Spinal and bulbar muscular atrophy: ligand-dependent pathogenesis and therapeutic perspectives

Spinal and bulbar muscular atrophy (SBMA) is a late-onset motor neuron disease characterized by proximal muscle atrophy, weakness, contraction fasciculations, and bulbar involvement. SBMA exclusively affects males, while females are usually asymptomatic. 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 histopathological hallmark is the presence of nuclear inclusions containing mutant truncated ARs with expanded polyQ tracts in the residual motor neurons in the brainstem and spinal cord, as well as in some other visceral organs. The AR ligand, testosterone, accelerates AR dissociation from heat shock proteins and thus its nuclear translocation. Ligand-dependent nuclear accumulation of mutant ARs has been implicated in the pathogenesis of SBMA. Transgenic mice carrying the full-length human AR gene with an expanded polyQ tract demonstrate neuromuscular phenotypes, which are profound in males. Their SBMA-like phenotypes are rescued by castration, and aggravated by testosterone administration. Leuprorelin, an LHRH agonist that reduces testosterone release from the testis, inhibits nuclear accumulation of mutant ARs, resulting in the rescue of motor dysfunction in the male transgenic mice. However, flutamide, an androgen antagonist promoting nuclear translocation of the AR, yielded no therapeutic effect. The degradation and cleavage of the AR protein are also influenced by the ligand, contributing to the pathogenesis. Testosterone thus appears to be the key molecule in the pathogenesis of SBMA, as well as main therapeutic target of this disease.     

Loss of endogenous androgen receptor protein accelerates motor neuron degeneration and accentuates androgen insensitivity in a mouse model of X-linked spinal and bulbar muscular atrophy

X-linked spinal and bulbar muscular atrophy (SBMA; Kennedy's disease) is a polyglutamine (polyQ) disease in which the affected males suffer progressive motor neuron degeneration accompanied by signs of androgen insensitivity, such as gynecomastia and reduced fertility. SBMA is caused by CAG repeat expansions in the androgen receptor (AR) gene resulting in the production of AR protein with an extended glutamine tract. SBMA is one of nine polyQ diseases in which polyQ expansion is believed to impart a toxic gain-of-function effect upon the mutant protein, and initiate a cascade of events that culminate in neurodegeneration. However, whether loss of a disease protein's normal function concomitantly contributes to the neurodegeneration remains unanswered. To address this, we examined the role of normal AR function in SBMA by crossing a highly representative AR YAC transgenic mouse model with 100 glutamines (AR100) and a corresponding control (AR20) onto an AR null (testicular feminization; Tfm) background. Absence of endogenous AR protein in AR100Tfm mice had profound effects upon neuromuscular and endocrine-reproductive features of this SBMA mouse model, as AR100Tfm mice displayed accelerated neurodegeneration and severe androgen insensitivity in comparison to AR100 littermates. Reduction in size and number of androgen-sensitive motor neurons in the spinal cord of AR100Tfm mice underscored the importance of AR action for neuronal health and survival. Promoter-reporter assays confirmed that AR transactivation competence diminishes in a polyQ length-dependent fashion. Our studies indicate that SBMA disease pathogenesis, both in the nervous system and the periphery, involves two simultaneous pathways: gain-of-function misfolded protein toxicity and loss of normal protein function.     

Transglutaminase potentiates ligand-dependent proteasome dysfunction induced by polyglutamine-expanded androgen receptor

Expansion of the CAG trinucleotide repeat encoding glutamine in the androgen receptor gene leads to spinobulbar muscular atrophy (SBMA), a neurodegenerative disorder in a family of polyglutamine diseases with enigmatic pathogenic mechanisms. One established property of glutamine residues is their ability to act as an amine accepter in a transglutaminase-catalyzed reaction, resulting in a proteolytically resistant glutamyl-lysine cross-link. To examine underlying disease mechanisms we investigated the relationship between polyglutamine-expanded androgen receptor and transglutaminase. We found androgen receptor N-terminal fragments are a substrate for transglutaminase. Western blots of the proteins following incubation with transglutaminase show that several different epitopes of the AR appear to be lost. We propose that this is due to the transglutaminase cross-linking of the AR, which interferes with antibody recognition. Furthermore, HEK GFP(u)-1 cells expressing polyglutamine-expanded androgen receptor and transglutaminase exhibit ligand-dependent proteasome dysfunction; this effect was not observed in the presence of cystamine, a transglutaminase inhibitor. In addition, transglutaminase-mediated isopeptide bonds were detected in brains of SBMA transgenic mice, but not in controls, suggesting involvement of transglutaminase-catalyzed reactions in polyglutamine disease pathogenesis. Our hypothesis is that cross-linked AR cannot to be degraded by the proteasome and obstructs the proteasome pore, preventing normal function. Because of the central role the ubiquitin-proteasome degradation system plays in fundamental cellular processes, any alteration in its function could cause cell death, ultimately contributing to SBMA pathogenesis.     

A mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease, caused by the expansion of a trinucleotide repeat (TNR) in exon 1 of the androgen receptor (AR) gene. This disorder is characterized by degeneration of motor and sensory neurons, proximal muscular atrophy, and endocrine abnormalities, such as gynecomastia and reduced fertility. We describe the development of a transgenic model of SBMA expressing a full-length human AR (hAR) cDNA carrying 65 (AR(65)) or 120 CAG repeats (AR(120)), with widespread expression driven by the cytomegalovirus promoter. Mice carrying the AR(120) transgene displayed behavioral and motor dysfunction, while mice carrying 65 CAG repeats showed a mild phenotype. Progressive muscle weakness and atrophy was observed in AR(120) mice and was associated with the loss of alpha-motor neurons in the spinal cord. There was no evidence of neurodegeneration in other brain structures. Motor dysfunction was observed in both male and female animals, showing that in SBMA the polyglutamine repeat expansion causes a dominant gain-of-function mutation in the AR. The male mice displayed a progressive reduction in sperm production consistent with testis defects reported in human patients. These mice represent the first model to reproduce the key features of SBMA, making them a useful resource for characterizing disease progression, and for testing therapeutic strategies for both polyglutamine and motor neuron diseases.     

Modulation of Hsp90 function in neurodegenerative disorders: a molecular-targeted therapy against disease-causing protein

Abnormal accumulation of disease-causing protein is a commonly observed characteristic in chronic neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and polyglutamine (polyQ) diseases. A therapeutic approach that could selectively eliminate would be a promising remedy for neurodegenerative disorders. Spinal and bulbar muscular atrophy (SBMA), one of the polyQ diseases, is a late-onset motor neuron disease characterized by proximal muscle atrophy, weakness, contraction fasciculations, and bulbar involvement. The pathogenic gene product is polyQ-expanded androgen receptor (AR), which belongs to the heat shock protein (Hsp) 90 client protein family. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a novel Hsp90 inhibitor, is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. 17-AAG is now in phase II clinical trials as a potential anti-cancer agent because of its ability to selectively degrade several oncoproteins. We have recently demonstrated the efficacy and safety of 17-AAG in a mouse model of SBMA. The administration of 17-AAG significantly ameliorated polyQ-mediated motor neuron degeneration by reducing the total amount of mutant AR. 17-AAG accomplished the preferential reduction of mutant AR mainly through Hsp90 chaperone complex formation and subsequent proteasome-dependent degradation. 17-AAG induced Hsp70 and Hsp40 in vivo as previously reported; however, its ability to induce HSPs was limited, suggesting that the HSP induction might support the degradation of mutant protein. The ability of 17-AAG to preferentially degrade mutant protein would be directly applicable to SBMA and other neurodegenerative diseases in which the disease-causing proteins also belong to the Hsp90 client protein family. Our proposed therapeutic approach, modulation of Hsp90 function by 17-AAG treatment, has emerged as a candidate for molecular-targeted therapies for neurodegenerative diseases. This review will consider our research findings and discuss the possibility of a clinical application of 17-AAG to SBMA and other neurodegenerative diseases.     

FUS is not dysregulated by the spinal bulbar muscular atrophy androgen receptor polyglutamine repeat expansion

Spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis are two distinct forms of motor neuron disease with different genetic causes, pathology, and clinical course. However, both disorders are characterized by the progressive loss of lower motor neurons and by a similar protective response to growth factors in animal models, therefore raising the possibility of an overlap in the final pathogenic cascade. Mutations in the FUS gene and fused in sarcoma (FUS) protein pathology have now been identified in some amyotrophic lateral sclerosis cases, while a CAG expansion in the androgen receptor gene is known to cause SBMA. Recently, multiple lines of evidence have identified FUS as a major target of the androgen receptor, suggesting that FUS could be dysregulated in SBMA motor neurons. We have investigated this possibility by using a well-established mouse model of SBMA and our analysis of primary motor neuron cultures, spinal cords, and microdissected motor neurons show no evidence for FUS dysregulation.     

JNK mediates pathogenic effects of polyglutamine-expanded androgen receptor on fast axonal transport

Expansion of the polyglutamine (polyQ) stretch in the androgen receptor (AR) protein leads to spinal and bulbar muscular atrophy (SBMA), a neurodegenerative disease characterized by lower motor neuron degeneration. The pathogenic mechanisms underlying SBMA remain unknown, but recent experiments show that inhibition of fast axonal transport (FAT) by polyQ-expanded proteins, including polyQ-AR, represents a new cytoplasmic pathogenic lesion. Using pharmacological, biochemical and cell biological experiments, we found a new pathogenic pathway that is affected in SBMA and results in compromised FAT. PolyQ-AR inhibits FAT in a human cell line and in squid axoplasm through a pathway that involves activation of cJun N-terminal kinase (JNK) activity. Active JNK phosphorylated kinesin-1 heavy chains and inhibited kinesin-1 microtubule-binding activity. JNK inhibitors prevented polyQ-AR-mediated inhibition of FAT and reversed suppression of neurite formation by polyQ-AR. We propose that JNK represents a promising target for therapeutic interventions in SBMA.     

Genetic modulation of polyglutamine toxicity by protein conjugation pathways in Drosophila

Spinal and bulbar muscular atrophy (SBMA) is a heritable neurodegenerative disease caused by the expansion of a polyglutamine [poly(Q)] repeat within the androgen receptor (AR) protein. We studied SBMA in Drosophila using an N-terminal fragment of the human AR protein. Expression of a pathogenic AR protein with an expanded poly(Q) repeat in Drosophila results in nuclear and cytoplasmic inclusion formation, and cellular degeneration, preferentially in neuronal tissues. We have studied the influence of ubiquitin-dependent modification and the proteasome pathway on neural degeneration and AR protein fragment solubility. Compromising the ubiquitin/proteasome pathway enhances degeneration and decreases poly(Q) protein solubility. Our data further suggest that Hsp70 and the proteasome act in an additive manner to modulate neurodegeneration. Through the over-expression of a mutant of the SUMO-1 activating enzyme Uba2, we further show that poly(Q)-induced degeneration is intensified when the cellular SUMO-1 protein conjugation pathway is altered. These data suggest that post-translational protein modification, including the ubiquitin/proteasome and the SUMO-1 pathways, modulate poly(Q) pathogenesis.     

Multiple founder effects in spinal and bulbar muscular atrophy (SBMA, Kennedy disease) around the world.

SBMA (spinal and bulbar muscular atrophy), also called Kennedy disease, is an X-chromosomal recessive adult-onset neurodegenerative disorder caused by death of the spinal and bulbar motor neurones and dorsal root ganglia. Patients may also show signs of partial androgen insensitivity. SBMA is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene on the X-chromosome. Our previous study suggested that all the Nordic patients with SBMA originated from an ancient Nordic founder mutation, but the new intragenic SNP marker ARd12 revealed that the Danish patients derive their disease chromosome from another ancestor. In search of relationships between patients from different countries, we haplotyped altogether 123 SBMA families from different parts of the world for two intragenic markers and 16 microsatellites spanning 25 cM around the AR gene. The fact that different SBMA founder haplotypes were found in patients from around the world implies that the CAG repeat expansion mutation has not been a unique event. No expansion-prone haplotype could be detected. Trinucleotide diseases often show correlation between the repeat length and the severity and earlier onset of the disease. The longer the repeat, the more severe the symptoms are and the onset of the disease is earlier. A negative correlation between the CAG repeat length and the age of onset was found in the 95 SBMA patients with defined ages at onset.     

Impaired motoneuronal retrograde transport in two models of SBMA implicates two sites of androgen action

Spinal and bulbar muscular atrophy (SBMA) impairs motor function in men and is linked to a CAG repeat mutation in the androgen receptor (AR) gene. Defects in motoneuronal retrograde axonal transport may critically mediate motor dysfunction in SBMA, but the site(s) where AR disrupts transport is unknown. We find deficits in retrograde labeling of spinal motoneurons in both a knock-in (KI) and a myogenic transgenic (TG) mouse model of SBMA. Likewise, live imaging of endosomal trafficking in sciatic nerve axons reveals disease-induced deficits in the flux and run length of retrogradely transported endosomes in both KI and TG males, demonstrating that disease triggered in muscle can impair retrograde transport of cargo in motoneuron axons, possibly via defective retrograde signaling. Supporting the idea of impaired retrograde signaling, we find that vascular endothelial growth factor treatment of diseased muscles reverses the transport/trafficking deficit. Transport velocity is also affected in KI males, suggesting a neurogenic component. These results demonstrate that androgens could act via both cell autonomous and non-cell autonomous mechanisms to disrupt axonal transport in motoneurons affected by SBMA.     

Cleavage, aggregation and toxicity of the expanded androgen receptor in spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of a polyglutamine repeat within the androgen receptor (AR). We have studied the mutant AR in an in vitro system, and find both aggregation and proteolytic processing of the AR protein to occur in a polyglutamine repeat length-dependent manner. In addition, we find the aberrant metabolism of expanded repeat AR to be coupled to cellular toxicity, indicating a likely molecular basis for the toxic gain of AR function that produces neuronal degeneration in SBMA.      

Huntingtin-associated protein 1 (HAP1) interacts with androgen receptor (AR) and suppresses SBMA-mutant-AR-induced apoptosis

Huntingtin-associated protein 1 (HAP1), an interactor of huntingtin, has been known as an essential component of the stigmoid body (STB) and recently reported to play a protective role against neurodegeneration in Huntington's disease (HD). In the present study, subcellular association between HAP1 and androgen receptor (AR) with a long polyglutamine tract (polyQ) derived from spinal-and-bulbar-muscular-atrophy (SBMA) was examined using HEp-2 cells cotransfected with HAP1 and/or normal ARQ25, SBMA-mutant ARQ65 or deletion-mutant AR cDNAs. The results provided the first clear evidence that HAP1 interacts with AR through its ligand-binding domain in a polyQ-length-dependent manner and forms prominent inclusions sequestering polyQ-AR, and that addition of dihydrotestosterone reduces the association strength of HAP1 with ARQ25 more dramatically than that with ARQ65. Furthermore, SBMA-mutant-ARQ65-induced apoptosis was suppressed by cotransfection with HAP1. Our findings strongly suggest that HAP1/STB is relevant to polyQ-length-dependent modification on subcellular AR functions and critically involved in pathogenesis of not only HD but also SBMA as an important intrinsic neuroprotectant determining the threshold for cellular vulnerability to apoptosis. Taking together with previous reports that HAP1/STB is selectively expressed in the brain regions spared from degenerative targets in HD and SBMA, the current study might explain the region-specific occurrence of neurodegeneration in both diseases, shedding light on common aspects of their molecular pathological mechanism and yet-to-be-uncovered diagnostic or therapeutic applications for HD and SBMA patients.     

Clinical Characteristics and Genotype-Phenotype Correlation of Korean Patients with Spinal and Bulbar Muscular Atrophy

Purpose

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuron disease characterized by proximal muscle weakness, muscle atrophy, and fasciculation. Although SBMA is not uncommon in Korea, there is only one study reporting clinical characteristics and genotype-phenotype correlation in Korean patients.

Materials and Methods

In this study, age at the onset of symptoms, the score of severity assessed by impairment of activities of daily living milestones, and rate of disease progression, and their correlations with the number of CAG repeats in the androgen receptor (AR) gene, as well as possible correlations among clinical characteristics, were analyzed in 40 SBMA patients.

Results

The median ages at onset and at diagnosis were 44.5 and 52.5 years, respectively, and median interval between onset and diagnosis and median rate of disease progression were 5.0 years and 0.23 score/year, respectively. The median number of CAG repeats in the AR gene was 44 and the number of CAG repeats showed a significant inverse correlation with the age at onset of symptoms (r=-0.407, p=0.009). In addition, patients with early symptom onset had slower rate of disease progression.

Conclusion

As a report with the largest and recent Korean cohort, this study demonstrates clinical features of Korean patients with SBMA and reaffirms the inverse correlation between the age at disease onset and the number of CAG repeats. Interestingly, this study shows a possibility that the rate of disease progression may be influenced by the age at onset of symptoms.     

CAG repeat contraction in the androgen receptor gene in three brothers with mental retardation.

We report on three brothers with mental retardation and a contracted CAG repeat in the androgen receptor (AR) gene. It is known that expansion of the CAG repeat in this gene leads to spinal and bulbar muscular atrophy (SBMA or Kennedy disease); however, contracted repeats have not yet been implicated in disease. As the range of the length of CAG repeats in the AR gene, like those of other genes associated with dynamic mutations, follows a normal distribution, the theoretical possibility of disease at both ends of the distribution should be considered.     

Sequencing analysis of the spinal bulbar muscular atrophy CAG expansion reveals absence of repeat interruptions

Trinucleotide repeat disorders are a heterogeneous group of diseases caused by the expansion, beyond a pathogenic threshold, of unstable DNA tracts in different genes. Sequence interruptions in the repeats have been described in the majority of these disorders and may influence disease phenotype and heritability. Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by a CAG trinucleotide expansion in the androgen receptor (AR) gene. Diagnostic testing and previous research have relied on fragment analysis polymerase chain reaction to determine the AR CAG repeat size, and have therefore not been able to assess the presence of interruptions. We here report a sequencing study of the AR CAG repeat in a cohort of SBMA patients and control subjects in the United Kingdom. We found no repeat interruptions to be present, and we describe differences between sequencing and traditional sizing methods.