Zuzana Krejciova

Propagation of prions causing synucleinopathies in cultured cells.

Significance Progressive supranuclear palsy (PSP) and multiple system atrophy (MSA) are neurodegenerative diseases caused by tau and α-synuclein prions, respectively. Prions, purified from human brains of deceased patients with PSP and MSA using phosphotungstic acid, were applied to cultured cell models that selectively form aggregates in the presence of tau or α-synuclein prions, respectively. Whereas brain homogenates prepared from two PSP and six MSA patients infected cultured cells, the same approach was unsuccessful with brain samples from three Parkinson’s disease patients. Our findings provide compelling evidence that PSP and MSA are prion diseases, and that MSA is caused by several distinct prion strains. Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)–YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson’s disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.

Human embryonic stem cells rapidly take up and then clear exogenous human and animal prions in vitro.

Susceptibility to prion infection involves interplay between the prion strain and host genetics, but expression of the host‐encoded cellular prion protein is a known prerequisite. Here we consider human embryonic stem cell (hESC) susceptibility by characterizing the genetics and expression of the normal cellular prion protein and by examining their response to acute prion exposure. Seven hESC lines were tested for their prion protein gene codon 129 genotype and this was found to broadly reflect that of the normal population. hESCs expressed prion protein mRNA, but only low levels of prion protein accumulated in self‐renewing populations. Following undirected differentiation, up‐regulation of prion protein expression occurred in each of the major embryonic lineages. Self‐renewing populations of hESCs were challenged with infectious human and animal prions. The exposed cells rapidly and extensively took up this material, but when the infectious source was removed the level and extent of intracellular disease‐associated prion protein fell rapidly. In the absence of a sufficiently sensitive test for prions to screen therapeutic cells, and given the continued use of poorly characterized human and animal bioproducts during hESC derivation and cultivation, the finding that hESCs rapidly take up and process abnormal prion protein is provocative and merits further investigation. Copyright

Human tonsil-derived follicular dendritic-like cells are refractory to human prion infection in vitro and traffic disease-associated prion protein to lysosomes.

The molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined. This is due, in part, to the absence of any well characterized and relevant cultured human cells susceptible to infection with human prions, such as those involved in Creutzfeldt-Jakob disease. In variant Creutzfeldt-Jakob disease, prion replication is thought to occur first in the lymphoreticular system and then spread into the brain. We have, therefore, examined the susceptibility of a human tonsil-derived follicular dendritic cell-like cell line (HK) to prion infection. HK cells were found to display a readily detectable, time-dependent increase in cell-associated abnormal prion protein (PrP(TSE)) when exposed to medium spiked with Creutzfeldt-Jakob disease brain homogenate, resulting in a coarse granular perinuclear PrP(TSE) staining pattern. Despite their high level of cellular prion protein expression, HK cells failed to support infection, as judged by longer term maintenance of PrP(TSE) accumulation. Colocalization studies revealed that exposure of HK cells to brain homogenate resulted in increased numbers of detectable lysosomes and that these structures immunostained intensely for PrP(TSE) after exposure to Creutzfeldt-Jakob disease brain homogenate. Our data suggest that human follicular dendritic-like cells and perhaps other human cell types are able to avoid prion infection by efficient lysosomal degradation of PrP(TSE).

Human stem cell-derived astrocytes replicate human prions in a PRNP genotype-dependent manner

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt–Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype–dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery.

Genotype-dependent Molecular Evolution of Sheep Bovine Spongiform Encephalopathy (BSE) Prions in Vitro Affects Their Zoonotic Potential

Background: The results of serial passage of BSE in sheep are unknown. Results: In vitro modeling shows a sheep genotype-dependent switch in prion protein type and loss of the ability to convert human prion protein. Conclusion: Molecular evolution of sheep BSE prions in vitro is genotype-dependent and affects zoonotic potential. Significance: Zoonotic risk might be predicted from cell-free modeling. Prion diseases are rare fatal neurological conditions of humans and animals, one of which (variant Creutzfeldt-Jakob disease) is known to be a zoonotic form of the cattle disease bovine spongiform encephalopathy (BSE). What makes one animal prion disease zoonotic and others not is poorly understood, but it appears to involve compatibility between the prion strain and the host prion protein sequence. Concerns have been raised that the United Kingdom sheep flock may have been exposed to BSE early in the cattle BSE epidemic and that serial BSE transmission in sheep might have resulted in adaptation of the agent, which may have come to phenotypically resemble scrapie while maintaining its pathogenicity for humans. We have modeled this scenario in vitro. Extrapolation from our results suggests that if BSE were to infect sheep in the field it may, with time and in some sheep genotypes, become scrapie-like at the molecular level. However, the results also suggest that if BSE in sheep were to come to resemble scrapie it would lose its ability to affect humans.

A novel vector for transgenesis in the rat CNS

The larger brain of the rat enables a much greater repertoire of complex behaviors than mice, likely making rats preferential for investigating neurodegeneration. Because molecular tools for specific expression of transgenes in the rat brain are sparse, we chose Prnp encoding the prion protein (PrP) to develop a novel vector to drive transgene expression in the rat brain. We compared the rat Prnp sequence with mouse and Syrian hamster Prnp sequences, identifying conserved genetic elements and hypothesizing that these elements would be able to drive neuronal transgene expression. We investigated this by generating a vector termed RaPrnp that encompasses portions of the rat Prnp gene. Importantly, we replaced the rat Prnp open reading frame (ORF) with a cloning site for rapid and seamless In-Fusion cloning. To validate the in vivo neuronal specificity of the RaPrnp vector in rats, we generated stable RaPrnp-LacZ/enhanced green fluorescent protein (EGFP) transgenic (Tg) rat lines, which led to robust LacZ activity and high EGFP fluorescence in the central nervous system of embryos and adult animals. Next, we restored the rat Prnp ORF and generated multiple Tg(RaPrnp-PrP) lines, demonstrating that overexpression of Prnp accelerates the onset of scrapie. While the incubation time in wild-type (WT) rats was 175xa0±xa03xa0days post inoculation (dpi), one line, Tg2919, overexpressed RaPrPC at 4.4-fold and exhibited a reduced incubation time of 149xa0±xa02 dpi. The second line, Tg2922, overexpressed RaPrPC atxa09.7-fold compared with WT animals and had an incubation time of 112xa0±xa00 dpi. Tg2922 rats inoculated with rat RML showed extensive vacuolation of the brainstem in contrast to WT and Tg2919 animals in which vacuolation was most prominent in the hippocampus and striatum as well as the motor and sensory cortices. It is possible that construction of Tg rats with modified phenotypes will prove more advantageous than mice for neurodegeneration studies.

Validating human stem cell derived neural cultures as a flexible model system in which to investigate neurodegenerative mechanisms

Until now, the 3-dimensional structure of infectious mammalian prions and how this differs from non-infectious amyloid fibrils remained unknown. Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity. One of the major challenges has been the production of highly homogeneous material of demonstrable high specific infectivity to allow direct correlation of particle structure with infectivity. nWe have recently developed novel methods to obtain exceptionally pure preparations of prions from prion-infected murine brain and have shown that pathogenic PrP in these high-titer preparations is assembled into rod-like assemblies (Wenborn et al. 2015. Sci. Rep. 10062). Our preparations contain very high titres of infectious prions which faithfully transmit prion strain-specific phenotypes when inoculated into mice making them eminently suitable for detailed structural analysis. We are now undertaking structural characterization of prion assemblies and comparing these to the structure of non-infectious PrP fibrils generated from recombinant PrP

Creutzfeldt-Jakob disease prion propagation in human iPS cells-derived astrocytes

The mechanism by which a random-coil polypeptide folds into the native structure may be critical for the regulation of misfolding diseases. However, how the folding pathway is related to the misfolding pathway remains unclear. Our recent study demonstrated that the native folding pathway of prion protein (PrP) involves at least 4 independent species, including native state (N), unfolded state (U), and 2 types of partially folded states (A and I) (Honda et al., Structure, 2015). Interestingly, one of the partially folded states (A state) readily formed a misfolded aggregate whose rates were strongly correlated with the initial population of the A state (Honda et al., J Biol Chem, 2014). This observation indicated that the formation of the A state may be the initial step in the misfolding pathway. We characterized the structure of the A state using circular dichroism, hydrogen/deuterium exchange coupled with NMR, etc. and found that the Strand 1Helix 1-Strand 2 segment was completely unfolded in the A state, whereas the Helix 2Helix 3 segment retained a native-like helical structure. Our studies revealed how the native structure is altered during the early stage of misfolding and how the misfolding pathway is related to the native folding pathway of PrP. O-02: HET-2s, an engineered, 4-rung b-solenoid protein as a model for the structure of PrP Holger Wille, Andrew Fang, Brian Tancowny, Xiongyao Wang, Lyudmyla Dorosh, and Maria Stepanova