Mutant AR activates the mitochondrial caspase pathway. (A) MN-1 cells expressing the normal and mutant AR were exposed to vehicle alone or androgen ligand (R1881), with and without caspase 9 inhibitor (Ac-LEHD) or CsA. The AR-65Q cells had an increase in caspase 9 activity in the presence of R1881. CsA and the caspase 9 inhibitor block this increase. (B) Caspase 3 activity was measured with a fluorometric assay in the AR-24Q and AR-65Q MN-1 cells. These cells were treated with vehicle alone, caspase 9 inhibitor (z-LEHD-FMK) or pan-caspase inhibitor (z-VAD). AR-65Q cells showed a ligand-independent increase in caspase 3 activity. This increase is partially blocked by z-LEHD-FMK. (C) We measured caspase 3 activity with another fluorometric assay in MN-1 cells expressing AR-24Q and AR-65Q cells. The cells were treated with either vehicle or CsA. There was an increase in caspase 3 activity in cells expressing the mutant AR. This increase is ligand-independent and partially blocked with CsA. The caspase activities are indicated as percent activity relative to untreated AR-24Q (assigned as 100%) and normalized to total protein content. (D) Immunoblotting of MN-1 total cellular lysates shows increased Bax relative to tubulin in AR-65Q cells. The first lane shows vehicle-treated parental MN-1 cells. (E) Immunoblotting of MN-1 cells for cytochrome C in mitochondrial and cytosolic fractions show no change in cytochrome C release in AR-65Q cells relative to parental MN-1 or AR-24Q cells. The last lane shows HeLa cells treated with staurosporine as a positive control. (F) Immunoblotting of MN-1 total cell lysates for phosphorylated Bad and total Bad. Quantitation showed no change in the ratio of phosphorylated and total Bad in AR-65Q cells relative to AR-24Q cells. The graphs (A–C) show data representative of three experiments each done in triplicate with the error bars indicating standard error of mean (SEM); *P ≤ 0.05.

Mutant AR is associated with decreased MtMP. (A) AR-24Q and AR-65Q MN-1 cells and (B) differentiated AR-10Q and AR-112Q PC12 cells induced to express AR were exposed to vehicle alone, and ligand (R1881 or DHT) or CsA or both. Using flow cytometry, MtMP was measured with a fluorescent indicator of MtMP and expressed relative to the vehicle-treated cells. Depolarization was significantly greater in both AR-65Q MN-1 (A) and AR-112Q PC12 cells (B) compared to the respective normal AR expressing cells. The depolarization is significantly increased by the addition of ligand to the mutant cells. In addition, CsA blocks this depolarization. The histograms represent five (A) and two (B) independent experiments each done in triplicate, with the error bars showing the SEM; *P ≤ 0.05.

Mutant AR alters expression of genes important for mitochondrial function. Total RNA from MN-1 cells (A), and hind limb muscle (B and C), spinal cord (D and E) and testis (F and G) of mice expressing AR-21Q, and AR-113Q was analyzed by quantitative RT–PCR as described in Materials and Methods. All transcript levels were normalized to the loading control, S18. (A) Gene expression of R1881-exposed MN-1 cells is shown relative to the respective vehicle treated controls (assigned as 1). (B–G) Transcript levels of pre-manifesting (B, D, F) and manifesting (C, E, G) AR-113Q mouse muscle (B, C), spinal cord (D, E) and testis (F, G) are shown relative to transcript levels in AR-21Q mice (assigned as 1). All experiments were done in triplicate, with the error bars indicating SEM; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001.

Cell death in AR-65Q MN-1 cells and AR-112Q PC12 cells is attenuated by treatment with mitochondrial modulators. (A) AR-24Q and AR-65Q MN-1 cells were analyzed for cell toxicity by propidium iodide staining and FACS. Relative to the AR-24Q cells (assigned as 100%), the vehicle-treated AR-65Q cells had increased toxicity. This increase was attenuated by treatment with CsA, CoQ10, idebenone and z-VAD. The histogram represents two experiments each done in triplicate, with error bars indicating the SEM; *P ≤ 0.05. (B) Differentiated PC12 cells induced to express AR-10Q and AR-112Q were exposed to vehicle control, R1881, the pan-caspase inhibitor, z-VAD, CoQ10, idebenone or CsA were analyzed for cell toxicity by propidium iodide staining and FACS. Vehicle-treated AR-112Q cells did not show toxicity relative to the vehicle-treated AR-10Q cells (assigned as 100%). PC12 cells expressing AR-112Q cells exposed to R1881 had increased cell toxicity. This increase was attenuated by treatment with CsA, CoQ10, idebenone and z-VAD. The histogram represents three experiments each done in triplicate, with error bars indicating the SEM; *P ≤ 0.001.

Reduced mitochondrial mass and number in cells expressing mutant AR. (A) AR-24Q and AR-65Q MN-1 cells and (B) differentiated AR-10Q and AR-112Q PC12 cells left uninduced or induced to express AR were treated with vehicle or the mitochondrial toxin, antimycin A for 48 h. The cells were then stained with a fluorescent marker and mitochondrial mass was analyzed by flow cytometry. AR-65Q (A) and AR-112Q cells (B) have ligand-independent decrease in mitochondrial mass. The data in both panels were normalized to the respective vehicle treated normal cells (set as 100%). Shown are the mean ± standard error of three experiments; *P ≤ 0.05. (C and D) Representative high magnification (10 000×) electron micrographs of AR-24Q (C) and AR-65Q MN-1 (D) cells exposed to vehicle for 24 h. The micrographs show more mitochondria (arrows in B) in AR-24Q compared to AR-65Q cells. The AR-65Q cells have vesiculated cristae (arrowheads in D). (E) The total number of mitochondria was counted in 10–12 cells per sample. The error bars indicate SEM; *P ≤ 0.05.

Mutant AR increases ROS (A) AR-24Q and AR-65Q MN-1 cells and (B) differentiated AR-10Q and AR-112Q PC12 cells induced to express AR were exposed to vehicle alone, ligand (R1881 or DHT) and CoQ10 or idebenone. ROS levels were measured with a fluorometric assay and are indicated relative to vehicle-treated AR-24Q cells (A) or vehicle-treated cells induced to express AR-10Q (B) (assigned as 100%). In both mutant MN-1 (A) and PC12 (B) cells, ROS levels increased with ligand and co-enzyme Q10 and idebenone blocked this increase. The graph shows representative results of one of the three independent experiments in (A), and the average of three experiments in (B), which were each done in triplicate. The error bars indicate the SEM; *P ≤ 0.05.

Altered subcellular distribution of androgen receptor. Cytosolic (A and D), mitochondrial (B and E) and nuclear (C) fractions were probed for AR in vehicle-treated and R1881-exposed parental, AR-24Q and AR-65Q MN-1 cells (A–C) and in differentiated vehicle-treated PC12 cells (D and E). To confirm the purity of the fractions, we used antibodies for tubulin (cytosol), porin (mitochondria) and HDAC1 (nucleus). HDAC1 and tubulin were not detected in the mitochondrial fractions, and porin was not detected in the cytosolic and nuclear fractions (latter not shown). Heart mitochondrial lysate and knock-in mouse testis tissue lysates were used as negative and positive controls for AR. The parental samples, which do not express AR, were also a negative control. Sample assignments in the gels (A–C) are: lane 1, parental vehicle-treated MN-1; lane 2, vehicle-treated AR-24Q; lane 3, AR-24Q+R1881; Lane 4, vehicle-treated AR-65Q; lane 5, AR-65Q+R1881; lane 6, heart mitochondrial lysate; lane 7, knock-in mouse testis tissue lysate. (D and E) Lane 1, vehicle-treated Dox-induced parental cells; lane 2, vehicle-treated uninduced AR-10Q; lane 3, vehicle-treated Dox-induced AR-10Q; lane 4, Dox-induced AR-10Q+R1881; lane 5, Dox-induced AR-10Q+DHT; lane 6, vehicle-treated uninduced AR-112Q; lane 7, vehicle-treated Dox-induced AR-112Q; lane 8, Dox-induced AR-112Q+R1881; lane 9, Dox-induced AR-112Q+DHT; lane 10, human heart mitochondrial lysate. (F–H) Electron dense immunogold particles indicate AR in AR-24Q (F) and AR-65Q (G) MN-1 cells exposed to vehicle alone. 10 000× electron micrographs (F and G) show mitochondria with (arrows) or without (arrowheads) associated AR in AR-24Q (F) and AR-65Q cells (G). (H) The percentage of mitochondria with associated AR is higher in AR-65Q cells. The histogram represents averages from 10 to 12 cells, with the error bars indicating SEM; *P ≤ 0.05.

Mechanism of mitochondrial dysfunction in SBMA. Mitochondrial mass, number and membrane potential are likely affected by mutant AR indirectly through ligand-dependent alterations in mitochondrial gene expression and directly through association of AR with mitochondria. The decrease in MtMP is blocked by CsA. Downstream from the mitochondrial dysfunction, mutant AR increases ROS levels, which can reversibly result in further deleterious effects on the mitochondrial membrane. The antioxidants CoQ10 and idebenone block the increase in ROS levels. Mitochondrial dysfunction increases caspase 9 activity, which in turn increases caspase 3 activity. Elevated ROS levels and caspase 3 activation result in cell death. Activation of the caspases is mitigated by CsA, and cell death is attenuated by CsA, CoQ10, idebenone and the caspase inhibitor, z-VAD. Thus, regardless of the cause of mitochondrial dysfunction, downstream events may be targets for therapeutic intervention, for example with CsA and antioxidants.