Challenges and Advances in Antemortem Diagnosis of Human Transmissible Spongiform Encephalopathies

Abstract

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, arise from the structural conversion of the monomeric, cellular prion protein (PrP^C) into its multimeric scrapie form (PrP^Sc). These pathologies comprise a group of intractable, rapidly evolving neurodegenerative diseases. Currently, a definitive diagnosis of TSE relies on the detection of PrP^Sc and/or the identification of pathognomonic histological features in brain tissue samples, which are usually obtained postmortem or, in rare cases, by brain biopsy (antemortem). Over the past two decades, several paraclinical tests for antemortem diagnosis have been developed to preclude the need for brain samples. Some of these alternative methods have been validated and can provide a probable diagnosis when combined with clinical evaluation. Paraclinical tests include in vitro cell-free conversion techniques, such as the real-time quaking-induced conversion (RT-QuIC), as well as immunoassays, electroencephalography (EEG), and brain bioimaging methods, such as magnetic resonance imaging (MRI), whose importance has increased over the years. PrP^Sc is the main biomarker in TSEs, and the RT-QuIC assay stands out for its ability to detect PrP^Sc in cerebrospinal fluid (CSF), olfactory mucosa, and dermatome skin samples with high sensitivity and specificity. Other biochemical biomarkers are the proteins 14-3-3, tau, neuron-specific enolase (NSE), astroglial protein S100B, α-synuclein, and neurofilament light chain protein (NFL), but they are not specific for TSEs. This paper reviews the techniques employed for definite diagnosis, as well as the clinical and paraclinical methods for possible and probable diagnosis, both those in use currently and those no longer employed. We also discuss current criteria, challenges, and perspectives for TSE diagnosis. An early and accurate diagnosis may allow earlier implementation of strategies to delay or stop disease progression.

Keywords: cell-free conversion assay; diagnostic; neurodegenerative disease; prion diseases; prion protein.

FIGURE 1

Prion protein structure and genetic variation. (A) Diglycosylated, full-length human PrP^C (huPrP^23–230) attached to the membrane (yellow brown) by a GPI anchor (purple). The globular C-terminal domain comprises three α-helices (blue) and two small β-strands (red), while the N-terminal is intrinsically flexible. Turns and random coils are represented in gray, and the glycosides are shown in salmon. A disulfide bond (yellow) connects the α-helices 2 and 3. The globular domain structure was determined by nuclear magnetic resonance (PDB code 1QLX). (B) Schematic of human PrP primary structure, with disease-associated mutations (below) and polymorphisms (above). Deleterious mutations include missense and nonsense changes, as well as octarepeat insertions and deletions. The octarepeat domain (green) is a copper-binding segment of 4–5 contiguous repeats of the sequence PHGGGWGQ. The 22-residue endoplasmic reticulum signal sequence (ER-SS) and 23-residue GPI signal sequence (GPI-SS) at the ends are shown in black. The other portions are colored as in panel (A).

FIGURE 2

PrP^Sc amplification from PrP^C. PrP^Sc particles grow by a nucleation process. A unit of PrP^C forms a complex with PrP^Sc, and the latter induces the refolding of the former into a β-sheet-rich structure which then becomes part of the PrP^Sc multimer. In this self-propagating process, the PrP^Sc particles act like seeds/nuclei, with PrP^C serving as a substrate. The secondary structures are colored as follows: α-helices in blue, β-strands in red, and unstructured portions in gray. The atomistic model of PrP^Sc was constructed by combining experimental data and molecular dynamics simulations (Spagnolli et al., 2019).

FIGURE 3

Protein misfolding cyclic amplification (PMCA) in automated fashion. Normal material homogenate (NMH), a source of PrP^C (substrate), is prepared from brain sections or cultured cells. Suspected infectious material homogenate (IMH), a possible source of PrP^Sc (seed), is prepared from brain sections, blood, urine, or olfactory mucosa brushings. Suspected IMH is diluted into NMH, and the resulting mixture is incubated with alternating cycles of sonication and rest. Subsequent rounds can be performed by diluting the product of the previous round into fresh NMH and repeating the incubation step. As a result, the amount of PrP^Sc is amplified after several sonication–rest cycles and even more after serial rounds of seeding and incubation. The products from different cycles and rounds are treated with proteinase K to eliminate PrP^C and then subjected to Western blotting to reveal the growing amount of ^resPrP. This result indicates a positive TSE diagnosis. The Western blot shown is republished with permission of American Society for Biochemistry and Molecular Biology, from Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification, Saá, Castilla, and Soto, 281, 46, 2006; permission conveyed through Copyright Clearance Center, Inc.

FIGURE 4

Real-time quaking-induced conversion (RT-QuIC). Recombinant PrP (rPrP) is expressed heterologously in E. coli and purified as folded protease-sensitive rPrP (^senrPrP), the substrate for RT-QuIC. Suspected infectious material homogenate, a possible source of PrP^Sc (seed), is prepared from brain sections, cerebrospinal fluid, olfactory mucosa brushings, and dermatome skin sections. High dilutions of a test sample are mixed with ^senrPrP, buffer, and thioflavin T (a fluorescent amyloid probe) to produce the conversion medium, which is loaded into a multiwell microplate and then incubated with alternating cycles of shaking and rest in a fluorescence microplate reader. This equipment records thioflavin T fluorescence emission throughout the incubation, thus revealing the kinetics of aggregate formation. A sigmoidal growth in thioflavin T fluorescence indicates a positive TSE diagnosis. The use of normal material homogenate does not lead to any increase in thioflavin T fluorescence, indicating that no conversion is happening. The positive result shown was obtained with brain homogenate from a patient with GSS.