Sporadic Creutzfeldt-Jakob disease: Real-Time Quaking Induced Conversion (RT-QuIC) assay represents a major diagnostic advance

Abstract

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare and fatal neurodegenerative disorder with an incidence of 1.5 to 2 cases per million population/year. The disease is caused by a proteinaceous infectious agent, named prion (or PrPSc), which arises from the conformational conversion of the cellular prion protein (PrPC). Once formed, PrPSc interacts with the normally folded PrPC coercing it to undergo similar structural rearrangement. The disease is highly heterogeneous from a clinical and neuropathological point of view. The origin of this variability lies in the aberrant structures acquired by PrPSc. At least six different sCJD phenotypes have been described and each of them is thought to be caused by a peculiar PrPSc strain. Definitive sCJD diagnosis requires brain analysis with the aim of identifying intracerebral accumulation of PrPSc which currently represents the only reliable biomarker of the disease. Clinical diagnosis of sCJD is very challenging and is based on the combination of several clinical, instrumental and laboratory tests representing surrogate disease biomarkers. Thanks to the advent of the ultrasensitive Real-Time Quaking-Induced Conversion (RT-QuIC) assay, PrPSc was found in several peripheral tissues of sCJD patients, sometimes even before the clinical onset of the disease. This discovery represents an important step forward for the clinical diagnosis of sCJD. In this manuscript, we present an overview of the current applications and future perspectives of RT-QuIC in the field of sCJD diagnosis.

Figure 1.

Creutzfeldt-Jakob disease, hallmark neuropathologic lesions. Spongiform changes may appear as small vacuoles (A) diffusely present in grey matter (H&E, cerebral cortex, 20x) or large, confluent vacuolar lesions (B) typical of the MM2-C (cortical) subtype (H&E, cerebral cortex, 20x). Kuru plaques (C) small aggregates of PrP with the tinctorial and optical properties of amyloid are typically found in the cerebellum in MV2 subtype (H&E, 60x). Astrogliosis (D) may be severe in all subtypes of Creutzfeldt-Jakob disease (glial fibrillary acidic protein immunohistochemistry, 10x). Neuronal loss (E) is usually very severe in the cerebral cortex, basal ganglia and cerebellum (H&E, cerebellum) but may be mild in some cases (F) (microtubule associated protein 2 immunohistochemistry; 10x).

Figure 2.

Creutzfeldt-Jakob disease, patterns of pathological PrP deposition (PrP immunohistochemistry using 3F4 monoclonal antibody). In MM/MV1 subtype PrP deposition is mainly of synaptic type and appears as homogeneous, finely granular immunoreactivity in the neuropil of the cerebral cortex (A) (10x), while in the cerebellum is finely granular in the molecular layer while forming coarser deposits in the granular layer (B) (10x). In VV2 subtype PrP deposition often decorates the boundaries of pyramidal neurons of the cerebral cortex (C) (10x), while in the cerebellum plaque-like deposition takes is common in the granular layer (D) (10x). In most of the patients with the MM2 subtype PrP deposition takes variable aspects in the cerebral cortex (E) (20x), while the typical feature is the presence of Kuru plaques, small aggregates of PrP with the tinctorial and optical properties of amyloid, in the cerebellum (F) (10x). MM2-C (cortical) subtype is characterized by PrP immunoreactivity more intense at the rims of the large vacuoles of spongiosis in the cerebral cortex (G) (20x), while the cerebellum is usually relatively spared (H) (10x).

Figure 3.

Schematic illustration of the RT-QuIC reaction. The RT-QuIC process is divided into three phases: (1) the lag phase, (2) the growth phase and (3) the plateau phase. The reaction mix is composed by recombinant PrP (recPrP) and Thioflavin T (ThT) which are dissolved in common buffers. The addition of PrP^Sc (pink triangle) to the reaction induces the convertion of recPrP (blue hexagon) into a misfolded form (red arrow) which starts to aggregate and form recPrP amyloid fibrils. In the absence of PrP^Sc, recPrP can aggregate (dotted line) following a well-defined kinetics. The formation of the aggregates induces the emission of a ThT fluorescence signal (yellow star). In the presence of PrP^Sc, the kinetics of recPrP aggregation is significantly accelerated (solid line). The increased kinetics of recPrP aggregation is known as seeding effect.