The solute carriers ZIP8 and ZIP14 regulate manganese accumulation in brain microvascular endothelial cells and control brain manganese levels

Abstract

Manganese supports numerous neuronal functions but in excess is neurotoxic. Consequently, regulation of manganese flux at the blood-brain barrier (BBB) is critical to brain homeostasis. However, the molecular pathways supporting the transcellular trafficking of divalent manganese ions within the microvascular capillary endothelial cells (BMVECs) that constitute the BBB have not been examined. In this study, we have determined that ZIP8 and ZIP14 (Zrt- and Irt-like proteins 8 and 14) support Mn²⁺ uptake by BMVECs and that neither DMT1 nor an endocytosis-dependent pathway play any significant role in Mn²⁺ uptake. Specifically, siRNA-mediated knockdown of ZIP8 and ZIP14 coincided with a decrease in manganese uptake, and kinetic analyses revealed that manganese uptake depends on pH and bicarbonate and is up-regulated by lipopolysaccharide, all biochemical markers of ZIP8 or ZIP14 activity. Mn²⁺ uptake also was associated with cell-surface membrane presentation of ZIP8 and ZIP14, as indicated by membrane protein biotinylation. Importantly, surface ZIP8 and ZIP14 biotinylation and Mn²⁺-uptake experiments together revealed that these transporters support manganese uptake at both the apical, blood and basal, brain sides of BMVECs. This indicated that in the BMVECs of the BBB, these two transporters support a bidirectional Mn²⁺ flux. We conclude that BMVECs play a critical role in controlling manganese homeostasis in the brain.

Keywords: ZIP14; ZIP8; blood brain barrier; brain metabolism; brain microvascular endothelial cells; endothelial cell; manganese; metal homeostasis; trafficking; transport metal.

Figure 1.

⁵⁴Mn²⁺ accumulation by hBMVECs is biphasic and does not proceed via an endocytosis pathway. hBMVECs were incubated with 100 nm ⁵⁴Mn²⁺ at 37 °C for 10–60 min (A) and for 0–30 h (B). The initial velocity of ⁵⁴Mn²⁺ accumulation was calculated for the first hour of ⁵⁴Mn²⁺ to be 0.125 ± 0.013 pmol/mg protein/min. The velocity of ⁵⁴Mn²⁺ accumulation from 3 to 24 h was 0.0224 ± 0.001 pmol/mg/min. The data are means ± S.D., with n ≥ 4 experimental replicates/time point. hBMVECs were loaded with 100 nm ⁵⁴Mn²⁺ for 24 h and then incubated in efflux medium at 37 °C for 0–3 h (C). The velocity of ⁵⁴Mn²⁺ efflux from 0 to 3 h was 0.049 ± 0.006 pmol/mg/min. hBMVECs were pretreated with 80 μm Dynasore in DMSO (D, open squares) or DMSO alone (D, closed circles) for 30 min prior to loading for 1 or 3 h with 100 nm ⁵⁴Mn²⁺, with n = 6 experimental replicates/sample. Statistical significance was provided by an unpaired t test. ****, p < 0.0001.

Figure 2.

⁵⁴Mn²⁺ accumulation by hBMVECs is pH-, LPS-, and HCO₃⁻-dependent. hBMVECs were loaded with 100 nm ⁵⁴Mn²⁺ for 1 h in HEPES buffers (11) with pH ranging from 6.2 to 7.8 (A). Statistical significance was tested by Tukey's multiple comparison test (n = 3 experimental replicates/pH). *, significant difference compared with pH 6.2; ***, p < 0.001; ∼, significant difference compared with pH 6.8; ∼∼, p < 0.01; ∼∼∼, p < 0.001; ∧, significant difference compared with pH 7.2; ∧∧∧, p < 0.001. hBMVECs were pretreated in the absence or presence of 1 μg/ml LPS in RPMI growth medium and then loaded with 100 nm ⁵⁴Mn for 1 h (B). Statistical significance was tested by unpaired t test (6 experimental replicates/condition). ***, p < 0.001. hBMVECs were loaded with 100 nm ⁵⁴Mn for 1 h in HBSS containing HCO₃⁻ (C). Statistical significance was tested by Tukey's multiple comparison test (6 experimental replicates/condition). *, significant difference compared with 0 mm HCO₃⁻; ***, p < 0.001; ∼, significant difference compared with 1 mm HCO₃⁻; ∼∼∼, p < 0.001; , significant difference compared with 10 mm HCO₃⁻; ∧∧∧, p < 0.001.

Figure 3.

ZIP8 and ZIP14 expression in hBMVECs. Transcript abundance of each protein was quantified by qPCR (A). RNA was collected, reverse-transcribed and resulting cDNA was quantified by qPCR. The relative fold expression of ZIP8 and ZIP14 presented was calculated by the standard ΔΔC_t method. ZIP8 and ZIP14 in hBMVECs were visualized by indirect immunofluorescence (B). The cells were grown on sterile coverslips to 85% confluency, fixed, blocked, and incubated overnight with primary antibodies to ZIP8 (top left panel) or ZIP14 (bottom left panel). Coverslips were incubated in anti-rabbit Alexa Fluor 647-conjugated secondary for 1 h followed by a 10-min nuclear stain with Hoescht 33342. Coverslips were mounted using Prolong Gold mounting media and sealed. Images were acquired at 63× magnification with oil immersion on a Leica TCS SP8 confocal microscope. Negative controls (right panel) were obtained by omitting the primary antibody incubation.

Figure 4.

Cell-surface localization of ZIP14, but not ZIP8 is enhanced with Dynasore, Mn²⁺, and LPS treatment. hBMVECs were treated with 80 μm Dynasore, 1 μm Fe²⁺-citrate (1 μm FeCl₃ + 250 μm citrate + 5 mm ascorbate), or 200 nm MnCl₂ for 3.5 h prior to cell-surface biotinylation. Input, unbound (flow-through) and bound fractions were collected and processed for Western blotting. Blots of the bound fractions were probed with ZIP8 (top panel) and ZIP14 (middle panel); the input was probed for β-actin (bottom panel) (A; see Fig. S2 to see all blots). The arrowheads denote the monomeric forms of the ZIP proteins; * denotes oligomeric forms. Quantification of ZIP8 and ZIP14 surface fractionation compared with untreated cells, normalized to β-actin (B). Statistically significant difference was calculated using Tukey's multiple comparison test, with n = 3 experimental replicates/condition. *, p < 0.05; ***, p < 0.001. n = 3 experimental replicates/condition. hBMVECs were treated with 1 μg/ml LPS for 3 or 24 h prior to cell surface biotinylation (C and D). Input and bound fractions were processed for Western blotting (see Fig. S3 to see blots). Band intensities for ZIP8 (C) and ZIP14 (D) expression were normalized to β-actin. Statistically significant difference was measured by Tukey's multiple comparison test. *, statistically significantly different compared with untreated cells. *, p < 0.05, with n = 3 or more replicates/condition.

Figure 5.

siRNA knockdown of ZIP8 and ZIP14 demonstrates each contribution to hBMVEC ⁵⁴Mn²⁺ accumulation. ZIP8 and ZIP14 transcript expression is reduced in siRNA knockdown samples (A and B). RNA was collected 2 days postelectroporation and reverse-transcribed, and the resulting cDNA was quantified by qPCR. The samples were compared with siGLO controls and normalized to β-actin transcript. ^#, significantly different compared with siGLO control; ^###, p < 0.001; ***, p < 0.001. n = 9 or more /siRNA condition. ZIP8 and ZIP14 protein expression is reduced in siRNA knockdown samples (C and D). Lysates of siRNA samples were collected 2 days postelectroporation, cell surface–biotinylated, and processed for Western blotting. The samples were compared with siGLO controls and normalized to β-actin transcript levels. *, significantly different compared with siGLO control. *, p < 0.05; **, p < 0.01. n = 3–4 samples/siRNA condition. Physiologic ⁵⁴Mn²⁺ accumulation is reduced in ZIP8 and ZIP14 knockdown hBMVECs (E). hBMVECs were loaded with ⁵⁴Mn²⁺ (200 nm) for 1 h with uptake normalized for protein concentration. Statistical significance was tested by Tukey's multiple comparison test. ^#, significantly different compared with siGLO control; ^###, p < 0.001; **, p < 0.01; ***, p < 0.001. n = 6 experimental replicates/sample. ⁵⁴Mn²⁺ accumulation is somewhat reduced in ZIP8 and ZIP14 knockdown hBMVECs challenged with a hypernormal [Mn] (F). hBMVECs were loaded with ⁵⁴Mn²⁺ (1 μm) for 1 h with uptake normalized for protein concentration. Statistical significance was tested by Tukey's multiple comparison test. ^#, significantly different compared with siGLO control; ^###, p < 0.001; **, p < 0.01. n = 6 experimental replicates/sample.

Figure 6.

ZIP8 and ZIP14 support ⁵⁴Mn²⁺ accumulation at both the apical and basolateral membranes of hBMVECs. hBMVECs grown in the apical chamber of Transwell Thincerts^TM were loaded with 100 nm ⁵⁴Mn²⁺ from the apical (A) or basal chamber (B), and the mass of ⁵⁴Mn²⁺ in each chamber was quantified over time. Statistical significance of change was tested by Tukey's multiple comparison test. *, statistically significantly different from 0.5 h; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ∧, difference from 1 h; ∧, p < 0.05; ∧∧∧, p < 0.001. ^#, difference from 3 h; ^#, p < 0.05; ^##, p < 0.01; ^###, p < 0.001. ⁺, difference from 6 h; ⁺⁺⁺, p < 0.001. Based on these data the relative ⁵⁴Mn²⁺ flux trajectory was calculated (C). After 24 h, the cells were lysed, and ⁵⁴Mn²⁺ accumulation was normalized for protein concentration (D). Statistically significant difference was tested by unpaired t test. *, p < 0.05; ****, p < 0.0001. hBMVECs were electroporated with siRNAs for ZIP8 and ZIP14 and grown in the apical chamber of Transwell Thincerts^TM (E and F). The cells were loaded with 100 nm ⁵⁴Mn²⁺ for 3 h from the apical (E) or basal chamber (F). Statistically significant difference was measured using Tukey's multiple comparison test. *, statistically significantly different compared with siGLO; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ∧∧, p < 0.01. n = 4 experimental replicates/condition.

Figure 7.

ZIP8 and ZIP14 are localized to the apical and basal surfaces of hBMVECs. hBMVECs were grown in Transwell inserts to confluency and tight junction integrity (quantified by TEER measurement), and the apical or basolateral membrane was surface-biotinylated by the addition of reagent to one or the other Transwell chamber. Cultures were processed for Western blotting analysis of biotinylated protein. Input and bound fractions were normalized to β-actin. n = 4 replicates/condition. The fractional distribution of cell-surface apical and basolateral ZIP8 (A, bottom panel) and ZIP14 (B, bottom panel) was quantified as a percentage of the total of each protein found to be membrane-associated. Statistically significant difference was analyzed using unpaired t test. *, p < 0.05. Note that the actin control is the same in A and B because the same blot was used to probe for ZIP8 and ZIP14 in the input fractions to which the bound fractions were normalized.