M. FLINT BEAL, M.D.
Weill Medical College of Cornell University
Q: You have made the case that oxidative damage plays a critical role in many different neurodegenerative diseases, and that mitochondrial dysfunction may be a common underlying mechanism. What leads you to this conclusion?
Beal: Mitochondria are the cell’s power plants. Their job is to produce energy for all the metabolic processing that goes on in a cell. So, you can imagine that if the cell’s power plants start to shut down, the cell is going to have a problem. Several lines of evidence suggest a critical role for oxidative damage—essentially the biological equivalent of rust—and mitochondrial dysfunction in many neurodegenerative diseases. For example, it’s been shown recently that genetic mutations affecting mitochondrial proteins or mitochondrial DNA (mtDNA), a short, specialized strand of DNA coiled within mitochondria, produce a number of damaging effects on cells that seem to accelerate normal aging. Since the incidence of most neurodegenerative diseases increases with age, it’s possible that these processes may set the stage for disease.
There is also strong evidence from genetics research and from transgenic animal models that mitochondrial dysfunction results in neurodegeneration, and may contribute to the pathogenesis of Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and other movement disorders that involve degeneration of certain nerve cell populations. And, we are now seeing increasing data from clinical trials suggesting that anti-oxidant therapies can ameliorate symptoms in some of these diseases, which also bolsters the case for a role for oxidative damage in their progression.
Q: To what degree do these diseases represent an acceleration of “normal” age-related events?
A:The most important risk factor for neurodegenerative diseases such as AD, PD and ALS is advancing age. A major theory of aging is that mutations in mtDNA may contribute to the aging process, based on the long-standing evidence that mitochondrial mutations accumulate with normal aging. Various experts have speculated that this age-related accumulation of mutations might tip the delicate balance of energy homeostasis in the cell, leading to increased cellular damage from free radicals.
The most definitive findings to date that mtDNA mutations acquired during normal aging can contribute to the aging process comes from studies of mice genetically engineered to carry mutations in an enzyme called mtDNA polymerase PolgA. This enzyme is involved in both copying and “proofreading” mtDNA— eliminating errors that it makes during replication— and is thought to participate in DNA repair processes. Mice lacking this enzyme accumulated mtDNA mutations faster than normal mice, and had a striking physiology. At about 25 weeks of age, which is early adulthood in a mouse, they started to show signs of premature aging: they stopped gaining weight, became bald, and developed curved spines, a sign of clinical osteoporosis, as a result of low bone mineral density. Half of the animals were dead by 48 to 61 weeks, roughly half the life expectancy of a typical mouse.
Further data favoring the oxidative damage theory of aging comes from a study of gene expression in frontal cortex of humans aged 26 to 106 years. In individuals older than 76, specific genes involved in learning, memory and neuronal survival were down-regulated, and the molecules that normally help turn these genes on had markedly increased DNA damage. In culture dishes, these same “gene promoters” were vulnerable to oxidative stress, and showed reduced capacity for DNA repair. In laboratory experiments, silencing the mitochondrial genes that were down-regulated in the elderly subjects resulted in increased DNA damage to vulnerable nuclear genes, consistent with the idea that dysfunctional mitochondria are a source of free radicals in the aging brain.
Q: How does mitochondrial dysfunction relate to oxidative damage?
A: Mitochondria generate energy for the cell through a metabolic process called oxidative phosphorylation, which occurs in the presence of oxygen. If this energy metabolism is disrupted, it sets off a number of problems within the cell. These include reduced production of ATP, a compound that serves as the primary energy transporter in the cell, as well as impaired ability to limit calcium influx (which can damage cells), and generation of reactive oxygen species, or free radicals.
The generation of free radicals is increasingly recognized as playing an important role in both aging and neurodegenerative diseases. Mitochondria are both targets and important sources of free radicals—in fact, they are probably the major source of free radicals in most cells. Generation of free radicals appears to be increased in damaged mitochondria, and in cells with compromised mitochondrial function.
Q: What do these possible mechanisms suggest in terms of therapeutic development?
A: There is strong evidence from genetics and transgenic mouse models that mitochondrial dysfunction results in neurodegeneration, and may contribute to the pathogenesis of AD, PD, HD, ALS, hereditary spastic paraplegia, and cerebellar degenerations. Therapeutic approaches targeting mitochondrial dysfunction and oxidative damage in these diseases therefore have great promise.
“Many dietary supplements that improve energy metabolism have been proposed as potentially therapeutic.”
A role for mitochondrial dysfunction and oxidative damage in both normal aging and neurodegenerative diseases has been greatly strengthened by recent findings. An accumulation of mitochondrial mutations leads to accelerated aging and oxidative damage. This likely contributes to the age-dependence of neurodegenerative diseases such as AD, HD, and ALS. Friedreich’s ataxia and two forms of hereditary spastic paraplegia are caused by mutations in nuclear-encoded proteins affecting mitochondria. Mutations in certain nuclear-encoded mitochondrial proteins (e.g., PINK1, DJ-1 and Parkin) also cause PD. These findings further strengthen the possibility that treatment with agents that improve mitochondrial function or that exert anti-oxidant activity may be beneficial in neurodegenerative diseases. Many dietary supplements that improve energy metabolism have been proposed as potentially therapeutic.
We and others have studied creatine and coenzyme Q-10 in a number of neurodegenerative diseases, including ALS, HD, and PD. Creatine appears to be a particularly promising therapy. Creatine is used by the body to replenish ATP stores in tissues, including the brain and muscles. Oral creatine supplementation seems to induce at least two actions that may be neuroprotective: it increases brain levels of creatine and phosphocreatine (a precursor to creatine) and inhibits activation of the mitochondrial permeability transition, a protein complex that is emerging as a central player in triggering cell damage and death. Baseline phosphocreatine levels in brain and muscle are decreased in normal aging and in HD, Friedreich's ataxia, cerebral ischemia, and muscular diseases. In animal models, creatine is protective against compounds that are toxic to mitochondria (including MPTP, which is used to induce Parkinson’slike symptoms in animal models). Many questions remain unanswered, but clinical trials are under way to determine whether or not creatine, coenzyme Q10, and other agents that enhance energy metabolism benefit patients with HD, PD, ALS, muscular dystrophy, and other neuromuscular and neurodegenerative disorders.
MICHAEL ZIGMOND, Ph.D.
University of Pittsburgh
Q: Your research group is investigating if structured, reasonably strenuous exercise can have an effect on Parkinson’s symptoms. What led you to believe that exercise might be helpful?
Zigmond: This actually began with Tim Schallert at the University of Texas, Austin. He was interested in how exercise—or more accurately, forced limb use— might influence the effect of brain injury. At the same time, he had a longstanding interest in animal models of Parkinson’s disease and had developed a battery of sensitive tests. He put those things together, and examined the effects of exercise in this animal model. In 2000, Schallert, together with his graduate student Jennifer Tillerson, showed that if you forced laboratory rats to exercise by placing one forelimb in a cast, they did not show the neurological deficits that would normally be associated with an injection of 6-hydroxydopamine [6-OHDA, a toxin that destroys dopamine neurons and is used to model Parkinson’s disease in humans].
Tim asked us to look at the brains of these animals for biological effects, because we have the tools to do that. Initially, I had no interest in this project. It turned out to be a very naïve perspective, but it just seemed to me that there was no way that exercise would alter the effects of this very powerful toxin. Eventually, we made the measurements, however, and we discovered, to our surprise, that in fact, the lesion was dramatically reduced. There were animals that showed no detectable lesions even after receiving a dose of toxin that would normally reduce the number of dopamine terminals by 75% or more.
We’ve since done a variety of experiments, showing, for example, that exercise can completely block the neurodegenerative response, and that exercise can also be protective if it occurs prior to the lesion. We know that there is a window of opportunity for this exercise effect, but we don’t yet know exactly what it is. We do know, however, that if you wait for seven days and then force the animals to use their limb, it’s no longer protective.
Q: Is exercise having some kind of antioxidant effect?
A: 6-OHDA kills nerve cells by causing oxidative stress within the dopamine neurons. It is a molecule that is taken up and concentrated in dopamine neurons, where it breaks down, forms free radicals, and kills the neurons. We think that there are a number of critical biochemical changes that occur in the brain as a result of using a limb that may block this pathway at some point or another, making the cells temporarily more resistant to oxidative stress.
We also want to know what kinds of exercise are useful. The initial experiments used a cast to force use of the other limb, which is not going to be very interesting clinically. We want to know if the animals will be protected if they do other things to use their limbs, such as daily exercise on a treadmill or running wheel. In other words, we want to know whether exercise that is closer to what you and I would call exercise affords any protection against oxidative stress. We’ve started those studies and will probably know that answer in 2005.
Q: What have you found out about the molecular mechanisms that might be underlying this protective effect?
A:We think that the mechanism involves a group of compounds called trophic factors, and we suspect that a number of trophic factors are involved. We have focused first on GDNF [glial cell line derived neurotrophic factor], mostly for historical reasons. GDNF was the first trophic factor shown to be quite potent in protecting dopamine neurons, both from the cell death that occurs early in the development of these cells and when you expose these cells to 6-OHDA.
We’re now looking at a wide range of trophic factors to see which ones are increased. There is a whole body of evidence that BDNF [brain-derived neurotrophic factor] is increased after exercise, much of it from Carl Cotman’s work at the University of California at Irvine. He has focused on a different brain region, the hippocampus, and is interested in it with respect to a different neurological condition, Alzheimer’s. We’re looking at the striatum, an area that has been very closely linked to movement disorders. His work and our work suggest a general theme—that these trophic factors, which were important in development, can become important again, and may be a key to addressing certain pathological conditions.
Furthermore, these trophic factors may not be there solely for maintenance of neurons, but may be there to serve neurons that are in trouble—to stabilize them and keep them from dying. It may be that in Parkinson’s disease, for whatever reasons, this protective defense doesn’t happen, and that the exercise might be somehow waking up the brain’s endogenous potential for self-protection. We have other data that also suggest nerve cells have a built-in capacity for self-protection against insults, which may make use of the same mechanisms that were critical to keeping nerve cells from dying during development.
Q: You’ve begun a pilot clinical trial to test exercise in Parkinson’s and are gearing up for a blinded clinical trial. What is the goal?
A: The goal is to take our animal model into the clinic. Tony Delitto, the principal investigator in this project here at the University of Pittsburgh, has shown that this approach is feasible in people with Parkinson’s: You can get patients to come in three times a week to spend an hour or an hour and a half doing reasonably intense exercise. He’s also shown that people seem to have at least symptomatic improvement, though he has only looked at them for 12 weeks. Now, if we get the funding, we’ll do a double-blind study and will use PET [Positron Emission Tomography] imaging as well as neurological and psychiatric examinations. This would be the first time we’ll have used PET imaging in the Parkinson’s patients who have exercised, and we’ll be comparing the images to a set of biomarkers previously developed by Nicolaas Bohnen here in our Parkinson Center that correlate with the severity of dopamine loss as judged by PET.