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

  1. Kuchuan Chen
  2. Tammy Szu-Yu Ho
  3. Guang Lin
  4. Kai Li Tan
  5. Matthew N Rasband
  6. Hugo J Bellen  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. Howard Hughes Medical Institute, Baylor College of Medicine, United States
  3. Jan and Dan Duncan Neurological Research Institute, United States
Research Advance
Cite this article as: eLife 2016;5:e20732 doi: 10.7554/eLife.20732
5 figures, 2 videos and 1 table

Figures

Figure 1 with 1 supplement
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.

https://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.

https://doi.org/10.7554/eLife.20732.003
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.

https://doi.org/10.7554/eLife.20732.006
Figure 3 with 2 supplements
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.

https://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.

https://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.

https://doi.org/10.7554/eLife.20732.009
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.

https://doi.org/10.7554/eLife.20732.010
Author response image 1
Sphingolipid levels of LacZ- and Fxn-sgRNA mice.
https://doi.org/10.7554/eLife.20732.012

Videos

Video 1
LacZ- and Fxn-sgRNA mice at P130.
https://doi.org/10.7554/eLife.20732.004
Video 2
Fxn-sgRNA mice at P130.
https://doi.org/10.7554/eLife.20732.005

Tables

Table 1

Clinical and genetic data of 6 FRDA patients.

https://doi.org/10.7554/eLife.20732.011

No.

Gender

Age of onset

Age of death

GAA1

GAA2

1

M

8

27

700

1070

2

M

4

37

674

674

3

F

5

25

800

1100

4

F

9

26

690

850

5

M

7

35

750

1100

6

F

18

67

621

766

  1. The sizes for two GAA trinucleotide repeat expansions in the first intron of FXN in FRDA patients are shown with the smaller expansion designated GAA1 and the larger expansion designated GAA2.

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