The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons

Abstract

Iron efflux in mammalian cells is mediated by the ferrous iron exporter ferroportin (Fpn); Fpn plasma membrane localization and function are supported by a multicopper ferroxidase and/or the soluble amyloid precursor protein (sAPP). Fpn and APP are ubiquitously expressed in all cell types in the central nervous system including neurons. In contrast, neuronal ferroxidase(s) expression has not been well characterized. Using primary cultures of hippocampal neurons, we examined the molecular mechanism of neuronal Fe efflux in detail. Developmental increases of Fpn, APP, and the ferroxidase hephaestin (Hp) were observed in hippocampal neurons. Iron efflux in these neurons depended on the level of Fpn localized at the cell surface; as noted, Fpn stability is supported by ferroxidase activity, an enzymatic activity that is required for Fe efflux. Iron accumulation increases and iron efflux decreases in Hp knockout neurons. In contrast, suppression of endogenous APP by RNAi knockdown does not affect surface Fpn stability or Fe efflux. These data support the model that the neuronal ferroxidase Hp plays a unique role in support of Fpn-mediated Fe efflux in primary hippocampal neurons. Our data also demonstrate that Hp ferroxidase activity relies on copper bioavailability, which suggests neuronal iron homeostasis will be modulated by cellular copper status.

Keywords: Amyloid precursor protein (APP); Ferroportin (Fpn); Ferroxidase; Hephaestin (Hp); Iron efflux; Primary hippocampal neurons.

Fig. 1

Ferroportin (Fpn), hephaestin (Hp), and amyloid precursor protein (APP) are expressed in rat primary hippocampal neurons. a Transcripts of Fpn and Hp. Total RNA was isolated from D11 primary hippocampal neurons, followed by one-step RT-PCR. The PCR product from the Fpn transcript was 357 bp; the Hp product was 516 bp. b Western blots of Fpn, APP, and Hp in rat hippocampal neurons cultured for 11, 15, or 20 days. Surface proteins were biotinylated and separated from the intracellular fraction. Samples loaded were total or intracellular protein (20 µg/per lane) and (per lane) the surface protein collected from 100 µg total protein sample. Arrow heads indicate the expected relative mass of each protein. Stars indicate oligomer or post-translationally modified forms. c Quantification of western blots, based on two biologic replicates. Band intensities at D15 and D20 were normalized to D11. Statistical significance was tested by one-way ANOVA with Bonferroni’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001

Fig. 2

Developmental changes of Fe efflux in rat primary hippocampal neurons. a ⁵⁹Fe efflux in primary hippocampal neurons grown for 11, 15, or 20 days. Neurons were incubated with 1 µM ⁵⁹Fe-NTA for 16 h at 37 °C. The ⁵⁹Fe-containing was removed and cells were washed with efflux medium containing 1 mM citrate. ⁵⁹Fe efflux at 37 °C was then monitored by the appearance of radionuclide in the medium as a function of time. Six aliquots were quantified at each time point; duplicate experiments were performed. b The ⁵⁹Fe-efflux rate as a function of neuronal development. The efflux data from (a) were analyzed by linear regression as represented by the connecting lines in (a). The R² values were D11, 0.9849; D15, 0.9866; and D20, 0.9691. The slopes of these fitted lines represent the velocity of ⁵⁹Fe efflux; in (b) these values are plotted as function of days in culture

Fig. 3

Endogenous APP plays a minor role in supporting surface Fpn localization in primary hippocampal neurons. a Western blot of APP in shRNA knockdown cells. Primary rat hippocampal neurons were infected by lentivirus containing scramble or APP knockdown shRNA. Lysates were isolated at 12 days after viral transduction. Band intensities were quantified as shown in (b); statistical significance was determined by unpaired t-test. **p < 0.01. c Surface Fpn, APP, and Hp abundance in APP knockdown neurons. Primary neurons were treated as in (a), followed by surface biotinylation and western blotting. sAPPα (14 nM) was added to APP knockdown cultures for 24 h. d Quantification of western blots in (c) derived from triplicate biologic samples. Statistical significance was tested by unpaired t-test. *p < 0.05; **p < 0.01; ns, no significant difference

Fig. 4

⁵⁹Fe accumulation does not change in the absence or presence of APP in primary hippocampal neurons. a ⁵⁹Fe accumulation. Primary rat hippocampal neurons treated by either scramble or APP knockdown RNAi were incubated with 1 µM ⁵⁹Fe-NTA for 16 h at 37 °C. One set of neurons with APP knockdown was loaded with ⁵⁹Fe in the presence of 14 nM human sAPPα. Cell-associated ⁵⁹Fe was quantified and normalized to the amount of total protein. Six replicates were included in each condition; experiments were repeated four times (biologic replicates). Statistical significance was tested by one-way ANOVA with Bonferroni’s multiple comparisons test. ns, no significant difference. b ⁵⁹Fe efflux in APP knockdown neurons. Neuronal cultures with either scramble or APP RNAi knockdown were loaded with 1 µM ⁵⁹Fe-NTA for 16 h at 37 °C. Extracellular ⁵⁹Fe was removed, the cultures were washed five times, and cells were then incubated in ⁵⁹Fe-free medium. The percentage of ⁵⁹Fe recovered in the medium over the initially accumulated ⁵⁹Fe was quantified as a function of incubation time. Six replicates were included in each condition; experiments were repeated four times. Statistical significance was tested by two-way ANOVA with Sidak’s multiple comparisons test. ns, no significant difference

Fig. 5

⁵⁹Fe efflux decreased in Hp knockout mouse hippocampal neurons. a Use of PCR to genotype neurons isolated from wild-type (564 bp) or HEPH knockout (363 bp) mice. b Quantitative RT-PCR for HEPH exon4 mRNA isolated from wild-type or HEPH knockout mouse neurons. Statistical significance was tested by unpaired t-test. ***p < 0.001. c Western blot of Hp in cultured hippocampal neurons isolated from wild-type or HEPH knockout mice. Arrow heads indicate Hp at the expected apparent molecular mass. d in-gel p-phenylenediamine (pPD) assay. Neuronal and Caco-2 lysates (80 μg total protein) were resolved in 4–12% native-PAGE. Human sCp (2 μg) was used as a positive control. The purple oxidized product was visible in the gel for sCp and the wild-type neuronal and Caco-2 lysates, and significantly reduced in the lysate prepared from HEPH knockout neurons. d HEPH knockout neurons accumulated more ⁵⁹Fe compared to wild-type neurons. Cells were loaded with ⁵⁹Fe-NTA (1 µM) for 17 h in the absence or presence of 0.25 μM sCp and cell-associated ⁵⁹Fe was quantified. Six replicates were used in each condition with data collected from duplicate biologic experiments. Statistical significance was tested by two-way ANOVA with Holm–Sidak’s multiple comparisons test. *p < 0.05; ****p < 0.0001; ns, no significant difference. e ⁵⁹Fe efflux in HEPH knockout neurons was reduced compared to wild-type neurons. Percent loss of total cell-associated ⁵⁹Fe was used to represent ⁵⁹Fe efflux. Statistical significance was tested by unpaired t-test. *p < 0.05. WT, wild-type mouse neurons; Hp KO, HEPH knockout mouse neurons

Fig. 6

Cu restriction decreased neuronal (ferro)oxidase activity and surface Fpn abundance. a pPD oxidase activity decreased in neuronal lysates treated with ammonium tetrathiomolybdate (TTM) and bathocuproine disulfonate (BCS). Primary hippocampal neurons were treated with 0.1% H₂O or 1 µM TTM and 200 µM BCS for 48 h, 72 h, or 90 h. Total neuronal lysates were isolated and resolved in 4–12% native-PAGE gels. b Quantification of relative band intensity in (a) based on triplicate experiments. Statistical significance was tested by unpaired t-test. *p < 0.05; **p < 0.01. c Surface Fpn, Hp, and APP abundance in primary hippocampal neurons after Cu restriction. Neurons were treated with 0.1% H₂O or 1 µM TTM/200 µM BCS for 48 h, then surface protein was biotinylated and processed for western blots analysis. d Quantification of Fpn, Hp, and APP blots, based on triplicate experiments. Statistical significance was tested by unpaired t-test. ***p < 0.001; ns, no significant difference

Fig. 7

Cu restriction affects ⁵⁹Fe accumulation and ⁵⁹Fe efflux in primary hippocampal neurons. a ⁵⁹Fe accumulation in neurons after Cu restriction. Neurons were treated with 1 µM TTM/200 µM BCS for 48 h. Control and TTM/BCS cultures were incubated with 1 µM ⁵⁹Fe-NTA for 24 h; TTM/BCS remained in the latter cultures. Cell-associated ⁵⁹Fe was quantified as described. Six replicates were used in each condition; experiments were repeated three times. Statistical significance of difference was quantified by unpaired t-test. ***p < 0.001. b ⁵⁹Fe efflux in neurons after Cu restriction. Neurons were treated with TMM/BCS and loaded with 1 µM ⁵⁹Fe-NTA as described in (a). After thorough washing, ⁵⁹Fe efflux into fresh medium was monitored over time in the absence or presence of TTM/BCS. The percentage of ⁵⁹Fe present in the medium relative to the total amount of ⁵⁹Fe in the culture represented the fractional Fe efflux. Six replicates were included in each condition; experiments were repeated twice. Statistical significance was tested by two-way ANOVA with Sidak’s multiple comparisons test. **p < 0.01; ****p < 0.0001; ns, no significant difference. c ⁵⁹Fe distribution after TTM/BCS treatment. Cell-associated ⁵⁹Fe was quantified after ⁵⁹Fe efflux in (b). Total ⁵⁹Fe was taken from the combination of cell- ⁵⁹Fe and medium-associated ⁵⁹Fe. The amount of each fraction relative to the total ⁵⁹Fe was calculated for each condition. Statistical significance was tested by two-way ANOVA with Bonferroni’s multiple comparisons test. *p < 0.05. ****p  <  0.0001. d Exogenous ferroxidase activity reduced ⁵⁹Fe retention in Cu-restricted neurons. Neurons were treated without or with TTM/BCS for 48 h and then incubated with 1 μM ⁵⁹Fe-NTA for 24 h in the absence or presence of 0.25 µM human sCp. Cell-associated ⁵⁹Fe was quantified; the ⁵⁹Fe-accumulation in the TTM/BCS-treated cultures (−/+sCp) is represented relative to the untreated control. Three replicates were included in each condition; experiments were repeated twice. Statistical significance was tested by two-way ANOVA with Bonferroni’s multiple comparisons test. *p < 0.05; **p < 0.01; ns, no significant difference