Are We Ready for Detecting α-Synuclein Prone to Aggregation in Patients? The Case of "Protein-Misfolding Cyclic Amplification" and "Real-Time Quaking-Induced Conversion" as Diagnostic Tools

Abstract

The accumulation and deposition of α-synuclein aggregates in brain tissue is the main event in the pathogenesis of different neurodegenerative disorders grouped under the term of synucleinopathies. They include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. To date, the diagnosis of any of these disorders mainly relies on the recognition of clinical symptoms, when the neurodegeneration is already in an advanced phase. In the last years, several efforts have been carried out to develop new diagnostic tools for early diagnosis of synucleinopathies, with special interest to Parkinson's disease. The Protein-Misfolding Cyclic Amplification (PMCA) and the Real-Time Quaking-Induced Conversion (RT-QuIC) are ultrasensitive protein amplification assays for the detection of misfolded protein aggregates. Starting from the successful application in the diagnosis of human prion diseases, these techniques were recently tested for the detection of misfolded α-synuclein in brain homogenates and cerebrospinal fluid samples of patients affected by synucleinopathies. So far, only a few studies on a limited number of samples have been performed to test PMCA and RT-QuIC diagnostic reliability. Neverthless, these assays have shown very high sensitivity and specificity in detecting synucleinopathies even at the pre-clinical stage. Despite the application of PMCA and RT-QuIC for α-synuclein detection in biological fluids is very recent, these techniques seem to have the potential for identifying subjects that will be likely to develop synucleinopathies.

Keywords: PMCA; RT-QuIC; early diagnosis; synucleinopathies; α-synuclein.

Figure 1

(A) Outline of PMCA and RT-QuIC kinetic assays. (i) An aliquot of tissue homogenate or a biological fluid containing a quantity of aggregates (blue cubes) is dissolved in a buffer containing abundant monomer in solution (red spheres). (ii) The sample is incubated for a defined time at specific temperature. In this phase the preformed aggregates undergo polymerization at their extremities and catalyze the formation of new nuclei on their surfaces. Monomers that undergo misfolding are depicted as red cubes. (iii) The number of available points for polymerization is increased by performing sonication or shaking of the sample, thus fragmenting the fibrils grown in the previous step. The steps (ii) and (iii) are repeated several times. (iv) At the end of the procedure the initial quantity of misfolded and aggregated protein is exponentially amplified at the expense of the monomer present in solution. (B) Simulation of a PMCA or RT-QuIC experiment. The simulation was performed by integrating differential equations describing polymerization, secondary catalyzed nucleation and fragmentation kinetics in presence of different quantities (20, 2, 0.2, 0.02, and 0.002 ng) of preformed aggregates (seeds). The simulation consisted in cycles of 30 min in which fragmentation kinetics was kept active only for 1 min (shaking) and turned off for 29 min (incubation). The cycles were repeated for a total time of 150 h. Normalized fluorescence intensity was calculated by considering it proportional to the total mass of fibrillary aggregates formed at a certain time.