Altered manganese homeostasis and manganese toxicity in a Huntington's disease striatal cell model are not explained by defects in the iron transport system

Abstract

Expansion of a polyglutamine tract in Huntingtin (Htt) leads to the degeneration of medium spiny neurons in Huntington's disease (HD). Furthermore, the HTT gene has been functionally linked to iron (Fe) metabolism, and HD patients show alterations in brain and peripheral Fe homeostasis. Recently, we discovered that expression of mutant HTT is associated with impaired manganese (Mn) uptake following overexposure in a striatal neuronal cell line and mouse model of HD. Here we test the hypothesis that the transferrin receptor (TfR)-mediated Fe uptake pathway is responsible for the HD-associated defects in Mn uptake. Western blot analysis showed that TfR levels are reduced in the mutant STHdh(Q111/Q111) striatal cell line, whereas levels of the Fe and Mn transporter, divalent metal transporter 1 (DMT1), are unchanged. To stress the Fe transport system, we exposed mutant and wild-type cells to elevated Fe(III), which revealed a subtle impairment in net Fe uptake only at the highest Fe exposures. In contrast, the HD mutant line exhibited substantial deficits in net Mn uptake, even under basal conditions. Finally, to functionally evaluate a role for Fe transporters in the Mn uptake deficit, we examined Mn toxicity in the presence of saturating Fe(III) levels. Although Fe(III) exposure decreased Mn neurotoxicity, it did so equally for wild-type and mutant cells. Therefore, although Fe transporters contribute to Mn uptake and toxicity in the striatal cell lines, functional alterations in this pathway are insufficient to explain the strong Mn resistance phenotype of this HD cell model.

FIG. 1.

Decrease levels of total TfR in HD striatal cells. Lysates harvested from STHdh^Q7/Q7 (dark gray bars) or STHdh^Q111/Q111 (light gray bars) cell lines after 3 h of the indicated level of Mn(II) chloride exposure and vehicle control were analyzed by Western blot for TfR and actin protein levels. Quantification of TfR protein expression in striatal cell lines, bar graph plotted as mean levels (±SEM) normalized to actin as a percent of the untreated wild-type control (n = 4 independent samples). Table shows statistical analysis of a two-factor univariate ANOVA for relative TfR protein levels, a significant effect of genotype is designated by shaded numbering.

FIG. 2.

Diminished response of Fe pathway to Mn in HD model. Lysates harvested from STHdh^Q7/Q7 (dark gray bars) or STHdh^Q111/Q111 (light gray bars) cell lines after 30 h of Mn(II) chloride exposure versus vehicle control were analyzed by Western blot for TfR and actin. Representative blots showing three samples of each genotype-exposure group are shown, Mn(II) exposure as indicated. Quantification of TfR protein expression in striatal cell lines, bar graph plotted as mean levels (±SEM) normalized to actin as a percent of the untreated wild-type control (n = 7). Table shows statistical analysis of a two-factor univariate ANOVA for relative TfR protein levels, significant effects of genotype and Mn(II) exposure are designated by shaded numbering. “*” Indicates significant differences between the four genotype-treatment groups (p < 0.05, post hoc t-test).

FIG. 3.

DMT1 levels unchanged in HD striatal cells. Lysates harvested from STHdh^Q7/Q7 (dark gray bars) or STHdh^Q111/Q111 (light gray bars) cell lines after 30 h of Mn(II) chloride exposure versus vehicle control were analyzed by Western blot for TfR and actin. Representative blot showing one set of samples analyzed, Mn(II) exposure as indicated. Quantification of TfR protein expression in striatal cell lines, bar graph plotted as mean levels (±SEM) normalized to actin as a percent of the untreated wild-type control (n = 3 independent samples). Table shows statistical analysis of a two-factor univariate ANOVA for relative DMT1 protein levels, no significant effects were observed, though genotype exhibited a trend toward increased DMT1 levels in mutant cells (p = 0.131).

FIG. 4.

Net Fe uptake is reduced in the HD striatal cells. (A) Measurement of total intracellular Fe levels in STHdh^Q7/Q7 or STHdh^Q111/Q111 striatal cell lines (as indicated) after application of the indicated concentrations of Fe(II) chloride for 30 h (n = 3 to 5). Mean values (±SEM) are plotted on a linear scale. (B) Comparisons of net Fe uptake versus net Mn uptake above basal levels (vehicle control) in STHdh^Q7/Q7 (blue) versus STHdh^Q111/Q111 (red) cells following a 30-h exposure to these metals. Calculations are derived from Fe data in (Fig. 4A) and previously published Mn data (Williams et al., 2010). Data expressed as the absolute difference (±SEM) between metal-exposed and vehicle-exposed cells (n = 3–5), plotted on log₁₀ scale. “*” Indicates a significant difference in net metal uptake (p < 0.05, using 95% confidence intervals) between wild-type and mutant cells.

FIG. 5.

Basal Mn deficiency in HD striatal cells. Measurement of total intracellular Fe and Mn by GFAAS of cells collected from two 10-cm² plates per sample of STHdh^Q7/Q7 or STHdh^Q111/Q111 cells (as indicated). Cells grown under standard culture conditions without supplemented Mn(II) or Fe(III). The mean total metal levels (±SEM) are plotted as indicated. “*” Indicates a significant difference in measured metal levels (p < 0.05, t-test) between wild-type and mutant cells.

FIG. 6.

Saturating levels of Fe have no significant effect on the HTT-Mn interaction. Wild-type STHdh^Q7/Q7 and mutant STHdh^Q111/Q111 cells (as indicated) were exposed for 30 h to Mn(II) chloride in the presence (dashed line) or absence (solid line) of 600μM Fe(III) chloride. (A) Cell survival by MTT assay was determined (n = 3 independent experiments, 4 wells per experiment) and graphed as mean survival (±SEM) normalized by the vehicle control (no Mn or Fe) for each genotype. (B) Table summarizes statistical analysis of three-way repeated measures ANOVA (genotype, Mn(II) exposure, Fe(III) exposure, and all two-way and three-way interaction terms); significant terms (p < 0.05) are indicated by shaded numbering. (C) Cell survival data for 100μM Mn(II) chloride exposure only, mean survival (±SEM) normalized by genotype and Fe(III) exposure to show the relative influence of Fe on Mn toxicity between wild-type and mutant cells (as indicated). Mutant cells show a significant increase in survival following Mn exposure both with and without Fe exposure. Note that the magnitude of the difference between genotypes is similar, irrespective of whether there was Fe present. “*” Indicates a significant difference in survival (p < 0.05, t-test) between wild-type and mutant cells.