Iron misregulation and neurodegenerative disease in mouse models that lack iron regulatory proteins

Abstract

Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are two cytosolic proteins that maintain cellular iron homeostasis by binding to RNA stem loops known as iron responsive elements (IREs) that are found in the untranslated regions of target mRNAs that encode proteins involved in iron metabolism. IRPs modify the expression of iron metabolism genes, and global and tissue-specific knockout mice have been made to evaluate the physiological significance of these iron regulatory proteins (Irps). Here, we will discuss the results of the studies that have been performed with mice engineered to lack the expression of one or both Irps and made in different strains using different methodologies. Both Irp1 and Irp2 knockout mice are viable, but the double knockout (Irp1(-/-)Irp2(-/-)) mice die before birth, indicating that these Irps play a crucial role in maintaining iron homeostasis. Irp1(-/-) mice develop polycythemia and pulmonary hypertension, and when these mice are challenged with a low iron diet, they die early of abdominal hemorrhages, suggesting that Irp1 plays an essential role in erythropoiesis and in the pulmonary and cardiovascular systems. Irp2(-/-) mice develop microcytic anemia, erythropoietic protoporphyria and a progressive neurological disorder, indicating that Irp2 has important functions in the nervous system and erythropoietic homeostasis. Several excellent review articles have recently been published on Irp knockout mice that mainly focus on Irp1(-/-) mice (referenced in the introduction). In this review, we will briefly describe the phenotypes and physiological implications of Irp1(-/-) mice and discuss the phenotypes observed for Irp2(-/-) mice in detail with a particular emphasis on the neurological problems of these mice.

Keywords: Amino cupric silver stain; Anemia; Axonal degeneration; Erythropoietic protoporphyria; Iron; Iron regulatory protein; Motor neuron; Neurodegeneration; Polycythemia; Pulmonary hypertension.

Fig. 1

Iron uptake and systemic distribution. Dietary iron, predominantly in the form of Fe³⁺ is absorbed in the enterocyte through a concerted action between DcytB, which reduces Fe³⁺ to Fe²⁺, and DMT1, which imports this Fe²⁺ into the enterocyte. After internalization, Fe²⁺ passes through a poorly characterized labile iron pool, from which it is stored in ferritin or exported across the basolateral membrane by FPN1. Exported Fe²⁺ is then oxidized by the membrane-bound ferroxidase, hephaestin, to Fe³⁺ which binds to Tf, and the diferric transferrin complex then circulates through the plasma. This holo-Tf binds to TfR1 on the plasma membrane of most cells, and the resulting complex is endocytosed. Fe³⁺ is released from the complex in the acidic environment of endosome, and is reduced by Steap3 to Fe²⁺ which is transported by DMT1 into cytosol. Fe²⁺ is utilized by several cellular organelles, or stored in ferritin, and unused Fe²⁺ is exported out by FPN1. This Fe²⁺ is then oxidized by hephaestin or ceruloplasmin to Fe³⁺ which then binds to Tf and is recycled back to plasma.

Fig. 2

Increased iron staining in the cerebellums of Irp2 deficient mice. Perls’DAB stain of brain sections of WT (Irp1^+/+Irp2^+/+), Irp1^+/+Irp2^−/−, Irp1^+/−Irp2^−/−, Irp1^−/−Irp2^+/+, Irp1^−/− Irp2^+/− mice showed iron accumulation in different parts of Irp1^+/+Irp2^−/− and Irp1^+/−Irp2^−/− mouse brains. One-year old mice were perfused with 4% paraformaldehyde/PBS. Gelatin-embedded brains were sectioned, and sequential sections were stained with DAB as described (LaVaute et al., 2001; Smith et al., 2004). Abbreviations: CDN - Cerebellar deep nuclei, W – White matter, G – Granular cell layer, P – Purkinje cell layer, M – Molecular layer, the blue dotted lines represent the Purkinje cell layers of cerebellar folia.

Fig. 3

Evidence for axonal degeneration in cerebellum of Irp2 deficient mice. Amino cupric silver stains of brain sections of WT (Irp1^+/+Irp2^+/+), Irp1^+/+Irp2^−/−, Irp1^+/−Irp2^−/−, Irp1^−/−Irp2^+/+, Irp1^−/−Irp2^+/− mice showed axonal degeneration in white matter tracts of Irp1^+/+Irp2^−/− and Irp1^+/−Irp2^−/− mouse brains, illustrated here in the cerebellum. One-year old mice were perfused with 4% paraformaldehyde/PBS. Gelatin-embedded brains were sectioned, and sequential sections were stained with amino cupric silver stain for detection of degenerating axons as previously described (LaVaute et al., 2001; Smith et al., 2004). Note the black strands, which represent axons that have lost integrity and allowed silver to stain their neurofilaments, particularly in the Irp1+/− Irp2−/− animals. Abbrebiations: CDN - Cerebellar deep nuclei, W – White matter, G – Granular cell layer, P – Purkinje cell layer, M – Molecular layer.

Fig. 4

Hypothetical model to explain axonal degeneration of Irp2 deficient mice. IRP2 is the predominant IRP protein in many neurons, where it stabilizes TfR1 mRNA and represses ferritin translation, thus maintaining intracellular iron homeostasis. IRP2 deficiency decreases iron absorption by reducing TfR1 expression and increases iron sequestration by enhancing ferritin translation, resulting in a deficiency of available cytosolic iron, even though much iron can be sequestered in ferritin, leading to “functional iron deficiency”. Deficiency of bioavailable iron impairs the synthesis of iron-sulfur clusters, heme and other iron proteins, which compromises mitochondrial function. Consequently, mitochondria cannot provide enough ATP to support activity of ion channels and pumps in axons, leading to the potential loss of electrical potential and inability to maintain axonal integrity and function.

Fig. 5

Both Irp1 and Irp2 bind to the HIF2α IRE. Band-shift experiments were done with MEF cells to measure the HIF2α IRE and ferritin IRE binding activities of Irp1 and Irp2 following a published method (Ghosh et al., 2008). HIF2α is highly expressed in brain endothelial cells (Tian et al., 1997).