Charles Weissmann

Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie

The ‘protein only’ hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N‐terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild‐type PrP or PrP lacking 26 or 49 amino‐proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild‐type and truncated PrPs. Within the framework of the ‘protein only’ hypothesis, this means that the amino‐proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).

Darwinian Evolution of Prions in Cell Culture

DNA-less Evolution Prions are proteinaceous infectious elements involved in a variety of neurodegenerative diseases, including scrapie in sheep and so-called mad cow disease in cattle. Now Li et al. (p. 869, published online 31 December) show that, when propagated in tissue culture cells, cloned prion populations become diverse by mutational events and can undergo selective amplification. Thus, even though devoid of a coding genome, prions, when propagated under a particular selection regime, can be subject to rapid evolution. When propagated in vitro, prion strains demonstrate adaptability and selection. Prions are infectious proteins consisting mainly of PrPSc, a β sheet–rich conformer of the normal host protein PrPC, and occur in different strains. Strain identity is thought to be encoded by PrPSc conformation. We found that biologically cloned prion populations gradually became heterogeneous by accumulating “mutants,” and selective pressures resulted in the emergence of different mutants as major constituents of the evolving population. Thus, when transferred from brain to cultured cells, “cell-adapted” prions outcompeted their “brain-adapted” counterparts, and the opposite occurred when prions were returned from cells to brain. Similarly, the inhibitor swainsonine selected for a resistant substrain, whereas, in its absence, the susceptible substrain outgrew its resistant counterpart. Prions, albeit devoid of a nucleic acid genome, are thus subject to mutation and selective amplification.

Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain

PrP knockout mice in which only the open reading frame was disrupted (‘Zürich I’) remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, Prnd. A new PrP knockout line, ‘Zürich II’, with a 2.9 kb Prnp deletion, developed this phenotype at ∼10 months (50% morbidity). A single Prnp allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a Prnd‐containing cosmid expressed Dpl at twice the level and became ataxic ∼5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.

Prions on the move.

Prions consist mainly, if not entirely, of PrPSc, an aggregated conformer of the host protein PrPC. Prions come in different strains, all based on the same PrPC sequence, but differing in their conformations. The efficiency of prion transmission between species is usually low, but increases after serial transmission in the new host, suggesting a process involving mutation and selection. Even within the same species, the transfer of prions between cell types entails a selection of favoured ‘substrains’, and propagation of prions in the presence of an inhibitory drug can result in the appearance of drug‐resistant prion populations. We propose that prion populations are comprised of a variety of conformers, constituting ‘quasi‐species’, from which the one replicating most efficiently in a particular environment is selected.

Birth of a Prion: Spontaneous Generation Revisited

The proposal that the transmissible agent in prion diseases can be a conformationally altered host protein that multiplies by autocatalytic conversion has gained wide acceptance. Recent work shows that the agent, the prion, can be replicated in a cell-free system, that it can be generated de novo, and that the strain-specific properties of prions are encoded by conformational variations of the underlying protein.

B lymphocyte-restricted expression of prion protein does not enable prion replication in prion protein knockout mice

Prion replication in spleen and neuroinvasion after i.p. inoculation of mice is impaired in forms of immunodeficiency where mature B lymphocytes are lacking. In spleens of wild-type mice, infectivity is associated with B and T lymphocytes and stroma but not with circulating lymphocytes. We generated transgenic prion protein knockout mice overexpressing prion protein in B lymphocytes and found that they failed to accumulate prions in spleen after i.p. inoculation. We conclude that splenic B lymphocytes are not prion-replication competent and that they acquire prions from other cells, most likely follicular dendritic cells with which they closely associate and whose maturation depends on them.

The Role of PrP in Pathogenesis of Experimental Scrapie

It was recognized early on that the agent responsible for transmissible spongiform encephalopathies such as scrapie in sheep or kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia (FFI) in man had quite extraordinary properties, such as unusually long incubation periods (measured in months to years) and uncommon resistance to high temperature, formaldehyde treatment, and UV irradiation. The agent has been designated as “prion” (see glossary, Table 1) to distinguish it from conventional pathogens such as bacteria and viruses (Prusiner 1982). n nIn recent years, a new form of prion disease emerged in Great Britain and to a lesser extent in other European countries, namely, bovine spongiform encephalopathy (BSE) or mad cow disease, which has been attributed to the consumption by cattle of feed supplements derived from scrapie-contaminated sheep and later from cattle offal (Wilesmith et al. 1992). It is, however, quite possible that BSE originated as a sporadic.

Differentiation and histological analysis of embryonic stem cell-derived neural transplants in mice.

We report here that neural transplantation of in vitro‐differentiated embryonic stem (ES) cells provides a versatile strategy for gene transfer into the central nervous system. ES cells were subjected to an optimized in vitro differentiation protocol to obtain embryoid bodies. These aggregates were stereotaxically transplanted into the brain of recipient adult mice, where they followed a strictly controlled differentiation pattern and eventually formed mature neural grafts. A marker gene, introduced into the ROSA26 locus allowed for precise determination of the fate of the descendants of the transplanted embryoid bodies and revealed that not only neurons but also astrocytes, oligodendrocytes and even microglial cells were graft‐derived. Evaluation of long‐term experiments showed viable grafts with a stable transgene expression and proved that this approach provides a tool for reliable gene expression within a spatially delimited area of neural tissue.

Assaying Prions in Cell Culture

Prions are usually quantified by bioassays based on intracerebral inoculation of animals, which are slow, imprecise, and costly. We have developed a cell-based prion assay that is based on the isolation of cell lines highly susceptible to certain strains (Rocky Mountain Laboratory and 22L) of mouse prions and a method for identifying individual, prion-infected cells and quantifying them. In the standard scrapie cell assay (SSCA), susceptible cells are exposed to prion-containing samples for 4 days, grown to confluence, passaged two or three times, and the proportion of rPrP(Sc)-containing cells is determined with automated counting equipment. The dose response is dynamic over 2 logs of prion concentrations. The SSCA has a standard error of +/-20-30%, is as sensitive as the mouse bioassay, 10 times faster, at least 2 orders of magnitude less expensive, and it is suitable for robotization. Assays performed in a more time-consuming end point titration format extend the sensitivity and show that infectivity titers measured in tissue culture and in the mouse are similar.

Genetic Informational RNA Is Not Required for Recombinant Prion Infectivity

ABSTRACT Whether a genetic informational nucleic acid is required for the infectivity of transmissible spongiform encephalopathies is central to the debate about the infectious agent. Here we report that an infectious prion formed with bacterially expressed recombinant prion protein plus synthetic polyriboadenylic acid and synthetic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol is competent to infect cultured cells and cause prion disease in wild-type mice. Our results show that genetic informational RNA is not required for recombinant prion infectivity.