ADVANCED ROBOTIC MICROSCOPE YIELDS CLEARER PICTURE OF HUNTINGTON’S
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ADVANCED ROBOTIC MICROSCOPE YIELDS CLEARER PICTURE OF HUNTINGTON’S
ATLANTA, October 16, 2006 - Researchers have gained deeper understanding of Huntington's and related diseases by using an advanced robotic microscope. A new model of the disease has given other researchers new insight into how the disease progresses, and other research has discovered that a drug already used to prevent seizures may help protect against the symptoms of Huntington's disease.
Huntington's disease (HD) is a hereditary neurodegenerative disease caused by a genetic mutation. It is among a group of lethal neurodegenerative diseases, called ataxias, that attack the regions of the brain included in coordinating movement and perhaps cognitive functions. As ataxia progresses, communication becomes increasingly difficult, either through speech, handwriting or both. Walking also becomes more difficult, usually leading to being wheelchair bound. In time, trouble with swallowing and breathing develop. Patients with HD usually die after 15 to 20 years following onset of symptoms.
"As the population of the United States ages, the neurodegenerative diseases are having an increasing impact on our health and quality of life," says Harry Orr, PhD, of the University of Minnesota. Orr will give a Presidential Special Lecture on neurodegenerative disorders at Neuroscience 2006 in Atlanta.
In work using a specially designed robotic microscope, researchers, for the first time, can see neurodegenerative processes as they occur within living cells. This critical advance allows scientists to answer questions about processes involved in a range of diseases, including Alzheimer's, Parkinson's, Huntington's diseases and amyotrophic lateral sclerosis. Combined with statistical analysis tools historically used in medical diagnostics, this important new technique also enables researchers to predict cell behavior and disease progression in vitro.
The first-generation system consists of a wide-field fluorescence microscope with customized robotics and software modifications that allow it to view activity inside individual cells at different time points. In a typical experiment, 300,000 cells are analyzed within minutes, a task that used to take well over a month.
The creator of the system, Steven Finkbeiner, MD, PhD, of the University of California, San Francisco, has used it to answer key questions about whether observed changes were detrimental, incidental, or beneficial. For example, for many years, protein aggregates called inclusion bodies that appear in Huntington's disease were assumed to be detrimental. Using the system, Finkbeiner was able to show that neurons that develop the aggregates lived longer than those that do not. Thus, the inclusion body is a beneficial reaction by the cell, and therapies focused on eliminating them might do more harm than good.
"Being able to view activity inside so many individual cells at different time points is like performing a clinical trial with millions of people. It's very sensitive, and we learn information we could not discover any other way," Finkbeiner says. "This approach provides a critical way to connect cellular changes to fate. The automated microscope system could be applied to answer many questions about cellular changes and abnormalities that affect disease."
In addition to providing greater understanding about disease processes, the system is also being used to search for therapies. The ability to rapidly determine the importance of a process to disease progression guides the selection of potential drug targets and facilitates the evaluation of thousands of candidate drugs for efficacy.
Using a well-known yeast model, researchers have discovered a genetic mutation in the development of Huntington's.
The yeast Saccharomyces cerevisiae, which has been used to brew beer and wine for millennia, is a commonly used laboratory organism. Numerous intracellular processes are well conserved between yeast and higher organisms, including humans. Studies in yeast have led to many significant, and often unexpected, advances in the knowledge of human diseases.
Research has recently developed a yeast model to study molecular mechanisms involved in Huntington's disease. When expressed in yeast, mutant huntingtin is toxic and forms inclusion bodies, as in neurons.
In a large-scale genetic screen in yeast, Paul Muchowski, PhD, of the Gladstone Institute of Neurological Disease in San Francisco, and colleagues identified mutations in 28 genes that suppress the toxicity of mutant huntingtin. Among the most potent was a mutation in the gene that encodes kynurenine 3-monooxygenase (KMO), which functions in the kynurenine pathway. In rodents, injection of quinolinic acid, a metabolite of this pathway, into the striatum reproduces the behavioral and pathological features of HD. The striatum is a region in the brain strongly affected by mutant huntingtin.
"This finding suggests that alterations in quinolinic acid metabolism may be central to HD pathophysiology," says Muchowski. "KMO is expressed predominantly in microglia, the immune cells of the brain, which are activated in HD patients and animal models of HD. Thus, our results suggest that some of the effects of mutant huntingtin may not be cell-autonomous and may also involve non-neuronal cell types."
Muchowski is now translating the studies from yeast into mouse models of HD. A high-affinity, small-molecule inhibitor of KMO developed by Roche Pharmaceuticals is available and is neuroprotective in models of brain ischemia and in a genetic model of paroxysmal dyskinesia. "Preliminary tests of the inhibitor in HD mice have shown promising results," says Muchowski. In addition to further analyzing the effects of this compound in HD mouse models, Muchowski's laboratory is also generating mice lacking KMO that will be crossed into HD mouse models.
In a preliminary study, Christopher Ross, MD, PhD, at Johns Hopkins University, found an FDA-approved anti-seizure drug, tiagabine, which extended survival by more than 15 percent and improved motor performance in a transgenic mouse model of HD.
"There is no currently available treatment which can delay the onset of Huntington's disease or slow its progression," says Ross. "Because tiagabine is an FDA-approved drug, it may be a promising candidate for further preclinical studies, and, if therapeutic levels are safely achievable in humans, perhaps clinical trials for the treatment of HD."
Ross and colleagues discovered the protective effects of tiagabine in Huntington's by screening a collection of FDA-approved drugs in a cell model of HD. HD is caused by a mutant form of the protein huntingtin. They previously developed a cell model of HD by inducing the expression of a fragment of mutant huntingtin, and screened a compound collection from the National Institute of Neurological Disorders and Stroke. In this screening, the researchers found that the inhibitory neurotransmitter Gamma-amino butyric acid (GABA) uptake inhibitor, nipecotic-acid, reduced mutant huntingtin induced toxicity.
"Because nipecotic-acid can not cross the blood-brain barrier, we studied its analogue, Tiagabine, which is able to cross the blood-brain barrier, in our transgenic HD mouse model," says Ross. After injecting tiagabine intraperitoneally in the HD mice, "the tiagabine showed the beneficial effect at a dose of 5 mg/kg, extended survival more than 15 percent, improved motor behavioral performance, and slowed body wasting," says Ross. The researchers also established a method to detect tiagabine levels both in brain tissues and blood samples of mice administered tiabagabine. They found that levels in serum are comparable to the effective levels in humans treated with tiagabine for seizure.
"We are currently investigating the effect of tiagabine on the transgenic mice by analyzing pathological changes in their brains," says Ross, "and we are also in the process of further investigating the mechanism by which tiagabine exerts its effect in this HD mice.
Because patients can be treated with tiagabine to control seizures for many years with few or no side effects, Ross hopes this class of anticonvulsants might be given to individuals who harbor HD-causing polyglutamine expansions in huntingtin before they become symptomatic.