Expression of the plant viral protease NIa in the brain of a mouse model of Alzheimer's disease mitigates Aβ pathology and improves cognitive function

Abstract

The plant viral protease, NIa, has a strict substrate specificity for the consensus sequence of Val-Xaa-His-Gln, with a scissoring property after Gln. We recently reported that NIa efficiently cleaved the amyloid-β (Aβ) peptide, which contains the sequence Val-His-His-Gln in the vicinity of the cleavage site by α-secretase, and that the expression of NIa using a lentiviral system in the brain of AD mouse model reduced plaque deposition levels. In the present study, we investigated whether exogenous expression of NIa in the brain of AD mouse model is beneficial to the improvement of cognitive deficits. To address this question, Lenti-NIa was intracerebrally injected into the brain of Tg-APPswe/ PS1dE9 (Tg-APP/PS1) mice at 7 months of age and behavioral tests were performed 15-30 days afterwards. The results of the water maze test indicated that Tg-APP/PS1 mice which had been injected with Lenti-GFP showed an increased latency in finding the hidden-platform and markedly enhanced navigation near the maze-wall, and that such behavioral deficits were significantly reversed in Tg-APP/PS1 mice injected with Lenti-NIa. In the passive avoidance test, Tg-APP/PS1 mice exhibited a severe deficit in their contextual memory retention, which was reversed by NIa expression. In the marble burying test, Tg-APP/PS1 mice buried marbles fewer than non-transgenic mice, which was also significantly improved by NIa. After behavioral tests, it was verified that the Tg-APP/PS1 mice with Lenti-NIa injection had reduced Aβ levels and plaque deposition when compared to Tg-APP/PS1 mice. These results showed that the plant viral protease, NIa, not only reduces Aβ pathology, but also improves behavioral deficits.

Figure 1

Spatial memory deficits displayed by Tg-APP/PS1 mice were improved by NIa expression. (A) Experimental design of Lenti-NIa injection and behavioral performance. Lenti-NIa was injected in Tg-APP/PS1 mice at 7 months of age and behavioral tests were started 15 days after the injection, for the two-week period, the sequence of which was the Morris water maze test (WM), passive avoidance test (PA), and marble burying test (MB). (B) Escape latency on a hidden platform of Tg-APP/PS1 mouse control (Tg-CON), Tg-APP/PS1 mice infused with Lenti-NIa (Tg+NIa), and their non-transgenic control (WT) in the Morris water maze test. Tg-APP/PS1 mice infused with Lenti-GFP are used as Tg-APP/PS1 mouse control (Tg-CON) throughout this work. Numbers of animals used: WT, 5 males and 5 females; Tg-CON, 5 males and 4 females; Tg+NIa, 5 males and 4 females. Two-way repeated measures ANOVA and Bonferroni post-hoc test: significant difference between animal groups [F_(2,204) = 19.77, P < 0.0001], significant effect of time [F_(4,204) = 32.94, P < 0.0001] and significant animal group × time interaction [F_(8,204) = 2.301, P < 0.05]. Data are presented as the means ± SEM. ^**denotes a difference between WT and Tg-CON at P < 0.01 at the indicated time point. Separate two-way repeated measures ANOVA for the data groups between Tg-CON and Tg+NIa showed significant effects of treatment (NIa) [F_(1,128) = 10.95, P < 0.01] and time [F_(4,128) = 14.10, P < 0.0001], but no significant treatment × time interaction [F_(4,128) = 0.8769, P = 0.4798]. Data are presented as the means ± SEM. (C) The percentage of time spent in the target quadrant in the spatial probe trial test. The dashed line represents the chance performance level (25%) at each quadrant. Tg-CON; Tg-APP/PS1 mouse control. Tg+NIa; Tg-APP/PS1 mice infused with Lenti-NIa. WT; non-transgenic control. Target (T), opposite (O), and adjacent quadrants (L, R) are depicted on the right panel. Two-way ANOVA and Bonferroni post-hoc test: no difference between animal groups [F_(2,96) = 0.0000, P = 1], but significant difference between zones [F_(3,96) = 14.92, P < 0.0001] and significant animal group × zone interaction [F_(6,96) = 3.997, P < 0.01]. ^* and ^** denote differences between indicated groups in the T zone, at P < 0.05 and P < 0.01, respectively. Data are presented as the means ± SEM. (D) The percentage of time spent in the center vs. periphery zones in the hidden platform version of the Morris water maze examined on day 5 (right panel). The center and periphery zones are depicted (left panels). The periphery zone is defined as the area between wall and the circle apart by 10-cm from the wall. Representative spatial navigations on the water maze pool for 30-sec period are presented for WT, Tg-CON, and Tg+NIa mice. One-way ANOVA and Newman-Keuls post-hoc test; significant difference between animal groups in the periphery zone [F_(2,24) = 8.693, P < 0.01] and in the center zone [F_(2,24) = 8.715, P < 0.01]. ^* and ^** denote differences between indicated groups at P < 0.05 and P < 0.01, respectively. Data are presented as the means ± SEM.

Figure 2

The contextual memory deficit of Tg-APP/PS1 mice was reversed by NIa expression. The entry latency into the dark chamber for Tg-APP/PS1 mouse control (Tg-CON), Tg-APP/PS1 mice infused with Lenti-NIa (Tg+NIa), and their non-transgenic control (WT) in the passive avoidance test. The latency to enter into the dark chamber after shock (post-shock) is used as an indicator of learning and memory. Two-way repeated measures ANOVA and Bonferroni post-hoc test for data groups at post-shock days: significant difference between animal groups [F_(2,46) = 11.34, P < 0.001], time [F_(2,46) = 10.08, P < 0.001], but no animal group × time interaction [F_(4,46) = 0.3358, P = 0.8524]; ^* and ^** denote differences between WT and Tg-CON at P < 0.05 and P < 0.01, respectively; ^# and ^## denote difference between Tg-CON and Tg+NIa at P < 0.05 and P < 0.01, respectively. Data are presented as the means ± SEM. Numbers of animals used: WT, 5 males and 5 females; Tg-CON, 4 males and 4 females; Tg+NIa, 4 males and 4 females.

Figure 3

Behavioral changes in the marble burying test were restored by NIa expression. The numbers of glass marbles buried under wood chips by Tg-APP/PS1 mouse control (Tg-CON), Tg-APP/PS1 mice infused with Lenti-NIa (Tg+NIa), and their non-transgenic control (WT) for the 30-min period are presented. NIa expression significantly reverted marble burying behavior. One-way ANOVA and Newman-Keuls post-hoc test: [F_(2,24) = 13.81, P = 0.0001]. ^* and ^** denote difference at P < 0.05 and P < 0.01, respectively. Data are presented as the means ± SEM. Numbers of animals used: WT, 5 males and 5 females; Tg-CON,4 males and 4 females; Tg+NIa, 4 males and 4 females.

Figure 4

Suppression of Aβ pathology by NIa expression in the brain of Tg-APP/PS1 mice. (A) RT-PCR showing the expression of NIa transcript in the brain of Tg-APP/PS1 mice infused with Lenti-NIa (Tg+NIa).The expression of NIa was analyzed on the parietal cortex of Tg-APP/PS1 mice and their control mice (Tg-CON). GAPDH is a control. (B) Western blots showing the expression of NIa protein in the brain of Tg-APP/PS1 mice infused with Lenti-NIa (Tg+NIa). The expression of NIa (molecular weight: 28 kD) in the prefrontal cortex was examined using anti-NIa. Tg-APP/PS1 mouse control (Tg-CON). α-tubulin was used as a loading control. (C-F) The amounts of Aβ (40) (C, E) and Aβ (42) (D, F) in Tris-buffer soluble (C, D) and formic acid-extractable (E, F) fractions in the brains of Tg-APP/PS1 mouse control (Tg-CON) and Tg-APP/PS1 mice infused with Lenti-NIa (Tg+NIa). (G, H) Photomicrographs showing anti-Aβ (Bam-10)-stained superior prefrontal cortex, parietal cortex and hippocampus, and piriform cortex of Tg-APP/PS1 mouse control (Tg-CON; G) and Tg-APP/PS1 mice infused with Lenti-NIa (Tg+NIa; H). (I, J) Quantification of the levels of plaques (mm²) in the prefrontal cortex, parietal cortex, and piriform cortex of Tg-APP/PS1 mouse control (Tg-CON) and Tg-APP/PS1 mice infused with Lenti-NIa. Plaque levels presented were counted using computer-aid image analysis program (I) and the 6-grade plaque photographic reference panels (J). All data were from female mice. Scale bar: 500 µm. Data are presented as the means ± SEM. ^*denotes difference between the indicated groups at P < 0.05 (Student t-test).