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Accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis, which is mediated by eIF2α-P and is associated with synaptic failure and neuronal loss in prion-diseased mice; promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Fine-tuning protein synthesis in prion disease Despite extensive research, the mechanisms leading to neuronal loss in neurodegenerative disease are still little understood, and no treatments or promising treatment strategies exist. Using prion-diseased mice as a model, this study demonstrates that the accumulation of misfolded prion protein during prion replication causes persistent translational repression of global protein synthesis. This is mediated by eIF2α-P and is associated with synaptic failure and neuronal loss in prion-diseased mice. Promoting translational recovery in the hippocampi of prion-infected mice is neuroprotective, suggesting that a generic approach involving the fine-tuning of protein synthesis may be worth pursuing in prion diseases, and perhaps in other neurodegenerative disorders involving protein misfolding. The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer’s, Parkinson’s and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the α-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2α-P levels are seen in patients with Alzheimer’s, Parkinson’s and prion diseases 1 , 2 , 3 , 4 , but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2α-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2α-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2α-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2α-P dephosphorylation 5 , increased eIF2α-P levels, exacerbating neurotoxicity and significantly reducing survival in prion-diseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders.

The prion hypothesis posits that a misfolded form of prion protein (PrP) is responsible for the infectivity of prion disease. Using recombinant murine PrP purified from Escherichia coli, we created a recombinant prion with the attributes of the pathogenic PrP isoform: aggregated, protease-resistant, and self-perpetuating. After intracerebral injection of the recombinant prion, wild-type mice developed neurological signs in approximately 130 days and reached the terminal stage of disease in approximately 150 days. Characterization of diseased mice revealed classic neuropathology of prion disease, the presence of protease-resistant PrP, and the capability of serially transmitting the disease; these findings confirmed that the mice succumbed to prion disease. Thus, as postulated by the prion hypothesis, the infectivity in mammalian prion disease results from an altered conformation of PrP.

Prions are infectious proteins composed of the abnormal disease-causing isoform PrPSc, which induces conformational conversion of the host-encoded normal cellular prion protein PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of PrPSc with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.

A definitive pre-mortem diagnosis of prion disease depends on brain biopsy for prion detection currently and no validated alternative preclinical diagnostic tests have been reported to date. To determine the feasibility of using skin for preclinical diagnosis, here we report ultrasensitive serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays of skin samples from hamsters and humanized transgenic mice (Tg40h) at different time points after intracerebral inoculation with 263K and sCJDMM1 prions, respectively. sPMCA detects skin PrP Sc as early as 2 weeks post inoculation (wpi) in hamsters and 4 wpi in Tg40h mice; RT-QuIC assay reveals earliest skin prion-seeding activity at 3 wpi in hamsters and 20 wpi in Tg40h mice. Unlike 263K-inoculated animals, mock-inoculated animals show detectable skin/brain PrP Sc only after long cohabitation periods with scrapie-infected animals. Our study provides the proof-of-concept evidence that skin prions could be a biomarker for preclinical diagnosis of prion disease. There are currently no validated methods for the diagnosis of prion disease at the preclinical stage. Here the authors show that serial protein misfolding cyclic amplification and real-time quaking-induced conversion can be used to detect prions in the skin of prion-inoculated hamsters and humanized transgenic mice at early preclinical stages.

Prions are infectious pathogens essentially composed of PrPSc, an abnormally folded form of the host-encoded prion protein PrPC. Constrained steric interactions between PrPSc and PrPC are thought to provide prions with species specificity and to control cross-species transmission into other host populations, including humans. We compared the ability of brain and lymphoid tissues from ovine and human PrP transgenic mice to replicate foreign, inefficiently transmitted prions. Lymphoid tissue was consistently more permissive than the brain to prions such as those causing chronic wasting disease and bovine spongiform encephalopathy. Furthermore, when the transmission barrier was overcome through strain shifting in the brain, a distinct agent propagated in the spleen, which retained the ability to infect the original host. Thus, prion cross-species transmission efficacy can exhibit a marked tissue dependence.

Key Points Since the early 1950s researchers have been studying slow and invariably fatal diseases such as scrapie (sheep), Creutzfeldt–Jakob disease and kuru (humans), bovine spongiform encephalopathy (cattle and sheep) and chronic wasting disease (deer) that are now known to be caused by transmissible agents dubbed 'prions'. After decades of investigation the precise structure of the prion is still debated. The 'protein-only' hypothesis posits that prions are congruent with PrP Sc , a misfolded form of the naturally occurring 'cellular prion protein' (PrP C ). PrP C is encoded by the Prnp locus and is normally attached to the cell surface by a glycosylphosphatidyl inositol (GPI) anchor. Replication of the prion is attributed to PrP Sc -catalysed conversion of PrP C to PrP Sc . The 'virus' and 'virino' hypotheses propose that the infectious agent contains an informational nucleic acid; however, despite the best efforts of many laboratories, no such molecule has been identified so far. Infectivity purified from infected brain material contains aggregates of PrP Sc as the major protein component, bundled together with other substances including glycosaminoglycans and polysaccharides. Solubilization of the aggregates by denaturants causes loss of infectivity, which so far is irreversible. The replication of prions is discussed, together with experimental evidence obtained from whole organisms, cell lines and cell-free in vitro systems. Research into so-called 'yeast prions', self-propagating conformational isoforms of certain yeast proteins, has added experimental support to the protein-only hypothesis. Differences in strains and factors that affect prion transmission are considered in light of the protein-only prion hypothesis. Expression of cellular PrP is essential for susceptibility to prion disease and for prion replication, but other genes also have a modulating role. The spread of prions within organisms also requires expression of the cellular form PrP C , both within and outside the central nervous system. Certain cells of the immune system serve as amplification sites in prion spread. The origin and evolution of prions are considered. Misfolded PrP may have evolved to serve a useful purpose and at the same time acquired a pathogenic potential that, in early evolutionary times, when the human life span was short, did not confer a selective disadvantage. Alternatively, prions could be derived from ancient exogenous pathogens that are now fully integrated into host chromosomes. More trivially, prions are misfolded proteins that by coincidence have the ability to invade a host through the digestive tract, make their way into the lymphoreticular system where they are amplified and transfer themselves into the central nervous system, which they then destroy. There is little doubt that the main component of the transmissible agent of spongiform encephalopathies — the prion — is a conformational variant of the ubiquitous host protein PrP C , and that the differing properties of various prion strains are associated with different abnormal conformations of this protein. The precise structure of the prion is not yet known, nor are the mechanisms of infection, conformational conversion and pathogenesis understood.

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrP C , into a plethora of assemblies, PrP Sc , associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrP Sc structures. However, the degree to which intra- and inter-strain PrP Sc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrP Sc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrP Sc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrP Sc particles. We found that each strain had a continuum of PrP Sc assemblies, with strong correlation between PrP Sc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrP Sc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrP Sc took place at a hydrodynamic radius (R h ) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrP Sc subpopulations and demonstrate that while PrP Sc quaternary structure is a major contributor to PrP Sc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrP Sc particles from maintaining proteinase K resistance below an R h of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.

Objective The goal of the research presented here is to determine if methods previously developed for the aqueous extraction of PrP Sc from formalin-fixed paraffin-embedded tissue (FFPET) are applicable to the detection PrP Sc by real-time quaking induced conversion (RT-QuIC). Previous work has utilized aqueous extraction of FFPET for detection of transmissible spongiform encephalopathies (TSEs) utilizing western blot and ELISA. This research extends the range of suitable methods for detection of TSEs in FFPET to RT-QuIC, which is arguably the most sensitive method to detect TSEs. Results We found complete agreement between the TSE status and the results from RT-QuIC seeded with the aqueous extract of FFPET samples. The method affords the diagnostic assessment TSE status by RT-QuIC of FFPET without the use of organic solvents that would otherwise create a mixed chemical-biological waste for disposal.

Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of β sheet-rich structures. Fibrils consisting of recMoPrP(89-230) were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89-231). The mice developed neurologic dysfunction between 380 and 660 days after inoculation. Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively. Neuropathological findings suggest that a novel prion strain was created. Our results provide compelling evidence that prions are infectious proteins.