Sci. Aging Knowl. Environ., 25 January 2006
Vol. 2006, Issue 4, p. pe4
[DOI: 10.1126/sageke.2006.4.pe4]


Prion 2005: Between Fundamentals and Society's Needs

Carina Treiber

The author is at the Free University of Berlin, Thielallee 63, 14195 Berlin, Germany. E-mail:treiber{at}

Key Words: prion disease • BSE • scrapie • transmission • Creutzfeldt-Jakob disease


Creutzfeldt-Jakob disease (CJD), characterized by deposits of abnormal prion protein in the brain of affected individuals, is a relatively rare disease that typically affects people later in life. It belongs to a family of conditions termed transmissible spongiform encephalopathies (TSE), which includes scrapie in sheep, chronic wasting disease (CWD) in deer and elk, and bovine spongiform encephalopathy (BSE) in cows. The main pathological characteristics of all prion diseases are spongiform changes and scrapie-associated fibrils in the brains of affected animals.

In contrast to other neurodegenerative diseases that affect the aging population, such as Alzheimer's disease (AD) or Parkinson's disease (PD), BSE can be transmitted, and it is believed to have crossed the species barrier from cows to people, causing the new variant of CJD (vCJD) (1). The ability for the disease to be transmitted has resulted in the generation of several animal models of the disease, providing scientists with invaluable tools to understand the molecular determinants of disease.

The agent that causes prion diseases seems to be a cellular protein, the prion protein (PrPC), which changes its conformation to a form containing high beta-sheet content called PrPSc (indicating the scrapie isoform of the prion protein). The molecular mechanism responsible for converting PrPC to PrPSc is still poorly understood. The most popular hypothesis, known as the "prion-protein only hypothesis," put forward by Nobel laureate Stanley B. Prusiner, states that heterodimerization of the two different protein isoforms drives the conversion of PrPC to PrPSc (2). As disease progresses, PrPSc is transmitted to neighboring cells, spreading further conversion of PrPC to PrPSc. It is thought that the aging-associated deficits in the cellular machinery, including disturbed metal-ion homeostasis (see Treiber Perspective ), might promote accumulation of the aberrant form of the prion protein in cells.

This Perspective summarizes findings presented at the "Prion 2005" conference held in Düsseldorf, Germany, from 19 to 21 October 2005. The meeting gave researchers the opportunity to exchange knowledge about prion disorders, as well as to address some pressing questions about improved diagnosis and therapy.

Biology of Prion Disease

A number of presentations delved into the normal functioning of the prion protein and mechanisms by which the abnormal PrPSc disrupts the normal functioning of neurons to cause disease. Stanley Prusiner of the University of California, San Francisco, and Melitta Schachner of the University of Hamburg, Germany, presented evidence that PrPC functions in signal transduction by interacting with neural-cell adhesion molecules (N-CAMs), which are expressed by neurons, astrocytes, oligodendrocytes, and Schwann cells (3, 4). Overnight exposure of cultured neurons to recombinant PrPC stimulated neurite outgrowth, axonal elongation, and synapse formation. These effects could be blocked by inhibitors of protein kinase C and Src kinases, including p59Fyn, all of which are molecules that act downstream of N-CAMs, suggesting that recombinant PrPC stimulates these signaling cascades. N-CAMs act in this process by interacting with PrP expressed by the same cell or different cells.

Another set of studies described by Prusiner indicated that neurite outgrowth is inhibited by PrPSc activating the Notch-1 pathway. Notch-1 signaling balances neurogenesis and dendritic growth. Accumulation of PrPSc in neocortical synapses coincides with the shedding of Notch-1 and the translocation of the Notch intracellular domain (NICD) to the nucleus (see Wolfe Perspective for additional description of these processes). Increasing concentrations of NICD in the nucleus correlates with regressive changes in dendrites in scrapie, suggesting a mechanism by which PrPSc contributes to neurodegeneration (5).

Most prion diseases target the CNS in aging individuals. Therefore, Giorgio Giaccone of the Instituto Nazionale Neurologico Carlo Besta in Milan, Italy, investigated whether changes present in the CNS of individuals with AD (see "Detangling Alzheimer's Disease"), such as abnormal forms of the microtubule-binding protein tau, were present in individuals suffering from prion diseases. He showed that aberrant and ectopic tau phosporylation occurs in the brains of vCJD patients, where amyloid precursor protein (APP) immunoreactive, enlarged cell processes were also detected. In contrast, immunostaining for the amyloid beta protein, which is derived from APP and found in deposits present in the brains of individuals with AD, was negative. Hyperphosphorylated tau is found in another prion disease, Gerstmann-Str´┐Żussler-Scheinker Syndrome, but not in sporadic CJD.

Molecular Determinants of Pathogenesis

The mechanism by which PrPC is converted to PrPSc is poorly understood. Several investigators tried to gain insights into this process by determining the regions of PrP necessary for this conversion.

In his keynote lecture, Kurt Wüthrich of the Eidgenössische Technische Hochschule Zürich, in Zürich, Switzerland, reviewed the structure of recombinant and natural prion proteins in their cellular forms, as determined by nuclear magnetic resonance spectroscopy. He pointed out that human and cow PrPC have an identical fold consisting of three {alpha} helices and two short antiparallel beta sheets, but different surface-charge distributions (6, 7). The loop region between a beta sheet and {alpha} helix 2 of the elk sequence was expressed in transgenic mice, resulting in the development of TSE-like symptoms, suggesting that this region is at least partly involved in CWD pathogenesis (8).

The next presentation, by Detlev Riesner from the Heinrich Heine University in Düsseldorf, provided evidence that PrPC is attached to the membrane by a glycosyl-phosphatidylinositol (GPI) anchor. He performed surface plasmon resonance studies to determine the equilibrium between membrane-bound PrPC and soluble PrPC, which can form {alpha}-helical monomers and dimers and undergo, depending on the solution conditions, a cooperative transition to beta-sheet-rich oligomers and large insoluble aggregates. For this purpose, native PrPC with a GPI anchor was isolated from Chinese hamster ovary cells and shown to bind to a lipid layer with a dissociation constant of 6.7 x 10—9 M, suggesting in vivo relevance for the interaction between PrP and lipid membranes. The concentration of PrPC that is free in solution was calculated to be 10—8 M for a PrPC-saturated membrane, a concentration at which the protein could form fibrils in the presence of minute amounts of PrPSc seeds.

Human Rezaei of the Institut National de Recherche Agronomique, Jouy-en-Josas, France, used heat to induce the oligomerization of full-length recombinant bovine PrP at pH 4.0. He showed the existence of oligomers consisting of 12, 24, and 36 PrP subunits by size-exclusion chromatography (9). To identify regions involved in the oligomerization process, Rezaei introduced double cysteine mutations in PrP, because these amino acids allow the protein to form disulfide bonds at regions that come in close contact to each other. The study revealed that different regions of PrP are involved in the formation of each oligomer species, suggesting that a 12 oligomer is not a precursor of a 24 or 36 oligomer.

Proteinase K-Resistant PrP

Proteinase K-resistant PrP (PrPres) is commonly regarded as a marker of infectivity, but atypical scrapie cases raise questions over the reliability of this marker. Ronna M. Barron of the Institute for Animal Health in Edinburgh, Scotland, attempted to quantify PrPres in brain homogenates (consisting of 1 g of brain per 100 mL of solution) of transgenic mice that are homozygous for a mutation in PrP equivalent to a disease-associated mutation in humans and are more susceptible to infection with PrP. After the mice were inoculated with different prion strains, Barron was unable to detect any PrPres in their brains, although the animals were 100% susceptible to transmission (10). This study shows that there is no correlation between PrPres concentrations and the titer of infectivity.

Armin Giese of the Zentrum fur Neuropathologie und Prionforschung in Munich, Germany, asked in his talk whether PrPSc is the infectious agent in TSE. He generated misfolded, infectious PrP in vitro by the technique of autocatalytic protein-misfolding cyclic amplification (PMCA), which involves the amplification of misfolded proteins through sonication and incubation and is conceptually similar to the polymerase chain reaction (11). A bioassay in hamsters indicated that the PMCA-derived agent was infectious but that infectivity was lower by a factor of 10 than that of brain-derived PrPSc. Coupling of the PMCA-derived agent to an inert carrier molecule, nitrocellulose, decreased the incubation time needed for infectivity, suggesting that the particle size of PrPres aggregates can modulate infectivity (e.g., the larger the aggregate, the higher the infectivity). The results of this study suggest that PrPres alone is the infectious agent, without requiring any other factors or a PrP intermediate. This knowledge could shed light on other protein-misfolding diseases that affect older people, such as amyotrophic lateral sclerosis (ALS), AD, and PD.

Mechanisms of Toxicity

Byron Caughey of the Rocky Mountain Laboratory in Hamilton, Montana, found that the most infectious scrapie particle isolated from the brains of hamsters inoculated with scrapie is of intermediate size and consists of aggregates of 14 to 28 PrP molecules, whereas particles smaller than hexamers are not infectious, as shown by inoculation of hamsters with different-sized particles (12). His group also generated transgenic mice expressing a form of PrP that lacks the GPI anchor (13) and that was, therefore, not attached to the membrane. The anchorless PrP showed reduced glycosylation and was secreted from cells. Inoculation of these animals with the scrapie agent revealed that the infectivity levels were lower by a factor of 10 than those of wild-type animals. This model demonstrates the requirement for membrane-anchored cellular PrP for a maximum toxic signal.

Albert Taraboulos of the Hebrew University-Hadassah Medical School in Jerusalem, Israel, described how both PrPC and PrPSc are located in lipid rafts--small platforms, composed of sphingolipids and cholesterol in the outer exoplasmic leaflet of the lipid bilayer; these assemblies are fluid but more ordered and tightly packed than the surrounding bilayer. The treatment of ScN2a cells (a neuroblastoma mouse cell line that can be infected with PrPSc) with cholesterol sulfate, the major sulphated sterol present in the human circulation, and dehydroepiandrosterone (DHEA), a neurosteroid, reduced PrPSc concentrations and altered membrane lipid composition by an unknown mechanism. This finding indicates that PrPSc formation requires optimal lipid composition of rafts. DHEA administration also increased incubation times after scrapie inoculation in mice, suggesting that it represents a new class of therapeutic that may also be of value in the elderly population.

The PrP mutations Gln160->Stop and Trp145->Stop, which are associated with inherited prion diseases in humans, result in proteins that are partly mistargeted to the cytosol. By attaching different signal peptides to wild-type PrP, the group of Jörg Tatzelt from the Max Planck Institute of Biochemistry in Martinsried, Germany, found that the protein adopts a misfolded and partially proteinase K-resistant conformation in different cellular compartments. When PrP is targeted to the cytosol, it induces apoptosis in cells exposed to proteosomal inhibitors, suggesting that proteosomal degradation prevents the toxic effects of cytosolic PrP. It was shown that the pathogenic mutants Gln160->Stop and Trp145->Stop interact with Bcl-2 (a protein that suppresses apoptosis by preventing the activation of caspases) and cause it to aggregate, thus causing apoptosis; aggregation and apoptosis can be suppressed by overexpressing Bcl-2. The heat-shock protein Hsp70/40 also interacts with cytosolic PrP and interferes with PrP-induced apoptosis, which is consistent with a protective role for molecular chaperones.

Prospects for Therapy

Therapeutic strategies for treating sporadic, familial, and infectious prion diseases are focusing on (i) reducing PrPC availability, (ii) preventing the binding of PrPC to PrPSc, (iii) interfering with the conversion of PrPC to PrPSc, (iv) decreasing PrPSc toxicity, and (v) increasing PrPSc degradation. Hans A. Kretzschmar of the Ludwig Maximilians University in Munich pointed out that reducing cellular concentrations of PrPC could be fatal. He found that infarct volumes--areas of tissue death in the brain due to low oxygen, in this case induced by treating animals with kainic acid, an agonist of a class of glutamate receptors, extensively used to model temporal-lobe epilepsy--were three times as large in PrP knockout mice as in wild-type mice; furthermore, the knockout mice are more susceptible to epilepsy. Kretzschmar's group also screened for compounds that interfere with the PrPC-PrPSc interaction, using dual-color scanning for intensely fluorescent targets, a general technique for quantifying and characterizing prion protein aggregates (14). Out of 10,000 drugs that were tested, six composed of a benzylidene benzohydrazide core microstructure were identified and blocked infectivity by interfering with PrPC-PrPSc interaction in scrapie-inoculated ScN2a cells. These six compounds also looked promising in the first set of experiments performed in mice.

Scrapie-infected mice treated with simvastatin [a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that blocks cholesterol synthesis] had delayed disease onset, suggesting that the compound induced neuroprotection. The study, presented by Ronit Sharon of the Hadassah University Hospital, showed that simvastatin treatment was associated with an increased accumulation of PrPSc in scrapie-infected mouse brain and a dramatic enhancement of reactive astrocytes, another pathologic hallmark of prion diseases; despite these observations, a neuroprotective effect, especially of Purkinje cells (a class of neurons in the cerebellum), was seen. The involvement of simvastatin in neuroprotective mechanisms will be further tested.

Mario Salmona of the Universitá degli Studi di Milano-Bicocca in Milan explained that tetracyclines prevent aggregation of PrP peptides, reduce proteinase K-resistance of PrPSc obtained from vCJD materials, and disrupt amyloid fibrils generated by the peptide 106-120 derived from PrP (15). On the basis of these results, hamsters were inoculated with the scrapie agent and tretracycline through the gastrocnemius muscle of the hind leg. There was a significant delay in the deaths of these animals as compared with controls inoculated with scrapie alone. Similar results were obtained by treating with doxycycline, an antibiotic drug belonging to the tetracycline class. The authors hypothesized that tetracyclines sensitize PrPSc to the activity of the proteosome.

Improving Diagnosis

At the present time, prion diseases cannot be cured. Early detection is desirable, because it may allow strategies to constrict infectivity and minimize damage to the brain. Currently, diagnostic assays are based on the postmortem detection of the proteinase K-resistant core of PrPSc. The occurrence of atypical cases that do not exhibit proteinase K-resistant protein has spurred efforts to develop more accurate, as well as more sensitive, diagnostic tools. Charles Weissmann of Scripps Florida in Miami reported the serial selection of a mouse neuroblastoma cell line that is highly susceptible to the scrapie agent, providing a new in vitro assay for prion infectivity (termed the standard scrapie cell assay), which is 10 times as fast as the mouse bioassay and as sensitive. By using this new assay, persistently infected cells could be divided into two categories: quiescent producers of PrPSc and dividing cells that do not produce PrPSc. The infectivity obtained from the first group of cells was largely proteinase-K sensitive. It was hypothesized that PrPSc is a proteinase K-resistant folding variant of PrPC, which is not infectious. Instead, an intermediate in the conversion from PrPC to PrPSc, termed PrP*, could be proteinase K-sensitive, but a pathogenic, replicating isoform.

The development of a new immunoassay for the detection of PrPres in human plasma was presented by Hervé Perron of bioMérieux SA in Lyon, France. The assay is based on the ability of certain molecules to bind PrPres in the blood. The first, streptomycine, aggregates PrPres in human plasma, and the second, calix-6-arene sulfonate, efficiently binds to PrPres aggregated by streptomycine, as a result of its "molecular basket" structure. Therefore, by coupling this second compound to microplates, circulating PrPres can be detected using a monoclonal antibody to PrP. Initial tests investigating 10 CJD cases showed promising results, with the identification of nine positives among the disease cases and none among 500 control samples.

Another approach for prion detection in blood uses PMCA. Paula Saá of the University of Texas Medical Branch in Austin (16) mixed small amounts of PrPSc in blood with larger amounts of PrPC. The mixture was incubated in several cycles of protein misfolding and refolding in order to propagate the PrPSc. This technique enables detection of PrPSc in blood samples of scrapie-afflicted hamsters 20 days after inoculation, with 89% sensitivity and 100% specificity. Thus, PMCA could offer a promising noninvasive method for early diagnosis of prion diseases.

The identification of PrPSc in blood by a misfolded protein diagnostic assay was introduced by Cindy Orser of Adlyfe, Inc., in Rockville, Maryland (17). The method uses fluorescent palindromic polypeptide ligands that bind PrP and mimic the folding of the prion protein. Any changes in the conformation of the protein will be revealed by the fluorescence given off by these ligands. At the moment, the assay is limited to the symptomatic stages of disease and needs to be further improved in sensitivity to recognize the preclinical stage of disease as well.


This meeting provided a state-of-the-art overview of the latest breakthroughs in TSE research. One of the highlights was that PrPres cannot be used as the only marker to diagnose prion diseases, because protease-sensitive PrPSc has been found in many tissues of affected animals, explaining the occurrence of atypical BSE and scrapie cases. Plenty of effort is being directed at understanding the process by which PrPC is converted into PrPSc. Elucidation of this mechanism will in turn result in new therapeutic approaches and sensitive and specific diagnostic tests, many of which are already on their way. Consolidated findings in the field of developing prion diseases may also help to understand the underlying mechanisms in other protein-misfolding diseases affecting mainly the aging population, such as AD, ALS, and PD.

January 25, 2006
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Citation: C. Treiber, Prion 2005: Between Fundamentals and Society's Needs. Sci. Aging Knowl. Environ. 2006 (4), pe4 (2006).

Science of Aging Knowledge Environment. ISSN 1539-6150