RNA editing regulates glutamatergic synapses in the frontal cortex of a molecular subtype of Amyotrophic Lateral Sclerosis.

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is a highly heterogenous neurodegenerative disorder that primarily affects upper and lower motor neurons, affecting additional cell types and brain regions. Underlying molecular mechanisms are still elusive, in part due to disease heterogeneity. Molecular disease subtyping through integrative analyses including RNA editing profiling is a novel approach for identification of molecular networks involved in pathogenesis. METHODS: We aimed to highlight the role of RNA editing in ALS, focusing on the frontal cortex and the prevalent molecular disease subtype (ALS-Ox), previously determined by transcriptomic profile stratification. We established global RNA editing (editome) and gene expression (transcriptome) profiles in control and ALS-Ox cases, utilizing publicly available RNA-seq data (GSE153960) and an in-house analysis pipeline. Functional annotation and pathway analyses identified molecular processes affected by RNA editing alterations. Pearson correlation analyses assessed RNA editing effects on expression. Similar analyses on additional ALS-Ox and control samples (GSE124439) were performed for verification. Targeted re-sequencing and qRT-PCR analysis targeting CACNA1C, were performed using frontal cortex tissue from ALS and control samples (n = 3 samples/group). RESULTS: We identified reduced global RNA editing in the frontal cortex of ALS-Ox cases. Differentially edited transcripts are enriched in synapses, particularly in the glutamatergic synapse pathway. Bioinformatic analyses on additional ALS-Ox and control RNA-seq data verified these findings. We identified increased recoding at the Q621R site in the GRIK2 transcript and determined positive correlations between RNA editing and gene expression alterations in ionotropic receptor subunits GRIA2, GRIA3 and the CACNA1C transcript, which encodes the pore forming subunit of a post-synaptic L-type calcium channel. Experimental data verified RNA editing alterations and editing-expression correlation in CACNA1C, highlighting CACNA1C as a target for further study. CONCLUSIONS: We provide evidence on the involvement of RNA editing in the frontal cortex of an ALS molecular subtype, highlighting a modulatory role mediated though recoding and gene expression regulation on glutamatergic synapse related transcripts. We report RNA editing effects in disease-related transcripts and validated editing alterations in CACNA1C. Our study provides targets for further functional studies that could shed light in underlying disease mechanisms enabling novel therapeutic approaches.

Gene targeting in amyotrophic lateral sclerosis using causality-based feature selection and machine learning.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a rare progressive neurodegenerative disease that affects upper and lower motor neurons. As the molecular basis of the disease is still elusive, the development of high-throughput sequencing technologies, combined with data mining techniques and machine learning methods, could provide remarkable results in identifying pathogenetic mechanisms. High dimensionality is a major problem when applying machine learning techniques in biomedical data analysis, since a huge number of features is available for a limited number of samples. The aim of this study was to develop a methodology for training interpretable machine learning models in the classification of ALS and ALS-subtypes samples, using gene expression datasets. METHODS: We performed dimensionality reduction in gene expression data using a semi-automated preprocessing systematic gene selection procedure using Statistically Equivalent Signature (SES), a causality-based feature selection algorithm, followed by Boosted Regression Trees (XGBoost) and Random Forest to train the machine learning classifiers. The SHapley Additive exPlanations (SHAP values) were used for interpretation of the machine learning classifiers. The methodology was developed and tested using two distinct publicly available ALS RNA-seq datasets. We evaluated the performance of SES as a dimensionality reduction method against: (a) Least Absolute Shrinkage and Selection Operator (LASSO), and (b) Local Outlier Factor (LOF). RESULTS: The proposed methodology achieved 85.18% accuracy for the classification of cerebellum or frontal cortex samples as C9orf72-related familial ALS, sporadic ALS or healthy samples. Importantly, the genes identified as the most determinative have also been reported as disease-associated in ALS literature. When tested in the evaluation dataset, the methodology achieved 88.89% accuracy for the classification of sporadic ALS motor neuron samples. When LASSO was used as feature selection method instead of SES, the accuracy of the machine learning classifiers ranged from 74.07 to 96.30%, depending on tissue assessed, while LOF underperformed significantly (77.78% accuracy for the classification of pooled cerebellum and frontal cortex samples). CONCLUSIONS: Using SES, we addressed the challenge of high dimensionality in gene expression data analysis, and we trained accurate machine learning ALS classifiers, specific for the gene expression patterns of different disease subtypes and tissue samples, while identifying disease-associated genes.

Photo-Fenton and TiO2 Photocatalytic Inactivation of Model Microorganisms under UV-A; Comparative Efficacy and Optimization.

Photocatalytic inactivation of pathogens in aqueous waste is gaining increasing attention. Several homogeneous and heterogeneous photocatalytic protocols exist using the Fenton's reagent and TiO2, respectively. A comprehensive study of homogeneous and heterogeneous photocatalysis on a range of microorganisms will significantly establish the most efficient method. Here, we report a comparative study of TiO2- and Fe+3-based photocatalytic inactivation under UV-A of diverse microorganisms, including Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, bacterial spores (Bacillus stearothermophilus spores) and viruses (MS2). We also present data on the optimization of TiO2 photocatalysis, including optimal catalyst concentration and H2O2 supplementation. Our results indicate that both photo-Fenton and TiO2 could be successfully applied for the management of microbial loads in liquids. Efficient microorganism inactivation is achieved with homogeneous photocatalysis (7 mg/L Fe+3, 100 mg/L H2O2, UV-A) in a shorter processing time compared to heterogeneous photocatalysis (0.5 g/L TiO2, UV-A), whereas similar or shorter processing is required when heterogenous photocatalysis is performed using microorganism-specific optimized TiO2 concentrations and H2O2 supplementation (100 mg/L); higher H2O2 concentrations further enhance the heterogenous photocatalytic inactivation efficiency. Our study provides a template protocol for the design and further application for large-scale photocatalytic approaches to inactivate pathogens in liquid biomedical waste.

RNA editing alterations define manifestation of prion diseases.

Prion diseases are fatal neurodegenerative disorders caused by misfolding of the normal prion protein into an infectious cellular pathogen. Clinically characterized by rapidly progressive dementia and accounting for 85% of human prion disease cases, sporadic Creutzfeldt-Jakob disease (sCJD) is the prevalent human prion disease. Although sCJD neuropathological hallmarks are well-known, associated molecular alterations are elusive due to rapid progression and absence of preclinical stages. To investigate transcriptome alterations during disease progression, we utilized tg340-PRNP129MM mice infected with postmortem material from sCJD patients of the most susceptible genotype (MM1 subtype), a sCJD model that faithfully recapitulates the molecular and pathological alterations of the human disease. Here we report that transcriptomic analyses from brain cortex in the context of disease progression, reveal epitranscriptomic alterations (specifically altered RNA edited pathway profiles, eg., ER stress, lysosome) that are characteristic and possibly protective mainly for preclinical and clinical disease stages. Our results implicate regulatory epitranscriptomic mechanisms in prion disease neuropathogenesis, whereby RNA-editing targets in a humanized sCJD mouse model were confirmed in pathological human autopsy material.

Regional and subtype-dependent miRNA signatures in sporadic Creutzfeldt-Jakob disease are accompanied by alterations in miRNA silencing machinery and biogenesis.

Increasing evidence indicates that microRNAs (miRNAs) are contributing factors to neurodegeneration. Alterations in miRNA signatures have been reported in several neurodegenerative dementias, but data in prion diseases are restricted to ex vivo and animal models. The present study identified significant miRNA expression pattern alterations in the frontal cortex and cerebellum of sporadic Creutzfeldt-Jakob disease (sCJD) patients. These changes display a highly regional and disease subtype-dependent regulation that correlates with brain pathology. We demonstrate that selected miRNAs are enriched in sCJD isolated Argonaute(Ago)-binding complexes in disease, indicating their incorporation into RNA-induced silencing complexes, and further suggesting their contribution to disease-associated gene expression changes. Alterations in the miRNA-mRNA regulatory machinery and perturbed levels of miRNA biogenesis key components in sCJD brain samples reported here further implicate miRNAs in sCJD gene expression (de)regulation. We also show that a subset of sCJD-altered miRNAs are commonly changed in Alzheimer's disease, dementia with Lewy bodies and fatal familial insomnia, suggesting potential common mechanisms underlying these neurodegenerative processes. Additionally, we report no correlation between brain and cerebrospinal fluid (CSF) miRNA-profiles in sCJD, indicating that CSF-miRNA profiles do not faithfully mirror miRNA alterations detected in brain tissue of human prion diseases. Finally, utilizing a sCJD MM1 mouse model, we analyzed the miRNA deregulation patterns observed in sCJD in a temporal manner. While fourteen sCJD-related miRNAs were validated at clinical stages, only two of those were changed at early symptomatic phase, suggesting that the miRNAs altered in sCJD may contribute to later pathogenic processes. Altogether, the present work identifies alterations in the miRNA network, biogenesis and miRNA-mRNA silencing machinery in sCJD, whereby contributions to disease mechanisms deserve further investigation.

A Closed Chemobrionic System as a Biochemical Delivery Platform.

Inorganic cells bearing calcium silicate membranes were prepared and resembled closed chemical gardens. It was demonstrated that these inorganic cells can successfully be loaded with natural products, proteins and plasmid DNA, and their cargo can be released in a controlled manner. These cells demonstrated the ability of chemical gardens to act as platforms for the sustained delivery of biomolecules and are expected to introduce chemical gardens in the field of biosciences.

Cerebrospinal fluid neurofilament light levels in neurodegenerative dementia: Evaluation of diagnostic accuracy in the differential diagnosis of prion diseases.

INTRODUCTION: Neurofilament light (NFL) levels in the cerebrospinal fluid are increased in several neurodegenerative dementias. However, their diagnostic accuracy in the differential diagnostic context is unknown. METHODS: Cerebrospinal fluid NFL levels were quantified in nonprimarily neurodegenerative neurological and psychiatric diseases (n = 122), mild cognitive impairment (n = 48), Alzheimer's disease (n = 108), dementia with Lewy bodies/Parkinson's disease dementia (n = 53), vascular dementia (n = 46), frontotemporal dementia (n = 41), sporadic Creutzfeldt-Jakob disease (sCJD, n = 132), and genetic prion diseases (n = 182). RESULTS: The highest NFL levels were detected in sCJD, followed by vascular dementia, frontotemporal dementia, dementia with Lewy bodies/Parkinson's disease dementia, Alzheimer's disease, and mild cognitive impairment. In sCJD, NFL levels correlated with cerebrospinal fluid tau and disease duration. NFL levels were able to differentiate sCJD from nonprimarily neurodegenerative neurological and psychiatric diseases (area under the curve = 0.99, 95% confidence interval: 0.99-1) and from the other diagnostic groups showing cognitive impairment/dementia of a non-CJD etiology (area under the curve = 0.90, 95% confidence interval: 0.87-0.92). Compared to nonprimarily neurodegenerative neurological and psychiatric diseases, NFL was also elevated in genetic prion diseases associated with the E200K, V210I, P102L, and D178N prion protein gene mutations. DISCUSSION: Increased NFL levels are a common feature in neurodegenerative dementias.

Evaluation of α-synuclein as a novel cerebrospinal fluid biomarker in different forms of prion diseases.

INTRODUCTION: Accurate diagnosis of prion diseases and discrimination from alternative dementias gain importance in the clinical routine, but partial overlap in cerebrospinal fluid (CSF) biomarkers impedes absolute discrimination in the differential diagnostic context. METHODS: We established the clinical parameters for prion disease diagnosis for the quantification of CSF α-synuclein in patients with sporadic (n = 234) and genetic (n = 56) prion diseases, in cases with cognitive impairment/dementia or neurodegenerative disease (n = 278), and in the neurologic control group (n = 111). RESULTS: An optimal cutoff value of 680 pg/mL α-synuclein results in 94% sensitivity and 96% specificity when diagnosing sporadic Creutzfeldt-Jakob disease (CJD). Genetic CJD cases showed increased CSF α-synuclein values. No increased α-synuclein levels were detected in non-CJD cases with rapid progression course. DISCUSSION: Detection of α-synuclein in the CSF of patients with suspected CJD is a valuable diagnostic test reaching almost full discrimination from non-prion disease cases. These data highlight the utility of CSF α-synuclein quantification in front of classical CSF biomarkers in clinical routine.

Perspectives of a scrapie resistance breeding scheme targeting Q211, S146 and K222 caprine PRNP alleles in Greek goats.

The present study investigates the potential use of the scrapie-protective Q211 S146 and K222 caprine PRNP alleles as targets for selective breeding in Greek goats. Genotyping data from a high number of healthy goats with special emphasis on bucks, revealed high frequencies of these alleles, while the estimated probabilities of disease occurrence in animals carrying these alleles were low, suggesting that they can be used for selection. Greek goats represent one of the largest populations in Europe. Thus, the considerations presented here are an example of the expected effect of such a scheme on scrapie occurrence and on stakeholders.

A Comparison of RML Prion Inactivation Efficiency by Heterogeneous and Homogeneous Photocatalysis.

Prions are proteinaceous pathogens responsible for a variety of devastating diseases in mammals, including scrapie in sheep and goats, chronic wasting disease in cervids, and Creutzfeldt-Jakob disease (CJD) in humans. They are characterized by their exceptional persistence to common inactivation procedures. This applies to all possible sources of prion contamination as prions may be present in the tissues and biological fluids of infected individuals. Hence, efficient prion inactivation procedures are still being sought to minimize the risk of intra- or inter-species transmission. In the past, photocatalytic treatment has been proven to be capable of efficiently oxidizing and inactivating prions. In the present study, the efficacy of homogeneous photo-Fenton-based photocatalysis as well as heterogeneous photocatalysis with TiO2 in reducing RML mouse scrapie infectivity was evaluated. Prion inactivation was assessed by means of a bioassay, and the results were confirmed by in vitro experiments. While the prion infectivity of the RML mouse scrapie was reduced after treatment with the photo-Fenton reagent, the heterogeneous photocatalytic treatment of the same prion strain completely eliminated prion infectivity.

SIMOA Diagnostics on Alzheimer's Disease and Frontotemporal Dementia.

BACKGROUND: Accurate diagnosis of Alzheimer's disease (AD) and frontotemporal dementia (FTD) represents a health issue due to the absence of disease traits. We assessed the performance of a SIMOA panel in cerebrospinal fluid (CSF) from 43 AD and 33 FTD patients with 60 matching Control subjects in combination with demographic-clinical characteristics. METHODS: 136 subjects (AD: n = 43, FTD: n = 33, Controls: n = 60) participated. Single-molecule array (SIMOA), glial fibrillary acidic protein (GFAP), neurofilament light (NfL), TAU, and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in CSF were analyzed with a multiplex neuro 4plex kit. Receiver operating characteristic (ROC) curve analysis compared area under the curve (AUC), while the principal of the sparse partial least squares discriminant analysis (sPLS-DA) was used with the intent to strengthen the identification of confident disease clusters. RESULTS: CSF exhibited increased levels of all SIMOA biomarkers in AD compared to Controls (AUCs: 0.71, 0.86, 0.92, and 0.94, respectively). Similar patterns were observed in FTD with NfL, TAU, and UCH-L1 (AUCs: 0.85, 0.72, and 0.91). sPLS-DA revealed two components explaining 19% and 9% of dataset variation. CONCLUSIONS: CSF data provide high diagnostic accuracy among AD, FTD, and Control discrimination. Subgroups of demographic-clinical characteristics and biomarker concentration highlighted the potential of combining different kinds of data for successful and more efficient cohort clustering.

Αnti-prion effects of anthocyanins.

Prion diseases, also known as Transmissible Spongiform Encephalopathies (TSEs), are protein-based neurodegenerative disorders (NDs) affecting humans and animals. They are characterized by the conformational conversion of the normal cellular prion protein, PrPC, into the pathogenic isoform, PrPSc. Prion diseases are invariably fatal and despite ongoing research, no effective prophylactic or therapeutic avenues are currently available. Anthocyanins (ACNs) are unique flavonoid compounds and interest in their use as potential neuroprotective and/or therapeutic agents against NDs, has increased significantly in recent years. Therefore, we investigated the potential anti-oxidant and anti-prion effects of Oenin and Myrtillin, two of the most common anthocyanins, using the most accepted in the field overexpressing PrPScin vitro model and a cell free protein aggregation model. Our results, indicate both anthocyanins as strong anti-oxidant compounds, upregulating the expression of genes involved in the anti-oxidant response, and reducing the levels of Reactive Oxygen Species (ROS), produced due to pathogenic prion infection, through the activation of the Keap1-Nrf2 pathway. Importantly, they showcased remarkable anti-prion potential, as they not only caused the clearance of pathogenic PrPSc aggregates, but also completely inhibited the formation of PrPSc fibrils in the Cerebrospinal Fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD). Therefore, Oenin and Myrtillin possess pleiotropic effects, suggesting their potential use as promising preventive and/or therapeutic agents in prion diseases and possibly in the spectrum of neurodegenerative proteinopathies.

A Systematic Review of Common and Brain-Disease-Specific RNA Editing Alterations Providing Novel Insights into Neurological and Neurodegenerative Disease Manifestations.

RNA editing contributes to transcriptome diversification through RNA modifications in relation to genome-encoded information (RNA-DNA differences, RDDs). The deamination of Adenosine (A) to Inosine (I) or Cytidine (C) to Uridine (U) is the most common type of mammalian RNA editing. It occurs as a nuclear co- and/or post-transcriptional event catalyzed by ADARs (Adenosine deaminases acting on RNA) and APOBECs (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like genes). RNA editing may modify the structure, stability, and processing of a transcript. This review focuses on RNA editing in psychiatric, neurological, neurodegenerative (NDs), and autoimmune brain disorders in humans and rodent models. We discuss targeted studies that focus on RNA editing in specific neuron-enriched transcripts with well-established functions in neuronal activity, and transcriptome-wide studies, enabled by recent technological advances. We provide comparative editome analyses between human disease and corresponding animal models. Data suggest RNA editing to be an emerging mechanism in disease development, displaying common and disease-specific patterns. Commonly edited RNAs represent potential disease-associated targets for therapeutic and diagnostic values. Currently available data are primarily descriptive, calling for additional research to expand global editing profiles and to provide disease mechanistic insights. The potential use of RNA editing events as disease biomarkers and available tools for RNA editing identification, classification, ranking, and functional characterization that are being developed will enable comprehensive analyses for a better understanding of disease(s) pathogenesis and potential cures.

Profiling Microglia through Single-Cell RNA Sequencing over the Course of Development, Aging, and Disease.

Microglia are macrophages present in the brain that function as the primary and most important source of immune response in the central nervous system (CNS). Regardless of their multitasking role, our knowledge regarding their molecular heterogeneity is limited; due to technical restrictions, it is only possible to measure gene expression in cell populations, not individual cells, with the results reflecting average mRNA levels. Therefore, recent scientific approaches have focused on single-cell techniques such as single-cell RNA sequencing (scRNAseq), a powerful technique that enables the delineation of transcriptomic cell-to-cell differences, revealing subpopulations with distinct molecular and functional characteristics. Here, we summarize recent studies that focused on transcriptomic microglial subpopulation clustering and classify them into three distinct groups based on age, spatial distribution, and disease. Additionally, we cross-compare populations from different studies to identify expressional and functional overlaps between them.

Carnosic Acid and Carnosol Display Antioxidant and Anti-Prion Properties in In Vitro and Cell-Free Models of Prion Diseases.

Prion diseases are transmissible encephalopathies associated with the conversion of the physiological form of the prion protein (PrPC) to the disease-associated (PrPSc). Despite intense research, no therapeutic or prophylactic agent is available. The catechol-type diterpene Carnosic acid (CA) and its metabolite Carnosol (CS) from Rosmarinus officinalis have well-documented anti-oxidative and neuroprotective effects. Since oxidative stress plays an important role in the pathogenesis of prion diseases, we investigated the potential beneficial role of CA and CS in a cellular model of prion diseases (N2a22L cells) and in a cell-free prion amplification assay (RT-QuIC). The antioxidant effects of the compounds were confirmed when N2a22L were incubated with CA or CS. Furthermore, CA and CS reduced the accumulation of the disease-associated form of PrP, detected by Western Blotting, in N2a22L cells. This effect was validated in RT-QuIC assays, indicating that it is not associated with the antioxidant effects of CA and CS. Importantly, cell-free assays revealed that these natural products not only prevent the formation of PrP aggregates but can also disrupt already formed aggregates. Our results indicate that CA and CS have pleiotropic effects against prion diseases and could evolve into useful prophylactic and/or therapeutic agents against prion and other neurodegenerative diseases.

RNA Editing Alterations Define Disease Manifestations in the Progression of Experimental Autoimmune Encephalomyelitis (EAE).

RNA editing is an epitranscriptomic modification, leading to targeted changes in RNA transcripts. It is mediated by the action of ADAR (adenosine deaminases acting on double-stranded (ds) RNA and APOBEC (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like) deaminases and appears to play a major role in the pathogenesis of many diseases. Here, we assessed its role in experimental autoimmune encephalomyelitis (EAE), a widely used non-clinical model of autoimmune inflammatory diseases of the central nervous system (CNS), which resembles many aspects of human multiple sclerosis (MS). We have analyzed in silico data from microglia isolated at different timepoints through disease progression to identify the global editing events and validated the selected targets in murine tissue samples. To further evaluate the functional role of RNA editing, we induced EAE in transgenic animals lacking expression of APOBEC-1. We found that RNA-editing events, mediated by the APOBEC and ADAR deaminases, are significantly reduced throughout the course of disease, possibly affecting the protein expression necessary for normal neurological function. Moreover, the severity of the EAE model was significantly higher in APOBEC-1 knock-out mice, compared to wild-type controls. Our results implicate regulatory epitranscriptomic mechanisms in EAE pathogenesis that could be extrapolated to MS and other neurodegenerative disorders (NDs) with common clinical and molecular features.

Germinant generation from δ-endotoxin of Bacillus thuringiensis strain 1.1.

The novel finding of this study is that the δ-endotoxin present in the spore coat of Bacillus thuringiensis strain 1.1 (Bt1.1), plays a central role in spore germination by generation of germinant via its ß-glucosidase activity and is based on the following: (i) the crystals of Bt1.1 consist of the 140 kDa δ-endotoxin which exhibits ß-glucosidase enzymatic activity. Besides crystals, δ-endotoxin is also located in the spore coat and at this site displays ß-glucosidase activity, resulting in glucose production; (ii) glucose is an efficient germinant of both Bt1.1 and acrystalliferous Bt4.1 strain; (iii) substrates of ß-glucosidase can activate the germination of Bt1.1 spores, but not those of the acrystalliferous Bt4.1 sister strain that do not contain the 140 kDa δ-endotoxin; (iv) Reduction or enhancement of enzymatic activity of δ-endotoxin, results in retardation or acceleration of germination and outgrowth, respectively. Bt1.1 cells secrete a 60 kDa polypeptide which displays ß-glucosidase activity as indicated by zymogram analysis and which is immunologically related to the 140 kDa δ-endotoxin.

Validation of Poly(Propylene Imine) Glycodendrimers Towards Their Anti-prion Conversion Efficiency.

Prion diseases, such as the sporadic Creutzfeldt-Jakob disease (sCJD), are a class of fatal neurodegenerative disorders. Currently, there is no efficient treatment or therapy available. Hence, the search for molecules that may inhibit the conversion of the cellular prion protein (PrPC) into its pathological counterpart PrPScrapie (PrPSc) is of great urgency. Here, we report the generation- and dose-dependent biological action of dense-shell poly(propylene imine) (PPI) glycodendrimers by using scrapie-infected neuroblastoma (ScN2a) cells and the real-time quaking-induced conversion assay (RT-QuIC) for validation of anti-prion efficiencies. Whereas the 2nd and 3rd generation of PPI glycodendrimers exhibited anti-prion conversion efficiency in ScN2a cells validated by RT-QuIC analysis, we observed that the 4th generation of glycodendrimers had shown no significant effect. Translational RT-QuIC studies conducted with human prions derived from sCJD patients indicated an anti-prion conversion effect (not on PrPRes degradation) of PPI glycodendrimers against human prions with the highest inhibitory activity of the 4th generation of PPI glycodendrimers towards prion aggregation compared to the 2nd and 3rd generation. In conclusion, our study highlights the potential of PPI glycodendrimers as therapeutic compounds due to their anti-conversion activity on human prions in a PrPSc strain depending manner.

Design and validation of a high-throughput assay to detect codon 146 polymorphisms in the caprine prion protein gene.

In sheep, scrapie susceptibility is so strongly associated with single nucleotide polymorphisms (SNPs) in the gene encoding the prion protein (PrP) that this linkage constitutes the basis for selective breeding strategies directed toward controlling the disease. For goats, in contrast, the association between scrapie susceptibility/resistance and variations in the PrP gene is far weaker, with only a few identified SNPs showing an influence on scrapie susceptibility. A recent survey of PrP genotypes in Cypriot goats, however, revealed the existence of a robust association between polymorphisms at codon 146 of the caprine PrP gene and resistance/susceptibility to natural scrapie. Here we describe here a high-throughput assay, based on homogeneous MassExtend technology coupled with mass spectrometry, for genotyping codon 146 of the caprine PrP gene. Our results demonstrate that this assay exhibits high accuracy, reproducibility, and repeatability, thereby making it suitable for large-scale SNP genotyping, as required for scrapie surveillance programs.

Cerebrospinal fluid neurofilament light in suspected sporadic Creutzfeldt-Jakob disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form of human prion disease. It is invariably fatal and displays a short clinical disease stage. The key event in sCJD is the propagation of a beta-sheet rich conformer of the physiological PrPC protein, known as PrPSc. Neuropathological disease characteristics include gliosis, neuronal loss and spongiform degeneration; disease clinical manifestations refer to mental and visual disabilities, cognitive impairment, gait or limb ataxia, myoclonus and mutism. Definite sCJD diagnosis requires post-mortem brain material histopathological examination. However, highly certain pre-mortem differential diagnosis is desired to exclude other treatable disorders and to reduce disease transmission risks. Detection and/or quantification of cerebrospinal fluid (CSF) biomarkers reflecting neuronal damage and PrPC misfolding in the diseased brain significantly enhance pre-mortem diagnosis. Previously established and newly identified biomarkers are used towards this direction. Increased CSF Neurofilament light chain (NFL) concentrations have been reported in several neurological disorders, including prion diseases. In the present study, we analyzed CSF NFL levels in two independent patient cohorts, consisting of highly suspected sCJD cases that were further classified as sCJD or non-CJD according to established diagnostic criteria. CSF NFL concentrations were increased in sCJD compared to non-CJD cases in both cohorts (area under the curve (with 95% confidence interval) equal to 0.89 (0.82 to 0.97) and 0.86 (0.77 to 0.96), respectively. CSF NFL was associated neither to age nor to sex but correlated with total-tau concentrations in both cohorts. Overall, our data provide independent validation of CSF NFL utility in sCJD differential diagnosis.