Archived Research Updates
September 25, 2010
Microarray Analysis of Gene Expression by Skeletal Muscle of Three Mouse Models of Kennedy Disease/Spinal Bulbar Muscular Atrophy
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.
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.
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.
Sept 23, 2010
Study links normal function of protein, not its build up inside cells, to death of neurons
A new study led by St. Jude Children's Research Hospital scientists signals hope for treatment of a neurodegenerative disease and a new model for understanding the mechanism at work in other more common neurodegenerative disorders
A study led by St. Jude Children's Research Hospital investigators links the muscle weakness and other symptoms of a rare neurodegenerative disease to a misstep in functioning of a normal protein, rather than its build-up inside cells. The finding offers insight into the mechanism driving common nervous system disorders like Parkinson's and Alzheimer's diseases.
The work advances understanding of how the inherited mistake at the heart of spinobulbar muscular atrophy (SBMA) leads to the death of neurons in the brain and spinal cord. Investigators showed that the underlying mutation caused an amplification of the protein's normal function. The work appears in the September 23 online edition of the scientific journal Neuron.
"The idea that toxicity is mediated by the native, or normal, function of the protein itself is a departure from conventional wisdom. This research adds to growing evidence the principle applies very broadly in other neurodegenerative disorders, including Alzheimer's and Parkinson's diseases," said J. Paul Taylor, M.D., Ph.D., an associate member in the St. Jude Department of Developmental Neurobiology and the paper's senior author.
The current neurodegenerative disease model links the disorders to a toxic build-up of improperly folded proteins inside cells. Taylor said: "Our findings suggest the focus on protein aggregation inside cells may be misplaced." Developing therapies that target the normal protein function will likely be easier and more effective, he added.
Medications are already available to block the androgen receptor (AR) protein, which is mutated in SBMA. Work is now underway in Taylor's laboratory to identify drugs that more selectively block AR functioning.
SBMA belongs to a family of eight disorders, including Huntington's disease, which stem from an overabundance of the same small, repeated sequence of DNA known as a trinucleotide. Such repetitions are common throughout the genome, but problems arise when they occur too frequently. That is what happens in the estimated 1 in 50,000 males with SBMA.
In the case of SBMA, the repeated sequence occurs in the gene for the androgen receptor. The repeated nucleotide sequence CAG is protein-production shorthand for an amino acid called glutamine. The resulting androgen receptor (AR) protein includes surplus glutamine.
After earlier work by other investigators showed that blocking testosterone prevented male mice with the SBMA mutation from developing the disease, Taylor and his colleagues set out to track what happened inside cells after the hormone bound to the mutated AR protein.
Working in a Drosophila fruit fly model of the disease, the scientists identified a small region of the AR protein, known as the AF-2 domain, which played a pivotal role.
Using a variety of techniques, researchers demonstrated they could rescue the cells by preventing certain members of a family of proteins called coregulators from binding to the AF-2 domain. Coregulators partner with AR and other transcription factors to regulate gene expression.
"In this study, we showed the ability of the mutant protein to interact with the normal binding partners is an essential step in the cascade of degeneration. By blocking it, we block degeneration," Taylor said. He added that the AF-2 domain is far from the mutated region of the AR protein. "That would be unexpected if the mechanism of toxicity were related to the protein aggregating," he explained.
Meanwhile, investigators are still studying why the protein's change in function is so deadly to cells. Taylor noted that research into inherited diseases like SBMA has historically provided important clues into the mechanisms at work in other more common neurodegenerative disorders, including Alzheimer's.
The findings also hold hope that treating or preventing SBMA by selectively disrupting AF-2 binding will soon be possible, Taylor said. "Selectively blocking the hormone will be key if we hope to prevent the side effects associated with androgen ablation in males," he said. The side effects include bone thinning, infertility or blocked sexual maturation.
The study also suggests the need to begin treatment earlier. If the damage to motor neurons begins with the hormone surge of puberty rather than the accumulation of mis-folded proteins, therapies must begin in childhood, Taylor said.
Natalia Nedelsky, of St. Jude and the University of Pennsylvania, is the study's first author. The co-authors are Maria Pennuto, Italian Institute of Technology, Genoa, Italy; Isabella Palazzolo, National Institute of Neurological Disorders and Stroke; Zhiping Nie, University of Pennsylvania; and Rebecca Smith, Jennifer Moore and Geoffrey Neale, all of St. Jude.
The study was funded in part by the Muscular Dystrophy Association, the Kennedy's Disease Association, the National Institutes of Health and ALSAC.
Efficacy and safety of leuprorelin in patients with spinal and bulbar muscular atrophy (JASMITT study): a multicentre, randomised, double-blind, placebo-controlled trial.
Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan; Institute for Advanced Research, Nagoya University, Nagoya, Japan.
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. FUNDI
NG: Large Scale Clinical Trial Network Project, Japan and Takeda Pharmaceuticals. Copyright © 2010 Elsevier Ltd. All rights reserved.