Microglia deficiency accelerates prion disease but does not enhance prion accumulation in the brain.

Prion diseases are transmissible, neurodegenerative disorders associated with misfolding of the prion protein. Previous studies show that reduction of microglia accelerates central nervous system (CNS) prion disease and increases the accumulation of prions in the brain, suggesting that microglia provide neuroprotection by phagocytosing and destroying prions. In Csf1rΔFIRE mice, the deletion of an enhancer within Csf1r specifically blocks microglia development, however, their brains develop normally and show none of the deficits reported in other microglia-deficient models. Csf1rΔFIRE mice were used as a refined model in which to study the impact of microglia-deficiency on CNS prion disease. Although Csf1rΔFIRE mice succumbed to CNS prion disease much earlier than wild-type mice, the accumulation of prions in their brains was reduced. Instead, astrocytes displayed earlier, non-polarized reactive activation with enhanced phagocytosis of neuronal contents and unfolded protein responses. Our data suggest that rather than simply phagocytosing and destroying prions, the microglia instead provide host-protection during CNS prion disease and restrict the harmful activities of reactive astrocytes.

Cerebrospinal fluid real-time quaking-induced conversion is a robust and reliable test for sporadic creutzfeldt-jakob disease: An international study.

Real-time quaking-induced conversion (RT-QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt-Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT-QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160-165.

Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease.

OBJECTIVE: Current cerebrospinal fluid (CSF) tests for sporadic Creutzfeldt-Jakob disease (sCJD) are based on the detection of surrogate markers of neuronal damage such as CSF 14-3-3, which are not specific for sCJD. A number of prion protein conversion assays have been developed, including real time quaking-induced conversion (RT-QuIC). The objective of this study is to investigate whether CSF RT-QuIC analysis could be used as a diagnostic test in sCJD. METHODS: An exploratory study was undertaken that analyzed 108 CSF samples from patients with neuropathologically confirmed sCJD or from control patients. Of the 108 CSF samples, 56 were from sCJD patients (30 female, 26 male; aged 31-84 years; mean age, 62.3 ± 13.5 years), and 52 were from control patients (26 female, 26 male; aged 43-84 years; mean age, 67.8 ± 10.4 years). A confirmatory group of 118 patients was subsequently examined that consisted of 67 cases of neuropathologically confirmed sCJD (33 female, 34 male; aged 39-82 years; mean age, 67.5 ± 9.0 years) and 51 control cases (26 female, 25 male; aged 36-87 years; mean age, 63.5 ± 11.6 years). RESULTS: The exploratory study showed that RT-QuIC analysis had a sensitivity of 91% and a specificity of 98% for the diagnosis of sCJD. These results were confirmed in the confirmatory study, which showed that CSF RT-QuIC analysis had a sensitivity and specificity of 87% and 100%, respectively. INTERPRETATION: This study shows that CSF RT-QuIC analysis has the potential to be a more specific diagnostic test for sCJD than current CSF tests.

Conservation of vCJD Strain Properties After Extraction and In Vitro Propagation of PrPSc from Archived Formalin-Fixed Brain and Appendix Tissues Using Highly Sensitive Protein Misfolding Cyclic Amplification.

Three retrospective lymphoreticular tissue studies (Appendix I, II, and III) aimed to estimate the UK prevalence of variant Creutzfeldt-Jakob disease (vCJD), following exposure of the population to the bovine spongiform encephalopathy (BSE) agent, in the late 1980s and 1990s. These studies evaluated the presence of abnormal prion protein aggregates, in archived formalin-fixed paraffin-embedded (FFPE) appendectomy samples, by immunohistochemical detection. Although there was concordance in the estimated prevalence of vCJD from these studies, the identification of positive specimens from pre- and post-BSE-exposure periods in Appendix III study has raised questions regarding the nature and origin of the detected abnormal prion protein. We applied a robust and novel approach in the extraction of disease-associated prion protein (PrPSc) present in frozen and FFPE samples of brain and appendix from a patient with pathologically confirmed vCJD. The extracted material was used to seed the highly sensitive protein misfolding cyclic amplification assay (hsPMCA) to investigate the in vitro and in vivo propagation properties of the extracted abnormal prion protein. We demonstrate that PrPSc can be successfully extracted from FFPE appendix tissue and propagated in vitro. Bioassay in wild-type and gene-targeted mouse models confirmed that the extracted and amplified product is infectious and retains strain properties consistent with vCJD. This provides a highly sensitive and reliable platform for subsequent analysis of the archived FFPE appendix tissue derived from the Appendix II and III surveys, to further evaluate the nature of the abnormal PrP detected in the positive samples.

Sensitive and specific detection of sporadic Creutzfeldt-Jakob disease brain prion protein using real-time quaking-induced conversion.

Real-time quaking-induced conversion (RT-QuIC) is an assay in which disease-associated prion protein (PrP) initiates a rapid conformational transition in recombinant PrP (recPrP), resulting in the formation of amyloid that can be monitored in real time using the dye thioflavin T. It therefore has potential advantages over analogous cell-free PrP conversion assays such as protein misfolding cyclic amplification (PMCA). The QuIC assay and the related amyloid seeding assay have been developed largely using rodent-passaged sheep scrapie strains. Given the potential RT-QuIC has for Creutzfeldt-Jakob disease (CJD) research and human prion test development, this study characterized the behaviour of a range of CJD brain specimens with hamster and human recPrP in the RT-QuIC assay. The results showed that RT-QuIC is a rapid, sensitive and specific test for the form of abnormal PrP found in the most commonly occurring forms of sporadic CJD. The assay appeared to be largely independent of species-related sequence differences between human and hamster recPrP and of the methionine/valine polymorphism at codon 129 of the human PrP gene. However, with the same conditions and substrate, the assay was less efficient in detecting the abnormal PrP that characterizes variant CJD brain. Comparison of these QuIC results with those previously obtained using PMCA suggested that these two seemingly similar assays differ in important respects.

Alpha-synuclein RT-QuIC in the CSF of patients with alpha-synucleinopathies.

We have developed a novel real-time quaking-induced conversion RT-QuIC-based assay to detect alpha-synuclein aggregation in brain and cerebrospinal fluid from dementia with Lewy bodies and Parkinson's disease patients. This assay can detect alpha-synuclein aggregation in Dementia with Lewy bodies and Parkinson's disease cerebrospinal fluid with sensitivities of 92% and 95%, respectively, and with an overall specificity of 100% when compared to Alzheimer and control cerebrospinal fluid. Patients with neuropathologically confirmed tauopathies (progressive supranuclear palsy; corticobasal degeneration) gave negative results. These results suggest that RT-QuiC analysis of cerebrospinal fluid is potentially useful for the early clinical assessment of patients with alpha-synucleinopathies.