Fig 2: Moderate loss of Mitochondrial Calcium Uniporter (MCU) does not worsen loss-of-frataxin conditions. (A–B) Compared to control flies (Repo-G4>w), 50% loss of MCU levels (fhRNAi-1;Repo-G4>MCU[1]) did not modified the negative geotaxis ability (A) as well as the levels of brain degeneration (B) of FRDA flies (fhRNAi-1;Repo-G4>w). (C–E) Representative paraffin brain sections and Bodipy staining of controls (C, Repo-G4>w), frataxin-deficient flies (D,fhRNAi-1;Repo-G4>w) or combination of frataxin deficiency and one MCU mutant allele (E,fhRNAi-1;Repo-G4>MCU[1]). Arrows indicated the presence of the vacuolization in frataxin-deficient brains with normal levels of MCU (D) and with 50% reduction of MCU expression (E). Bodipy staining of whole fly brains showed complete absence of lipid deposits in controls (C) and a strong accumulation in frataxin deficient flies (D) that was not modified by 50% inactivation of MCU (E). Graphs represent means ± SEM In A and B data was analyzed by One-way ANOVA with post-hoc Tukey Multiple Comparison test. **, P < 0.01 ***, P < 0.001, Scale bars: 50 µm.

Fig 3: Dose response to carbachol in SH-SY5Y and FXN-138.2 cells. (A) FRET analysis of mitochondrial calcium levels measured through 4mtD3cpv chameleon. 25 µM, 250 µM, 500 µM and 900 µM carbachol (Cch) were used to stimulate Ca2+ release from the ER through IP3R channels. FXN-138.2 cell line did not change in response to 25 µM carbachol. 250 µM carbachol was chosen as the most suitable concentration that gave the maximum response. Results (n = 3) are represented as normalized levels of fluorescence intensity over carbachol loading value. A range of 11–31 cells were analyzed in each condition per replicate. (B) Area Under curve (AUC) analysis of the mitochondrial response to the different concentrations of carbachol.

Fig 4: Antioxidants rescue MAMs connections in frataxin-deficiency cells. (A) Representative microscopy images of the PLA evaluating the interactions between VDAC1 and GRP75 in FXN-138.1 and FXN-138.2 cells after 1 mM Trolox treatment compared to untreated conditions. (B) Normalized number of VDAC1-GRP75 interactions (puncta) per cell in each frataxin-silenced clone treated with Trolox compared to untreated conditions. (C) Representative microscopy images of the PLA evaluating the interactions between VDAC1 and IP3R in FXN-138.1 and FXN-138.2 cells after 1 mM Trolox treatment compared to untreated conditions. (D) Normalized number of VDAC1-IP3R interactions (puncta) per cell in each frataxin-silenced clone treated with Trolox compared to untreated conditions. (E) Representative microscopy images of the PLA evaluating the interactions between VDAC1 and GRP75 in FXN-138.2 cells after 5 mM NAC treatment compared to untreated conditions. (F) Normalized number of VDAC1-GRP75 interactions (puncta) per cell in FXN-138.2 cells treated with NAC compared to untreated conditions. Results (n = 4) are represented as mean ± SEM. A minimum of 500 cells were analyzed in each independent experiment. Student's t-test significance: *p = 0.05; **p = 0.01 over untreated cells.

Fig 5: Antioxidants recover mitochondrial calcium uptake and alleviate lipid peroxidation in FXN-138.2 cells. (A) FRET analysis of mitochondrial calcium levels measured through 4mtD3cpv chameleon. 250 µM Carbachol was used to stimulate Ca2+ release from the ER through IP3R channels. Cells were then treated either with 1 mM Trolox or 5 mM NAC for 24 h. Results (n = 3) are represented as normalized levels of fluorescence intensity over carbachol loading value. A range of 5–34 cells were analyzed in each condition per replicate. (B) Area Under curve (AUC) analysis of the mitochondrial response to 250 µM carbachol. (C) Flow cytometry detection of lipid peroxidation. SH-SY5Y cells were treated with 500 µM H2O2 as positive control, and FXN-138.2 were treated either with 1 mM Trolox or 5 mM NAC for 24 h. Cells were stained with 5 µM Bodipy C11 (581/591) for 30 min. Results (n = 4) are represented as 481/591 ratio of the fluorescence intensity of the dye (mean ± SEM). ANOVA significance: *p = 0.05; **p = 0.01; ****p = 0.0001.