Gender Differences in Neurodegeneration, Neuroinflammation and Na+-Ca2+ Exchangers in the Female A53T Transgenic Mouse Model of Parkinson's Disease

Abstract

Twelve-month-old male mice expressing the human A53T variant of α-synuclein (A53T) develop dopamine neuron degeneration, neuroinflammation, and motor deficits, along with dysfunctions of the mitochondrial Na⁺-Ca²⁺ exchanger (NCX) isoforms 1 (NCX1) and 3 (NCX3) in the nigrostriatal system. Since gender is thought to play a role in the etiology of Parkinson's disease (PD), we characterized neurochemical and behavioral alterations in 12-month-old female A53T transgenic mice. We investigated the presence of dopaminergic degeneration, astrogliosis and microgliosis using immunohistochemistry for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (IBA-1) in both the substantia nigra pars compacta (SNc) and striatum. In the same regions, we also evaluated the co-localization of NCX1 in cells positive for IBA-1 and the co-localization of NCX3 in TH-positive neurons and fibers. Furthermore, in both male and female mice, we performed motor (beam walking and pole tests) and memory [novel object recognition (NOR) and spontaneous alternation] tasks, together with tests to evaluate peripheral deficits (olfactory and stool collection tests). Female A53T transgenic mice displayed degeneration of nigral dopaminergic neurons, but neither microgliosis nor astrogliosis in the SNc and striatum. Moreover, female A53T transgenic mice displayed co-localization between NCX1 and IBA-1 positive cells in the striatum but not SNc, whereas NCX3 did not co-localize with either TH-positive terminals or neuronal bodies in the nigrostriatal system. Furthermore, female A53T transgenic mice showed increased crossing time in the beam walking test, but no impairments in the pole or memory tests, and in tests that evaluated peripheral deficits, whereas male A53T transgenic mice displayed motor, memory and peripheral deficits. Immunohistochemical and behavioral results obtained here in the female mice differ from those previously observed in males, and suggest a dissimilar influence of NCX1 and NCX3 on dopaminergic function in female and male A53T transgenic mice, strengthening the validity of these mice as a model for studying the etiological factors of PD.

Keywords: GFAP; IBA-1; NCXs; constipation; dopamine; memory; midbrain; striatum.

Figure 1

Tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra pars compacta (SNc) and striatum of female A53T transgenic and wild type (WT) mice. Representative sections and histograms of the SNc and striatum immunostained for TH of female A53T transgenic and WT mice. The histograms for the SNc show the total number of TH-positive neurons, calculated with stereological analysis, expressed as mean ± SEM. The histograms for the striatum show the density of TH-positive fibers, expressed as mean ± SEM. The number of mice per group is as follows: A53T transgenic mice n = 5–17; WT mice n = 6–16 for both the SNc and striatum. **p < 0.005 compared with WT mice. Scale bars are 500 μm for the SNc and 50 μm for the striatum.

Figure 2

Ionized calcium binding adaptor molecule-1 (IBA-1) and glial fibrillary acidic protein (GFAP) immunoreactivity in the SNc and striatum of female A53T transgenic and WT mice. Representative histograms of the SNc and striatum immunostained for IBA-1 (A,B) or GFAP (C,D) of female A53T transgenic and WT mice. The histograms show the number of IBA-1- or GFAP-positive cells, expressed as mean ± SEM. The number of mice per group is as follows: A53T transgenic mice n = 7; WT mice n = 7 for both the SNc and striatum immunostained for IBA-1; A53T transgenic mice n = 14; WT mice n = 16 for both the SNc and striatum immunostained for GFAP.

Figure 3

TH+NCX3 co-localization in the SNc and striatum of female A53T transgenic and WT mice. Representative histograms of the SNc and striatum immunostained for TH and Na⁺-Ca²⁺ exchanger 3 (NCX3) of female A53T transgenic and WT mice. The histograms show the values of Pearson’s coefficients, expressed as mean ± SEM. The number of mice per group is: A53T transgenic mice n = 7; WT mice n = 7 for both the SNc and striatum.

Figure 4

IBA-1 + NCX1 co-localization in the SNc and striatum of female A53T transgenic and WT mice. Representative sections and histograms of the SNc and striatum immunostained for IBA-1 (red) and NCX1 (green) of female A53T transgenic and WT mice. The histograms show the values of Pearson’s coefficients, expressed as mean ± SEM. Arrowheads indicate brain regions where IBA-1 and NCX1 signals co-localized (yellow). The number of mice per group is as follows: A53T transgenic mice n = 7; WT mice n = 7 for both the SNc and striatum. **p < 0.005 compared with WT mice. The scale bar is 50 μm.

Figure 5

Motor tests in female A53T transgenic and WT mice. Representative histograms for the beam walking test (A) and pole test (B) evaluated in female A53T transgenic and WT mice. The histogram in (A) shows the average time to cross the beam (s), the average number of steps to cross the beam and the average number of errors per step. The histogram in (B) shows the average time to descend the pole. Data are expressed as mean ± SEM across five trials in (A), and three trials in (B). The number of mice per group is as follows: A53T transgenic mice n = 17; WT mice n = 16 for (A), A53T transgenic mice n = 10; WT mice n = 9 for (B). *p < 0.05 compared with WT mice.

Figure 6

Memory and peripheral tasks in male and female A53T transgenic and WT mice. Representative histograms for the novel object recognition (NOR; A), spontaneous alternation behavior in a Y-maze (SAB; B), olfactory (C) and 1-h stool collection (D’,D”) tests performed in male and female A53T transgenic and WT mice. Data in (A) show the mean ± SEM of the percentage of time spent exploring the novel object. Data in (B) show the mean ± SEM of the percentage of spontaneous alternation. Data in (C) show the mean ± SEM of the time required to retrieve a pellet (sec). Data in (D’) show the mean ± SEM of the dry stool weight (mg), whereas data in (D”) show the mean ± SEM of the stool water content (mg). The number of mice per group in (A) is as follows: male A53T transgenic mice n = 5; male WT mice n = 5; female A53T transgenic mice n = 11; female WT mice n = 14. The number of mice per group in (B) is as follows: male A53T transgenic mice n = 6; male WT mice n = 7; female A53T transgenic mice n = 11; female WT mice n = 14. The number of mice per group in (C) is as follows: male A53T transgenic mice n = 8; male WT mice n = 7; female A53T transgenic mice n = 11; female WT mice n = 12. The number of mice per group in (D’,D”) is as follows: male A53T transgenic mice n = 4; male WT mice n = 7; female A53T transgenic mice n = 12; female WT mice n = 14. *p < 0.05 compared with WT mice by Newman–Keuls post hoc test.