Loss of Frataxin activates the iron/sphingolipid/PDK1/Mef2 pathway in mammals

Abstract

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by mutations in Frataxin (FXN). Loss of FXN causes impaired mitochondrial function and iron homeostasis. An elevated production of reactive oxygen species (ROS) was previously proposed to contribute to the pathogenesis of FRDA. We recently showed that loss of frataxin homolog (fh), a Drosophila homolog of FXN, causes a ROS independent neurodegeneration in flies (Chen et al., 2016). In fh mutants, iron accumulation in the nervous system enhances the synthesis of sphingolipids, which in turn activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2) to trigger neurodegeneration of adult photoreceptors. Here, we show that loss of Fxn in the nervous system in mice also activates an iron/sphingolipid/PDK1/Mef2 pathway, indicating that the mechanism is evolutionarily conserved. Furthermore, sphingolipid levels and PDK1 activity are also increased in hearts of FRDA patients, suggesting that a similar pathway is affected in FRDA.

Keywords: Frataxin; Friedreich's ataxia; Mef2; PDK1; human; iron; mouse; neuroscience; sphingolipid.

Figure 1.. Phenotypic characterization of Fxn-sgRNA mice.

(A) Body size of LacZ- and Fxn-sgRNA mice at P130. Fxn-sgRNA mice exhibit a hunchback phenotype. (B) Hindlimb reflexes of LacZ- and Fxn-sgRNA mice at P130. (C) Rotarod test of LacZ- and Fxn-sgRNA mice at P50 (n = 5) and P130 (n = 6). (D) Wire hang test of LacZ- and Fxn-sgRNA mice at P130. n = 6. Data are presented as mean ± SEM. ***, p<0.001, Student’s t-test. DOI: http://dx.doi.org/10.7554/eLife.20732.002

Figure 1—figure supplement 1.. Removal of Fxn using AAV and CRISPR/Cas9.

(A) T7 endonuclease assay to detect Cas9 induced mutations. Red arrows indicate the mismatched genomic Fxn DNA fragments which are cleaved by T7 endonuclease. (B) Immunoblot of FXN protein levels of Neuro-2a cells with LacZ- or Fxn-sgRNA expression. (C) AAV infection efficiency. AAV carrying Fxn-sgRNA with a GFP marker was delivered into the P1 mouse brain. GFP fluorescence indicates cells with AAV infection (green). Nuclei are labeled with DAPI (blue). Scale bar, 50 μm. (D) mRNA levels of Fxn in LacZ- and Fxn-sgRNA mice. Data are presented as mean ± SEM. ***, p<0.001, Student’s t-test. DOI: http://dx.doi.org/10.7554/eLife.20732.003

Figure 2.. Loss of Fxn leads to neuronal insults.

(A) Immunostaining of cerebral cortex layer I (top) to VI (bottom) of LacZ- and Fxn-sgRNA mice at P130. ATF3 is labeled in red, and nuclei are marked by Hoechst in blue. n = 3. Quantification is on the right. Scale bar, 50 μm. (B) Immunostaining of cortical neurons of LacZ- and Fxn-sgRNA mice at P130. Axon initial segments are marked by anti-AnkG antibody (green), and ATF3 is marked in red. n = 3. Quantification is on the right. Scale bar, 10 μm. Data are presented as mean ± SEM. **, p<0.01, ***, p<0.001, Student’s t-test. DOI: http://dx.doi.org/10.7554/eLife.20732.006

Figure 3.. Loss of Fxn leads to an activation of the iron/sphingolipid/PDK1/Mef2 pathway.

(A) Perls’ blue staining with DAB enhancement of the cerebral cortex layer I (top) to VI (bottom) of LacZ- and Fxn-sgRNA mice at P130. n = 3. Scale bar, 100 and 10 μm. (B) RPA and RPAC staining of mouse cortical neurons of LacZ- and Fxn-sgRNA mice at P60. RPA and RPAC fluorescence is in red, and nuclei are labeled by DAPI in green. n = 3. Scale bar: 20 µm. (C) Immunoblot of PDK1 phosphorylation levels of brains of LacZ- and Fxn-sgRNA mice at P60. (D) mRNA levels of Mef2 downstream targets of LacZ- and Fxn-sgRNA mice at P60. n = 3. Data are presented as mean ± SEM. *, p<0.05. **, p<0.01, Student’s t-test. DOI: http://dx.doi.org/10.7554/eLife.20732.007

Figure 3—figure supplement 1.. Fe³⁺ distribution in brains of LacZ- and Fxn-sgRNA mice.

Perls’/DAB staining with neuronal and glial markers of the cerebral cortex of LacZ- and Fxn-sgRNA mice at P130. Neurons are marked by NeuroTrace (red), astrocytes are marked by anti-GFAP antibody (green), and microglia are marked by anti-Iba1 antibody (cyan). DAB signals are marked by circles in other images. Scale bar: 12 µm. DOI: http://dx.doi.org/10.7554/eLife.20732.008

Figure 3—figure supplement 2.. Fe³⁺ staining and immunoblot of LacZ- and Fxn-sgRNA mice.

(A) Perls’ blue staining without DAB enhancement of cerebral cortex layer I (top) to VI (bottom) of LacZ- and Fxn-sgRNA mice at P130. Scale bar: 100 µm. (B) Immunoblot of 4-HNE levels in brain tissues of LacZ- and Fxn-sgRNA mice at P130. (C) Immunoblot of PDK1 levels in brain tissues of LacZ- and Fxn-sgRNA mice at P60. DOI: http://dx.doi.org/10.7554/eLife.20732.009

Figure 4.. Sphingolipid levels and PDK1 activity are increased in heart tissues of FRDA patients.

(A) Sphingolipid profiling of heart tissues from controls (n = 5) and FRDA patients (n = 6). Data are presented as mean ± SEM. (B) Immunoblot of PDK1 phosphorylation levels of heart tissues from controls (n = 5) and FRDA patients (n = 6). Box plot of PDK1 phosphorylation is on the right. DOI: http://dx.doi.org/10.7554/eLife.20732.010

Author response image 1.. Sphingolipid levels of LacZ- and Fxn-sgRNA mice.

DOI: http://dx.doi.org/10.7554/eLife.20732.012