Interferon gamma upregulates frataxin and corrects the functional deficits in a Friedreich ataxia model

Abstract

Friedreich's ataxia (FRDA) is the most common hereditary ataxia, affecting ∼3 in 100 000 individuals in Caucasian populations. It is caused by intronic GAA repeat expansions that hinder the expression of the FXN gene, resulting in defective levels of the mitochondrial protein frataxin. Sensory neurons in dorsal root ganglia (DRG) are particularly damaged by frataxin deficiency. There is no specific therapy for FRDA. Here, we show that frataxin levels can be upregulated by interferon gamma (IFNγ) in a variety of cell types, including primary cells derived from FRDA patients. IFNγ appears to act largely through a transcriptional mechanism on the FXN gene. Importantly, in vivo treatment with IFNγ increases frataxin expression in DRG neurons, prevents their pathological changes and ameliorates the sensorimotor performance in FRDA mice. These results disclose new roles for IFNγ in cellular metabolism and have direct implications for the treatment of FRDA.

Figures

Figure 1.

IFNγ induces frataxin accumulation in multiple cell types. HeLa cells (A), U937 cells (B), U118 cells (C) and PBMC isolated from healthy donors (D) were cultured for 24h in the presence of the indicated concentrations of IFNγ, and then whole cell lysates were analyzed by SDS–PAGE and blotted with anti-frataxin and anti-actin mAbs. Representative blots are shown, three to six independent experiments for each cell type were performed.

Figure 2.

IFNγ induces frataxin accumulation in FRDA cells. FRDA fibroblasts (GM03816 cells) (A) and PBMC freshly isolated from an FRDA patient (B) were cultured for 24h in the presence of the indicated concentrations of IFNγ, and then whole cell lysates were analyzed by SDS–PAGE and blotted with anti-frataxin and anti-actin mAbs. The amount of frataxin present in the PBMC of a healthy sibling of the patient is also shown for comparison (HC). Nine out of 10 different FRDA patients (6 males and 4 females, GAA triplets range 350–915, age range 14–56) tested gave similar results. (C) IFNγ induces accumulation of frataxin mRNA in FRDA cells. FRDA fibroblasts were cultured for the indicated times in the presence of 500 ng/ml of IFNγ, and then frataxin mRNA was quantitated by RT–PCR. The means ± 1 SD from three independent experiments is shown. The increase in frataxin mRNA in IFNγ-treated cells, versus control-treated cells, was significant at 1h (**P < 0.001) and at 2h (*P < 0.05). (D). Actinomycin D blocks IFNγ-induced frataxin mRNA accumulation in FRDA cells. FRDA fibroblasts were pre-treated for 30 min with 5 μg/ml actinomycin D, cultured for 2h in the presence of 500 ng/ml of IFNγ and then frataxin mRNA (black columns) and PA28alpha mRNA (grey columns) were quantitated by RT–PCR. The means ± 1SD from two independent experiments is shown.

Figure 3.

In vivo IFNγ treatment improves locomotor and motor coordination performances in FRDA mice. Two groups of 13 eight-week-old FRDA mice were used, one group injected subcutaneously with 40 μg/kg IFNγ, three times/week for 14 weeks and the other with PBS. Every 2 weeks, functional evaluation of locomotor and coordination activity was performed. Mean values for each functional parameter measured from all 13 FRDA mice of each group are shown at the indicated time points. Both locomotor parameters, such as ambulatory distance (A, P < 0.01), average velocity (B, P < 0.01), vertical counts (C, P < 0.001) and coordination parameters such as rotarod performance (D, P < 0.001) gave significantly different results between the two groups. Body weight was also measured at every time point (E). Squares: IFNγ-treated animals, triangles: PBS-treated animals.

Figure 4.

In vivo IFNγ treatment upregulates human frataxin in DRG of FRDA mice. Immunohistochemistry of DRG tissue from PBS-treated FRDA mice (A and C) and IFNγ-treated FRDA mice (B and D), after 14 weeks of treatment. Immunostaining (see Materials and Methods) was performed in the presence (A and B) or absence (C and D) of anti-frataxin mAb, followed by anti-mouse IgG secondary antibody treatment and DAB staining. All four panels were counterstained with hematoxylin. (E). SDS–PAGE analysis of cell extracts from pooled DRG neurons from male (M, n = 3) and female (F, n = 2) FRDA mice, treated with either PBS or 40 μg/kg IFNγ three times/week for 14 weeks. Blots were analyzed for the mitochondrial protein VDAC (as negative control), for the IFNγ-inducible PA28alpha (as positive control), and for human frataxin. (F). Densitometric analysis of the results shown in (E). Data are presented as % fold induction of frataxin accumulation in DRG neurons of IFNγ-treated FRDA mice compared with frataxin levels in DRG neurons of PBS-treated FRDA mice.

Figure 5.

In vivo IFNγ treatment prevents degeneration of DGR neurons of FRDA mice. HE staining of DRG sections from FRDA mice treated for 14 weeks with PBS (A) or IFNγ (B). Arrows show vacuolar degeneration in the cytoplasm of three DRG neurons from PBS-treated FRDA mice. (C). Quantitation and statistical analysis of DRG neurons shown in (A) and (B). Six DRG HE sections from each of the three mice for each group (IFNγ-treated and PBS-treated, total of 18 sections per group) were analyzed and vacuolated neurons were quantitated as a percentage of total neurons counted. A significant (*P < 0.01) reduction was detected in the IFNγ-treated group compared with the PBS-treated group.