Detection of Prions

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

1) Human prion diseases have long incubation times followed typically by rapid clinical decline. RT-QuIC seeding assays and neurodegeneration biofluid biomarkers are very useful in the clinical phase, but their potential to predict clinical onset in healthy people remains unclear. Therefore, we aided a collaborative study of the accrual of a longitudinal biofluid resource in patients, controls and healthy people at risk of prion diseases, to which ultrasensitive techniques such as RT-QuIC and Simoa digital immunoassays were applied for preclinical biomarker discovery. We studied 648 CSF and plasma samples, including 16 people who had samples taken when healthy but later developed inherited prion disease (IPD) (converters; range from 9.9 prior to, and 7.4 years after onset). Symptomatic IPD CSF samples were screened by RT-QuIC assay variations before testing the entire collection of at-risk samples using the most sensitive assay. Glial fibrillary acidic protein (GFAP), neurofilament light (NfL), tau and UCH-L1 levels were measured in plasma and CSF. Our IQ-CSF RT-QuIC proved 100% sensitive and specific for sporadic Creutzfeldt-Jakob disease (sCJD), iatrogenic and familial CJD phenotypes, and subsequently detected seeding activity in four presymptomatic CSF samples from 3 E200K carriers; one converted in under 2 months while two remain asymptomatic after at least 3 years follow-up. A bespoke HuPrP P102L RT-QuIC showed partial sensitivity for P102L disease. Plasma GFAP and NfL, and CSF NfL levels emerged as proximity markers of neurodegeneration in the typically slow IPDs (e.g. P102L), with significant differences in mean values segregating healthy control from IPD carriers (within 2 years to onset) and symptomatic IPD cohorts; plasma GFAP appears to change before NfL, and before clinical conversion. In conclusion, we showed distinct biomarker trajectories in fast and slow IPDs. Specifically, we identify several years of presymptomatic seeding positivity in E200K, a new proximity marker (plasma GFAP) and sequential neurodegenerative marker evolution (plasma GFAP followed by NfL) in slow IPDs. We suggest a preclinical staging system featuring clinical, seeding and neurodegeneration aspects for validation with larger prion at-risk cohorts, and with potential application to other neurodegenerative proteopathies. 2) Amyloid kuru plaques are a pathological hallmark of sporadic (s) CJD of the MV2K subtype. Recently, PrP plaques (p) have been described in the white matter of a small group of CJD (p-CJD) cases with the 129MM genotype and carrying resPrPD type 1 (T1). Despite the different histopathological phenotype, the gel mobility and molecular features of p-CJD resPrPD T1 mimic those of sCJDMM1, the most common human prion disease. Our collaborative study described the clinical features, histopathology, and molecular properties of two distinct PrP plaque phenotypes affecting the gray matter (pGM) or the white matter (pWM) of sCJD cases with the PrP 129MM genotype (sCJDMM). Prevalence of pGM- and pWM-CJD was estimated to be 0.6% among sporadic prion diseases and 1.1% among the sCJDMM group. PrP plaques were mostly confined to the cerebellar cortex in pGM-CJD, but were ubiquitous in pWM-CJD. Typing of resPrPD T1 showed an unglycosylated fragment of 20 kDa (T120) in pGM-CJD and sCJDMM1 patients, while a doublet of 21-20 kDa (T121-20) was a molecular signature of pWM-CJD in subcortical regions. In addition, conformational characteristics of pWM-CJD resPrPD T1 differed from those of pGM-CJD and sCJDMM1. Inoculation of pWM-CJD and sCJDMM1 brain extracts to transgenic mice expressing human PrP reproduced the histotype with PrP plaques only in mice challenged with pWM-CJD. Furthermore, T120 of pWM-CJD, but not T121, was propagated in mice. These data suggest that T121 and T120 of pWM-CJD, and T120 of sCJDMM1 are distinct prion strains. 3) Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are distinct clinicopathological subtypes of frontotemporal lobar degeneration. They both have atypical parkinsonism, and they usually have distinct clinical features. The most common clinical presentation of PSP is Richardson syndrome, and the most common presentation of CBD is corticobasal syndrome. We participated in describing a patient with a 5-y history of Richardson syndrome and a family history of PSP in her mother and sister. A tau PET scan revealed low-to-moderate uptake in the substantia nigra, globus pallidus, thalamus and posterior cortical areas, including temporal, parietal and occipital cortices. Neuropathological evaluation revealed widespread neuronal and glial tau pathology in cortical and subcortical structures, including tufted astrocytes in the motor cortex, striatum and midbrain tegmentum. The subthalamic nucleus had mild-to-moderate neuronal loss with globose neurofibrillary tangles, consistent with PSP. On the other hand, there were also astrocytic plaques, a pathological hallmark of CBD, in the neocortex and striatum. To further characterize the mixed pathology, we applied two machine learning-based diagnostic pipelines. These models suggested diagnoses of PSP and CBD depending on the brain region, i.e., PSP in the motor cortex and superior frontal gyrus and CBD in caudate nucleus. Western blots of insoluble tau from motor cortex showed a banding pattern consistent with mixed features of PSP and CBD, whereas tau from the superior frontal gyrus showed a pattern consistent with CBD. RT-QuIC using brain homogenates from the motor cortex and superior frontal gyrus showed ThT maxima consistent with PSP, while reaction kinetics were consistent with CBD. There were no pathogenic variants in MAPT with whole genome sequencing. We concluded that this patient had an unclassified tauopathy and features of both PSP and CBD. 4) Misfolded -Synuclein deposition is a hallmark of Parkinsons disease. The GI tract may be an initial site of a-synuclein aggregation, and its detection might be useful in the early diagnosis of Parkinsons disease. We used a rapid, ultrasensitive seed amplification assay (RT-QuICR) to show that pathologic -Syn aggregates with prion-like self-propagating activity are in the upper intestine (duodenum) of Parkinsons disease patients. Our intra vitam detection of a-synuclein seeding activity in duodenum biopsies gave high diagnostic accuracy. Quantitation revealed high levels of seeds in duodenal tissue. Thus, our findings suggest that a-synuclein seeds in the upper intestine might be both an early accurate biomarker for Parkinsons disease and cause of gut dysfunction.