Prion Disease Susceptibility Research Articles

The First Genetic Characterization of the SPRN Gene in Pekin Ducks (Anas platyrhynchos domesticus).

Prion diseases are fatal neurodegenerative disorders characterized by an accumulation of misfolded prion protein (PrPSc) in brain tissues. The shadow of prion protein (Sho) encoded by the shadow of prion protein gene (SPRN) is involved in prion disease progress. The interaction between Sho and PrP accelerates the PrPSc conversion rate while the SPRN gene polymorphisms have been associated with prion disease susceptibility in several species. Until now, the SPRN gene has not been investigated in ducks. We identified the duck SPRN gene sequence and investigated the genetic polymorphisms of 184 Pekin ducks. We compared the duck SPRN nucleotide sequence and the duck Sho protein amino acid sequence with those of several other species. Finally, we predicted the duck Sho protein structure and the effects of non-synonymous single nucleotide polymorphisms (SNPs) using computational programs. We were the first to report the Pekin duck SPRN gene sequence. The duck Sho protein sequence showed 100% identity compared with the chicken Sho protein sequence. We found 27 novel SNPs in the duck SPRN gene. Four amino acid substitutions were predicted to affect the hydrogen bond distribution in the duck Sho protein structure. Although MutPred2 and SNPs&GO predicted that all non-synonymous polymorphisms were neutral or benign, SIFT predicted that four variants, A22T, G49D, A68T, and M105I, were deleterious. To the best of our knowledge, this is the first report about the genetic and structural characteristics of the duck SPRN gene.

Recombinant ovine prion protein can be mutated at position 136 to improve its efficacy as an inhibitor of prion propagation

Prion diseases are progressive neurodegenerative disorders with no effective therapeutics. The central event leading to the pathology in the diseases is the conversion of PrPC into PrPSc and its accumulation in the central nervous system. Previous studies demonstrated that recombinant PrP (rPrP) and PrP peptides can inhibit the formation of PrPSc. Here, the effectiveness of ovine rPrP mutants at codon 136 and peptides derived from this region were assessed for their ability to inhibit PrPSc replication, using protein misfolding cyclic amplification (PMCA). Based on a rPrP VRQ (rVRQ) genotype background (positions 136, 154 and 171) and mutations at position 136, the most effective inhibitors were V136R, V136K and V136P mutants, with IC50 values of 1 to 2 nM; activities much more potent than rVRQ (114 nM). rRRQ and rKRQ were also shown to effectively inhibit multiple ruminant prion amplification reactions that used distinct prion strain seeds and substrate PRNP genotypes. rRRQ, rKRQ and rPRQ were also shown to effectively protect Rov9 cells from scrapie infection when applied at 250 nM. The study demonstrates for the first time that the rPrP sequence can be mutated at sites known to be involved in prion disease susceptibility, to produce inhibitors with improved efficacy.

Investigating CRISPR/Cas9 gene drive for production of disease-preventing prion gene alleles.

Prion diseases are a group of fatal neurodegenerative disorders that includes chronic wasting disease, which affects cervids and is highly transmissible. Given that chronic wasting disease prevalence exceeds 30% in some endemic areas of North America, and that eventual transmission to other mammalian species, potentially including humans, cannot be ruled out, novel control strategies beyond population management via hunting and/or culling must be investigated. Prion diseases depend upon post-translational conversion of the cellular prion protein, encoded by the Prnp gene, into a disease-associated conformation; ablation of cellular prion protein expression, which is generally well-tolerated, eliminates prion disease susceptibility entirely. Inspired by demonstrations of gene drive in caged mosquito species, we aimed to test whether a CRISPR/Cas9-based gene drive mechanism could, in principle, promote the spread of a null Prnp allele among mammalian populations. First, we showed that transient co-expression of Cas9 and Prnp-directed guide RNAs in RK13 cells generates indels within the Prnp open-reading frame, indicating that repair of Cas9-induced double-strand breaks by non-homologous end-joining had taken place. Second, we integrated a ~1.2 kb donor DNA sequence into the Prnp open-reading frame in N2a cells by homology-directed repair following Cas9-induced cleavages and confirmed that integration occurred precisely in most cases. Third, we demonstrated that electroporation of Cas9/guide RNA ribonucleoprotein complexes into fertilised mouse oocytes resulted in pups with a variety of disruptions to the Prnp open reading frame, with a new coisogenic line of Prnp-null mice obtained as part of this work. However, a technical challenge in obtaining expression of Cas9 in the male germline prevented implementation of a complete gene drive mechanism in mice.

Excessive replacement changes drive evolution of global sheep prion protein (PRNP) sequences.

Sheep prion protein (PRNP) is the major host genetic factor responsible for susceptibility to scrapie. We aimed to understand the evolutionary history of sheep PRNP, and primarily focused on breeds from Turkey and Ethiopia, representing genome-wise ancient sheep populations. Population molecular genetic analyses are extended to European, South Asian, and East Asian populations, and for the first time to scrapie associated haplotypes. 1178 PRNP coding region nucleotide sequences were analyzed. High levels of nucleotide diversity driven by extensive low-frequency replacement changes are observed in all populations. Interspecific analyses were conducted using mouflon and domestic goat as outgroup species. Despite an abundance of silent and replacement changes, lack of silent or replacement fixations was observed. All scrapie-associated haplotype analyses from all populations also showed extensive low-frequency replacement changes. Neutrality tests did not indicate positive (directional), balancing or strong negative selection or population contraction for any of the haplotypes in any population. A simple negative selection history driven by prion disease susceptibility is not supported by the population and haplotype based analyses. Molecular function, biological process enrichment, and protein-protein interaction analyses suggested functioning of PRNP protein in multiple pathways, and possible other functional constraint selections. In conclusion, a complex selection history favoring excessive replacement changes together with weak purifying selection possibly driven by frequency-dependent selection is driving PRNP sequence evolution. Our results is not unique only to the Turkish and Ethiopian samples, but can be generalized to global sheep populations.

How an Infection of Sheep Revealed Prion Mechanisms in Alzheimer's Disease and Other Neurodegenerative Disorders.

Although it is not yet universally accepted that all neurodegenerative diseases (NDs) are prion disorders, there is little disagreement that Alzheimer’s disease (AD), Parkinson’s disease, frontotemporal dementia (FTD), and other NDs are a consequence of protein misfolding, aggregation, and spread. This widely accepted perspective arose from the prion hypothesis, which resulted from investigations on scrapie, a common transmissible disease of sheep and goats. The prion hypothesis argued that the causative infectious agent of scrapie was a novel proteinaceous pathogen devoid of functional nucleic acids and distinct from viruses, viroids, and bacteria. At the time, it seemed impossible that an infectious agent like the one causing scrapie could replicate and exist as diverse microbiological strains without nucleic acids. However, aggregates of a misfolded host-encoded protein, designated the prion protein (PrP), were shown to be the cause of scrapie as well as Creutzfeldt–Jakob disease (CJD) and Gerstmann–Sträussler–Scheinker syndrome (GSS), which are similar NDs in humans. This review discusses historical research on diseases caused by PrP misfolding, emphasizing principles of pathogenesis that were later found to be core features of other NDs. For example, the discovery that familial prion diseases can be caused by mutations in PrP was important for understanding prion replication and disease susceptibility not only for rare PrP diseases but also for far more common NDs involving other proteins. We compare diseases caused by misfolding and aggregation of APP-derived Aβ peptides, tau, and α-synuclein with PrP prion disorders and argue for the classification of NDs caused by misfolding of these proteins as prion diseases. Deciphering the molecular pathogenesis of NDs as prion-mediated has provided new approaches for finding therapies for these intractable, invariably fatal disorders and has revolutionized the field.

The First Report of the Prion Protein Gene (PRNP) Sequence in Pekin Ducks (Anas platyrhynchos domestica): The Potential Prion Disease Susceptibility in Ducks.

Pathogenic prion protein (PrPSc), converted from normal prion protein (PrPC), causes prion disease. Although prion disease has been reported in several mammalian species, chickens are known to show strong resistance to prion diseases. In addition to chickens, the domestic duck occupies a large proportion in the poultry industry and may be regarded as a potential resistant host against prion disease. However, the DNA sequence of the prion protein gene (PRNP) has not been reported in domestic ducks. Here, we performed amplicon sequencing targeting the duck PRNP gene with the genomic DNA of Pekin ducks. In addition, we aligned the PrP sequence of the Pekin duck with that of various species using ClustalW2 and carried out phylogenetic analysis using Molecular Evolutionary Genetics Analysis X (MEGA X). We also constructed the structural modeling of the tertiary and secondary structures in avian PrP using SWISS-MODEL. Last, we investigated the aggregation propensity on Pekin duck PrP using AMYCO. We first reported the DNA sequence of the PRNP gene in Pekin ducks and found that the PrP sequence of Pekin ducks is more similar to that of geese than to that of chickens and mallards (wild ducks). Interestingly, Pekin duck PrP showed a high proportion of β-sheets compared to that of chicken PrP, and a high aggregation propensity compared to that of avian PrPs. However, Pekin duck PrP with substitutions of chicken-specific amino acids showed reduced aggregation propensities. To the best of our knowledge, this is the first report on the genetic characteristics of the PRNP sequence in Pekin ducks.

The Effects of Immune System Modulation on Prion Disease Susceptibility and Pathogenesis.

Prion diseases are a unique group of infectious chronic neurodegenerative disorders to which there are no cures. Although prion infections do not stimulate adaptive immune responses in infected individuals, the actions of certain immune cell populations can have a significant impact on disease pathogenesis. After infection, the targeting of peripherally-acquired prions to specific immune cells in the secondary lymphoid organs (SLO), such as the lymph nodes and spleen, is essential for the efficient transmission of disease to the brain. Once the prions reach the brain, interactions with other immune cell populations can provide either host protection or accelerate the neurodegeneration. In this review, we provide a detailed account of how factors such as inflammation, ageing and pathogen co-infection can affect prion disease pathogenesis and susceptibility. For example, we discuss how changes to the abundance, function and activation status of specific immune cell populations can affect the transmission of prion diseases by peripheral routes. We also describe how the effects of systemic inflammation on certain glial cell subsets in the brains of infected individuals can accelerate the neurodegeneration. A detailed understanding of the factors that affect prion disease transmission and pathogenesis is essential for the development of novel intervention strategies.

Cellular prion protein gene polymorphisms linked to differential scrapie susceptibility correlate with distinct residue connectivity between secondary structure elements

The conformational conversion of the cellular prion protein (PrPC) to the misfolded and aggregated isoform, termed scrapie prion protein (PrPSc), is key to the development of a group of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). Although the conversion mechanism is not fully understood, the role of gene polymorphisms in varying susceptibilities to prion diseases is well established. In ovine, specific gene polymorphisms in PrPC alter prion disease susceptibility: the Valine136–Glutamine171 variant (Susceptible structure) displays high susceptibility to classical scrapie while the Alanine136–Arginine171 variant (Resistant structure) displays reduced susceptibility. The opposite trend has been reported in atypical scrapie. Despite the differentiation between classical and atypical scrapie, a complete understanding of the effect of polymorphisms on the structural dynamics of PrPC is lacking. From our structural bioinformatics study, we propose that polymorphisms locally modulate the network of residue interactions in the globular C-terminus of the ovine recombinant prion protein while maintaining the overall fold. Although the two variants we examined exhibit a densely connected group of residues that includes both β-sheets, the β2–α2 loop and the N-terminus of α-helix 2, only in the Resistant structure do most residues of α-helix 2 belong to this group. We identify the structural role of Valine136Alanine and Glutamine171Arginine: modulation of residue interaction networks that affect the connectivity between α-helix 2 and α-helix 3. We propose blocking interactions of residue 171 as a potential target for the design of therapeutics to prevent efficient PrPC misfolding. We discuss our results in the context of initial PrPC conversion and extrapolate to recently proposed PrPSc structures.Communicated by Ramaswamy H. Sarma

The First Report of Polymorphisms and Genetic Features of the prion-like Protein Gene (PRND) in a Prion Disease-Resistant Animal, Dog.

Prion disease has displayed large infection host ranges among several species; however, dogs have not been reported to be infected and are considered prion disease-resistant animals. Case-controlled studies in several species, including humans and cattle, indicated a potent association of prion protein gene (PRNP) polymorphisms in the progression of prion disease. Thus, because of the proximal location and similar structure of the PRNP gene among the prion gene family, the prion-like protein gene (PRND) was noted as a novel candidate gene that contributes to prion disease susceptibility. Several case-controlled studies have confirmed the relationship of the PRND gene with prion disease vulnerability, and strong genetic linkage disequilibrium blocks were identified in prion-susceptible species between the PRNP and PRND genes. However, to date, polymorphisms of the dog PRND gene have not been reported, and the genetic linkage between the PRNP and PRND genes has not been examined thus far. Here, we first investigated dog PRND polymorphisms in 207 dog DNA samples using direct DNA sequencing. A total of four novel single nucleotide polymorphisms (SNPs), including one nonsynonymous SNP (c.149G>A, R50H), were identified in this study. We also found two major haplotypes among the four novel SNPs. In addition, we compared the genotype and allele frequencies of the c.149G>A (R50H) SNP and found significantly different distributions among eight dog breeds. Furthermore, we annotated the c.149G>A (R50H) SNP of the dog PRND gene using in silico tools, PolyPhen-2, PROVEAN, and PANTHER. Finally, we examined linkage disequilibrium between the PRNP and PRND genes in dogs. Interestingly, we did not find a strong genetic linkage between these two genes. To the best of our knowledge, this was the first genetic study of the PRND gene in a prion disease-resistant animal, a dog.

Oral Prion Neuroinvasion Occurs Independently of PrPC Expression in the Gut Epithelium.

The early replication of certain prion strains within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain after oral exposure. Our data show that orally acquired prions utilize specialized gut epithelial cells known as M cells to enter Peyer's patches. M cells express the cellular isoform of the prion protein, PrPC, and this may be exploited by some pathogens as an uptake receptor to enter Peyer's patches. This suggested that PrPC might also mediate the uptake and transfer of prions across the gut epithelium into Peyer's patches in order to establish infection. Furthermore, the expression level of PrPC in the gut epithelium could influence the uptake of prions from the lumen of the small intestine. To test this hypothesis, transgenic mice were created in which deficiency in PrPC was specifically restricted to epithelial cells throughout the lining of the small intestine. Our data clearly show that efficient prion neuroinvasion after oral exposure occurred independently of PrPC expression in small intestinal epithelial cells. The specific absence of PrPC in the gut epithelium did not influence the early replication of prions in Peyer's patches or disease susceptibility. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease pathogenesis and susceptibility. However, our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences the risk of oral prion disease susceptibility.IMPORTANCE The accumulation of orally acquired prions within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. Little is known of how the prions initially establish infection within Peyer's patches. Some gastrointestinal pathogens utilize molecules, such as the cellular prion protein PrPC, expressed on gut epithelial cells to enter Peyer's patches. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease susceptibility. We used transgenic mice to determine whether the uptake of prions into Peyer's patches was dependent upon PrPC expression in the gut epithelium. We show that orally acquired prions can establish infection in Peyer's patches independently of PrPC expression in gut epithelial cells. Our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences oral prion disease susceptibility.

Chapter 5 - The role of the immune system in prion infection

Prion diseases are a unique group of chronic neurodegenerative diseases that affect humans and certain domestic and free-ranging animal species. Many natural prion diseases are acquired peripherally, such as by ingestion of contaminated food or pasture. Although the pathology during prion disease appears to be restricted to the central nervous system, where it causes extensive neurodegeneration, some prion diseases accumulate to high levels within the secondary lymphoid tissues of the host's immune system as they make their journey from the site of infection to the brain. The replication of prions within these tissues is essential for the efficient spread of disease to the brain. Moreover, the immune system has a profound influence on the development of disease within the central nervous system. This chapter describes the interactions between prions and the host's immune system. Particular emphasis is given to studies which have helped to identify the key tissues, cells, and molecules which the prions exploit to facilitate their propagation from peripheral sites of exposure (such as the intestine) to the brain. This chapter also describes how prion disease pathogenesis and susceptibility may be influenced by inflammation, co-infection with other pathogens, and aging. A thorough understanding of the factors which influence prion disease susceptibility is important as it may help to identify important targets for therapeutic intervention and to help determine the risk of susceptibility to novel peripherally acquired prion diseases.

How do PrPSc Prions Spread between Host Species, and within Hosts?

Prion diseases are sub-acute neurodegenerative diseases that affect humans and some domestic and free-ranging animals. Infectious prion agents are considered to comprise solely of abnormally folded isoforms of the cellular prion protein known as PrPSc. Pathology during prion disease is restricted to the central nervous system where it causes extensive neurodegeneration and ultimately leads to the death of the host. The first half of this review provides a thorough account of our understanding of the various ways in which PrPSc prions may spread between individuals within a population, both horizontally and vertically. Many natural prion diseases are acquired peripherally, such as by oral exposure, lesions to skin or mucous membranes, and possibly also via the nasal cavity. Following peripheral exposure, some prions accumulate to high levels within the secondary lymphoid organs as they make their journey from the site of infection to the brain, a process termed neuroinvasion. The replication of PrPSc prions within secondary lymphoid organs is important for their efficient spread to the brain. The second half of this review describes the key tissues, cells and molecules which are involved in the propagation of PrPSc prions from peripheral sites of exposure (such as the lumen of the intestine) to the brain. This section also considers how additional factors such as inflammation and aging might influence prion disease susceptibility.

Prion disease pathogenesis in the absence of the commensal microbiota.

Prion diseases are a unique group of transmissible, typically sub-acute, neurodegenerative disorders. During central nervous system (CNS) prion disease, the microglia become activated and are thought to provide a protective response by scavenging and clearing prions. The mammalian intestine is host to a large burden of commensal micro-organisms, especially bacteria, termed the microbiota. The commensal microbiota has beneficial effects on host health, including through the metabolism of essential nutrients, regulation of host development and protection against pathogens. The commensal gut microbiota also constitutively regulates the functional maturation of microglia in the CNS, and microglial function is impaired when it is absent in germ-free mice. In the current study, we determined whether the absence of the commensal gut microbiota might also affect prion disease pathogenesis. Our data clearly show that the absence of the commensal microbiota in germ-free mice did not affect prion disease duration or susceptibility after exposure to prions by intraperitoneal or intracerebral injection. Furthermore, the magnitude and distribution of the characteristic neuropathological hallmarks of terminal prion disease in the CNS, including the development of spongiform pathology, accumulation of prion disease-specific protein (PrP), astrogliosis and microglial activation, were similar in conventionally housed and germ-free mice. Thus, although the commensal gut microbiota constitutively promotes the maintenance of the microglia in the CNS under steady-state conditions in naïve mice, our data suggest that dramatic changes to the abundance or complexity of the commensal gut microbiota are unlikely to influence CNS prion disease pathogenesis.

Oral Prion Disease Pathogenesis Is Impeded in the Specific Absence of CXCR5-Expressing Dendritic Cells.

ABSTRACTAfter oral exposure, the early replication of certain prion strains upon stromal cell-derived follicular dendritic cells (FDC) in the Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. However, little is known of how prions are initially conveyed from the gut lumen to establish infection on FDC. Our previous data suggest that mononuclear phagocytes such as CD11c+ conventional dendritic cells play an important role in the initial propagation of prions from the gut lumen into Peyer's patches. However, whether these cells conveyed orally acquired prions toward FDC within Peyer's patches was not known. The chemokine CXCL13 is expressed by FDC and follicular stromal cells and modulates the homing of CXCR5-expressing cells toward the FDC-containing B cell follicles. Here, novel compound transgenic mice were created in which a CXCR5 deficiency was specifically restricted to CD11c+ cells. These mice were used to determine whether CXCR5-expressing conventional dendritic cells propagate prions toward FDC after oral exposure. Our data show that in the specific absence of CXCR5-expressing conventional dendritic cells the early accumulation of prions upon FDC in Peyer's patches and the spleen was impaired, and disease susceptibility significantly reduced. These data suggest that CXCR5-expressing conventional dendritic cells play an important role in the efficient propagation of orally administered prions toward FDC within Peyer's patches in order to establish host infection.IMPORTANCE Many natural prion diseases are acquired by oral consumption of contaminated food or pasture. Once the prions reach the brain they cause extensive neurodegeneration, which ultimately leads to death. In order for the prions to efficiently spread from the gut to the brain, they first replicate upon follicular dendritic cells within intestinal Peyer's patches. How the prions are first delivered to follicular dendritic cells to establish infection was unknown. Understanding this process is important since treatments which prevent prions from infecting follicular dendritic cells can block their spread to the brain. We created mice in which mobile conventional dendritic cells were unable to migrate toward follicular dendritic cells. In these mice the early accumulation of prions on follicular dendritic cells was impaired and oral prion disease susceptibility was reduced. This suggests that prions exploit conventional dendritic cells to facilitate their initial delivery toward follicular dendritic cells to establish host infection.

Immunology of Prion Protein and Prions.

Many natural prion diseases are acquired peripherally, such as following the oral consumption of contaminated food or pasture. After peripheral exposure many prion isolates initially accumulate to high levels within the host's secondary lymphoid tissues. The replication of prions within these tissues is essential for their efficient spread to the brain where they ultimately cause neurodegeneration. This chapter describes our current understanding of the critical tissues, cells, and molecules which the prions exploit to mediate their efficient propagation from the site of exposure (such as the intestine) to the brain. Interactions between the immune system and prions are not only restricted to the secondary lymphoid tissues. Therefore, an account of how the activation status of the microglial in the brain can also influence progression of prion disease pathogenesis is provided. Prion disease susceptibility may also be influenced by additional factors such as chronic inflammation, coinfection with other pathogens, and aging. Finally, the potential for immunotherapy to provide a means of safe and effective prophylactic or therapeutic intervention in these currently untreatable diseases is considered.

Increased Abundance of M Cells in the Gut Epithelium Dramatically Enhances Oral Prion Disease Susceptibility.

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure, the early accumulation and replication of prions within small intestinal Peyer’s patches is essential for the efficient spread of disease to the brain. To replicate within Peyer’s patches, the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer’s patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling, and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice, the accumulation of prions within Peyer’s patches and the spread of disease to the brain was blocked, demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens, inflammatory stimuli and aging can modify M cell-density in the gut, these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice, resulting in shortened survival times and increased disease susceptibility, equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection, whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases.

The influence of the commensal and pathogenic gut microbiota on prion disease pathogenesis.

Prion diseases are a unique group of transmissible, chronic, neurodegenerative disorders. Following peripheral exposure (e.g. oral), prions often accumulate first within the secondary lymphoid tissues before they infect the central nervous system (CNS). Prion replication within secondary lymphoid tissues is crucial for the efficient spread of disease to the CNS. Once within the CNS, the responses of innate immune cells within it can have a significant influence on neurodegeneration and disease progression. Recently, there have been substantial advances in our understanding of how cross-talk between the host and the vast community of commensal microorganisms present at barrier surfaces such as the gut influences the development and regulation of the host's immune system. These effects are evident not only in the mucosal immune system in the gut, but also in the CNS. The actions of this microbial community (the microbiota) have many important beneficial effects on host health, from metabolism of nutrients and regulation of host development to protection from pathogen infection. However, the microbiota can also have detrimental effects in some circumstances. In this review we discuss the many and varied interactions between prions, the host and the gut microbiota. Particular emphasis is given to the ways by which changes to the composition of the commensal gut microbiota or congruent pathogen infection may influence prion disease pathogenesis and/or disease susceptibility. Understanding how these factors influence prion pathogenesis and disease susceptibility is important for assessing the risk to infection and the design of novel opportunities for therapeutic intervention.

The Gut-Associated Lymphoid Tissues in the Small Intestine, Not the Large Intestine, Play a Major Role in Oral Prion Disease Pathogenesis.

ABSTRACTPrion diseases are infectious neurodegenerative disorders characterized by accumulations of abnormally folded cellular prion protein in affected tissues. Many natural prion diseases are acquired orally, and following exposure, the early replication of some prion isolates upon follicular dendritic cells (FDC) within gut-associated lymphoid tissues (GALT) is important for the efficient spread of disease to the brain (neuroinvasion). Prion detection within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, the relative contributions of the small and large intestinal GALT to oral prion pathogenesis were unknown. To address this issue, we created mice that specifically lacked FDC-containing GALT only in the small intestine. Our data show that oral prion disease susceptibility was dramatically reduced in mice lacking small intestinal GALT. Although these mice had FDC-containing GALT throughout their large intestines, these tissues were not early sites of prion accumulation or neuroinvasion. We also determined whether pathology specifically within the large intestine might influence prion pathogenesis. Congruent infection with the nematode parasite Trichuris muris in the large intestine around the time of oral prion exposure did not affect disease pathogenesis. Together, these data demonstrate that the small intestinal GALT are the major early sites of prion accumulation and neuroinvasion after oral exposure. This has important implications for our understanding of the factors that influence the risk of infection and the preclinical diagnosis of disease.IMPORTANCE Many natural prion diseases are acquired orally. After exposure, the accumulation of some prion diseases in the gut-associated lymphoid tissues (GALT) is important for efficient spread of disease to the brain. However, the relative contributions of GALT in the small and large intestines to oral prion pathogenesis were unknown. We show that the small intestinal GALT are the essential early sites of prion accumulation. Furthermore, congruent infection with a large intestinal helminth (worm) around the time of oral prion exposure did not affect disease pathogenesis. This is important for our understanding of the factors that influence the risk of prion infection and the preclinical diagnosis of disease. The detection of prions within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, our data suggest that using these biopsy specimens may miss individuals in the early stages of oral prion infection and significantly underestimate the disease prevalence.

The influence of PRNP polymorphisms on human prion disease susceptibility: an update.

Two normally occurring polymorphisms of the human PRNP gene, methionine (M)/valine (V) at codon 129 and glutamic acid (E)/lysine (K) at codon 219, can affect the susceptibility to prion diseases. It has long been recognized that 129M/M homozygotes are overrepresented in sporadic Creutzfeldt-Jakob disease (CJD) patients and variant CJD patients, whereas 219E/K heterozygotes are absent in sporadic CJD patients. In addition to these pioneering findings, recent progress in experimental transmission studies and worldwide surveillance of prion diseases have identified novel relationships between the PRNP polymorphisms and the prion disease susceptibility. For example, although 219E/K heterozygosity confers resistance against the development of sporadic CJD, this genotype is not entirely protective against acquired forms (iatrogenic CJD and variant CJD) or genetic forms (genetic CJD and Gerstmann-Sträussler-Scheinker syndrome) of prion diseases. In addition, 129M/V heterozygotes predispose to genetic CJD caused by a pathogenic PRNP mutation at codon 180. These findings show that the effects of the PRNP polymorphisms may be more complicated than previously thought. This review aims to summarize recent advances in our knowledge about the influence of the PRNP polymorphisms on the prion disease susceptibility.

Rare structural genetic variation in human prion diseases

Prion diseases are a diverse group of neurodegenerative conditions, caused by the templated misfolding of prion protein. Aside from the strong genetic risk conferred by multiple variants of the prion protein gene (PRNP), several other variants have been suggested to confer risk in the most common type, sporadic Creutzfeldt-Jakob disease (sCJD) or in the acquired prion diseases. Large and rare copy number variants (CNVs) are known to confer risk in several related disorders including Alzheimer's disease (at APP), schizophrenia, epilepsy, mental retardation, and autism. Here, we report the first genome-wide analysis for CNV-associated risk using data derived from a recent international collaborative association study in sCJD (n = 1147 after quality control) and publicly available controls (n = 5427). We also investigated UK patients with variant Creutzfeldt-Jakob disease (n = 114) and elderly women from the Eastern Highlands of Papua New Guinea who proved highly resistant to the epidemic prion disease kuru, who were compared with healthy young Fore population controls (n = 395). There were no statistically significant alterations in the burden of CNVs >100, >500, or >1000 kb, duplications, or deletions in any disease group or geographic region. After correction for multiple testing, no statistically significant associations were found. A UK blood service control sample showed a duplication CNV that overlapped PRNP, but these were not found in prion disease. Heterozygous deletions of a 3′ region of the PARK2 gene were found in 3 sCJD patients and no controls (p = 0.001, uncorrected). A cell-based prion infection assay did not provide supportive evidence for a role for PARK2 in prion disease susceptibility. These data are consistent with a modest impact of CNVs on risk of late-onset neurologic conditions and suggest that, unlike APP, PRNP duplication is not a causal high-risk mutation.