Activation of NRF2 and FXN in Friedreich Ataxia

Jennifer S. Sun, PhD

FRIEDREICH ATAXIA (FRDA) is a frequently occurring degenerative disease that affects the brain, cardiac myocytes, and pancreatic β cells.^1,2 Progressive neurodegeneration results in cognitive deficits.^3,4 Patients consequently experience loss of coordination and ambulation, fatigue, cardiomyopathy, metabolic disturbances, scoliosis, and other skeletal abnormalities.¹ The average age at disease onset is 10 to 15 years, after which the accumulation of pathological changes results in death at a young age.^1,4,5 No cures or effective treatments are available for FRDA.^3,4,6,7 One roadblock in developing FRDA therapeutics is that presymptomatic defects may determine the onset and worsening of disease phenotypes.⁴ Evaluating early pathological changes is thus essential for identifying novel therapeutic targets.⁴

FRDA pathogenesis is attributed to oxidative stress.^4-7 One pathway through which cells cope with this stress (FIGURE 1⁸) is regulated by NRF2, which binds to antioxidant responsive elements in the promoter of the target gene FXN to control its expression.^1,3,4,6

Analysis of skin biopsies from patients with FRDA has identified genes of which messenger RNA (mRNA) and protein expression are decreased in FRDA: glutathione (GSH), FXN, NRF2, and the NRF2 target genes NQO1, HO-1, and γGCS.⁶ Upregulation of these genes could balance cellular redox.6 Activation of NRF2 signaling has been successful as a neuroprotective strategy in Parkinson disease and multiple sclerosis.^3,4,6 One caveat of this approach is that NRF2 regulates various transcriptional networks; thus, NRF2 modulation risks off-target effects.⁶

Click the image to enlarge.

FIGURE 1. TNRF2 Pathway Regulation⁸

The NRF2 pathway regulates cytoprotective pathways by activating antioxidant defenses upon NRF2 binding ARE sequences in target genes. Omav promotes NRF2 activity by preventing Keap1 from ubiquitinating NRF2 and targeting it for degradation.

ARE, antioxidant responsive element; Keap1, Kelch-like ECH-associated protein 1; MAF, musculoaponeurotic fibrosarcoma oncogene homolog; NRF2, nuclear factor erythroid 2-related factor 2; Omav, omaveloxolone; ROS, reactive oxygen species.

Figure reprinted with permission from Abeti R, Baccaro A, Esteras N, and Giunti P. Novel Nrf2-inducer prevents mitochondrial defects and oxidative stress in Friedreich’s ataxia models. Front Cell Neurosci. 2018;12:188. doi:10.3389/fncel.2018.00188

FXN plays an early, critical role in iron-sulfur cluster (ISC) synthesis and mitochondrial antioxidant defense (FIGURE 2⁹).^2,7,9 Impairment of ISC biogenesis, due to NRF2 or FXN deficiencies, results in a disrupted mitochondrial electron transport chain, accumulation of iron free radicals, and increased ferroptosis as a consequence of high mitochondrial iron accumulation, all of which are predicted to contribute to FRDA symptoms.^1-3,5,7-9 Most cases of FRDA are caused by expansion of an intronic GAA repeat in FXN, although some patients have an expansion plus a point mutation in the opposite allele.^2,3,9 Indeed, the FXN gene defect leads to FXN protein depletion and corresponds to an impaired GSH system that culminates in increased reactive oxygen species (ROS) generation and lipid peroxidation.^1,3 As FXN itself is sensitive to mitochondrial iron accumulation and ROS, this impairment of ISC biogenesis inevitably exacerbates FXN turnover.⁹

FXN deficiency and NRF2 dysfunction co-occur in FRDA.^3,7 Indeed, the phenotypic defects from FXN deficiency are partially restored by drug-driven NRF2 induction, providing evidence of upstream neurogenesis defects occurring in FRDA.⁴ NRF2 normally regulates cytoprotective pathways by inhibiting inflammation and activating antioxidant defenses, and maintaining protein homeostasis.^3,6 NRF2 also regulates neural stem proliferation and self-renewal.⁴ Moreover, NRF2 modulates cellular levels of GSH to maintain the cellular redox balance with GSSG.¹ Consequently, NRF2 suppression in FRDA contributes to excessive oxidative stress, mitochondrial dysfunction, impaired GSH levels, reduced adenosine triphosphate production, and impaired neurogenesis.^1,3,5,8

A case study examined FRDA in a complex family that included a symptomatic proband and relatives who were asymptomatic carriers.¹ A GAAGGA repeat identified in asymptomatic individuals within this cohort appeared to be a benign variant, although FXN mRNA and protein levels were reduced in those carriers.¹ This is because FRDA carriers are asymptomatic until the levels of FXN fall below 50%.^6,8 Symptomatic individuals, on the other hand, displayed significant depletion of FXN and NRF2.^1,6 Strategies to increase FXN abundance above the pathological threshold could be curative.^1,6 Widespread upregulation of NRF2 expression may provide protection for susceptible tissues against the progressive oxidative damage in FRDA.^1,6

Iron chelators could be effective at limiting mitochondrial iron, although there is the concern of concurrent inhibition of ISC synthesis, which would exacerbate oxidative stress.⁹ Potential therapeutic approaches could thus regulate mitochondrial iron accumulation by concurrently increasing FXN expression and boosting ISC synthesis.⁹

Click the image to enlarge.

FIGURE 2. FXN and ISC Assembly in Mitochondria⁹

FXN, a transcriptional target of NRF2, is critical for maintaining ISC assembly in the mitochondria. In FRDA, the characteristically diminished FXN levels result from aberrant NRF2 regulation. Compounds that increase FXN levels in FRDA cells can decrease ROS and increase ISC synthesis to delay disease progression.

Fe, iron; FRDA, Friedreich ataxia; FXN, frataxin; ISC, iron-sulfur cluster; Iscu, iron-sulfur cluster assembly enzyme; Nfs1, L-cysteine desulfurase; NRF2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; WT, wild-type.

Figure reprinted with permission from Hackett PT, Jia X, Li L, Ward DM. Posttranslational regulation of mitochondrial frataxin and identification of compounds that increase frataxin levels in Friedreich’s ataxia. J Biol Chem. 2022;298(6):101982. doi:10.1016/j.jbc.2022.101982

The 2019 Petrillo et al study of patient skin biopsies examined variable gene expression as a consequence of treatment with a panel of redox drugs—sulforaphane, dimethyl fumarate, N-acetylcysteine, EPI-743, idebenone, and the cyclic cyanoenone RTA 408 (omaveloxolone, Omav)—several of which are being investigated as treatments for neurodegenerative and mitochondrial diseases.⁶ These drugs exhibited differential effects on FXN, NRF2, and NRF2 target genes.⁶ NRF2 levels increased rapidly within 2 hours of administration, peaking at 6 hours and stabilizing by 24 hours.^4,6 The restoration of NRF2 expression was correlated with restored balance to cellular redox.⁶ NRF2 autoregulates its own expression to ensure appropriate cellular redox balance after reaching maximal activation.⁶ Importantly, such compounds function via multiple mechanisms (eg, reduced autophagy) to increase FXN levels, suggesting that personalized, targeted therapies for preventing inflammation, lipid peroxidation, or redox imbalance are feasible.^6,9

The global, phase 2 MOXIe study (NCT02255435) conducted by Lynch et al from 2015 to 2017 assessed the safety, pharmacodynamics, and potential benefit of Omav in patients with FRDA.⁵ Omav is a specific NRF2 activator and NF-κB suppressor.⁵ NRF2 turnover is normally regulated through ubiquitination and proteasomal degradation.^3,6 Omav prevents this ubiquitination to preserve NRF2 levels.⁶ The 2-part MOXIe study included a double-blind, randomized, placebo-controlled, dose-ranging, multicenter trial.⁵ A total of 69 patients with FRDA were randomly assigned 3:1 to Omav (2.5-300 mg/d) or placebo, administered once daily for 12 weeks.⁵ Omav was well tolerated.⁵ Mild adverse events included upper respiratory tract infections, nasopharyngitis, and alanine transaminase and aspartate transferase level increases that were not associated with liver dysfunction.⁵ Dose-dependent improvements in oxidative stress prevention and mitochondrial function were observed.⁵ Ferritin levels and glucose metabolism improved.⁵ The optimal doses were found to be at 80 and 160 mg/d.⁵ Across all doses, Omav produced no significant changes in peak workload during exercise (primary outcome); however, dose-dependent neurological improvements were observed via the modified Friedreich Ataxia Rating Scale (mFARS, a key secondary outcome).⁵ Importantly, benefits were observed irrespective of GAA repeat length, disease duration, or patient’s age.⁵ The 160-mg/d dose of Omav is being further examined for FRDA.⁵ Although the anatomical site of action of Omav is not entirely clear,⁵ if Omav can enter the central and peripheral nervous systems, immediate and sustained management of neurodegeneration may be possible.^5,8

For correspondence: jennsun@rutgers.edu
Rutgers University, New Brunswick, NJ

REFERENCES

1. Petrillo S, Santoro M, La Rosa P, et al. Nuclear factor erythroid 2-related factor 2 activation might mitigate clinical symptoms in Friedreich’s ataxia: clues of an “out-brain origin” of the disease from a family study. Front Neurosci. 2021;15:638810. doi:10.3389/fnins.2021.638810

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3. La Rosa P, Bertini ES, Piemonte F. The NRF2 signaling network defines clinical biomarkers and therapeutic opportunity in Friedreich’s ataxia. Int J Mol Sci. 2020;21(3):916. doi:10.3390/ijms21030916

4. La Rosa P, Russo M, D’Amico J, et al. Nrf2 induction re-establishes a proper neuronal differentiation program in Friedreich’s ataxia neural stem cells. Front Cell Neurosci. 2019;13:356. doi:10.3389/fncel.2019.00356

5. Lynch DR, Farmer J, Hauser L, et al. Safety, pharmacodynamics, and potential benefit of omaveloxolone in Friedreich ataxia. Ann Clin Transl Neurol. 2018;6(1):15-26. doi:10.1002/acn3.660

6. Petrillo S, D’Amico J, La Rosa P, Bertini ES, Piemonte F. Targeting NRF2 for the treatment of Friedreich’s ataxia: a comparison among drugs. Int J Mol Sci. 2019;20(20):5211. doi:10.3390/ijms20205211

7. La Rosa P, Petrillo S, Turchi R, et al. The Nrf2 induction prevents ferroptosis in Friedreich’s ataxia. Redox Biol. 2021;38:101791. doi:10.1016/j.redox.2020.101791

8. Abeti R, Baccaro A, Esteras N, Giunti P. Novel Nrf2-inducer prevents mitochondrial defects and oxidative stress in Friedreich’s ataxia models. Front Cell Neurosci. 2018;12:188. doi:10.3389/fncel.2018.00188

9. Hackett PT, Jia X, Li L, Ward DM. Posttranslational regulation of mitochondrial frataxin and identification of compounds that increase frataxin levels in Friedreich’s ataxia. J Biol Chem. 2022;298(6):101982. doi:10.1016/j.jbc.2022.101982