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.      

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.     

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.     

Bone marrow transplantation attenuates the myopathic phenotype of a muscular mouse model of spinal muscular atrophy

Bone marrow (BM) transplantation was performed on a muscular mouse model of spinal muscular atrophy that had been created by mutating the survival of motor neuron gene (Smn) in myofibers only. This model is characterized by a severe myopathy and progressive loss of muscle fibers leading to paralysis. Transplantation of wild-type BM cells following irradiation at a low dose (6 Gy) improved motor capacity (+85%). This correlated with a normalization of myofiber number associated with a higher number of regenerating myofibers (1.6-fold increase) and an activation of CD34 and Pax7 satellite cells. However, BM cells had a very limited capacity to replace or fuse to mutant myofibers (2%). These data suggest that BM transplantation was able to attenuate the myopathic phenotype through an improvement of skeletal muscle regeneration of recipient mutant mice, a process likely mediated by a biological activity of BM-derived cells. This hypothesis was further supported by the capacity of muscle protein extracts from transplanted mutant mice to promote myoblast proliferation in vitro (1.6-fold increase). In addition, a tremendous upregulation of hepatocyte growth factor (HGF), which activates quiescent satellite cells, was found in skeletal muscle of transplanted mutants compared with nontransplanted mutants. Eventually, thanks to the Cre-loxP system, we show that BM-derived muscle cells were strong candidates harboring this biological activity. Taken together, our data suggest that a biological activity is likely involved in muscle regeneration improvement mediated by BM transplantation. HGF may represent an attractive paracrine mechanism to support this activity.     

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

Human Molecular     

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.     

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.     

Developmental and degenerative cardiac defects in the Taiwanese mouse model of severe spinal muscular atrophy

Spinal muscular atrophy (SMA), an autosomal recessive disease caused by a decrease in levels of the survival motor neuron (SMN) protein, is the most common genetic cause of infant mortality. Although neuromuscular pathology is the most severe feature of SMA, other organs and tissues, including the heart, are also known to be affected in both patients and animal models. Here, we provide new insights into changes occurring in the heart, predominantly at pre‐ and early symptomatic ages, in the Taiwanese mouse model of severe SMA. Thinning of the interventricular septum and dilation of the ventricles occurred at pre‐ and early symptomatic ages. However, the left ventricular wall was significantly thinner in SMA mice from birth, occurring prior to any overt neuromuscular symptoms. Alterations in collagen IV protein from birth indicated changes to the basement membrane and contributed to the abnormal arrangement of cardiomyocytes in SMA hearts. This raises the possibility that developmental defects, occurring prenatally, may contribute to cardiac pathology in SMA. In addition, cardiomyocytes in SMA hearts exhibited oxidative stress at pre‐symptomatic ages and increased apoptosis during early symptomatic stages of disease. Heart microvasculature was similarly decreased at an early symptomatic age, likely contributing to the oxidative stress and apoptosis phenotypes observed. Finally, an increased incidence of blood retention in SMA hearts post‐fixation suggests the likelihood of functional defects, resulting in blood pooling. These pathologies mirror dilated cardiomyopathy, with clear consequences for heart function that would likely contribute to potential heart failure. Our findings add significant additional experimental evidence in support of the requirement to develop systemic therapies for SMA capable of treating non‐neuromuscular pathologies.     

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.     

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.      

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.     

Analysis of a read-through promoting compound in a severe mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is the leading genetic cause of infantile death and caused by the loss of functional Survival Motor Neuron 1 (SMN1). The remaining copy gene, SMN2, is unable to rescue from disease because the primary gene product lacks the final coding exon, exon 7, due to an alternative splicing event. While SMNΔ7 is a rapidly degraded protein, exon 7 is not specifically required in a sequence-specific manner to confer increased functionality to this truncated protein. Based upon this molecular observation, aminoglycosides have been examined to artificially elongate the C-terminus of SMNΔ7 by "read-through" of the stop codon. An SMNΔ7 read-through event benefits intermediate mouse models of SMA. Here we demonstrate that delivery of a read-through inducing compound directly to the CNS can partially lessen the severity of a severe model of SMA (Smn(-/-); SMN2(+/+)), albeit not to the extent seen in the less severe model. This further demonstrates the utility of read-through inducing compounds in SMA.     

Neurotransmitter release in motor nerve terminals of a mouse model of mild spinal muscular atrophy

Spinal muscular atrophy is a genetic disease which severity depends on the amount of SMN protein, the product of the genes SMN1 and SMN2. Symptomatology goes from severe neuromuscular impairment leading to early death in infants to slow progressing motor deficits during adulthood. Much of the knowledge about the pathophysiology of SMA comes from studies using genetically engineered animal models of the disease. Here we investigated one of the milder models, the homozygous A2G SMA mice, in which the level of the protein is restored to almost normal levels by the addition of a mutated transgene to the severe SMN-deficient background. We examined neuromuscular function and found that calcium-dependent neurotransmitter release was significantly decreased. In addition, the amplitude of spontaneous endplate potentials was decreased, the morphology of NMJ altered, and slight changes in short-term synaptic plasticity were found. In spite of these defects, excitation contraction coupling was well preserved, possibly due to the safety factor of this synapse. These data further support that the quasi-normal restoration of SMN levels in severe cases preserves neuromuscular function, even when neurotransmitter release is significantly decreased at motor nerve terminals. Nevertheless, this deficit could represent a greater risk of motor impairment during aging or after injuries.     

Therapeutic benefits of cardiotrophin-1 gene transfer in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a recessive autosomal disorder characterized by degeneration of lower motor neurons caused by mutations of the survival motor neuron gene (SMN1). No curative treatment is known so far. Mutant mice carrying homozygous deletion of Smn exon 7 directed to neurons display skeletal muscle denervation, moderate loss of motor neuron cell bodies and severe axonal degeneration. These features, similar to those found in human SMA, strongly suggest the involvement of a dying back process of motor neurons and led us to test whether neurotrophic factors might have a protective role in SMA. We report here the therapeutic benefits of systemic delivery of cardiotrophin-1 (CT-1), a neurotrophic factor belonging to the IL-6 cytokine family. Intra-muscular injection of adenoviral vector expressing CT-1, even at very low dose, improves median survival, delays motor defect of mutant mice and exerts protective effect against loss of proximal motor axons and aberrant cytoskeletal organization of motor synaptic terminals. In spite of the severity of SMA phenotype in mutant mice, CT-1 is able to slow down disease progression. Neuroprotection could be regarded as valuable therapeutic approach in SMA.