Prion diseases are a group of fatal neurodegenerative disorders such as Creutzfeldt-Jabob disease (CJD), scrapie or bovine spongiforme encepahlophathy (BSE). In contrast to other neurodegenerative disorders like Alzheimer disease or Chorea Hungtinton they are caused not only sporadically or genetically but also by infection (?transmissible?). Histopathology shows spongiform degeneration and astrocytic gliosis (figure 1). Clinical symptoms are coordinative malfunctions (Ataxia) and dementia.

The oldest known prion disease, was described 1759 (Leopold) and is characterized by the abnormal walk and scratching of infected sheep and goats. The srapie agent was adapted to mice and hamster, which are used as model systems in basic research.

CJD is the most common form of human prion diseases and occurs with an estimated incidence of one case per 1 million population per year. The mean age of onset is about 60-65 years. The mean illness duration is about 7-8 month; death occurs within 12 months of illness in 85-90 % of cases. Usual symptoms are rapid dementia, visual abnormalities, cerebellar dysfunctions (incoordination, gait, speech abnormalities) and EEG tracing shows characteristic signals. However confirmation still requires postmortem histopathology and/or PrPres detection.

About 85 % of all CJD cases are caused sporadically, no genetic or infectious reason could be found (see also replication model). About 10-15 % are caused by mutations (genetic). The mean age of onset is depending on the mutation slightly decreased. Besides genetic CJD Gerstmann-Straussler-Scheinker (GSS) and fatal familiar insomnia (FFI) are genetic caused prion diseases. About 20 different mutations in the prion protein gene are associated with human prion diseases. Depending on the mutation the symptoms and the affected area of the brain can vary.

Less than 1 % of all CJD cases are caused by infection. Infection was caused by massive medical treatment as neurosurgery, cornea or dura-mater transplantations (> 60 cases) or human growth hormone (> 85 cases).

Another infectious human prion disease is called kuru or translated laughing death. Among the Fore people in Papua New Guinea the spread of kuru was caused by a ritual cannibalism. Besides the coordinative malfunctions the cerebellar deficits were often associated by uncontrollable and inapropiate episodes of laughter.

Since 1985 prion diseases attracted economic attention by the recognition of BSE epidemic in the UK. The offal of scrapie infected sheep is thought to be responsible for the BSE epidemic. In the late 70ies the process of meat and bone meal (MBM) production was altered and the scrapie agent was not inactivated anymore. After the feed-ban in 1988 the BSE epidemic reached its maximum in 1992 with more than 37000 cases (figure 3). A mean incubation time of 4-5 years and still more than 1000 cases in 2000 in the UK suggests other, secondary infection routes, which still have to be examined. In 2000 the first cases of BSE occurred in Germany, the infection route is also still unknown, but MBM seems to be unlikely.

In 1996 prion diseases again attracted public attention because a new variant of CJD was reported in the UK (figure 3). The vCJD can be distinguished from CJD by its unusual youg age at onset (mean: 28 years) and prolonged duration (mean: 14 month). Most of the patients show psychiatric symptoms first and no EEG characteristics are found. So far all vCJD patients were Methionin homozygot at Codon 129 (see Polymorphism Codon 129).

The causative agent was characterized by its abnormal resistance against conventional decontamination methods. Methods which modify or destroy nucleicacids like radiation or could not destroy the infectivity. In contrast there to methods which modify or destroy the structure of proteins like urea or guanidinium hydrochlorid could destroy the infectivity. This is contradictory to the fact that conventional viruses use nucleicacids for replication (figure 4). In 1982 Stanley B. Prusiner coined the term prion for proteinaceous infectious particle. Highly purified prion-rods consist mainly if not exclusively of an abnormal isoform of the prion protein. The prion protein is a host encode protein with yet unknown function. Chemical differences between the cellular prion protein (PrPC) and the scrapie isoform (PrPSc) are not found. They differ in their biochemical and biophysical properties. is in contrast to PrPC insoluble, resistant against proteinase K digestion and infectious. Its secondary structure is beta-sheeted while PrPC is mainly alpha-helical. PrPC is a monomeric, GPI-anchored and N-glycosylated membrane protein. Figure 5 shows the NMR structure of recombinant hamster PrP and a structure model of PrPSc. Besides PrP only lipids and sugars have been found in prion rods yet. Nucleic acids could not just be found yet, but nucleic acids longer than 80 nucleotides could also be excluded as an essential part of an infectious unit.

According to the template assisted heterodimer model (Prusiner and Cohen, 1998) converted to PrPSc by interaction of an metastable form (PrP*) with PrPSc and a necessary secondary factor so called protein X or factor X. This was the first model which could explain all three forms of prion disease manifestation. In case of an infection PrPSc is added by an extra cellular incident and converts cellular The sporadic form can be explained by a spontaneous conversion of PrPC. This is a rare event, which explains the small number of cases and the high age at onset, the possibility of a spontaneous conversion increases with lifetime. Mutations in the prion protein (genetic form) also increase the possibility of a spontaneous conversion.

The exact mechanism of the conversion from the cellular PrPC to the infectious form PrPSc is still unknown. The "normal", cellular isoform of the prion protein has exactly the same aminoacid sequence as the scrapie isoform, but it does have different characteristics:

PrPC is a monomeric protein which is soluble in so-called "soft" detergents and is sensitive to digestion with proteinase K. In contrast, PrPSc is insoluble, and the N-terminus (AA 90-231) is unusually resistent to PK digestion.

The infectious agent of the scrapie disease is thought to consist mainly, if not entirely, of protein: the prion-protein. In order to prove this hypothesis, it would be necessary to simulate the conversion from the cellular form into the infectious form in the test tube (in vitro). So far, nobody has achieved this in vitro conversion, although many labs are working on it.

In our group, we have developed a method for an in vitro conversion which can induce at least some features of PrPSc from either solubilized PrP27-30 (prion rods) or recombinant PrP of the same amino acid sequence. This system uses the anionic detergent SDS (sodium dodecyl sulfate) to induce a soluble, monomeric and PK sensitiv form of PrP from either the recombinant protein or prion rods. Dilution of the SDS leads to aggregation which is concomitant with a structural change. Those aggregates or multimers are not significantly more infectious than the soluble form, but they have a different structure (increased beta-sheet content) and they are partially resistent to proteinase K.

Fig. 5a: Conversion model.

The differences between the two states can be examined by biophysical methods:

Circular dichroism measurements show the secondary structure of the protein and allow the determination of an increased beta-sheet content. The multimerisation can be followed by the use of fluorescence correlation spectroscopy. This rather new technique measures the diffusion time of molecules in solution. Because multimers move much slower in a solution than the smaller monomers, the aggregation process can be detected.

At the moment, the in vitro conversion system using SDS is used for different purposes:

	diagnostics: A new and sensitive test for BSE might be developed using FCS
	other components: One reason for the failing of all attemps to generate new infectivity from non infectious material might be that additional factors (for example polysaccherides) must be added to the protein.
	more detailed analysis of the involved transitions

Fig. 5b: Structural model of the dimer. (model: J. Warwicker Group, Manchester, UK)

The exact conditions for the formation of large, highly structured fibrils is one important direction of current research. Another is the mechanism of the structural transition. For example, what intermediates are there in the structural transition? Can PrP dimers be found as a first step in the formation of larger multimers, and what do they look like?

Besides spontaneous aggregation (see "The conversion from PrPC to PrPS") also seeded multimerization was analysed. It was found that addition of soluble PrP into pre-existing PrP-multimers, either natural and infectious ones or synthetic, was much faster and could be studied by FCS in a time window smaller than 30 minutes in which spontaneous aggregation did not lead to multimers of comparable size. (Post et al. 1998) One hallmark of amyloid diseases like AD and TSE is the presence of amyloid fibrils. So the principle of the seeded aggregation was applied to the diagnostic of Alzheimer disease (AD) in the cerebrospinal fluid (CSF) of AD-patients (Pitschke et al., 1998). By the incooperation of fluorescence labeld antibodies or Aß-peptides highly labeled aggregates are formed which have at least by a factor of 2 a higher fluorescence than the background and can be easliy detected as fluorescence bursts. The occurence and amount of amyloid fibrils can be determined by the amount of fluorescence bursts. This is true for either for labeld Aß-peptides incoorporated into Aß-fibrils or labeled PrP into prion-rods (as shown in fig. 6a).

The basic approach for the diagnostic system based on the assumption that extracellular deposits of Aß-aggregates, primarily existing in the central nervous system also occur in the cerebrospinal fluid in the case of AD. That this is true was shown succesfully in initial experiments with 15 AD patients and 16 controls as it its shown in fig. 6b.

J. Schell, K. Jansen, O. Schäfer
Last modified: Thu Mar 20 15:16:27 CET 2003