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An early and accurate in vivo diagnosis of rapidly progressive dementia remains challenging, despite its critical importance for the outcome of treatable forms, and the formulation of prognosis. Real-Time Quaking-Induced Conversion (RT-QuIC) is an in vitro assay that, for the first time, specifically discriminates patients with prion disease. Here, using cerebrospinal fluid (CSF) samples from 239 patients with definite or probable prion disease and 100 patients with a definite alternative diagnosis, we compared the performance of the first (PQ-CSF) and second generation (IQ-CSF) RT-QuIC assays, and investigated the diagnostic value of IQ-CSF across the broad spectrum of human prions. Our results confirm the high sensitivity of IQ-CSF for detecting human prions with a sub-optimal sensitivity for the sporadic CJD subtypes MM2C and MM2T, and a low sensitivity limited to variant CJD, Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia. While we found no difference in specificity between PQ-CSF and IQ-CSF, the latter showed a significant improvement in sensitivity, allowing prion detection in about 80% of PQ-CSF negative CJD samples. Our results strongly support the implementation of IQ-CSF in clinical practice. By rapidly confirming or excluding CJD with high accuracy the assay is expected to improve the outcome for patients and their enrollment in therapeutic trials.

The cellular prion protein (PrPC) is studied in prion diseases, where its misfolded isoform (PrPSc) leads to neurodegeneration. PrPC has also been implicated in several physiological functions. The protein is abundant in the nervous system, and it is critical for cell signaling in cellular communication, where it acts as a scaffold for various signaling molecules. The Reelin signaling pathway, implicated both in Alzheimer’s and prion diseases, engages Dab1, an adaptor protein influencing APP processing and amyloid beta deposition. Here, we show, using Prnp knockout models (Prnp0/0), that PrPC modulates Reelin signaling, affecting Dab1 activation and downstream phosphorylation in both neuronal cultures and mouse brains. Notably, Prnp0/0 mice showed reduced responsiveness to Reelin, associated with altered Dab1 phosphorylation and Fyn kinase activity. Even though no direct interaction between PrPC and Reelin/ApoER2 was found, Prnp0/0 neurons showed lower NCAM levels, a well-established PrPC interactor. Prion infection further disrupted the Reelin signaling pathway, thus downregulating Dab1 and Reelin receptors and altering Reelin processing, like Alzheimer’s disease pathology. These findings emphasize PrPC indirect role in Dab1 signaling via the NCAM and Fyn pathways, which influence synaptic function and neurodegeneration in prion diseases.

The diagnosis of prion-associated diseases such as variant Creutzfeldt–Jakob disease remains difficult. In this study, cases of variant Creutzfeldt–Jakob disease were identified by means of a technique that amplifies minute quantities of the prion protein in urine. Prion diseases are fatal neurodegenerative disorders for which no therapy or definitive noninvasive intravital diagnosis is available. 1 These diseases affect humans and animals. Creutzfeldt–Jakob disease in humans and scrapie, bovine spongiform encephalopathy, and chronic wasting disease in animals are the most common forms of transmissible spongiform encephalopathies. The infectious agent in transmissible spongiform encephalopathies appears to be composed exclusively of the misfolded form of the prion protein (termed PrPSc), which self-propagates in the absence of nucleic acid. 2 , 3 PrPSc replicates in infected persons by acting as a template for the misfolding of the cellular prion protein (PrPC). When . . .

Serpins represent the most broadly distributed superfamily of proteases inhibitors. They contribute to a variety of physiological functions and any alteration of the serpin-protease equilibrium can lead to severe consequences. SERPINA3 dysregulation has been associated with Alzheimer’s disease (AD) and prion diseases. In this study, we investigated the differential expression of serpin superfamily members in neurodegenerative diseases. SERPIN expression was analyzed in human frontal cortex samples from cases of sporadic Creutzfeldt-Jakob disease (sCJD), patients at early stages of AD–related pathology, and age-matched controls not affected by neurodegenerative disorders. In addition, we studied whether Serpin expression was dysregulated in two animal models of prion disease and AD. Our analysis revealed that, besides the already observed upregulation of SERPINA3 in patients with prion disease and AD, SERPINB1 , SERPINB6 , SERPING1 , SERPINH1 , and SERPINI1 were dysregulated in sCJD individuals compared to controls, while only SERPINB1 was upregulated in AD patients. Furthermore, we analyzed whether other serpin members were differentially expressed in prion-infected mice compared to controls and, together with SerpinA3n , SerpinF2 increased levels were observed. Interestingly, SerpinA3n transcript and protein were upregulated in a mouse model of AD. The SERPINA3/SerpinA3nincreased anti-protease activity found in post-mortem brain tissue of AD and prion disease samples suggest its involvement in the neurodegenerative processes. A SERPINA3/SerpinA3n role in neurodegenerative disease-related protein aggregation was further corroborated by in vitro SerpinA3n-dependent prion accumulation changes. Our results indicate SERPINA3/SerpinA3n is a potential therapeutic target for the treatment of prion and prion-like neurodegenerative diseases.

Prion diseases are neurodegenerative and invariably fatal conditions that affect humans and animals. In particular, Creutzfeldt-Jakob disease (CJD) and bovine spongiform encephalopathy (BSE) are paradigmatic forms of human and animal prion diseases, respectively. Human exposure to BSE through contaminated food caused the appearance of the new variant form of CJD (vCJD). These diseases are caused by an abnormal prion protein named PrPSc (or prion), which accumulates in the brain and leads to the onset of the disease. Their definite diagnosis can be formulated only at post-mortem after biochemical and neuropathological identification of PrPSc. Thanks to the advent of an innovative technique named protein misfolding cyclic amplification (PMCA), traces of PrPSc, undetectable with the standard diagnostic techniques, were found in peripheral tissues of patients with vCJD, even at preclinical stages. The technology is currently being used in specialized laboratories and can be exploited for helping physicians in formulating an early and definite diagnosis of vCJD using peripheral tissues. However, this assay is currently unable to detect prions associated with the sporadic CJD (sCJD) forms, which are more frequent than vCJD. This review will focus on the most recent advances and applications of PMCA in the field of vCJD and other human prion disease diagnosis.

Prion diseases are fatal neurodegenerative disorders affecting several mammalian species, characterized by the accumulation of the misfolded form of the prion protein, which is followed by the induction of endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). GRP78, also called BiP, is a master regulator of the UPR, reducing ER stress levels and apoptosis due to an enhancement of the cellular folding capacity. Here, we studied the role of GRP78 in prion diseases using several in vivo and in vitro approaches. Our results show that a reduction in the expression of this molecular chaperone accelerates prion pathogenesis in vivo . In addition, we observed that prion replication in cell culture was inversely related to the levels of expression of GRP78 and that both proteins interact in the cellular context. Finally, incubation of PrP Sc with recombinant GRP78 led to the dose-dependent reduction of protease-resistant PrP Sc in vitro . Our results uncover a novel role of GRP78 in reducing prion pathogenesis, suggesting that modulating its levels/activity may offer a novel opportunity for designing therapeutic approaches for these diseases. These findings may also have implications for other diseases involving the accumulation of misfolded proteins.

Prion diseases are neurodegenerative disorders caused by a change in conformation of the prion protein from the cellular form (PrP C ) to a misfolded isoform (PrP Sc ). PrP C is a copper binding protein via histidine residues in the octapeptide repeats (OR) and the non-OR region located at the N-terminus. Although the functional implication of copper binding to PrP C is still under investigation, copper may play a role in prion disease. In this study, we describe transgenic mice expressing mouse prion protein replacing histidine 95 by tyrosine (PrP H95Y) to disrupt the non-OR copper-binding site. Transgenic mice overexpressing PrP H95Y showed clinical signs and died at about 100 days with spongiform degeneration and PK-resistant PrP. Inoculation of brain homogenate from mice overexpressing PrP H95Y to Tga20 mice expressing wild-type PrP also causes lethal, spongiform encephalopathy. We conclude that this substitution could promote PrP C -PrP Sc conversion and induce spontaneous prion disease in vivo .

Chronic wasting disease (CWD) is an emerging prion disease in Nordic countries and has been detected in reindeer, moose, and red deer since 2016. CWD sporadically detected in moose and red deer in 3 Nordic countries demonstrated pathologic and strain characteristics different from CWD in reindeer, including an unexpected lack of prions outside the central nervous system as measured by standard diagnostic tests. Using protein misfolding cyclic amplification, we detected prions in the lymphoreticular system of moose and red deer with CWD in Norway and, remarkably, in muscles of both of those species and in CWD-infected reindeer. One moose lymph node and 1 moose muscle sample showed infectivity when experimentally transmitted to bank voles. Our findings highlight the systemic nature of CWD strains in Europe and raise questions regarding the risk of human exposure through edible tissues.

Background Parkinson’s disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal α-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either α-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Definite diagnosis of all these diseases can be formulated only neuropathologically by detection and localization of α-synuclein or tau aggregates in the brain. Compelling evidence suggests that trace-amount of these proteins can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM). Methods We have set and standardized the experimental conditions to extend the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for OM analysis. In particular, by using human recombinant α-synuclein as substrate of reaction, we have assessed the ability of OM collected from patients with clinical diagnoses of PD and MSA to induce α-synuclein aggregation, and compared their seeding ability to that of OM samples collected from patients with clinical diagnoses of CBD and PSP. Results Our results showed that a significant percentage of MSA and PD samples induced α-synuclein aggregation with high efficiency, but also few samples of patients with the clinical diagnosis of CBD and PSP caused the same effect. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples owned peculiar biochemical and morphological features potentially enabling their discrimination. Conclusions Our study provide the proof-of-concept that olfactory mucosa samples collected from patients with PD and MSA possess important seeding activities for α-synuclein. Additional studies are required for (i) estimating sensitivity and specificity of the technique and for (ii) evaluating its application for the diagnosis of PD and neurodegenerative parkinsonisms. RT-QuIC analyses of OM and cerebrospinal fluid (CSF) can be combined with the aim of increasing the overall diagnostic accuracy of these diseases, especially in the early stages.

Background Detection of the pathological and disease-associated alpha-synuclein (αSyn D ) in the brain is required to formulate the definitive diagnosis of multiple system atrophy (MSA) and Parkinson’s disease (PD). We recently showed that αSyn D can be detected in the olfactory mucosa (OM) of MSA and PD patients. For this reason, we have performed the first interlaboratory study based on α-synuclein Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC) analysis of OM samples collected from PD and MSA patients with the parkinsonian (MSA-P) and cerebellar (MSA-C) phenotypes. Methods OM samples were prospectively collected from patients with a probable diagnosis of MSA-P ( n  = 20, mean disease duration 4.4 years), MSA-C ( n  = 10, mean disease duration 4 years), PD ( n  = 13, mean disease duration 8 years), and healthy control subjects (HS) ( n  = 11). Each sample was analyzed by αSyn_RT-QuIC in two independent specialized laboratories, one located in Italy (ITA-lab) and one located in the USA (USA-lab). Both laboratories have developed and used harmonized αSyn_RT-QuIC analytical procedures. Results were correlated with demographic and clinical data. Results The αSyn_RT-QuIC analysis reached a 96% interrater agreement of results (IAR) between laboratories (Kappa = 0.93, 95% CI 0.83–1.00). In particular, αSyn_RT-QuIC seeding activity was found in the OM of 9/13 patients with PD (sensitivity 69%, IAR 100%) and 18/20 patients with MSA-P (sensitivity 90%, IAR 100%). Interestingly, samples collected from patients with MSA-C did not induce αSyn_RT-QuIC seeding activity, except for one subject in USA-lab. Therefore, we found that MSA-P and MSA-C induced opposite effects. Regardless of disease diagnosis, the αSyn_RT-QuIC seeding activity correlated with some clinical parameters, including the rigidity and postural instability. Conclusions Our study provides evidence that OM-αSyn D may serve as a novel biomarker for accurate clinical diagnoses of PD, MSA-P, and MSA-C. Moreover, αSyn_RT-QuIC represents a reliable assay that can distinguish patients with MSA-P from those with MSA-C, and may lead to significant advancements in patients stratification and selection for emerging pharmacological treatments and clinical trials.