Frataxin deficiency induces Schwann cell inflammation and death

Abstract

Mutations in the frataxin gene cause dorsal root ganglion demyelination and neurodegeneration, which leads to Friedreich's ataxia. However the consequences of frataxin depletion have not been measured in dorsal root ganglia or Schwann cells. We knocked down frataxin in several neural cell lines, including two dorsal root ganglia neural lines, 2 neuronal lines, a human oligodendroglial line (HOG) and multiple Schwann cell lines and measured cell death and proliferation. Only Schwann cells demonstrated a significant decrease in viability. In addition to the death of Schwann cells, frataxin decreased proliferation in Schwann, oligodendroglia, and slightly in one neural cell line. Thus the most severe effects of frataxin deficiency were on Schwann cells, which enwrap dorsal root ganglia neurons. Microarray of frataxin-deficient Schwann cells demonstrated strong activations of inflammatory and cell death genes including interleukin-6 and Tumor Necrosis Factor which were confirmed at the mRNA and protein levels. Frataxin knockdown in Schwann cells also specifically induced inflammatory arachidonate metabolites. Anti-inflammatory and anti-apoptotic drugs significantly rescued frataxin-dependent Schwann cell toxicity. Thus, frataxin deficiency triggers inflammatory changes and death of Schwann cells that is inhibitable by inflammatory and anti-apoptotic drugs.

Fig.1

The effects of frataxin depletion by siRNA on proliferation and viability at day 3. A, Frataxin depletion by siRNA in human Schwann cell line T265 (Sch), human oligodendroglioma cell line (HOG) and Human neuronal cell line (NT2), rat neuronal cell line (PC12), rat/mouse DRG cell line (ND7/23) and rat DRG cell line (50B11). Frataxin mRNA levels were measured by quantitative PCR and represented as percentage of the basal expression in each cell line transfected with the scrambled siRNA (scr). B, frataxin depletion leads to significantly decreased viability uniquely in Schwann cells. C, Frataxin depletion causes about 5-fold increase in floating cells in frataxin-depleted Schwann cells. D, frataxin depletion causes proliferation inhibition in Schwann cells, HOG cells and NT2 cells, but not in PC12 and two DRG neuron lines. Error Bars represent averages ± standard deviations (n=3-8, P<0.05*, P<0.01**, P<0.001***).

Fig.1

The effects of frataxin depletion by siRNA on proliferation and viability at day 3. A, Frataxin depletion by siRNA in human Schwann cell line T265 (Sch), human oligodendroglioma cell line (HOG) and Human neuronal cell line (NT2), rat neuronal cell line (PC12), rat/mouse DRG cell line (ND7/23) and rat DRG cell line (50B11). Frataxin mRNA levels were measured by quantitative PCR and represented as percentage of the basal expression in each cell line transfected with the scrambled siRNA (scr). B, frataxin depletion leads to significantly decreased viability uniquely in Schwann cells. C, Frataxin depletion causes about 5-fold increase in floating cells in frataxin-depleted Schwann cells. D, frataxin depletion causes proliferation inhibition in Schwann cells, HOG cells and NT2 cells, but not in PC12 and two DRG neuron lines. Error Bars represent averages ± standard deviations (n=3-8, P<0.05*, P<0.01**, P<0.001***).

Fig.2

Inhibition of cell proliferation and IL6 cytokine induction precede death in frataxin-deficient Schwann cells. The floating cells and the adherent cells in the same well were collected separately, and counted by trypan blue exclusion assay using the Beckman Coulter Vi-Cell cell counter. Total viable cell number was divided by total cell number from both parts to obtain the viability. Frataxin depletion results in a significant decrease in proliferation 1 day after siRNA transfection and becomes more severe at day 2 and day 3 (A). Frataxin depletion only causes significantly increased floating cells (B) and significantly decreased viability (C) at day 2, and both becomes more severe at day 3. IL6 levels are presented as fold change over the basal levels in scrambled controls (D). E, frataxin protein levels at day1, 2 and 3 upon siRNA transfection, shown here are representative western blotting data out of three independent experiments and bar graph from densitometry analysis of 3 independent experiments (frataxin protein levels were normalized to beta-actin levels). Bars represent averages ± standard deviations (n=3, P<0.05*, P<0.01**, P<0.001***).

Fig.2

Inhibition of cell proliferation and IL6 cytokine induction precede death in frataxin-deficient Schwann cells. The floating cells and the adherent cells in the same well were collected separately, and counted by trypan blue exclusion assay using the Beckman Coulter Vi-Cell cell counter. Total viable cell number was divided by total cell number from both parts to obtain the viability. Frataxin depletion results in a significant decrease in proliferation 1 day after siRNA transfection and becomes more severe at day 2 and day 3 (A). Frataxin depletion only causes significantly increased floating cells (B) and significantly decreased viability (C) at day 2, and both becomes more severe at day 3. IL6 levels are presented as fold change over the basal levels in scrambled controls (D). E, frataxin protein levels at day1, 2 and 3 upon siRNA transfection, shown here are representative western blotting data out of three independent experiments and bar graph from densitometry analysis of 3 independent experiments (frataxin protein levels were normalized to beta-actin levels). Bars represent averages ± standard deviations (n=3, P<0.05*, P<0.01**, P<0.001***).

Fig. 3

Frataxin deficiency leads to cell proliferation inhibition and cell cycle profile alteration in both Schwann and HOG cells. Quantification of cell-cycle distribution at day 3 and statistical analysis is presented in the bar graph. Bars represent averages ± standard deviations of three independent assay samples (n=3 P<0.05*, P<0.01**, P<0.001***).

Fig.4

Frataxin depletion causes a morphological change in T265 Schwann cells (Magnification, 20X). A, image of the scramble control of Schwann cells at day 3. B, image of frataxin-depleted Schwann cells at day 3.

Fig.5

Inflammatory and death transcripts are induced by frataxin deficiency in T265 Schwann cells by quantitative RT-PCR. Error Bars represent averages ± standard deviations (n=3 P<0.05*, P<0.01**, P<0.001***).

Fig.6

Cytokine protein levels are induced by frataxin deficiency in Schwann cells by multiplex ELISA assay. Cytokine levels are presented as fold change over the basal levels in scrambled controls. The average basal level (pg/ml/million cells) of each cytokine: IL6= 349.55, IL1a= 1.16, GCSF= 4.73, IL1b= 0.74, IL8= 5784, TNFa= 24.64, GMCSF= 92.27. Error Bars represent averages ± standard deviations (n=4, P<0.05*, P<0.01**, P<0.001***).

Fig.7

Inflammatory arachidonate metabolites increase in frataxin-deficient Schwann Cells. (A), A schematic of arachidonate pathway, (B and C),15 Inflammatory Lipoxygenase metabolites are increased significantly (with P value < 0.02) in frataxin-deficient Schwann Cells (if F-test was significant (p < 0.05, TTEST for two-sample unequal variance was applied, otherwise, TTEST for two-sample equal variance was applied ).

Fig.7

Inflammatory arachidonate metabolites increase in frataxin-deficient Schwann Cells. (A), A schematic of arachidonate pathway, (B and C),15 Inflammatory Lipoxygenase metabolites are increased significantly (with P value < 0.02) in frataxin-deficient Schwann Cells (if F-test was significant (p < 0.05, TTEST for two-sample unequal variance was applied, otherwise, TTEST for two-sample equal variance was applied ).

Fig.8

Dexamethasone and SB203580 (a specific inhibitor of p38 MAP kinase) partially rescue cell death in frataxin-depleted Schwann cells. Dexamethasone (250nM) and SB203580 (20μM) treatment significantly decrease the floating cells in frataxin-depletion Schwann cells (A), Dexamethasone and SB203580 treatment significantly increase the viability in frataxin-depletion Schwann cells (B) Error Bars represent averages ± standard deviations (n=3 for dexamethasone treatment, n=4 for SB203580 treatment, P<0.05*, P<0.01**, P<0.001***).

Fig.9

The effect of Q-VD-OPH, Dexamethasone and SB203580 treatments on the cytokine protein expressions. Q-VD-OPH (5μM) does not decrease TNF and IL6 protein levels at all, Dexamethasone significantly decrease IL6 and IL1A protein levels and decrease TNF protein levels non-significantly, SB203580 decreases IL1A protein levels significantly and decreases IL6 protein levels non-significantly. Error Bars represent averages ± standard deviations (n=3-8, P<0.05*, P<0.01**, P<0.001***).

Fig.10

Frataxin depletion results in an insignificant increase in mean phosphorylation of p38 MAP kinase at day 3. Western results of MAP kinase (P38, JNK and P42/44) in scrambled controls (SCR) and frataxin-depleted Schwann cells (siFxn) at 3 days post-knockdown (A). Bar graph represents the densitometry results of the western blots (B). Error Bars represent averages ± standard deviations (n=4). The difference in phosphorylation of p38 MAP kinase between scrambled controls (SCR) and frataxin-depleted Schwann cells (siFxn) is not significant.