Mitochondrial Dysfunction in Friedreich's Ataxia: From Pathogenesis to Treatment Perspectives

Friedreich's ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein, which is severely reduced in FRDA patients. The demonstration that deficit of frataxin in FRDA is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of cardiac and skeletal muscle tissue energy metabolism, has established FRDA as a "new" nuclear encoded mitochondrial disease. Pilot studies have shown the potential effect of antioxidant therapy based on idebenone or coenzyme Q 10 plus Vitamin E administration in this condition and provide a strong rationale for designing larger randomized clinical trials.     

Mitochondrial dysfunction in Friedreich's ataxia: from pathogenesis to treatment perspectives

Friedreich's ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein, which is severely reduced in FRDA patients. The demonstration that deficit of frataxin in FRDA is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of cardiac and skeletal muscle tissue energy metabolism, has established FRDA as a "new" nuclear encoded mitochondrial disease. Pilot studies have shown the potential effect of antioxidant therapy based on idebenone or coenzyme Q 10 plus Vitamin E administration in this condition and provide a strong rationale for designing larger randomized clinical trials.     

Blood cells from Friedreich ataxia patients harbor frataxin deficiency without a loss of mitochondrial function

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by GAA triplet expansions or point mutations in the FXN gene on chromosome 9q13. The gene product called frataxin, a mitochondrial protein that is severely reduced in FRDA patients, leads to mitochondrial iron accumulation, Fe-S cluster deficiency and oxidative damage. The tissue specificity of this mitochondrial disease is complex and poorly understood. While frataxin is ubiquitously expressed, the cellular phenotype is most severe in neurons and cardiomyocytes. Here, we conducted comprehensive proteomic, metabolic and functional studies to determine whether subclinical abnormalities exist in mitochondria of blood cells from FRDA patients. Frataxin protein levels were significantly decreased in platelets and peripheral blood mononuclear cells from FRDA patients. Furthermore, the most significant differences associated with frataxin deficiency in FRDA blood cell mitochondria were the decrease of two mitochondrial heat shock proteins. We did not observe profound changes in frataxin-targeted mitochondrial proteins or mitochondrial functions or an increase of apoptosis in peripheral blood cells, suggesting that functional defects in these mitochondria are not readily apparent under resting conditions in these cells.     

Mitochondrial dysfunction in friedreich's ataxia

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disorder caused in the vast majority of cases by a GAA triplet expansion in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein which is severely reduced in FRDA patients. Loss of the homologue of frataxin in yeast is associated with mitochondrial iron overload, increased sensitivity to oxidative stress and profound deficit of oxidative phosphorylation. The demonstration that the human pathology of FRDA is also characterised by mitochondrial iron accumulation, deficit of respiratory chain complex activities and in vivo deficit of tissue energy metabolism establishes FRDA as a 'new' nuclear encoded mitochondrial disease.     

Structure-function analysis of Friedreich's ataxia mutants reveals determinants of frataxin binding and activation of the Fe-S assembly complex

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k(cat)/K(M) higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k(cat)/K(M) of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.     

Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.     

Activating frataxin expression by single-stranded siRNAs targeting the GAA repeat expansion

Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by reduced expression of the mitochondrial protein frataxin (FXN). The genetic cause of the disease is an expanded GAA repeat within the FXN gene. Agents that increase expression of FXN protein are a potential approach to therapy. We previously described anti-trinucleotide GAA duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) that activate FXN protein expression in multiple patient derived cell lines. Here we test two distinct series of compounds for their ability to increase FXN expression. ASOs with butane linkers showed low potency, which is consistent with the low T_m values and suggesting that flexible conformation impairs activity. By contrast, single-stranded siRNAs (ss-siRNAs) that combine the strengths of dsRNA and ASO approaches had nanomolar potencies. ss-siRNAs provide an additional option for developing nucleic acid therapeutics to treat FRDA.     

Novel Frataxin Isoforms May Contribute to the Pathological Mechanism of Friedreich Ataxia

Friedreich ataxia (FRDA) is an inherited neurodegenerative disease caused by frataxin (FXN) deficiency. The nervous system and heart are the most severely affected tissues. However, highly mitochondria-dependent tissues, such as kidney and liver, are not obviously affected, although the abundance of FXN is normally high in these tissues. In this study we have revealed two novel FXN isoforms (II and III), which are specifically expressed in affected cerebellum and heart tissues, respectively, and are functional in vitro and in vivo. Increasing the abundance of the heart-specific isoform III significantly increased the mitochondrial aconitase activity, while over-expression of the cerebellum-specific isoform II protected against oxidative damage of Fe-S cluster-containing aconitase. Further, we observed that the protein level of isoform III decreased in FRDA patient heart, while the mRNA level of isoform II decreased more in FRDA patient cerebellum compared to total FXN mRNA. Our novel findings are highly relevant to understanding the mechanism of tissue-specific pathology in FRDA.     

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich’s ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.     

Genotype and phenotype analysis of Friedreich's ataxia compound heterozygous patients

Friedreich's ataxia is caused by mutations in the FRDA gene that encodes frataxin, a nuclear-encoded mitochondrial protein. Most patients are homozygous for the expansion of a GAA triplet repeat within the FRDA gene, but a few patients show compound heterozygosity for a point mutation and the GAA-repeat expansion. We analyzed DNA samples from a cohort of 241 patients with autosomal recessive or isolated spinocerebellar ataxia for the GAA triplet expansion. Patients heterozygous for the GAA expansion were screened for point mutations within the FRDA coding region. Molecular analyses included the single-strand conformation polymorphism analysis, direct sequencing, and linkage analysis with FRDA locus flanking markers. Seven compound heterozygous patients were identified. In four patients, a point mutation that predicts a truncated frataxin was detected. Three of them associated classic early-onset Friedreich's ataxia with an expanded GAA allele greater than 800 repeats. The other patient associated late-onset disease at the age of 29 years with a 350-GAA repeat expansion. In two patients manifesting the classical phenotype, no changes were observed by single-strand conformation polymorphism (SSCP) analysis. Linkage analysis in a family with two children affected by an ataxic syndrome, one of them showing heterozygosity for the GAA expansion, confirmed no linkage to the FRDA locus. Most point mutations in compound heterozygous Friedreich's ataxia patients are null mutations. In the present patients, clinical phenotype seems to be related to the GAA repeat number in the expanded allele. Complete molecular definition in these patients is required for clinical diagnosis and genetic counseling.     

Normal and Friedreich ataxia cells express different isoforms of frataxin with complementary roles in iron-sulfur cluster assembly

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by insufficient expression of frataxin (FXN), a mitochondrial iron-binding protein required for Fe-S cluster assembly. The development of treatments to increase FXN levels in FRDA requires elucidation of the steps involved in the biogenesis of functional FXN. The FXN mRNA is translated to a precursor polypeptide that is transported to the mitochondrial matrix and processed to at least two forms, FXN(42-210) and FXN(81-210). Previous reports suggested that FXN(42-210) is a transient processing intermediate, whereas FXN(81-210) represents the mature protein. However, we find that both FXN(42-210) and FXN(81-210) are present in control cell lines and tissues at steady-state, and that FXN(42-210) is consistently more depleted than FXN(81-210) in samples from FRDA patients. Moreover, FXN(42-210) and FXN(81-210) have strikingly different biochemical properties. A shorter N terminus correlates with monomeric configuration, labile iron binding, and dynamic contacts with components of the Fe-S cluster biosynthetic machinery, i.e. the sulfur donor complex NFS1·ISD11 and the scaffold ISCU. Conversely, a longer N terminus correlates with the ability to oligomerize, store iron, and form stable contacts with NFS1·ISD11 and ISCU. Monomeric FXN(81-210) donates Fe(2+) for Fe-S cluster assembly on ISCU, whereas oligomeric FXN(42-210) donates either Fe(2+) or Fe(3+). These functionally distinct FXN isoforms seem capable to ensure incremental rates of Fe-S cluster synthesis from different mitochondrial iron pools. We suggest that the levels of both isoforms are relevant to FRDA pathophysiology and that the FXN(81-210)/FXN(42-210) molar ratio should provide a useful parameter to optimize FXN augmentation and replacement therapies.     

Somatic instability of the expanded GAA triplet-repeat sequence in Friedreich ataxia progresses throughout life

Friedreich ataxia (FRDA) patients are homozygous for expanded GAA triplet-repeat alleles in the FXN gene. Primary neurodegeneration involving the dorsal root ganglia (DRG) results in progressive ataxia. While it is known that DRG are inherently sensitive to frataxin deficiency, recent observations also indicate that they show age-dependent, further expansion of the GAA triplet-repeat mutation. Whether somatic instability is progressive has not been systematically investigated in FRDA patients. "Small-pool" PCR analysis of approximately 2300 individual molecules from tissues of an 18-week fetus homozygous for expanded alleles revealed very low levels of instability compared with adult-derived tissues (4.2% versus 30.6%, p<0.0001). Mutation load in blood samples from multiple patients and carriers increased significantly with age, ranging from 7.5% at 18-weeks gestation to 78.7% at 49 years of age (R=0.91; p=0.0001). Therefore, somatic instability in FRDA occurs mostly after early embryonic development and progresses throughout life, lending further support to the role of postnatal somatic instability in disease pathogenesis.