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.     

Founder effect in spinal and bulbar muscular atrophy (SBMA)

We analyzed the polymorphic (CAG)n and (GGC)n repeats of the androgen receptor gene in 113 unrelated X-linked spinal and bulbar muscular atrophy (SBMA) X chromosomes and 173 control X chromosomes in Japanese males. The control chromosomes had an average CAG repeat number of 21 +/- 3 with a range from 14-32 repeat units, and SBMA chromosomes had a range from 40-55 with a median of 47 +/- 3 copies. The control chromosomes had seven different alleles of the (GGC)n repeat with the range of 11 to 17; the most frequent size of (GGC)n was 16 (79%), while (GGC)17 was very rare (1%). However, in SBMA chromosomes only two alleles were seen; the most frequent size of (GGC)n was 16 (61%) followed by 17 (39%). (GGC)n size distribution was significantly different between SBMA and control chromosomes (P < 0.0001), indicating the presence of linkage disequilibrium. There was no allelic association between the (CAG)n and (GGC)n microsatellites among control subjects as well as SBMA patients, which suggests that a founder effect makes a more significant contribution to generation of Japanese SBMA chromosomes than new mutations.      

Founder effect in spinal and bulbar muscular atrophy (SBMA) in Scandinavia

We haplotyped 13 Finnish, 10 Swedish, 12 Danish and 2 Norwegian SBMA (spinal and bulbar muscular atrophy, Kennedy disease) families with a total of 45 patients and 7 carriers for 17 microsatellite markers spanning a 25.2 cM region around the androgen receptor gene on chromosome Xq11-q12 in search of a genetic founder effect. In addition, the haplotypes of 50 Finnish, 20 Danish and 22 Swedish control males were examined. All the Scandinavian SBMA families shared the same 18 repeat allele for the intragenic GGC repeat, which was present in only 24% of the controls. Linkage disequilibrium was also seen for the closest microsatellite markers. In addition, extended haplotypes of the Finnish, Swedish and Danish SBMA families revealed country-specific common founder haplotypes, which over time became gradually shortened by recombinations. No common haplotype was found among the controls. The data suggest that the SBMA mutation was introduced into western Finland 20 generations ago. Haplotype analysis implies a common ancestor for the majority of Scandinavian SBMA patients.     

Tremor in X-linked recessive spinal and bulbar muscular atrophy (Kennedy's disease)

OBJECTIVE:

To study tremor in patients with X-linked recessive spinobulbar muscular atrophy or Kennedy''s disease.

METHODS:

Ten patients (from 7 families) with a genetic diagnosis of Kennedy''s disease were screened for the presence of tremor using a standardized clinical protocol and followed up at a neurology outpatient clinic. All index patients were genotyped and showed an expanded allele in the androgen receptor gene.

RESULTS:

Mean patient age was 37.6 years and mean number of CAG repeats 47 (44-53). Tremor was present in 8 (80%) patients and was predominantly postural hand tremor. Alcohol responsiveness was detected in 7 (88%) patients with tremor, who all responded well to treatment with a β-blocker (propranolol).

CONCLUSION:

Tremor is a common feature in patients with Kennedy''s disease and has characteristics similar to those of essential tremor.     

Clearance of the mutant androgen receptor in motoneuronal models of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease caused by an abnormal expansion of a tandem CAG repeat in exon 1 of the androgen receptor (AR) gene that results in an abnormally long polyglutamine tract (polyQ) in the AR protein. As a result, the mutant AR (ARpolyQ) misfolds, forming cytoplasmic and nuclear aggregates in the affected neurons. Neurotoxicity only appears to be associated with the formation of nuclear aggregates. Thus, improved ARpolyQ cytoplasmic clearance, which indirectly decreases ARpolyQ nuclear accumulation, has beneficial effects on affected motoneurons. In addition, increased ARpolyQ clearance contributes to maintenance of motoneuron proteostasis and viability, preventing the blockage of the proteasome and autophagy pathways that might play a role in the neuropathy in SBMA. The expression of heat shock protein B8 (HspB8), a member of the small heat shock protein family, is highly induced in surviving motoneurons of patients affected by motoneuron diseases, where it seems to participate in the stress response aimed at cell protection. We report here that HspB8 facilitates the autophagic removal of misfolded aggregating species of ARpolyQ. In addition, though HspB8 does not influence p62 and LC3 (two key autophagic molecules) expression, it does prevent p62 bodies formation, and restores the normal autophagic flux in these cells. Interestingly, trehalose, a well-known autophagy stimulator, induces HspB8 expression, suggesting that HspB8 might act as one of the molecular mediators of the proautophagic activity of trehalose. Collectively, these data support the hypothesis that treatments aimed at restoring a normal autophagic flux that result in the more efficient clearance of mutant ARpolyQ might produce beneficial effects in SBMA patients.     

Somatic stability of the expanded CAG trinucleotide repeat in X-linked spinal and bulbar muscular atrophy

Expansion of trinucleotide repeats has now been associated with eight inherited diseases: X-linked spinal and bulbar muscular atrophy, two fragile X syndromes, myotonic dystrophy, Huntington's disease, spinocerebellar ataxia type I, dentatorubral pallidoluysian atrophy and Machado-Joseph disease. It has been shown that these expanded DNA repeats are unstable in number when transmitted from parents to offspring (“meiotic instability”), while somatic variation in repeat number has also been found in the fragile X syndrome and myotonic dystrophy. Moderate meiotic instability has been demonstrated in X-linked spinal and bulbar muscular atrophy (SBMA, Kennedy's disease). In order to determine if the expanded CAG repeat in SBMA also shows somatic instability, we compared different tissues from two patients with SBMA. We then examined the in vitro stability of the CAG repeat expansion by analyzing fibroblast cell cultures. Length comparison of expanded CAG repeats from all these materials clearly demonstrates that the CAG trinucleotide repeat in SBMA does not exhibit somatic variation. © 1996 Wiley-Liss, Inc.     

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.      

Aggregation and proteasome: the case of elongated polyglutamine aggregation in spinal and bulbar muscular atrophy

Aggregates, a hallmark of most neurodegenerative diseases, may have different properties, and possibly different roles in neurodegeneration. We analysed ubiquitin-proteasome pathway functions during cytoplasmic aggregation in polyglutamine (polyQ) diseases, using a unique model of motor neuron disease, the SpinoBulbar Muscular Atrophy. The disease, which is linked to a polyQ tract elongation in the androgen receptor (ARpolyQ), has the interesting feature that ARpolyQ aggregation is triggered by the AR ligand, testosterone. Using immortalized motor neurons expressing ARpolyQ, we found that a proteasome reporter, YFPu, accumulated in absence of aggregates; testosterone treatment, which induced ARpolyQ aggregation, allowed the normal clearance of YFPu, suggesting that aggregation contributed to proteasome de-saturation, an effect not related to AR nuclear translocation. Using AR antagonists to modulate the kinetic of ARpolyQ aggregation, we demonstrated that aggregation, by removing the neurotoxic protein from the soluble compartment, protected the proteasome from an excess of misfolded protein to be processed.     

Mitotic and meiotic stability of the CAG repeat in the X-linked spinal and bulbar muscular atrophy gene

X-linked spinal and bulbar muscular atrophy (SBMA) occurs due to an expansion of the trinucleotide repeat (CAG)_n in the androgen receptor gene. Anticipation is relatively rare in SBMA in contrast to spinocerebellar ataxia type 1 (SCA1), and dentatorubral and pallidoluysian atrophy (DRPLA) which show obvious paternal anticipation. The differences in the CAG repeat number were compared among sperm, leukocytes and skeletal muscles of SBMA patients. In SBMA, the sperm of most patients and the skeletal muscle of all patients showed the same repeat number as their leukocytes, whereas the increase in the repeat number from leukocytes to sperm was evident in SCA1 and DRPLA patients. The higher mosaicism level in sperm compared with leukocytes was common in SBMA, SCA1 and DRPLA, and the level of sperm was lower in SBMA than in SCA1 and DRPLA. Thus, spermatogenesis was suggested to be strongly associated with paternal anticipation. The mosaicism level was smaller in SBMA than in other (CAG)_n expanded disorders, and smallest in the SBMA carrier females. These findings demonstrate that the CAG repeat in SBMA is relatively stable in mitotic and meiotic processes, and there is a possibility that the lower mosaicism level of the carrier females compared with the SBMA patients is associated with X-linked recessive inheritance.     

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.     

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.     

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.     

Animal models of spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) is the second most common autosomal recessive inherited disorder in humans. It is the most common genetic cause of infant mortality. As yet, there is no cure for this neuromuscular disorder which affects the lower motor neurons and proximal muscles of the limbs and trunk. In the last decade, significant advances have been made in understanding this disease, from linkage analysis to isolating the defective gene and identifying its protein product. This review summarizes the most recent advance in SMA research: the development of animal models of the disease, in particular mouse models of SMA. The SMA mice that we describe here present with symptoms similar to those seen in SMA patients. They promise to further the understanding of the molecular basis of this disease and demonstrate the feasibility of using the intact SMN2 gene, found in all SMA patients, as a means of treating this disorder.     

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.     

Gene deletions in spinal muscular atrophy.

Two candidate genes (NAIP and SMN) have recently been reported for childhood onset spinal muscular atrophy (SMA). Although affected subjects show deletions of these genes, these deletions can lead to either a very mild or a severe phenotype. We have analysed a large number of clinically well defined patients, carriers, and normal controls to assess the frequency and extent of deletions encompassing both of these genes. A genotype analysis indicates that more extensive deletions are seen in the severe form of SMA than in the milder forms. In addition, 1 center dot 9% of phenotypically normal carriers are deleted for the NAIP gene; no carriers were deleted for the SMN gene. Our data suggest that deletions in both of these genes, using the currently available assays, are associated with both a severe and very mild phenotype.