In the first study, Adriano Aguzzi and coworkers at the University Hospital of Zurich in Switzerland show that the cellular prion protein (PrPc) plays a critical role in the maintenance of peripheral nerve myelin. Expression of PrPc in neurons and its regulated cleavage are both necessary for normal myelination and function of peripheral nerves, the study shows. How this relates to the toxicity seen in prion diseases is not clear, however. By Aguzzi’s results, PrPc does not seem to play the same role in the central nervous system, where myelin appears normal in the knockout mice. The study was published January 28 in Nature Neuroscience online.

A second paper, published online in Science January 28 addresses the latter question, offering the strongest evidence to date to support the infectious protein hypothesis of prion disease. In the study, Jiyan Ma and colleagues at Ohio State University in Columbus show that, under the right conditions, recombinant prion protein can twist into an infectious shape capable of transmitting prion disease in mice. The recipe includes a dose of lipid, which seems to facilitate the production of pathogenic prions in vitro.

A Force for Good?
To answer the question of what PrPc is doing in its normal shape, Aguzzi used a veritable zoo of prion knockout mice, conditional knockouts, and transgenic mice to probe the physiological role of the protein. In two prion protein knockout strains, researchers previously described late-onset peripheral neuropathy (Bueler et al, 1992; Nishida et al., 1999). Therefore, first author Juliane Bremer and coworkers looked more closely at myelin in those strains plus two additional PrP knockout strains. In all four, the researchers noted a peripheral neuropathy involving axon demyelination in 60-week-old mice. The damage began even earlier, though, as the researchers saw macrophages ingesting myelin debris from degenerating nerve fibers as early as 10 weeks. The mice showed decreased nerve fiber conduction, grip strength, and heat responses, indicating that the nerves were functionally affected. Reintroducing the prion protein gene by crossing knockouts with prion transgenic mice prevented the neuropathy.

Further study suggested that PrPc is required for myelin maintenance, rather than deposition. Young knockout mice appeared normal until the first signs of demyelination appeared at around 10 weeks, corresponding to the time when active myelination is complete. The PrPc also acted from the neuronal side, because specifically removing PrPc from neurons, but not Schwann cells, triggered the neuropathy. Conversely, restoring expression of PrPc to neuronal cells, but not Schwann cells, prevented demyelination. Together, the results suggest that PrPc is the previously unknown signal that axons send to Schwann cells to maintain myelin sheaths.

The actions of PrPc required its regulated cleavage, as indicated by the failure of non-cleavable mutants to correct the neuropathy. Specifically, there appeared to be an association between the presence of an N-terminally truncated cleavage fragment, C1, and normal myelin maintenance, as only mice without C1 experienced neuropathy.

It is not clear what signaling pathways might be triggered by PrPc to support myelination. PrPc regulates β-secretase (see ARF related news story on Parkin et al., 2007), which itself has been implicated in both peripheral and central nervous system myelination via processing of neuregulin type III (see ARF related news story on Willem et al., 2006, and also Hu et al., 2006). The authors write, however, that they did not find any difference in neuregulin gene expression between PrPc knockout and normal mice, suggesting that PrPc does not act via that pathway. In addition, Aguzzi told ARF in an e-mail, it does not appear that BACE is responsible for the cleavage of PrPc.

The β-secretase is, of course, also essential for production of Aβ, and PrPc has been implicated in Aβ toxicity on CNS neurons (see ARF related news story on Lauren et al., 2009, and a more recent ARF related news story). Nonetheless, Aguzzi writes, based on the current work, “There is little reason to speculate that the role of PrP in peripheral nerves would be of relevance to AD.”

And, a Protein Gone Bad
If the prion hypothesis of disease is correct, then prion protein alone should be able to cause and propagate the disease. Several years ago, work in the lab of prion discoverer Stanley Prusiner at the University of California, San Francisco, showed that an amyloid fiber derived from recombinant PrP could cause prion disease in mice that overexpress a prion protein fragment (Legname et al., 2004). As yet, no synthetic prion had been shown to cause disease in normal mice.

Since the prion protein exists in cells as a GPI (glycosylphosphatidylinositol)-linked membrane protein, Ma and colleagues reasoned that lipids might facilitate pathogenic folding. To test that idea, first authors Fei Wang and Xinhe Wang used the protein misfolding cycling amplification (PMCA) technique, a process conceptually similar to PCR that involves subjecting proteins to repeated cycles of folding and sonication, to break up growing fiber chains into smaller seeds. They subjected mixtures of recombinant prion protein to PMCA in the presence of a variety of lipids plus RNA (already known to help fibril formation in vitro). In one condition, a combination of synthetic phospholipids and RNA promoted the formation of an abundant protease-resistant aggregate of 15 kDa apparent molecular weight that resembled PrPsc, the prion that causes scrapie disease in sheep.

The recombinant prion was infective, as confirmed by its ability to propagate a proteinase-resistant conformation to endogenous PrPc in mouse cells in culture. In mice, the investigators found that 15 of 15 wild-type animals infected with recombinant PrP aggregate developed signs of prion disease after 130 days. After developing neurological symptoms, the animals progressed quickly and died within a few weeks (average survival, 150 days). Spongiform encephalitis was confirmed by histological analysis, and aggregated prion protein was detected in all the brains. None of the control mice (that received inoculums of recombinant protein that had not been seeded or exposed to folding PCMA) came down with neurological disease. Finally, the researchers showed that brain homogenates from the infected mice could serially transmit the prion disease to healthy mice.

Aguzzi has praise for Ma’s work, calling it “superb.” In an e-mail to ARF, he said the study opens the way to very important structural work.

Ma told ARF that he is very interested in the lipid-protein interaction that results in infectious prion. “Our experiments do not prove this happens in vivo, but in vitro these interactions seem crucial to generate the infectious conformation.” From here, he wants to use the synthetic prion to understand exactly what the infectious conformation is, and explore potential means to block its formation.—Pat McCaffrey.

References:
Wang F, Wang X, Yuan CG, Ma J. Generating a Prion with Bacterially Expressed Recombinant Prion Protein. Science. 2010 Jan 28. Abstract

Bremer J, Baumann F, Tiberi C, Wessig C, Fischer H, Schwarz P, Steele AD, Toyka KV, Nave KA, Weis J, Aguzzi A. Axonal prion protein is required for peripheral myelin maintenance. Nat Neurosci. 2010 Jan 24. Abstract