Absence of iron-responsive element-binding protein 2 causes a novel neurodegenerative syndrome

Abstract

Disruption of cellular iron homeostasis can contribute to neurodegeneration. In mammals, two iron-regulatory proteins (IRPs) shape the expression of the iron metabolism proteome. Targeted deletion of Ireb2 in a mouse model causes profoundly disordered iron metabolism, leading to functional iron deficiency, anemia, erythropoietic protoporphyria, and a neurodegenerative movement disorder. Using exome sequencing, we identified the first human with bi-allelic loss-of-function variants in the gene IREB2 leading to an absence of IRP2. This 16-year-old male had neurological and haematological features that emulate those of Ireb2 knockout mice, including neurodegeneration and a treatment-resistant choreoathetoid movement disorder. Cellular phenotyping at the RNA and protein level was performed using patient and control lymphoblastoid cell lines, and established experimental assays. Our studies revealed functional iron deficiency, altered post-transcriptional regulation of iron metabolism genes, and mitochondrial dysfunction, as observed in the mouse model. The patient's cellular abnormalities were reversed by lentiviral-mediated restoration of IRP2 expression. These results confirm that IRP2 is essential for regulation of iron metabolism in humans, and reveal a previously unrecognized subclass of neurodegenerative disease. Greater understanding of how the IRPs mediate cellular iron distribution may ultimately provide new insights into common and rare neurodegenerative processes, and could result in novel therapies.

Keywords: IREB2; chorea; iron metabolism; mitochondrial dysfunction; neurodegeneration.

Figure 1

Schematic representation of the mechanism of cellular iron sensing mediated by the IRE-IRP system. In iron-deficient cells (−Fe), iron regulatory proteins 1 and 2 (IRPs) enhance iron uptake and decrease iron sequestration by binding to iron-responsive elements (IREs) present at the 5′-UTRs of transcripts like the iron storage protein ferritin, blocking their translation, and at the 3′-UTRs of mRNAs such as transferrin receptor (TFRC), responsible for iron uptake, where binding confers protection against endonucleolytic cleavage. Conversely, under iron-replete conditions (+Fe), IREs remain unoccupied, iron uptake is reduced, and storage is promoted.

Figure 2

Selected brain MRI images and clinical photographs of an individual with bi-allelic loss-of-function variants in IREB2. Selected images from serial brain imaging of the patient (A–D). Sagittal T₁-weighted MRI (A) demonstrates prominent extra-axial CSF spaces suggestive of cerebral volume loss in the patient at age 18 months. The brainstem and posterior fossa structures are normal. Axial FLAIR (B) MRI obtained at the same time demonstrates prominent ventricles and mildly delayed myelin maturation, with moderate loss of white matter volume that appears most marked in the frontal regions. Matching subsequent T₁-weighted and FLAIR MRI at age 12 years reveals (C) mild prominence of the cerebellar interfoliate fissures and (D) progression of cerebral atrophy and white matter volume loss. Portrait and side profile photographs (E and F) taken at age 16 years 11 months demonstrate ptosis and mild non-specific facial dysmorphisms, including a short philtrum, low-set ears, and subtle midface hypoplasia. The scalp hair and body hair (not pictured) was thick, unruly, and wiry in texture, and differed from the hair of either parent (see also the fur of mice in Supplementary Video 1).

Figure 3

Complete loss of IRP2 in patient-derived lymphoblasts caused altered post-transcriptional regulation of iron metabolism genes and mitochondrial dysfunction. (A) Control (CTRL; obtained from the patient’s father, who carries one normal allele and a G357X allele) and patient (PT) lymphoblasts grown for 16 h in unsupplemented medium (lanes C), medium supplemented with 100 μM of the iron chelator deferoxamine mesilate (lanes D), with 300 μM ferric ammonium citrate (lanes F), or with 300 μM of the stable nitroxide molecule Tempol (lanes T) were analyzed by western blot or gel-shift assay. Immunoblots and gel-shift assay confirmed the absence of IRP2 protein in the patient cells and showed significant downregulation of transferrin receptor and upregulation of H-ferritin (FTH), even though IRP1 protein levels increased ∼2-fold in the patient cells. As expected, IRE-binding activities of IRP1 and IRP2 were increased in iron-depleted cells (lanes D) or in cells treated with Tempol, which shifted the equilibrium of IRP1 from the iron-sulfur cluster-containing cytosolic aconitase towards the IRE-binding apoform. Conversely, IRE-binding activities of IRP1 and IRP2 were decreased under iron-replete conditions (lanes F). (B) Immunoblots to NDUFS1 and NDUFV2 (complex I iron-sulfur subunits), SDHB (complex II iron-sulfur subunit), MTCO1 (complex IV subunit) and total OXPHOS showed decreased levels of subunits of the respiratory complexes in PT cells. Consistent with loss of constituent subunits, in-gel activity assays showed significantly decreased complexes I, II, and IV activities in the patient cells. Levels of subunits of respiratory complexes as well as activities were restored by re-expression of IRP2 in the patient cells. For quantification and statistical analysis, see Supplementary Fig. 1. (C) The altered phenotype of the patient cells was rescued by lentiviral-mediated transduction of IRP2 (PT+IRP2). TFRC protein levels were increased to levels comparable to CTRL and FTH levels were conversely effectively repressed under iron deficient conditions (lanes D). Gel shift assay confirmed restoration of IRP2 IRE-binding activity in the patient cells transduced with wild type IRP2 (PT+IRP2).

Figure 4

Biochemical characterization of IRP2-patient derived lymphoblasts. (A–E) mRNA levels of IRP2, IRP1, TFRC, H-ferritin (FTH1) and L-ferritin (FTL) in control and patient-derived lymphoblasts grown for 16 h in unsupplemented medium (lanes C), or in medium supplemented with deferoxamine mesilate (lanes D), ferric ammonium citrate (lanes F), or Tempol (lanes T). (A) IRP2 mRNA levels were significantly lower in the patient cells compared to control (CTRL versus PT, P < 0.0001). (B) IRP1 mRNA levels were significantly higher in the patient cells (CTRL versus PT, P < 0.0001). (C) TFRC mRNA levels were lower in the patient cells compared to control (CTRL C versus PT C, P = 0.0031; CTRL + D versus PT + D, P < 0.0001; CTRL + F versus PT + F, non significant; CTRL + T versus PT + T, P < 0.0001). (F) In-gel aconitase activity assay in control and patient lymphoblasts showed decreased cytosolic aconitase activity (ACO1) in the patient cells, consistent with abnormally elevated IRE-binding activity of IRP1 (Figs 3A, B and 4G) in the patient cells lacking IRP2. (G) The altered phenotype of the IRP2-patient cells was rescued by lentiviral-mediated transduction of C-terminally FLAG-tagged IRP2 (PT + IRP2-FLAG). TFRC protein levels were increased to levels comparable to CTRL and FTH and FTL levels were conversely effectively repressed under iron deficient conditions (lanes D). Gel shift assay confirmed restoration of IRP2 IRE-binding activity in the patient cells transduced with wild type recombinant IRP2-FLAG (PT + IRP2-FLAG). (H) Labile iron pool (LIP) measurements showed a significant reduction (≅2.6 fold) in the patient cells compared to control (CTRL versus PT, P < 0.0001), without significant changes in total cellular iron content, as assessed by inductively coupled plasma mass spectrometry (ICPMS). Total cellular iron content in control cells was 25.43 ± 2.56 ng of iron per 10⁹ cells. Total cellular iron content in IREB2-deficient patient cells was 28.74 ± 1.63 ng of iron per 10⁹ cells. Results in A–E and H are reported as mean ± SD. For quantification and statistical analysis of immunoblots in G, see Supplementary Fig. 2.