR into the nucleus activates an intracellular feedback loop by enhancing
In the present experiment, we measured one GR phosphorylated level in the hippocampus. However, further study measuring phosphorylated GR levels at different sites is needed to corroborate the findings. The GR is phosphorylated at three major sites (S203, S211, and S226) on the N-terminal side, and an interaction between multiple phosphorylation sites is reported to be essential for GR activation and repression. For example, the phosphorylated GR level at S203 was higher when no GR phosphorylation occurred at S226 and vice versa, indicative of intersite dependency . Moreover, GR transcriptional activation increases when S211 phosphorylation exceeds S226 .
Most studies using
This work was supported by grants from the Bio & Medical Technology Development Program of the National Research Foundation (NRF) and was funded by the Korean government (Ministry of Science and ICT) (2020M3E5D9080734 to J.-S.H.).
The authors declare no conflicts of interest.