New shapes of prions

Modification of a system for rapid amplification of misfolded prion proteins allows de novo generation of these infectious molecules and provides a glimpse of the diverse range of possible misfolded prion strains.

Of the three origins of prion diseases—familial, infectious and what is called 'spontaneous'—the latter is most common in humans. In this case, a normal prion protein spontaneously 'flips' to a misfolded form that kills neurons and then autocatalytically replicates, causing neurodegenerative disease.

Though many studies have shown that misfolded prion protein (PrP^Sc) induces misfolding of the normal cellular prion protein (PrP^C), the idea that PrP^Sc replicates in the absence of nucleic acids, contrary to the dogma of molecular biology, is known as the 'prion hypothesis'. As a step toward convincing the remaining critics of this hypothesis, Claudio Soto at the University of Texas Medical School in Houston wanted to reproduce this spontaneous prion conversion process in vitro.

His group had previously described protein misfolding cyclical amplification (PMCA), a technique in which PrP^Sc induces the misfolding of normal PrP^C in a test tube in a process catalyzed by ultrasonic waves (Saborio et al., 2001). “So something that in vivo takes many years—or in humans can take decades—to happen, in a test tube we can do it in a matter of hours,” says Soto, further reasoning that “if the normal protein becomes transformed into the misfolded form in vivo spontaneously at a very low frequency, this process of PMCA is very powerful and should detect spontaneous formation of prions.”

To this end, Soto's group modified the PMCA assay, essentially increasing the number of sonication cycles from 144 to 240 to detect these low-frequency events (Barria et al., 2009). Starting with brain homogenate from disease-free hamsters, mice and humans in the extended PMCA assay, they observed PrP^Sc formation in 2 and 1 out of 10 hamster and mouse samples, respectively—but not in any of the human samples. Notably, even after modifying the conditions, the group saw no spontaneous conversion in the human samples or in samples from transgenic mice expressing human PrP^C, suggesting that the human version of the protein misfolds at a lower rate.

Interestingly, the extended PMCA yielded novel forms of misfolded prions that caused new disease phenotypes. When injected into wild-type hamsters, the PMCA-generated hampster PrP^Sc caused disease that was distinct from known hamster prion diseases, suggesting that their work yielded a new prion. Thus, Soto proposes that “the universe of possible prions is not restricted to what we know in nature..., and the sequence of the protein can accommodate many more prions that we [know of] today, and some of these could be potentially more virulent or more transmissible,” analogous to today's situation with the influenza virus. But unlike with the flu, for which the new culprit mutant can be identified using existing technologies, protein-to-protein transmission complicates the study of prion disease. PMCA, however, is a powerful tool for this task: the original PMCA conditions can be used as a diagnostic assay to detect preformed prions in samples, and the extended PMCA is a model for studying the sporadic origin of prions.

The question of the molecular basis for this phenomenon still remains: “How can one single protein without changes in the amino acid sequence encode all the diversity that you have in prions? What are the differences between them, and how they produce the diseases? We are now studying this with natural prions and de novo–produced prions,” says Soto.

So new insights into these confounding diseases are on the way, and PMCA is an adaptable tool for the task.