Investigating Friedreich ataxia disease mechanisms and therapy

Abstract

Friedreich ataxia (FRDA) is an autosomal recessive, neurodegenerative disease caused by the excessive pathological expansion of an unstable GAA trinucleotide repeat within intron 1 of the FXN gene. FRDA occurs due to a pathological GAA repeat ranging from 70-1200 repeats, whereas normal individuals have up to 40 repeats. The prevalence of FRDA in Caucasians is 1:50,000 and it is the most common inherited ataxia. The presence of this large GAA repeat leads to silencing of the FXN gene, resulting in severely diminished levels of the essential mitochondrial protein, frataxin. Frataxin is required throughout development. Therefore, decreased frataxin levels affect the onset and progression of FRDA due to mitochondrial iron accumulation and an increase in oxidative damage, leading to detrimental cellular defects. The pathology of FRDA predominantly affects the cerebellum. Various FRDA cellular and mouse models have been developed to represent the characteristics of this debilitating disease. Data has revealed a positive correlation to exist between an increase in the number of GAA repeats and the severity of the disease. The analysis of these models has shown the expansion of the GAA repeat to be unstable, resulting in both somatic and intergenerational instability. In recent times, a single nucleotide polymorphism (SNP), Asn/Ser46, in the Sirt6 protein of FRDA patients has been associated with a better outcome of the disease. Therefore, Sirt6 heterozygote knockout mice obtained from the Jackson Laboratory were bred with YG8sR FRDA mice, to observe the effects of Sirt6 status on expanded GAA repeat instability, compared to Sirt6 wild type YG8sR FRDA mice. Densitometry analysis showed decreased levels of somatic GAA repeat instability and an increase in FXN protein in 6-month old heterozygous Sirt6 KO mice. However, increased levels of GAA repeat instability and decreased levels of FXN protein was seen in 12-month heterozygous Sirt6 KO mice. The SNP and decreased Sirt6 function may be beneficial to begin with, but not in the long run. A major aim of scientific research is to pave way for the generation of effective therapy. Therefore, attention turned to therapy to increase frataxin protein levels in YG8sR transgenic mice. The effects of the Src kinase inhibitor dasatinib, which had previously been shown to increase frataxin in cell culture, were investigated in YG8sR mice. Dasatinib was administered over a 3-month period, during which time motor coordination ability and frataxin protein levels were investigated. No effect on motor coordination was seen over the course of the treatment. Although, there was a non-significant decrease in frataxin protein levels in the brain in comparison to the vehicle-treated mice. In addition, further analysis by immunohistochemistry showed a decrease of frataxin protein expression in the dorsal root ganglia (DRG). Furthermore, the mean body weight of the dasatinib-treated mice was shown to decrease over time in comparison to vehicle-treated mice. Overall, these studies do not support the use of dasatinib for FRDA therapy. Further studies investigated a new line of GAA repeat expansion-containing FRDA transgenic mice, designated ‘YG8LR’. These mice contain approximately 410 GAA repeats, which is significantly more than the previous ‘YG8sR’ line of mice which contained approximately 220 GAA repeats. As result of this larger GAA repeat, a further decrease of frataxin mRNA and protein was detected in the cerebellum and heart of YG8LR mice compared to YG8sR mice. Further investigations were undertaken to study the epigenetic status and the effects of lowered frataxin protein on the phenotype of the YG8LR model. Increased levels of histone deacetylation and methylation, together with DNA methylation, were detected at the FXN transgenic locus. The YG8LR mice also showed somatic and intergenerational GAA repeat instability as previously detected in earlier FRDA mouse models. Immunohistochemistry analysis of DRG sections showed a decreased level of frataxin protein in YG8LR mice. However, there was an absence of vacuoles in the DRG of the YG8LR mice as previously seen in YG8sR mice, although this particular phenotype is not actually seen in FRDA patients. Motor coordination ability and locomotor activity were significantly decreased as assessed by accelerating rotarod, beam-walk and beam-breaker open-field locomotor analysis. YG8LR mice showed increased glucose intolerance and insulin hypersensitivity in comparison to YG8sR and Y47R mice, investigated by glucose and insulin tolerance tests. Moreover, the most common cause of death in FRDA patients is caused by cardiomyopathy, therefore heart weights were obtained from B6, Y47R, YG8sR and YG8LR mice and a slight increase in mean heart weight was observed in YG8LR mice compared to the other models. This could suggest the lower levels of frataxin leads to the pathology of the heart as seen in FRDA patients. These investigations may help to provide an insight into and confirm molecular mechanisms of the disease, as well as providing a great mouse model for testing future FRDA therapies.