MPTP neurotoxicity is highly concordant between the sexes among BXD recombinant inbred mouse strains

Abstract

Continuing our previous work in which we showed wide-ranging strain differences in MPTP neurotoxicity in male mice among ten BXD recombinant inbred strains, we replicated our work in females from nine of the same strains. Mice received a single s.c. injection of 12.5mg/kg MPTP or saline. Forty-eight hours later the striatum was dissected for neurochemical analysis. Striatal dopamine (DA) and its metabolites, DOPAC and HVA, striatal serotonin (5-HT) and its metabolite, 5-HIAA, were analyzed using HPLC. Tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP), an astrocytic protein that increases during the astroglial response to neural injury, were measured using ELISA. There were wide genetic variations in the DA, DOPAC, HVA, TH and GFAP responses to MPTP. We also performed principal component analysis (PCA) on the difference values, saline minus MPTP, for DA, DOPAC, HVA and TH and mapped the dominant principal component to a suggestive QTL on chromosome 1 at the same location that we observed previously for males. Moreover, there were significant correlations between the sexes for the effect of MPTP on DA, HVA, and TH. Our findings suggest that the systems genetic approach as utilized here can help researchers understand the role of sex in individual differences. The same approach can pave the way to understand and pinpoint the genetic bases for individual differences in pathology attributable to toxicants. Such systems genetics approach has broad implications for elucidating gene-environment contributions to neurodegenerative diseases.

Keywords: QTL; Recombinant inbred mice; Sex differences.

Fig. 1

Left panel Effect of MPTP on DA concentration in the striatum in 9 strains of BXD recombinant inbred (RI) female mice. The mice were injected with 12.5 mg/kg MPTP (vs. saline) and tissue collected 48 h later. DA content in the striatum was determined by HPLC, normalized to tissue wet weight and expressed as mean ± s.e.m. Right panel. Effect of MPTP in BXD mice expressed as% control, for both sexes. BXD40 showed the greatest MPTP-related DA loss for both sexes. ANOVA revealed a significant main effect for strain, sex and treatment (F_8,140 = 17.21, F_1,140 = 7.77, F_1,140 = 849.86 respectively, p < 0.01 for each) and significant strain × sex, strain × treatment and strain × sex × treatment interaction (F_8,140 = 5.49, F_8,140 = 17.37 respectively, p < 0.01 for both, F_8,140 = 2.2, p < 0.05 respectively). (n = 5 per strain and treatment, b p < 0.05, showing significant differences between saline and MPTP treated groups).

Fig. 2

Left panel Effect of MPTP on striatal DOPAC in females. Mice were injected with 12.5 mg/kg MPTP (vs. saline) and tissue harvested 48 h later. DOPAC content in striatum was determined by HPLC, normalized to tissue wet weight and expressed as mean ± s.e.m. Right panel. The effect of MPTP on DOPAC expressed as % control for both sexes. BXD40 showed the greatest MPTP-related DOPAC loss for both sexes. ANOVA revealed a significant main effect of strain, sex, treatment (F_8,139 = 42.42, F_1,139 = 125.81, F_1,139 = 142.24, respectively, p < 0.01 for each) and significant strain × sex interaction (F_8,139 = 24.39, p < 0.01) and strain × treatment interaction (F_8,139 = 2.38, p < 0.05), (n = 5 per strain and treatment, b p <0.05, showing significant differences between saline and MPTP treated groups).

Fig. 3

Left panel Effect of MPTP on homovanillic acid (HVA) concentration in the striatum in females. The mice were injected with12.5 mg/kg MPTP or saline and tissue harvested 48 h later. HVA content in striatum was determined by HPLC, normalized to tissue wet weight and expressed as mean ± s.e.m. Significant differences in basal levels of HVA were observed with strain BXD32 and BXD9 having the lowest and highest HVA concentration in females respectively. Right panel. The effect of MPTP on HVA expressed as % control in both sexes. BXD 40 showed the greatest reduction in both sexes. ANOVA revealed a significant main effect for strain and treatment (F_8,140 = 11.49, F_1,140 = 111.26 respectively, p < 0.01 for each). ANOVA revealed a significant strain × treatment interaction (F_8,140 = 6.70, p < 0.01), (n = 5 per strain and treatment, b p < 0.05, showing significant differences between saline and MPTP treated groups).

Fig. 4

Left panel Effect of MPTP on DA turnover as determined by the ratio of DOPAC/DA in the striatum in female BXD mice. Mice were injected with 12.5 mg/kg MPTP (vs. saline) and tissue harvested 48 h later. Experimental and control values normalized to tissue wet weight are expressed as mean ± s.e.m. Right panel. The effect of MPTP on DOPAC/DA expressed as% control for both sexes. ANOVA revealed significant main effects for strain, sex and treatment (F_8,140 = 4.42, F_1,140 = 13.11, F_1,140 = 6.98 respectively, p <0.01 for each), and significant effect for strain × sex interaction (F_8,140 = 3.62, p < 0.01). (n = 5 per strain and treatment, b p < 0.05, showing significant differences between saline and MPTP treated groups).

Fig. 5

Left panel Effect of MPTP on DA turnover as determined by the ratio of HVA/DA in the striatum of female BXD RI mice. Female mice were injected with 12.5 mg/kg MPTP (vs. saline) and tissue harvested 48 h later. Experimental and control values normalized to tissue wet weight are expressed as mean ± s.e.m. Right panel. The effect of MPTP on HVA/DA expressed as % control in both sexes. ANOVA revealed significant main effects for strain, sex and treatment (F_8,140 = 24.48, F_1,140 = 8.53, F_1,140 = 128.89 respectively, p <0.01 for each).The sex × strain, strain × treatment, and strain × sex × treatment interactions were significant (F_8,140 = 8.99, F_8,140 = 19.97, F_8,140 = 5.33, p < 0.01 for each). The sex × treatment interaction was also significant (F_1,140 = 4.64, p < 0.05), (n = 5 per strain and treatment, b p < 0.05, showing significant differences between saline and MPTP treated groups).

Fig. 6

Left panel The effects of MPTP on tyrosine hydroxylase (TH) concentration in the striatum in females. Mice were injected with 12.5 mg/kg MPTP (vs. saline) and tissue harvested 48 h later. TH content was determined by sandwich ELISA according to the method of O’Callaghan (1991) and normalized to total protein. Data are expressed as mean ± s.e.m. Right panel. Effect of MPTP on TH expressed as % control in both sexes. ANOVA revealed significant main effect of strain and treatment (F_8,141 = 19.07, F_1,141 = 641.67, p < 0.01 for each). Sex × strain, strain × treatment and sex × treatment interactions were all significant (F_8,141 = 5.71, F_8,141 = 14.40, F_1,141 = 9.85, p < 0.01 for each). The sex × treatment × strain interaction was also significant (F_8,141 = 3.31, p < 0.05), (n = 5 per strain and treatment, a p < 0.05, showing significant differences between saline and MPTP treated groups).

Fig. 7

Left panel Effect of MPTP on the concentration of glial fibrillary acidic protein (GFAP) in the striatum in females. The mice were injected with 12.5 mg/kg MPTP (vs. saline) and tissue harvested 48 h later. GFAP content was determined by sandwich ELISA (O’Callaghan, 1991; O’Callaghan et al., 2014). GFAP concentrations were normalized to total protein and expressed as mean ± s.e.m. Right panel. Effects of MPTP on striatal GFAP expressed as % control in both sexes. ANOVA revealed significant strain, sex and strain × treatment effects (F_8,138 = 20.19, F_8,138 = 10.65, respectively, p < 0.01 for each), (n = 5 per strain and treatment, a p < 0.05, showing significant differences between saline and MPTP treated groups).