Masahisa Katsuno

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.

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.

Leuprorelin rescues polyglutamine-dependent phenotypes in a transgenic mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease that affects males. It is caused by the expansion of a polyglutamine (polyQ) tract in androgen receptors. Female carriers are usually asymptomatic. No specific treatment has been established. Our transgenic mouse model carrying a full-length human androgen receptor with expanded polyQ has considerable gender-related motor impairment. This phenotype was abrogated by castration, which prevented nuclear translocation of mutant androgen receptors. We examined the effect of androgen-blockade drugs on our mouse model. Leuprorelin, a lutenizing hormone–releasing hormone (LHRH) agonist that reduces testosterone release from the testis, rescued motor dysfunction and nuclear accumulation of mutant androgen receptors in male transgenic mice. Moreover, leuprorelin treatment reversed the behavioral and histopathological phenotypes that were once caused by transient increases in serum testosterone. Flutamide, an androgen antagonist promoting nuclear translocation of androgen receptors, yielded no therapeutic effect. Leuprorelin thus seems to be a promising candidate for the treatment of SBMA.

Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis.

The causative pathomechanism of sporadic amyotrophic lateral sclerosis (ALS) is not clearly understood. Using microarray technology combined with laser‐captured microdissection, gene expression profiles of degenerating spinal motor neurons isolated from autopsied patients with sporadic ALS were examined. Gene expression was quantitatively assessed by real‐time reverse transcription polymerase chain reaction and in situ hybridization. Spinal motor neurons showed a distinct gene expression profile from the whole spinal ventral horn. Three percent of genes examined were downregulated, and 1% were upregulated in motor neurons. Downregulated genes included those associated with cytoskeleton/axonal transport, transcription, and cell surface antigens/receptors, such as dynactin, microtubule‐associated proteins, and early growth response 3 (EGR3). In contrast, cell death–associated genes were mostly upregulated. Promoters for cell death pathway, death receptor 5, cyclins A1 and C, and caspases‐1, ‐3, and ‐9, were upregulated, whereas cell death inhibitors, acetyl‐CoA transporter, and NF‐κB were also upregulated. Moreover, neuroprotective neurotrophic factors such as ciliary neurotrophic factor (CNTF), Hepatocyte growth factor (HGF), and glial cell line–derived neurotrophic factor were upregulated. Inflammation‐related genes, such as those belonging to the cytokine family, were not, however, significantly upregulated in either motor neurons or ventral horns. The motor neuron–specific gene expression profile in sporadic ALS can provide direct information on the genes leading to neurodegeneration and neuronal death and are helpful for developing new therapeutic strategies. Ann Neurol 2005;57:236–251

Overexpression of IGF-1 in Muscle Attenuates Disease in a Mouse Model of Spinal and Bulbar Muscular Atrophy

Expansion of a polyglutamine tract in the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA). We previously showed that Akt-mediated phosphorylation of AR reduces ligand binding and attenuates the mutant AR toxicity. Here, we show that in culture insulin-like growth factor 1 (IGF-1) reduces AR aggregation and increases AR clearance via the ubiquitin-proteasome system through phosphorylation of AR by Akt. In vivo, SBMA transgenic mice overexpressing a muscle-specific isoform of IGF-1 selectively in skeletal muscle show evidence of increased Akt activation and AR phosphorylation and decreased AR aggregation. Augmentation of IGF-1/Akt signaling rescues behavioral and histopathological abnormalities, extends the life span, and reduces both muscle and spinal cord pathology of SBMA mice. This study establishes IGF-1/Akt-mediated inactivation of mutant AR as a strategy to counteract disease in vivo and demonstrates that skeletal muscle is a viable target tissue for therapeutic intervention in SBMA.

CHIP Overexpression Reduces Mutant Androgen Receptor Protein and Ameliorates Phenotypes of the Spinal and Bulbar Muscular Atrophy Transgenic Mouse Model

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine tract within the androgen receptor (AR). The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem and diffuse nuclear accumulation and nuclear inclusions of the mutant AR in the residual motor neurons and certain visceral organs. Many components of the ubiquitin-proteasome and molecular chaperones are also sequestered in the inclusions, suggesting that they may be actively engaged in an attempt to degrade or refold the mutant AR. C terminus of Hsc70 (heat shock cognate protein 70)-interacting protein (CHIP), a U-box type E3 ubiquitin ligase, has been shown to interact with heat shock protein 90 (Hsp90) or Hsp70 and ubiquitylates unfolded proteins trapped by molecular chaperones and degrades them. Here, we demonstrate that transient overexpression of CHIP in a neuronal cell model reduces the monomeric mutant AR more effectively than it does the wild type, suggesting that the mutant AR is more sensitive to CHIP than is the wild type. High expression of CHIP in an SBMA transgenic mouse model also ameliorated motor symptoms and inhibited neuronal nuclear accumulation of the mutant AR. When CHIP was overexpressed in transgenic SBMA mice, mutant AR was also preferentially degraded over wild-type AR. These findings suggest that CHIP overexpression ameliorates SBMA phenotypes in mice by reducing nuclear-localized mutant AR via enhanced mutant AR degradation. Thus, CHIP overexpression would provide a potential therapeutic avenue for SBMA.

Phase 2 trial of leuprorelin in patients with spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a hereditary motor neuron disease caused by the expansion of a polyglutamine tract in the androgen receptor (AR). Animal studies have shown that the pathogenesis of SBMA is dependent on serum testosterone level. This study is aimed at evaluating the efficacy and safety of androgen deprivation by leuprorelin acetate in patients with SBMA.

Efficacy and safety of leuprorelin in patients with spinal and bulbar muscular atrophy (JASMITT study): a multicentre, randomised, double-blind, placebo-controlled trial

BACKGROUND Spinal and bulbar muscular atrophy is a hereditary motor neuron disease caused by the expansion of a polyglutamine tract in the androgen receptor. At present there are no treatments for spinal and bulbar muscular atrophy, although leuprorelin suppressed the accumulation of pathogenic androgen receptors in a phase 2 trial. We aimed to assess the efficacy and safety of leuprorelin for spinal and bulbar muscular atrophy. METHODS The Japan SBMA Interventional Trial for TAP-144-SR (JASMITT) was a 48-week, randomised, double-blind, placebo-controlled trial done at 14 hospitals between August, 2006, and March, 2008. Patients with spinal and bulbar muscular atrophy were randomly assigned (1:1) by minimisation to subcutaneous 11.25 mg leuprorelin or identical placebo every 12 weeks. Patients and investigators were masked to treatment allocation. The primary endpoint was pharyngeal barium residue, which indicates incomplete bolus clearance, measured at week 48 by videofluorography. All patients who were randomly assigned and who were assessed with videofluorography at least once were included in the analyses. This study is registered with the JMACCT clinical trials registry, number JMA-IIA00009, and the UMIN clinical trials registry, number UMIN000000465. FINDINGS 204 patients were randomly assigned and 199 started treatment: 100 with leuprorelin and 99 with placebo. At week 48, the pharyngeal barium residue after initial swallowing had changed by -5.1% (SD 21.0) in the leuprorelin group and by 0.2% (18.2) in the placebo group (difference between groups -5.3%; 95% CI -10.8 to 0.3; p=0.063). The mean difference in pharyngeal barium residue after piecemeal deglutition at week 48 was -3.2% (-6.4 to 0.0; p=0.049), but there was no significant difference between the groups after covariate adjustment for the baseline data (-4.1 to 1.6; p=0.392). In a predefined subgroup analysis, leuprorelin treatment was associated with a greater reduction in barium residue after initial swallowing than was placebo in patients with a disease duration less than 10 years (difference between groups -9.8, -17.1 to -2.5; p=0.009). There were no significant differences in the number of drug-related adverse events between groups (57 of 100 in the leuprorelin group and 54 of 99 in the placebo group; p=0.727). INTERPRETATION 48 weeks of treatment with leuprorelin did not show significant effects on swallowing function in patients with spinal and bulbar muscular atrophy, although it was well tolerated. Disease duration might influence the efficacy of leuprorelin and thus further clinical trials with sensitive outcome measures should be done in subpopulations of patients. FUNDING Large Scale Clinical Trial Network Project, Japan and Takeda Pharmaceuticals.

Disulfide bond mediates aggregation, toxicity, and ubiquitylation of familial amyotrophic lateral sclerosis-linked mutant SOD1

Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathological hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insoluble SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermolecular disulfide bonds. Here we show that a non-physiological intermolecular disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high molecular weight aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degradation. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys6- and Cys111-disulfide cross-linked form and targeted it for proteasomal degradation.

Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions

FUS is an RNA-binding protein that regulates transcription, alternative splicing, and mRNA transport. Aberrations of FUS are causally associated with familial and sporadic ALS/FTLD. We analyzed FUS-mediated transcriptions and alternative splicing events in mouse primary cortical neurons using exon arrays. We also characterized FUS-binding RNA sites in the mouse cerebrum with HITS-CLIP. We found that FUS-binding sites tend to form stable secondary structures. Analysis of position-dependence of FUS-binding sites disclosed scattered binding of FUS to and around the alternatively spliced exons including those associated with neurodegeneration such as Mapt, Camk2a, and Fmr1. We also found that FUS is often bound to the antisense RNA strand at the promoter regions. Global analysis of these FUS-tags and the expression profiles disclosed that binding of FUS to the promoter antisense strand downregulates transcriptions of the coding strand. Our analysis revealed that FUS regulates alternative splicing events and transcriptions in a position-dependent manner.