Frataxin knockdown causes loss of cytoplasmic iron–sulfur cluster functions, redox alterations and induction of heme transcripts

Abstract

Frataxin protein deficiency causes the neurodegenerative disease Friedreich ataxia. We used inducible siRNA to order the consequences of frataxin deficiency that we and others have previously observed. The earliest consequence of frataxin deficiency was a defect in cytoplasmic iron-sulfur proteins. In the second phase, protein oxidative damage increased, and CuZnSOD was induced, as was the unfolded protein response (UPR), long before any decline in mitochondrial aconitase activity. In the third phase, mitochondrial aconitase activity declined. And in the fourth phase, coincident with the decrease in heme-containing cytochrome c protein, a transcriptional induction of the heme-dependent transcripts ALAS1 and MAOA occurred. These observations suggest that the earliest consequences of frataxin deficiency occur in ISC proteins of the cytoplasm, resulting in oxidative damage and stress and activation of the unfolded protein response which has been associated with neurological disease, and that later consequences involve mitochondrial iron-sulfur cluster deficiency, heme deficiency, and then increased heme biosynthesis.

Cell functions impaired by frataxin deficiency are restored by drug-mediated iron relocation

“…20 As shown in Figure 1A, frataxin levels steadily declined after exposure of T-rex 293 cells to Tet for 2 days or more. The cells were comprehensively characterized in terms of the following biophysical and biochemical parameters: LIPs in cytosol and mitochondria, ROS production, mitochondrial redox potential, MMP, ATP levels, respiration, and susceptibility to ROS and apoptotic stimuli.…”

“…Cells included the following: T-Rex-293 cells (Invitrogen, Carlsbad, CA) stably expressing the tetracycline repressor and tetracycline-inducible shRNA against human frataxin were prepared as described. 20 The cells were grown in Dulbecco modified Eagle medium supplemented with 10% tetracycline-free fetal calf serum (FCS; BD Clontech, Mountain View, CA), 20 mM glutamine, 4.5 g/L glucose, and antibiotics and induced with tetracycline (1 g/mL) to repress frataxin expression. Cells were transfected with CaPi used as recommended.…”

Hydrogen peroxide scavenging rescues frataxin deficiency in a Drosophila model of Friedreich's ataxia

“…Discovering how this deficiency leads to the clinical outcomes of the disease is urgent. Understanding the etiology of the disease has been aided by two salient findings, namely, that frataxin deficiency leads in turn to impaired intracellular iron homeostasis (7,17,(40)(41)(42) and that oxidative stress plays a role as either a primary or secondary effector of FRDA pathology (for review, see refs. 25 and 26).…”

Lateral-flow immunoassay for the frataxin protein in Friedreich’s ataxia patients and carriers

“…The decrease in frataxin protein has broad, far-reaching effects because the protein is an essential iron chaperone required for the biogenesis of iron-sulfur clusters, aconitase activation, and heme biosynthesis [18][19][20][21], and further performs a critical role in iron detoxification and anti-oxidant protection [22][23][24]. Thus a loss of frataxin leads to widespread impairment of energy metabolism, increased oxidative stress, and a generally dysregulated iron metabolism, including accumulation of iron in the heart and nervous system [25].…”