ARTICLE IN BRIEF
Two research teams, working with induced pluripotent stem cells from patients with amyotrophic lateral sclerosis (ALS), were able to decipher new mechanisms for ALS progression and potential therapeutic targets.
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By using induced pluripotent stem cells (iPSCs) derived from actual patients, researchers at Harvard and the University of Wisconsin have revealed new insights about the mechanisms that drive the advancing muscle paralysis of amyotrophic lateral sclerosis (ALS).
The researchers achieved their results by building on their own past discoveries.
In a 2008 paper in Science, Kevin C. Eggan, PhD, an associate professor in the Harvard University department of stem cell and regenerative biology, and colleagues reported that they had produced induced pluripotent stem (iPS) cells from an 82-year-old woman diagnosed with a familial form of ALS. The researchers then directed these iPS cells, which contained the patient's own DNA, to differentiate into the type of motor neurons destroyed by ALS, thereby providing a model containing the underlying genetic defects that drive the disease.
In their current work, reported in the April 10 Cell Reports, the Harvard researchers produced iPS cells from an ALS patient, and then used gene targeting to correct the mutation causing symptoms, thereby creating “an ideal control,” according to Dr. Eggan. They made motor neurons from these control cells as well as from the patient's cells.
“Using a combination of classical and multi-electrode array recordings, we found that the mutant cells were more active in both evoked and spontaneous settings,” said Dr. Eggan, who collaborated on the recordings with the paper's first author, Brian Wainger, MD, PhD, an instructor at Harvard Medical School, and Clifford Woolf, MD, PhD, professor in neurology and neurobiology at Harvard Medical School and Boston Children's Hospital.
“Careful analysis of currents in the motor neurons revealed a smaller rectifying potassium current in these neurons, which was corrected by the potassium channel opener retigabine.”
The drug, used to treat the hyperexcitability of neurons in people with epilepsy, improved the survival of the mutant motor neurons. “Importantly, we found that this increased activity was also found in a larger cohort of motor neurons similarly produced from additional ALS patients and also blocked by retigabine,” Dr. Eggan said.
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The hyperexcitability of motor neurons in ALS detected by the Harvard researchers has prompted a phase I clinical trial of retigabine, an anticonvulsive already approved by the Food and Drug Administration that acts on potassium channels to quell excessive firing of neurons.
The clinical trial, which will test the safety of the drug in ALS patients, will be conducted by Dr. Wainger and Merit E. Cudkowicz, MD, chief of the neurology service and director of the Amyotrophic Lateral Sclerosis Clinic and of the Neurological Clinical Research Institute at Massachusetts General Hospital.
Much work remains to be done before ALS is fully understood and effective treatments are available, said Dr. Eggan. “The big problem in ALS is that there are more than 100 mutations in dozens of genes that all cause the disease,” he said. “But almost all of the therapeutics that have gone forward in the clinic have done so for just one of those mutations, superoxide dismutase 1 (SOD1), which almost everyone studies in mice.”
However, many mutations in ALS patients appear to trigger hyperexcitability in motor neurons, which seems to contribute to ALS rather than result from it, according to Dr. Wainger.
“If the motor neuron hyperexcitability were more of an upstream event, we'd expect that blocking it might affect motor neuron survival, which is precisely what we saw,” Dr. Wainger told Neurology Today. “If it were a downstream coincident observation, then blocking the hyperexcitability would be less likely to reduce cell death.”
DEFECTS IN NEUROFILAMENTS
The research at the University of Wisconsin, led by Su-Chun Zhang, MD, PhD, a professor of neuroscience and neurology, found defects in neurofilaments, which transport neurotransmitters and other substances from the cell body to the terminal of very long motor neurons.
A decade ago, Dr. Zhang and colleagues developed a method of growing motor neurons from human embryonic stem cells. In the current study, reported in the April 3 online edition of Cell Stem Cell, he used a variation of that method to transform skin cells from ALS patients into motor neurons that contain the same mutations found in the donor. Like the Harvard researchers, he and colleagues also created control cells by correcting the SOD1 D90A mutation and by expressing a SOD1 D90A in a wild-type, or non-disease background.
By comparing the cells, they found that spinal motor neurons exhibited neurofilament aggregation early in the disease process. Gene editing to correct the mutation rescued the motor neurons from neurofilament breakdown. Further investigation revealed that mutant SOD1 altered the transcription of neurofilament subunits, causing breakdown and aggregation, as well as neurite degeneration.
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Dr. Zhang and his colleagues showed that misregulation of neurofilament protein causes neurofilaments to become disorganized. The same process might be involved in Alzheimer's, Parkinson's, and other neurodegenerative diseases, he added, and appears to begin when the cells are still young. This suggests it is a cause rather than a consequence of pathology.
Mutant SOD1 leads to transcriptional disruption of one of three proteins that make up neurofilaments, the researchers explained. This results in the disorganization of neurofilaments, followed by their aggregation and neurite degeneration. The cells were rescued when the researchers edited the gene that produces the defective protein, resulting in normal cells. Scientists at the Small Molecule Screening and Synthesis Facility at the University of Wisconsin are already testing candidate drugs that may rescue cells from this genetic defect.
The research would have been impossible without the use of iPS cells, according to Dr. Zhang.
“The ability to derive human motor neurons is critical for our finding about the change in neurofilament subunits,” Dr. Zhang told Neurology Today. “If enriched motor neurons can be similarly isolated from transgenic mice, one might also reveal a similar change. The problem may be that the high level of expression of mutant proteins — for example, SOD1 — overshadows the change in neurofilaments in animal studies.”
Other researchers agree that the use of iPS cells from ALS patients will certainly provide much more reliable information about the mechanisms that drive the disease process.
“There are many significant differences in the mouse model of ALS,” said Ole Isacson, MD, a professor of neurology at Harvard and director of the Center for Neuroregeneration Research Institute at McLean Hospital in Belmont, MA. “For example, SOD1 mice may have as many as 35–40 copies of the human mutated gene. Obviously, in ALS there is a mutation in only one allele, so the animal model is an exaggeration of the disease. So SOD mice for ALS have reputation for being sort of non-predictive. People have been fooled more than once into believing that a mouse model represents the disease.”
Arnold Kriegstein, MD, PhD, said iPS cells from ALS patients allow the investigation of disease mechanisms even when the precise mutation driving the pathology isn't known.
“What can be done with patient iPS cells that isn't possible with mouse models is to examine diseases for which we don't know the exact genetic cause,” said Dr. Kriegstein, director of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, and professor of neurology at the University of California, San Francisco. “In the mouse you have to mutate a particular gene, creating a genetically modified animal, but in humans you can create a disease cell by using iPS cell technology, and then study the mechanisms of the disease without even knowing exactly which genes might be causative. I feel the use of iPS cells is most exciting for this type of application — for unraveling disease mechanisms, and for discovering potential drug targets.”
And Mahendra Rao, MD, PhD, believes the research at Harvard and the University of Wisconsin sets the stage for “big advances” in the use of iPS cells to investigate ALS and other neurodegenerative diseases.
“The issue has always been, when you make iPS cells, will they reflect the disease, which is chronic and occurs late in life?” said Dr. Rao, former director of the Intramural Center for Regenerative Medicine at the National Institutes of Health who, in April, joined Q Therapeutics, Inc. as chief strategy officer and chairman of the scientific advisory board. “The data from both of these groups suggest there is some phenotype, which is a big step forward, and that phenotype mimics what people have seen in animals. That says that all this effort to make iPS lines is a good thing. In fact, the more people who have access to these cell lines the better, because then you have many minds working on something that's very useful.”
Dr. Eggan, who is also affiliated with the Howard Hughes Medical Institute, was the lead author of a 2008 Science paper that envisioned the possibility of differentiating iPS cells from ALS patients into motor neurons, but he acknowledges that the technique has limitations.
“Nerve cells inside of an animal or inside of a patient have many other checks on their activity we can't model in vitro,” he told Neurology Today. “For instance, a change in the absolute amount of activity in a neuron in the body can be governed by a variety of factors — the glial cells around it, and the number of inhibitory inputs it has on it. Eventually there will have to be human tests of these hypotheses. Nevertheless we feel strongly that a detailed understanding of how these ALS-causing mutations proximally change the most sensitive cell types in the disease is valuable information. We firmly believe that understanding those proximal changes and inhibiting them is bound to be useful in patients.”
The cost of working with iPSCs is also a challenge, according to Dr. Eggan, although the cost of animal models is not trivial. “Having large mouse colony is expensive too. I think people underestimate the number of mice needed to do these experiments well. The cost (of iPSCs) is not astronomically beyond mouse modeling.”
Besides, drug companies, frustrated by findings in animal models that fail to hold up in human trials, have become far more interested in research involving iPSCs, according to Dr. Wainger. “Drug companies are concerned about the failure to translate successes from mice to humans,” he said. “There's a tremendous advantage in working with human cells where human proteins are functioning in their native environment. It allows for modeling additional complexities, such as genetic variation, that are hard to model in mice. There are no ways to model non-SOD1 ALS and sporadic variants in mice. iPS neurons give an opportunity to do that.”
STEM CELL EXPERTS COMMENT ON THE ALS FINDINGS
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