Parkinson’s disease is one of the most prevalent neurodegenerative disorders and is characterized by the loss of dopaminergic neurons and widespread α-Synuclein aggregation. Recent advances in α-Synuclein research has enabled more accurate assessments of disease progression. However, the precise mechanisms of aggregation, and how that translates to downstream cellular damage, remain poorly understood.

While assays designed to measure aggregated and phosphorylated (S129) human α-Synuclein protein are commonly used to assess pathological burden in patients with synucleinopathy disorders, detecting low levels of the protein remains a challenge.

Development of novel quantification tools for pS129 detection

In a recent study¹, reformulated immunoassay tools were developed for quantifying total, phospho-Ser129, and aggregated α-Synuclein to characterize disease mechanisms and regional distribution of α-Synuclein. Researchers reformulated existing AlphaLISA™ SureFire® Ultra™ assay kits to improve their sensitivity, as well as increase compatibility and cross-reactivity with mouse α-Synuclein.

The AlphaLISA SureFire Ultra assay is a highly sensitive, bead-based immunoassay designed for the quantitative detection of phosphorylated proteins in cellular lysates. It utilizes Alpha technology to bring donor and acceptor beads into proximity, generating a signal proportional to the amount of target protein present.

These advancements can improve efficiency in studying α-Synuclein pathology in neurodegenerative diseases, aiding our understanding of synucleinopathies and the development of therapeutic strategies.

Using these reformulated tools, the authors described robust quantification capabilities with a lower limit of quantification ≤ 0.5 pg/mL, for both mouse and human α-Synuclein. These tools were also effective in assessing the relationship between α-Synuclein phosphorylation and aggregation in various mouse brain tissues and regions.

Additionally, the LEGEND MAX™ Human α-Synuclein Aggregate ELISA Kit from BioLegend, a part of Revvity, was also used to detect aggregated α-Synuclein and assess the relationship between α-Synuclein S129 phosphorylation and aggregation in different mouse brain tissue preparations.

Profiling changes to the regional abundance of total and pS129 α-Synuclein

The authors found that phosphorylation of α-Synuclein at Ser129 and its aggregation were regionally distinct in the mouse brain. High levels of phosphorylated α-Synuclein were observed in the substantia nigra and striatum, while aggregation was more prominent in the cortex and hippocampus. These findings suggest that phosphorylation and aggregation of α-Synuclein may contribute differentially to pathology in various brain regions.

For additional resources on immunoassay research applications and sample preparation, view our app notes on biomarker quantification in a kidney toxicity model and a workflow for tissue lysate preparation and simultaneous quantification of 13 key biomarkers relevant to Cytokine Release Syndrome (CRS). This app note features the LEGENDplex™ Mouse CRS Panel, which can quantify IFN-γ, IL-10, CXCL9, CXCL10, TNF-α, IL-6, IL-4, CCL3, and GM-CSF. LEGENDplex bead-based immunoassays work on the same principle as sandwich ELISAs, with the advantage that they can simultaneously quantify up to 14 targets in a single sample on common flow cytometers.

In summary, the authors aimed to create sensitive and robust assays for quantifying total, phosphorylated, and aggregated α-Synuclein for use in mouse models, which are widely studied as pre-clinical models, and crucial for synucleinopathy research. Low level detection and quantification of α-Synuclein and its phosphorylated and aggregated forms will greatly increase our understanding of α-Synuclein pathology and aid in the development of novel diagnostic and therapeutic modalities.

Read the paper to learn more about the tools developed and their applications in the study, or visit our neuroscience page to explore Revvity’s solutions for cell analysis and immunoassay-based neurodegeneration research.