Abnormal iron metabolism in fibroblasts from a patient with the neurodegenerative disease hereditary ferritinopathy

Abstract

Background: Nucleotide duplications in exon 4 of the ferritin light polypeptide (FTL) gene cause the autosomal dominant neurodegenerative disease neuroferritinopathy or hereditary ferritinopathy (HF). Pathologic examination of patients with HF has shown abnormal ferritin and iron accumulation in neurons and glia in the central nervous system (CNS) as well as in cells of other organ systems, including skin fibroblasts. To gain some understanding on the molecular basis of HF, we characterized iron metabolism in primary cultures of human skin fibroblasts from an individual with the FTL c.497_498dupTC mutation.

Results: Compared to normal controls, HF fibroblasts showed abnormal iron metabolism consisting of increased levels of ferritin polypeptides, divalent metal transporter 1, basal iron content and reactive oxygen species, and decreased levels of transferrin receptor-1 and IRE-IRP binding activity.

Conclusions: Our data indicates that HF fibroblasts replicate the abnormal iron metabolism observed in the CNS of patients with HF. We propose that HF fibroblasts are a unique cellular model in which to study the role of abnormal iron metabolism in the pathogenesis of HF without artifacts derived from over-expression or lack of endogenous translational regulatory elements.

Figure 1

Accumulation of ferritin in cultured HF skin fibroblasts. Confocal immunofluorescence microscopy was performed on cultured confluent wild-type (non-HF) (a-c) and HF (d-f) fibroblasts using antibodies against alpha-tubulin (a, d) and mutant FTL (b, e). Nuclei were stained by DAPI (blue). A merge of the alpha-tubulin (red) and mutant FTL (green) images is shown in c and f. Mutant FTL was observed only in HF fibroblasts, both in the cytoplasm and nucleus. Scale bars: 30 μm.

Figure 2

Increased levels of ferritin polypeptides in HF skin fibroblasts. a) Representative western blot showing the expression of ferritin subunits in non-HF and HF fibroblasts. Western blot was carried out using antibodies against ferritin heavy chain (FTH), the amino terminus of ferritin light chain (FTL) that recognizes wild-type and mutant FTL subunits, and abs specific for the mutant subunit (MT-FTL). b) Densitometric analysis of ferritin subunit protein levels shows increased levels of FTH and wild-type and mutant FTL. Results are the means and SD of three independent experiments (*p < 0.05).

Figure 3

Expression of proteins of iron metabolism. Representative western blots showing the expression of: a) TfR-1, b) DMT1 (+/-) IRE, and c) FPN, in non-HF and HF fibroblasts. Results are the means and SD of three independent experiments. Densitometric analysis shows a significant decrease in TfR1 levels (d) and a significant increase in total levels of DMT1 isoforms (e) in HF compared to non-HF fibroblasts.(*p < 0.05). No significant changes in the levels of FPN (f) were observed.

Figure 4

Cellular iron levels. a) Fibroblasts were cultured under basal conditions (BC) and total iron content was analyzed by the ferrozine method. A statistically significant difference (*p < 0.05) in the levels of iron content (nmol Fe/mg protein) was observed in HF compared to non-HF fibroblasts. b) Analysis of the labile iron pool (LIP) in basal conditions and after treatment with 100 μM FAC for 72 h. The LIP was measured using the metal-sensitive fluorescence probe calcein. No statistically significant differences were observed under basal conditions. After FAC treatment, LIP levels increased significantly in both non-HF and HF fibroblasts. However, FAC-induced levels of LIP were significantly lower in HF compared to non-HF fibroblasts. *p < 0.05

Figure 5

IRE-IRP binding activity is decreased in HF skin fibroblasts. a) Cytoplasmic extracts from non-HF and HF fibroblasts were analyzed for IRE-IRP binding activity using a gel-shift assay with a ³²P-labelled RNA probe containing a ferritin IRE sequence in the absence or presence of 2% 2-mercaptoethanol (2% 2-ME) to promote maximal IRE-IRP1 binding. Fibroblasts were cultured in the presence (+) or absence (-) of 100 μM ferric ammonium citrate (FAC) for 72 h. A lower binding activity of IRP1 was observed under basal conditions (no FAC) in HF fibroblasts. After FAC treatment, HF fibroblasts showed significantly less binding activity than controls (HF: 30 ± 7%; non-HF: 62 ± 4%). *p < 0.05.

Figure 6

Oxidative stress in HF skin fibroblasts. Fibroblasts were grown in standard culture medium and then cultured for 72 h in medium containing 100 μM FAC. Cells were subsequently loaded with DHCF-DA and DCF fluorescence was determined. Significantly higher levels of ROS were observed in HF fibroblasts under basal conditions. After FAC treatment, HF fibroblasts showed significantly higher levels of ROS than wild-type fibroblasts. *p < 0.05.

Figure 7

Iron-induced ferritin aggregation and oxidative stress in HF. The mutant FTL polypeptide acts as a dominant negative mutant, leading to a failure of ferritin in its iron storage function and an increase in the levels of intracellular (ic) iron, resulting in the release of the IRP proteins from the ferritin IRE and degradation of TfR1 mRNA, generating a positive feed-back loop that leads to over-production of ferritin polypeptides. The C-termini of the mutant FTL polypeptides may extend above the spherical shell allowing them to crosslink with other ferritin molecules through iron bridging, promoting iron-mediated aggregation of ferritin. Free iron leads to the generation of ROS and oxidative stress. Ferritin aggregates and oxidative stress may lead to neurodegeneration in HF.