Elucidation of the mechanism of mitochondrial iron loading in Friedreich's ataxia by analysis of a mouse mutant

Abstract

We used the muscle creatine kinase (MCK) conditional frataxin knockout mouse to elucidate how frataxin deficiency alters iron metabolism. This is of significance because frataxin deficiency leads to Friedreich's ataxia, a disease marked by neurologic and cardiologic degeneration. Using cardiac tissues, we demonstrate that frataxin deficiency leads to down-regulation of key molecules involved in 3 mitochondrial utilization pathways: iron-sulfur cluster (ISC) synthesis (iron-sulfur cluster scaffold protein1/2 and the cysteine desulferase Nfs1), mitochondrial iron storage (mitochondrial ferritin), and heme synthesis (5-aminolevulinate dehydratase, coproporphyrinogen oxidase, hydroxymethylbilane synthase, uroporphyrinogen III synthase, and ferrochelatase). This marked decrease in mitochondrial iron utilization and resultant reduced release of heme and ISC from the mitochondrion could contribute to the excessive mitochondrial iron observed. This effect is compounded by increased iron availability for mitochondrial uptake through (i) transferrin receptor1 up-regulation, increasing iron uptake from transferrin; (ii) decreased ferroportin1 expression, limiting iron export; (iii) increased expression of the heme catabolism enzyme heme oxygenase1 and down-regulation of ferritin-H and -L, both likely leading to increased "free iron" for mitochondrial uptake; and (iv) increased expression of the mammalian exocyst protein Sec15l1 and the mitochondrial iron importer mitoferrin-2 (Mfrn2), which facilitate cellular iron uptake and mitochondrial iron influx, respectively. Our results enable the construction of a model explaining the cytosolic iron deficiency and mitochondrial iron loading in the absence of frataxin, which is important for understanding the pathogenesis of Friedreich's ataxia.

Fig. 1.

Microarray analysis of 4- and 10-week-old WT and mutant [frataxin (Fxn) knockout] mice. (A) Principal component plot showing the overall differential expression profile between WT and mutants of different ages. (B and C) Functional clustering of significantly (P < .05) altered genes in 10-week-old mutants showing various biological processes significantly (P < .05) up-regulated (B) or down-regulated (C).

Fig. 2.

RT-PCR analysis of iron metabolism–related genes selected from Affymetrix GeneChips that were significantly (P < .05) differentially expressed between 4- and 10-week-old WT and mutant (Fxn knockout) mice. (A) RT-PCR confirming significant differential expression of genes observed in Affymetrix GeneChips. (B) Densitometry of RT-PCR analysis. *, P < .05; **, P < .01; ***, P < .001. Results shown in (A) are representative of 3–6 experiments, and those in (B) are the mean ± SD of 3–6 experiments.

Fig. 3.

Western blot analysis of significantly (P < .05) differentially expressed iron metabolism–related genes and densitometric analysis from 4- and 10-week-old WT and mutant (Fxn knockout) mice. (A) Western blot confirming the differential gene expression from the RT-PCR analysis shown in Fig. 2. (B) Densitometric analysis. *, P < .05; **, P < .01; ***, P < .001. Results shown in (A) are representative of 3–6 experiments, and those in (B) are the mean ± SD of 3–6 experiments.

Fig. 4.

Alterations in frataxin mRNA expression in extracardiac tissues in the MCK mutant (Fxn knockout) mice relative to the WT mice are not due to Fxn deletion. (A) Conditional deletion of mouse frataxin exon 4 as reported previously (7). From top to bottom: WT allele, loxP-flanked Fxn exon 4 allele (L3), and Cre-mediated exon 4 deleted allele (Δ). The Δ allele was derived from the exon 4 deletion of the L3 allele via a cross with a CMV-Cre line (7). Flag, loxP site; B, BamHI; E, exon; P, primer; X, XbaI; X^o, modified XbaI. (B) The L3 allele was identified in the liver, kidney, and spleen, but not the heart, of the mutants, demonstrating the muscle-specific deletion of exon 4. This results in the Δ allele in the mutant heart only. PCR conditions were as described previously (7, 11). (C) RT-PCR showing pronounced down-regulation of frataxin mRNA in the heart and less marked reductions in the liver and kidney of 10-week-old mutants relative to WT mice of the same age. Densitometry data are reported as the mean ± SD of 3 experiments. *, P < .05; **, P < .01; ***, P < .001.

Fig. 5.

Model of altered iron metabolism due to frataxin deficiency in MCK mutant hearts. Frataxin deficiency leads to increased mitochondrial-targeted iron uptake and cytosolic iron deficiency, facilitated by (i) Tfr1 up-regulation, increasing transferrin iron uptake; (ii) Fpn1 down-regulation, preventing iron release; (iii) Hmox1 up-regulation, increasing cytosolic heme catabolism; and (iv) Sec15l1 up-regulation, potentially aiding iron uptake. Iron is then taken up more avidly by the mitochondrion via increased Mfrn2. Moreover, there is down-regulation of 3 major pathways of mitochondrial iron utilization—ISC synthesis, heme synthesis from iron incorporation into protoporphyrin IX (PIX), and mitochondrial iron storage (Ftmt). The decreased iron utilization in these pathways reduces iron export from the mitochondrion as heme and ISCs. This suppression, together with increased iron uptake, decreased iron release, and iron targeting to the mitochondrion, leads to the marked mitochondrial iron loading (1).