By David N. Leff

"The field of mitochondrial diseases is quite big nowadays."

So says molecular biologist Carlos Moraes at the University of Miami. His own field of research covers the functioning and malfunctioning of mammalian mitochondria, especially human.

Among the maladies laid partly at the door of mitochondria these days are Alzheimer's disease, diabetes and epilepsy. Of diseases due wholly to mutations in mitochondrial genes, Moraes observed, the best known is Leber's hereditary optic neuropathy. "It's a form of blindness," he told BioWorld Today, "caused by gene mutations on Complex I of the mitochondrial respiratory chain."

That chain, or cascade, is the stepwise transfer of electrons that powers the cell's source of energy.

"Another example of an apparently pure mitochondrial disease," Moraes went on, "rarer but very interesting, is mitochondrial DNA depletion syndrome. Babies born with extremely low levels of mitochondrial DNA usually die very early." He added that "this syndrome is inherited in Mendelian fashion, which means that the primary problem is in the cell's nuclear DNA, but still causes the organelle's DNA level to be very low. And that's what's killing the patient."

If disease-causing mitochondria were invading pathogenic bacteria, rather than indispensable organelles in every cell, infectious-disease specialists would go after them with antibiotics. Ironically, mitochondria are now thought to be reformed bacteria, which entered cells millions of years ago and dropped their life of crime in favor of symbiosis.

"The mitochondrion is the only organelle in the cell — with the exception, of course, of the nucleus — that has DNA," Moraes said. "It's a very small DNA, 16,569 base pairs long, which encodes only 13 polypeptides. Because it's such a limited set of genes," he noted, "more than 100 proteins encoded by the genome in the cell's nucleus have to be imported into the mitochondrion, just to control its gene expression."

Both sets of DNA cooperate to express genes that control the activity of the energy-converting mitochondrial enzymes. This genetic symbiosis is so vital that mitochondrial DNA (mtDNA) must have evolved in step with nuclear DNA.

This strict condition led Moraes and his co-author, molecular biologist Lesley Kenyon, to explore how far back in primate evolution such compatibility could exist. Their findings appear in the current issue of The Proceedings of the National Academy of Sciences (PNAS), dated Aug. 19, 1997. The report's title: "Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids."

Cybrids are artificial constructs obtained by fusing one cell, in which the 2,000 or so mitochondria have all been stripped of their DNA, with another cell deprived of its nucleus."

Climbing The Great Apes' Family Tree

Moraes continued: "We and others have been using this system to study mutations in mtDNA that cause mitochondrial diseases. It's one way of transferring the mtDNA from the patient to this cell line, then studying the mtDNA mutation in a common nuclear background."

Working back up the primate family tree, the co-authors compared the compatibility of mtDNA from chimpanzees, pygmy chimps, gorillas and orangutans with nuclear DNA from humans. Only mtDNA from chimpanzees, which diverged genomically from humans six million years ago, and gorillas, two million years earlier, he said, "were able to function well enough to restore growth under the selective condition that requires mitochondrial respiratory chain activity."

Orangutans, which diverged 18 million years ago, were a different story. Chimpanzees and humans share 97 percent of their mitochondrial DNA. Orangs weigh in at 95 percent, with gorillas in between. "The orangutan DNA," Moraes recounted, "is very similar to ours; still, the difference was large enough to give some kind of incompatibility, so we could not rescue any growing clone after the fusion." He added, "This has relevance to diseases."

Diagnosing Mitochondrial Maladies

Moraes explained: "A big problem that researchers in the field have now is this: You suspect that the patient is suffering from a mitochondrial disease, that there might be a mutation in his mtDNA. So you go and sequence all or most of that mitochondrial genome, and you find differences from the standard published sequence for the human mtDNA.

"So how do you know," he pointed out, "which differences have functional significance, and which do not?"

One answer: "Looking at the database of polymorphisms observed in normal people. The number of differences in the mtDNA between chimps and gorillas is huge compared with the different races of humans."

Since submitting his paper to PNAS last March, the Miami researcher has been analyzing every complex of the mitochondrial respiratory chain for normal or abnormal functions. "So that's the main contribution of this paper," he observed.

Moraes cited a specific example.

"About four months ago, there was a paper in PNAS describing mtDNA point mutations in patients with Alzheimer's disease. [See BioWorld Today, April 29, 1997, p. 1.] In a footnote, its authors cited similarities between human mutant genes encoding a a potentially defective cytochrome oxidase — a terminal enzyme of the mitochondrial electron transport chain — and normal wild-type gorilla and chimp sequences."

Moraes continued, "We finished the analysis of this enzyme, and found that it seems to be completely normal. So those variations that they were saying are causing cytochrome oxidase deficiency, and therefore Alzheimer's, are very unlikely to be true. Because those mutations do not affect the activity of enzymes in this selection system.

"So that's just one example," he concluded, "of how an expanded database would be used." *