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. 2013 Nov 1;19(13):1481-93.
doi: 10.1089/ars.2012.4537. Epub 2013 Mar 28.

Frataxin Deficiency Leads to Defects in Expression of Antioxidants and Nrf2 Expression in Dorsal Root Ganglia of the Friedreich's Ataxia YG8R Mouse Model

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Frataxin Deficiency Leads to Defects in Expression of Antioxidants and Nrf2 Expression in Dorsal Root Ganglia of the Friedreich's Ataxia YG8R Mouse Model

Yuxi Shan et al. Antioxid Redox Signal. .
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Abstract

Aims: Oxidative stress is thought to be involved in Friedreich's ataxia (FRDA), yet it has not been demonstrated in the target neurons that are first to degenerate. Using the YG8R mouse model of FRDA, microarray and neuritic growth experiments were carried out in the dorsal root ganglion (DRG), the primary site of neurodegeneration in this disease.

Results: YG8R hemizygous mice exhibited defects in movement, and DRG neurites had growth defects. Microarray of DRG tissue identified decreased transcripts encoding the antioxidants, including peroxiredoxins, glutaredoxins, and glutathione S-transferase, and these were confirmed by immunoblots and quantitative real-time PCR. Because the decreased gene transcripts are the known targets of the antioxidant transcription factor nuclear factor-E2-related factor-2 (Nrf2), Nrf2 expression was measured; it was significantly decreased at the transcript and protein level in both the DRG and the cerebella of the YG8R hemizygous mouse; further, frataxin expression was significantly correlated with Nrf2 expression. Functionally, in YG8R hemizygous DRG, the total glutathione levels were reduced and explanted cells were more sensitive to the thioredoxin reductase (TxnRD) inhibitor auranofin, a thiol oxidant. In cell models of FRDA, including Schwann and the DRG, frataxin deficiency caused a decreased expression of the Nrf2 protein level in the nucleus, but not a defect in its translocation from the cytosol. Further, frataxin-deficient cells had decreased enzyme activity and expression of TxnRD, which is regulated by Nrf2, and were sensitive the TxnRD inhibitor auranofin.

Innovation and conclusion: These results support a mechanistic hypothesis in which frataxin deficiency decreases Nrf2 expression in vivo, causing the sensitivity to oxidative stress in target tissues the DRG and the cerebella, which contributes to the process of neurodegeneration.

Figures

FIG. 1.
FIG. 1.
Functional and neuritic defects in UC Davis YG8R mice. (A) Accelerating rotarod performance (4 mice/group); (B) horizontal activity in open-field performance test (4 mice/genotype); (C) frataxin expression in DRG tissue; each lane represents a different mouse. No expression is observed in WT samples, as the antibody is human-specific; (D, E) DRGs from YG8R mice exhibit decreased neurite extension, WT=34.02±1.53 versus homo=34.48±1.29 versus hemi=29.17±1.15 μm, n=3 mice per genotype, 48 neurites counted per mouse. Error bars=SEM. *p<0.05, **p<0.005, ***p<0.0005. Using a 1-tailed Student's t-test. DRG, dorsal root ganglion; WT, wild type.
FIG. 2.
FIG. 2.
Quantitative real time-PCR verification of select microarray hits. One nanogram of total RNA was used for amplification, and the values were normalized to GAPDH or Lmnb1 expression. n=3 mice per genotype. Error bars=SEM. *p<0.05 versus WT; **p<0.01 versus WT; #p<0.05 versus homo; ##p<0.01 versus homo using a 1-tailed Student's t-test.
FIG. 3.
FIG. 3.
Hemizygous YG8R DRGs exhibit reduced levels of thiol antioxidant proteins. (A) Representative immunoblots of Gstm1, Glrx1, and Prx3; (B) densitometry of immunoblots shown in (A). Each lane contains 30 μg protein from a different mouse. Error bars=SEM. *p<0.05 versus WT; **p<0.01 versus WT; using a 1-tailed paired Student's t-test [the bottom blot of (A) is the same blot used in Fig. 1C]. Glrx1, glutaredoxin 1; Gstm1, glutathione S-transferase mu 1.
FIG. 4.
FIG. 4.
Transcripts encoding Nrf2 and downstream targets are decreased in YG8R DRGs and correlated with frataxin levels. (A–H) Correlation analysis of the frataxin transcript levels with Nrf2 (A), Cat (B) Gclc (C), Gclm (D), Hmox (E), Sod1 (F), Sod2 (G), and TxnRD1 (H). n=12 mice. Statistics were generated using GraphPad Prism software v4.02 for Windows (GraphPad Software). Cat, catalase; Gclc, glutamate–cysteine ligase, catalytic subunit; Gclm, glutamate–cysteine ligase, modifier subunit; Hmox, heme oxygenase; Nrf2, nuclear factor-E2-related factor-2; Sod1, Cu/Zn superoxide dismutase; Sod2, Mn superoxide dismutase; TxnRD1, thioredoxin reductase 1.
FIG. 5.
FIG. 5.
Nrf2 and downstream target proteins are decreased in YG8R DRGs. (A) Representative immunoblots of Nrf2, Hmox, Nqo1, and Sod2 in the YG8R DRGs. Each lane represents an individual mouse. (B) Densitometry of blots shown in A, normalized to tubulin. Each lane contains 30 μg protein from a different mouse. Error bars=SEM. *p<0.05 versus WT; #p<0.05 versus homo using a 1-tailed Student's t-test. Nqo1, NAD(P)H dehydrogenase, quinone 1.
FIG. 6.
FIG. 6.
Glutathione levels are altered in YG8R DRGs. Total glutathione, GSH, and GSSG were measured in a pool of 16 DRGs from more than 7 mice per genotype. (A) Total glutathione (GSH+GSSG); (B) GSH; (C) GSSG; (D) GSH/GSSG. Error bars=SEM. *p<0.05 using a one-tailed Student's t-test. GSH, reduced glutathione; GSSG, oxidized glutathione.
FIG. 7.
FIG. 7.
Nrf2 is decreased in frataxin-deficient cells. (A) Nrf2 protein decreased in HeLa and T265 frataxin-depleted cells. (B) No Nrf2 nuclear translocation defect in frataxin-deficient cells. Nuclear extraction and immunopurified anti-Nrf2 antibody demonstrate a substantial nuclear localization of Nrf2 protein, which is inducible by tBHQ, and deficient induction of Nrf2 in the nucleus, but no defect in cytosolic transport to nucleus is evident. Immunoblots contain 30 μg proteins per lane. tBHQ, tert-butylhydroquinone.
FIG. 8.
FIG. 8.
TxnRD activity and protein are decreased in frataxin-deficient cells. (A) TxnRD activity analysis in HeLa or lymphoblast cells. HeLa cells were transfected with control or frataxin A11 siRNA; after 72 h, cells were used to TxnRd activity assay. For lymphoblasts, healthy (GM15851) and patient (GM15850) cells were used in this assay. (B) TxnRd immunoblot in HeLa cells and lymphoblasts. The densitometries were normalized with tubulin. (C) Frataxin deficiency shifts the redox state of Prdx2 toward the oxidized isoform. HeLa cells were treated with increasing concentrations of auranofin for 1 h, then treated with an N-ethylmaleimide-containing buffer for 15 min before harvest. A, DMSO (vehicle); B, 10 μM; C, 20 μM; D, 40 μM; E, 80 μM. Immunoblots contain 30 μg protein per lane. Error bars=SEM. *p<0.05 using Student's t-test. Ctrl=control siRNA. siFxn=siRNA oligonucleotide directed against frataxin A11. Prdx2, peroxiredoxin 2.
FIG. 9.
FIG. 9.
Frataxin-deficient cells exhibit increased sensitivity to the TxnRD inhibitor auranofin. (A) Left panel: Frataxin-knockdown HeLa cells treated with 4 μM auranofin. Data indicate means of 6 replicates; ***p<0.001. Right panel: Frataxin-knockdown ND7/23 cells treated with 2.5 μM auranofin. Data indicate means of three replicates; **p<0.01. (B) Auranofin-treated YG8R DRG cells. Data indicate means of three replicates per dosage over five experiments, each experiment using one mouse per genotype. Error bars=SEM. **p<0.01 versus WT; #p<0.05 versus homo using a 1-tailed Student's t-test.
FIG. 10.
FIG. 10.
A model for frataxin deficiency with neurodegeneration in the DRG cells.

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