The cellular prion protein has received a lot of attention recently for its potential role in amyloid-β (Aβ) toxicity (see ARF related news story), but that is merely the latest wrinkle in the prion story. Two reports out in the past week solve a pair of longstanding questions about the prion protein; namely, what does it normally do, and is it capable of transmitting diseases, including Creutzfeldt-Jakob disease in humans and mad cow disease in bovines, on its own?

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


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Comments on News and Primary Papers

  1. The generation of a pathogenic molecule through protein misfolding cycling amplification (PMCA) in the presence of phospholipid is reminiscent of the "globulomer" complex formed from Aβ and specific lipids (Barghorn et al., 2005). There may also be some relationship to the role of gangliosides in creation of a toxic Aβ moiety (Kakio et al., 2002; Yamamoto et al., 2007). Perhaps lipid-protein interactions play a general and critical role in the development of peptide misfolding disorders.


    . Globular amyloid beta-peptide oligomer - a homogenous and stable neuropathological protein in Alzheimer's disease. J Neurochem. 2005 Nov;95(3):834-47. PubMed.

    . Interactions of amyloid beta-protein with various gangliosides in raft-like membranes: importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid. Biochemistry. 2002 Jun 11;41(23):7385-90. PubMed.

    . A ganglioside-induced toxic soluble Abeta assembly. Its enhanced formation from Abeta bearing the Arctic mutation. J Biol Chem. 2007 Jan 26;282(4):2646-55. PubMed.

  2. In this paper, Wang et al. report on how they were able to manufacture, with short incubation times, prions capable of infecting wild-type mice. This is an important finding. Looking at the prion field, and the body of research produced over the years, the major efforts were focused on using recombinant proteins to produce infective prions. In our 2004 paper (Legname et al., 2004), we demonstrated that it was possible to create low levels of infectivity using only recombinant prion protein produced in Escherichia coli. After that, there was a major push in research to find out how to enhance these low levels. The main advance came from Surachai Supattapone, formerly of Stanley Prusiner’s lab, when he employed RNA molecules to enhance the production of PrPSc in vitro. Around the same time, Claudio Soto’s group perfected protein misfolding cyclic amplification (PMCA), another important contribution. From then on it was clear that cofactors were probably needed to enhance PrP conversion, and that is basically what the group of Jiyan Ma has just described in their Science paper. They used lipids and RNA, combined with PMCA, to produce highly infectious samples.

    This is an important piece of work because it once again confirms that PrP is definitely necessary to create prions, and, perhaps most importantly, it shows that it is actually possible to induce high infectivity with the addition of other cofactors, which are still not well defined but nevertheless necessary to increase the infectivity in the samples. As a matter of fact, these prions are very similar to the mouse-adapted scrapie prions that we already know about, such as the Rocky Mountain Laboratory (RML) strain and others. But the neuropathology that they show is clearly different from RML and any other mouse-adapted prions. All these wild-type prions in mice rarely lead to widespread vacuolation, but the authors here do see vacuolation in many different areas of the brain. A contamination with RML would not explain this.

    One puzzling piece of information that is presented in the work of Ma and coworkers is the delayed incubation time upon second passage of these novel prions to the same wild-type recipient mice. Usually, subsequent passages lead to abbreviated incubation times.

    In addition, it would be interesting to receive additional information about the stability of their novel prion strain, and more biochemical characterization beyond proteinase K (PK) digestion. One of the things we did with our synthetic prions was to show that they were a completely new set of prions, because they possessed higher stability in terms of resistance to chemical or chaotropic agent denaturation. We found that with synthetic prions, in addition to the neuropathology and to all the other major indications that this is a different prion, the biochemical stability is completely different from any wild-type prion. The authors here did not provide stability data, unfortunately, because it would have given additional indication that they are actually handling something completely different. In our recent work, we also showed how incubation times vary based on the conformation and stability of different synthetic prions (Legname et al., 2006; Colby et al., 2009).

    One suggestion of Ma and coworkers’ paper is that whenever you have PK resistance, you have prions, but that’s not always true, because some prions are sensitive to PK attack (Colby et al., 2010).


    . Synthetic mammalian prions. Science. 2004 Jul 30;305(5684):673-6. PubMed.

    . Continuum of prion protein structures enciphers a multitude of prion isolate-specified phenotypes. Proc Natl Acad Sci U S A. 2006 Dec 12;103(50):19105-10. PubMed.

    . Design and construction of diverse mammalian prion strains. Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20417-22. Epub 2009 Nov 13 PubMed.

    . Protease-sensitive synthetic prions. PLoS Pathog. 2010 Jan;6(1):e1000736. PubMed.

    View all comments by Giuseppe Legname

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News Citations

  1. Model Shows Oligomers Impair Memory, Questions Role of Prion Protein
  2. Prion Protein Keeps β-secretase in Check
  3. Double Paper Alert—A Function for BACE, a Basis for Amyloid
  4. Keystone: Partners in Crime—Do Aβ and Prion Protein Pummel Plasticity?

Paper Citations

  1. . Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature. 1992 Apr 16;356(6370):577-82. PubMed.
  2. . A mouse prion protein transgene rescues mice deficient for the prion protein gene from purkinje cell degeneration and demyelination. Lab Invest. 1999 Jun;79(6):689-97. PubMed.
  3. . Cellular prion protein regulates beta-secretase cleavage of the Alzheimer's amyloid precursor protein. Proc Natl Acad Sci U S A. 2007 Jun 26;104(26):11062-7. PubMed.
  4. . Control of peripheral nerve myelination by the beta-secretase BACE1. Science. 2006 Oct 27;314(5799):664-6. PubMed.
  5. . Bace1 modulates myelination in the central and peripheral nervous system. Nat Neurosci. 2006 Dec;9(12):1520-5. Epub 2006 Nov 12 PubMed.
  6. . Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Nature. 2009 Feb 26;457(7233):1128-32. PubMed.
  7. . Synthetic mammalian prions. Science. 2004 Jul 30;305(5684):673-6. PubMed.

Further Reading

Primary Papers

  1. . Generating a prion with bacterially expressed recombinant prion protein. Science. 2010 Feb 26;327(5969):1132-5. PubMed.
  2. . Axonal prion protein is required for peripheral myelin maintenance. Nat Neurosci. 2010 Jan 24; PubMed.