Novel method for classification of prion diseases by detecting PrPres signal patterns from formalin-fixed paraffin-embedded samples.

Prion disease is an infectious and fatal neurodegenerative disease. Western blotting (WB)-based identification of proteinase K (PK)-resistant prion protein (PrPres) is considered a definitive diagnosis of prion diseases. In this study, we aimed to detect PrPres using formalin-fixed paraffin-embedded (FFPE) specimens from cases of sporadic Creutzfeldt-Jakob disease (sCJD), Gerstmann-Sträussler-Scheinker disease (GSS), glycosylphosphatidylinositol-anchorless prion disease (GPIALP), and V180I CJD. FFPE samples were prepared after formic acid treatment to inactivate infectivity. After deparaffinization, PK digestion was performed, and the protein was extracted. In sCJD, a pronounced PrPres signal was observed, with antibodies specific for type 1 and type 2 PrPres exhibited a strong or weak signals depending on the case. Histological examination of serial sections revealed that the histological changes were compatible with the biochemical characteristics. In GSS and GPIALP, prion protein core-specific antibodies presented as PrPres bands at 8-9 kDa and smear bands, respectively. However, an antibody specific for the C-terminus presented as smears in GSS, with no PrPres detected in GPIALP. It was difficult to detect PrPres in V180I CJD. Collectively, our findings demonstrate the possibility of detecting PrPres in FFPE and classifying the prion disease types. This approach facilitates histopathological and biochemical evaluation in the same sample and is safe owing to the inactivation of infectivity. Therefore, it may be valuable for the diagnosis and research of prion diseases.

Anti-prion drugs do not improve survival in novel knock-in models of inherited prion disease.

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions, as well as anti-prion strategies that are not strain-dependent.

Detection of prions in matching post-mortem skin and cerebrospinal fluid samples using second-generation real-time quaking-induced conversion assay.

Real-time quaking-induced conversion assay (RT-QuIC) exploits templating activity of pathogenic prion protein for ultrasensitive detection of prions. We have utilized second generation RT-QuIC assay to analyze matching post-mortem cerebrospinal fluid and skin samples of 38 prion disease patients and of 30 deceased neurological controls. The analysis of cerebrospinal fluid samples led to 100% sensitivity and 100% specificity, but some samples had to be diluted before the analysis to alleviate the effect of present RT-QuIC inhibitors. The analysis of the corresponding skin samples provided 89.5% sensitivity and 100% specificity. The median seeding dose present in the skin was one order of magnitude higher than in the cerebrospinal fluid, despite the overall fluorescent signal of the skin samples was comparatively lower. Our data support the use of post-mortem cerebrospinal fluid for confirmation of prion disease diagnosis and encourage further studies of the potential of skin biopsy samples for intra-vitam prion diseases´ diagnostics.

[Clinical characteristics and diagnostics of human spongiform encephalopathies: an update]. / Klinik und Diagnostik humaner spongiformer Enzephalopathien: ein Update.

Human spongiform encephalopathies are rare transmissible neurodegenerative diseases of the brain and the nervous system that are caused by misfolding of the physiological prion protein into a pathological form and its deposition in the central nervous system (CNS). Prion diseases include Creutzfeldt-Jakob disease (CJD, sporadic or familial), Gerstmann-Straussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI). Prion diseases can be differentiated into three etiological categories: spontaneous (sporadic CJD), inherited (familial CJD, FFI, and GSS) and acquired (variant CJD and iatrogenic CJD). Most cases occur sporadically. Prion diseases can lead to a variety of neurological symptoms and always have an inevitably fatal course. Cerebrospinal fluid analysis and magnetic resonance imaging (MRI) play a crucial role in the diagnostics of prion diseases and may facilitate an early and reliable clinical diagnosis. A causal treatment or specific therapeutic agents are not yet available. In general, a palliative therapeutic concept is indicated.

Infectious Diseases of the Brain and Spine: Parasitic and Other Atypical Transmissible Diseases.

Atypical infections of the brain and spine caused by parasites occur in immunocompetent and immunosuppressed hosts, related to exposure and more prevalently in endemic regions. In the United States, the most common parasitic infections that lead to central nervous system manifestations include cysticercosis, echinococcosis, and toxoplasmosis, with toxoplasmosis being the most common opportunistic infection affecting patients with advanced HIV/AIDS. Another rare but devastating transmittable disease is prion disease, which causes rapidly progressive spongiform encephalopathies. Familiarity and understanding of various infectious agents are a crucial aspect of diagnostic neuroradiology, and recognition of unique features can aid timely diagnosis and treatment.

Targeting Neuroinflammation by Pharmacologic Downregulation of Inflammatory Pathways Is Neuroprotective in Protein Misfolding Disorders.

Neuroinflammation plays a crucial role in the development of neurodegenerative protein misfolding disorders. This category of progressive diseases includes, but is not limited to, Alzheimer's disease, Parkinson's disease, and prion diseases. Shared pathogenesis involves the accumulation of misfolded proteins, chronic neuroinflammation, and synaptic dysfunction, ultimately leading to irreversible neuronal loss, measurable cognitive deficits, and death. Presently, there are few to no effective treatments to halt the advancement of neurodegenerative diseases. We hypothesized that directly targeting neuroinflammation by downregulating the transcription factor, NF-κB, and the inflammasome protein, NLRP3, would be neuroprotective. To achieve this, we used a cocktail of RNA targeting therapeutics (SB_NI_112) shown to be brain-penetrant, nontoxic, and effective inhibitors of both NF-κB and NLRP3. We utilized a mouse-adapted prion strain as a model for neurodegenerative diseases to assess the aggregation of misfolded proteins, glial inflammation, neuronal loss, cognitive deficits, and lifespan. Prion-diseased mice were treated either intraperitoneally or intranasally with SB_NI_112. Behavioral and cognitive deficits were significantly protected by this combination of NF-κB and NLRP3 downregulators. Treatment reduced glial inflammation, protected against neuronal loss, prevented spongiotic change, rescued cognitive deficits, and significantly lengthened the lifespan of prion-diseased mice. We have identified a nontoxic, systemic pharmacologic that downregulates NF-κB and NLRP3, prevents neuronal death, and slows the progression of neurodegenerative diseases. Though mouse models do not always predict human patient success and the study was limited due to sample size and number of dosing methods utilized, these findings serve as a proof of principle for continued translation of the therapeutic SB_NI_112 for prion disease and other neurodegenerative diseases. Based on the success in a murine prion model, we will continue testing SB_NI_112 in a variety of neurodegenerative disease models, including Alzheimer's disease and Parkinson's disease.

A Protein Misfolding Shaking Amplification-based method for the spontaneous generation of hundreds of bona fide prions.

Prion diseases are a group of rapidly progressing neurodegenerative disorders caused by the misfolding of the endogenous prion protein (PrPC) into a pathogenic form (PrPSc). This process, despite being the central event underlying these disorders, remains largely unknown at a molecular level, precluding the prediction of new potential outbreaks or interspecies transmission incidents. In this work, we present a method to generate bona fide recombinant prions de novo, allowing a comprehensive analysis of protein misfolding across a wide range of prion proteins from mammalian species. We study more than 380 different prion proteins from mammals and classify them according to their spontaneous misfolding propensity and their conformational variability. This study aims to address fundamental questions in the prion research field such as defining infectivity determinants, interspecies transmission barriers or the structural influence of specific amino acids and provide invaluable information for future diagnosis and therapy applications.

Foiling deadly prions.

Can the course of fatal prion diseases be changed by removing the protein before it goes bad?

Detection limitations of prion seeding activities in blood samples from patients with sporadic prion disease.

BACKGROUND: Human prion diseases (HPDs) are fatal neurodegenerative disorders characterized by abnormal prion proteins (PrPSc). However, the detection of prion seeding activity in patients with high sensitivity remains challenging. Even though real-time quaking-induced conversion (RT-QuIC) assay is suitable for detecting prion seeding activity in a variety of specimens, it shows lower accuracy when whole blood, blood plasma, and blood-contaminated tissue samples are used. In this study, we developed a novel technology for the in vitro amplification of abnormal prion proteins in HPD to the end of enabling their detection with high sensitivity known as the enhanced quaking-induced conversion (eQuIC) assay. METHODS: Three antibodies were used to develop the novel eQUIC method. Thereafter, SD50 seed activity was analyzed using brain tissue samples from patients with prion disease using the conventional RT-QUIC assay and the novel eQUIC assay. In addition, blood samples from six patients with solitary prion disease were analyzed using the novel eQuIC assay. RESULTS: The eQuIC assay, involving the use of three types of human monoclonal antibodies, showed approximately 1000-fold higher sensitivity than the original RT-QuIC assay. However, when this assay was used to analyze blood samples from six patients with sporadic human prion disease, no prion activity was detected. CONCLUSION: The detection of prion seeding activity in blood samples from patients with sporadic prion disease remains challenging. Thus, the development of alternative methods other than RT-QuIC and eQuIC will be necessary for future research.

Single nucleotide polymorphisms (SNPs) in the open reading frame (ORF) of prion protein gene (PRNP) in Nigerian livestock species.

BACKGROUND: Prion diseases, also known as transmissible spongiform encephalopathies (TSEs) remain one of the deleterious disorders, which have affected several animal species. Polymorphism of the prion protein (PRNP) gene majorly determines the susceptibility of animals to TSEs. However, only limited studies have examined the variation in PRNP gene in different Nigerian livestock species. Thus, this study aimed to identify the polymorphism of PRNP gene in Nigerian livestock species (including camel, dog, horse, goat, and sheep). We sequenced the open reading frame (ORF) of 65 camels, 31 village dogs and 12 horses from Nigeria and compared with PRNP sequences of 886 individuals retrieved from public databases. RESULTS: All the 994 individuals were assigned into 162 haplotypes. The sheep had the highest number of haplotypes (n = 54), and the camel had the lowest (n = 7). Phylogenetic tree further confirmed clustering of Nigerian individuals into their various species. We detected five non-synonymous SNPs of PRNP comprising of G9A, G10A, C11G, G12C, and T669C shared by all Nigerian livestock species and were in Hardy-Weinberg Equilibrium (HWE). The amino acid changes in these five non-synonymous SNP were all "benign" via Polyphen-2 program. Three SNPs G34C, T699C, and C738G occurred only in Nigerian dogs while C16G, G502A, G503A, and C681A in Nigerian horse. In addition, C50T was detected only in goats and sheep. CONCLUSION: Our study serves as the first to simultaneously investigate the polymorphism of PRNP gene in Nigerian livestock species and provides relevant information that could be adopted in programs targeted at breeding for prion diseases resistance.

Variably protease-sensitive prionopathy with methionine homozygosity at codon 129 in the prion protein gene.

Variably protease-sensitive prionopathy (VPSPr) is a recently characterised rare subtype of sporadic prion disease, mainly affecting individuals with valine homozygosity at codon 129 in the prion protein gene, with only seven methionine homozygote cases reported to date. This case presents clinical, neuropathological and biochemical features of the eighth VPSPr case worldwide with methionine homozygosity at codon 129 and compares the features with the formerly presented cases.The patient, a woman in her 70s, presented with cognitive decline, impaired balance and frequent falls. Medical history and clinical presentation were suggestive of a rapidly progressive dementia disorder. MRI showed bilateral thalamic hyperintensity. Cerebrospinal fluid real-time quaking-induced conversion was negative, and the electroencephalogram was unremarkable. The diagnosis was established through post-mortem pathological examinations. VPSPr should be suspected in rapidly progressive dementia lacking typical features or paraclinical results of protein misfolding diseases.

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.

Ultrasensitive Detection of Lipid-Induced Misfolding of the Prion Protein at the Aqueous-Liquid Crystal Interface.

The misfolding of the α-helical cellular prion protein into a self-propagating ß-rich aggregated form is a key pathogenic event in fatal and transmissible neurodegenerative diseases collectively known as prion diseases. Herein, we utilize the interfacial properties of liquid crystals (LCs) to monitor the lipid-membrane-induced conformational switching of prion protein (PrP) into ß-rich amyloid fibrils. The lipid-induced conformational switching resulting in aggregation occurs at the nanomolar protein concentration and is primarily mediated by electrostatic interactions between PrP and lipid headgroups. Our LC-based methodology offers a potent and sensitive tool to detect and delineate molecular mechanisms of PrP misfolding mediated by lipid-protein interactions at the aqueous interface under physiological conditions.

Creutzfeldt-Jakob disease and other prion diseases.

Prion diseases share common clinical and pathological characteristics such as spongiform neuronal degeneration and deposition of an abnormal form of a host-derived protein, termed prion protein. The characteristic features of prion diseases are long incubation times, short clinical courses, extreme resistance of the transmissible agent to degradation and lack of nucleic acid involvement. Sporadic and genetic forms of prion diseases occur worldwide, of which genetic forms are associated with mutations in PRNP. Human to human transmission of these diseases has occurred due to iatrogenic exposure, and zoonotic forms of prion diseases are linked to bovine disease. Significant progress has been made in the diagnosis of these disorders. Clinical tools for diagnosis comprise brain imaging and cerebrospinal fluid tests. Aggregation assays for detection of the abnormally folded prion protein have a clear potential to diagnose the disease in peripherally accessible biofluids. After decades of therapeutic nihilism, new treatment strategies and clinical trials are on the horizon. Although prion diseases are relatively rare disorders, understanding their pathogenesis and mechanisms of prion protein misfolding has significantly enhanced the field in research of neurodegenerative diseases.

Spectral Fluorescence Pathology of Protein Misfolding Disorders.

Protein misfolding has been extensively studied in the context of neurodegenerative disorders and systemic amyloidoses. Due to misfolding and aggregation of proteins being highly heterogeneous and generating a variety of structures, a growing body of evidence illustrates numerous ways how the aggregates contribute to progression of diseases such as Alzheimer's disease, Parkinson's disease, and prion disorders. Different misfolded species of the same protein, commonly referred to as strains, appear to play a significant role in shaping the disease clinical phenotype and clinical progression. The distinct toxicity profiles of various misfolded proteins underscore their importance. Current diagnostics struggle to differentiate among these strains early in the disease course. This review explores the potential of spectral fluorescence approaches to illuminate the complexities of protein misfolding pathology and discusses the applications of advanced spectral methods in the detection and characterization of protein misfolding disorders. By examining spectrally variable probes, current data analysis approaches, and important considerations for the use of these techniques, this review aims to provide an overview of the progress made in this field and highlights directions for future research.

The Role of PET Imaging in Patients with Prion Disease: A Literature Review.

Prion diseases are rare, rapidly progressive, and fatal incurable degenerative brain disorders caused by the misfolding of a normal protein called PrPC into an abnormal protein called PrPSc. Their highly variable clinical presentation mimics various degenerative and non-degenerative brain disorders, making diagnosis a significant challenge for neurologists. Currently, definitive diagnosis relies on post-mortem examination of nervous tissue to detect the pathogenic prion protein. The current diagnostic criteria are limited. While structural magnetic resonance imaging (MRI) remains the gold standard imaging modality for Creutzfeldt-Jakob disease (CJD) diagnosis, positron emission tomography (PET) using 18fluorine-fluorodeoxyglucose (18F-FDG) and other radiotracers have demonstrated promising potential in the diagnostic assessment of prion disease. In this context, a comprehensive and updated review exclusively focused on PET imaging in prion diseases is still lacking. We review the current value of PET imaging with 18F-FDG and non-FDG tracers in the diagnostic management of prion diseases. From the collected data, 18F-FDG PET mainly reveals cortical and subcortical hypometabolic areas in prion disease, although fails to identify typical pattern or laterality abnormalities to differentiate between genetic and sporadic prion diseases. Although the rarity of prion diseases limits the establishment of a definitive hypometabolism pattern, this review reveals some more prevalent 18F-FDG patterns associated with each disease subtype. Interestingly, in both sporadic and genetic prion diseases, the hippocampus does not show significant glucose metabolism alterations, appearing as a useful sign in the differential diagnosis with other neurodegenerative disease. In genetic prion disease forms, PET abnormality precedes clinical manifestation. Discordant diagnostic value for amyloid tracers among different prion disease subtypes was observed, needing further investigation. PET has emerged as a potential valuable tool in the diagnostic armamentarium for CJD. Its ability to visualize functional and metabolic brain changes provides complementary information to structural MRI, aiding in the early detection and confirmation of CJD.

Large-scale validation of skin prion seeding activity as a biomarker for diagnosis of prion diseases.

Definitive diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) relies on the examination of brain tissues for the pathological prion protein (PrPSc). Our previous study revealed that PrPSc-seeding activity (PrPSc-SA) is detectable in skin of sCJD patients by an ultrasensitive PrPSc seed amplification assay (PrPSc-SAA) known as real-time quaking-induced conversion (RT-QuIC). A total of 875 skin samples were collected from 2 cohorts (1 and 2) at autopsy from 2-3 body areas of 339 cases with neuropathologically confirmed prion diseases and non-sCJD controls. The skin samples were analyzed for PrPSc-SA by RT-QuIC assay. The results were compared with demographic information, clinical manifestations, cerebrospinal fluid (CSF) PrPSc-SA, other laboratory tests, subtypes of prion diseases defined by the methionine (M) or valine (V) polymorphism at residue 129 of PrP, PrPSc types (#1 or #2), and gene mutations in deceased patients. RT-QuIC assays of the cohort #1 by two independent laboratories gave 87.3% or 91.3% sensitivity and 94.7% or 100% specificity, respectively. The cohort #2 showed sensitivity of 89.4% and specificity of 95.5%. RT-QuIC of CSF available from 212 cases gave 89.7% sensitivity and 94.1% specificity. The sensitivity of skin RT-QuIC was subtype dependent, being highest in sCJDVV1-2 subtype, followed by VV2, MV1-2, MV1, MV2, MM1, MM1-2, MM2, and VV1. The skin area next to the ear gave highest sensitivity, followed by lower back and apex of the head. Although no difference in brain PrPSc-SA was detected between the cases with false negative and true positive skin RT-QuIC results, the disease duration was significantly longer with the false negatives [12.0 ± 13.3 (months, SD) vs. 6.5 ± 6.4, p < 0.001]. Our study validates skin PrPSc-SA as a biomarker for the detection of prion diseases, which is influenced by the PrPSc types, PRNP 129 polymorphisms, dermatome sampled, and disease duration.

Prion science and its unsung heroes.

My first encounter with prion diseases dates to 1986. As a clinical resident in neuropathology, I was tasked with performing autopsies of patients who died of mysterious brain diseases. In his early 60s, my patient had developed a form of dementia that progressed at a terrifyingly rapid pace and eventually led to his death. I sampled the patient's brain and processed it for histological examination. The microscope revealed an eerie landscape of destruction. All that was left in the patient's cortex were astrocytes and microglia, and the few remaining neurons showed extensive vacuolation of their bodies and processes. Such blazing destruction of the brain was indicative of just one diagnosis: Creutzfeldt-Jakob disease, a spongiform encephalopathy caused by enigmatic infectious agents called prions.