Exp Neurobiol 2019; 28(2): 158-171
Published online April 30, 2019
© The Korean Society for Brain and Neural Sciences
Juli Choi1, Yoon-Keun Kim2, and Pyung-Lim Han1,3*
1Department of Brain and Cognitive Sciences, Ewha Womans University, Seoul 03760, Korea.
2MD Healthcare Inc., Seoul 03923, Korea.
3Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Korea.
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-3277-4130, FAX: 82-2-3277-3419
Gut microbiota play a role in regulating mental disorders, but the mechanism by which gut microbiota regulate brain function remains unclear. Gram negative and positive gut bacteria release membrane-derived extracellular vesicles (EVs), which function in microbiota-host intercellular communication. In the present study, we investigated whether
Gut microbiota have an influence on cognition and behavior [1,2]. Recent studies have reported that patients with depression had reduced composition of
Several studies have attempted to explore the mechanisms of how gut microbiota influence brain function. Gut microbiota appear to maintain host homeostasis by communicating directly and indirectly with the nerve system [1,12,13]. Several underlying mechanisms have been proposed to explain how gut bacteria affect neural function; (i) bacterial metabolites including short chain fatty acids, carbohydrates, bile acids [14,15], and kynurenine , (ii) cytokines such as IL-6, MCP-1, TNFα, and INFα which were secreted from monocytes after stimulation with gut microbiota [16,17,18], and (iii) bacterial neurometabolites including dopamine, GABA, tryptophan or 5-HT precursors [19,20,21]. These products and metabolites are believed to enter the circulatory system and affect brain function. In contrast to this view, (iv) retrograde transport of bacterial metabolites directly through the vagus nerves innervating gut epithelial cells could occur and thereby change neural function [22,23,24].
Recent studies demonstrated that gut bacteria release membrane-derived extracellular vesicles (EVs) . EVs carry nucleic acids, lipids, proteins, and bacterial metabolites, which can affect various cellular pathways in the host . For example,
In this study, we investigated whether EVs produced by
Bacterial culture and EV isolation were carried out as described previously .
HT22 cells were cultured as described previously [37,38]. In brief, HT22 cells were grown in Dulbecco's modified Eagle's medium (DMEM; LM001-05, Welgene, Gyeongan-si, Korea) containing 10% heat-inactivated fetal bovine serum (FBS; FB02-500, Serum Source International, Charlotte, NC, USA) and penicillin (20 units/ml)/ streptomycin (20 mg/ml) (LS020-02, Welgene) at 37℃ and 5% CO2 supply conditions. At 70~80% confluency, HT22 cells were trypsinized and counted using a trypan blue (0.4%) staining method. They were plated at a density of 1.0×105 cells/well on a 6-well plate (SPL Life Science, Pocheon-si, Gyeonggi-do), or 1.0×106 cells on a 100-mm dish in DMEM media supplemented with 10% FBS and antibiotics. After 24 h of culture, cells were treated with glucocorticoid (GC; corticosterone, 400 ng/ml) or
Transfection of siRNA into the HT22 cells was performed as described previously [37,39]. HT22 cells were plated at a concentration of 1.0×105 cells/well in DMEM supplemented with 10% FBS in a 6-well plate (SPL Life Science). After 24 h, the media was changed with DMEM containing 1% FBS and siRNA was transfected using Lipofectamine-2000 (13778-075; Invitrogen). To prepare the siRNA and Lipofectamine-2000 mixture, 20 µM siRNA (3 µl; final concentration, 50 pM) and Lipofectamine-2000 (9 µl; final concentration, 7.5 µl/well) were separately diluted in 150 µl of Opti-MEM® Medium (31985070, Gibco, Thermo Fisher Scientific, Paisley, Scotland, UK). Each mixture was diluted at 1:1 ratio, mixed slowly and then incubated for 5 min at room temperature. The siRNA and Lipofectamine-2000 mixture (250 µl/well) was carefully dripped onto HT22 cells while culturing in fresh DMEM containing 1% FBS with or without 20 µg/well of
Mice were treated with restraints as described previously [40,41]. Male C57BL/6J mice (7-weeks old) were purchased from Daehan BioLink, Inc. (Eumsung, Chungbuk, Korea). Mice were housed in pairs per cage allowing an ad libitum access to water and food at a temperature (23℃) and humidity (50~60%)-controlled room under a 12-h light/dark cycle (lights on at 07:00~19:00 h). Animals were handled in accordance with the animal care guidelines of Ewha Womans University and restraint treatment procedures in this study were approved by the Ewha Womans University Animal Care and Use Committee (IACUC 15-012). To deliver restraint, mice were placed in a 50-ml polypropylene tube carrying many punched holes for ventilation and were restrained for 2-h daily for 14 days. Control mice housed in pairs were maintained in home cages without disturbance.
Quantitative real-time PCR (qPCR) analysis was carried out as described previously [40,41]. Briefly, hippocampus tissue was obtained and ground using pellet pestles (Z359971, Sigma-Aldrich) in TRI-zol reagent (15596-018, Invitrogen). Harvested HT22 cells were homogenized with TRI-zol reagent. Total RNA was isolated from homogenates and eluted in RNase free water (129112, Qiagen, Hilden, Germany). Two µg of total RNA was treated with DNase I to remove genomic DNA contamination and then converted to cDNA using a reverse transcriptase system (Promega, Madison, WI, USA).
Four µl of 1/8 diluted cDNA, 10 µl of 2X iQTM SYBR Green Supermix (Bio-Rad Laboratories, Foster City, CA, USA), and 1 µl each of 5 pmol/µl forward and reverse primers were mixed to a total volume of 20 µl. The qPCR reaction was carried out using the CFX 96 Real-Time PCR System Detector (Bio-Rad Laboratories). The transcript expression levels were normalized relative to
The primers used in this study were: t
Western blot analysis was carried out as described previously . HT22 cells were washed with 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and harvested. Harvested HT22 cells or hippocampal tissue was homogenized in homogenization buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.1% sodium deoxycholate) containing a protease inhibitor cocktail (Roche) by sonicating on ice using an Epishear probe sonicator, with two rounds of 15-sec pulses and 30-sec rest intervals at 40% power outlet (Active Motif). The supernatant of homogenates was obtained after centrifugation at 13,000 ×g at 4℃ for 15 min.
The amount of protein was determined by the Bradford method (500-0006, Bio-Rad Laboratories). Twenty µg of tissue or cell sample was mixed with 6X gel loading buffer and boiled for 5 min. The proteins were resolved on SDS-PAGE and transferred onto PVDF membrane using Trans-Blot® SD semi-dry Electronic transfer cell and power supply system (1703848, Bio-Rad Laboratories). The blots were incubated with 5% skim milk or 1% bovine serum albumin (BSA) in 1X TBST (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.1% Tween 20) to block a non-specific binding. Blots were incubated with a primary antibody in blocking solution followed by a secondary antibody in 1X TBST. Specific bands were visualized using Enhanced ChemiLuminescence (ECL)TM Western Blot Analysis System (RPN2109, Amersham, GE Healthcare, UK). Quantification of blot images was processed using the Image-Pro-Premier 6.0 software (MediaCybernetics, Rockville, MD, USA).
The primary antibodies used were: anti-proBDNF (ANT-006; 1:1,000, Alomone Labs, Hadassah Ein Kerem, Jerusalem, Israel), BDNF (ab108319; 1:2,000, Abcam, Cambridge, UK), and anti-β-actin (sc-47778; 1:1,000, Santa Cruz). The secondary antibodies used were: anti-mouse IgG-HRP (GTX213111-01; 1:1,000, Gene-Tex, Irvine, CA, USA) and anti-rabbit IgG-HRP (GTX213110-01; 1:1,000, GeneTex).
The behavioral tests were carried out as described previously [40,41]. Mice were acclimated in the testing room for at least 30 min prior to the start of each behavioral test. All behavioral tests were performed in the light cycle phase (9 a.m.~3 p.m.) and monitored with a computerized video tracking system (SMART; Panlab S.I., Barcelona, Spain) or a webcam recording system (HD Webcam C210, Logitech, Newark, CA, USA).
The sociability test was performed as described previously [40,42]. Briefly, the U-shaped two-choice field was prepared by partitioning an open field (40×40 cm2) with a wall (20-cm wide and 20-cm high). Circular grid cages (12 cm in diameter×33 cm height) were presented on each side of the U-shaped two-choice field. For habituation, a subject mouse was allowed to freely explore the U-shaped two-choice field with empty circular grid cages on each side for 5 min and returned to the home cage. After 10 min, a social target mouse was loaded to a circular grid cage at one side and the subject mouse was placed in the center of the U-shaped two-choice field where the subject mouse was able to see both grid cages. The subject mouse was allowed to explore both fields for 10 min while the trajectory spent in the fields was recorded by a video tracking system (SMART, Panlab S.I.) The field with a circular grid cage carrying a social target mouse and the field containing an empty circular grid cage were defined as the target field and non-target field, respectively. Social target mice with same age and sex as the subject mice were prepared. Social targets had never been exposed to subject mice from the acclimation stage.
The tail suspension test (TST) was carried out as described previously . Mice were suspended individually by fixing their tails with adhesive tape to the ceiling of a shelve 50 cm above the bottom floor. The subject mouse was suspended for 6 min and the cumulative immobility time was measured. Behavioral performances were recorded with a webcam recording system (HD Webcam C210, Logitech) and subsequently analyzed.
The forced swim test was performed as described previously . Mice were placed in a Plexiglas cylinder (15 cm in diameter×27 cm height) holding water at 24℃ with a depth of 15 cm. Mice were placed in the cylinder for 6 min and the cumulative immobility time was measured for the final 5 min. Immobility was defined as the time when a mouse was floating with all limbs motionless. The performance during the test was recorded using a webcam recording system and then analyzed.
Multiple comparisons were performed by one-way ANOVA followed by the Newman-Keuls
Administration of microbiota products in mice increased BDNF in hippocampus [13,43]. Treatment with probiotics mixture in aged rats increased BDNF levels in the hippocampus . Administration of a probiotic formulation (
BDNF expression is regulated by transcription and/or epigenetic factors, including cAMP response element (CRE) binding protein (CREB1) , histone acetyltransferase 2 (HDAC2) , and Sirtuin1 (Sirt1) . HDAC2 negatively regulates BDNF expression in stress-induced depression models , whereas CREB1 and Sirt1 enhance BDNF expression [47,48,49].
GC treatment in HT22 cells decreased
Next, we examined whether
The body weight of mice decreased during restraint treatment, but was not affected by
These results suggest that
Next, we examined whether
Mice treated with CRST showed reduced social interaction in the sociability test (Fig. 3E and 3F) and increased immobility time in the TST and FST (Fig. 3G and 3H). In contrast, post-stress treatment with
Next, we examined whether antidepressant-like effects of
In the present study, we demonstrated that
The results of EVs-induced increase of BDNF and other genes in HT22 cells (Fig. 1) indicate that EVs can induce genomic responses by directly acting on cells. Considering that intraperitoneally (i.p.) injected
BDNF expression is reduced in the hippocampus of postmortem samples from major depressive disorder patients and in mice with stress-induced depression [58,59]. In contrast, depressive behavior is reversed by administration of recombinant BDNF in mice . BDNF expression is regulated by epigenetic factors such as Sirt1, HDACs and MeCP2 in stress-induced depression models [61,62]. Sirt1 has a deacetylase activity . Sirt1 indirectly regulates BDNF and CREB expression by decreasing miR-134 expression [48,63]. Sirt1 can promote axon development and dendritic arborization [63,64,65]. Sirt1 expression is conversely regulated by miRNAs or other factors [48,63,66]. Sirt1 is reduced in the hippocampus of mice displaying depressive-like behaviors induced by chronic unpredictable mild stress (CUMS), whereas its activation reversed depression-like behaviors . In rats exposed to CUMS, depression-like behaviors were reversed by resveratrol, which increased the expression of Sirt1, CREB, and BDNF in the hippocampus, while decreasing miR-134 . As demonstrated in the present study, Sirt1 was also reduced in the hippocampus of mice exposed to chronic restraint stress, whereas
Which factor contained in