Activation of BDNF by transcription factor Nrf2 contributes to antidepressant-like actions in rodents

The transcription factor erythroid 2-related factor 2 (Nrf2) and brain-derived neurotrophic factor (BDNF) play a key role in depression. However, the molecular mechanisms underlying the crosstalk between Nrf2 and BDNF in depression remain unclear. We examined whether Nrf2 regulates the transcription of Bdnf by binding to its exon I promoter. Furthermore, the role of Nrf2 and BDNF in the brain regions from mice with depression-like phenotypes was examined. Nrf2 regulated the transcription of Bdnf by binding to its exon I promoter. Activation of Nrf2 by sulforaphane (SFN) showed fast-acting antidepressant-like effects in mice by activating BDNF as well as by inhibiting the expression of its transcriptional repressors (HDAC2, mSin3A, and MeCP2) and revising abnormal synaptic transmission. In contrast, SFN did not affect the protein expression of BDNF and its transcriptional repressor proteins in the medial prefrontal cortex (mPFC) and hippocampus, nor did it reduce depression-like behaviors and abnormal synaptic transmission in Nrf2 knockout mice. In the mouse model of chronic social defeat stress (CSDS), protein levels of Nrf2 and BDNF in the mPFC and hippocampus were lower than those of control and CSDS-resilient mice. In contrast, the protein levels of BDNF transcriptional repressors in the CSDS-susceptible mice were higher than those of control and CSDS-resilient mice. These data suggest that Nrf2 activation increases the expression of Bdnf and decreases the expression of its transcriptional repressors, which result in fast-acting antidepressant-like actions. Furthermore, abnormalities in crosstalk between Nrf2 and BDNF may contribute to the resilience versus susceptibility of mice against CSDS.

Mice were monitored using a video tracking system (Ethovision XT 14.0) for 6 minutes.
SPT: The mice were habituated to a 1% sucrose solution for 48 h before the test day.
And then the mice were deprived of water and food for 4 h, followed by a preference test with water and 1% sucrose for 1 h. The bottles containing water and sucrose were weighed before and at the end of this period and the sucrose preference (%) was determined [5][6][7].

Quantitative real-time PCR assay
Levels of Nrf2 and Bdnf mRNA were analyzed by quantitative real-time PCR. RNA was extracted by using Eastep® Super Kit (Promega), and then reverse transcription was performed with GoScript TM Reverse Transcriptase Mix, Oligo (dT) (Promega). All real-time PCR reactions were performed by using the 788BR05175 Real-Time PCR System and ChamQ TM SYBR® qPCR Master Mix Kit (Vazyme). The target gene expression was calculated as the 2−ΔΔCt method. Forty cycles of PCR amplification were performed as follows: denature at 95°C for 30 s, annealing at 55°C for 30 s, and extend for 30 s at 72°C.

Western blotting assay
Cells or brain samples were lysed in RIPA buffer (20 mM pH 7.5 Tris-HCl, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na 3 VO 4 , 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). The concentrations of total proteins were examined by Bradford assay. 30 µg Proteins were resolved on 7.5%, 10%, or 15% polyacrylamide gels, according to each marker's molecular weight, and then transferred to polyvinylidenedifluoride (PVDF) membrane. For the immunodetection, the blots were blocked with 2% BSA plus 5% nonfat dry milk in TBST (TBS + 0.1% Tween-20) for 1 h at room temperature (RT) and then incubated with primary antibodies (The concentration is selected by the manufacturer's instructions) overnight at 4°C. The next day, the blot was washed three times in TBST and incubated with a horseradish peroxidase-conjugated anti-rabbit antibody (1:5000) or anti-mouse antibody (1:5000) for 1hour, at RT. After the 3 time washes with TBST, the bands were detected by using enhanced chemiluminescence (ECL) detection reagents (GE Healthcare) and exposed 3 to Tanon-5200CE imaging system (Tanon, Shanghai, China). The quantification was carried out with ImageJ software.

Luciferase assay
Cells in 6-wells plates were transfected with BDNF exon I, II, and IV luciferase reporter together, pRL-TK Renilla luciferase plasmid (Promega), and different kinds of plasmids or siRNA. Following transfection for 48 h, the cells were collected and subjected to the dual-luciferase reporter assay kit (Promega) according to the manual.

Chromatin immunoprecipitation (ChIP) assay
After transfection or treatment with certain plasmids or drugs, the cells were subjected to the ChIP assay according to the procedures described in the manual of the SimpleChIP® Enzymatic Chromatin IP Kit (Cell signaling). For the Nrf2 antibody, 7.5 µg of Nrf2 antibody (Abcam) was added to the homogenate for the sample. For the PCR analysis. Specific primers were for the amplification of the promoter region of 150 bp 5' of BDNF exon I, which contains a putative Nrf2 binding site. The primer sequences were: forward 5'TGATCATCACTCACGACCACG 3'; reverse 5'CAGCCTCTCTGAGCCAGTTACG3' based on previously published results [8].
The 35 cycle PCR was performed as follows: denature at 95°C for 30 s, annealing at 58°C for 30 s and extend for 30 s at 72°C, and then the PCR sample was resolved on a 2% agarose gel and sequenced.

Immunofluorescence staining
After LPS administration 24 hours, mice were anesthetized with sodium pentobarbital and perfused transcardially with 10 ml of isotonic saline, followed by 40 ml of ice-cold 4% paraformaldehyde in 0.1-M phosphate buffer (pH 7.4). After the perfused the brain samples were collected and postfixed overnight at 4°C. On the next day, 50-µm thick serial coronal sections of brain tissue were cut in ice-cold, 0.01-M phosphate-buffered saline (pH 7.5), using a vibrating blade microtome (VT1000S, Leica Microsystems AG, Wetzlar, Germany). Brain sections were identified according to the previously reported [7]. For the immunofluorescence staining, the slides with cells were fixed by 4% paraformaldehyde. And then the slides with cells or mice brain sections were incubated with 3% hydrogen peroxide at room temperature for 10 minutes followed by blocking and incubation with primary antibodies for 48 h at 4°C. On the third day, the slides with cells or brian sections were incubated with an Alexa Fluor 488 or 568 conjugated isotype-specific secondary antibody for 1 h at room temperature. Images were then collected with an Olympus confocal microscope. The fluorescence intensity was quantified using Image J.

Electrophysiological recordings
For acute slice preparation, all experiments were performed as previously reported [9,10]. After LPS or SFN administration twenty-four hours, mice were deeply anesthetized with isoflurane and then decapitated, mice brains were quickly and carefully removed and subsequently transferred for ice-cold oxygenated ACSF containing 120 mM NaCl, 2.5 mM KCl, 1.