Toxic and non-toxic aggregates from the SBMA and normal forms of androgen receptor have distinct oligomeric structures

Hormone-dependent aggregation of the androgen receptor (AR) with a polyglutamine (polyQ) stretch amplification (>38) is considered to be the causative agent of the neurodegenerative disorder spinal and bulbar muscular atrophy (SBMA), consistent with related neurodegenerative diseases involving polyQ-extended proteins. In spite of the widespread acceptance of this common causal hypothesis, little attention has been paid to its apparent incompatibility with the observation of AR aggregation in healthy individuals with no polyQ stretch amplification. Here we used atomic force microscopy (AFM) to characterize sub-micrometer scale aggregates of the wild-type (22 glutamines) and the SBMA form (65 glutamines), as well as a polyQ deletion mutant (1 glutamine) and a variant with a normal length polyQ stretch but with a serine to alanine double mutation elsewhere in the protein. We used a baculovirus-insect cell expression system to produce full-length proteins for these structural analyses. We related the AFM findings to cytotoxicity as measured by expression of the receptors in Drosophila motoneurons or in neuronal cells in culture. We found that the pathogenic AR mutants formed oligomeric fibrils up to 300-600nm in length. These were clearly different from annular oligomers 120-180nm in diameter formed by the nonpathogenic receptors. We could also show that melatonin, which is known to ameliorate the pathological phenotype in the fly model, caused polyQ-extended AR to form annular oligomers. Further comparative investigation of these reproducibly distinct toxic and non-toxic oligomers could advance our understanding of the molecular basis of the polyQ pathologies.     

Genistein,a natural product derived from soybeans,ameliorates polyglutamine‐mediated motor neuron disease

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ) tract within the androgen receptor (AR) gene. The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem, and diffuse nuclear accumulation and nuclear inclusions of mutant AR in residual motor neurons and certain visceral organs. AR‐associated coregulator 70 (ARA70) was the first coregulator of AR to be identified, and it has been shown to interact with AR and increase its protein stability. Here, we report that genistein, an isoflavone found in soy, disrupts the interaction between AR and ARA70 and promotes the degradation of mutant AR in neuronal cells and transgenic mouse models of SBMA. We also demonstrate that dietary genistein ameliorates behavioral abnormalities, improves spinal cord and muscle pathology, and decreases the amounts of monomeric AR and high‐molecular‐weight mutant AR protein aggregates in SBMA transgenic mice. Thus, genistein treatment may be a potential therapeutic approach for alleviating the symptoms of SBMA by disrupting the interactions between AR and ARA70.     

Autophagy and access: Understanding the role of androgen receptor subcellular localization in SBMA

Ridding neurons of toxic misfolded proteins is a critical feature of many neurodegenerative diseases. We have recently reported that lack of access of nuclear polyglutamine-expanded androgen receptor (AR) to the autophagic degradation pathway is a critical point in pathogenesis. When mutant AR is contained within the cytoplasm, it can be degraded by autophagy, resulting in amelioration of its toxic effects, as has been observed in other polyglutamine expansion diseases involving cytoplasmic mutant proteins. However, we have also found that pharmacological induction of autophagy protects SBMA motor neurons from the toxic effects of even nuclear localized mutant AR, albeit without affecting mutant nuclear AR levels. Thus, we have further investigated the mechanism by which autophagy elicits therapeutic benefit in cell culture. We found that endogenous autophagy only slightly alters nuclear mutant AR aggregation compared to substantial effects on cytoplasmic AR aggregation. Interestingly, pharmacological activation of mTOR-dependent autophagy did not significantly alter nuclear AR aggregation, whereas we observed that it protects SBMA motor neurons. Our findings indicate that therapeutic intervention to induce autophagy represents a potential potent benefit for SBMA, and that it likely does so by protecting SBMA motor neurons independent of a direct effect on mutant AR.     

In vivo expansion of trinucleotide repeats yields plasmid and YAC constructs for targeting and transgenesis

Production of mouse models of inherited neurodegenerative diseases is an important step towards understanding the mechanism of neurotoxicity and for testing potential therapies. We are interested in creating a mouse model for X-linked spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by expansion of a CAG repeat within the androgen receptor (AR) gene. To permit generation of mice that will show a SBMA phenotype within their life span, we decided to obtain a yeast artificial chromosome (YAC) carrying the AR gene and introduce CAG repeat mutations numbering 100 or more triplets. SBMA patients with more than 70 CAGs have never been observed; therefore, we chose to expand a 59 CAG repeat tract in vivo in Escherichia coli. Although we set out to expand this repeat tract using a recombination paradigm involving two plasmid co-propagation, we did not observe large expansions. We were instead able to incrementally generate repeat tracts from 100 to 200 CAGs in a yeast integrating plasmid vector by taking advantage of replication instability. In the course of our experiments that yielded these CAG repeat tracts, we evaluated the role of repeat orientation, vector co-propagation, and recA function on the expansion process. We then used one of the yeast integrating vectors to successfully produce an AR YAC construct carrying 100 CAG repeats. AR YAC CAG100 will serve as a valuable reagent for the production of a SBMA mouse.     

Human Adipose-Derived Mesenchymal Stem Cells as a New Model of Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) or Kennedy''s disease is an X-linked CAG/polyglutamine expansion motoneuron disease, in which an elongated polyglutamine tract (polyQ) in the N-terminal androgen receptor (ARpolyQ) confers toxicity to this protein. Typical markers of SBMA disease are ARpolyQ intranuclear inclusions. These are generated after the ARpolyQ binds to its endogenous ligands, which promotes AR release from chaperones, activation and nuclear translocation, but also cell toxicity. The SBMA mouse models developed so far, and used in preclinical studies, all contain an expanded CAG repeat significantly longer than that of SBMA patients. Here, we propose the use of SBMA patients adipose-derived mesenchymal stem cells (MSCs) as a new human in vitro model to study ARpolyQ toxicity. These cells have the advantage to express only ARpolyQ, and not the wild type AR allele. Therefore, we isolated and characterized adipose-derived MSCs from three SBMA patients (ADSC from Kennedy''s patients, ADSCK) and three control volunteers (ADSCs). We found that both ADSCs and ADSCKs express mesenchymal antigens, even if only ADSCs can differentiate into the three typical cell lineages (adipocytes, chondrocytes and osteocytes), whereas ADSCKs, from SBMA patients, showed a lower growth potential and differentiated only into adipocyte. Moreover, analysing AR expression on our mesenchymal cultures we found lower levels in all ADSCKs than ADSCs, possibly related to negative pressures exerted by toxic ARpolyQ in ADSCKs. In addition, with proteasome inhibition the ARpolyQ levels increased specifically in ADSCKs, inducing the formation of HSP70 and ubiquitin positive nuclear ARpolyQ inclusions. Considering all of this evidence, SBMA patients adipose-derived MSCs cultures should be considered an innovative in vitro human model to understand the molecular mechanisms of ARpolyQ toxicity and to test novel therapeutic approaches in SBMA.     

Ligand promotes intranuclear inclusions in a novel cell model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA, Kennedy's disease) is one of a group of progressive neurodegenerative diseases resulting from a polyglutamine repeat expansion. In SBMA the polymorphic trinucleotide CAG repeat in exon 1 of the androgen receptor (AR) gene is increased, resulting in expansion of a polyglutamine tract. Patient autopsy material reveals neuronal intranuclear inclusions (NII) in affected regions that contain only amino-terminal epitopes of the AR. Cell models have previously been unable to produce intranuclear inclusions containing only a portion of the AR. We report here the creation of an inducible cell model of SBMA that reproduces this important characteristic of disease pathology. PC12 cells expressing highly expanded AR form ubiquitinated intranuclear inclusions containing amino-terminal epitopes of the AR as well as heat shock proteins. Inclusions appear as distinct granular electron-dense structures in the nucleus by immunoelectron microscopy. Dihydrotestosterone treatment of mutant AR-expressing cells results in increased inclusion load. This model mimics the formation of ubiquitinated intranuclear inclusions containing the amino-terminal portion of AR observed in patient tissue and reveals a role for ligand in the pathogenesis of SBMA.     

17-AAG, an Hsp90 inhibitor, ameliorates polyglutamine-mediated motor neuron degeneration

Heat-shock protein 90 (Hsp90) functions as part of a multichaperone complex that folds, activates and assembles its client proteins. Androgen receptor (AR), a pathogenic gene product in spinal and bulbar muscular atrophy (SBMA), is one of the Hsp90 client proteins. We examined the therapeutic effects of 17-allylamino-17-demethoxygeldanamycin (17-AAG), a potent Hsp90 inhibitor, and its ability to degrade polyglutamine-expanded mutant AR. Administration of 17-AAG markedly ameliorated motor impairments in the SBMA transgenic mouse model without detectable toxicity, by reducing amounts of monomeric and aggregated mutant AR. The mutant AR showed a higher affinity for Hsp90-p23 and preferentially formed an Hsp90 chaperone complex as compared to wild-type AR; mutant AR was preferentially degraded in the presence of 17-AAG in both cells and transgenic mice as compared to wild-type AR. 17-AAG also mildly induced Hsp70 and Hsp40. 17-AAG would thus provide a new therapeutic approach to SBMA and probably to other related neurodegenerative diseases.     

Enhanced Aggregation of Androgen Receptor in Induced Pluripotent Stem Cell-derived Neurons from Spinal and Bulbar Muscular Atrophy

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.     

Transgenic mouse models of spinal and bulbar muscular atrophy (SBMA)

Spinal and bulbar muscular atrophy (SBMA) is a late-onset motor neuron disease characterized by proximal muscle atrophy, weakness, contraction fasciculations, and bulbar involvement. Only males develop symptoms, while female carriers usually are asymptomatic. A specific treatment for SBMA has not been established. The molecular basis of SBMA is the expansion of a trinucleotide CAG repeat, which encodes the polyglutamine (polyQ) tract, in the first exon of the androgen receptor (AR) gene. The pathologic hallmark is nuclear inclusions (NIs) containing the mutant and truncated AR with expanded polyQ in the residual motor neurons in the brainstem and spinal cord as well as in some other visceral organs. Several transgenic (Tg) mouse models have been created for studying the pathogenesis of SBMA. The Tg mouse model carrying pure 239 CAGs under human AR promoter and another model carrying truncated AR with expanded CAGs show motor impairment and nuclear NIs in spinal motor neurons. Interestingly, Tg mice carrying full-length human AR with expanded polyQ demonstrate progressive motor impairment and neurogenic pathology as well as sexual difference of phenotypes. These models recapitulate the phenotypic expression observed in SBMA. The ligand-dependent nuclear localization of the mutant AR is found to be involved in the disease mechanism, and hormonal therapy is suggested to be a therapeutic approach applicable to SBMA.     

ASC-J9 ameliorates spinal and bulbar muscular atrophy phenotype via degradation of androgen receptor

Motor neuron degeneration resulting from the aggregation of the androgen receptor with an expanded polyglutamine tract (AR-polyQ) has been linked to the development of spinal and bulbar muscular atrophy (SBMA or Kennedy disease). Here we report that adding 5-hydroxy-1,7-bis(3,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one (ASC-J9) disrupts the interaction between AR and its coregulators, and also increases cell survival by decreasing AR-polyQ nuclear aggregation and increasing AR-polyQ degradation in cultured cells. Intraperitoneal injection of ASC-J9 into AR-polyQ transgenic SBMA mice markedly improved disease symptoms, as seen by a reduction in muscular atrophy. Notably, unlike previous approaches in which surgical or chemical castration was used to reduce SBMA symptoms, ASC-J9 treatment ameliorated SBMA symptoms by decreasing AR-97Q aggregation and increasing VEGF164 expression with little change of serum testosterone. Moreover, mice treated with ASC-J9 retained normal sexual function and fertility. Collectively, our results point to a better therapeutic and preventative approach to treating SBMA, by disrupting the interaction between AR and AR coregulators.     

Testosterone reduction prevents phenotypic expression in a transgenic mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is a polyglutamine disease caused by the expansion of a CAG repeat in the androgen receptor (AR) gene. We generated a transgenic mouse model carrying a full-length AR containing 97 CAGs. Three of the five lines showed progressive muscular atrophy and weakness as well as diffuse nuclear staining and nuclear inclusions consisting of the mutant AR. These phenotypes were markedly pronounced in male transgenic mice, and dramatically rescued by castration. Female transgenic mice showed only a few manifestations that markedly deteriorated with testosterone administration. Nuclear translocation of the mutant AR by testosterone contributed to the phenotypic difference with gender and the effects of hormonal interventions. These results suggest the therapeutic potential of hormonal intervention for SBMA.     

Viral delivery of miR-196a ameliorates the SBMA phenotype via the silencing of CELF2

Spinal and bulbar muscular atrophy (SBMA) is an inherited neurodegenerative disorder caused by the expansion of the polyglutamine (polyQ) tract of the androgen receptor (AR-polyQ). Characteristics of SBMA include proximal muscular atrophy, weakness, contraction fasciculation and bulbar involvement. MicroRNAs (miRNAs) are a diverse class of highly conserved small RNA molecules that function as crucial regulators of gene expression in animals and plants. Recent functional studies have shown the potent activity of specific miRNAs as disease modifiers both in vitro and in vivo. Thus, potential therapeutic approaches that target the miRNA processing pathway have recently attracted attention. Here we describe a novel therapeutic approach using the adeno-associated virus (AAV) vector–mediated delivery of a specific miRNA for SBMA. We found that miR-196a enhanced the decay of the AR mRNA by silencing CUGBP, Elav-like family member 2 (CELF2). CELF2 directly acted on AR mRNA and enhanced the stability of AR mRNA. Furthermore, we found that the early intervention of miR-196a delivered by an AAV vector ameliorated the SBMA phenotypes in a mouse model. Our results establish the proof of principle that disease-specific miRNA delivery could be useful in neurodegenerative diseases.     

Disruption of nongenomic testosterone signaling in a model of spinal and bulbar muscular atrophy

As one of the nine hereditary neurodegenerative polyQ disorders, spinal and bulbar muscular atrophy (SBMA) results from a polyQ tract expansion in androgen receptor (AR). Although protein aggregates are the pathological hallmark of many neurodegenerative diseases, their direct role in the neurodegeneration is more and more questioned. To determine the early molecular mechanisms causing motor neuron degeneration in SBMA, we established an in vitro system based on the tetracycline-inducible expression of normal (AR20Q), the mutated, 51 glutamine-extended (AR51Q), or polyQ-deleted (AR0Q) AR in NSC34, a motor neuron-like cell line lacking endogenous AR. Although no intracellular aggregates were formed, the expression of the AR51Q leads to a loss of function characterized by reduced neurite outgrowth and to a toxic gain of function resulting in decreased cell viability. In this study, we show that both AR20Q and AR51Q are recruited to lipid rafts in response to testosterone stimulation. However, whereas testosterone induces the activation of the c-jun N-terminal kinase/c-jun pathway via membrane-associated AR20Q, it does not so in NSC34 expressing AR51Q. Phosphorylation of c-jun N-terminal kinase plays a crucial role in AR20Q-dependent survival and differentiation of NSC34. Moreover, c-jun protein levels decrease more slowly in AR20Q- than in AR51Q-expressing NSC34 cells. This is due to a rapid and transient inhibition of glycogen synthase kinase 3α occurring in a phosphatidylinositol 3-kinase-independent manner. Our results demonstrate that the deregulation of nongenomic AR signaling may be involved in SBMA establishment, opening new therapeutic perspectives.     

Microarray analysis of gene expression by skeletal muscle of three mouse models of Kennedy disease/spinal bulbar muscular atrophy

Background

Emerging evidence implicates altered gene expression within skeletal muscle in the pathogenesis of Kennedy disease/spinal bulbar muscular atrophy (KD/SBMA). We therefore broadly characterized gene expression in skeletal muscle of three independently generated mouse models of this disease. The mouse models included a polyglutamine expanded (polyQ) AR knock-in model (AR113Q), a polyQ AR transgenic model (AR97Q), and a transgenic mouse that overexpresses wild type AR solely in skeletal muscle (HSA-AR). HSA-AR mice were included because they substantially reproduce the KD/SBMA phenotype despite the absence of polyQ AR.

Methodology/Principal Findings

We performed microarray analysis of lower hindlimb muscles taken from these three models relative to wild type controls using high density oligonucleotide arrays. All microarray comparisons were made with at least 3 animals in each condition, and only those genes having at least 2-fold difference and whose coefficient of variance was less than 100% were considered to be differentially expressed. When considered globally, there was a similar overlap in gene changes between the 3 models: 19% between HSA-AR and AR97Q, 21% between AR97Q and AR113Q, and 17% between HSA-AR and AR113Q, with 8% shared by all models. Several patterns of gene expression relevant to the disease process were observed. Notably, patterns of gene expression typical of loss of AR function were observed in all three models, as were alterations in genes involved in cell adhesion, energy balance, muscle atrophy and myogenesis. We additionally measured changes similar to those observed in skeletal muscle of a mouse model of Huntington''s Disease, and to those common to muscle atrophy from diverse causes.

Conclusions/Significance

By comparing patterns of gene expression in three independent models of KD/SBMA, we have been able to identify candidate genes that might mediate the core myogenic features of KD/SBMA.     

PCR片段测序和基因分型两种方法对Kennedy遗传病的诊断

X连锁脊延髓肌萎缩症（SBMA）或肯尼迪病是一种成年人发病的神经变性疾病,以肌无力与慢性、进行性肌萎缩为特征. 通过PCR片段测序和基因分型法准确检测雄激素受体（AR）基因CAG复制数目,兄弟俩（来自同一个中国家庭）被确诊为隐性遗传性SBMA. 为了得到该中国家庭SMBA家系人员AR基因的CAG复制数目,我们采用了PCR片段测序和基因分型两种方法. 在该SMBA家系中有两个已发病的成年男性、未发病的年轻男性,及女性基因携带者. 两个已发病男性患者AR基因中CAG三核苷酸串重复数目分别是48和45. 以前的研究表明特定三核苷酸串重复数目的扩增可导致人类遗传性神经障碍疾病发病。我们的研究结果完全支持这一观点,SMBA中国家系的三核苷酸CAG拷贝数目检测结果表明,AR基因CAG扩增数目与SMBA发病相关. 关键词雄性激素受体; CAG多重三核苷酸重复; 肯尼迪病; 脊延髓肌萎缩症; X连锁     

Identification of neuron selective androgen receptor inhibitors

AIM To identify neuron-selective androgen receptor(AR) signaling inhibitors, which could be useful in the treatment of spinal and bulbar muscular atrophy(SBMA), or Kennedy's disease, a neuromuscular disorder in which deterioration of motor neurons leads to progressive muscle weakness. METHODS Cell lines representing prostate, kidney, neuron, adipose, and muscle tissue were developed that stably expressed the CFP-AR-YFP FRET reporter. We used these cells to screen a library of small molecules for cell typeselective AR inhibitors. Secondary screening in luciferase assays was used to identify the best cell-type specific AR inhibitors. The mechanism of action of a neuronselective AR inhibitor was examined in vitro using luciferase reporter assays, immunofluorescence microscopy, and immunoprecipitations. Rats were treated with the most potent compound and tissue-selective AR inhibition was examined using RT-q PCR of AR-regulated genes and immunohistochemistry.RESULTS We identified the thiazole class of antibiotics as com-pounds able to inhibit AR signaling in a neuronal cell line but not a muscle cell line. One of these antibiotics, thiostrepton is able to inhibit the activity of both wild type and polyglutamine expanded AR in neuronal GT1-7 cells with nanomolar potency. The thiazole antibiotics are known to inhibit FOXM1 activity and accordingly, a novel FOXM1 inhibitor FDI-6 also inhibited AR activity in a neuron-selective fashion. The selective inhibition of AR is likely indirect as the varied structures of these compounds would not suggest that they are competitive antagonists. Indeed, we found that FOXM1 expression correlates with cell-type selectivity, FOXM1 co-localizes with AR in the nucleus, and that sh RNA-mediated knock down of FOXM1 reduces AR activity and thiostrepton sensitivity in a neuronal cell line. Thiostrepton treatment reduces FOXM1 levels and the nuclear localization of beta-catenin, a known co-activator of both FOXM1 and AR, and reduces the association between beta-catenin and AR. Treatment of rats with thiostrepton demonstrated AR signaling inhibition in neurons, but not muscles. CONCLUSION Our results suggest that thiazole antibiotics, or other inhibitors of the AR-FOXM1 axis, can inhibit AR signaling selectively in motor neurons and may be useful in the treatment or prevention of SBMA symptoms.