Real-time quaking-induced conversion

We recently developed a new in vitro amplification technology, designated “real-time quaking-induced conversion (RT-QUIC)”, for detection of the abnormal form of prion protein (PrPSc) in easily accessible specimens such as cerebrospinal fluid (CSF). After assessment of more than 200 CSF specimens from Japanese and Australian patients, we found no instance of a false positive, and more than 80% accuracy for the correct diagnosis of sporadic Creutzfeldt–Jakob disease (sCJD). Furthermore, the RT-QUIC can be applied to other prion diseases, including scrapie, chronic wasting disease (CWD), and bovine spongiform encephalopathy (BSE), and is able to quantify prion seeding activity when combined with an end-point dilution of samples. These results indicate that the RT-QUIC, with its high sensitivity and specificity, will be of great use as an early, rapid and specific assay for prion diseases.     

Case Report: A patient with spinocerebellar ataxia type 31 and sporadic Creutzfeldt-Jakob disease

We report a Japanese patient with spinocerebellar ataxia type 31 (SCA31) and sporadic Creutzfeldt-Jakob disease (sCJD). A 52-year-old man developed progressive cognitive impairment after the appearance of cerebellar symptoms. Brain MR diffusion-weighted imaging (DWI) demonstrated a slowly expanding hyperintense lesion in the cerebral cortex. The patient was finally diagnosed as having both SCA31 and sCJD by identification of genetic mutations and by real-time quaking-induced conversion (RT-QUIC) analysis of the cerebrospinal fluid (CSF), respectively. Here, we report the clinical details of this rare combined case, with particular reference to the association between prion protein and the early onset of SCA31.     

Early Detection of Abnormal Prion Protein in Genetic Human Prion Diseases Now Possible Using Real-Time QUIC Assay

Introduction

The definitive diagnosis of genetic prion diseases (gPrD) requires pathological confirmation. To date, diagnosis has relied upon the finding of the biomarkers 14-3-3 protein and total tau (t-tau) protein in the cerebrospinal fluid (CSF), but many researchers have reported that these markers are not sufficiently elevated in gPrD, especially in Gerstmann-Sträussler-Scheinker syndrome (GSS). We recently developed a new in vitro amplification technology, designated “real-time quaking-induced conversion (RT-QUIC)”, to detect the abnormal form of prion protein in CSF from sporadic Creutzfeldt-Jakob disease (sCJD) patients. In the present study, we aimed to investigate the presence of biomarkers and evaluate RT-QUIC assay in patients with gPrD, as the utility of RT-QUIC as a diagnostic tool in gPrD has yet to be determined.

Method/Principal Findings

56 CSF samples were obtained from gPrD patients, including 20 cases of GSS with P102L mutation, 12 cases of fatal familial insomnia (FFI; D178N), and 24 cases of genetic CJD (gCJD), comprising 22 cases with E200K mutation and 2 with V203I mutation. We subjected all CSF samples to RT-QUIC assay, analyzed 14-3-3 protein by Western blotting, and measured t-tau protein using an ELISA kit. The detection sensitivities of RT-QUIC were as follows: GSS (78%), FFI (100%), gCJD E200K (87%), and gCJD V203I (100%). On the other hand the detection sensitivities of biomarkers were considerably lower: GSS (11%), FFI (0%), gCJD E200K (73%), and gCJD V203I (67%). Thus, RT-QUIC had a much higher detection sensitivity compared with testing for biomarkers, especially in patients with GSS and FFI.

Conclusion/Significance

RT-QUIC assay is more sensitive than testing for biomarkers in gPrD patients. RT-QUIC method would thus be useful as a diagnostic tool when the patient or the patient''s family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing.     

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

The development of technologies for the in vitro amplification of abnormal conformations of prion protein (PrP(Sc)) has generated the potential for sensitive detection of prions. Here we developed a new PrP(Sc) amplification assay, called real-time quaking-induced conversion (RT-QUIC), which allows the detection of ≥1 fg of PrP(Sc) in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate. Moreover, we assessed the technique first in a series of Japanese subjects and then in a blind study of 30 cerebrospinal fluid specimens from Australia, which achieved greater than 80% sensitivity and 100% specificity. These findings indicate the promising enhanced diagnostic capacity of RT-QUIC in the antemortem evaluation of suspected CJD.     

An autopsy-verified case of steroid-responsive encephalopathy with convulsion and a false-positive result from the real-time quaking-induced conversion assay

We report an autopsy-verified case of steroid-responsive encephalopathy with convulsion and a false-positive result from the real-time quaking-induced conversion (RT-QUIC) assay. A 61-year-old Japanese man presented with acute onset of consciousness disturbance, and convulsions, but without a past medical or family history of progressive dementia, epilepsy, or prion disease. Brain diffusion and fluid-attenuated inverted recovery MR images revealed edematous cortical hyper-intensity, which diminished after the acute phase. Steroid pulse therapy was partially effective, although he continued to have dementia with myoclonus and psychiatric symptoms, despite resolution of the consciousness disturbance. Cerebrospinal fluid (CSF) analysis revealed a normal cell count, with significantly elevated levels of 14–3–3 protein and total tau protein. In addition, prion protein in the CSF was slowly amplified by the RT-QUIC assay. PRNP gene analysis revealed methionine homozygosity at codon 129 without mutation. The patient died of sudden cardiac arrest at 3 months after the onset of symptoms.

The positive result from the RT-QUIC assay led us to suspect involvement of prion disease, although a postmortem assessment revealed that he had pathological changes after convulsion, and no prion disease. This case indicates that convulsion may cause false-positive RT-QUIC results, and that a postmortem evaluation remains the gold standard for diagnosing similar cases.     

Structural conservation of prion strain specificities in recombinant prion protein fibrils in real-time quaking-induced conversion

ABSTRACT

A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrP^Sc). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrP^Sc results in the formation of rPrP-fibrils seeded with various strains. We demonstrated that there are differences in the secondary structures, especially in the β-sheets, and conformational stability between 2 rPrP-fibrils seeded with either Chandler or 22L strains in the first round of RT-QUIC. In particular, the differences in conformational properties of these 2 rPrP-fibrils were common to those of the original PrP^Sc. However, the strain specificities of rPrP-fibrils seen in the first round were lost in subsequent rounds. Instead, our findings suggest that nonspecific fibrils became the major species, probable owing to their selective growth advantage in the RT-QUIC. This study shows that at least some strain-specific conformational properties of the original PrP^Sc can be transmitted to rPrP-fibrils in vitro, but further conservation appears to require unknown cofactors or environmental conditions or both.     

An autopsy-verified case of FTLD-TDP type A with upper motor neuron-predominant motor neuron disease mimicking MM2-thalamic-type sporadic Creutzfeldt-Jakob disease

Here we report an autopsy-verified case of frontotemporal lobar degeneration (FTLD)-transactivation responsive region (TAR) DNA binding protein (TDP) type A with upper motor neuron-predominant motor neuron disease mimicking MM2-thalamic-type sporadic Creutzfeldt-Jakob disease (sCJD). A 69-year-old woman presented with an 11-month history of progressive dementia, irritability, insomnia, and gait disturbance without a family history of dementia or prion disease. Neurological examination revealed severe dementia, frontal signs, and exaggerated bilateral tendon reflexes. Periodic sharp-wave complexes were not observed on the electroencephalogram. Brain diffusion MRI did not reveal abnormal changes. An easy Z score (eZIS) analysis for ^99mTc-ECD-single photon emission computed tomography (^99mTc-ECD-SPECT) revealed a bilateral decrease in thalamic regional cerebral blood flow (rCBF). PRNP gene analysis demonstrated methionine homozygosity at codon 129 without mutation. Cerebrospinal fluid (CSF) analysis showed normal levels of both 14-3-3 and total tau proteins. Conversely, prion protein was slowly amplified in the CSF by a real-time quaking-induced conversion assay. Her symptoms deteriorated to a state of akinetic mutism, and she died of sudden cardiac arrest, one year after symptom onset.

?Despite the SPECT results supporting a clinical diagnosis of MM2-thalamic-type sCJD, a postmortem assessment revealed that this was a case of FTLD-TDP type A, and excluded prion disease. Thus, this case indicates that whereas a bilateral decreasing thalamic rCBF detected by ^99mTc-ECD-SPECT can be useful for diagnosing MM2-thalamic-type sCJD, it is not sufficiently specific. Postmortem diagnosis remains the gold standard for the diagnosis of this condition.     

14-3-3 and enolase abundances in the CSF of Prion diseased rats

ABSTRACT

Creutzfeldt-Jakob disease (CJD) is characterized by an extended asymptomatic preclinical phase followed by rapid neurodegeneration. There are no effective treatments. CJD diagnosis is initially suspected based upon the clinical presentation of the disease and the exclusion of other etiologies. Neurologic symptoms are assessed in combination with results from cerebrospinal fluid (CSF) biomarker abundances, electroencephalography (EEG), magnetic resonance imaging (MRI), and in some countries, real-time quaking-induced conversion (RT-QuIC). Inconsistencies in sensitivities and specificities of prion disease biomarker abundance in CSF have been described, which can affect diagnostic certainty, but the utility of biomarkers for prognosis has not been fully explored. The clinical presentation of CJD is variable, and factors such as prion protein polymorphic variants, prion strain, and other genetic or environmental contributions may affect the disease progression, confounding the appearance or abundance of biomarkers in the CSF. These same factors may also affect the appearance or abundance of biomarkers, further confounding diagnosis. In this study, we controlled for many of these variables through the analysis of serial samples of CSF from prion-infected and control rats. Prion disease in laboratory rodents follows a defined disease course as the infection route and time, prion strain, genotype, and environmental conditions are all controlled. We measured the relative abundance of 14-3-3 and neuron-specific enolase (NSE) in CSF during the course of prion infection in rats. Even when disease-related, environmental and genetic variables were controlled, CSF 14-3-3 and NSE abundances were variable. Our study emphasizes the considerable diagnostic and prognostic limitations of these prion biomarkers.     

New generation QuIC assays for prion seeding activity

The ability of abnormal TSE-associated forms of PrP to seed the formation of amyloid fibrils from recombinant PrP^Sen has served as the basis for several relatively rapid and highly sensitive tests for prion diseases. These tests include rPrP-PMCA (rPMCA), standard quaking-induced conversion (S-QuIC), amyloid seeding assay (ASA), real-time QuIC (RT-QuIC) and enhanced QuIC (eQuIC). Here, we summarize recent improvements in the RT-QuIC-based assays that enhance the practicality, sensitivity and quantitative attributes of assays QuIC and promote the detection of prion seeding activity in dilute, inhibitor-laden fluids such as blood plasma.     

Re-Assessment of PrPSc Distribution in Sporadic and Variant CJD

Human prion diseases are fatal neurodegenerative disorders associated with an accumulation of PrP^Sc in the central nervous system (CNS). Of the human prion diseases, sporadic Creutzfeldt-Jakob disease (sCJD), which has no known origin, is the most common form while variant CJD (vCJD) is an acquired human prion disease reported to differ from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. Peripheral tissue involvement in prion disease, as judged by PrP^Sc accumulation in the tonsil, spleen, and lymph node has been reported in vCJD as well as several animal models of prion diseases. However, this distribution of PrP^Sc has not been consistently reported for sCJD. We reexamined CNS and non-CNS tissue distribution and levels of PrP^Sc in both sCJD and vCJD. Using a sensitive immunoassay, termed SOFIA, we also assessed PrP^Sc levels in human body fluids from sCJD as well as in vCJD-infected humanized transgenic mice (Tg666). Unexpectedly, the levels of PrP^Sc in non-CNS human tissues (spleens, lymph nodes, tonsils) from both sCJD and vCJD did not differ significantly and, as expected, were several logs lower than in the brain. Using protein misfolding cyclic amplification (PMCA) followed by SOFIA, PrP^Sc was detected in cerebrospinal fluid (CSF), but not in urine or blood, in sCJD patients. In addition, using PMCA and SOFIA, we demonstrated that blood from vCJD-infected Tg666 mice showing clinical disease contained prion disease-associated seeding activity although the data was not statistically significant likely due to the limited number of samples examined. These studies provide a comparison of PrP^Sc in sCJD vs. vCJD as well as analysis of body fluids. Further, these studies also provide circumstantial evidence that in human prion diseases, as in the animal prion diseases, a direct comparison and intraspecies correlation cannot be made between the levels of PrP^Sc and infectivity.     

Phosphatidylinositol-Glycan-Phospholipase D Is Involved in Neurodegeneration in Prion Disease

PrP^Sc is formed from a normal glycosylphosphatidylinositol (GPI)-anchored prion protein (PrP^C) by a posttranslational modification. Most GPI-anchored proteins have been shown to be cleaved by GPI phospholipases. Recently, GPI-phospholipase D (GPI-PLD) was shown to be a strictly specific enzyme for GPI anchors. To investigate the involvement of GPI-PLD in the processes of neurodegeneration in prion diseases, we examined the mRNA and protein expression levels of GPI-PLD in the brains of a prion animal model (scrapie), and in both the brains and cerebrospinal fluids (CSF) of sporadic and familial Creutzfeldt-Jakob disease (CJD) patients. We found that compared with controls, the expression of GPI-PLD was dramatically down-regulated in the brains of scrapie-infected mice, especially in the caveolin-enriched membrane fractions. Interestingly, the observed decrease in GPI-PLD expression levels began at the same time that PrP^Sc began to accumulate in the infected brains and this decrease was also observed in both the brain and CSF of CJD patients; however, no differences in expression were observed in either the brains or CSF specimens from Alzheimer’s disease patients. Taken together, these results suggest that the down-regulation of GPI-PLD protein may be involved in prion propagation in the brains of prion diseases.     

Relationships between Clinicopathological Features and Cerebrospinal Fluid Biomarkers in Japanese Patients with Genetic Prion Diseases

A national system for surveillance of prion diseases (PrDs) was established in Japan in April 1999. Here, we analyzed the relationships among prion protein gene (PRNP) mutations and the clinical features, cerebrospinal fluid (CSF) markers, and pathological characteristics of the major genotypes of genetic PrDs (gPrDs). We retrospectively analyzed age at onset and disease duration; the concentrations and incidences of 14-3-3 protein, tau protein, and abnormal prion protein (PrP^Sc) in the CSF of 309 gPrD patients with P102L, P105L, E200K, V180I, or M232R mutations; and brain pathology in 32 autopsied patients. Three clinical phenotypes were seen: rapidly progressive Creutzfeldt-Jakob disease (CJD), which included 100% of E200K cases, 70% of M232R, and 21% of P102L; slowly progressive CJD, which included 100% of V180I and 30% of M232R; and Gerstmann-Sträussler-Scheinker disease, which included 100% of P105L and 79% of P102L. PrP^Sc was detected in the CSF of more than 80% of patients with E200K, M232R, or P102L mutations but in only 39% of patients with V180I. V180I was accompanied by weak PrP immunoreactivity in the brain. Patients negative for PrP^Sc in the CSF were older at disease onset than positive patients. Patients with mutations associated with high 14-3-3 protein levels in the CSF typically had synaptic deposition of PrP in the brain and a rapid course of disease. The presence of small PrP protein fragments in brain homogenates was not correlated with other clinicopathological features. Positivity for PrP^Sc in the CSF may reflect the pathological process before or at disease onset, or abnormality in the secretion or metabolism of PrP^Sc. The amount of 14-3-3 protein in the CSF likely indicates the severity of the pathological process and accompanying neuronal damage. These characteristic features of the CSF in cases of gPrD will likely facilitate accurate diagnosis and clinicopathological study of the various disease subtypes.     

Immediate and Ongoing Detection of Prions in the Blood of Hamsters and Deer following Oral,Nasal, or Blood Inoculations

Infectious prions traverse epithelial barriers to gain access to the circulatory system, yet the temporal parameters of transepithelial transport and persistence in the blood over time remain unknown. We used whole-blood real-time quaking-induced conversion (wbRT-QuIC) to analyze whole blood collected from transmissible spongiform encephalopathy (TSE)-inoculated deer and hamsters throughout the incubation period for the presence of common prion protein-conversion competent amyloid (PrP^C-CCA). We observed PrP^C-CCA in the blood of TSE-inoculated hosts throughout the disease course from minutes postexposure to terminal disease.     

Exploring Physical and Chemical Factors Influencing the Properties of Recombinant Prion Protein and the Real-Time Quaking-Induced Conversion (RT-QuIC) Assay

Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrP^d) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease ante mortem. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay’s sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an in vitro-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test.     

Early detection of chronic wasting disease prions in urine of pre-symptomatic deer by real-time quaking-induced conversion assay

Chronic wasting disease (CWD) is a prion disease of captive and free-ranging deer (Odocoileus spp), elk (Cervus elaphus nelsonii) and moose (Alces alces shirasi). Unlike in most other prion diseases, in CWD prions are shed in urine and feces, which most likely contributes to the horizontal transmission within and between cervid species. To date, CWD ante-mortem diagnosis is only possible by immunohistochemical detection of protease resistant prion protein (PrP^Sc) in tonsil or recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsies, which requires anesthesia of animals. We report on detection of CWD prions in urine collected from pre-symptomatic deer and in fecal extracts by using real time quaking-induced conversion (RT-QuIC). This assay can be useful for non-invasive pre-symptomatic diagnosis and surveillance of CWD.     

H-Type Bovine Spongiform Encephalopathy: Complex Molecular Features and Similarities with Human Prion Diseases

We previously reported that some cattle affected by bovine spongiform encephalopathy (BSE) showed distinct molecular features of the protease-resistant prion protein (PrP^res) in Western blot, with a 1–2 kDa higher apparent molecular mass of the unglycosylated PrP^res associated with labelling by antibodies against the 86–107 region of the bovine PrP protein (H-type BSE). By Western blot analyses of PrP^res, we now showed that the essential features initially described in cattle were observed with a panel of different antibodies and were maintained after transmission of the disease in C57Bl/6 mice. In addition, antibodies against the C-terminal region of PrP revealed a second, more C-terminally cleaved, form of PrP^res (PrP^res #2), which, in unglycosylated form, migrated as a ≈ 14 kDa fragment. Furthermore, a PrP^res fragment of ≈7 kDa, which was not labelled by C-terminus-specific antibodies and was thus presumed to be a product of cleavage at both N- and C-terminal sides of PrP protein, was also detected. Both PrP^res #2 and ≈7 kDa PrP^res were detected in cattle and in C57Bl/6 infected mice. These complex molecular features are reminiscent of findings reported in human prion diseases. This raises questions regarding the respective origins and pathogenic mechanisms in prion diseases of animals and humans.Key Words: prion, BSE, Creutzfeldt-Jakob, Gerstmann-Sträussler-Scheinker, Western blot, amyloid     

A simple in vitro assay for assessing the efficacy,mechanisms and kinetics of anti-prion fibril compounds

ABSTRACT

Prion diseases are caused by the conversion of normal cellular prion proteins (PrP) into lethal prion aggregates. These prion aggregates are composed of proteinase K (PK) resistant fibrils and comparatively PK-sensitive oligomers. Currently there are no anti-prion pharmaceuticals available to treat or prevent prion disease. Methods of discovering anti-prion molecules rely primarily on relatively complex cell-based, tissue slice or animal-model assays that measure the effects of small molecules on the formation of PK-resistant prion fibrils. These assays are difficult to perform and do not detect the compounds that directly inhibit oligomer formation or alter prion conversion kinetics. We have developed a simple cell-free method to characterize the impact of anti-prion fibril compounds on both the oligomer and fibril formation. In particular, this assay uses shaking-induced conversion (ShIC) of recombinant PrP in a 96-well format and resolution enhanced native acidic gel electrophoresis (RENAGE) to generate, assess and detect PrP fibrils in a high throughput fashion. The end-point PrP fibrils from this assay can be further characterized by PK analysis and negative stain transmission electron microscopy (TEM). This cell-free, gel-based assay generates metrics to assess anti-prion fibril efficacy and kinetics. To demonstrate its utility, we characterized the action of seven well-known anti-prion molecules: Congo red, curcumin, GN8, quinacrine, chloropromazine, tetracycline, and TUDCA (taurourspdeoxycholic acid), as well as four suspected anti-prion compounds: trans-resveratrol, rosmarinic acid, myricetin and ferulic acid. These findings suggest that this in vitro assay could be useful in identifying and comprehensively assessing novel anti-prion fibril compounds.

Abbreviations: PrP, prion protein; PK, proteinase K; ShIC, shaking-induced conversion; RENAGE, resolution enhanced native acidic gel electrophoresis; TEM, transmission electron microscopy; TUDCA, taurourspdeoxycholic acid; BSE, bovine spongiform encephalopathy; CWD, chronic wasting disease; CJD, Creutzfeldt Jakob disease; GSS, Gerstmann–Sträussler–Scheinker syndrome; FFI, fatal familial insomnia; PrP^c, cellular prion protein; recPrP^C, recombinant monomeric prion protein; PrP^Sc, infectious particle of misfolded prion protein; RT-QuIC, real-time quaking-induced conversion; PMCA, Protein Misfolding Cyclic Amplification; LPS, lipopolysaccharide; EGCG, epigallocatechin gallate; GN8, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide; DMSO, dimethyl sulfoxide; ScN2A, scrapie infected neuroblastoma cells; IC50, inhibitory concentration for 50% reduction; recMoPrP ^23?231, recombinant full-length mouse prion protein residues 23-231; EDTA; PICUP, photo-induced cross-linking of unmodified protein; BSA, bovine serum albumin;; PMSF, phenylmethanesulfonyl fluoride.     

Cellular prion protein and Alzheimer disease

Soluble oligomeric amyloid-β (Aβ) has been suggested to impair synaptic and neuronal function, leading to neurodegeneration that is clinically observed as the memory and cognitive dysfunction characteristic of Alzheimer disease, while the precise mechanism(s) whereby oligomeric Aβ causes neurotoxicity remains unknown. Recently, the cellular prion protein (PrP^C) was reported to be an essential co-factor in mediating the neurotoxic effect of oligomeric Aβ. Our recent study showed that Prnp^−/− mice are resistant to the neurotoxic effect of oligomeric Aβ in vivo and in vitro. Furthermore, application of an anti-PrP^C antibody or PrP^C peptide was able to block oligomeric Aβ-induced neurotoxicity. These findings demonstrate that PrP^C may be involved in neuropathologic conditions other than conventional prion diseases, i.e., Creutzfeldt-Jakob disease.