Age-Dependent Neuropsychiatric Symptoms in the NF-κB/c-Rel Knockout Mouse Model of Parkinson's Disease

Abstract

Non-motor symptoms are frequently observed in Parkinson's disease (PD) and precede the onset of motor deficits by years. Among them, neuropsychiatric symptoms, including anxiety, depression, and apathy, are increasingly considered as a major challenge for patients with PD and their caregivers. We recently reported that mice lacking the nuclear factor-κB (NF-κB)/c-Rel protein (c-rel^-/- mice) develop an age-dependent PD-like pathology and phenotype characterized by the onset of non-motor symptoms, including constipation and hyposmia, starting at 2 months of age, and motor deficits at 18 months. To assess whether c-rel^-/- mice also suffer from neuropsychiatric symptoms, in this study we tested different cohorts of wild-type (wt) and c-rel^-/- mice at 3, 6, 12, and 18-20 months with different behavioral tests. Mice lacking c-Rel displayed anxiety and depressive-like behavior starting in the premotor phase at 12 months, as indicated by the analysis with the open field (OF) test and the forced swim test with water wheel (FST), respectively. A deficit in the goal-oriented nesting building test was detected at 18-20 months, suggesting apathetic behavior. Taken together, these results indicate that c-rel^-/- mice recapitulate the onset and the progression of PD-related neuropsychiatric symptoms. Therefore, this animal model may represent a valuable tool to study the prodromal stage of PD and for testing new therapeutic strategies to alleviate neuropsychiatric symptoms.

Keywords: NF-κB/c-Rel; Parkinson’s disease; anxiety; apathy; depression; mouse model; non-motor symptoms.

FIGURE 1

Different cohorts of wild-type (wt) and c-Rel protein (c-rel^–/–) mice were tested for anxiety-like behavior in the open field (OF) at 3, 6, 12, and 18–20 months of age. (A) Representative track plots of 18–20-month-old wt and c-rel^–/– mice at the end of a 5-min OF test. The distance travelled by the mice is represented by the purple lines. The total distance covered (B), the time spent, the distance covered, and the average speed calculated in the center of the apparatus (C–E, respectively), the time spent, the distance covered, and the number of entries registered in the peripheral area (F–H, respectively) are shown. No significant differences were found between wt and c-rel^–/– mice at 3 and 6 months in any of the described parameters. At 12 months, c-rel^–/– mice covered a higher total distance (B, p < 0.05), spent less time and walked a lower distance but at a higher speed in the central area (C–E, p < 0.05), and spent more time and entered more often in the peripheral area (F, p < 0.05 and H, p < 0.01, respectively). At 18–20 months, the differences between wt and c-rel^–/– mice became significant for the distance covered in the peripheral area (G, p < 0.05), remained significant for the time spent and distance covered in the central area, and the time spent in the peripheral area (C,D,F, p < 0.05), and further increased for the total distance covered (B, p < 0.001), the average speed in the central area (E, p < 0.01), and the number of entries in the peripheral area (H, p < 0.001). Moreover, the total distance traveled by wt mice, but not c-rel^–/– mice, decreased with aging (B: p < 0.05, wt 3 months vs. wt 18–10 months). Similarly, the number of entries in the peripheral area decreased with aging only in wt groups (H: p < 0.0001, wt 3 months vs. wt 12 months; p < 0.001, wt 3 months vs. wt 18–20 months). Finally, we observed an age-dependent decline in the time spent and the distance covered in the peripheral area for both wt and c-rel^–/– mice (F,G: p < 0.0001, wt 3 months vs. wt 12 months; p < 0.001, wt 3 months vs. wt 18–20 months; p < 0.001, c-rel^–/– 3 months vs. c-rel^–/– 12 months; p < 0.05, c-rel^–/– 3 months vs. c-rel^–/– 18–20 months). 3-month-old wt: 12 mice; 3-month-old c-rel^–/–: 16 mice; 6-month-old wt: 13 mice; 6-month-old c-rel^–/–: 13 mice; 12-month-old wt: 13 mice; 12-month-old c-rel^–/–: 15 mice; 18–20-month-old wt: 14 mice; and 18–20-month-old c-rel^–/–: 16 mice. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. Two-way ANOVA followed by Sidak’s multiple comparison test in (B–D,H); the Kruskal–Wallis test followed by Dunn’s multiple comparison test in (E–G). Data are expressed as mean ± SEM in (B–D,H) or as median ± interquartile range in (E–G).

FIGURE 2

Different cohorts of wt and c-rel^–/– mice were tested for depression-like behavior with the FST at 3, 6, 12, and 18–20 months of age. The immobility time and latency to immobility are shown (A,B, respectively). A higher, age-dependent, immobility time was observed in 12-month-old (A: p < 0.05, wt 12 months vs. c-rel^–/– 12 months) and 18–20-month-old c-rel^–/– mice (A: p < 0.01, wt 18–20 months vs. c-rel^–/– 18–20 months; p < 0.0001, c-rel^–/– 3 months vs. c-rel^–/– 18–20 months; p < 0.001, c-rel^–/– 6 months vs. c-rel^–/– 18–20 months). Moreover, 18–20-month-old c-rel^–/– mice displayed a shorter, age-dependent, latency to immobility compared with age-matched wt mice (B, p < 0.01, wt 18–20 months vs. c-rel^–/– 18–20 months; p < 0.01, c-rel^–/– 3 months vs. c-rel^–/– 18–20 months). 3-month-old wt: 13 mice; 3-month-old c-rel^–/–: 16 mice; 6-month-old wt: 19 mice; 6-month-old c-rel^–/–: 20 mice; 12-month-old wt: 14 mice; 12-month-old c-rel^–/–: 14 mice; 18–20-month-old wt: 15 mice; and 18–20-month-old c-rel^–/–: 21 mice. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The Kruskal–Wallis test followed by Dunn’s multiple comparison test. Data are expressed as median ± interquartile range.

FIGURE 3

Different cohorts of wt and c-rel^–/– mice were tested for apathetic behavior with the nest building test at 3, 6, 12, and 18–20 months of age. Cages containing 2–4 mice per cage were used to evaluate the latency to build the nest. A small cellulose bag containing corncobs (Mucedola) was placed in the cage, and every 30 min, the nest building was monitored. (A) Representative images of 18–20-month-old wt and c-rel^–/– mice nest building at time 0 and after 3 h following the introduction of bags containing corncobs. (B) The latency to build the nest is shown. c-rel^–/– mice took a longer time to prepare the nest at 18–20 months of age (p < 0.05). Moreover, the nest building latency of 18–20-month-old c-rel^–/– mice was significantly higher than the values scored for c-rel^–/– mice at younger ages (p < 0.0001, c-rel^–/– 3 months vs. c-rel^–/– 18–20 months; p < 0.001, c-rel^–/– 6 months vs. c-rel^–/– 18–20 months; p < 0.05 c-rel^–/– 12 months vs. c-rel^–/– 18–20 months). 3-month-old wt: 6 cages; 3-month-old c-rel^–/–: 6 cages; 6-month-old wt: 5 cages; 6-month-old c-rel^–/–: 4 cages; 12-month-old wt: 5 cages; 12-month-old c-rel^–/–: 5 cages; 18–20-month-old wt: 7 cages; and 18–20-month-old c-rel^–/–: 10 cages; *p < 0.05; ***p < 0.001; ****p < 0.0001. The Kruskal–Wallis test followed by Dunn’s multiple comparison test. Data are expressed as median ± interquartile range.

FIGURE 4

Two different cohorts of wt and c-rel^–/– male mice were tested for working and spatial short-term memory with Y-maze and novel object recognition (NOR) tests at 18–20 months of age. (A) In the Y-maze, spontaneous alternation behavior (SAB) percentage did not vary between wt and c-rel^–/– male mice. wt: 18 mice; c-rel^–/–: 18 mice; p > 0.05, t-test. (B) On trial 2 of the NOR test, recognition index (RI) did not change between the groups. wt: 8 mice; c-rel^–/–: 10 mice; p > 0.05, t-test. Data are expressed as mean ± SEM.