Animals
The 14-3-3γ deficient mouse was generated at Kumamoto University by random insertion of the gene trap vector pU-21 W into the exon 2 region of the Ywhag gene (Entrez Gene ID: 22628) located on the mouse chromosome 5qG2 [28]. In this study, we used the 14-3-3γ HET mice and CTL mice. The mouse card ID was 1462, and the strain name was B6;CB YwhagGt(pU-21W)266Car. The website address of the database for exchangeable gene trap clones is http://egtc.jp/action/access/clone_detail?id=21-W266. The mice were housed in polycarbonate cages under standard laboratory conditions (12-h light/dark cycle, 22 ± 2 °C, and 50 ± 5% humidity). Food and water were provided ad libitum. After performing all the behavioral tests, the mice were sacrificed using CO2 inhalation, and the brain tissue was collected. Collected samples were stored at 80 °C until analysis. All animal experiments followed the guidelines of the Institutional Animal Care and Use Committee of Sejong University and Korea University.
Genotyping
For mouse genotyping using polymerase chain reaction (PCR), the following primer sequences were used: Knockout (KO) forward primer 5′-CTCAGGTGGTCTGATGGCAG-3′ and KO reverse primer 5′-TGGGGTTCTCCACATTGAGC-3′ for Ywhag knockout mice, and wild-type (WT) forward primer 5′-TCATCAGCAGCATCGAGCAG-3′ and WT reverse primer 5′-ATGGCGTCGTCGAAGGC-3′ for CTL mice. PCR products were detected using 2% agarose gel electrophoresis in tris acetate ethylene diamine tetra acetic acid (EDTA) buffer.
Quantitative reverse transcription-polymerase chain reaction
Total RNA was extracted from the mouse brains using a Hybrid-RTM kit (Cat. No. 305-101, GeneAll, Seoul, Korea). The concentration and purity of RNA were measured at 260 nm and 280 nm, respectively, using Nanodrop 2000 spectrophotometer (RRID: SCR_018042, Thermo Scientific, Waltham, MA, USA). The complementary DNA was reverse transcribed from the RNA using the EcoDryTM premix cDNA synthesis kit (Cat. No. 639543, TAKARA, SHIGA, JAPAN). The primer sequences were as follows: Ywhag reverse transcriptase (RT) forward primer 5′-CCTACCGGGAGAAGATCGAG-3′ and Ywhag RT reverse primer 5′-TAGTTGTCCAGCAGGCTCAG-3′ for Ywhag (NM_018871) gene and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RT forward primer 5′-AATGTGTCCGTCGTGGATCT-3′ and GAPDH RT reverse primer 5′-AGACAACCTGGTCCTCAGTG-3′ for GAPDH (NM_001289726) gene. Reverse transcription-polymerase chain reaction (RT-PCR) was performed in 48-well plates using StepOne Real-Time PCR System (RRID: SCR_014281, Applied Biosystems, Waltham, MA, USA). Each sample was analyzed in triplicate. The expression levels of the target genes were normalized with GAPDH as an endogenous reference. The results were calculated using the 2−ΔΔCt method [29].
Western blotting
Brain tissues were homogenized in 4 mL RIPA buffer, 40 μL protease inhibitor cocktail, and 40 μL phosphate inhibitor cocktail and centrifuged at 4 °C at 13,000 rpm for 10 min. Protein concentrations were determined using the Bradford assay. Proteins were diluted to 3 mg/mL with 5X sample buffer and RIPA buffer and then boiled at 95 °C for 5 min. Proteins were separated using 10–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk in tris buffered saline buffer with Tween 20 for 50 min at 22 °C. They were then incubated overnight at 4 °C with anti-14-3-3γ (1:1000, Cat. No. sc-398423, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Tyrosine Hydroxylase (TH) (1:1000, Cat. No. MAB318, Millipore, Burlington, MA, USA), anti- phosphorylated TH (S31) (1:1000, Cat. No. 12041, Cell Signaling Technology, Danvers, MA, USA), anti-Leucine-rich repeat kinase 2 (LRRK2) (1:500, Cat. No. ab133474, Abcam, Cambridge, UK), anti-phosphorylated LRRK2 (S910) (1:1000, Cat. No. ab133449, Abcam, Cambridge, UK), anti-phosphorylated LRRK2 (S935) (1:1000, Cat. No. ab133450, Abcam, Cambridge, UK), anti-dopamine transporter (DAT) (1:1000, Cat. No. DAT14-A, ALPHA DIAGNOSTIC, San Antonio, TX, USA), anti-glial fibrillary acidic protein (GFAP) (1:1000, Cat. No. 12389S, Cell Signaling Technology, Danvers, MA, USA), and anti-β actin (1:1000, Cat. No. A2066, Sigma-Aldrich, St. Louis, MO, USA). After incubation with the primary antibodies, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h. Protein bands were visualized using Fusion Solo software (RRID: SCR_016305, Vilber Lourmat, Collégien, France). Protein expressions were quantified using ImageJ software (RRID: SCR_003070, National Institutes of Health, Bethesda, MD, USA) and normalized by β-actin.
Immunohistochemistry
For immunohistochemical staining, 40-week-old 14-3-3γ HET and CTL mice were used. Mouse brains were fixed with 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS, pH 7.4) via intracardiac perfusion and dehydrated by immersing in 30% sucrose dissolved in PBS. Brain slices were obtained using CM1950 cryostat (RRID; SCR_018061, Leica Biosystems, Wetzlar, Germany). The brain sections were permeabilized in PBS containing 0.5% Triton X-100 for 45 min after antigen retrieval in Tris–EDTA (10 mM Tris, 1 mM EDTA, 0.02% Tween 20; pH 9.0) solution at 80 °C for 30 min. After washing, the brain sections were blocked with PBS containing 10% normal goat serum and 5% bovine serum albumin at 22 °C for 2 h. They were incubated overnight at 4 °C with the following primary antibodies: anti-YWHAG (1:200, Cat. No. HPA026918, ATLAS antibodies, Bromma, Sweden), anti-neuronal nuclei (NeuN) (1:1000, Cat. No. ab104224, Abcam, Cambridge, UK), anti-glial fibrillary acidic protein (GFAP) (1:500, Cat. No. PA1-10004, Invitrogen, Waltham, MA, USA), anti-Dopamine transporter (DAT) (1:500, Cat. No. DAT14-A, Alpha Diagnostic, San Antonio, TX, USA), anti-Tyrosine Hydroxylase (TH) (1:500, Cat. No. MAB318, Millipore, Burlington, MA, USA), anti-phosphorylated TH (S31) (1:500, Cat. No. 13041, Cell Signaling Technology, Danvers, MA, USA). The brain sections were washed and were incubated with appropriate Alexa Fluor 488-, 594-, or 647-conjugated secondary antibodies (1:400, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at 22 °C. The washed brain sections were further incubated with 4,6-diamidino-2-phenylindole staining solution for 15 min, placed on a microscope slide, and fixed with a mounting solution (Cat. No. H-1400, Vector Laboratories, Burlingame, CA, USA). Images were obtained using Nikon Eclipse Ti2 confocal microscope (RRID: SCR_021068, Nikon, Tokyo, JAPAN). Immuno-intensity was quantified using ImageJ software (RRID: SCR_003070, National Institutes of Health, Bethesda, MD, USA).
Quantitative Dopamine measurement assay
Dopamine ELISA Kit (Cat. No. ab285238, K4219, Abcam, Cambridge, UK) was used for quantitative identification of dopamine in brain homogenate. The experimental procedure follows the protocol suggested in the kit. Briefly summarized as follows; Reconstitute the lyophilized dopamine standard with Standard/Sample dilution buffer to make serial dilutions. The proposed standard points of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0 ng/mL of dopamine solution were reacted simultaneously with the sample to draw the standard curve. Brains are extracted from mice, and the tissue is rinsed with ice-cold phosphate-buffered saline with a protease inhibitor to remove excess hemolytic blood. Homogenize the brain tissue using a glass homogenizer on ice. Centrifuge the brain homogenate at 5000 g for 5 min to recover the supernatant. Allow the reagents to be prepared immediately before use to room temperature. Wash the plates provided in the kit with 1X wash buffer. Add 50 µL of standards, samples, and controls into the appropriate wells. Immediately add 50 µL of Biotin-detection antibody working solution into each well. Cover with a plate sealer and gently pat to mix thoroughly, then incubate at 37 °C for 45 min. Discard the solution and wash it several times with 1 × wash solution. After completely removing the washing solution, add 0.1 mL of HRP-Streptavidin conjugate working solution into each well and incubate at 37 °C for 30 min. Discard the solution and wash it several times with 1X wash solution. After completely removing the wash solution, add 90 µL of TMB substrate into each well and incubate in the dark at 37 °C. After 15 min, add 50 µL of stop solution into each well. Read the results at 450 nm using a microplate reader Tecan infinite m200 pro (Tecan, Männedorf, Switzerland).
Behavioral tests
Hindlimb clasping test
The hindlimb clasping test (HCT) was performed to assess motor coordination. The mice were suspended by their tail, and the behavior of the hindlimb was recorded on video for 15 s. The clasping score of the mice was estimated by at least two persons watching the videos. The standard score was based on a previous study [30]. Briefly, score 0 was given when both hindlimbs and toes were splayed; score 1, when one hindlimb was splayed and the other was not; score 2, when both hindlimbs were partially retracted; and score 3, when both hindlimbs moved completely close to the abdomen and the toes shrank.
Balance beam test
The balance beam test (BBT) was performed to assess motor coordination. The apparatus consisted of beams and a goal box. The wide beam was 12 mm wide and 100 cm long, while the narrow beam was 6 mm wide and 100 cm long. The start and finish lines were located 10 cm from the ends of the beams, and the distance between the lines was 80 cm. A black goal box (5 cm × 4.5 cm × 5.5 cm) with fist-sized bedding and two or three chows was perceived as a safe place rather than the beams for the mice. The beams were placed 50 cm above the floor. The fabric was installed 7.5 cm above the bottom to prevent injury when the mice fell from the beams. The goal box was placed on the opposite side of the start line. The mice were expected to cross the beams from the start line to the finish line with a maximum cut-off of 60 s. Tests were conducted for three trials for each beam with minimum 3-min intervals. When the mouse did not move on, the mouse’s rump was tapped to urge them to cross. Fifteen seconds after reaching the goal box, the mice were left to return to their cage. The tests were repeated until every mouse succeeded in three trials on the wide beam, and that day was considered a test day. Several mice failed to cross on the narrow beam, and the graphs were created, excluding the failed trials. The following cases were considered a failure: falling off the beam, failing to cross in 60 s, hanging upside down when crossing a beam, crawling with the abdomen attached to the beam, and returning to the start line.
Rota-rod test
A rota-rod test (RRT) was performed to assess basal motor activity. Before the training session began, the mice were placed for 60 s on a rotating rod (4 rpm, constant speed) for adaptation and were returned to their home cage for at least 5 min. These adaptation sessions were repeated until none of the mice had fallen. After achieving adaptation, the mice were placed on the rotating rod for 300 s (4–40 rpm, gradual accelerating speed) for training. They were subjected to four trials with at least 5-min intervals and returned to their home cage between the trials. After 24 h, the tests were conducted again. The test conditions were identical to those of the training sessions. The latency to fall off the rod was recorded with a maximum cut-off of 300 s. For example, if the mouse withstood the rotating rod for 300 s, the latency to fall was recorded as 300 s. The recorded times were then averaged.
U-shaped social interaction test
The U-shaped social interaction test (USIT) was used to assess social ability. An open-field apparatus was used for the USIT with modifications. The apparatus comprised a square arena (40 cm long) with 30 cm high walls, and a small wall (20 cm × 30 cm) was erected in the middle of one high wall. Thus, this small wall created two partitioned square arenas (20 × 20 cm) and a neutral section (40 × 20 cm). Of the two partitioned arenas, one was considered an interaction zone and the other an empty zone. The two partitioned arenas contained an empty “holding cell” (10.16 cm in diameter and 13.97 cm in height) at the corner during the habituation phase. Each mouse was placed in the neutral section and allowed to explore the entire apparatus for 5 min. After the habituation session, the mouse was returned to the cage, while a partner male mouse was placed under the holding cell (on a randomly selected side), and a triangle-shaped object was placed under the other holding cell. The interaction zone was identified as the arena with the partner mouse, and the empty zone was identified as the arena with the object. During the test phase, each mouse was placed in the neutral section and allowed to explore the entire apparatus for 5 min. The ANY-maze video tracking system recorded the time spent by the test mouse in investigating the novel/stranger mouse or object. The investigation was defined as the test mouse’s nose touching the holding cell or sniffing within 1 cm of the target.
Nestlet shredding test
The nestlet shredding test (NST) was performed to investigate nesting behavior indicative of PD. The animals were given one nestlet in their home cage weekly for acclimation. For the test, the mice were isolated in a single cage with clean bedding and received one nestlet, which was weighed beforehand. The other conditions were maintained similarly to those in their home cage. The next day, the nestlets were collected and weighed to assess the shredding. The mice were tested for two trials, and the data from the last trial was used for evaluation.
Nest building test
The nest building test (NBT) was performed to investigate nesting behaviors indicative of PD. The NST was considered as the training for NBT. The mice were subjected to NBT a day after the NST. The conditions for the NBT were the same as those for the NST, except for the bedding. For the accuracy of measurement, the bedding material used in the NBT was made with smaller particles than the daily bedding to prevent nesting using bedding and to induce nesting using only nestlets. After overnight incubation, each mouse was returned to its home cage. In the test cage, the height of the nest from the bottom to the top and the NBT score were measured. A standard score indicates how well the nest was built based on a previous study [31].
Statistical analysis
GraphPad Prism 8.0.2 (GraphPad Software Inc., La Jolla, California, CA, USA) was used for statistical analysis. Levene’s test was used to determine whether the two groups had similar variances. Statistical significances were analyzed using a two-tailed unpaired student’s t-test or Welch’s t-test, followed by post hoc Tukey’s multi-comparison test. According to the tests, P-values < 0.05, < 0.01, or < 0.001 were considered statistically significant.