Mitochondrial-dependent manganese neurotoxicity in rat primary astrocyte cultures

Abstract

Chronic exposure to excessive levels of Mn results in a movement disorder termed manganism, which resembles Parkinson's disease (PD). The pathogenic mechanisms underlying this disorder are not fully understood. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity. In the present study, we investigated the effects of Mn on mitochondrial function. Primary astrocyte cultures were prepared from cerebral cortices of one-day-old Sprague-Dawley rats. We have examined the cellular toxicity of Mn and its effects on the phosphorylation of extracellular signal-regulated kinase (ERK) and activation of the precursor protein of caspase-3. The potentiometric dye, tetramethyl rhodamine ethyl ester (TMRE), was used to assess the effect of Mn on astrocytic mitochondrial inner membrane potential (DeltaPsi(m)). Our studies show that, in a concentration-dependent manner, Mn induces significant (p<0.05) activation of astrocyte caspase-3 and phosphorylated extracellular signal-regulated kinase (p-ERK). Mn treatment (1 and 6 h) also significantly (p<0.01) dissipates the DeltaPsi(m) in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. These results suggest that activations of astrocytic caspase-3 and ERK are involved in Mn-induced neurotoxicity via mitochondrial-dependent pathways.

Figures

Figure 1. Effects of Mn on LDH release from primary astrocyte cultures

Rat primary astrocyte cultures were incubated at 37°C in the absence or presence of Mn (100, 500 and 1000 μM) and LDH release to the media was quantified at the indicated times. Figures 1A and 1B depict the concentration-dependent effects of Mn exposure for 30 min (A) and 2 h (B). Figures 1C and 1D show the time course from 30 min to 24 h at Mn concentrations of 100 μM (C) and 500 μM (D). Statistical analysis was carried out by one-way ANOVA followed by Bonferroni multi-comparison tests. (* p

Figure 1. Effects of Mn on LDH release from primary astrocyte cultures

Rat primary astrocyte cultures were incubated at 37°C in the absence or presence of Mn (100, 500 and 1000 μM) and LDH release to the media was quantified at the indicated times. Figures 1A and 1B depict the concentration-dependent effects of Mn exposure for 30 min (A) and 2 h (B). Figures 1C and 1D show the time course from 30 min to 24 h at Mn concentrations of 100 μM (C) and 500 μM (D). Statistical analysis was carried out by one-way ANOVA followed by Bonferroni multi-comparison tests. (* p

Figure 2. Effects of Mn on astrocyte viability by MTT assay in primary astrocyte cultures

Astrocytes were treated with different concentrations of Mn (0, 100, 500 and 1000 μM) and cell viability by MTT assay was quantified at the indicated times. Figures show exposure to Mn for 30 min (A) or 2 h (B) in a concentration-dependent manner and Mn exposure from 30 min to 24 h at 100 (C) or 500 μM (D) in a time course. The values are mean ± S.E.M. of 4 different experiments. * p

Figure 2. Effects of Mn on astrocyte viability by MTT assay in primary astrocyte cultures

Astrocytes were treated with different concentrations of Mn (0, 100, 500 and 1000 μM) and cell viability by MTT assay was quantified at the indicated times. Figures show exposure to Mn for 30 min (A) or 2 h (B) in a concentration-dependent manner and Mn exposure from 30 min to 24 h at 100 (C) or 500 μM (D) in a time course. The values are mean ± S.E.M. of 4 different experiments. * p

Figure 3. Effect of Mn on ERK phosphorylation in cultured astrocytes as determined by immunoblotting

Values are mean ± SEM of 4–7 experiments in each group. Statistical analysis was carried out by one-way ANOVA followed by Bonferroni comparison tests; * p

Figure 3. Effect of Mn on ERK phosphorylation in cultured astrocytes as determined by immunoblotting

Values are mean ± SEM of 4–7 experiments in each group. Statistical analysis was carried out by one-way ANOVA followed by Bonferroni comparison tests; * p

Figure 4. Cleavage of caspase-3 precursor in cultured astrocytes exposed to Mn (1 hr and 24 hrs) as determined by immunoblotting

Values are mean ± SEM of 4–7 experiments in each group. Statistical analysis was carried out by one way ANOVA followed by Bonferroni multiple comparison tests. * p

Figure 4. Cleavage of caspase-3 precursor in cultured astrocytes exposed to Mn (1 hr and 24 hrs) as determined by immunoblotting

Values are mean ± SEM of 4–7 experiments in each group. Statistical analysis was carried out by one way ANOVA followed by Bonferroni multiple comparison tests. * p

Figure 5. Effect of Mn on mitochondrial inner membrane potential (ΔΨm)

Control astrocytes show a prominent fluorescence indicating polarized mitochondria; astrocytes treated with Mn (100, 500 μM and 1 mM ) for 1 hr (A) show decreased TMRE fluorescence consistent with the depolarization of the ΔΨm. The fluorescent quantification (B) was determined as described in Section 2. Values are expressed as mean ± S.E.M. of 24 random fields in each group. * p< 0.05, *** p< 0.001 versus control.