Animals and treatment
All animal treatments, procedures, and care were approved by the National Institute of Mental Health (NIMH) Animal Care and Use Committee and followed the Guide for the Care and Use of Laboratory Animals
(ISBN 0-309-05377-3). Rats were chronically treated with lithium and VPA using a clinically relevant regimen established by our laboratory (Einat et al, 2003
). In brief, male Kyoto Wistar rats (starting weight 150–200 g) were housed two to four per cage in a 12-hour light/dark cycle with water and food ad libitum. After a one-week accommodation period, the rats received drug treatments. Control (regular) chow and lithium carbonate- or VPA-containing chows were custom-produced by Bio-Serve (Frenchtown, NJ, USA). Chows were identical with the exception of the drug. Half-dose chows (1.2 g/kg lithium carbonate or 10g/kg VPA) were used in the first week, and full-dose (2.4 g/kg lithium carbonate or 20g/kg VPA) chows for the remaining three weeks. Lithium-treated rats were provided with saline to prevent any potential electrolyte imbalance due to lithium treatment. The rats were euthanized by decapitation, rat brains were dissected on ice, and hippocampi rapidly frozen in dry ice and stored at −80°C until further tissue processing.
miRNAs from rat hippocampus were separated with mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer’s manual.
Screening miRNA targets of chronic lithium and VPA treatment
miRNA microarray was used to screen potential targets. The array slides were made in the Microarray CORE Facility of NHGRI, NIH. In brief, mirVana miRNA Probe Set 1564V2, which is a collection of 662 amine-modified DNA oligonucleotides targeting a comprehensive selection of human, mouse, and rat miRNAs, was purchased from Ambion (Austin, TX). These probes were printed on Corning Epoxide Coated Slides (Corning Inc, Corning, NY) in triplicate. miRNA labeling and hybridization were carried out using Ambion’s miRNA Labeling Kit and Bioarray Essential Kit, respectively, per manufacturer’s instructions (Ambion, Austin, TX). miRNA samples were pooled from two animals of the same treatment group for one slide. Samples from control animals and lithium- or VPA-treated animals were measured on the same slide. A second round of array experiments was conducted with another set of pooled samples from different animals to serve as a biological repeat in screening. Assay consistency was analyzed and tested via three replicates of the same miRNA on the slide.
The following criteria were used to select targets for further validation: (1) the detection was reliable, i.e. the difference between the control and drug-treated animal groups were detected with three repeats per group on the same slide, and this difference had a p-value of ≤0.05 (Student’s t-test); (2) the detected difference was biologically repeatable, i.e. similar and significant results were also obtained in the second round of array screening; (3) the expression differences were similar for the lithium and VPA treatment groups, i.e. significant changes occurred in the same direction for both lithium and VPA.
Validation of microarray findings with quantitative real-time PCR (qRT-PCR)
qRT-PCR was carried out using Ambion’s qRT-PCR miRNA detection kit per the manufacturer’s instructions (Ambion, Austin, TX). In brief, hippocampal miRNAs from each individual animal were reverse-transcribed with the corresponding RT primer. The RT product (cDNA) then entered a 40-cycle PCR with the corresponding PCR primers, in which SYBR Green I was used as the signal fluorescence and ROX as the control fluorescence. miRNA reverse transcription was on an Applied Biosystems’ GeneAmp 9700 thermal cycler, and real-time PCR on an Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Samples from all treatment groups were processed at the same time to avoid inter-experiment variances.
miRNA effector prediction
Selection of miRNA effectors for follow-up investigation
The effectors of lithium- and VPA-targeted miRNAs were selected for follow-up investigation based on presumed illness pathophysiology (admittedly in its infancy), as well as from targets implicated in the whole genome association dataset generated by the Wellcome Trust Case Control Consortium (WTCCC) (Consortium, 2007
). The WTCCC list contains all genes for which the single nucleotide polymorphisms (SNPs) have strong or moderate (e.g. p-value less than 0.0001) associations with BPD.
Immunoblotting (Western blot)
Immunoblotting was conducted as previously described (Chen et al, 1998
), with modifications. In brief, rat hippocampal samples were suspended in ice-cold lysing buffer. The buffer contained 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1% Triton X-100, 150 mM NaCl and 1 mM β-glycerophosphate, as well as the following ingredients added into the buffer immediately before use to the final concentrations of 5 mM DTT: 1% Phosphatase Inhibitor Cocktail I, 1% Phosphatase Inhibitor Cocktail II, and 10% Phosphatase Inhibitor Cocktail (Sigma, St. Louis, MO). Samples were homogenized by sonication (setting 2.5, 1 sec × 10 times, VirSonic ultrasonic cell disrupter (Virtis, Gardiner, NY)). Samples were then centrifuged (15 sec at 14000 × g) to remove undissolvable debris. Protein concentrations were determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL).
Immunobloting was performed using amounts of protein demonstrated to be within the linear range for the analysis. Equal amounts of proteins were loaded to 8–16% SDS-PAGE gels and separated by electrophoresis. Proteins were then electrophoretically transferred to nitrocellulose membranes. Nonspecific binding on the nitrocellulose was blocked with Tris Buffered Saline plus Tween−20 (TBST), 5% nonfat dry milk. The antibodies for beta-actin (ACTB) (Cell signaling, Boston, MA), calpain 6 (Abcam, Cambridge, MA), Dipeptidyl peptidase 10 (DPP10) (Abcam, Cambridge, MA), estrogen-related receptor gamma (ESRRG) (Imgenex, San Diego, CA), metabotropic glutamate receptor 7 (GRM7) (Abcam, Cambridge, MA), and thyroid hormone receptor beta (THRB) (Abcam, Cambridge, MA) were diluted according to the manufacturer’s recommendations and, if necessary, further adjusted according to initial test results. The secondary antibodies were horseradish peroxidase (HRP) conjugated anti-rabbit antibody. The immunocomplex was detected with an ECL plus kit (Amersham Biosciences, Piscataway, NJ). Quantitation of the immunoblots was performed by densitometric scanning of the film using the Kodak IS4400CF image analysis system and the corresponding software (Eastman Kodak, Rochester, NY).
Primary hippocampal neuron culture, treatments and transfections
Primary hippocampal neuron cultures were prepared as previously described (Du et al, 2008
; Du et al, 2004
). The neurons were treated with 1 mM (final concentration) of lithium or VPA for one or five days. RNAs were delivered into neurons using i-Fect reagent according to the manufacturer’s specification. A pilot experiment was conducted to test the dosage of RNAs (10, 20, 30 and 40 nmol/L) as well as incubation time (one, two, and three days) to assess earliest optimal outcomes. Forty nmol/L of RNAs and a two-day incubation period were selected for the rest of experiments. All RNA reagents were from Ambion (Austin, TX) including Pre-miR™ miRNA-34a precursor (PM11030), Pre-miR™ miRNA-34a precursor negative control (AM17110), anti-miR™ miRNA-34a inhibitor (AM11050), and anti-miR™ miRNA-34a inhibitor negative control (AM17010).