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.

Fluorescence resonance energy transfer links membrane ferroportin, hephaestin but not ferroportin, amyloid precursor protein complex with iron efflux

“…Our conclusion that sAPP and not APP functions in iron trafficking contrasts with one report suggesting that cell APP (the unprocessed, native protein) stimulated Fpn-dependent iron efflux in hippocampal neurons (24), a result, however, that recently has been challenged (19). On the other hand, the results reported here in HEK293T cells are similar to those reported in brain microvascular endothelial cells (6,25) and hippocampal neurons (19), i.e. stimulation of iron efflux upon addition of sAPP or the ferroportin targeting peptide from its E2 domain.…”

“…This result suggests that cellular, full-length APP likely plays a minor if any role in the localization or activity of Fpn in iron efflux at the plasma membrane. A similar finding was described in primary hippocampal neurons in which APP was knockeddown by an iRNA approach (19). Note that the C-terminal, cytoplasmic domains to which the fluors were appended in Fpn, Heph, and the APP proteins were of comparable length (35-45 residues); thus, the lack of FRET between the latter species and Fpn-CFP cannot readily be ascribed to a difference in linkerlength between Heph and the APP proteins.…”

“…Fpn membrane occupancy has been linked to the iron and copper status of the cell as well. Thus, in cells made copperlimited by treatment with Cu(I) chelating agents like bathocuproine disulfonate (BCS) or tetrathiomolybdate membrane Fpn declines as does iron efflux as a result of the decrease in copperreplete, active Heph (6,19,20). A similar Fpn down-regulation has been reported in cells from Cp knockout mice (21).…”

Marked Age-Related Changes in Brain Iron Homeostasis in Amyloid Protein Precursor Knockout Mice

“…Iron accumulates in several brain regions with normal aging [16,19,30], and this is exaggerated in particular regions in several neurodegenerative diseases including AD, where it may contribute to oxidative burden and possibly even pathogenesis [31][32][33][34]. A recent in vitro study implicated a major role for hephaestin rather than APP in regulating ferroportinmediated iron efflux [35]. In our current study, we provide evidence that APP plays a major role in maintaining normal iron levels in the brain and liver during aging and that its deletion is sufficient to induce iron increase in the brain and liver in vivo.…”

Brain iron transport

“…Transcriptional changes in Fpn1 mRNA were found to be mediated by hepcidin in differentiated neuronal‐like PC12 cells subjected to iron challenge (Helgudottir et al, ), and iron export through Fpn1 is modulated by the iron chaperone poly(rC)‐binding protein 2 (PCBP2) (Yanatori et al, ). CP, a critical ferroxidase, has been found in neurons (Loeffler et al, ; Loeffler, Sima & LeWitt, ) and astrocytes (Klomp et al, ; Klomp & Gitlin, ; Loeffler et al, ; Loeffler, Sima, & Lewitt, ; Jeong & David, , ), while another ferroxidase Heph is expressed in neurons (Hudson et al, ; Ji et al, ), oligodendrocytes (Schulz et al, ; Zarruk et al, ) and microglia (Zarruk et al, ). These findings suggest that iron efflux from the neuron is mediated by the Fpn1/CP and/or Fpn1/Heph pathways, from astrocytes by Fpn1/CP, and from oligodendrocytes and microglia by the Fpn1/Heph route.…”