Final Report Summary - POLYQ MUTANT AR/SBMA (SBMA as a model of polyglutamine diseases: generation of a suitable cell system to study the post-transcriptional modifications of mutant androgen receptor and to discover potential therapeutic drugs)
SBMA as a model of polyglutamine diseases: generation of a suitable cell system to study the post-transcriptional modifications of mutant androgen receptor and to discover potential therapeutic drugs
Spinal and bulbar muscular atrophy (SBMA) is an untreatable neurodegenerative disease caused by expansion of a polyglutamine (polyQ) tract in the gene coding for Androgen Receptor (AR) (La Spada et al., 1991). SBMA is characterized by the selective loss of lower motor neurons in the brainstem and spinal cord, together with weakness, fasciculations and atrophy of skeletal muscle in males (Katsuno et al, 2006). The sex-specificity of SBMA is due to the fact that polyglutamine-expanded AR (polyQ-AR) becomes toxic when it binds its natural ligands (testosterone and dihydrotestosterone, DHT) and translocates into the nucleus. Ligand binding induces several post-translational modifications in the AR, such as phosphorylation which play a critical role in disease pathogenesis (reviewed by Parodi and Pennuto, 2011).
In this project, we explored the role of AR phosphorylation by activation of protein kinase A (PKA) pathway in the context of disease pathogenesis. We initially observed that in motor neuron-derived MN-1 cells expressing polyQ-AR (AR65Q and AR100Q), which showed increased caspase 3 activity compared to cells expressing normal AR (AR24Q) (Fig. 1a e 1b), overexpression of wtPKA or constitutively active PKA (caPKA) reduced caspase 3 activity. Conversely, pharmacologic inhibition of endogenous PKA by the PKA inhibitor PKI had opposite effect (Fig. 1a). Similarly to PKA overexpression, pharmacologic stimulation of endogenous PKA by a neuropeptide, pituitary adenylate cyclase activating peptide (PACAP) or by forskolin reduced the caspase 3 cleavage in stable MN-1 cells expressing AR100Q (Fig. 1c).
Upon synthesis, non-expanded AR has been shown to undergo extensive phosphorylation and the phosphorylated isoform of AR runs in western blotting as a 110-112 kDa doublet (Gioeli et al., 2002). To investigate the effect of activation of PKA signaling on the biology of polyQ-AR, we expressed AR55Q together with wild type PKA (wtPKA), constitutively active PKA (caPKA) and dominant negative PKA (dnPKA) (Fig.1d). Surprisingly, we found that both wtPKA and caPKA, but not dnPKA, decreased the accumulation of the 112 kDa phosphorylated isoform of polyQ-AR (Fig.1d). Activation of PKA pathway through PACAP or forskolin affected AR phosphorylation also in transfected Hek293T and stable MN-1 cells respectively (Fig. 1e e 1f). Moreover, treatment with forskolin reduced the accumulation of the upper isoform of the AR in presence and absence of DHT also in induced pluripotent stem cells (iPSCs) obtained from SBMA patient fibroblasts (Fig.1g).
To investigate if activation of PACAP/PKA signaling affects ligand-induced nuclear translocation, we performed an immunofluorescence assay in COS7 over-expressing AR55Q upon PACAP treatment. In the absence of DHT, the AR was present in the cytosol of transfected cells; after 24h of DHT treatment, the receptor translocated to the nucleus, also in presence of PACAP. Therefore, activation of PACAP/PKA signaling do not affect DHT-dependent nuclear translocation of polyQ-AR.
We noticed that activation of PACAP/PKA pathway reduced the accumulation of monomeric AR. In order to test a faster turnover of expanded AR upon activation of this pathway, we performed the cycloheximide degradation assay in presence of forskolin and PACAP. We actually observed that forskolin and PACAP increased the degradation of polyQ-AR in MN-1, treated with DHT (Fig.1e).
Altogether our results indicate that the reduced phosphorylation of polyQ-AR due to PACAP/PKA signaling activation, attenuates the ligand-induced cell toxicity in motor neuron-like (MN1) cells through increased degradation of polyQ AR.
- Gioeli, D., Ficarro, S.B. Kwiek, J.J. Aaronson, D., Hancock, M., Catling, A.D. White, F.M. Christian, R.E. Settlage, R.E. Shabanowitz, J., et al. (2002). Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites. J Biol Chem 277, 29304-29314.
- Katsuno, M., Adachi, H., Waza, M., Banno, H., Suzuki, K., Tanaka, F., Doyu, M., and Sobue, G. (2006). Pathogenesis, animal models and therapeutics in spinal and bulbar muscular atrophy (SBMA). Exp Neurol 200, 8-18.
- La Spada, A.R. Wilson, E.M. Lubahn, D.B. Harding, A.E. and Fischbeck, K.H. (1991). Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 352, 77-79.
- Parodi, S. and Pennuto, M. (2011) Neurotoxic effects of androgens in spinal and bulbar muscular atrophy. Frontiers Endocr 32: 416-425.
LEGEND
SBMA as a model of polyglutamine diseases: generation of a suitable cell system to study the post-transcriptional modifications of mutant androgen receptor and to discover potential therapeutic drugs
a) Mouse motor neuron–like cells (MN-1) stably expressing AR24Q or AR65Q were transiently transfected with empty vector or vector expressing either wtPKA or constitutively active PKA (caPKA). To inhibit endogenous PKA, the cells were treated with the PKA inhibitor PKI. The cells were then processed for fluorometric caspase 3 assay.* p = 0.001 n = 3.
b) Western blotting analysis of caspase 3 cleavage in MN-1 cells stably expressing AR24Q and AR100Q treated with DHT for 72h. Graph, mean ± sem (n = 6), * p = 0.04.
c) Western blotting analysis of caspase 3 cleavage in MN-1 DHT stably expressing AR100Q treated as indicated. Graph, mean ± sem (n = 3), * p = 0.05
d) Hek293T cells were transfected with vector expressing AR55Q alone or with wtPKA, caPKA, and dominant negative PKA (dnPKA), treated with or without DHT and processed for western blotting.
e) HEK293T cells expressing AR55Q were treated as indicated and processed for western blotting.
f) Western blotting analysis of AR in stable MN-1 cells treated with forskolin (Forsk, 10 μM) for 5 hours.
g) iPSCs derived from SBMA patient were treated as indicated (F = forskolin) and processed for western blotting. Top panel, lower exposure and middle panel higher exposure of AR. Bottom panel, Calnexin is shown as loading control.
h) Cos1 cells transfected with AR55Q were treated with vehicle or DHT and processed for immunocytochemistry. AR was detected with anti-AR antibody (green), and nuclei were stained with DAPI (blue).
i) Western blotting analysis of AR100Q turnover in MN-1 cells treated with vehicle, PACAP, and forskolin in the presence of cycloheximide (CHX). Graph, mean ± sem (n = 4), * p = 0.04.
Spinal and bulbar muscular atrophy (SBMA) is an untreatable neurodegenerative disease caused by expansion of a polyglutamine (polyQ) tract in the gene coding for Androgen Receptor (AR) (La Spada et al., 1991). SBMA is characterized by the selective loss of lower motor neurons in the brainstem and spinal cord, together with weakness, fasciculations and atrophy of skeletal muscle in males (Katsuno et al, 2006). The sex-specificity of SBMA is due to the fact that polyglutamine-expanded AR (polyQ-AR) becomes toxic when it binds its natural ligands (testosterone and dihydrotestosterone, DHT) and translocates into the nucleus. Ligand binding induces several post-translational modifications in the AR, such as phosphorylation which play a critical role in disease pathogenesis (reviewed by Parodi and Pennuto, 2011).
In this project, we explored the role of AR phosphorylation by activation of protein kinase A (PKA) pathway in the context of disease pathogenesis. We initially observed that in motor neuron-derived MN-1 cells expressing polyQ-AR (AR65Q and AR100Q), which showed increased caspase 3 activity compared to cells expressing normal AR (AR24Q) (Fig. 1a e 1b), overexpression of wtPKA or constitutively active PKA (caPKA) reduced caspase 3 activity. Conversely, pharmacologic inhibition of endogenous PKA by the PKA inhibitor PKI had opposite effect (Fig. 1a). Similarly to PKA overexpression, pharmacologic stimulation of endogenous PKA by a neuropeptide, pituitary adenylate cyclase activating peptide (PACAP) or by forskolin reduced the caspase 3 cleavage in stable MN-1 cells expressing AR100Q (Fig. 1c).
Upon synthesis, non-expanded AR has been shown to undergo extensive phosphorylation and the phosphorylated isoform of AR runs in western blotting as a 110-112 kDa doublet (Gioeli et al., 2002). To investigate the effect of activation of PKA signaling on the biology of polyQ-AR, we expressed AR55Q together with wild type PKA (wtPKA), constitutively active PKA (caPKA) and dominant negative PKA (dnPKA) (Fig.1d). Surprisingly, we found that both wtPKA and caPKA, but not dnPKA, decreased the accumulation of the 112 kDa phosphorylated isoform of polyQ-AR (Fig.1d). Activation of PKA pathway through PACAP or forskolin affected AR phosphorylation also in transfected Hek293T and stable MN-1 cells respectively (Fig. 1e e 1f). Moreover, treatment with forskolin reduced the accumulation of the upper isoform of the AR in presence and absence of DHT also in induced pluripotent stem cells (iPSCs) obtained from SBMA patient fibroblasts (Fig.1g).
To investigate if activation of PACAP/PKA signaling affects ligand-induced nuclear translocation, we performed an immunofluorescence assay in COS7 over-expressing AR55Q upon PACAP treatment. In the absence of DHT, the AR was present in the cytosol of transfected cells; after 24h of DHT treatment, the receptor translocated to the nucleus, also in presence of PACAP. Therefore, activation of PACAP/PKA signaling do not affect DHT-dependent nuclear translocation of polyQ-AR.
We noticed that activation of PACAP/PKA pathway reduced the accumulation of monomeric AR. In order to test a faster turnover of expanded AR upon activation of this pathway, we performed the cycloheximide degradation assay in presence of forskolin and PACAP. We actually observed that forskolin and PACAP increased the degradation of polyQ-AR in MN-1, treated with DHT (Fig.1e).
Altogether our results indicate that the reduced phosphorylation of polyQ-AR due to PACAP/PKA signaling activation, attenuates the ligand-induced cell toxicity in motor neuron-like (MN1) cells through increased degradation of polyQ AR.
- Gioeli, D., Ficarro, S.B. Kwiek, J.J. Aaronson, D., Hancock, M., Catling, A.D. White, F.M. Christian, R.E. Settlage, R.E. Shabanowitz, J., et al. (2002). Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites. J Biol Chem 277, 29304-29314.
- Katsuno, M., Adachi, H., Waza, M., Banno, H., Suzuki, K., Tanaka, F., Doyu, M., and Sobue, G. (2006). Pathogenesis, animal models and therapeutics in spinal and bulbar muscular atrophy (SBMA). Exp Neurol 200, 8-18.
- La Spada, A.R. Wilson, E.M. Lubahn, D.B. Harding, A.E. and Fischbeck, K.H. (1991). Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 352, 77-79.
- Parodi, S. and Pennuto, M. (2011) Neurotoxic effects of androgens in spinal and bulbar muscular atrophy. Frontiers Endocr 32: 416-425.
LEGEND
SBMA as a model of polyglutamine diseases: generation of a suitable cell system to study the post-transcriptional modifications of mutant androgen receptor and to discover potential therapeutic drugs
a) Mouse motor neuron–like cells (MN-1) stably expressing AR24Q or AR65Q were transiently transfected with empty vector or vector expressing either wtPKA or constitutively active PKA (caPKA). To inhibit endogenous PKA, the cells were treated with the PKA inhibitor PKI. The cells were then processed for fluorometric caspase 3 assay.* p = 0.001 n = 3.
b) Western blotting analysis of caspase 3 cleavage in MN-1 cells stably expressing AR24Q and AR100Q treated with DHT for 72h. Graph, mean ± sem (n = 6), * p = 0.04.
c) Western blotting analysis of caspase 3 cleavage in MN-1 DHT stably expressing AR100Q treated as indicated. Graph, mean ± sem (n = 3), * p = 0.05
d) Hek293T cells were transfected with vector expressing AR55Q alone or with wtPKA, caPKA, and dominant negative PKA (dnPKA), treated with or without DHT and processed for western blotting.
e) HEK293T cells expressing AR55Q were treated as indicated and processed for western blotting.
f) Western blotting analysis of AR in stable MN-1 cells treated with forskolin (Forsk, 10 μM) for 5 hours.
g) iPSCs derived from SBMA patient were treated as indicated (F = forskolin) and processed for western blotting. Top panel, lower exposure and middle panel higher exposure of AR. Bottom panel, Calnexin is shown as loading control.
h) Cos1 cells transfected with AR55Q were treated with vehicle or DHT and processed for immunocytochemistry. AR was detected with anti-AR antibody (green), and nuclei were stained with DAPI (blue).
i) Western blotting analysis of AR100Q turnover in MN-1 cells treated with vehicle, PACAP, and forskolin in the presence of cycloheximide (CHX). Graph, mean ± sem (n = 4), * p = 0.04.
