Enhanced aggregation of androgen receptor in induced pluripotent stem cell-derived neurons from spinal and bulbar muscular atrophy

Abstract

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuron disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. Ligand-dependent nuclear accumulation of mutant AR protein is a critical characteristic of the pathogenesis of SBMA. SBMA has been modeled in AR-overexpressing animals, but precisely how the polyglutamine (polyQ) expansion leads to neurodegeneration is unclear. Induced pluripotent stem cells (iPSCs) are a new technology that can be used to model human diseases, study pathogenic mechanisms, and develop novel drugs. We established SBMA patient-derived iPSCs, investigated their cellular biochemical characteristics, and found that SBMA-iPSCs can differentiate into motor neurons. The CAG repeat numbers in the AR gene of SBMA-iPSCs and also in the atrophin-1 gene of iPSCs derived from another polyQ disease, dentato-rubro-pallido-luysian atrophy (DRPLA), remain unchanged during reprogramming, long term passage, and differentiation, indicating that polyQ disease-associated CAG repeats are stable during maintenance of iPSCs. The level of AR expression is up-regulated by neuronal differentiation and treatment with the AR ligand dihydrotestosterone. Filter retardation assays indicated that aggregation of ARs following dihydrotestosterone treatment in neurons derived from SBMA-iPSCs increases significantly compared with neurological control iPSCs, easily recapitulating the pathological feature of mutant ARs in SBMA-iPSCs. This phenomenon was not observed in iPSCs and fibroblasts, thereby showing the neuron-dominant phenotype of this disease. Furthermore, the HSP90 inhibitor 17-allylaminogeldanamycin sharply decreased the level of aggregated AR in neurons derived from SBMA-iPSCs, indicating a potential for discovery and validation of candidate drugs. We found that SBMA-iPSCs possess disease-specific biochemical features and could thus open new avenues of research into not only SBMA, but also other polyglutamine diseases.

FIGURE 1.

Generation of KAS01 #2 and #3 iPSCs and determination of their pluripotency. A–J, both KAS01 #2 and #3 iPSC lines exhibit markers of pluripotency (A–E, KAS01 #2; F–J, KAS01 #3). All iPSCs expressed the pluripotency markers Tra-1–60, Tra-1–81, SSEA3, and SSEA4. SSEA1 is used as a negative surface marker for undifferentiated stem cells. The nuclei were stained with DAPI. Scale bar, 200 μm. K, RT-PCR analysis of the transgenes OCT3/4, SOX2, KLF4, and the endogenous human embryonic stem cell marker genes. Patient fibroblasts 6 days after retroviral transduction are positive for the transgenes. hFib, healthy control fibroblast; RT(−), healthy control samples without RT-PCR. L–Q, embryoid bodies derived from KAS01 #2 and KAS01 #3 iPSCs express germ layer-specific markers, including α-fetoprotein (AFP, endoderm), α-smooth muscle actin (αSMA, mesoderm), and βIII-tubulin (βIIITub, ectoderm). L–N, KAS01 #2, O–Q: KAS01 #3. Scale bar, 100 μm. R–W, teratomas derived from SCID mice injected with KAS01 #2 and KAS01 #3 iPSCs. Representative images of hematoxylin and eosin staining of teratoma sections are shown (R–T, KAS01 #2; U–W, KAS01 #3). Tissues representing all three embryonic germ layers, including glandular structure (endoderm), cartilage (mesoderm), and pigmented epithelium (ectoderm), are visible in cells of both iPSC lines. Scale bar, 50 μm.

FIGURE 2.

Motor neuron differentiation of SBMA-iPSCs and AR expression in differentiated neurons. A and B, expression of the motor neuron progenitor marker islet-1 (A, green) and GFP under the control of the HB9 promoter for the mature neuron marker (green) in βIII-tubulin-positive (βIII-Tub, red) cells (B). Scale bar, 100 μm. C and D, differentiated neurons were double-stained with anti-MAP2 and anti-AR antibodies. ARs are localized primarily in the nuclei. Scale bar in C, 200 μm. Scale bar in D, 20 μm.

FIGURE 3.

The CAG repeat number in the AR gene is stable during reprogramming and differentiation. GeneScan analyses indicating that fibroblasts have 47 and 49 CAG repeats in the AR gene are shown. Both KAS01 #2 and #3 iPSCs had only 47 repeats, even after long term passage. Differentiation (differentiated neurons and teratomas) does not influence the number of CAG repeats. DRP01 #7, which contains the normal 20 CAG repeats in the AR gene, was used as a control iPSC line. P indicates passage number.

FIGURE 4.

The number of CAG repeats in the atrophin-1 gene is stable during reprogramming. Shown are GeneScan analyses indicating that DRP-01 fibroblasts and DRP-01 #6 and DRP-01 #7 iPSCs had the same number of CAG repeats: 12 (normal allele) and 68 (mutant allele).

FIGURE 5.

DHT enhances aggregation of AR in SBMA-iPSC-derived neurons. A, analysis of AR expression in KAS01 fibroblasts, iPSCs, and differentiated neurons. Differentiated neurons were cultured with/without 50 nm DHT (DHT (+) or (−)) for 7 days. iPSC lines derived from an idiopathic PD patient (PD01 #24 or #26) were used as a control. AR expression was up-regulated by neuronal differentiation and DHT treatment in all iPSCs. B and C, results of filter retardation assays. The cellulose acetate membrane traps insoluble aggregated AR, whereas the nitrocellulose membrane traps total AR. Levels of aggregated AR (upper membrane) were determined using densitometry and normalized to the levels of total AR (lower membrane). The histogram shows the ratio of the level of aggregated AR in neurons treated with DHT to that in untreated neurons (means ± S.D.). Note that the level of aggregated AR in both KAS01 #2 and #3 neurons subjected to DHT treatment was significantly higher than the level of aggregated AR in PD01 #24 and PD01 #26 neurons. Differences were assessed using the Student's t test. *, versus KAS01 #2; p < 0.05. †, versus KAS01 #3, p < 0.05.

FIGURE 6.

DHT does not enhance AR aggregation in SBMA-fibroblasts and iPSCs. A, analysis of AR expression in KAS01, PD01, healthy control fibroblasts, and iPSCs. Differentiated neurons were cultured with/without 50 nm DHT (DHT (+) or (−)) for 7 days. iPSC lines derived from an idiopathic PD patient (PD01 #17 or #24) and an iPSC line derived from a healthy volunteer were used as controls. Total AR expression was up-regulated by DHT treatment in all fibroblasts, but not in all iPSCs. B and C, results of filter retardation assays. The cellulose acetate membrane traps insoluble aggregated AR, whereas the nitrocellulose membrane traps total AR. Levels of aggregated AR (upper membrane) were determined using densitometry and normalized to the levels of total AR (lower membrane). The histogram shows the ratio of the level of aggregated AR in neurons treated with DHT to that in untreated neurons (means ± S.D.). Significant up-regulation of aggregated AR by addition of DHT was not observed with any fibroblasts. hc., healthy control. *, versus healthy control fibroblast (hc. fib.), p < 0.05.

FIGURE 7.

Effect of 17-AAG on the mutant AR in SBMA-iPSCs. A, KAS01 #2 neurons were cultured with/without 50 nm DHT and/or 165 nm 17-AAG. Note that treatment with 17-AAG resulted in a decrease in AR expression, regardless of the presence of DHT. B and C, filter retardation assay results demonstrated that 17-AAG effectively reduces the level of aggregated AR, even in the presence of DHT. The histogram in C shows the level of aggregated AR as determined by densitometric analysis in DHT- and/or 17-AAG-treated neurons and untreated neurons (means ± S.D.). Differences were assessed using the Student's t test. *, versus none, p < 0.05. †, versus DHT, p < 0.05.