▪ Abstract Many neurodegenerative diseases, including Alzheimer's and Parkinson's and the transmissible spongiform encephalopathies (prion diseases), are characterized at autopsy by neuronal loss and protein aggregates that are typically fibrillar. A convergence of evidence strongly suggests that protein aggregation is neurotoxic and not a product of cell death. However, the identity of the neurotoxic aggregate and the mechanism by which it disables and eventually kills a neuron are unknown. Both biophysical studies aimed at elucidating the precise mechanism of in vitro aggregation and animal modeling studies support the emerging notion that an ordered prefibrillar oligomer, or protofibril, may be responsible for cell death and that the fibrillar form that is typically observed at autopsy may actually be neuroprotective. A subpopulation of protofibrils may function as pathogenic amyloid pores. An analogous mechanism may explain the neurotoxicity of the prion protein; recent data demonstrates that the disease-associated, infectious form of the prion protein differs from the neurotoxic species. This review focuses on recent experimental studies aimed at identification and characterization of the neurotoxic protein aggregates.

A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP^C to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD₅₀). Brain tissue from 263K scrapie-affected hamsters gave SD₅₀ values of 10¹¹-10¹²/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD₅₀ values of 10^3.5–10^5.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD₅₀ values of 10^2.0–10^2.9/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity. Prion diseases are deadly infectious neurodegenerative disorders of mammals which involve the misfolding of host prion protein. To better manage these diseases, we need to be able to detect and quantify the infectious particles, or prions, in biological samples. However, current tests lack the sensitivity, speed and/or quantitative capabilities required for many important applications in medicine, agriculture, wildlife biology and research. To address this problem, we have developed a new prion assay that is highly sensitive, rapid, and quantitative. This assay takes advantage of the ability of miniscule amounts of infectious prions to seed the misfolding of large excesses of normal prion protein in test tube reactions. Quantitation is achieved by testing a range of sample dilutions and determining loss of seeding activity, i.e. the end-point dilution. Similar analyses have long been used to quantify prions by inoculation into animals; however, such bioassays take months or years to perform and are both animal-intensive and expensive. Our new method provides a more practical means of detecting and quantifying prions. So far, we have applied this assay to prions from sheep, deer, and hamsters, and have found surprisingly high levels of prions in the nasal and cerebral spinal fluids of infected hamsters.

The diagnosis and treatment of synucleinopathies such as Parkinson disease and dementia with Lewy bodies would be aided by the availability of assays for the pathogenic disease-associated forms of α-synuclein (αSyn^D) that are sufficiently sensitive, specific, and practical for analysis of accessible diagnostic specimens. Two recent αSyn^Dseed amplification tests have provided the first prototypes for ultrasensitive and specific detection of αSyn^Din patients’ cerebrospinal fluid. These prototypic assays require 5–13 days to perform. Here, we describe an improved α-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay that has similar sensitivity and specificity to the prior assays, but can be performed in 1–2 days with quantitation. Blinded analysis of cerebrospinal fluid from 29 synucleinopathy cases [12 Parkinson’s and 17 dementia with Lewy bodies] and 31 non-synucleinopathy controls, including 16 Alzheimer’s cases, yielded 93% diagnostic sensitivity and 100% specificity for this test so far. End-point dilution analyses allowed quantitation of relative amounts of αSyn^Dseeding activity in cerebrospinal fluid samples, and detection in as little as 0.2 μL. These results confirm that αSyn^Dseeding activity is present in cerebrospinal fluid. We also demonstrate that it can be rapidly detected, and quantitated, even in early symptomatic stages of synucleinopathy.

Definite diagnosis of sporadic Creutzfeldt–Jakob disease in living patients remains a challenge. A test that detects the specific marker for Creutzfeldt–Jakob disease, the prion protein (PrP^CJD), by means of real-time quaking-induced conversion (RT-QuIC) testing of cerebrospinal fluid has a sensitivity of 80 to 90% for the diagnosis of sporadic Creutzfeldt–Jakob disease. We have assessed the accuracy of RT-QuIC analysis of nasal brushings from olfactory epithelium in diagnosing sporadic Creutzfeldt–Jakob disease in living patients. We collected olfactory epithelium brushings and cerebrospinal fluid samples from patients with and patients without sporadic Creutzfeldt–Jakob disease and tested them using RT-QuIC, an ultrasensitive, multiwell plate–based fluorescence assay involving PrP^CJD-seeded polymerization of recombinant PrP into amyloid fibrils. The RT-QuIC assays seeded with nasal brushings were positive in 30 of 31 patients with Creutzfeldt–Jakob disease (15 of 15 with definite sporadic Creutzfeldt–Jakob disease, 13 of 14 with probable sporadic Creutzfeldt–Jakob disease, and 2 of 2 with inherited Creutzfeldt–Jakob disease) but were negative in 43 of 43 patients without Creutzfeldt–Jakob disease, indicating a sensitivity of 97% (95% confidence interval [CI], 82 to 100) and specificity of 100% (95% CI, 90 to 100) for the detection of Creutzfeldt–Jakob disease. By comparison, testing of cerebrospinal fluid samples from the same group of patients had a sensitivity of 77% (95% CI, 57 to 89) and a specificity of 100% (95% CI, 90 to 100). Nasal brushings elicited stronger and faster RT-QuIC responses than cerebrospinal fluid (P<0.001 for the between-group comparison of strength of response). Individual brushings contained approximately 10⁵ to 10⁷ prion seeds, at concentrations several logs₁₀ greater than in cerebrospinal fluid. In this preliminary study, RT-QuIC testing of olfactory epithelium samples obtained from nasal brushings was accurate in diagnosing Creutzfeldt–Jakob disease and indicated substantial prion seeding activity lining the nasal vault. (Funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases and others.)

The prion (infectious protein) concept has evolved with the discovery of new self-propagating protein states in organisms as diverse as mammals and fungi. The infectious agent of the mammalian transmissible spongiform encephalopathies (TSE) has long been considered the prototypical prion, and recent cell-free propagation and biophysical analyses of TSE infectivity have now firmly established its prion credentials. Other disease-associated protein aggregates, such as some amyloids, can also have prion-like characteristics under certain experimental conditions. However, most amyloids appear to lack the natural transmissibility of TSE prions. One feature that distinguishes the latter from the former is the glycophosphatidylinositol membrane anchor on prion protein, the molecule that is corrupted in TSE diseases. The presence of this anchor profoundly affects TSE pathogenesis, which involves major membrane distortions in the brain, and may be a key reason for the greater neurovirulence of TSE prions relative to many other autocatalytic protein aggregates.