Immunobiology of Prion Diseases

Prion diseases are invariably lethal neurodegenerative diseases, associated with the structural conversion of the cellular isoform of the prion protein to its pathological, disease-associated isoform. The cellular isoform of the prion protein is highly conserved and virtually ubiquitously expressed; nevertheless, its physiological role remains unclear. Mounting evidence suggests its involvement in the regulation and function of the immune system. At the same time, the immune system is heavily involved in the pathogenesis of the diseases, playing a major role in the peripheral replication of the infectious agent and spread toward the central nervous system. On the other hand, immunotherapies are among the most promising means of intervention. This chapter deals with these fascinating and sometimes contrasting aspects of prion biology, with an emphasis on the immunization protocols developed for prophylaxis and treatment of prion diseases

MicroRNA alterations in the brain and body fluids of human and animal príon disease models: current status and perspectives

Prion diseases are transmissible progressive neurodegenerative conditions characterized by rapid neuronal loss accompanied by a heterogeneous neuropathology, including spongiform degeneration, gliosis and protein aggregation. The pathogenic mechanisms and the origins of prion diseases remain unclear on the molecular level. Even though neurodegenerative diseases, including prion diseases, represent distinct entities, their pathogenesis shares a number of features including disturbed protein homeostasis, an overload of protein clearance pathways, the aggregation of pathological altered proteins, and the dysfunction and/or loss of specific neuronal populations. Recently, direct links have been established between neurodegenerative diseases and miRNA dysregulated patterns. miRNAs are a class of small non-coding RNAs involved in the fundamental post-transcriptional regulation of gene expression. Studies of miRNA alterations in the brain and body fluids in human prion diseases provide important insights into potential miRNA-associated disease mechanisms and biomarker candidates. miRNA alterations in prion disease models represent a unique tool to investigate the cause-consequence relationships of miRNA dysregulation in prion disease pathology, and to evaluate the use of miRNAs in diagnosis as biomarkers. Here, we provide an overview of studies on miRNA alterations in human prion diseases and relevant disease models, in relation to pertinent studies on other neurodegenerative diseases. KEYWORDS: CSF; blood; brain; microRNAs; neurodegenerative disorders; prion diseases; prion diseases animal models; sCJ

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

MicroRNA Alterations in the Brain and Body Fluids of Humans and Animal Prion Disease Models: Current Status and Perspectives

Prion diseases are transmissible progressive neurodegenerative conditions characterized by rapid neuronal loss accompanied by a heterogeneous neuropathology, including spongiform degeneration, gliosis and protein aggregation. The pathogenic mechanisms and the origins of prion diseases remain unclear on the molecular level. Even though neurodegenerative diseases, including prion diseases, represent distinct entities, their pathogenesis shares a number of features including disturbed protein homeostasis, an overload of protein clearance pathways, the aggregation of pathological altered proteins, and the dysfunction and/or loss of specific neuronal populations. Recently, direct links have been established between neurodegenerative diseases and miRNA dysregulated patterns. miRNAs are a class of small non-coding RNAs involved in the fundamental post-transcriptional regulation of gene expression. Studies of miRNA alterations in the brain and body fluids in human prion diseases provide important insights into potential miRNA-associated disease mechanisms and biomarker candidates. miRNA alterations in prion disease models represent a unique tool to investigate the cause-consequence relationships of miRNA dysregulation in prion disease pathology, and to evaluate the use of miRNAs in diagnosis as biomarkers. Here, we provide an overview of studies on miRNA alterations in human prion diseases and relevant disease models, in relation to pertinent studies on other neurodegenerative diseases

Ykl-40 in the brain and cerebrospinal fluid of neurodegenerative dementias

Background: YKL-40 (also known as Chitinase 3-like 1) is a glycoprotein produced by inflammatory, cancer and stem cells. Its physiological role is not completely understood but YKL-40 is elevated in the brain and cerebrospinal fluid (CSF) in several neurological and neurodegenerative diseases associated with inflammatory processes. Yet the precise characterization of YKL-40 in dementia cases is missing. Methods: In the present study, we comparatively analysed YKL-40 levels in the brain and CSF samples from neurodegenerative dementias of different aetiologies characterized by the presence of cortical pathology and disease-specific neuroinflammatory signatures. Results: YKL-40 was normally expressed in fibrillar astrocytes in the white matter. Additionally YKL-40 was highly and widely expressed in reactive protoplasmic cortical and perivascular astrocytes, and fibrillar astrocytes in sporadic Creutzfeldt-Jakob disease (sCJD). Elevated YKL-40 levels were also detected in Alzheimer's disease (AD) but not in dementia with Lewy bodies (DLB). In AD, YKL-40-positive astrocytes were commonly found in clusters, often around beta-amyloid plaques, and surrounding vessels with beta-amyloid angiopathy; they were also distributed randomly in the cerebral cortex and white matter. YKL-40 overexpression appeared as a pre-clinical event as demonstrated in experimental models of prion diseases and AD pathology. CSF YKL-40 levels were measured in a cohort of 288 individuals, including neurological controls (NC) and patients diagnosed with different types of dementia. Compared to NC, increased YKL-40 levels were detected in sCJD (p 0.05, AUC = 0.71) or in DLB/Parkinson's disease dementia (PDD) (p > 0.05, AUC = 0.70). Further, two independent patient cohorts were used to validate the increased CSF YKL-40 levels in sCJD. Additionally, increased YKL-40 levels were found in genetic prion diseases associated with the PRNP-D178N (Fatal Familial Insomnia) and PRNP-E200K mutations. Conclusions: Our results unequivocally demonstrate that in neurodegenerative dementias, YKL-40 is a disease-specific marker of neuroinflammation showing its highest levels in prion diseases. Therefore, YKL-40 quantification might have a potential for application in the evaluation of therapeutic intervention in dementias with a neuroinflammatory component

Detection of molecules and polymorphisms possibly affecting the pathogenesis of neurodegenerative disorders, including Prion diseases

Scrapie is a fatal neurodegenerative disease of sheep and goats, characterized by the conformational conversion of the normal PrPC protein (encoded by the prnp gene). This conversion is the result of PrPC interaction with its β-sheet rich isoform, termed PrPSc. The latter is considered as the infectious agent of scrapie. Single Nucleotide Polymorphisms (SNPs) in the caprine prnp gene have been associated with some degree of protection against goat scrapie. However, scrapie resistance selective breeding schemes have not been applied in goat populations in Greece and any other country, due to the scarcity of available data on caprine prnp genetic variability and corresponding protective alleles. The present study aimed: a) at the assessment of the caprine prnp genetic variability in Greek goats, through the detection of SNPs in the gene's coding region and b) at the study of selected polymorphisms in a cell culture model of scrapie, in order to correlate polymorphisms with resistance or susceptibility to the disease and to detect possible mechanisms, through which the resistance associated SNPs may confer their protective effect. For the study of the caprine prnp genetic variability, the prnp coding region was analyzed through Sanger-based sequencing (436 samples from herds located in Northern Greece were studied), sequencing data were aligned to the reference sequence and the detected polymorphisms' frequencies were determined. For the in vitro study of selected polymorphisms, caprine PrP variants encompassing the polymorphisms of interest, were expressed in scrapie affected murine neuroblastoma cells (22L-scN2a) and the percentage of these variants that was converted to PrPSc was assessed. Variants displaying a lower conversion efficiency compared to the wild type caprine PrP were associated with increased protection against scrapie. The present study enhanced existing knowledge concerning prnp variability in Greek goats, by detecting new polymorphic positions and determining the frequencies of scrapie-protective alleles in a representative population of indigenous dairy Greek goats. The results of this study suggest that the caprine prnp alleles S146, Κ222 and Q211 should be considered for a selection goal in the design and implementation of scrapie resistance breeding programs for dairy goats in Greece; their protective effect in the examined population should be confirmed by large-scale studies considering also their association with production traits. Moreover, the in vitro study of selected caprine prnp polymorphisms in a cell model of scrapie, confirmed the protective effect of the S146 polymorphism and enabled the study of some of its biosynthesis properties, revealing the potentially protective mechanisms of its protective action.Η scrapie αποτελεί λοιμώδες νευροεκφυλιστικό νόσημα των προβάτων και των αιγών. Η νόσος χαρακτηρίζεται από τη δομική τροποποίηση της φυσιολογικής PrPC πρωτεΐνης (προϊόν του prnp γονιδίου), που προκύπτει ως αποτέλεσμα της αλληλεπίδρασης της PrPC με μια πλούσια σε β-ελάσματα ισομορφή της (PrPSc). Η τελευταία πιστεύεται ότι αντιστοιχεί στον παθογόνο παράγοντα της scrapie. Πολυμορφισμοί του prnp γονιδίου αιγών έχουν συσχετιστεί με ορισμένο βαθμό προστασίας έναντι της scrapie. Ωστόσο, τα περιορισμένα διαθέσιμα δεδομένα σχετικά με τη γενετική ποικιλομορφία του prnp γονιδίου αιγών και των αλληλομόρφων του που σχετίζονται με ανθεκτικότητα έναντι της scrapie, δεν επέτρεψαν το σχεδιασμό και την εφαρμογή προγραμμάτων επιλεκτικών διασταυρώσεων που αποσκοπούν στην αύξηση των συχνοτήτων των προστατευτικών αλληλομόρφων σε πληθυσμούς αιγών, τόσο στην Ελλάδα όσο και σε άλλες χώρες. Στόχος της παρούσας διδακτορικής διατριβής ήταν: α) η ανίχνευση πολυμορφισμών στην κωδικοποιούσα περιοχή του prnp γονιδίου ελληνικών αιγών, με σκοπό την εκτίμηση της γενετικής ποικιλομορφίας του prnp γονιδίου και β) η μελέτη επιλεγμένων πολυμορφισμών σε κυτταρικό μοντέλο της scrapie, προκειμένου να διερευνηθεί η συσχέτισή τους με τη νόσο και να προσδιοριστούν πιθανοί μηχανισμοί που σχετίζονται με προστασία έναντι της scrapie. Για τη μελέτη της ποικιλομορφίας του prnp γονιδίου, εφαρμόστηκε αλληλούχιση κατά Sanger στο επιθυμητό τμήμα του prnp γονιδίου αιγών (436 δείγματα από εκτροφές της Βορείου Ελλάδος), σύγκριση των αποτελεσμάτων αλληλούχισης με την αντίστοιχη ακολουθία αναφοράς και προσδιορισμός της συχνότητας των πολυμορφισμών στο σύνολο του πληθυσμού που μελετήθηκε. Για την in vitro μελέτη επιλεγμένων πολυμορφισμών, ποικιλίες της PrP αιγών που έφεραν τους πολυμορφισμούς ενδιαφέροντος εκφράστηκαν σε μολυσμένα με το στέλεχος 22L της scrapie κύτταρα νευροβλαστώματος ποντικού (22L-scN2a) και εκτιμήθηκε το ποσοστό τροποποίησής τους προς PrPSc. Ποικιλίες με μικρότερο βαθμό τροποποίησης σε σχέση με την αγρίου τύπου PrP αιγών συσχετίστηκαν με ορισμένο βαθμό προστασίας έναντι της scrapie. Η παρούσα διδακτορική διατριβή συνέβαλλε σημαντικά στην ενίσχυση της υπάρχουσας γνώσης που αφορά την ποικιλομορφία του prnp γονιδίου στις Ελληνικές αίγες, μέσω της ανίχνευσης νέων πολυμορφικών θέσεων και του προσδιορισμού των συχνοτήτων προστατευτικών έναντι της scrapie αλληλομόρφων, σε αντιπροσωπευτικό δείγμα Ελληνικών αιγών. Τα αποτελέσματα που προέκυψαν από την έρευνα αυτή συνηγορούν υπέρ της επιλογής των αλληλομόρφων S146, Κ222 και Q211 του prnp γονιδίου αιγών για το σχεδιασμό και την εφαρμογή προγραμμάτων επιλεκτικών διασταυρώσεων με σκοπό την αντιμετώπιση της scrapie στις Ελληνικές αίγες, εφόσον επιβεβαιωθεί η προστατευτική τους δράση στις Ελληνικές αίγες μέσω μεγάλης κλίμακας μελετών και εκτιμηθεί η συσχέτισή τους με παραγωγικά χαρακτηριστικά των ζώων. Επιπρόσθετα, η μελέτη ορισμένων από τους φυσικά απαντώμενους πολυμορφισμούς του prnp γονιδίου αιγών που μελετήθηκαν στα πλαίσια της παρούσας διδακτορικής διατριβής σε κυτταρικό μοντέλο της νόσου, επιβεβαίωσε την προστατευτική δράση του αλληλομόρφου S146 και παράλληλα επέτρεψε τη μελέτη χαρακτηριστικών της βιοσύνθεσης του, που πιθανόν να συμβάλλουν στην κατανόηση της προστατευτικής του δράσης

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

International audienceThe 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

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

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