CaBP-9k knockout mice and TUDCA treatment
CaBP-9k KO mice were generated as previously described [18, 19]. The genotypes of offspring were determined by PCR analysis, as described previously . The Mice were housed in a cage with 12:12h light-dark cycle. No more than 5 mice were housed in a cage. Mice were handled according to a protocol approved by the Ethics Committee of the Chungbuk National University. TUDCA-treated mice were fed a diet of standard laboratory chow (Purina Mills, USA) supplemented with either 0.4 % (wt/wt) TUDCA (sodium salt; Matrix Scientific, USA). Treatment was started when the mice were 5 months old and continued for 8 months. Behavioral testing started at 7 months of age, where CaBP-9k KO mice start to display memory deﬁcits and lasted for a month. Weight was measured at the beginning of the treatment TUDCA and general activity was monitored during experiments.
Real-time reverse transcription PCR
Quantitative real-time PCR was performed as described previously . Total RNA was extracted from wild-type and calbindin mutant brains using Trizol reagent (Invitrogen). First-strand complementary DNA (cDNA) was prepared by reverse transcription using the MMLV cDNA synthesis kit (Thermo Fisher Scientific). To determine the conditions for the logarithmic phase during PCR amplification with target mRNA, aliquots (1 mg) were amplified using different numbers of cycles. A linear relationship between PCR product band visibility and the number of amplification cycles was observed for target mRNAs. Real-time PCR was performed with 1 ml of the cDNA template added to 10 ml of 2x SYBR Premix Ex Taq (TaKaRa Bio) and specific primers (10 pM each). Primer sequences are presented in supplementary table 1. Real-time PCR (Applied Biosystems) was carried out for 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, and extension at 72°C for 15 sec. Target gene expression was quantified relative to that of an internal control gene (Gapgh) based on the comparison of the threshold cycle (CT) at constant fluorescence intensity. The amount of transcript was inversely related to the observed CT and the CT was expected to increase by 1 for every two-fold dilution of the transcript. Relative expression (R) was calculated using the equation R = 2-[ΔCT sample-ΔCT control]. All data were normalized relative to Gapgh as well as to the respective controls.
Western blot analysis
Western blotting was performed as described previously [20, 21]. Brain lysates were extracted with RIPA buffer (Invitrogen). Proteins (40 μg/lane) were separated on 10~15% SDS-PAGE gel and transferred to an immobilon-P membrane (Millipore) by a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories). The resulting blot was blocked in TBS (tris-buffed saline) containing 5% BSA for 60 minutes, then incubated with a primary antibody rabbit anti-calbindin-D9k (Invitrogen, PA5-68289, 1:1000), rabbit anti-BACE (Cell Signaling Technology, #5606, 1:1000), rabbit anti-Tau (Cell Signaling Technology, #4019, 1:1000), p-SAPK/JNK (Cell Signaling Technology, #9251, 1:1000), mouse anti-Bax (Santa Cruz, sc-7480, 1:500), mouse anti-Bcl-2 (Santa Cruz, sc-7382, 1:500), rabbit anti-cleaved caspase-3 (Cell Signaling Technology, #9664, 1:1000), rabbit anti-cleaved caspase-9 (Cell Signaling Technology, #7237, 1:1000), PARP (Cell Signaling Technology, #9532, 1:1000), mouse anti-GADD153/CHOP (Cell Signaling Technology, #2895, 1:1000), rabbit anti-p-PERK (Cell Signaling Technology, #3179, 1:1000), rabbit anti-PERK (Cell Signaling Technology, #3192, 1:1000), rabbit anti-p-IRE1α (Thermo Fisher Scientific, PA1-16927, 1:1000), rabbit anti-IRE1α (Cell Signaling Technology, #3294, 1:1000), rabbit anti-p-eIF2α (Cell Signaling Technology, #3597, 1:1000), rabbit anti-caspase-12 (Cell Signaling Technology, #2202, 1:1000) and GAPDH (Santa Cruz, sc-25778, 1:500) for overnight at 4°C. After washing in TBS containing 0.1% Tween 20, the membrane was incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (1:2500, Cell Signaling Technology) for 1 hour at room temperature. After washing, the membrane was developed by using ECL Western-blotting reagents (Pierce Biotechnology). Immunoreactive proteins were visualized by exposure to Agpa CP-BU X-ray film (Agfa-Gevaert NV). Protein bands were visualized by image-scanning, and optical density was measured by using ImageJ analysis software 1.37 after the data were corrected by background subtraction
Immunostaining of brain sections or dissociated cells was performed as described previously [18, 20, 21]. Primary antibodies used were mouse anti-APP/b-Amyroid (Cell Signaling Technology, #2450, 1:500), mouse anti-Tau (Cell Signaling Technology, #4019, 1:300), rabbit anti-PARP (Cell Signaling Technology, #9532, 1:500), rabbit anti-cleaved caspase-3 (Cell Signaling Technology, #9664, 1:500), mouse anti-BrdU (BD Biosciences, #555627, 1:800), rabbit anti-Ki67 (Cell Signaling Technology, #9129, 1:500), mouse anti-a-synuclein (Cell Signaling Technology, #2647, 1:500), chicken anti-tyrosine hydroxylase (AVES, TYH, 1:800), chicken anti-GFP (Invitrogen, A10262, 1:800), rabbit anti-calbindin-D9k (Invitrogen, PA5-68289, 1:800) antibodies. Appropriate secondary antibodies conjugated with Alexa dyes (Invitrogen) were used to detect primary antibodies. DAPI (Sigma-Aldrich) was used to stain nuclei.
For evaluation of CaBP-9k knockdown by shRNA constructs, Neuro2a cells maintained in DMEM/10% FBS were transiently transfected using Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions. Transfections were allowed to proceed for 4–5 hours, and then cells were cultured in 10% FBS/DMEM for 24 hours.
The primary neuronal cells were isolated from E14.5-16.5 CaBP-9k KO mice. Meninges were removed, and primary neuronal cells were dissociated with trituration after trypsin/EDTA treatment. The cells were plated onto poly-D-lysine/laminin-coated coverslips and cultured in the medium containing Neurobasal medium (Invitrogen), 2 mM glutamine, 2% (v/v) B27 supplement (Invitrogen), 1% (v/v) N2 supplement (Invitrogen), and 50 U/mL penicillin/streptomycin (Invitrogen).
As described previously [18, 20], tissue sections and cell cultures were fixed with 4% PFA, and in situ detection of cells with DNA-strand breaks was performed using In Situ Cell Death Detection Kit (Roche Diagnostics) according to the manufacturer's instruction.
Measurement of intracellular calcium
The primary neuronal cells were plated in 96 well plates. DIV7 neuronal cells were incubated with calcium dye, Rhod-4 (Abcam), for 1h at 37°C. The plates were then placed into a Lionheart™ FX Automated Microscope (BioTeK) for the calcium response to monitor cell fluorescence (Rhod-4: Excitation = 540 nm and Emission = 590 nm). Images were acquired using a 4x objective at a rate of 3 frames per second. In-line injectors were used to dispense 20 μL of glutamate (30 µM, Sigma-Aldrich) into the wells, and cells were imaged for an additional 60 seconds to monitor response.
All behavioral assays were done during the light cycle. Health conditions including weights, activity and feeding were checked prior to assays. We used male and female mice for most behavioral assays. All behavioral assays were done blind to genotypes with age-matched littermates of mice.
Novel object recognition test: As described previously , a test mouse was first habituated to an open field arena (60 × 60 cm) for 5 min. Following habituation, the test mouse was removed from the arena and two identical objects with size (10.5 × 4.5 × 2.5 cm) were placed in the opposite corners of the arena, 15 cm from the side walls. Then the test mouse was reintroduced into the center of the arena and allowed to explore the arena including the two novel objects for 10 min. After 6 h, one object was replaced with another novel object, which was of similar size but different shape and color than the previous object. The same test mouse was placed in the arena to explore the arena and the two objects. The movement of mice was recorded by a camera for 10 min and further analyzed by the video tracking EthoVision XT 14 software (Noldus).
Passive avoidance test: Mice were individually habituated to the lighted compartment before a test. During the training session, each mouse was placed into the lighted compartment and the latency to enter the dark compartment was recorded. When the mouse entered the dark compartment with all four paws, a foot shock (2 mA, 3 sec) was delivered. During retention session 24 h later, each mouse was placed into lighted compartment again and the latency to enter the dark compartment was recorded.
Morris water maze test: Mice were introduced into the perimeter of a circular water-filled tank 90 cm in diameter and 42 cm in depth with visual cues that were present on the tank walls as spatial references. The tank was divided into four equal quadrants (Q1-4) by lines drawn on the floor. A circular plexiglass platform was submerged 1 cm deep in Q2 and as such hidden from the mice. The mice started the task from one of three quadrants Q1, Q3 and Q4, varied by day of testing. Four trials were performed per mouse per day for ten days. Each trial lasted 1 minute and ended when the mouse climbed onto and remained on the hidden platform for ten seconds. The mouse was given 20 seconds to rest on the platform between trials. The time taken by the mouse to reach the platform was recorded as its latency. The time for four trials was averaged and recorded as a result for each mouse. On day 10, the mice were subjected to a single 60-second probe trial without a platform to test memory retention. The mice started the trial from Q4, the number of annulus crossings was counted, and the swimming path was recorded and analyzed using the Ethovision XT 14 tracking software (Noldus).
Cylinder test: Each mouse was placed in a transparent acrylic cylinder (10 cm in diameter and 14 cm high). The number of wall contacts with each forelimb when rearing in at least 15 rearing cycles was computed. Animals that did not meet this criterion were excluded from this assay. The cylinder test score was determined as follows: (use of the affected forepaw (contralateral) – intact forepaw (ipsilateral)/total (contralateral + ipsilateral + both).
Rotarod test: Using an accelerating rotarod apparati (PanLab) the rotarod test was performed by placing mice on rotating drums (2.5 cm diameter) and measuring retention time on the rod. The speed of the rotarod accelerated from 4 to 40 rpm over a 5 min period. At least 20 min recovery time was allowed between trials.
Pole test: Mice were placed head-up on top of a metal pole (50 cm high and 1 cm wide) that has been wrapped in wooden wire. The base of the pole was placed in the home cage. When placed on the pole, mice orient themselves downward and descend the length of the pole back into their home cage. The time to orient downward (t-turn) and the total time to descend (t-total) were measured.
Nesting behavior: Each animal was provided with a Nestlet (5 × 5 cm2 piece of cotton; Ancare). After 24h, the next morning remaining Nestlets were scored on a scale from 1-5. (1) nesting material unmodiﬁed; (2) ﬂat nest with partially shredded nesting material; (3) shallow nest with shredded material but lacking fully formed walls; (4) nest with well-developed walls; and (5) nest in a shape of a cocoon with a partial or complete roof. Pictures were taken of all nests.
Open field test: A mouse was placed near the wall-side of a 60 × 60 cm open-field arena, and the movement of the mouse was recorded by a camera for 5 min. The recorded video file was further analyzed using EthoVision XT 14 software (Noldus). The number of entries into and the overall time spent in the center of the arena (30 × 30 cm imaginary square) were measured.
Tail suspension test: A mouse was suspended from the hook of a tail suspension test box, 60 cm above the surface of a table using adhesive tape placed 1 cm away from the tip of the tail. After 1 min acclimatization, immobility duration was recorded by a camera for 5 min. Mice were considered immobile only when they hung passively and were completely motionless.
Normal distribution was tested using the Kolmogorov-Smirnov test and variance was compared. Unless otherwise stated, statistical significance was determined using two-tailed unpaired Student’s t-tests for two population comparison followed by the Bonferroni's post hoc test for multiple comparisons. Data were analyzed using the GraphPad Prism and presented as means ± SEM. P values for each comparison were described in the legends or supplementary information section. To determine and confirm sample sizes (n), we performed a power analysis. The values for the power (1-b) and the type I error rate (a) were 0.8 and 0.05 (or 0.01), respectively. Each experiment in this study was performed blind and randomized. Mice were assigned randomly to the various experimental groups, and data were collected and processed randomly. The allocation, treatment, and handling of mice were the same across study groups. Control mice were selected from the same litter as the test group. The individuals conducting the experiments were blinded to group allocation and the allocation sequence. Exclusion criteria for mice were based on abnormal health conditions including weights below 15g at 6 weeks and noticeably reduced activity or feeding as used in previous studies [22, 23]. Statistical data and n numbers for all behavioral assays were described in the legends.