Prion proof? Evidence grows for mad cow protein.

Even as scientist Stanley B. Prusiner was accepting a Nobel prize in 1997 for linking misfolded proteins to certain brain diseases, doubters were pointing out that no one had ever actually shown that these proteins--which Prusiner dubbed prions--could cause infection.

Prusiner, a neurologist and biochemist at the University of California, San Francisco (UCSF), and his colleagues now report results that could silence many of his critics. The study, published in the July 30 Science, shows that purified prions can cause disease when injected into the brains of genetically engineered mice.

Previous work by Prusiner and others had implicated prions in human-brain ailments that include Creutzfeldt-Jakob disease and kuru, as well as mad cow disease, chronic wasting disease in deer and elk, and scrapie in sheep.

Suggesting that proteins, misfolded or not, can be infectious is "a radical notion," says Nell R. Cashman of the University of Toronto. Nevertheless, to Cashman and others, the new research supplies the proof.

Previous animal experiments with infectious preparations of natural prions hadn't provided strong evidence of infectivity. Nucleic acids, such as viral DNA or RNA, might have tainted the preparations, says Chih-Yen King of Florida State University in Tallahassee.

To avoid this uncertainty, Prusiner and his colleagues genetically engineered bacteria to make a synthetic prion that could be exhaustively purified and that folds differently from known prions. The synthetic prions would presumably cause brain changes in test animals unlike any seen in typical prion diseases. The researchers injected either their synthetic prions or a saline solution into mice genetically engineered to make vast quantities of a normal protein called PrP. According to Prusiner's theory of prion-based brain diseases, prions target normal PrP and turn it into a form that folds and clumps, ultimately causing brain cells to rupture.

Animals getting the prions showed signs of brain damage between 380 and 660 days later, while those getting the saline injection didn't. The damaged brain tissue had the holes characteristic of prion-implicated diseases, which can take years to develop, but the holes were larger titan usual, says Prusiner's UCSF colleague and coauthor Giuseppe Legname. The brains also contained an abundance of PrP that was resistant to breakdown by natural enzymes, an indication that the PrP molecules were misfolded.

The scientists then took some of this prion-insulted brain tissue and injected it into normal mice, which became sick within 90 to 150 days. The researchers are now conducting the same procedure using brain tissue from the mice that received the saline placebo, Legname says.

Byron Caughey of the National Institute of Allergy and Infectious Diseases Rocky Mountain Laboratories in Hamilton, Mont., notes that the latter test could rule out hidden infections in the genetically engineered mice.

Caughey also says that to guarantee that lab contamination didn't influence the infections, other labs will have to replicate the experiments. However, he adds, "my operating hypothesis is that the protein can [cause infection], and the data suggest that."

Other scientists aren't convinced. "If it's the real thing, they should be able to give it directly to [normal] mice, and not some freak transgenic mouse," says Laura Manuelidis of Yale University School of Medicine.

Legname agrees that much remains to be clarified. For one thing, no one knows just how prions induce normal PrP proteins to misfold into a specific, disease-causing shape. "There must be other factors or proteins involved in this process," Legname says.