Mesenchymal stem cells restore frataxin expression and increase hydrogen peroxide scavenging enzymes in Friedreich ataxia fibroblasts

Abstract

Dramatic advances in recent decades in understanding the genetics of Friedreich ataxia (FRDA)--a GAA triplet expansion causing greatly reduced expression of the mitochondrial protein frataxin--have thus far yielded no therapeutic dividend, since there remain no effective treatments that prevent or even slow the inevitable progressive disability in affected individuals. Clinical interventions that restore frataxin expression are attractive therapeutic approaches, as, in theory, it may be possible to re-establish normal function in frataxin deficient cells if frataxin levels are increased above a specific threshold. With this in mind several drugs and cytokines have been tested for their ability to increase frataxin levels. Cell transplantation strategies may provide an alternative approach to this therapeutic aim, and may also offer more widespread cellular protective roles in FRDA. Here we show a direct link between frataxin expression in fibroblasts derived from FRDA patients with both decreased expression of hydrogen peroxide scavenging enzymes and increased sensitivity to hydrogen peroxide-mediated toxicity. We demonstrate that normal human mesenchymal stem cells (MSCs) induce both an increase in frataxin gene and protein expression in FRDA fibroblasts via secretion of soluble factors. Finally, we show that exposure to factors produced by human MSCs increases resistance to hydrogen peroxide-mediated toxicity in FRDA fibroblasts through, at least in part, restoring the expression of the hydrogen peroxide scavenging enzymes catalase and glutathione peroxidase 1. These findings suggest, for the first time, that stem cells may increase frataxin levels in FRDA and transplantation of MSCs may offer an effective treatment for these patients.

Figure 1. Fibroblasts derived from patients with Friedreich ataxia have low frataxin expression.

(A) Immunoblotting of human frataxin in FRDA and control (CON) fibroblasts. Upper panels correspond to frataxin (FXN); lower panel corresponds to the loading control GAPDH. (B) Densitometic analysis of frataxin expression of western blot bands. Data are given using arbitrary units of integrated density. (C) The relative frataxin mRNA expression in control and FRDA fibroblasts . Results are expressed as the mean +/− (SEM). (*p<0.05, comparing test condition to control; n = 3 independent experiments).

Figure 2. Mesenchymal stem cell conditioned medium increases frataxin protein expression.

Immunoblotting of human frataxin in (A) FRDA fibroblasts and (B) control fibroblasts after exposure to minimal medium (MIN) or MSC conditioned medium (MSC CM) for 24 hours. Upper panels correspond to frataxin (FXN); lower panel corresponds to the loading control GAPDH. Western blot densitometic analysis of frataxin expression in fibroblasts derived from patients with Friedreich ataxia at (C) 24 hours and (D) over a 72 hour period. (E) Western blot densitometic analysis of frataxin expression in fibroblasts derived from healthy controls. (F) The dipstick immunoassay of human frataxin in FRDA fibroblasts (or MSC CM alone) after exposure to minimal medium (MIN) or MSC CM for 24 hours. Upper panels correspond to internal control; lower panel corresponds to human frataxin (FXN). (G) The dipstick immunoassay densitometic analysis of frataxin expression in fibroblasts derived from patients with Friedreich ataxia. Data are given using arbitrary units of integrated density. Results are expressed as the mean +/− (SEM). (*p<0.05, comparing test condition to control; n = 3 independent experiments).

Figure 3. Mesenchymal stem cell conditioned medium increases frataxin gene expression.

The relative frataxin mRNA expression in (A) FRDA and (B) control fibroblasts after exposure to minimal medium (MIN) or MSC conditioned medium (MSC CM) for 2, 6 and 24 hours. The mean maximal relative frataxin mRNA expression in (C) FRDA and (D) control fibroblasts evident throughout the 24 hour exposure to minimal medium (MIN) or MSC CM. Results are expressed as the mean +/− (SEM). (*p<0.05, comparing test condition to control; n = 3 independent experiments).

Figure 4. Mesenchymal stem cell conditioned medium increases both catalase and glutathione peroxidase 1 expression in fibroblasts-derived from FRDA patients.

Immunoblotting of human catalase, glutathione peroxidase 1 (GPX1) and loading control GAPDH in (A) FRDA/control fibroblasts and (C) control fibroblasts after exposure to minimal medium (MIN) or MSC conditioned medium (MSC CM) for 24 hours. Western blot densitometic analysis of catalase and GPX1expression in fibroblasts derived from (B) patients with FRDA/controls and (D) control fibroblasts at 24 hours. Data are given using arbitrary units of integrated density. Results are expressed as the mean +/− (SEM). (*p<0.05, comparing test condition to control; n = 3 independent experiments).

Figure 5. Enhanced frataxin expression in FXN-transfected FRDA fibroblasts increases catalase expression.

(A) FRDA fibroblast culture transfected with a GFP-tagged FXN gene. (B) Immunoblotting of human frataxin in GFP-tagged frataxin transfected (right) and untransfected (left) FRDA fibroblasts. Upper panels correspond to frataxin; lower panel corresponds to the loading control GAPDH. Immunoblotting of human catalase, glutathione peroxidase 1 (GPX1) and loading control GAPDH in (C) FRDA fibroblasts and (E) control fibroblasts after transfection with the GFP-tagged FXN gene. Western blot densitometic analysis of catalase and GPX1expression in fibroblasts derived from (D) patients with FRDA and (F) control fibroblasts at 24 hours. Data are given using arbitrary units of integrated density. Results are expressed as the mean +/− (SEM). (*p<0.05, comparing test condition to control; n = 3 independent experiments).

Figure 6. Mesenchymal stem cell conditioned medium or FXN-transfection increases resistance to hydrogen peroxide mediated toxicity in fibroblasts derived from FRDA patients.

The effect of hydrogen peroxide (600 µM) on FRDA and control fibroblast cell survival in vitro post exposure to minimal medium (MIN) or MSC-conditioned medium (MSC CM) for 24hours, with/without the addition of the catalase inhibitor 3-AminoTriazole (CATIN), catalase (CAT) and glutathione peroxidase (GPX1). FXN indicates fibroblasts have been transfected with the GFP-tagged FXN gene. Cell survival was assesed using the MTT cell viability assay (MTT). Cell survival is expressed as a percentage of cell survival compared to cells grown in minimal medium alone. Results are expressed as the mean +/− (SEM). (*p<0.05, comparing test condition to control; n = 3 independent experiments).