Plasma a-Synuclein Aggregation Seeding Activity as a Novel Biomarker for Neurodegeneration Disease

Overview

Synuclein disease, also known as Synucleinopathies, is a general term for a class of degenerative diseases. It mainly includes Parkinson's disease (PD), Dementia with lewy bodies (DLB), Multiple system atrophy (MSA) and so on. The disease is characterized by the abnormal folding of alpha-synuclein (α-syn) in the peripheral and central nervous system, resulting in the production of lewy bodies, lewy neurites, neurons, and glial cytoplasmic inclusion bodies in neurons or glial cells. The intracellular location and accumulation pattern of α-syn vary in different synuclein diseases. There are overlapping conditions between synuclein diseases, which also makes the accuracy of clinical diagnosis of synuclein diseases very low. A definitive diagnosis can be made only after the patient has died and brain samples are evaluated using immunohistochemical staining. Therefore, in order to intervene and save synuclein disease in advance, it is necessary to develop more accurate and less invasive diagnostic methods. Real-Time Quaking Induced Conversion (RT-QuIC) is a method for in vitro amplification of pathogenic protein seeds, which can enable seed proteins with in vitro amplification ability to transform substrates into misfolded seeds under specific conditions. RT-QuIC was originally used in the diagnosis of patients with prion protein. The technique can identify the prion protein in the misfolded form in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease with a specificity of 100% and a sensitivity of 95-98%, so it has been incorporated into the clinical diagnostic criteria for sporadic Creutzfeldt-Jakob disease. Studies have shown that pathogenic seeds extracted from patients' cerebrospinal fluid or other biological fluids or tissues are incubated with recombinant protein substrates, and the use of intermittent shaking to promote the interaction between the seed and substrate can force the substrate to transform into pathogenic protein seeds. The RT-QuIC reaction process is monitored in real time by a detector inside the machine for thioflavin, a fluorescent dye that fluoresces when bound to the beta lamellar layer, which is also typical of amyloid fibrils. Studies have shown that testing patients' cerebrospinal fluid with RT-QuIC can accurately distinguish lewy body dementia from non-Parkinson's dementia In this study, RT-QuIC technique was used to amplify the misfolded α-syn in clinical samples to explore the differences in protein characteristics among synuclein diseases, and to try to diagnose synuclein diseases by this method. Firstly, clinical cases of synucleoprotein disease were enrolled, and plasma of 100 control subjects, 221 cases of PD patients and 127 cases MSA patients, 10 cases of PSP patients, were established. Using RT-QuIC technology, we used aSyn (successfully prepared in our laboratory) to amplify the plasma of normal subjects and Synucleinopathies patients. It was found that the maximum fluorescence intensity (extreme value), peak time, K/2 (slope at extreme value/2) and Tmax/2 (time of reaching extreme value/2) can be used as diagnostic and differential diagnostic indicators for Synucleinopathies.