Molecular Signature in Human and Animal Prion Disorders

In human and animal transmissible spongiform encephalopathies (TSEs) or prion disorders, biochemical analysis of disease-associated prion protein (PrPTSE) is a first-line approach for large scale routine testing and for a rapid molecular typing. This characterization is based on conformational properties of PrPTSE enciphered in its secondary and tertiary structures and on glycosylation profile. Several biochemical approaches are helpful in distinguishing PrPTSE forms in human prion diseases. In particular, in sporadic Creutzfedlt-Jakob disease (CJD), PrPTSE is characterized by two main glycotypes conventionally named PrPTSE type 1 and PrPTSE type 2 based on the apparent gel migration at 19 kDa and 17.5 kDa and glycofrom ratio. Further, there are PrPTSE low molecular weight fragments which correlate to distinct phenotypes of sCJD. Finally, by using two-dimensional PAGE analysis, which separates PrPTSE on both isoelectric point and molecular size, we were able to detect two distinct migration pattern in PrPTSE type 2, one in subjects with MM at codon 129 and another in MV, VV. We here provide an extensive PrPTSE biochemical analysis in humans and animals affected with prion disorders. Further, we showed that PrPTSE glycotypes observed in CJD shared similarities with PrPTSE in bovine spongiform encephalopathies (BSEs). These signature similarities obtained by a biochemical analysis had been further confirmed by experimental transmission

CSF alpha-synuclein aggregates by seed amplification and clinical presentation of AD

Introduction: Accumulating evidence suggests that α-synuclein (αSyn) can modulate Alzheimer's disease (AD) pathology. The aim of this study was to evaluate the prevalence and clinical features associated with cerebrospinal fluid (CSF) αSyn detected by seed amplification assay (SAA) in AD. Methods: Eighty AD patients with CSF AT(N) biomarker positivity (mean age 70.3 ± 7.3 years) and 28 non-AD age-matched controls were included. All subjects underwent standardized clinical assessment; CSF αSyn aggregates were detected by SAA. Results: CSF was αSyn-SAA positive (αSyn+) in 36/80 AD patients (45%) and in 2/28 controls (7.1%). AD αSyn+ and αSyn- patients were comparable for age, disease severity, comorbidity profile, and CSF core biomarkers. AD αSyn+ presented a higher prevalence of atypical phenotypes and symptoms. Conclusions: Our findings demonstrate that concomitant CSF αSyn pathology is present in a significant proportion of AD patients starting in the early stages and can affect clinical presentation. Longitudinal studies are warranted to evaluate the significance for the disease course

High diagnostic accuracy of RT-QuIC assay in a prospective study of patients with suspected sCJD

The early and accurate in vivo diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) is essential in order to differentiate CJD from treatable rapidly progressive dementias. Diagnostic investigations supportive of clinical CJD diagnosis include magnetic resonance imaging (MRI), electroencephalogram (EEG), 14-3-3 protein detection, and/or real-time quaking-induced conversion (RT-QuIC) assay positivity in the cerebrospinal fluid (CSF) or in other tissues. The total CSF tau protein concentration has also been used in a clinical setting for improving the CJD diagnostic sensitivity and specificity. We analyzed 182 CSF samples and 42 olfactory mucosa (OM) brushings from patients suspected of having sCJD with rapidly progressive dementia (RPD), in order to determine the diagnostic accuracy of 14-3-3, the total tau protein, and the RT-QuIC assay. A probable and definite sCJD diagnosis was assessed in 102 patients. The RT-QuIC assay on the CSF samples showed a 100% specificity and a 96% sensitivity, significantly higher compared with 14-3-3 (84% sensitivity and 46% specificity) and tau (85% sensitivity and 70% specificity); however, the combination of RT-QuIC testing of the CSF and OM samples resulted in 100% sensitivity and specificity, proving a significantly higher accuracy of RT-QuIC compared with the surrogate biomarkers in the diagnostic setting of patients with RPD. Moreover, we showed that CSF blood contamination or high protein levels might interfere with RT-QuIC seeding. In conclusion, we provided further evidence that the inclusion of an RT-QuIC assay of the CSF and OM in the diagnostic criteria for sCJD has radically changed the clinical approach towards the diagnosis

Neurodegeneration-associated proteins in human olfactory neurons collected by nasal brushing

The olfactory neuroepithelium is located in the upper vault of the nasal cavity, lying on the olfactory cleft and projecting into the dorsal portion of the superior and middle turbinates beyond the mid-portion of the nasal septum. It is composed of a variety of cell types including olfactory sensory neurons, supporting glial-like cells, microvillar cells, and basal stem cells. The cells of the neuroepithelium are often intermingled with respiratory and metaplastic epithelial cells. Olfactory neurons undergo a constant self-renewal in the timespan of 2\u20133 months; they are directly exposed to the external environment, and thus they are vulnerable to physical and chemical injuries. The latter might induce metabolic perturbations and ultimately be the cause of cell death. However, the lifespan of olfactory neurons is biologically programmed, and for this reason, these cells have an accelerated metabolic cycle leading to an irreversible apoptosis. These characteristics make these cells suitable for research related to nerve cell degeneration and aging. Recent studies have shown that a non-invasive and painless olfactory brushing procedure allows an efficient sampling from the olfactory neuroepithelium. This approach allows to detect the pathologic prion protein in patients with sporadic Creutzfeldt\u2013Jakob disease, using the real-time quaking-induced conversion assay. Investigating the expression of all the proteins associated to neurodegeneration in the cells of the olfactory mucosa is a novel approach toward understanding the pathogenesis of human neurodegenerative diseases. Our aim was to investigate the expression of \u3b1-synuclein, \u3b2-amyloid, tau, and TDP-43 in the olfactory neurons of normal subjects. We showed that these proteins that are involved in neurodegenerative diseases are expressed in olfactory neurons. These findings raise the question on whether a relationship exists between the mechanisms of protein aggregation that occur in the olfactory bulb during the early stage of the neurodegenerative process and the protein misfolding occurring in the olfactory neuroepithelium

Biochemical and neuropathological findings in a Creutzfeldt-Jakob Disease patient with the rare Val180Ile-129Val haplotype in the prion protein gene

Genetic Creutzfeldt-Jakob disease (gCJD) associated with the V180I mutation in the prion protein (PrP) gene (PRNP) in phase with residue 129M is the most frequent cause of gCJD in East Asia, whereas it is quite uncommon in Caucasians. We report on a gCJD patient with the rare V180I-129V haplotype, showing an unusually long duration of the disease and a characteristic pathological PrP (PrPSc) glycotype. Family members carrying the mutation were fully asymptomatic, as commonly observed with this mutation. Neuropathological examination showed a lesion pattern corresponding to that commonly reported in Japanese V180I cases with vacuolization and gliosis of the cerebral cortexes, olfactory areas, hippocampus and amygdala. PrP was deposited with a punctate, synaptic-like pattern in the cerebral cortex, amygdala and olfactory tract. Western blot analyses of proteinase-K-resistant PrP showed the characteristic two-banding pattern of V180I gCJD, composed of mono- and un-glycosylated isoforms. In line with reports on other V180I cases in the literature, Real-Time Quaking Induced Conversion (RT-QuIC) analyses did not demonstrate the presence of seeding activity in the cerebrospinal fluid and olfactory mucosa, suggesting that this haplotype also may result in a reduced seeding efficiency of the pathological PrP. Further studies are required to understand the origin, penetrance, disease phenotype and transmissibility of 180I-129V haplotype in Caucasians

Detection of TDP-43 seeding activity in the olfactory mucosa from patients with frontotemporal dementia

Introduction: We assessed TAR DNA-binding protein 43 (TDP-43) seeding activity and aggregates detection in olfactory mucosa of patients with frontotemporal lobar degeneration with TDP-43-immunoreactive pathology (FTLD-TDP) by TDP-43 seeding amplification assay (TDP43-SAA) and immunocytochemical analysis. Methods: The TDP43-SAA was optimized using frontal cortex samples from 16 post mortem cases with FTLD-TDP, FTLD with tau inclusions, and controls. Subsequently, olfactory mucosa samples were collected from 17 patients with FTLD-TDP, 15 healthy controls, and three patients carrying MAPT variants. Results: TDP43-SAA discriminated with 100% accuracy post mortem cases presenting or lacking TDP-43 neuropathology. TDP-43 seeding activity was detectable in the olfactory mucosa, and 82.4% of patients with FTLD-TDP tested positive, whereas 86.7% of controls tested negative (P < 0.001). Two out of three patients with MAPT mutations tested negative. In TDP43-SAA positive samples, cytoplasmatic deposits of phosphorylated TDP-43 in the olfactory neural cells were detected. Discussion: TDP-43 aggregates can be detectable in olfactory mucosa, suggesting that TDP43-SAA might be useful for identifying and monitoring FTLD-TDP in living patients

Detection of TDP-43 seeding activity in the olfactory mucosa from patients with frontotemporal dementia

INTRODUCTION: We assessed TAR DNA-binding protein 43 (TDP-43) seeding activity and aggregates detection in olfactory mucosa of patients with frontotemporal lobar degeneration with TDP-43-immunoreactive pathology (FTLD-TDP) by TDP-43 seeding amplification assay (TDP43-SAA) and immunocytochemical analysis. METHODS: The TDP43-SAA was optimized using frontal cortex samples from 16 post mortem cases with FTLD-TDP, FTLD with tau inclusions, and controls. Subsequently, olfactory mucosa samples were collected from 17 patients with FTLD-TDP, 15 healthy controls, and three patients carrying MAPT variants. RESULTS: TDP43-SAA discriminated with 100% accuracy post mortem cases presenting or lacking TDP-43 neuropathology. TDP-43 seeding activity was detectable in the olfactory mucosa, and 82.4% of patients with FTLD-TDP tested positive, whereas 86.7% of controls tested negative (P < 0.001). Two out of three patients with MAPT mutations tested negative. In TDP43-SAA positive samples, cytoplasmatic deposits of phosphorylated TDP-43 in the olfactory neural cells were detected. DISCUSSION: TDP-43 aggregates can be detectable in olfactory mucosa, suggesting that TDP43-SAA might be useful for identifying and monitoring FTLD-TDP in living patients

PMCA-based detection of prions in the olfactory mucosa of patients with Sporadic Creutzfeldt-Jakob Disease

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder caused by the conformational conversion of the prion protein (PrPC) into an abnormally folded form, named prion (or PrPSc). The combination of the polymorphism at codon 129 of the PrP gene (coding either methionine or valine) with the biochemical feature of the proteinase-K resistant PrP (generating either PrPSc type 1 or 2) gives rise to different PrPSc strains, which cause variable phenotypes of sCJD. The definitive diagnosis of sCJD and its classification can be achieved only post-mortem after PrPSc identification and characterization in the brain. By exploiting the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, traces of PrPSc were found in the olfactory mucosa (OM) of sCJD patients, thus demonstrating that PrPSc is not confined to the brain. Here, we have optimized another technique, named protein misfolding cyclic amplification (PMCA) for detecting PrPSc in OM samples of sCJD patients. OM samples were collected from 27 sCJD and 2 genetic CJD patients (E200K). Samples from 34 patients with other neurodegenerative disorders were included as controls. Brains were collected from 26 sCJD patients and 16 of them underwent OM collection. Brain and OM samples were subjected to PMCA using the brains of transgenic mice expressing human PrPC with methionine at codon 129 as reaction substrates. The amplified products were analyzed by Western blot after proteinase K digestion. Quantitative PMCA was performed to estimate PrPSc concentration in OM. PMCA enabled the detection of prions in OM samples with 79.3% sensitivity and 100% specificity. Except for a few cases, a predominant type 1 PrPSc was generated, regardless of the tissues analyzed. Notably, all amplified PrPSc were less resistant to PK compared to the original strain. In conclusion, although the optimized PMCA did not consent to recognize sCJD subtypes from the analysis of OM collected from living patients, it enabled us to estimate for the first time the amount of prions accumulating in this biological tissue. Further assay optimizations are needed to faithfully amplify peripheral prions whose recognition could lead to a better diagnosis and selection of patients for future clinical trials

Are Cerebrospinal Fluid Biomarkers Useful in Predicting the Prognosis of Multiple Sclerosis Patients?

Multiple sclerosis (MS) is the prototypical inflammatory demyelinating disorder of the central nervous system (CNS). Although many advances have been made in the comprehension of its pathogenesis, the etiology is still unknown. The complexity of MS reflects in the extreme variability of the clinical manifestations and clinical course both between and within patients, in addition to immunopathological mechanisms and response to treatment. Several prognostic factors have been suggested in large scale studies, but predictions in individual cases are difficult to make. Cerebrospinal fluid (CSF) biomarkers, such as 14-3-3, tau, and cystatin C are promising sources of prognostic information with a good potential of quantitative measure, sensitivity, and reliability. However, none has shown sufficient reproducibility to be applied in clinical practice. Here we review the current literature addressing the above mentioned biomarkers as MS severity predictors at an early stage

Detection of pathological prion protein by RT-QuIC analysis of cerebrospinal fluid and olfactory neuroepithelium of patients with sporadic Creutzfeldt-Jakob disease

ITALIAN SUMMARY PREMESSA: La diagnosi definitiva della forma sporadica di Creutzfeldt-Jakob (sCJD) in pazienti ancora in vita rimane una sfida. La Real time quaking-induced conversion (RT-QuIC) testata nel fluido cerebro- spinale (CSF) ha permesso l\u2019identificazione di pazienti sCJD con una sensibilit\ue0 del 80-90%. Poich\ue8 la CJD \ue8 una malattia trasmissibile, non trattabile e fatale, \ue8 importante effettuare una diagnosi corretta. Uno studio precedente ha individuato la presenza della proteina prionica patologica (PrP) nell\u2019 epitelio olfattivo di pazienti sCJD. Questo studio ci ha spinto ha verificare se l\u2019impiego dell\u2019RT-QuIC utilizzando campioni di brushings nasale pu\uf2 migliorarne la diagnosi pre-mortem nei casi di sCJD. METODI: Abbiamo testato i brushings di neuroepitelio olfattivo da pazienti con diagnosi di sospetta sCJD e non-CJD utilizzando l\u2019RT-QuIC. L\u2019RT-QuIC \ue8 una tecnica di amplificazione in vitro che utilizza una proteina prionica ricombinante (rPrPc) come substrato per la PrP patologica. Il substrato viene convertito in fibrille amiloidee che possono essere monitorate in tempo reale utilizzando il marcatore fluorescente thioflavina T (ThT). RISULTATI: Abbiamo osservato una forte positivit\ue0 all\u2019RT-QuIC in 7 campioni su 7 di brushings nasale di pazienti con probabile sCJD, e non negli 11 controlli negativi. Questo risultato ha determinato una sensibilit\ue0 e una specificit\ue0 del 100%. In confronto abbiamo osservato che 6 campioni su 7 di CSF appartenenti allo stesso gruppo di pazienti sCJD erano positivi all\u2019RT-QuIC, determinando quindi una sensibilit\ue0 dell\u201986%. Inoltre la quantificazione con RT-QuIC dei campioni di brushing nasali ha mostrato che contengono un\u2019 attivit\ue0 di seeding di ~105-107. CONCLUSIONI: L\u2019 utilizzo dei campioni di brushings nasale insieme al test RT-QuIC potrebbero facilitare e rafforzare la diagnosi pre-mortem di sCJD. Inoltre, gli alti livelli di attivit\ue0 di seeding della proteina prionica trovati in questi campioni suggeriscono un alto grado di trasmissibilit\ue0 del prione attraverso la mucosa olfattiva.ABSTRACT BACKGROUND: Definitive diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) in living patients remains a challenge. Real time quaking-induced conversion (RT-QuIC) testing of cerebrospinal fluid (CSF) has allowed identification of sCJD patients with 80-90% sensitivity. However, because CJD is transmissible, untreatable and fatal, it is important to eliminate missed diagnoses. Previous work identified abnormal prion protein (PrP) in olfactory neuroepithelium of sCJD patients, prompting us to investigate whether RT-QuIC analysis of easily accessible nasal brushings might improve sCJD diagnosis. METHODS: We tested olfactory neuroepithelium brushings from sCJD and non-CJD patients using RT-QuIC, which is an ultrasensitive, multi-well plate-based fluorescence assay involving prion-seeded polymerization of recombinant PrP into amyloid fibrils. RESULTS: We observed strong positive RT-QuIC reactions seeded with nasal brushings from 7 of 7 probable sCJD patients, but none of 11 negative controls, providing 100% sensitivity and 100% specificity. By comparison, 6 out of 7 CSF samples from the same group of sCJD patients was RT-QuIC-positive, giving 86% sensitivity. Quantitative RT-QuIC showed that olfactory brushings contained ~105-107 prion seeds. CONCLUSIONS: Nasal brushing-based RT-QuIC may markedly facilitate and strengthen diagnosis of sCJD. Moreover, the high levels of prion seeding activity found in these samples raises concerns about transmissible sCJD prion shedding from olfactory mucosa