17β-estradiol and tamoxifen protect mice from manganese-induced dopaminergic neurotoxicity

Abstract

Chronic exposure to manganese (Mn) causes neurotoxicity, referred to as manganism, with common clinical features of parkinsonism. 17β-estradiol (E2) and tamoxifen (TX), a selective estrogen receptor modulator (SERM), afford neuroprotection in several neurological disorders, including Parkinson's disease (PD). In the present study, we tested if E2 and TX attenuate Mn-induced neurotoxicity in mice, assessing motor deficit and dopaminergic neurodegeneration. We implanted E2 and TX pellets in the back of the neck of ovariectomized C57BL/6 mice two weeks prior to a single injection of Mn into the striatum. One week later, we assessed locomotor activity and molecular mechanisms by immunohistochemistry, real-time quantitative PCR, western blot and enzymatic biochemical analyses. The results showed that both E2 and TX attenuated Mn-induced motor deficits and reversed the Mn-induced loss of dopaminergic neurons in the substantia nigra. At the molecular level, E2 and TX reversed the Mn-induced decrease of (1) glutamate aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1) mRNA and protein levels; (2) transforming growth factor-α (TGF-α) and estrogen receptor-α (ER-α) protein levels; and (3) catalase (CAT) activity and glutathione (GSH) levels, and Mn-increased (1) malondialdehyde (MDA) levels and (2) the Bax/Bcl-2 ratio. These results indicate that E2 and TX afford protection against Mn-induced neurotoxicity by reversing Mn-reduced GLT1/GLAST as well as Mn-induced oxidative stress. Our findings may offer estrogenic agents as potential candidates for the development of therapeutics to treat Mn-induced neurotoxicity.

Keywords: 17β-estradiol; GLAST; GLT-1; Manganese; Tamoxifen; Tyrosine hydroxylase.

Fig. 1

Effects of E2 and TX on Mn-induced decrease in locomotor activity in mice. (A) Experimental paradigm. C57BL/6 ovariectomized female mice were implanted with E2 or TX pellets in the back of the neck of mice prior to striatal administration of Mn in the right side of mouse brain as described in the Methods section. (B) Locomotor activity was assessed in an open-field on 21 days after treatment of E2, TX, and Mn. Total distances traveled were measured in each group of mice. ^##, p<0.01; ^*, p<0.05 (two-way ANOVA followed by Tukey’s post hoc test; n = 8).

Fig. 2

Effects of E2 and TX on Mn-induced decrease in TH protein levels and dopaminergic neurons in the mouse brain. (A), (B) At the end of treatment with Mn, E2, and TX, striatal tissues were isolated from the ipsilateral side of mouse brain from each experimental group as described in the Methods section. Mouse brain tissues were analyzed for TH mRNA (A) and protein (B) levels. GAPDH and β-actin were used for loading controls of mRNA and protein, respectively. ^###, p<0.001; ^***, p<0.001 (two-way ANOVA followed by Tukey’s post hoc test; n = 3). (C) Coronal sections were used for immunohistochemistry to assess the level of TH-positive dopaminergic neurons in the substantia nigra pars compacta (white arrows), as described in the Methods section. Images are captured under 10× magnification (scale bar = 100 µm). (D) Quantification of TH-positive dopaminergic neurons are shown as a percentage of the control. The imaging data are representatives of 6 samples in each group and derived from two animals.

Fig. 3

Effects of E2 and TX on Mn-induced decrease of GLAST and GLT-1 in the mouse striatum. At the end of treatment with Mn, E2, and TX, striatal tissues were isolated from the ipsilateral side of the brain from each experimental group. Tissues were analyzed for GLAST (A and B) and GLT-1 (C and D) mRNA and protein levels as described in the Methods section. GAPDH and β-actin were used for loading controls of mRNA and protein, respectively. ^##, p<0.01; ^###, p<0.001; ^**, p<0.01; ^***, p<0.001; ^@, p<0.001 compared to control (two-way ANOVA followed by Tukey’s post hoc test; n = 3).

Fig. 4

Effects of E2 and TX on Mn-induced decrease of GLAST and GLT-1 in the cerebellar region of the mouse brain. At the end of treatment with Mn, E2, and TX, cerebellar tissues were prepared from the mouse brain as described in the Methods section. Tissues were analyzed for mRNA and protein levels of GLAST (A and B) and GLT-1 (C and D). GAPDH and β-actin were used as loading controls of mRNA and protein, respectively. ^#, p<0.05; ^###, p<0.001; ^*, p<0.05; ^**, p<0.01; ^***, p<0.001; ^@, p<0.05;, p<0.01; ^@, p<0.001 compared to control (two-way ANOVA followed by Tukey’s post hoc test; n = 3).

Fig. 5

Effects of E2 and TX on Mn-induced decrease of TGF-α and ER-α in the mouse brain. At the end of treatment with Mn, E2, and TX, brain tissues were analyzed for TGF-α (A) and ER-α (B) protein levels. β-actin was used as a loading control. ^#, p<0.05; ^**, p<0.01; ^***, p<0.001 (two-way ANOVA followed by Tukey’s post hoc test; n = 3).

Fig. 6

Effects of E2 and TX on Mn-induced oxidative stress in the mouse brain. At the end of treatment with Mn, E2, and TX, tissue samples were prepared from the mouse brain as described in the Methods section. Tissues were analyzed for CAT protein levels (A) and CAT activity (B), GSH levels (C) and MDA levels (D). ^##, p<0.01; ^###, p<0.001; ^* p<0.05; ^**, p<0.01; ^***, p<0.001 (two-way ANOVA followed by Tukey’s post hoc test; n = 3).

Fig. 7

Effects of E2 and TX on Mn-induced apoptosis in the mouse brain. At the end of treatment with Mn, E2, and TX, tissue samples were prepared from the mouse brain as described in the Methods section. Tissues were analyzed for pro-apoptotic Bax and antiapoptotic Bcl-2 protein (A), and the levels of Bax/Bcl-2 ratio (B). β-actin was used as a loading control. ^###, p<0.001; ^*, p<0.05 (two-way ANOVA followed by Tukey’s post hoc test; n = 3).

Fig. 8

Schematic diagram of the proposed mechanism of neuroprotective effects of E2/TX against Mn-induced dopaminergic neurotoxicity in the mouse brain. E2 and TX increase expression of astrocytic glutamate transporters GLT-1/GLAST, and reverse Mn-reduced GLT-1/GLAST. E2/TX also attenuate Mn-induced oxidative stress by reversing Mn-reduced CAT protein levels, CAT activity and GSH levels, as well as reversing Mn-induced lipid peroxidation. These events may lead to impairment of GLT-1/GLAST as well as apoptosis, which might be directly associated with dopaminergic cell death and impairment of motor function. ↓↑ Mn effects, ↓⊥ E2/TX effects.