Sex-related differences in phenotype and nigro-striatal degeneration of c-rel-/- mouse model of Parkinson's disease

Abstract

Background: Sex is an important factor in the development and symptom expression of Parkinson's disease (PD). Risk of developing PD, motor and non-motor symptoms and response to treatment differ between men and women, with women showing lower disease incidence, later onset of motor deficits and generally milder symptoms than men. We previously reported that male mice lacking the NF-κB/c-Rel protein (c-rel^-/- mice) undergo age-related accumulation of α-synuclein, and loss of dopaminergic neurons, in the substantia nigra (SN). In addition, c-rel^-/- male mice present a progressive PD-like phenotype characterized by both motor deficits and non-motor symptoms (such as constipation, hyposmia, anxiety, depressive-like behavior and apathy). In this study, we give evidence that female mice reproduce only part of the parkinsonian pathology and do not show behavioral manifestations.

Methods: Nigro-striatal alterations as well as motor and non-motor symptoms were assessed in aged c-rel^-/- and wild-type (wt) male and female mice through histological techniques and behavioral tests.

Results: Likewise c-rel^-/- males, c-rel^-/- females displayed significant reduction of dopaminergic neurons in the SN at 18 months of age, but only minor reduction of striatal TH-positive (TH+) and DAT-positive (DAT+) dopaminergic fibers compared to wt littermates. Besides, c-rel^-/- females did not develop significant motor deficits and non-motor symptoms, as constipation, hyposmia, depressive-like and apathetic behaviors.

Conclusions: Our results show that, differently from aged males, c-rel^-/- females do not develop a parkinsonian behavior, in line with evidence from the human PD. The phenotype mice display a nigral dopaminergic neuron degeneration but conserved nigrostriatal fiber density. The degeneration and PD-like symptoms are compatible with the sex-related differences on incidence and symptoms progression observed in PD patients.

Keywords: DAT; Motor symptoms; NF-κB/c-Rel; Nigrostriatal degeneration; Non-motor symptoms; Parkinson’s disease; Sexual dimorphism.

Fig. 1

Aged c-rel^-/- male and female mice display loss of SNpc dopaminergic neurons. Representative images of TH staining in SN dopaminergic neurons of 18–19-month-old wt males (a), c-rel^-/- males (b), wt females (c), c-rel^-/- females (d). The stereology analysis data of T+ cells are presented in e. A significant drop of the number of T+ neurons was observed in both male and female c-rel^-/- mice. ****p < 0.0001, two way ANOVA followed by Sidak’s multiple comparison test (wt males: 5 mice; c-rel^-/- males: 7; wt females: 6; c-rel^-/- females: 6). Representative images of Nissl staining in SN of 18–19-month-old wt males (f), c-rel^-/- males (g), wt females (h), c-rel^-/- females (i). Data from counts of Nissl-stained neurons are presented in j. Both male and female c-rel^-/- mice showed a significant reduction in Nissl+ neurons. *p < 0.05, **p < 0.01, two way ANOVA followed by Sidak’s multiple comparison test (wt males: 4 mice; c-rel^-/- males: 3; wt females: 4; c-rel^-/- females: 4). Scale bar in a and f = 100 μm

Fig. 2

Sexually dimorphic changes in the dorsal striatum of aged c-rel^-/- male and female mice. Illustrative pictures of TH immunostaining in caudate putamen of wt males (a), c-rel^-/- males (b), wt females (c), c-rel^-/- females (d). The results from the analysis of TH+ area are shown in e. A significant decrease of TH immunoreactivity was observed in c-rel^-/- male mice when compared to wt males, but not in c-rel^-/- female mice. *p < 0.05, Kruskal-Wallis test followed by Dunn’s multiple comparison test. Representative pictures of DAT immunostaining in caudate putamen of wt males (f), c-rel^-/- males (g), wt females (h), c-rel^-/- females (i). Analysis of DAT+ area is presented in j. Wt males displayed significantly higher DAT levels than c-rel^-/- males and wt females. *p < 0.05, two way ANOVA followed by Sidak’s multiple comparison test. For both TH and DAT immunostaining the data are presented in µm² as mean ± SEM (n = 10–11 animals per group). Scale bar in a = 20 μm

Fig. 3

Motor deficits in aged c-rel^-/- male and female mice. CatWalk gait analysis was performed in 20–23-month-old wt and c-rel^-/- male and female mice (wt males: 11 mice; c-rel^-/- males: 6; wt females: 9; c-rel^-/- females: 6). c-rel^-/- male mice displayed lower run speed (a) and swing speed (b), higher stand duration (c), and lower stride length (d) than sex-matched wt mice. Conversely, c-rel^-/- females did not significantly differ from wt females in any of the considered gait parameters. *p < 0.05; ***p < 0.001; ****p < 0.0001, two-way ANOVA followed by Sidak’s multiple comparison test

Fig. 4

Intestinal dysfunctions in aged c-rel^-/- male and female mice. Stool frequency normalized to 30 g of bw (a) and stool water content percentage (b) of 18–19-month-old wt and c-rel^-/- mice, males and females, are displayed (wt males: 18 mice; c-rel^-/- males: 15 mice; wt females: 13 mice; c-rel^-/- females: 15 mice). Stool frequency and water content percentage are decreased in c-rel^-/- male mice, but not in c-rel^-/- females. Moreover, both wt and c-rel^-/- females showed lower stool frequency and stool water % than corresponding male siblings. (c) Colon shortening was investigated in wt and c-rel^-/- males and females at 18–20 months. While aged c-rel^-/- males displayed a reduction in colon length, c-rel^-/- females did not. Wt males: n = 16 mice; c-rel^-/- males: n = 10 mice; wt females: n = 9 mice; c-rel^-/- females: n = 8 mice. *p < 0.05; **p < 0.01; ****p < 0.0001, two-way ANOVA followed by Sidak’s multiple comparisons test. Results were presented as mean ± SEM

Fig. 5

Olfactory deficits in aged c-rel^-/- male and female mice. Odor detection test was conducted on 18–19-month-old wt and c-rel^-/- males and females (wt males: 13 mice; c-rel^-/- males: 13 mice; wt females: 9 mice; c-rel^-/- females: 15 mice). The percentage of time sniffing the odor at concentration 1:10⁴ is presented. Aged wt male mice were able to localise the odor, as indicated by the percentage of time sniffing the odor significantly higher than the 50% chance level (#p < 0.05, one-sample t-test). On the contrary, age-matched c-rel^-/- males could not target the fragrance (p > 0.05, one-sample t-test), and exhibited a significant odor detection impairment compared to wt males (*p < 0.05; **p < 0.01, two-way ANOVA followed by Sidak’s multiple comparison test). Both wt and c-rel^-/- females recognized the cartridge filled with the vanilla extract (# #p < 0.01 and #p < 0.05, respectively, one-sample t-test)

Fig. 6

Neuropsychiatric symptoms in aged c-rel^-/- male and female mice. Anxiety-like behavior was assessed by the OF test (a-g) in 18–19-month-old wt and c-rel^-/- mice, males and females (wt males: 15 mice; c-rel^-/- males: 15 mice; wt females: 20 mice; c-rel^-/- females: 23 mice). The total distance covered (a), the time spent, the distance covered, and the average speed scored in the center of the apparatus (b-d, respectively), the time spent, the distance covered, and the number of entries calculated in the peripheral area (e-g, respectively) are displayed. c-rel^-/- male and female mice shared a similar behavior in comparison with sex-matched controls, covering a higher total distance (a, males and females p < 0.0001), and travelling a lower distance (c, males and females p < 0.05) but at a higher speed (d, males p < 0.001, females p < 0.05) in the central area. Conversely, while c-rel^-/- males spent less time in the central area (b, p < 0.05), and spent more time, covered a higher distance and moved into the peripheral area more frequently (e: p < 0.05, f: p < 0.05, g: p < 0.001, respectively) in comparison to wt males, c-rel^-/- females did not differ significantly from wt females in these parameters (b, e-g: p > 0.05). Depressive-like behavior was measured with the FST in wt and c-rel^-/- mice aged 18–19 months, males and females (wt males: 15 mice; c-rel^-/- males: 21 mice; wt females: 10 mice; c-rel^-/- females: 11 mice). The latency to immobility (h) and the immobility time (i) are shown. A higher immobility time and a shorter latency to immobility were observed in c-rel^-/- male mice compared with sex-matched wt (h: p < 0.05, i: p < 0.01, respectively), but not in c-rel^-/- females vs. wt females (h, i: p > 0.05). Apathetic behavior was measured as latency to build the nest in the nest building test (j) in 18–19-month-old wt and c-rel^-/- mice, males and females (wt males: 9 cages; c-rel^-/- males: 9 cages; wt females: 7 cages; c-rel^-/- females: 8 cages). c-rel^-/- male mice took more time to build the nest in comparison to sex-matched wt (j: p < 0.05), while c-rel^-/- females did not (j: p > 0.05). c-rel^-/- females were faster than corresponding males in building the nest (j: p < 0.0001, respectively). *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. Two-way ANOVA followed by Sidak’s multiple comparison test in (a-d, f); Kruskal-Wallis test followed by Dunn’s multiple comparison test in (e, g-j)