Selected missense mutations impair frataxin processing in Friedreich ataxia

Abstract

Objective: Frataxin (FXN) is a highly conserved mitochondrial protein. Reduced FXN levels cause Friedreich ataxia, a recessive neurodegenerative disease. Typical patients carry GAA repeat expansions on both alleles, while a subgroup of patients carry a missense mutation on one allele and a GAA repeat expansion on the other. Here, we report that selected disease-related FXN missense mutations impair FXN localization, interaction with mitochondria processing peptidase, and processing.

Methods: Immunocytochemical studies and subcellular fractionation were performed to study FXN import into the mitochondria and examine the mechanism by which mutations impair FXN processing. Coimmunoprecipitation was performed to study the interaction between FXN and mitochondrial processing peptidase. A proteasome inhibitor was used to model traditional therapeutic strategies. In addition, clinical profiles of subjects with and without point mutations were compared in a large natural history study.

Results: FXN^I154F and FXN^G130V missense mutations decrease FXN ^81-210 levels compared with FXN^WT, FXN^R165C, and FXN^W155R, but do not block its association with mitochondria. FXN^I154F and FXN^G130V also impair FXN maturation and enhance the binding between FXN ^42-210 and mitochondria processing peptidase. Furthermore, blocking proteosomal degradation does not increase FXN ^81-210 levels. Additionally, impaired FXN processing also occurs in fibroblasts from patients with FXN^G130V. Finally, clinical data from patients with FXN^G130V and FXN^I154F mutations demonstrates a lower severity compared with other individuals with Friedreich ataxia.

Interpretation: These data suggest that the effects on processing associated with FXN^G130V and FXN^I154F mutations lead to higher levels of partially processed FXN, which may contribute to the milder clinical phenotypes in these patients.

Figure 1

Selected FRDA‐associated missense mutations decrease FXN ^81–210 levels. A. Western blot of whole cell lysates collected from HEK 293 cells transfected with FXN^WT, FXN^{R 165C}, FXN^{W 155R}, FXN^{I 154F}, FXN^{G 130V}, FXN^{G 137V}, and FXN^{L 106S}. An anti‐FXN antibody was used to detect both exogenous FXN ^81–210 (15 kD) and endogenous FXN ^81–210 (14 kD) levels after transfection. B. Quantification of exogenous FXN levels was normalized to FXN^WT and endogenous FXN. Endogenous FXN serves as a loading control. (***) = P < 0.0005.

Figure 2

Selected FRDA‐associated missense mutations do not impair FXN association with mitochondria. Confocal microscopy images of HEK 293 cells cotransfected with mutant FXN constructs and mito‐GFP, fixed, and stained using a primary anti‐HA antibody to detect exogenous FXN only. Secondary antibodies included Alexa Fluor 568 (FXN) and Alexa Fluor 488 (mito‐GFP). DAPI was also used as a nuclear stain. A. FXN^WT, FXN^{R 165C}, and FXN^{W 155R}. B. FXN ^154F, FXN^{G 130V}, and FXN^{G 137V}. C. FXN^{L 106S}. Pearson's correlation scatter plots of red and green signal intensities for each mutant were generated using Image J Software.

Figure 3

Selected FRDA‐associated missense mutations impair processing from FXN ^42–210 to FXN ^81–210. Following transfection of mutant constructs in HEK 293 cells, whole cell lysates were centrifuged to perform subcellular fractionation of soluble mitochondria fraction and insoluble mitochondrial pellet. A. FXN levels were detected by western blot using an anti‐FXN antibody. Anti‐SDHA antibody was used to detect SDHA as a mitochondria marker and loading control. The soluble mitochondria fraction includes: exogenous FXN ^42–210 (19 kD), exogenous FXN ^81–210 (15 kD), endogenous FXN ^81–210 (14 kD), and SDHA (70 kD). The insoluble mitochondria pellet includes: exogenous FXN ^42–210 (19 kD) and SDHA (70 kD). B. Percent FXN ^81–210 of total FXN. C. Percent ^{FXN 42–210} of total FXN. D. Total FXN. E. Percent soluble FXN ^42–210. F. Percent insoluble FXN ^42–210. G. Total FXN ^42–210. (*) = P < 0.05 and (***) = P < 0.005.

Figure 4

Missense mutations FXN^{I 154F}and FXN^{G 130V} enhance the association of FXN ^42–210 with MPP. Whole cell lysates from transfected HEK 293 cells were immunoprecipitated with anti‐MPP antibody and immunoblotted with primary anti‐FXN antibody. The same whole cell lysates were used as inputs for quantification analysis. Western blot was used to detect FXN pulled down by anti‐MPP. The Co‐IP 19 kD blot represents immunoprecipitated FXN ^42‐210. The lysate 19 kD blot represents total FXN ^42‐210 in whole cell lysate. The graph represents fold change of FXN mutant interaction with MPP compared to FXN^WT.

Figure 5

Increasing FXN^{G 130V} and FXN^{I 154F} FXN ^1–210 levels does not increase FXN ^81–210 levels. Following transfection of HEK 293 cells with mutant FXN constructs, cells were treated with 10 μmol/L MG132 proteasome inhibitor for 5 h followed by cell lysis. Exogenous FXN^1–210 (23 kD), FXN^42–210 (19 kD), and FXN^81–210 (15 kD) levels, before and after treatment, were detected by western blot using a primary anti‐HA antibody.

Figure 6

Impaired FXN processing from FXN ^42–210 to FXN ^81–210 occurs in fibroblasts from FRDA patients with FXN^{G 130V}. FXN levels were quantified by western blot using whole cell extracts from control (CTRL), FRDA, and G130V patient fibroblasts. CTRL= 5 fibroblast lines (n = 13), G130V =3 lines (n = 17), and Typical = 7 lines (n = 8). A. FXN ^42–210 (18 kD), and FXN ^81–210 (13 kD) levels from control (CTRL), G130V, and typical FRDA were detected from whole cell extracts by western blot using an anti‐FXN antibody. Detection of GAPDH serves as a loading control. FXN levels are quantified and expressed as a ratio of FXN ^42–210 to FXN ^81–210. (*) = P < 0.05. B. Confocal microscopy images of patient fibroblasts (CTRL, FRDA, and G130V) that were fixed and stained using primary anti‐FXN and primary anti‐mitofusin antibodies. Secondary antibodies included Alexa Fluor 568 (FXN) and Alexa Fluor 488 (mitofusin). DAPI was also used as a nuclear stain.