Significance Converging evidence points to the build-up of phosphorylated α-synuclein (α-syn) at residue serine 129 (pS129) in Lewy body disease, suggesting its central role in the regulation of α-syn aggregation and neuronal degeneration. However, a comprehensive understanding of the role of α-syn phosphorylation at pS129 in α-synuclenopathies pathogenesis is still lacking. Herein, we study the phosphorylation incidence and its effect on α-syn aggregation propensity and cellular toxicity. Collectively, our data suggest that pS129 occurred subsequent to initial α-syn aggregation, lessened aggregation propensity, and attenuated cytotoxicity through diverse assays. Our findings highlight major implications for a better understanding of the role of a molecular modification on protein aggregation.
A BS TRACT: Background: Tangible efforts have been made to identify biomarkers for Parkinson's disease (PD) diagnosis and progression, with α-synuclein (α-syn) related biomarkers being at the forefront. Objectives: The objectives of this study were to explore whether cerebrospinal fluid (CSF) levels of total, oligomeric, phosphorylated Ser 129 α-synuclein, along with total tau, phosphorylated tau 181, and β-amyloid 1-42 are (1) informative as diagnostic markers for PD, (2) changed over disease progression, and/or (3) correlated with motor and cognitive indices of disease progression in the longitudinal De Novo Parkinson cohort. Methods: A total of 94 de novo PD patients and 52 controls at baseline and 24-and 48-month follow-up were included, all of whom had longitudinal lumbar punctures and clinical assessments for both cognitive and motor functions. Using our in-house enzymelinked immunosorbent assays and commercially available assays, different forms of α-synuclein, tau, and β-amyloid 1-42 were quantified in CSF samples from the De Novo Parkinson cohort.Results: Baseline CSF total α-synuclein was significantly lower in early de novo PD compared with healthy controls, whereas the ratio of oligomeric/total and phosphorylated/total were significantly higher in the PD group. CSF oligomeric-α-synuclein longitudinally increased over the 4-year follow-up in the PD group and correlated with PD motor progression. Patients at advanced stages of PD presented with elevated CSF oligomeric-α-synuclein levels compared with healthy controls. Conclusions: Longitudinal transitions of CSF biomarkers over disease progression might not occur linearly and are susceptible to disease state. CSF oligomeric-α-synuclein levels appear to increase with diseases severity and reflect PD motor rather than cognitive trajectories.
Background and objectives:There’s an unmet need to identify robust diagnostic biomarker that can mirror Parkinson’s disease (PD) clinical course. Here we present a novel approach to investigate disease associated αSyn aggregates as biomarkers of PD clinical stage.Methods:We combined both seed amplification assay (SAA) and enzyme-linked immunosorbent assay (ELISA) to provide a quantitative test readout that reflects the clinical severity of PD patients. To attain this goal, we initially explored the potential of our test using two sets of human brain homogenates (pilot and validation sets), and then verified it with two independent human CSF cohorts; discovery (62 PD, and 34 control) and validation (49 PD and 48 control).Results:We showed that oligomers-specific ELISA robustly quantified SAA end product from subjects with PD or DLB with high sensitivity and specificity scores (100%). Analysis also demonstrated that seeding activity could be detected earlier with oligomeric ELISA as the test readout rather than SAA alone. More importantly, multiplexing the assays provided robust information about the patients’ clinical disease stage. In the discovery cohort, levels of CSF seeded αSyn oligomers correlated with the severity of the clinical symptoms of PD as measured by UPDRS-motor (r= 0.58, p <0.001) and H&Y scores (r= 0.43, p <0.01). Similar correlations were observed in the validation cohort between the concentrations of CSF seeded αSyn oligomers and both UPDRS-motor (r= 0.50, p <0. 01) and H&Y scores (r= 0.49, p <0.01). At 20 h, ROC analysis yielded a sensitivity of 91.9% (95% CI, 82.4%–96.5%) and a specificity of 85.3% (95% CI,69.8 %–93.5%), with an area under the curve of 0.969 for CSF seeded αSyn oligomers differentiating PD from controls in the Discovery CSF cohort, whereas, a sensitivity of 80.7% (95% CI, 69.1 %–88.5%), a specificity of 76.5% (95% CI, 60.0 %–87.5%), and area under the curve of 0.860 were generated with ThT Imax at the same time-point.Discussion:We showed that combining SAA and ELISA assays is more promising diagnostic tool than SAA alone, providing information about the disease stage by correlating with clinical measures of disease severity.
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