Mitochondria: Cellular Energy Co.

Volume 16 | Issue 13 | 30 | Jun. 24, 2002 Previous | Next Mitochondria: Cellular Energy Co. Researchers strive to keep the energy pipeline open in the face of damaging cellular insults | By Amy Adams Where fossil fuels power the world, mitochondria power the cell. They provide the energy that allows your eyes to scan this page, and fuel each nerve impulse as your brain processes the words. Mu

Amy Adams
Jun 23, 2002
lab consumer
Volume 16 | Issue 13 | 30 | Jun. 24, 2002

Mitochondria: Cellular Energy Co.

Researchers strive to keep the energy pipeline open in the face of damaging cellular insults | By Amy Adams

Where fossil fuels power the world, mitochondria power the cell. They provide the energy that allows your eyes to scan this page, and fuel each nerve impulse as your brain processes the words. Mutations that impair mitochondrial proteins, encoded by both nuclear and mitochondrial genes,1 cause widespread disturbances in muscle, nerve, kidney, and other high-energy cells.

In the most severe cases, these mutations can cause childhood death; less severe cases result in degenerative diseases such as diabetes and Parkinson disease. "As you start to look at these large important diseases, sure enough there is a mitochondrial component," says James Dykens, associate director of business and corporate development at San Diego-based MitoKor. Researchers are actively pursuing new treatments for mitochondria-related conditions; to identify drug targets, they must learn how mitochondrial impairment harms the cell and find ways to protect mitochondria from damage.

THE BUCKET BRIGADE Mitochondria provide cells with energy in the form of adenosine triphosphate (ATP). The process starts when fuel from fat or sugar is broken down into two-carbon units called acetyl-CoA, which enter the Krebs cycle in the mitochondria and are oxidized to water and CO2. The resulting high-energy electrons transfer to NAD+ and FADH+ to produce NADH and FADH2, respectively. These reducing equivalents then transfer their electrons to protein complexes in the inner mitochondrial membrane.2

From there, the electrons are transported along the electron transport chain, which acts like a bucket brigade for electrons. The chain consists of three protein complexes and two smaller electron carriers that shuttle electrons between the complexes. As each pair of electrons is passed to the next complex, protons are pumped from the mitochondria to the cytoplasm, generating energy in the form of a proton gradient across the mitochondrial membrane.2 The protons reenter the mitochondria through ATP synthetase, driving the conversion of ADP to ATP.

As with any bucket brigade, some water--or electrons--inevitably gets spilled. "There is a slight leak in the insulation in at least two places, where a small proportion of electrons can leak out," says David Nicholls, professor of mitochondrial physiology at the Buck Institute for Age Research. In one case, electrons seep into the cytoplasm, while in the other, electrons flow back into the mitochondria.

Electrons combining with oxygen form superoxide,2 a molecular loose cannon that oxidizes molecules within the cell, causing DNA mutations and irreparable damage to proteins. To combat this problem, enzymes called superoxide dismutases (SODs) convert the superoxide into the moderately less dangerous hydrogen peroxide (H2O2). The antioxidant glutathione then converts H2O2 into water.2

Nicholls describes this as "the normal everyday crisis that every mitochondrion faces." But, any mutations that affect proteins in the electron transport chain will make the process less effective, meaning more electron spillage, increased generation of reactive oxygen species (ROS), and decreased ATP production.

DEATH OF A NEURON Several cellular insults, including mutations, toxins, and lack of oxygen, can induce mitochondrial failure, but the end result is the same: too little energy to meet the cell's demands. Most cells store glycogen and can derive some energy from glycolysis, but neurons are particularly susceptible to damage from low levels of ATP, owing to their high energy needs and almost exclusive reliance on mitochondria for energy.

ATP-driven ion pumps maintain neurons in a low-calcium state; the resulting calcium gradient is essential for the calcium signaling that plays such an important role in neuronal function.3 When calcium enters the cell, the mitochondria act as temporary reservoirs until the ion pumps can lower intracellular calcium to their usual levels. Without sufficient ATP to drive the pumps, however, mitochondrial calcium levels become excessive,4 further impairing the mitochondria and leading to intracellular accumulation of ROS. "Mitochondria cannot work in a high calcium environment," Nicholls explains.

ATP also functions in sequestering neurotransmitters such as glutamate in synaptic vesicles. When the neuron receives an impulse, these vesicles release glutamate into the synapse, where it triggers a calcium influx. The cell then quickly takes up the glutamate, thus removing the stimulation from the synapse and allowing neuronal calcium levels to normalize. But in a low-ATP state, glutamate remains in the synapse indefinitely, which exacerbates already-elevated calcium levels, increases ROS levels, and further impairs the mitochondrion.5

Eventually the calcium overload and excess ROS kill the cell. Tim Greenamyre, professor of neurology and pharmacology at Emory University, says that whether the cell dies by apoptosis or necrosis depends on how quick and severe the damage is. "It's like most insults. If you slam it they die from necrosis," he says. "If you don't, they get their affairs in order and commit suicide."

THE WEAKEST LINK When mutations severely impair mitochondrial proteins, the resulting diseases appear early and impact almost every cell type. That is not the case, however, with milder mutations. "It's a threshold process," Dykens explains. "As long as there is minimal energy [from ATP] then the person may be impaired but not ill." In these cases, he says, the disease may not appear until later in life, as in Parkinson and Alzheimer diseases. "In one cell or another, mitochondrial capacity can't keep pace with demand," he says, and disease results.

STUDYING MITOCHONDRIA Most tools for studying mitochondrial function are also used to study apoptosis, although not all cells with impaired mitochondria die by that mechanism. These tools include kits for isolating mitochondria, monitoring permeability, and tracking intracellular levels of calcium and reactive oxygen species (ROS). (See table)

A range of antibodies for mitochondrial proteins exists, though these are commonly marketed for apoptosis. Likewise, many companies offer fluorescent dyes that are targeted to mitochondria. Eugene, Ore.-based Molecular Probes offers a selection of MitoTracker dyes that are sequestered by the mitochondria and retained during cell fixation. Palo Alto, Calif.-based BD Biosciences-Clontech offers enhanced green fluorescent protein (EGFP) variants in cyan, yellow, and red that are targeted to the mitochondrial membrane.

Some kits for studying apoptosis detect the initial depolarization of the mitochondrial membrane; researchers can also use these to study depolarization in mitochondrial disease. These kits generally use the dye JC-1 to detect changes in membrane potential. Under normal membrane potentials, JC-1 aggregates in the mitochondria and produces red fluorescence. In the absence of membrane potential, JC-1 exists as monomers in the cytosol where it fluoresces green. This type of assay can by used for high-throughput screening of cells exposed to different conditions.

Cells with impaired mitochondria generally have higher ROS levels coupled with decreased levels of glutathione and superoxide dismutase to deal with that problem. Many kits are available to detect these cellular changes as they occur in diseased cells. Scientists can use these kits in high-throughput assays to detect cellular changes due to mitochondrial impairment.

Together, these tools help researchers better understand the pathogenesis of mitochondrial diseases. They also provide a way to screen potential drug candidates that preserve the mitochondrial membrane potential, reduce ROS, or protect the cell from calcium influx or excess glutamate.

Researchers discovered the link between mitochondria and Parkinson disease in 1982 when symptoms very much like Parkinson disease developed in drug addicts.6 The heroin they had injected contained a compound called MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridin),which is metabolized into a strong inhibitor of the first protein complex in the electron transport chain. This led researchers to track down complex I mutations in patients with Parkinson disease.

To better understand the association of complex I with Parkinson disease, Greenamyre administered a complex I inhibitor called rotenone to rats. Although the rotenone was systemic and therefore affected the mitochondria in all cells, the rats exhibited symptoms similar to Parkinson disease. Further, they had nerve cell degradation in the substantia nigra, the same brain region affected in patients with Parkinson.7 The rats also had neuronal protein aggregates similar to those found in people who have died from Parkinson disease.

Why cells in the substantia nigra are so affected by complex I mutations is "a million dollar question," says Greenamyre. He points out that both the synthesis and degradation of the neurotransmitter used by these cells produce H2O2 as a byproduct. As these cells exist in a constant state of oxidative stress, additional stress, whether genetic or pharmacological, could overload the cells. He suspects that many cellular defects associated with Parkinson disease result from the damage to critical proteins and harmful mutations caused by long-term exposure to superoxide. Nevertheless, if the cause is enigmatic, the effect is clear: the cell is unable to function and dies.

When this happens, says Greenamyre, the firing patterns of nearby neurons are affected. These neurons use the neurotransmitter glutamate. Because the substantia nigra cells are impaired, they cannot tolerate the excessive glutamate stimulation; calcium accumulates and further impairs mitochondrial function, accelerating the cell's demise.

The complicated nature of the system makes for interesting science, says Greenamyre, "but it's hard to find good targets for pharmacology." Researchers have not yet characterized all the proteins in mitochondria, and they certainly don't know what all those proteins are doing. Each protein has a complicated web of functions; inhibiting one disease-causing aspect may damage a protein's essential roles. "Mucking around can have really unanticipated consequences," Greenamyre concludes.

The first order of business in drug discovery, therefore, is to identify all of the mitochondrial proteins, says Dykens. "It's clear that there are a lot of proteins we don't know about," Dykens says. Of the roughly 2,000 known mitochondrial proteins, researchers can identify only 300. MitoKor intends to compare the proteomes of normal and diseased mitochondria to identify potential drug targets.

IN SEARCH OF NEW DRUGS Because the end result of mitochondrial damage in neurons is the same regardless of the disease process, similar drugs are being tested for many different diseases, including Parkinson, Alzheimer, and Huntington diseases, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), and stroke.

Though stroke is not normally listed with neurodegenerative diseases, Nicholls says the mechanism of damage is the same. "In stroke a region of brain has no blood," he explains. "Broadly speaking there is nothing you can do to save those nerve cells." Without oxygen those neurons can't produce enough ATP to retain potentially toxic glutamate, which then spills out into the synapse. "The problem," Nicholls says, "is that this massive glutamate flood spreads out to other regions even where there is still blood flow." This causes a calcium increase in surrounding cells, damaging mitochondria and spreading the damage beyond those neurons whose oxygen supply was occluded.

According to Nicholls, a small fortune has been spent trying to inhibit the glutamate receptor that allows calcium into the cell. The hope is that these drugs can preserve those neurons that still have adequate blood supply and weren't directly impacted by the stroke. Researchers are also looking into drugs that could block calcium-induced damage; these drugs could help stem cellular damage associated with other diseases as well.

MitoKor has estrogen analogs in Phase I clinical trials for Parkinson disease and in Phase III trials for Alzheimer disease. These are based on findings that postmenopausal women who take estrogen have a lower risk of Alzheimer disease. But as the scientific community is split over the efficacy of estrogen replacement therapy, and since half the population cannot benefit from this option, estrogen itself is not a good treatment candidate. MitoKor has developed nonfeminizing estrogen analogs that do not appear to produce estrogen's side effects. Flint Beal, professor of neuroscience at Cornell University Medical School, calls the preclinical data for estrogen analogs promising, but says it's too soon to tell how effective they will be.

Another compound under investigation is creatine. Beal has done several studies using creatine supplements in mouse models of Huntington disease and ALS.8 In these studies creatine reduced glutamate levels and buffered the calcium within the cell. Creatine supplementation is also being tested in early human trials for Parkinson, Huntington, and ALS. "We don't know for sure what the mechanism is," Beal says, but creatine's normal role is to increase energy levels. If creatine increased ATP, the cell would be better able to regulate calcium levels and sequester glutamate. As with the estrogen analogs, Beal says it is too early to know if creatine will be a useful treatment for human disease.

MitoKor tests these and other drugs in cell lines carrying diseased mitochondria, called cybrids. MitoKor's researchers deplete immortalized cell lines of their mitochondria and fuse these cells with anuclear platelets from a person with a mitochondrial disease. The resulting cybrid contains a nucleus from a stable cell line and the diseased mitochondria under study. The investigators can then expose these cells to different compounds and screen for a lower rate of cell death.

Additional drugs under investigation include coenzyme Q, gingko biloba, nicotinamide, riboflavin, carnitine, lipoic acid, and dichloroacetate.8 All of these reduce glutamate levels, buffer calcium, or act as antioxidants in some way. Although their modes of action are different, each has some potential to ameliorate the effects of impaired mitochondria. And that could help your brain keep processing thoughts and images well into old age.

Amy Adams ( is a freelance writer in Mountain View, Calif.

1. S. Anderson et al., "Sequence and organization of the human mitochondrial genome," Nature, 290:457-65, 1981.

2. D.G. Nicholls, S.J. Ferguson, Bioenergetics 2, San Diego: Academic Press, 1992.

3. A. Adams, "Untangling neuronal calcium signaling," The Scientist, 16[2]:36-9, Jan. 21, 2002.

4. D.G. Nicholls, M.W. Ward, "Mitochondrial membrane potential and neuronal glutamate excitotoxicity: Mortality and millivolts," Trends in Neurosciences, 23:166-74, 2000.

5. I.J. Reynolds, T.G. Hastings, "Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor activation," Journal of Neuroscience, 15:3318-27, 1995.

6. J.T. Greenamyre et al., "Complex I and Parkinson disease," IUBMB (International Union of Biochemistry and Molecular Biology) Life, 52:135-41, 2001.

7. R. Betarbet et al., "Chronic systemic pesticide exposure reproduces features of Parkinson disease," Nature Neuroscience, 3:1301-6, 2000.

8. M.A. Tarnopolsky, M.F. Beal, "Potential for creatine and other therapies targeting cellular energy dysfunction in neurological disorders," Annals of Neurology, 49:561-74, 2001.
©2002, The Scientist Inc.
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