Overexpression of human and fly frataxins in Drosophila provokes deleterious effects at biochemical, physiological and developmental levels

Abstract

Background: Friedreich's ataxia (FA), the most frequent form of inherited ataxias in the Caucasian population, is caused by a reduced expression of frataxin, a highly conserved protein. Model organisms have contributed greatly in the efforts to decipher the function of frataxin; however, the precise function of this protein remains elusive. Overexpression studies are a useful approach to investigate the mechanistic actions of frataxin; however, the existing literature reports contradictory results. To further investigate the effect of frataxin overexpression, we analyzed the consequences of overexpressing human (FXN) and fly (FH) frataxins in Drosophila.

Methodology/principal findings: We obtained transgenic flies that overexpressed human or fly frataxins in a general pattern and in different tissues using the UAS-GAL4 system. For both frataxins, we observed deleterious effects at the biochemical, histological and behavioral levels. Oxidative stress is a relevant factor in the frataxin overexpression phenotypes. Systemic frataxin overexpression reduces Drosophila viability and impairs the normal embryonic development of muscle and the peripheral nervous system. A reduction in the level of aconitase activity and a decrease in the level of NDUF3 were also observed in the transgenic flies that overexpressed frataxin. Frataxin overexpression in the nervous system reduces life span, impairs locomotor ability and causes brain degeneration. Frataxin aggregation and a misfolding of this protein have been shown not to be the mechanism that is responsible for the phenotypes that have been observed. Nevertheless, the expression of human frataxin rescues the aconitase activity in the fh knockdown mutant.

Conclusion/significance: Our results provide in vivo evidence of a functional equivalence for human and fly frataxins and indicate that the control of frataxin expression is important for treatments that aim to increase frataxin levels.

Figure 1. Effect of FXN overexpression in the embryonic development.

(A) Detection of the FXN protein in da-GAL4>UAS-FXN (+,+) larvae in the mitochondrial (mito) and the cytosolic (cyto) fractions. The control genotypes of the larvae were da-GAL4>yw (+,−) and yw>UAS-FXN (−,+). The human frataxin protein was localized in the mitochondria. Anti-actin was used as a control for cytosolic contamination. (B–G) Muscular and nervous system defects in da-GAL4>UAS-FXN and actin-GAL4>UAS-FXN embryos at stage 16. In these panels, anterior is toward the left, and all of the views are lateral views. Anti-myosin staining revealed abnormalities in the junctions of lateral transversal muscles 1, 2 and 3 and the ventral longitudinal muscle 1 (C) compared with the control (B). Moreover, a few embryos exhibited abnormalities in the muscular development of mutant (E) versus control (D) embryos. Staining with 22C10 detected strong abnormalities in the axonal path finding of the sensory nerves (G) with respect to the control (F).

Figure 2. Physiological and behavioral defects induced by frataxin overexpression in nervous system.

(A–C) Life span under normoxia conditions. Overexpression of human (black square) or Drosophila (black circle) frataxin in a pan-neural fashion (A), in the sensory organs and their precursors (B) or the glial cells (C) dramatically shortens the mean and maximum life span compared to control flies (white square). (D–F) Negative geotaxis experiment with 5- and 10-day-old individuals. Overexpression of frataxin in all 3 nervous system cell types strongly reduced the walking ability of the flies. The strongest effect was observed when the PNS driver (neur-GAL4) was applied. The statistical differences between the survival curves in A, B and C were analyzed using the Kapplan-Meier test, and both of the frataxins exhibited a statistically significant reduction (p<0.001) compared to that of the control individuals. The level of significance in D, E and F was determined using a one-way ANOVA with the post hoc Newman-Keuls test (* p<0.05). The error bars represent the standard error.

Figure 3. Strong degeneration and lipid droplet accumulation in glial cells overexpressing frataxin.

(A–C) 25-day-old Repo-GAL4 / + controls; (D–F) 25-day-old Repo-GAL4 / UAS-FXN. (D) Overexpression of human frataxin induced a strong degeneration in the cortex (white arrow and 3X magnification box). (E, F) The electron microscopy analysis revealed an accumulation of lipid droplets (denoted by asterisks; E) in the glial cells of the frataxin-overexpressing brains and revealed mitochondria with altered morphologies (arrows; F) and internal vacuolization (arrow heads; F). The scale bar represents 50 µm (A, D) and 2.5 µm (B, C, E, F).

Figure 4. Molecular effects of frataxin overexpression and the involvement of oxidative stress.

(A) The negative effects of human-frataxin overexpression on aconitase activity under normoxia and hyperoxia (99.5% O₂) conditions. (B) Human frataxin overexpression triggered a reduction in the synthesis of the complex I subunit (amount normalized to the internal control α-tubulin). (C–E) Increased susceptibility to hyperoxia-mediated oxidative damage in flies overexpressing human and fly frataxin in the nervous system. (F,G) Constitutive expression of mitochondrial catalase (mitoCat) led to an extension of the mean and maximum life span of the flies with increased frataxin expression. This effect was strong in the peripheral nervous system (neur-GAL4) and moderate in the glial cells (repo-GAL4). (H) Co-expression of mitochondrial catalase (mitoCat) rescues (5d) and alleviates (10d) the locomotor deficits in the flies with an increased level of frataxin expression in the glial cells. The survival curves were analyzed using the Kapplan-Meier test. The level of significance in A, B and H was determined using a one-way ANOVA with a post hoc Newman-Keuls test (*p<0.05). The error bars represent the standard error.

Figure 5. FXN does not form aggregates in Drosophila, and its expression is not diluted when it is coexpressed with the interference of fh.

(A) Mitochondrial cell extracts were obtained from actin-GAL4>UAS-FXN larvae and were size fractionated. The fractions were subsequently analyzed using SDS-PAGE and western blotting with an anti-human frataxin antibody. The positions of ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (44 kDa) and the estimated position for the frataxin monomer and dimer are indicated as arrowheads and arrows, respectively. (B) Detection of FXN protein in actin-GAL4>UAS-FXN and actin-GAL4>UDIR2; UAS-FXN larvae. The FXN protein is not diluted when it is co-expressed with an RNA interference construct of fh. α-tubulin was used as a loading control.