All animal procedures were approved by the Animal Care and Ethical Committee at Xi’an Medical University (Permit Number: 2012-8, 7 March 2012). Male C57BL/6 mice of 1 month, 3 months and 18 months were supplied by the Experimental Animal Center of Xi’an Jiao Tong University. Efforts were made to reduce the number of animals used in the study by following the 3Rs (reduction, refinement and replacement). The total number of mice used in our experiments was 180.

TAT-PEP (PirB extracellular peptide) construction, expression and purification were carried out as previously reported (Deng et al., 2016). Briefly, cDNA of extracellular domain of PirB was synthesized and cloned into expression vector pTAT-HA-6xHis. For protein expression, pTAT-PEP was transformed into BL21 (DE3). Protein production was induced with 100 mM isopropyl β-D-1-thiogalactopyranoside (TaKaRa, Tokyo, Japan), and purified by Ni-NTA-agarose chromatography (Merck, Darmstadt, Germany). The size and purity were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). As a control peptide, the scrambled PEP fusion protein named as TAT-mPEP was expressed and purified according to the same procedure used for TAT-PEP (Deng et al., 2016).

PC12 cell lines derived from rat adrenal gland pheochromocytoma were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% horse serum, 5% FBS and 1% penicyline/streptomycine at 37°C and 5% CO₂. Cells with overexpressed PirB were obtained through lentiviral transduction of mouse PirB, which was constructed by HANBIO company (Shanghai, China), and then selected with puromycin (8 μg/ml) according to our recently published procedures (Chen et al., 2015). These cells were named PC12^PirB cells.

PC12 cells and PC12^PirB cells were plated on glass coverslips, and one half of PC12^PirB cells were treated with 150 μg/L TAT-PEP. After 24 h, cells were stained with the anti-β-tubulin antibody (Catalog#: MA5-16308, 1:200, Thermo Scientific) and then the Alexa-594-labeled donkey anti-mouse IgG secondary antibody (1:800, Thermo Scientific). Cells were imaged using a fluorescence microscope (OLYMPUS IX73). The length of an axon was determined as the linear distance from the point of exit to the end of the longest branch of the neurite according to a previous report (Richardson et al., 2007). In every experimental group, 150–200 cells were analyzed.

A scheme for animal studies was shown in Figure 4A. Mice at the selected ages were randomly divided into two groups, with 12 mice in each group. One group received treatment of TAT-PEP in saline, another group received TAT-mPEP in saline as control. Treatments were carried out through intraperitoneal administration of TAT-PEP or TAT-mPEP at 8 mg/kg/injection, twice a day, for 30 or 60 days, as indicated in Figure 4A. Behavior training and evaluation were performed according to the time points indicated in Figure 4A.

Water maze tests were performed as previously described (Torres et al., 2015). The diameter and the depth of the circular pool were 90 cm and 50 cm, respectively. The inner wall was carefully cleaned to eliminate any local cues. The temperature of the room and water was kept at 23 ± 1°C, and the pool was filled with water to a depth of 40 cm and rendered opaque by the addition of milk to hide the escape platform. The plexiglas platform of 9 cm in diameter was placed in the pool 1 cm below the water surface. The recording camera was connected to a digital tracking device, and the water-maze software was used to process the tracking information. The platform position remained constant throughout the training period.

In the training period, each animal performed four trials per session, one session per day, for 4 days. The intertrial interval (ITI) was 1 min. During each session, the mouse started from a random position in the pool, and the maximum time allowed for swimming and searching was 60 s. Mice were allowed to remain on the platform for 10 s after they had found it. The subjects that failed to find the platform were gently guided to it and placed on the platform for 10 s. The average distance traveled to the platform per day was recorded. On the fifth day, probe test was performed with the platform removed from the pool. Mice were allowed to swim for 60 s. The distance traveled and the time spent in target quadrant were recorded, and the percentages were calculated from the recorded data.

On the next day after the probe test, a weight (5% body weight) was attached to the tail of each mouse for the swim-to-exhaustion test (You et al., 2015). The swimming exercise was carried out in a tank filled with water in 38 cm depth and at 34 ± 1°C. Mice were defined to be exhausted when they failed to rise to the surface of the water to breathe within 5–7 s period. Swimming time was recorded for each mouse.

Total RNA was isolated using the Trizol reagent (Invitrogen), and treated with RNase-free DNase I (Roche) to remove residual DNA. cDNA was obtained using ReverTra Ace quantitative real-time PCR (qPCR) RT Kit (TOYOBO). qPCR was carried out using ABI Stepone plus and Realtime PCR Master Mix (SYBR Green; TOYOBO). Primers for mouse PirB were: 5′-TACAAGGAAGTACCACGCCC-3′ (forward) and 5′-GGTTCAGCCTTGATGGTTGG-3′ (reverse). GAPDH was used as the endogenous control.

Proteins were prepared using RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime Biotechnology). Protein concentrations were determined using BCA protein assay (Thermo Scientific). Proteins were separated using 8% SDS-PAGE and transferred to the polyvinylidene fluoride membranes (Millipore). The membranes were blocked for 1 h, followed by incubation with the primary antibodies overnight, and then incubated with the HRP-conjugated anti-mouse antibody afterwards (1:10,000, Roche). The primary antibodies were anti-6xHis antibody (1:1000, Abcam) and anti-PirB (1:500, Thermo Scientific). GAPDH was used as loading control and determined using the anti-GAPDH antibody (1:2000, Santa Cruz). Blots were detected using BM Chemiluminescence Western Blotting kit (Roche). Densitometry quantification was analyzed using IPP6.0 software.

The cell surface expression level of PirB in transduced cells was determined using flow cytometry. Briefly, the cells were centrifuged and washed with phosphate buffered saline (PBS), followed by incubation with the primary antibody against PirB (1:200, Thermo Scientific) for 1 h on ice. After washing 3 times with PBS, the cells were treated with a 1:50 dilution of FITC-conjugated secondary antibody (1:800, Thermo Scientific). The cells were then washed 3 times with PBS and analyzed using a Accuri C6 flow cytometer (BD Biosciences), and data analysis was performed with Flowjo software.

BDNF concentration was determined using the BDNF Emax ImmunoAssay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. First, a 96-well microplate was sealed, incubated with anti-BDNF antibody (1:1000) overnight at 4°C, and washed with Tris-buffered saline (TBS). On the next day, the plate was blocked at RT for 1 h and washed. Samples and BDNF standards were added into the plate and incubated at RT for 2 h. After an extensive wash, the anti-BDNF antibody (1:500) was added to each well and the plate was incubated at RT for 2 h. After an additional wash, the HRP-conjugated anti-IgY (1:200) was added. One hour later, the plate was washed and incubated with TMB One solution for 10 min at RT. The reaction was ended by adding 1 M HCl. The absorbance was measured at 450 nm. The same procedures were used to determine the level of nerve growth factor (NGF).

The data is expressed as the mean ± SD and analyzed with SPSS 13.0 statistical software. The data in Figures 4C–E was analyzed using two-way ANOVA with Bonferroni post-test analysis, with the treatments as the between-subject factor and the trial days as the within-subject factors. The data in Figures 1B, 2C were analyzed using One-way ANOVA with Dunnett’s test. The data in Figures 2B, 4B,F,G, 5 were analyzed using Student’s t-test.

To determine the temporal and spatial expression pattern of endogenous PirB in the brain, we harvested the cerebral cortex, cerebellum and hippocampus from mice at the age of 1 month, 3 months and 18 months. The expression of PirB in different regions was determined by qPCR and western blot. Results in Figure 1 showed that the expression of PirB increased gradually with age in all compartments at both the mRNA and protein levels, suggesting that PirB is associated with aging.

We studied the impact of PirB on axon outgrowth using PirB-overexpressed PC12 cells, PC12^PirB, which were generated through lentiviral transduction. The results from the flow cytometry showed that the expression level of PirB in PC12^PirB cells was 2.4-fold of that in PC12 cells (Figures 2A,B). As shown in Figure 2C, overexpression of PirB significantly inhibits axon outgrowth. The average length of an axon in PC12^PirB cells is 7 μm, compared to 53 μm in control PC12 cells. This result is consistent with previous reports that PirB inhibits axon outgrowth (Gou et al., 2014; Liu et al., 2015).

Next, we investigated whether blocking PirB activity could reverse the axon outgrowth inhibition caused by PirB overexpression. TAT-PEP, a recombinant protein consisting of PirB extracellular motif fused with TAT, which demonstrated the ability to rescue neurite outgrowth inhibition induced by Nogo, MAG and OMgp in stroke (Deng et al., 2016), was used to treat PC12^PirB. Results in Figure 2C showed that treatment with TAT-PEP effectively prolonged the growth of axon.

In our previous work, we demonstrated that the TAT peptide mediated efficient delivery of a 40 aa peptide, NEP1-40, to the brain (Han et al., 2016). To evaluate whether the conjugation of TAT enhances brain penetration of PEP, we injected TAT-PEP intraperitoneally into mice. At 0.5 h, 2 h, 12 h, 24 h, 48 h and 72 h after injection, the hippocampus was collected and processed. The presence of TAT-PEP was detected by western blot. Results in Figure 3 showed that TAT-PEP was detectable at 0.5 h after administration. The concentration of TAT-PEP in the hippocampus peaked at 12 h. The protein sample of the first lane was TAT-PEP synthesized in vitro, and here it was used as a positive control.

Motor deficit is one of the major results of aging. As the PirB expression is associated with aging, we set to study whether suppression of PirB activity by TAT-PEP could enhance mouse motor capacity. Based on the pharmacokinetic result described in Figure 3, TAT-PEP or TAT-mPEP was administered in mice every 12 h. Treatment lasted for 30 days for 1 month, 3 months and 18 months groups. Exhaustive swimming capacities in the three groups of mice were examined (Figure 4A). Compared to TAT-mPEP treatment, the time of swimming significantly increased in both 1 month and 3 months groups mice that received the 30-day TAT-PEP treatment (Figure 4B). With the same treatment regimen, the difference in the 18 months group between mice with TAT-PEP and TAT-mPEP treatments was insignificant (data not shown). However, the difference reached significance when the 60-day treatment was carried out (Figure 4B).

The spatial learning ability and memory in mice was examined using the Morris water maze evaluation method. Figures 4C–E showed the performance of both TAT-PEP and TAT-mPEP treated animals on the spatial reference memory tests. The group of 1 month mice injected with TAT-PEP exhibited no significant difference in the acquisition of the spatial reference memory task, as revealed by distance traveled to the platform in Figure 4C. For 3 months and 18 months groups, the TAT-PEP treatment notably increased the acquisition of the spatial reference memory task (Figures 4D,E). Figures 4F,G showed that treatment with TAT-PEP significantly improved the percentage of time and distance traveled in the target quadrant for 3 months and 18 months groups. Similarly, the data for 18 months groups in the Morris water maze evaluation was presented as a 60-day treatment of TAT-PEP, because no significant difference was detected for the 30-day treatment (data not shown).

We explored the potential mechanism for TAT-PEP-mediated motor and cognitive behavior enhancement by analyzing the expression of brain-derived neurotrophic factor (BDNF) and NGF in the brain of 18 months mice. Both BDNF and NGF are known to play major roles in promoting neuronal survival, neurite growth and cognitive ability (Lu et al., 2014; Lin et al., 2015). At the end of the behavioral test, the cerebral cortex, cerebellum and hippocampus were harvested and subjected to an ELISA assay. Results in Figure 5 showed that the 60-day TAT-PEP treatment significantly enhanced the level of BDNF in all three sub-regions. By contrast, no significant difference in NGF expression was seen between mice with TAT-PEP and TAT-mPEP treatment. These results suggest that the motor and cognitive behavior enhancement effect of TAT-PEP is likely mediated by BDNF.