Pathological Prion Research Articles

Characterization of variably protease-sensitive prionopathy by capillary electrophoresis

Variably Protease Sensitive Prionopathy (VPSPr) is a rare human prion disease that, like Creutzfeldt-Jakob disease (CJD), results in the deposition of abnormally folded prion protein aggregates in the brain and is ultimately fatal. Neuropathology and clinical features of VPSPr are heterogeneous. However, the key discriminating feature is the relative sensitivity of the pathological prion protein to proteinase digestion compared to that typically seen in other human prion cases. Three major fragments of 23, 17 and 7 kDa are characteristic of the disease following digestion with proteinase K. We recently reported the utility of the highly adaptive and reproducible ProteinSimple™ capillary electrophoresis (CE) system to perform protein separation of PK digested prion protein in CJD. Consequently, we explored capillary-based electrophoresis (CE) technology as a sensitive method to detect and characterize VPSPr in a cohort of 29 cases. The unique 7 kDa fragment has high intensity, particularly in cases with the codon 129 VV genotype, but can be missed by regular Western blotting due to the small size. However, this fragment is readily detected by CE in all cases. In addition, the flexibility of CE produced highly reproducible, semi-quantitative data for determining relative proteinase K sensitivity and epitope mapping of representative cases from each codon 129 genotype (VV, MV and MM).

Characterisation of European Field Goat Prion Isolates in Ovine PrP Overexpressing Transgenic Mice (Tgshp IX) Reveals Distinct Prion Strains.

After the detection of bovine spongiform encephalopathy (BSE), and a zoonotic transmissible spongiform encephalopathy (TSE) caused by the pathological prion protein (PrPSc) in two goats, the investigation of goat prions became of greater interest. Therefore, a broad collection of European goat TSE isolates, including atypical scrapie, CH1641 and goat BSE as reference prion strains were biochemically characterised and subsequently inoculated into seven rodent models for further analysis (already published results of this comprehensive study are reviewed here for comparative reasons). We report here the histopathological and immunohistochemical data of this goat TSE panel, obtained after the first passage in Tgshp IX (tg-shARQ) mice, which overexpress the ovine prion protein. In addition to the clear-cut discrimination of all reference prion strains from the classical scrapie (CS) isolates, we were further able to determine three categories of CS strains. The investigation further indicates the occurrence of sub-strains that slightly resemble distant TSE strains, such as BSE or CH1641, reinforcing the theory that CS is not a single strain but a mixture of sub-strains, existing at varying extents in one isolate. This study further proved that Tgshp IX is a potent and reliable tool for the in-depth characterisation of prion strains.

Genetic prion disease – fatal familial insomnia (clinical case)

Background. Fatal familial insomnia is a rare genetically determined neurodegenerative disorder from the group of prion diseases. Its main cause is the autosomal dominant D178N mutation of the PRNP gene, which leads to the synthesis of the pathological prion protein PrP.The aim. Using the example of a clinical case to describe an example of the early onset of fatal familial insomnia in a teenager, a clinical example of its management.Materials and methods. Female patient V., 16 years old, of hyposthenic constitution, undernourished, with negative family history (multiple sclerosis in her paternal grandmother) for the first time consulted a neurologist in Tver for the complaints of superficial sleep, shortened to 4–5 hours, unspecific pain all over the body, periodic numbness in the upper limbs. Six months later, retardation of speech and movements, changes in gait, and intentional tremor occurred; sleep was shortened to 2 hours. In the future, the teenager lost the ability to independently maintain the vertical body position, the ability to walk without assistance, speech was reduced to syllable answers to questions. In order to verify the diagnosis and to carry out differential diagnosis with other neurodegenerative diseases, the girl underwent auxiliary research methods: detection of antibodies to nuclear antigens, magnetic resonance imaging, computer electroencephalography, polyexomal genome sequencing.Results. Based on the anamnesis, complaints, clinical picture and results of genetic research the final diagnosis of fatal familial insomnia was made. Due to the lack of etiological and pathogenetic therapy, the patient was subsequently provided with palliative medical care. The fatal outcome occurred 19 months after the onset of the disease.Conclusions. The presented clinical case reflects the complexity of managing patients with rare genetic diseases, confirms the need for mandatory polyexomal genome sequencing in order to verify the diagnosis, which allows timely palliative care

NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling

Oligodendrocyte-lineage cells, including NG2 glia, undergo prominent changes in various neurodegenerative disorders. Here, we identify a neuroprotective role for NG2 glia against prion toxicity. NG2 glia were activated after prion infection in cerebellar organotypic cultured slices (COCS) and in brains of prion-inoculated mice. In both model systems, depletion of NG2 glia exacerbated prion-induced neurodegeneration and accelerated prion pathology. Loss of NG2 glia enhanced the biosynthesis of prostaglandin E2 (PGE2) by microglia, which augmented prion neurotoxicity through binding to the EP4 receptor. Pharmacological or genetic inhibition of PGE2 biosynthesis attenuated prion-induced neurodegeneration in COCS and mice, reduced the enhanced neurodegeneration in NG2-glia-depleted COCS after prion infection, and dampened the acceleration of prion disease in NG2-glia-depleted mice. These data unveil a non-cell-autonomous interaction between NG2 glia and microglia in prion disease and suggest that PGE2 signaling may represent an actionable target against prion diseases.

Assessment of the Zoonotic Potential of Atypical Scrapie Prions in Humanized Mice Reveals Rare Phenotypic Convergence but Not Identity With Sporadic Creutzfeldt-Jakob Disease Prions.

Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from classical bovine spongiform encephalopathy (C-BSE) may copropagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, 1 isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. The results obtained suggest a low zoonotic potential for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.

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.

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.

The APOE4 Allele Exacerbates Pathology in a Mouse Model of Prion Disease

AbstractBackgroundThe APOE4 allele is the strongest genetic risk factor in sporadic Alzheimer’s disease (AD). In allele‐dose dependent manner APOE4 increases AD risk, lowers age of onset, and accelerates the rate of dementia progression. Like AD, prionoses are conformational neurodegenerative disorders, which neuropathological hallmarks include accumulation of disease specific misfolded protein(s) and presence of reactive astrocytes and microglia. Accumulation of PrPSc, a toxic conformer of the cellular prion protein‐PrPC, is the initial culprit in prion pathogenesis. PrPSc buildup is associated with the presence of A1 neurotoxic astrocytes and neurodegenerative phenotype microglia (MGnD), which proinflammatory character contributes importantly to neurodegeneration in prionoses. Despite similarities between AD and prionoses, and established effects of APOE4 on misfolded protein accumulation and MGnD activation in AD, effects of the APOE genotype on prion pathology remain uninvestigated.MethodAPOE2, APOE3, and APOE4 targeted replacement mice were inoculated with 22L mouse adapted scrapie strain or normal brain homogenate (NBH) and subjected to repetitive behavioral testing. They were sacrificed at 15 or 23 weeks post inoculation (WPI), when they remained presymptomatic or showed overt neurological symptoms. Neuropathological and biochemical analyses were performed at either timepoint, while NanoStringTM analysis of microglia and astrocyte transcriptomic markers was done at 23WPI.ResultAPOE4 mice feature significantly shorter incubation period and more severe burden of neurological symptoms following prion inoculation than APOE2 and APOE3 mice. Neuropathological analysis reveals significantly higher burden of spongiforms changes in the brain cortex and greater load of reactive astrocytes and microglia in APOE4 mice compared to other APOE genotypes. APOE4 mice also show the highest level of total PrP and PrPSc and insoluble/soluble PrP index. Transcriptomic analysis reveals prion inoculation effects marked elevation of PAN and A1 reactive astrocyte markers, MGnD markers, and in pro‐inflammatory chemokines and cytokine receptors transcripts across all APOE genotypes but the NBH‐relative increase is the highest in APOE4 mice.ConclusionThe APOE4 allele promotes prion neurodegeneration through two separate mechanisms. It is associated with greater PrPSc accumulation and toxicity suggesting its role in the PrPSc metabolism and clearance. It also promotes acquisition of A1 and MGnD neurotoxic phenotypes by astrocytes and microglia, respectively.

Characterization of the First Case of Classical Scrapie in a Sheep in Tunisia

Classical scrapie is a contagious prion disease of sheep and goats. It is endemic in many countries in Europe, North America, and Asia. In Africa, imported scrapie cases have been described in South Africa and Kenia in the past. More recently, several cases have been reported from different regions of Libya, based on clinical signs and histological lesions. Here, we report the results of thorough investigations carried out on a suspect case of scrapie in a 6-year-old Barbarine sheep, born, and bred in Tunisia, showing behavioral changes, weight loss, itching, skin lesions, wool loss, and motor incoordination. Histopathology and immunohistochemistry revealed spongiform change in several brain areas with associated pathological prion protein deposition. Western blotting confirmed the diagnosis and showed a classical scrapie-like molecular pattern of PrPres, different from atypical scrapie and bovine spongiform encephalopathy (BSE) in small ruminants. Sequence analysis of the prion protein gene showed that the animal carried the ARQ/ARQ genotype, one of the most susceptible to classical scrapie. The inoculation of sheep brain homogenate in a susceptible rodent model proved the experimental transmissibility of the disease. These results demonstrate the circulation of classical scrapie in Tunisia and confirm its presence in North Africa, indicating the need to improve epidemiological surveillance and diagnostic capacity for prion diseases in the region.

Heterogeneity of pathological prion protein accumulation in the brain of moose (Alces alces) from Norway, Sweden and Finland with chronic wasting disease

Prion diseases are a group of neurodegenerative, transmissible, and fatal disorders that affect several animal species. They are characterized by the conformational conversion of the cellular prion protein (PrPC) into the pathological prion protein (PrPSc). In 2016, chronic wasting disease (CWD) gained great importance at European level due to the first disease detection in a wild reindeer (Rangifer tarandus) in Norway. The subsequent intensive CWD surveillance launched in cervids resulted in the detection of CWD in moose (Alces alces), with 11 cases in Norway, 3 in Finland and 4 in Sweden. These moose cases differ considerably from CWD cases in North American and reindeer in Norway, as PrPSc was detectable in the brain but not in lymphoid tissues. These facts suggest the occurrence of a new type of CWD. Here, we show some immunohistochemical features that are clearly different from CWD cases in North American and Norwegian reindeer. Further, the different types of PrPSc deposits found among moose demonstrate strong variations between the cases, supporting the postulation that these cases could carry multiple strains of CWD.

A Presentation of Rare Heidenhain-Variant Creutzfeldt-Jakob Disease

Heidenhain-variant Creutzfeldt-Jakob disease (HV-CJD) is a rare pathologic prion disease characterized by visual symptoms preceding progressive neurodegeneration and death. In HV-CJD, pathologic prions display demyelinating neurotropism for the occipital lobe, yielding characteristic visual changes and hallucinations. Herein, we describe the case of a patient who presented with antecedent visual hallucinations followed by declining neuropsychiatric status, startle myoclonus, gait ataxia, and personality change. Clinical symptoms, electroencephalography, magnetic resonance imaging, and lumbar puncture supported a diagnosis of HV-CJD. This case emphasizes the importance of multidisciplinary management for ruling out alternative medical diagnoses, performing appropriate diagnostic studies, and implementing patient-centered management.

The natural history study of preclinical genetic Creutzfeldt-Jakob Disease (CJD): a prospective longitudinal study protocol

BackgroundCreutzfeldt-Jakob Disease (CJD) is the most common prion disease in humans causing a rapidly progressive neurological decline and dementia and is invariably fatal. The familial forms (genetic CJD, gCJD) are caused by mutations in the PRNP gene encoding for the prion protein (PrP). In Israel, there is a large cluster of gCJD cases, carriers of an E200K mutation in the PRNP gene, and therefore the largest population of at-risk individuals in the world. The mutation is not necessarily sufficient for the formation and accumulation of the pathological prion protein (PrPsc), suggesting that other, genetic and non-genetic factors affect the age at symptoms onset. Here we present the protocol of a cross-sectional and longitudinal natural history study of gCJD patients and first-degree relatives of gCJD patients, aiming to identify biological markers of preclinical CJD and risk factors for phenoconversion.MethodsThe study has two groups: Patients diagnosed with gCJD, and first-degree healthy relatives (HR) (both carriers and non-carriers of the E200K mutation in the PRNP gene) of patients diagnosed with gCJD. At baseline, and at the end of every year, healthy participants are invited for an “in-depth” visit, which includes a clinical evaluation, blood and urine collection, gait assessment, brain MRI, lumbar puncture (LP), and Polysomnography (PSG). At 6 months from baseline, and then halfway through each year, participants are invited for a “brief” visit, which includes a clinical evaluation, short cognitive assessment, and blood and urine collection. gCJD patients will be invited for one “in-depth” visit, similar to the baseline visit of healthy relatives.DiscussionThis continuous follow-up of the participants and the frequent assessments will allow early identification and diagnosis in case of conversion into disease. The knowledge generated from this study is likely to advance the understanding of the underlying clinicopathological processes that occur at the very beginning of CJD, as well as potential genetic and environmental risk factors for the development of the disease, therefore advancing the development of safe and efficient interventions.Trial registrationThe study is an observational study. It has registered retrospectively in https://clinicaltrials.gov/ and has been assigned an identification number NCT05746715.

Susceptibility of Ovine Bone Marrow-Derived Mesenchymal Stem Cell Spheroids to Scrapie Prion Infection

In neurodegenerative diseases, including prion diseases, cellular in vitro models appear as fundamental tools for the study of pathogenic mechanisms and potential therapeutic compounds. Two-dimensional (2D) monolayer cell culture systems are the most used cell-based assays, but these platforms are not able to reproduce the microenvironment of in vivo cells. This limitation can be surpassed using three-dimensional (3D) culture systems such as spheroids that more effectively mimic in vivo cell interactions. Herein, we evaluated the effect of scrapie prion infection in monolayer-cultured ovine bone marrow-derived mesenchymal stem cells (oBM-MSCs) and oBM-MSC-derived spheroids in growth and neurogenic conditions, analyzing their cell viability and their ability to maintain prion infection. An MTT assay was performed in oBM-MSCs and spheroids subjected to three conditions: inoculated with brain homogenate from scrapie-infected sheep, inoculated with brain homogenate from healthy sheep, and non-inoculated controls. The 3D conditions improved the cell viability in most cases, although in scrapie-infected spheroids in growth conditions, a decrease in cell viability was observed. The levels of pathological prion protein (PrPSc) in scrapie-infected oBM-MSCs and spheroids were measured by ELISA. In neurogenic conditions, monolayer cells and spheroids maintained the levels of PrPSc over time. In growth conditions, however, oBM-MSCs showed decreasing levels of PrPSc throughout time, whereas spheroids were able to maintain stable PrPSc levels. The presence of PrPSc in spheroids was also confirmed by immunocytochemistry. Altogether, these results show that a 3D culture microenvironment improves the permissiveness of oBM-MSCs to scrapie infection in growth conditions and maintains the infection ability in neurogenic conditions, making this model of potential use for prion studies.

Comparative analysis of the structural dynamics of the Doppel protein and the C-terminus of the cellular prion protein.

The Doppel protein is the closest related protein on the phylogenetic tree to the prion protein. While the misfolding and aggregation of the prion protein is the hallmark of prion diseases, there is no reported evidence of an equivalent pathological process for the Doppel protein. Although the two proteins share only 25% sequence similarity, the folded C-terminus of the prion protein demonstrates a consistent topology with the Doppel protein. Despite the resemblance in topology, previous studies suggest that the Doppel protein has a higher energy barrier against misfolding than prion protein. However, the factors that mold the energy barrier remain unknown. Our goal is to decipher the role of structural dynamics and primary residue sequence of the native fold in shaping the misfolding energy barrier of each protein. Understanding the driving forces that stabilize similar native topologies will provide insight into subtle interactions that potentially trigger pathological prion protein misfolding. To perform a comparative analysis of the structural dynamics of the proteins, we used structural bioinformatics techniques. Our preliminary results indicate that the pattern of backbone local mobility of both proteins is similar: a flexible α-helix 1, and a somewhat rigid cluster that includes the β-sheets and α-helices 2 and 3. We also observed that the main modes of deformations are qualitatively similar between the two proteins. We will discuss the key features of the residue connectivity that determine the common topology despite differences in primary structure and propose target druggable residue interactions that may prevent prion protein misfolding.

The manifold role of octapeptide repeats in prion protein assembly.

Prion protein misfolding is associated with fatal neurodegenerative disorders such as kuru, Creutzfeldt-Jakob disease, and several animal encephalopathies. While the C-terminal 106-126 peptide has been well studied for its role in prion replication and toxicity, the octapeptide repeat (OPR) sequence found within the N-terminal domain has been relatively under explored. Recent findings that the OPR has both local and long-range effects on prion protein folding and assembly, as well as its ability to bind and regulate transition metal homeostasis, highlights the important role this understudied region may have in prion pathologies. This review attempts to collate this knowledge to advance a deeper understanding on the varied physiologic and pathologic roles the prion OPR plays, and connect these findings to potential therapeutic modalities focused on OPR-metal binding. Continued study of the OPR will not only elucidate a more complete mechanistic model of prion pathology, but may enhance knowledge on other neurodegenerative processes underlying Alzheimer's, Parkinson's, and Huntington's diseases.

Systematic Review of Clinical and Pathophysiological Features of Genetic Creutzfeldt-Jakob Disease Caused by a Val-to-Ile Mutation at Codon 180 in the Prion Protein Gene.

Genetic Creutzfeldt-Jakob disease (gCJD) is a subtype of genetic prion diseases (gPrDs) caused by the accumulation of mutated pathological prion proteins (PrPSc). gCJD has a phenotypic similarity with sporadic CJD (sCJD). In Japan, gCJD with a Val to Ile substitution at codon 180 (V180I-gCJD) is the most frequent gPrD, while the mutation is extremely rare in countries other than Japan and Korea. In this article, we aim to review previously elucidated clinical and biochemical features of V180I-gCJD, expecting to advance the understanding of this unique subtype in gCJD. Compared to classical sCJD, specific clinical features of V180I-gCJD include older age at onset, a relatively slow progression of dementia, and a lower positivity for developing myoclonus, cerebellar, pyramidal signs, and visual disturbance. Diffuse edematous ribboning hyperintensity of the cerebral cortex, without occipital lobes in diffusion-weighted magnetic resonance imaging, is also specific. Laboratory data reveal the low positivity of PrPSc in the cerebrospinal fluid and periodic sharp wave complexes on an electroencephalogram. Most patients with V180I-gCJD have been reported to have no family history, probably due to the older age at onset, and clinical and biochemical features indicate the specific phenotype associated with the prion protein gene mutation.

An arrayed genome-wide perturbation screen identifies the ribonucleoprotein Hnrnpk as rate-limiting for prion propagation.

A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting cellular host-factors that can modify prion propagation. We exposed prion-infected cells in high-density microplates to 35,364 ternary pools of 52,746 siRNAs targeting 17,582 genes representing the majority of the mouse protein-coding transcriptome. We identified 1,191 modulators of prion propagation. While 1,151 modified the expression of both the pathological prion protein, PrPSc , and its cellular counterpart, PrPC , 40 genes selectively affected PrPSc . Of the latter 40 genes, 20 augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrPC . Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Further, the unexpected identification of a prion-controlling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.

Sporadic Creutzfeldt-Jakob disease VM1: phenotypic and molecular characterization of a novel subtype of human prion disease

The methionine (M)—valine (V) polymorphic codon 129 of the prion protein gene (PRNP) plays a central role in both susceptibility and phenotypic expression of sporadic Creutzfeldt-Jakob diseases (sCJD). Experimental transmissions of sCJD in humanized transgenic mice led to the isolation of five prion strains, named M1, M2C, M2T, V2, and V1, based on two major conformations of the pathological prion protein (PrPSc, type 1 and type 2), and the codon 129 genotype determining susceptibility and propagation efficiency. While the most frequent sCJD strains have been described in codon 129 homozygosis (MM1, MM2C, VV2) and heterozygosis (MV1, MV2K, and MV2C), the V1 strain has only been found in patients carrying VV. We identified six sCJD cases, 4 in Catalonia and 2 in Italy, carrying MV at PRNP codon 129 in combination with PrPSc type 1 and a new clinical and neuropathological profile reminiscent of the VV1 sCJD subtype rather than typical MM1/MV1. All patients had a relatively long duration (mean of 20.5 vs. 3.5 months of MM1/MV1 patients) and lacked electroencephalographic periodic sharp-wave complexes at diagnosis. Distinctive histopathological features included the spongiform change with vacuoles of larger size than those seen in sCJD MM1/MV1, the lesion profile with prominent cortical and striatal involvement, and the pattern of PrPSc deposition characterized by a dissociation between florid spongiform change and mild synaptic deposits associated with coarse, patch-like deposits in the cerebellar molecular layer. Western blot analysis of brain homogenates revealed a PrPSc type 1 profile with physicochemical properties reminiscent of the type 1 protein linked to the VV1 sCJD subtype. In summary, we have identified a new subtype of sCJD with distinctive clinicopathological features significantly overlapping with those of the VV1 subtype, possibly representing the missing evidence of V1 sCJD strain propagation in the 129MV host genotype.

The Use of Real-Time Quaking-Induced Conversion for the Diagnosis of Human Prion Diseases.

Prion diseases are rapidly progressive, invariably fatal, transmissible neurodegenerative disorders associated with the accumulation of the amyloidogenic form of the prion protein in the central nervous system (CNS). In humans, prion diseases are highly heterogeneous both clinically and neuropathologically. Prion diseases are challenging to diagnose as many other neurologic disorders share the same symptoms, especially at clinical onset. Definitive diagnosis requires brain autopsy to identify the accumulation of the pathological prion protein, which is the only specific disease biomarker. Although brain post-mortem investigation remains the gold standard for diagnosis, antemortem clinical, instrumental, and laboratory tests showing variable sensitivities and specificity, being surrogate disease biomarkers, have been progressively introduced in clinical practice to reach a diagnosis. More recently, the ultrasensitive Real-Time Quaking-Induced Conversion (RT-QuIC) assay, exploiting, for the first time, the detection of misfolded prion protein through an amplification strategy, has highly improved the “in-vitam” diagnostic process, reaching in cerebrospinal fluid (CSF) and olfactory mucosa (OM) around 96% sensitivity and close to 100% specificity. RT-QuIC also improved the detection of the pathologic prion protein in several peripheral tissues, possibly even before the clinical onset of the disease. The latter aspect is of great interest for the early and even preclinical diagnosis in subjects at genetic risk of developing the disease, who will likely be the main target population in future clinical trials. This review presents an overview of the current knowledge and future perspectives on using RT-QuIC to diagnose human prion diseases.

The engineered peptide construct NCAM1-Aβ inhibits fibrillization of the human prion protein (PrP).

In prion diseases, the prion protein (PrP) becomes misfolded and forms fibrillar aggregates that are responsible for prion infectivity and pathology. So far, no drug or treatment procedures have been approved for prion disease treatment. We have previously shown that engineered cell-penetrating peptide constructs can reduce the amount of prion aggregates in infected cells. However, the molecular mechanism underlying this effect is unknown. Here, we use atomic force microscopy (AFM) imaging to show that the amyloid aggregation and fibrillization of the human PrP protein can be inhibited by equimolar amounts of the 25 residues long engineered peptide construct NCAM1-Aβ.