Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons

Abstract

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell-derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co-localize, indicating these two proteins may function in Fe(3+) reduction prior to Fe(2+) permeation. Zip8, DMT1, and Steap2 co-localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin-mediated iron assimilation. In brain interstitial fluid, transferring-bound iron (TBI) and non-transferrin-bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin-(59) Fe(3+)) and NTBI, whether presented as (59) Fe(2+) -citrate or (59) Fe(3+) -citrate; reductase-independent (59) Fe(2+) uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn(2+) inhibition of Fe(2+) uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of (59) Fe from TBI relies at least in part on an endocytosis-independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons. Analysis of the expression and localization of known iron uptake transporters demonstrated that Zip8 makes a major contribution to iron accumulation in primary cultures of rat embryonic hippocampal neurons. These cells exhibit uptake pathways for ferrous and ferric iron (non-transferrin-bound iron, NTBI in figure) and for transferrin-bound iron; the ferrireductases Steap2 and SDR2 support the uptake of ferric iron substrates. Zip8 and Steap2 are strongly expressed in the plasma membrane of both soma and processes, implying a crucial role in iron accumulation from NTBI and transferrin-bound iron (TBI) by hippocampal neurons.

Keywords: NTBI; TBI; Zip8; ferrireductase; permease; primary hippocampal neurons.

Fig. 1

Expression of iron transport proteins in primary hippocampal neurons. (a) RT-PCR detects the Zip8, Zip14 and 1B-DMT1 transcripts. (b) RT-PCR detects the Steap2 and SDR2 transcripts. (c) Western blot of Zip8, Zip14 and DMT1. Arrowheads highlight species that correlate with the calculated molecular mass of rat Zip8 (NP_001011952.1, ~50 kDa) and Zip14 (NP_001100745.1, ~54 kDa). In the Zip8 immunoblots, a higher molecular mass species (100 kDa, *) was detected and likely represents the Zip8 dimer. In Zip14 immunoblots, a higher molecular mass species (150 kDa, *) was detected and may represent a glycosylated form of Zip14. In DMT1 immunoblots, several bands near the predicted molecular mass of DMT1 (~62 – 64 kDa) were observed which likely represent the different isoforms of DMT1 expressed in these cells. (d) Western blot of Steap2 and SDR2. In SDR2 immunoblots, the arrowhead indicates the band that correlate with the calculated molecular mass of rat SDR2 (XP_008773478.1, ~66 kDa); the lower band (*) may represent a degraded form of SDR2 associated with the acetone precipitation method used to prepare a concentrated protein lysate. (e) TfR expression in primary hippocampal neurons. In c-e, neuronal lysates were collected as a function of days post plating.

Fig. 2

Localization of Zip8, Zip14, Steap2 and SDR2 in primary hippocampal neurons. (a) Confocal images indicate Zip8 predominates on the cell surface, while Zip14 has an intracellular localization. (b) Confocal images of Steap2 and SDR2 indicate that Steap2 has both cell surface and intracellular localization, while SDR2 is visualized only on the cell surface. MAP2 is a neuron-specific marker. The images are representative of 5 cells examined in each of three independent experiments. Scale bar: 10 μm.

Fig. 3

Co-localization of ferrous iron transporters with TfR or Tf-594. (a) Confocal analysis of endogenous Zip8, Zip14 or DMT1 with TfR. Zip8 immunofluorescence signal has a strong overlap with TfR on the cell surface of both neuronal soma and processes. Zip14 shows intracellular co-localization with TfR primarily on the cell body. DMT1 and TfR partially co-localize with each other on both soma and processes. (b) Immunofluorescence analysis of Zip8, Zip14 or DMT1 with Alexa Fluor 594 conjugated Tf (Tf-594). Tf-594 (50 μM) was incubated with primary hippocampal neurons at 37°C for 5 min, then excess Tf-594 removed by washing. Cells were fixed and immunostained with anti-Zip8, anti-Zip14 or anti-DMT1. Zip8 staining overlaps the Tf-594 fluorescence signal associated with neuronal processes. Zip14 signal does not co-localize with Tf-594. DMT1 co-localizes with Tf-594 in the soma. The images are representative of 5 cells examined in two independent experiments. Scale bar: 10 μm.

Fig. 4

Co-localization of ferrireductase and iron transport proteins. (a) Confocal analysis of endogenous Steap2 and Zip8 indicates that Steap2 co-localizes with Zip8 on the cell surface of neuronal soma and processes. (b) Immunofluorescence analysis of Steap2 or SDR2 with Tf-594 [method as in Fig. 3(b)]. Steap2 is partially co-localized with Tf-594 at the neuronal soma and processes. SDR2 immunofluorescence partially overlapped with Tf-594 in the neuronal soma. The images are representative of 5 cells in examined in two independent experiments. Scale bar: 10 μm.

Fig. 5

⁵⁹Fe uptake from NTBI and TBI is active in primary hippocampal neurons. (a) ⁵⁹Fe²⁺ uptake is time and temperature-dependent. ⁵⁹Fe²⁺ (1 μM as the citrate complex) was incubated with neurons at 37 °C or 4 °C. (b) Fe²⁺ uptake is concentration-dependent. Initial velocity of Fe²⁺ uptake was calculated from the rate of ⁵⁹Fe²⁺ accumulation in first 10 minutes; the Michaelis-Menten equation was used to fit the data (r² = 0.9662). (c) ⁵⁹Fe³⁺ uptake is time-dependent and temperature-dependent in primary hippocampal neurons. ⁵⁹Fe³⁺-citrate (1 μM) was incubated with neurons at 37°C or 4°C. (d) Fe³⁺ uptake is concentration-dependent (method as directly above). Note that the lack of velocity data at [Fe] < K_M strongly limits the significance of this fit (r² = 0.3661). (e) Time course of ⁵⁹Fe uptake from ⁵⁹Fe-transferrin (⁵⁹Fe-Tf). ⁵⁹Fe-Tf (0.5 μM Tf, 1.0 μM ⁵⁹Fe) was incubated with neurons at 37°C or 4°C. All experiments were performed twice; in each experiment, 4 technical replicates were taken at each experimental point.

Fig. 6

⁵⁹Fe uptake from NTBI is inhibited by divalent metal ions in primary hippocampal neurons. (a) Fe²⁺ uptake is inhibited by Zn²⁺, Mn²⁺ and Cd^{2+. 59}Fe²⁺-citrate (1 μM) was added along with 50 μM Zn²⁺, Mn²⁺ or Cd²⁺. Experiments were performed twice; in each experiment, 3 replicates were observed in each condition. Statistical significance was tested by one-way ANOVA followed by Bonferroni pair-wise comparison post hoc tests. ***, p < 0.001. None of the metal ion competitors exhibited cytotoxicity at the concentrations employed (Fig. S1). (b) Kinetic analyses of Zn²⁺ inhibition of ⁵⁹Fe²⁺ uptake. ⁵⁹Fe²⁺ uptake rates were determined in the presence of different Zn²⁺ concentrations; the data are presented in reciprocal form based on fitting the Michaelis-Menten equation to each data set. Data are from one experiment with 4 technical replicates for each data point. (c) A linear regression analysis of the K_m(app) values derived from the data shown in 6(b) (r² = 0.9690). This replot provided a K_i value for Zn²⁺, 7.6 μM. (d) ⁵⁹Fe³⁺ uptake was inhibited by Zn^2+. Fe³⁺-citrate (1 μM) was incubated with neurons in the absence or presence of 40 μM Zn²⁺. Data are from two separate experiments with 4 technical replicates for each data point. Significance was tested by the unpaired t test: **, p < 0.01. (e) Primary hippocampal neurons exhibit ferrireductase activity when incubated with ferric iron substrates: TBI (Tf-Fe³⁺), or NTBI (Fe³⁺-NTA, Fe³⁺-Citrate). Data are from two separate experiments with 3 technical replicates for each data point.

Fig. 7

Zip8 knockdown results in a decrease of Fe²⁺ uptake in primary hippocampal neurons. (a) Zip8 transcript is decreased in Zip8 shRNA knockdown. (b) Zip8 expression is reduced at the protein level in neurons with Zip8 shRNA knockdown. Decreases of Zip8 expression were observed with respect to both 50 kDa (arrowhead) and 100 kDa (*) bands. (c) Fe²⁺ uptake is decreased in Zip8 knockdown neurons. ⁵⁹Fe²⁺-citrate (1 μM) was incubated with neurons at 37°C for 30 min. Data are from one experiment with 5 technical replicates for each data point. Statistical significance was tested by the unpaired t test. *, p < 0.05, ** p < 0.01.

Fig. 8

TBI uptake is not sensitive to the inhibition of endocytosis or endosomal acidification in primary hippocampal neurons. (a) Dynasore inhibits Tf-594 internalization. Neurons are pre-incubated in the absence or presence of 80 μM dynasore for 30 min at room temperature followed by incubation with 50 μM Tf-594 for 2 min. After a brief wash with Tf-free buffer, the cultures were returned to 37°C and incubated for the time period indicated. DAPI stains the nuclei. The images are representative of 5 cells in two independent experiments. Scale bar: 10 μm. (b) Dynasore does not significantly inhibit ⁵⁹Fe-Tf iron uptake. Neurons were pre-incubated in the absence or presence of 80 μM dynasore for 30 min, followed by incubation with ⁵⁹Fe-Tf (0.5 μM Tf, 1.0 μM ⁵⁹Fe) at 37°C for 1 hour. Experiments were performed twice; 4 replicates were observed in each condition. Significance was tested by the unpaired t test, n.s. (c) Neither inhibition of H⁺-ATPase (Bifilomycin A1 or Concanamycin A), nor ablation of endosomal acidification (NH₄Cl) significantly affects Fe uptake from ⁵⁹Fe-Tf. Neurons were pretreated with 500 nM Bifilomycin A1, or 50 nM Concanamycin A, or 10 mM NH₄Cl for 30 min, followed by incubation with ⁵⁹Fe-Tf (0.5 μM Tf, 1.0 μM ⁵⁹Fe) at 37°C for 1 h. Data are from two experiments with 5 technical replicates for each data point. Significance was tested by one-way ANOVA, n.s.