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.

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.

Prion meeting 2023: implications of a growing field.

The history of human prion diseases began with the original description, by Hans Gerhard Creutzfeldt and by Alfons Maria Jakob, of patients with a severe brain disease that included speech abnormalities, confusion, and myoclonus, in a disease that was then named Creutzfeldt Jakob disease (CJD). Later, in Papua New Guinea, a disease characterized by trembling was identified, and given the name "Kuru". Neuropathological examination of the brains from CJD and Kuru patients, and of brains of sheep with scrapie disease revealed significant similarities and suggested a possible common mode of infection that, at the time, was thought to derive from an unknown virus that caused slow infections. John Stanley Griffith hypothesized that the agent causing these diseases was "probably a protein without nucleic acid" and, in 1982, Stanley Prusiner reported the identification of a proteinaceous infectious particle (coining the term prion) that was resistant to inactivation methods that were at the time standard for nucleic acids, and identified PrP as the major protein component of the infectious agent in scrapie and in Creutzfeldt-Jakob disease, classifying this also as a prion disease. Interestingly, the prion concept had been previously expanded to yeast proteins capable of replicating their conformation, seeding their own aggregation and transmitting phenotypic information. The prion concept has been more recently expanded to refer to misfolded proteins that are capable of converting a normal form of a protein into an abnormal form. The quest to understand and treat prion diseases has united a specific research community around the topic, and regular meetings (Prion Meetings) have taken place over the years to enable discussions, train junior researchers, and inspire research in the field.

New Light on Prions: Putative Role of PrPc in Pathophysiology of Mood Disorders.

Mood disorders are highly prevalent and heterogenous mental illnesses with devastating rates of mortality and treatment resistance. The molecular basis of those conditions involves complex interplay between genetic and environmental factors. Currently, there are no objective procedures for diagnosis, prognosis and personalization of patients' treatment. There is an urgent need to search for novel molecular targets for biomarkers in mood disorders. Cellular prion protein (PrPc) is infamous for its potential to convert its insoluble form, leading to neurodegeneration in Creutzfeldt-Jacob disease. Meanwhile, in its physiological state, PrPc presents neuroprotective features and regulates neurotransmission and synaptic plasticity. The aim of this study is to integrate the available knowledge about molecular mechanisms underlying the impact of PrPc on the pathophysiology of mood disorders. Our review indicates an important role of this protein in regulation of cognitive functions, emotions, sleep and biological rhythms, and its deficiency results in depressive-like behavior and cognitive impairment. PrPc plays a neuroprotective role against excitotoxicity, oxidative stress and inflammation, the main pathophysiological events in the course of mood disorders. Research indicates that PrPc may be a promising biomarker of cognitive decline. There is an urgent need of human studies to elucidate its potential utility in clinical practice.

[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.

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.

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.

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.

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.

A systemic analysis of Creutzfeldt Jakob disease cases in Asia.

Creutzfeldt Jakob Disease (CJD) is a rapidly progressive, fatal neurodegenerative disorder, also known as a subacute spongiform encephalopathy. There are three major subtypes of CJD i.e. Sporadic CJD, which occurs for reasons unbeknown to science (85% of known cases), Genetic or Familial CJD which is characterized by the presence of mutations in the human prion protein (PRNP) gene (10-15% cases) and Iatrogenic CJD that occurs via accidental transmission through medical and surgical procedures (1-2% cases). CJD cases occur globally with 1 case per one million population/year. Considerable data is available related to the incidence and prevalence of CJD in Europe and America. However, the global surveillance database is yet to include Asia even though several Asian countries have their own CJD monitoring units. sCJD is the highest among all CJD cases in Asia. China (1957) and Japan (1705) have reported more cases of sCJD than any Asian country and Hong Kong (1) has reported the least. On the other hand, gCJD is highest in Japan (370) and least in India (2). Our analysis establishes the presence of all variants of CJD across Asia. However, in most Asian countries in general and Southeast Asian countries in particular, CJD cases are misdiagnosed and often underreported. Since Asia is the most populated continent in the world, the actual global prevalence of CJD cannot be estimated until and unless these countries are accounted for. Concrete and reliable surveillance networks are needed across Asia to evaluate the prevalence and incidence of CJD in the region. [Figure: see text]The graphical abstract demonstrates the prevalence of CJD cases in the world and systematically analyses the incidence of CJD in Asian countries between the year 1986-2022. Highest number of cases were reported in Japan followed by China. The study emphasizes the need for assimilation of Asian data in global prevalence.

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.

Development of statistical auto-segmentation method for diffusion restriction gray matter lesions in patients with newly diagnosed sporadic Creutzfeldt-Jakob disease.

Quantification of diffusion restriction lesions in sporadic Creutzfeldt-Jakob disease (sCJD) may provide information of the disease burden. We aim to develop an automatic segmentation model for sCJD and to evaluate the volume of disease extent as a prognostic marker for overall survival. Fifty-six patients (mean age ± SD, 61.2 ± 9.9 years) were included from February 2000 to July 2020. A threshold-based segmentation was used to obtain abnormal signal intensity masks. Segmented volumes were compared with the visual grade. The Dice similarity coefficient was calculated to measure the similarity between the automatic vs. manual segmentation. Cox proportional hazards regression analysis was performed to evaluate the volume of disease extent as a prognostic marker. The automatic segmentation showed good correlation with the visual grading. The cortical lesion volumes significantly increased as the visual grade aggravated (extensive: 112.9 ± 73.2; moderate: 45.4 ± 30.4; minimal involvement: 29.6 ± 18.1 mm3) (P < 0.001). The deep gray matter lesion volumes were significantly higher for positive than for negative involvement of the deep gray matter (5.6 ± 4.6 mm3 vs. 1.0 ± 1.3 mm3, P < 0.001). The mean Dice similarity coefficients were 0.90 and 0.94 for cortical and deep gray matter lesions, respectively. However, the volume of disease extent was not associated with worse overall survival (cortical extent: P = 0.07; deep gray matter extent: P = 0.12).

Iatrogenic Alzheimer's disease in recipients of cadaveric pituitary-derived growth hormone.

Alzheimer's disease (AD) is characterized pathologically by amyloid-beta (Aß) deposition in brain parenchyma and blood vessels (as cerebral amyloid angiopathy (CAA)) and by neurofibrillary tangles of hyperphosphorylated tau. Compelling genetic and biomarker evidence supports Aß as the root cause of AD. We previously reported human transmission of Aß pathology and CAA in relatively young adults who had died of iatrogenic Creutzfeldt-Jakob disease (iCJD) after childhood treatment with cadaver-derived pituitary growth hormone (c-hGH) contaminated with both CJD prions and Aß seeds. This raised the possibility that c-hGH recipients who did not die from iCJD may eventually develop AD. Here we describe recipients who developed dementia and biomarker changes within the phenotypic spectrum of AD, suggesting that AD, like CJD, has environmentally acquired (iatrogenic) forms as well as late-onset sporadic and early-onset inherited forms. Although iatrogenic AD may be rare, and there is no suggestion that Aß can be transmitted between individuals in activities of daily life, its recognition emphasizes the need to review measures to prevent accidental transmissions via other medical and surgical procedures. As propagating Aß assemblies may exhibit structural diversity akin to conventional prions, it is possible that therapeutic strategies targeting disease-related assemblies may lead to selection of minor components and development of resistance.

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.

Performance of a seed amplification assay for misfolded alpha-synuclein in cerebrospinal fluid and brain tissue in relation to Lewy body disease stage and pathology burden.

The development of in vitro seed amplification assays (SAA) detecting misfolded alpha-synuclein (αSyn) in cerebrospinal fluid (CSF) and other tissues has provided a pathology-specific biomarker for Lewy body disease (LBD). However, αSyn SAA diagnostic performance in early pathological stages or low Lewy body (LB) pathology load has only been assessed in small cohorts. Moreover, the relationship between SAA kinetic parameters, the number of αSyn brain seeds and the LB pathology burden assessed by immunohistochemistry has never been systematically investigated. We tested 269 antemortem CSF samples and 138 serially diluted brain homogenates from patients with and without neuropathological evidence of LBD in different stages by the αSyn Real-Time Quaking-Induced Conversion (RT-QuIC) SAA. Moreover, we looked for LB pathology by αSyn immunohistochemistry in a consecutive series of 604 Creutzfeldt-Jakob disease (CJD)-affected brains. αSyn CSF RT-QuIC showed 100% sensitivity in detecting LBD in limbic and neocortical stages. The assay sensitivity was significantly lower in patients in early stages (37.5% in Braak 1 and 2, 73.3% in Braak 3) or with focal pathology (50% in amygdala-predominant). The average number of CSF RT-QuIC positive replicates significantly correlated with LBD stage. Brain homogenate RT-QuIC showed higher sensitivity than immunohistochemistry for the detection of misfolded αSyn. In the latter, the kinetic parameter lag phase (time to reach the positive threshold) strongly correlated with the αSyn seed concentration in serial dilution experiments. Finally, incidental LBD prevalence was 8% in the CJD cohort. The present results indicate that (a) CSF RT-QuIC has high specificity and sufficient sensitivity to detect all patients with LB pathology at Braak stages > 3 and most of those at stage 3; (b) brain deposition of misfolded αSyn precedes the formation of LB and Lewy neurites; (c) αSyn SAA provides "quantitative" information regarding the LB pathology burden, with the lag phase and the number of positive replicates being the most promising variables to be used in the clinical setting.

A review of the enhanced CJD surveillance feasibility study in the elderly in Scotland, UK.

BACKGROUND: Variant Creutzfeldt - Jakob disease (vCJD) arose from dietary contamination with bovine-spongiform-encephalopathy (BSE). Because of concerns that vCJD-cases might be missed in the elderly, a feasibility study of enhanced CJD surveillance on the elderly was begun in 2016. Recruitment was lower than predicted. We describe a review of the challenges encountered in that study: identification, referral, and recruitment, and the effects of actions based on the results of that review. METHODS: Review was conducted in 2017. Study data for all eligible cases identified and referred from one participating service (Anne Rowling clinic (ARC)) was curated and anonymised in a bespoke database. A questionnaire was sent out to all the clinicians in medicine of the elderly, psychiatry of old age and neurology (including ARC) specialties in NHS Lothian, exploring possible reasons for low recruitment. RESULTS: Sixty-eight cases were referred from the ARC (March 2016-September 2017): 25% were recruited. Most cases had been referred because of diagnostic uncertainty. No difference was seen between those recruited and the non-recruited, apart from age and referrer. Twelve of 60 participating clinicians completed the questionnaire: only 4 had identified eligible cases. High workload, time constraints, forgetting to refer, unfamiliarity with the eligibility criteria, and the rarity of eligible cases, were some of the reasons given. Suggestions as to how to improve referral of eligible cases included: regular email reminders, feedback to referrers, improving awareness of the study, visible presence of the study team, and integration of the study with other research oriented services. These results were used to increase recruitment but without success. CONCLUSION: Recruitment was lower than predicted. Actions taken following a review at 21 months did not lead to significant improvement; recruitment remained low, with many families/patients declining to take part (75%). In assessing the failure to improve recruitment, two factors need to be considered. Firstly, the initial referral rate was expected to be higher because of existing patients already known to the clinical services, with later referrals being only newly presenting patients. Secondly, the unplanned absence of a dedicated study nurse. Searching digital records/anonymised derivatives to identify eligible patients could be explored.

Punctate Diffuse Cortex Signals in Creutzfeldt-Jakob Disease.

This case report describes 2 patients with genetic Creutzfeldt-Jakob disease with atypical changes on diffusion-weighted imaging.