A disruption in iron-sulfur center biogenesis via inhibition of mitochondrial dithiol glutaredoxin 2 may contribute to mitochondrial and cellular iron dysregulation in mammalian glutathione-depleted dopaminergic cells: implications for Parkinson's disease

Abstract

Parkinson's disease (PD) is characterized by early glutathione depletion in the substantia nigra (SN). Among its various functions in the cell, glutathione acts as a substrate for the mitochondrial enzyme glutaredoxin 2 (Grx2). Grx2 is involved in glutathionylation of protein cysteine sulfhydryl residues in the mitochondria. Although monothiol glutathione-dependent oxidoreductases (Grxs) have previously been demonstrated to be involved in iron-sulfur (Fe-S) center biogenesis, including that in yeast, here we report data suggesting the involvement of mitochondrial Grx2, a dithiol Grx, in iron-sulfur biogenesis in a mammalian dopaminergic cell line. Given that mitochondrial dysfunction and increased cellular iron levels are two important hallmarks of PD, this suggests a novel potential mechanism by which glutathione depletion may affect these processes in dopaminergic neurons. We report that depletion of glutathione as substrate results in a dose-dependent Grx2 inhibition and decreased iron incorporation into a mitochondrial complex I (CI) and aconitase (m-aconitase). Mitochondrial Grx2 inhibition through siRNA results in a corresponding decrease in CI and m-aconitase activities. It also results in significant increases in iron-regulatory protein (IRP) binding, likely as a consequence of conversion of Fe-S-containing cellular aconitase to its non-Fe-S-containing IRP1 form. This is accompanied by increased transferrin receptor, decreased ferritin, and subsequent increases in mitochondrial iron levels. This suggests that glutathione depletion may affect important pathologic cellular events associated with PD through its effects on Grx2 activity and mitochondrial Fe-S biogenesis.

FIG. 1.

Effect of total glutathione depletion on mitochondrial Grx2 activity in mammalian midbrain-derived dopaminergic N27 cells. (A) Measurement of Grx2 activity in mitochondrial fractions isolated from N27 cells treated with increasing concentrations of BSO for 24 h (0–40 μM). Activities are reported as percentage of control. *p < 0.05, BSO treated compared with control. Experiments were repeated three times with n = 3. (B) Representative Western blot analyses verifying purity of mitochondrial fractions isolated from experimental versus control cell populations and used for assay of Grx2 enzyme activity and all subsequent experiments.

FIG. 2.

siRNA-mediated knockdown of Grx2 in dopaminergic N27 cells. N27 cells transfected with three different primer sets (97, 206, 396) via Lipofectamine for 24 h. Control cells were transfected with scrambled siRNA (C) and mock transfected (-ve). (A) Representative RT-PCR analysis of Grx2 mRNA; actin RT-PCR was included as a control. (B) Representative dual Grx2 immunocytochemistry (Grx2) and mitotracker red staining (Mito marker) in cells transfected with 396 primer set (SiRNA) or scrambled (Control). (C) Grx2 activity after transfection with Grx2 siRNA primer sets 97, 206, and 396 versus scrambled control set in N27 cells. Activities are reported as percentage of control. (D) Representative RT-PCR analysis of Grx1 and Grx2 mRNA; GAPDH RT-PCR was included as a control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 3.

Decreased Grx2 activity in N27 cells after siRNA transfection and reduced iron incorporation into mitochondria Fe-S–containing enzymes after GSH depletion. (A) Grx2 activity in N27 cells transfected with 396 primers for 24 h (CONT, mock transfected; siRNA, Grx2 396 siRNA-treated). Activities are reported as percentage of control. *p < 0.0001, siRNA treated compared with control (n = 3). (B) N27 cells treated with 10 μM BSO for 72 h, and mitochondria were harvested from these cells for immunoprecipitation of CI, m-aconitase, and c-aconitase for measurement of iron incorporation. ICP-MS analysis of ⁵⁷Fe levels in immunoprecipitated proteins; reported as percentage of control. *p < 0.05, BSO treated compared with control (n = 3). All experiments were repeated at least 3 times. (C) Representative examples of Sypro-Ruby gels verifying efficiency of IP of m-ACON (upper panel) and CI (lower panel) from control (CONT) and Grx2 siRNA-transfected (siRNA) cells. Molecular weight markers (M) are shown in lane 1.

FIG. 4.

Effect of Grx2 siRNA mitochondrial Fe-S center-containing enzyme activities. N27 cells were treated with siRNA TS-396 for 24 h before exchange with normal RPMI media for another 48 h before measurement of corresponding enzyme activities. (A) Mitochondrial aconitase (m-ACON) and (B) complex I. *p < 0.0001 (n = 7) and *p < 0.05 (n = 4), respectively, compared with control (CONT, mock-transfected; siRNA, 396 transfected, reported as percentage of control). (C) Representative IRP binding in mock transfected (C) or 396-transfected (siRNA); densitometric quantification was performed by using β-mercaptoethanol (+bME) as a normalizing control. Experiments were repeated 3 times with n = 3.

FIG. 5.

Increased TFR1 and ferritin protein levels after Grx2 knockdown. (A) Representative Western blot analysis of TfR1 (TfR) and ferritin (FerrH) proteins from mock-transfected (CONT) controls versus 396-transfected (siRNA) cells. Actin is shown as loading control. (B) Band density (integrated density value) is expressed graphically as a percentage ratio of optical density of the TFR1 and ferritin proteins versus actin from three different Western blots. *p < 0.05, siRNA treated compared with controls.

FIG. 6.

Effect of Grx2 knockdown on labile iron pool content in N27 cells. (A) Mitochondrial (white bars) and cellular LIP levels were measured with Calcein dequenching after 5-min SIH treatment. *p < 0.008 siRNA treated compared with control. **p < 0.05 siRNA treated compared with control. Experiments were repeated three times with n = 5. (B) Mitochondrial (white bars) and cellular total iron content (black bars) as measured by ferrozine assay after mock-transfection (CONT), BSO, or 396 siRNA treatments. Experiments were repeated three times with n = 3.