Infectious Prion Protein Research Articles

The PINK1/Parkin pathway of mitophagy exerts a protective effect during prion disease.

The PINK1/Parkin pathway of mitophagy has been implicated in the pathogenesis of Parkinson's disease. In prion diseases, a transmissible neurodegenerative disease caused by the misfolded and infectious prion protein (PrPSc), expression of both PINK1 and Parkin are elevated, suggesting that PINK1/Parkin mediated mitophagy may also play a role in prion pathogenesis. Using mice in which expression of either PINK1 (PINK1KO) or Parkin (ParkinKO) has been ablated, we analyzed the potential role of PINK1 and Parkin in prion pathogenesis. Prion infected PINK1KO and ParkinKO mice succumbed to disease more rapidly (153 and 150 days, respectively) than wild-type control C57Bl/6 mice (161 days). Faster incubation times in PINK1KO and ParkinKO mice did not correlate with altered prion pathology in the brain, altered expression of proteins associated with mitochondrial dynamics, or prion-related changes in mitochondrial respiration. However, the expression level of mitochondrial respiration Complex I, a major site for the formation of reactive oxygen species (ROS), was higher in prion infected PINK1KO and ParkinKO mice when compared to prion infected control mice. Our results demonstrate a protective role for PINK1/Parkin mitophagy during prion disease, likely by helping to minimize ROS formation via Complex I, leading to slower prion disease progression.

The Prion Basis of Progressive Neurodegenerative Disorders.

The discovery of proteinaceous infectious agents by Prusiner in 1982 was sensational. All previously known pathogens contained nucleic acids, the code of life, that enabled them to reproduce. In contrast, the proteinaceous agents of disease, called prion proteins (PrP), lacked nucleic acids and propagated by binding to the functional, endogenous form of cellular prion protein (referred to as PrPC) and altering its conformation to produce the infectious disease-causing misfolded protein (referred to as PrPSc). The accumulation and aggregation of these infectious prion proteins within the brain cause destruction of neural tissue and lead to fatal spongiform encephalopathies. In this review, we present the molecular pathology of prion-based diseases. These insights are of particular importance since the principles of prion pathogenesis apply to other neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Collectively, the global prevalence of these diseases is rapidly increasing while effective therapies against them are still lacking. Thus, the need to understand their etiology and pathogenesis is urgent, and it holds profound implications for societal health.

Movement of Chronic Wasting Disease Prions in Prairie, Boreal and Alpine Soils.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy negatively impacting cervids on three continents. Soil can serve as a reservoir for horizontal transmission of CWD by interaction with the infectious prion protein (PrPCWD) shed by diseased individuals and from infected carcasses. We investigated the pathways for PrPCWD migration in soil profiles using lab-scale soil columns, comparing PrPCWD migration through pure soil minerals (quartz, illite and montmorillonite), and diverse soils from boreal (Luvisol, Brunisol) and prairie (Chernozem) regions. We analyzed the leachate of the soil columns by immunoblot and protein misfolding cyclic amplification (PMCA) and detected PrP in the leachates of columns composed of quartz, illite, Luvisol and Brunisol. Animal bioassay confirmed the presence of CWD infectivity in the leachates from quartz, illite and Luvisol columns. Leachates from columns with montmorillonite and prairie Chernozems did not contain PrP detectable by immunoblotting or PMCA; bioassay confirmed that the Chernozemic leachate was not infectious. Analysis of the solid phase of the columns confirmed the migration of PrP to lower layers in the illite column, while the strongest signal in the montmorillonite column remained close to the surface. Montmorillonite, the prevalent clay mineral in prairie soils, has the strongest prion binding ability; by contrast, illite, the main clay mineral in northern boreal and tundra soils, does not bind prions significantly. This suggests that in soils of North American CWD-endemic regions (Chernozems), PrPCWD would remain on the soil surface due to avid binding to montmorillonite. In boreal Luvisols and mountain Brunisols, prions that pass through the leaf litter will continue to move through the soil mineral horizon, becoming less bioavailable. In light-textured soils where quartz is a dominant mineral, the majority of the infectious prions will move through the soil profile. Local soil properties may consequently determine the efficiency of environmental transmission of CWD.

Adipose-derived mesenchymal stromal cells decrease prion-induced glial inflammation in vitro

Prion diseases are characterized by the cellular prion protein, PrPC, misfolding and aggregating into the infectious prion protein, PrPSc, which leads to neurodegeneration and death. An early sign of disease is inflammation in the brain and the shift of resting glial cells to reactive astrocytes and activated microglia. Few therapeutics target this stage of disease. Mesenchymal stromal cells produce anti-inflammatory molecules when exposed to inflammatory signals and damaged tissue. Here, we show that adipose-derived mesenchymal stromal cells (AdMSCs) migrate toward prion-infected brain homogenate and produce the anti-inflammatory molecules transforming growth factor β (TGFβ) and tumor necrosis factor-stimulated gene 6 (TSG-6). In an in vitro model of prion exposure of both primary mixed glia and BV2 microglial cell line, co-culturing with AdMSCs led to a significant decrease in inflammatory cytokine mRNA and markers of reactive astrocytes and activated microglia. This protection against in vitro prion-associated inflammatory responses is independent of PrPSc replication. These data support a role for AdMSCs as a beneficial therapeutic for decreasing the early onset of glial inflammation and reprogramming glial cells to a protective phenotype.

Molecular insights into the critical role of gallate moiety of green tea catechins in modulating prion fibrillation, cellular internalization, and neuronal toxicity

Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases with no approved therapeutics. TSE pathology is characterized by abnormal accumulation of amyloidogenic and infectious prion protein conformers (PrPSc) in the central nervous system. Herein, we examined the role of gallate group in green tea catechins in modulating the aggregation of human prion protein (HuPrP) using two green tea constituents i.e., epicatechin 3-gallate (EC3G; with intact gallate ring) and epigallocatechin (EGC; without gallate ring). Molecular docking indicated distinct differences in hydrogen bonding and hydrophobic interactions of EC3G and EGC at the β2-α2 loop of HuPrP. These differences were substantiated by 44-fold higher KD for EC3G as compared to EGC with the former significantly reducing Thioflavin T (ThT) binding aggregates of HuPrP. Conformational alterations in HuPrP aggregates were validated by particle sizing, AFM analysis and A11 and OC conformational antibodies. As compared to EGC, EC3G showed relatively higher reduction in toxicity and cellular internalization of HuPrP oligomers in Neuro-2a cells. Additionally, EC3G also displayed higher fibril disaggregating properties as observed by ThT kinetics and electron microscopy. Our observations were supported by molecular dynamics (MD) simulations that showed markedly reduced α2-α3 and β2-α2 loop mobilities in presence of EC3G that may lead to constriction of HuPrP conformational space with lowered β-sheet conversion. In totality, gallate moiety of catechins play key role in modulating HuPrP aggregation, and toxicity and could be a new structural motif for designing therapeutics against prion diseases and other neurodegenerative disorders.

Cryo-EM structure of disease-related prion fibrils provides insights into seeding barriers.

One of the least understood aspects of prion diseases is the structure of infectious prion protein aggregates. Here we report a high-resolution cryo-EM structure of amyloid fibrils formed by human prion protein with the Y145Stop mutation that is associated with a familial prion disease. This structural insight allows us not only to explain previous biochemical findings, but also provides direct support for the conformational adaptability model of prion transmissibility barriers.

Prion/virus the danger of biological weapons

If the attacking force has a vaccine before the attack, will inflict a crippling damage on the enemy. Thus, virus emerges as potential danger since they can be used in the development of such weapons. The reported of a neurotropic strain influenza A virus (IAV) induced the conversion of normal prion protein (PrPC) into infectious prion protein (PrPSc) as well as formation of infectious prions bring out the danger of a type of prion/virus as unprecedented infectious pathogens that cause fatal neurodegenerative diseases. Due to their singular characteristics a virus inducer of prion disease can be a stealth advantage for the attacker if he has an effective vaccine. Therefore, lethal prion/virus can be developed by malicious researchers who could use it to attack political enemies.

A new hope: Mitochondria, a critical factor in the war against prions

Prion diseases encompass a group of incurable neurodegenerative disorders that occur due to the misfolding and aggregation of infectious proteins. The most well-known prion diseases are Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (also known as mad cow disease), and kuru. It is estimated that around 1–2 persons per million worldwide are affected annually by prion disorders. Infectious prion proteins propagate in the brain, clustering in the cells and rapidly inducing tissue degeneration and death. Prion disease alters cell metabolism and energy production damaging mitochondrial function and dynamics leading to a fast accumulation of damage. Dysfunction of mitochondria could be considered as an early precursor and central element in the pathogenesis of prion diseases such as in sporadic CJD. Preserving mitochondria function may help to resist the rapid spread and damage of prion proteins and even clearance. In the war against prions and other degenerative diseases, studying how to preserve the function of mitochondria by using antioxidants and even replacing them with artificial mitochondrial transfer/transplant (AMT/T) may bring a new hope and lead to an increase in patients' survival. In this perspective review, we provide key insights about the relationship between the progression of prion disease and mitochondria, in which understanding how protecting mitochondria function and viability by using antioxidants or AMT/T may help to develop novel therapeutic interventions.

Prion-like strain effects in tauopathies.

Tau is a microtubule-associated protein that plays crucial roles in physiology and pathophysiology. In the realm of dementia, tau protein misfolding is associated with a wide spectrum of clinicopathologically diverse neurodegenerative diseases, collectively known as tauopathies. As proposed by the tau strain hypothesis, the intrinsic heterogeneity of tauopathies may be explained by the existence of structurally distinct tau conformers, “strains”. Tau strains can differ in their associated clinical features, neuropathological profiles, and biochemical signatures. Although prior research into infectious prion proteins offers valuable lessons for studying how a protein-only pathogen can encompass strain diversity, the underlying mechanism by which tau subtypes are generated remains poorly understood. Here we summarize recent advances in understanding different tau conformers through in vivo and in vitro experimental paradigms, and the implications of heterogeneity of pathological tau species for drug development.

Prion protein with a mutant N-terminal octarepeat region undergoes cobalamin-dependent assembly into high–molecular weight complexes

The cellular prion protein (PrPC) has a C-terminal globular domain and a disordered N-terminal region encompassing five octarepeats (ORs). Encounters between Cu(II) ions and four OR sites produce interchangeable binding geometries; however, the significance of Cu(II) binding to ORs in different combinations is unclear. To understand the impact of specific binding geometries, OR variants were designed that interact with multiple or single Cu(II) ions in specific locked coordinations. Unexpectedly, we found that one mutant produced detergent-insoluble, protease-resistant species in cells in the absence of exposure to the infectious prion protein isoform, scrapie-associated prion protein (PrPSc). Formation of these assemblies, visible as puncta, was reversible and dependent upon medium formulation. Cobalamin (Cbl), a dietary cofactor containing a corrin ring that coordinates a Co3+ ion, was identified as a key medium component, and its effect was validated by reconstitution experiments. Although we failed to find evidence that Cbl interacts with Cu-binding OR regions, we instead noted interactions of Cbl with the PrPC C-terminal domain. We found that some interactions occurred at a binding site of planar tetrapyrrole compounds on the isolated globular domain, but others did not, and N-terminal sequences additionally had a marked effect on their presence and position. Our studies define a conditional effect of Cbl wherein a mutant OR region can act in cis to destabilize a globular domain with a wild type sequence. The unexpected intersection between the properties of PrPSc's disordered region, Cbl, and conformational remodeling events may have implications for understanding sporadic prion disease that does not involve exposure to PrPSc.

Monitoring of chronic wasting disease in deer in Poland between 2018 and 2020

Chronic wasting disease (CWD) is a lethal infectious neurodegenerative disease, the etiological factor of which is the infectious prion protein. Infections affect animals of the deer family (Cervidae). The aim of the study was to analyze the results of monitoring for the presence of CWD in the years 2018-2020 carried out by the Veterinary Inspectorate in Poland. The aim of the monitoring program in force in 2018-2020 was to assess the epizootic situation of CWD in Poland and other European countries, where no cases of the disease have been detected so far. Six species of deer were monitored: Eurasian tundra reindeer, Finnish forest reindeer, roe deer, elk, red deer, and white-tailed deer. Samples for the study were collected by veterinarians and trained persons and came from a latch within the medulla, retropharyngeal lymph nodes, tonsils, or other lymph nodes in the head. During the three-year CWD monitoring in Poland, a total of 3541 samples from animals from the deer family were examined. With regard to farmed and captive animals, the samples came from 225 red deer and 1 clump, and in the case of wild animals, the study included 192 elk, 2703 roe deer, and 420 red deer. In 2018, 1141 animals were tested: 23 farm-kept noble deer and 42 elk, 886 roe deer, and 190 wild-living noble deer. In 2019, the monitoring covered 1246 animals: 115 red deer and 1 female elk were farm animals, and the remaining 83 elk, 902 roe deer, and 145 red deer were free-living. In the last year of the program, 1154 animals were tested: 87 farmed red deer and 67 wild elk, 915 roe deer, and 85 wild deer. During the monitoring period, no cases of chronic wasting disease (CWD) of cervids were detected in Poland. The program officially ended at the end of December 2020 and was not extended because of the absence of CWD-positive results.

The Role of the Nasal Cavity in the Pathogenesis of Prion Diseases.

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a class of fatal neurodegenerative diseases caused by the entry and spread of infectious prion proteins (PrPSc) in the central nervous system (CNS). These diseases are endemic to certain mammalian animal species that use their sense of smell for a variety of purposes and therefore expose their nasal cavity (NC) to PrPSc in the environment. Prion diseases that affect humans are either inherited due to a mutation of the gene that encodes the prion protein, acquired by exposure to contaminated tissues or medical devices, or develop without a known cause (referred to as sporadic). The purpose of this review is to identify components of the NC that are involved in prion transport and to summarize the evidence that the NC serves as a route of entry (centripetal spread) and/or a source of shedding (centrifugal spread) of PrPSc, and thus plays a role in the pathogenesis of the TSEs.

Magnetic microparticle-based multimer detection system for the electrochemical detection of prion oligomers in sheep using a recyclable BDD electrode

There is a strong need of sensitive and accurate diagnostic tests of infectious prion proteins (PrPSc) in pre-symptomatic individuals and animals. This work associates, for the first time, the magnetic microparticle multimer detection system (MDS) with a recyclable boron doped diamond (BDD) electrode for the electrochemical detection of PrPSc in plasma samples from scrapie-infected sheep with clinical signs. The electrochemical results are in agreement with the chemiluminescence results. The main advantages of the electrochemical fluidic system are its recyclability and the use of low quantity of functional magnetic beads.

Fundamentals of Chemical Reactor Engineering: A Multi-Scale Approach.

There is a strong need of sensitive and accurate diagnostic tests of infectious prion proteins (PrPSc) in pre-symptomatic individuals and animals. This work associates, for the first time, the magnetic microparticle multimer detection system (MDS) with a recyclable boron doped diamond (BDD) electrode for the electrochemical detection of PrPSc in plasma samples from scrapie-infected sheep with clinical signs. The electrochemical results are in agreement with the chemiluminescence results. The main advantages of the electrochemical fluidic system are its recyclability and the use of low quantity of functional magnetic beads.

Problems of ante mortem diagnostics of prion diseases

The review presents the state-of-the-art on the problem of diagnosis of prion diseases (PD) in humans and animals with a brief description of their etiology and pathogenesis. We pointed out that understanding the nature of the etio logical agent of PD determined their zoonotic potential and led to the development of highly specific immunological diagnostic methods aimed at identifying the infectious isoform of prion protein (PrPd) as the only marker of the disease. In this regard, we briefly summarize the results of studies, including our own, concerning the conversion of normal prion protein molecules (PrPc) to PrPd, the production of monoclonal antibodies and their application as immunodiagnostic reagents for the post-mortem detection of PrPd in various formats of immunoassay. We also emphasize the issues related to the development of methods for ante mortem diagnostics of PD. In this regard, a method for amplifying amino acid sequences using quacking-induced conversion of PrPc to PrPd in real time (RTQuIC) described in details. The results of recent studies on the assessment of the sensitivity, specificity and reproducibility of this method, carried out in various laboratories around the world, are presented. The data obtained indicate that RT-QuIC is currently the most promising laboratory assay for detecting PrPd in biological material at the preclinical stage of the disease. The significant contribution of US scientists to the introduction of this method into clinical practice on the model of diagnosis of chronic wasting disease of wild Cervidae (CWD) is noted. The possible further spread of CWD in the population of moose and deer in the territories bordering with Russia, as well as the established fact of alimentary transmission of CWD to macaques, indicate the threat of the appearance of PD in our country. In conclusion, the importance of developing new hypersensitive and/or selective components of known methods for PrPd identification from the point of view of assessing the risks of creating artificial infectious prion proteins in vivo or in vitro, primarily new pathogenic isoforms ("strains") and synthetic prions, was outlined.

Understanding and exploiting interactions between cellular proteostasis pathways and infectious prion proteins for therapeutic benefit.

Several neurodegenerative diseases of humans and animals are caused by the misfolded prion protein (PrPSc), a self-propagating protein infectious agent that aggregates into oligomeric, fibrillar structures and leads to cell death by incompletely understood mechanisms. Work in multiple biological model systems, from simple baker's yeast to transgenic mouse lines, as well as in vitro studies, has illuminated molecular and cellular modifiers of prion disease. In this review, we focus on intersections between PrP and the proteostasis network, including unfolded protein stress response pathways and roles played by the powerful regulators of protein folding known as protein chaperones. We close with analysis of promising therapeutic avenues for treatment enabled by these studies.

A Novel, Reliable and Highly Versatile Method to Evaluate Different Prion Decontamination Procedures.

Transmissible spongiform encephalopathies (TSEs) are a group of invariably fatal neurodegenerative disorders. The causal agent is an aberrantly folded isoform (PrPSc or prion) of the endogenous prion protein (PrPC) which is neurotoxic and amyloidogenic and induces misfolding of its physiological counterpart. The intrinsic physical characteristics of these infectious proteinaceous pathogens makes them highly resistant to the vast majority of physicochemical decontamination procedures used typically for standard disinfection. This means prions are highly persistent in contaminated tissues, the environment (surfaces) and, of great concern, on medical and surgical instruments. Traditionally, decontamination procedures for prions are tested on natural isolates coming from the brain of infected individuals with an associated high heterogeneity resulting in highly variable results. Using our novel ability to produce highly infectious recombinant prions in vitro we adapted the system to enable recovery of infectious prions from contaminated materials. This method is easy to perform and, importantly, results in highly reproducible propagation in vitro. It exploits the adherence of infectious prion protein to beads of different materials allowing accurate and repeatable assessment of the efficacy of disinfectants of differing physicochemical natures to eliminate infectious prions. This method is technically easy, requires only a small shaker and a standard biochemical technique and could be performed in any laboratory.

Prion Protein Gene (PRNP) Sequences Suggest Differing Vulnerability to Chronic Wasting Disease for Florida Key Deer (Odocoileus virginianus clavium) and Columbian White-Tailed Deer (O. v. leucurus).

Chronic wasting disease (CWD) is a fatal, highly transmissible spongiform encephalopathy caused by an infectious prion protein. CWD is spreading across North American cervids. Studies of the prion protein gene (PRNP) in white-tailed deer (WTD; Odocoileus virginianus) have identified non-synonymous substitutions associated with reduced CWD frequency. Because CWD is spreading rapidly geographically, it may impact cervids of conservation concern. Here, we examined the genetic vulnerability to CWD of 2 subspecies of WTD: the endangered Florida Key deer (O. v. clavium) and the threatened Columbian WTD (O. v. leucurus). In Key deer (n = 48), we identified 3 haplotypes formed by 5 polymorphisms, of which 2 were non-synonymous. The polymorphism c.574G>A, unique to Key deer (29 of 96 chromosomes), encodes a non-synonymous substitution from valine to isoleucine at codon 192. In 91 of 96 chromosomes, Key deer carried c.286G>A (G96S), previously associated with substantially reduced susceptibility to CWD. Key deer may be less genetically susceptible to CWD than many mainland WTD populations. In Columbian WTD (n = 13), 2 haplotypes separated by one synonymous substitution (c.438C>T) were identified. All of the Columbian WTD carried alleles that in other mainland populations are associated with relatively high susceptibility to CWD. While larger sampling is needed, future management plans should consider that Columbian WTD are likely to be genetically more vulnerable to CWD than many other WTD populations. Finally, we suggest that genetic vulnerability to CWD be assessed by sequencing PRNP across other endangered cervids, both wild and in captive breeding facilities.

Expanding spectrum of prion diseases.

Prions were initially discovered in studies of scrapie, a transmissible neurodegenerative disease (ND) of sheep and goats thought to be caused by slow viruses. Once scrapie was transmitted to rodents, it was discovered that the scrapie pathogen resisted inactivation by procedures that modify nucleic acids. Eventually, this novel pathogen proved to be a protein of 209 amino acids, which is encoded by a chromosomal gene. After the absence of a nucleic acid within the scrapie agent was established, the mechanism of infectivity posed a conundrum and eliminated a hypothetical virus. Subsequently, the infectious scrapie prion protein (PrPSc) enriched for β-sheet was found to be generated from the cellular prion protein (PrPC) that is predominantly α-helical. The post-translational process that features in nascent prion formation involves a templated conformational change in PrPC that results in an infectious copy of PrPSc. Thus, prions are proteins that adopt alternative conformations, which are self-propagating and found in organisms ranging from yeast to humans. Prions have been found in both Alzheimer's (AD) and Parkinson's (PD) diseases. Mutations in APP and α-synuclein genes have been shown to cause familial AD and PD. Recently, AD was found to be a double prion disorder: both Aβ and tau prions feature in this ND. Increasing evidence argues for α-synuclein prions as the cause of PD, multiple system atrophy, and Lewy body dementia.

Periplasmic production of pernisine in Escherichia coli and determinants for its high thermostability.

Pernisine is a subtilisin-like serine proteinase secreted by the hyperthermophilic archaeon Aeropyrum pernix. The significant properties of this proteinase are remarkable stability and ability to degrade the infectious prion proteins. Here we show the production of pernisine in the periplasm of Escherichia coli. This strategy prevented the aggregation of pernisine in the cytoplasm and increased the purity of the isolated pernisine. The thermostability of this recombinant pernisine was significantly increased compared with previous studies. In addition, several truncated pernisine variants were constructed and expressed in E. coli to identify the minimally active domain. The catalytic domain of pernisine consists of the αẞα structurally similar core flanked by the N-terminal and C-terminal outer regions. The deletion of the C-terminal α helix did not affect the pernisine activity at 90°C. However, the complete deletion of the C-terminal outer region resulted in loss of proteolytic activity. The pernisine variant, in which the N-terminal outer region was deleted, had a reduced activity at 90°C. These results underline the importance of the Ca2+ binding sites predicted in these outer regions for stability and activity of pernisine. KEY POINTS: • Aggregation of produced pernisine was prevented by translocation into periplasm. • Thermostability of mature pernisine was increased. • The outer regions of the catalytic core are required for pernisine thermostability.