Peter-Christian Klöhn

A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions.

Prions are usually quantified by bioassays based on intracerebral inoculation of mice that are slow, imprecise, and costly. We have isolated neuroblastoma N2a sublines highly susceptible to mouse prions, as evidenced by accumulation of infectivity and the scrapie form of prion protein (PrPSc), and developed quantitative in vitro assays for prion infectivity. In the scrapie cell (SC) assay, susceptible N2a cells are exposed to prion-containing samples for 3 days, grown to confluence, and split 1:10 three times, and the proportion of PrPSc-containing cells is determined with automated counting equipment. In a log/log plot, the dose–response is linear over two logs of prion concentrations. The SC assay is about as sensitive as the mouse bioassay, 10 times faster, >2 orders of magnitude less expensive, and suitable for robotization. SC 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.

Transmission of Prions

The “protein only” hypothesis states that the infectious agent causing transmissible spongiform encephalopathies is a conformational isomer of PrP, a host protein predominantly expressed in brain, and is strongly supported by many lines of evidence. Prion diseases are so far unique among conformational diseases in that they are transmissible, not only experimentally but also by natural routes, mainly by ingestion. A striking feature of prions is their extraordinary resistance to conventional sterilization procedures, and their capacity to bind to surfaces of metal and plastic without losing infectivity. This property, first observed in a clinical setting, is now being investigated in experimental settings, both in animals and in cell culture.

Disease-related prion protein forms aggresomes in neuronal cells leading to caspase activation and apoptosis

The molecular basis for neuronal death in prion disease is not established, but putative pathogenic roles for both disease-related prion protein (PrPSc) and accumulated cytosolic PrPC have been proposed. Here we report that only prion-infected neuronal cells become apoptotic after mild inhibition of the proteasome, and this is strictly dependent upon sustained propagation of PrPSc. Whereas cells overexpressing PrPC developed cytosolic PrPC aggregates, this did not cause cell death. In contrast, only in prion-infected cells, mild proteasome impairment resulted in the formation of large cytosolic perinuclear aggresomes that contained PrPSc, heat shock chaperone 70, ubiquitin, proteasome subunits, and vimentin. Similar structures were found in the brains of prion-infected mice. PrPSc aggresome formation was directly associated with activation of caspase 3 and 8, resulting in apoptosis. These data suggest that neuronal propagation of prions invokes a neurotoxic mechanism involving intracellular formation of PrPSc aggresomes. This, in turn, triggers caspase-dependent apoptosis and further implicates proteasome dysfunction in the pathogenesis of prion diseases.

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.

PrP Antibodies Do Not Trigger Mouse Hippocampal Neuron Apoptosis

Potentially therapeutic monoclonal antibodies do not kill neurons. Intraperitoneal administration of ICSM18 and 35, monoclonal antibodies against prion protein (PrP), has been shown to significantly delay the onset of prion disease in mice, and humanized versions are candidate therapeutics for prion and Alzheimer’s diseases. However, a previous report of severe and widespread apoptosis after intracerebral injection of anti-PrP monoclonal antibodies raised concerns about such therapy and led to an influential model of prion neurotoxicity via cross-linking of cell surface PrP by disease-related PrP aggregates. In extensive studies including ICSM18 and 35, fully humanized ICSM18, and the previously reported proapoptotic antibodies, we found no evidence of apoptosis, thereby questioning this model of prion neurotoxicity.

Plasmacytoid Dendritic Cells Sequester High Prion Titres at Early Stages of Prion Infection

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.

Identification of a gene regulatory network associated with prion replication

Prions consist of aggregates of abnormal conformers of the cellular prion protein (PrPC). They propagate by recruiting host‐encoded PrPC although the critical interacting proteins and the reasons for the differences in susceptibility of distinct cell lines and populations are unknown. We derived a lineage of cell lines with markedly differing susceptibilities, unexplained by PrPC expression differences, to identify such factors. Transcriptome analysis of prion‐resistant revertants, isolated from highly susceptible cells, revealed a gene expression signature associated with susceptibility and modulated by differentiation. Several of these genes encode proteins with a role in extracellular matrix (ECM) remodelling, a compartment in which disease‐related PrP is deposited. Silencing nine of these genes significantly increased susceptibility. Silencing of Papss2 led to undersulphated heparan sulphate and increased PrPC deposition at the ECM, concomitantly with increased prion propagation. Moreover, inhibition of fibronectin 1 binding to integrin α8 by RGD peptide inhibited metalloproteinases (MMP)‐2/9 whilst increasing prion propagation. In summary, we have identified a gene regulatory network associated with prion propagation at the ECM and governed by the cellular differentiation state.

Exosome release from infected dendritic cells: A clue for a fast spread of prions in the periphery?

Prion diseases are incurable transmissible neurological disorders. In many natural and experimental prion diseases, infectious prions can be detected in the lymphoreticular system (LRS) long before they reach the brain where they cause a fatal rapidly progressive degeneration. Although major cell types that contribute to prion accumulation have been identified, the mode of prion dissemination in the LRS remains elusive. Recent evidence of a remarkably fast splenic prion accumulation after peripheral infection of mice, resulting in high prion titers in dendritic cells (DCs) and a release of prions from infected DCs via exosomes suggest that intercellular dissemination may contribute to rapid prion colonization in the LRS. A vast body of evidence from retroviral infections shows that DCs and other antigen-presenting cells (APCs) share viral antigens by intercellular transfer to warrant immunity against viruses if APCs remain uninfected. Evolved to adapt the immune response to evading pathogens, these pathways may constitute a portal for unimpeded prion dissemination owing to the tolerance of the immune system against host-encoded prion protein. In this review we summarize current paradigms for antigen-sharing pathways which may be relevant to better understand dissemination of rogue neurotoxic proteins.