Optimized Alpha Synuclein Monomers for Seed Amplification

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, characterized by motor dysfunctions such as tremor, rigidity, and bradykinesia, as well as depression and cognitive decline. Accurately diagnosing PD is challenging, since clinical symptoms are common to other neurodegenerative conditions, including multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and diffuse Lewy body disease (DLBD). In recent years, alpha synuclein seed amplification assays (SAAs) have emerged as promising tools to detect PD in its early stages, monitor disease progression, and evaluate therapeutic effectiveness. Now, efforts are focused on standardizing protocols to improve assay reliability and advance patient care.

StressMarq Biosciences is a leading provider of protein constructs for neurodegenerative disease research. This article outlines the core scientific principles behind the seed amplification assay, and its growing significance in advancing PD research, as well as highlights StressMarq’s expertise and ongoing commitment to innovation. Notably, StressMarq’s development of an optimized monomeric alpha synuclein substrate for the SAA complements an extensive portfolio of fibrillar, oligomeric, and monomeric alpha synuclein proteins—exclusively developed for research applications.*

The link between alpha synuclein and Parkinson’s disease was first established in 1997, when a point mutation in the alpha synuclein gene (SNCA) was connected to the familial disease.¹ At around the same time, alpha synuclein was found to be a major component of Lewy bodies, the abnormal clumps of protein observed in the brains of PD patients.² Since then, it has been established that PD pathogenesis involves the aggregation of alpha synuclein monomers into neurotoxic oligomers and fibrils, which act as seeds for further aggregate formation. Today, the alpha synuclein seeding mechanism is being investigated for its potential to support early diagnosis of PD, which can currently only be confirmed through post-mortem analysis of brain tissue.

The first seed amplification assay was developed by Kocisko et al. in 1994, who showed that an aggregated, protease-resistant prion protein (PrP^Sc), derived from scrapie brains, could convert the normal, protease-sensitive protein (PrP^C) into PrP^Sc in a cell-free system.³ However, the need to use radiolabeled PrP^C, coupled with the technical complexity and low conversion rate of this approach led to the development of protein misfolding cyclic amplification (PMCA) by Sarborio et al. in 2001 as a more efficient alternative.⁴

During PMCA, high-frequency ultrasound pulses are used to fragment newly formed PrP^Sc aggregates, thereby generating more seeds to accelerate the conversion of normal substrate to misfolded product. Through multiple cycles of incubation and sonication, the concentration of the pathogenic protein is exponentially increased, enabling its detection via Western blot or other immunoassay methods.

In 2011, Atarashi et al. developed a new PrP^Sc amplification assay, called real-time quaking-induced conversion (RT-QuIC).⁵ This differs from PMCA in that it uses shaking instead of sonication as the energy source, as well as allows for dynamic monitoring of aggregate formation by measuring Thioflavin T fluorescence upon fibril binding.

The first-generation PMCA and RT-QuIC assays each had distinct advantages and disadvantages, as covered in a recent publication by Riu et al. and summarized in Table 1.⁶ Over the years, both methods have undergone multiple optimizations to bolster their sensitivity, detection speed, and clinical applicability, and it is now possible for researchers to detect attogram quantities of PrP^Sc in sample types including cerebral spinal fluid (CSF), blood, and urine. Additionally, PMCA and RT-QuIC have been adapted for the amplification and detection of other proteins than infectious prions, including seed-competent alpha synuclein, tau, and amyloid beta.

Table 1. Comparison of classic PMCA and RT-QuIC. Adapted from Liu et al. 2025.⁶

RT-QuIC assays are widely used for studying alpha synuclein seed amplification, typically aided by instruments such as the FLUOstar^® Omega, a multi-mode microplate reader from BMG Laboratories that can automatically perform shaking and fluorescence readings to simply the experimental workflow. To ensure accurate and reliable results, the recombinant soluble alpha synuclein monomers must be of high purity and free of aggregates, and the fibrillar alpha synuclein seeds should be rigorously characterized for activity, stability, and disease relevance. As experimental protocols are highly sensitive to the type of equipment and reagents being used, researchers must diligently optimize their own protocols for the resources at their disposal, using scientific publications and established data as a starting point.

A growing number of studies is available to demonstrate the diagnostic and prognostic value of alpha synuclein SAA for Parkinson’s disease. For example, a 2023 publication from Concha-Marambio et al. describes a protocol that can differentiate alpha synuclein aggregates in CSF samples from patients with PD versus those from patients with MSA.⁷ Additionally, a longitudinal cohort study published by Orrú et al. in 2025 shows that genetic status in PD determines the profile of alpha synuclein seeding kinetics, while the SAA kinetic measure, time to threshold (TTT), could potentially be used as a clinical trial stratification tool to recruit PD cohorts with similar rates of clinical disease progression.⁸

StressMarq Biosciences offers a diverse and comprehensive range of alpha synuclein monomers, oligomers, and pre-formed fibrils (PFFs) to accelerate preclinical drug discovery and Parkinson’s disease modelling. As seeded aggregation assays and RT-QuIC continue to gain recognition as powerful tools in neurodegeneration research, StressMarq is actively developing and refining optimized alpha synuclein materials tailored for these applications.

1. Polymeropoulos MH, Lavedan C, Leroy E, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science. 1997;276(5321):2045-2047. doi:10.1126/science.276.5321.2045

2. Spillantini MG, Schmidt ML, Lee VM, et al. Alpha-synuclein in Lewy bodies. Nature. 1997;388(6645):839-840. doi:10.1038/42166

3. Kocisko DA, Come JH, Priola SA, et al. Cell-free formation of protease-resistant prion protein. Nature. 1994;370(6489):471-474. doi:10.1038/370471a0

4. Saborio GP, Permanne B, Soto C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature. 2001;411(6839):810-813. doi:10.1038/35081095

5. Atarashi R, Satoh K, Sano K, et al. Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion. Nat Med. 2011;17(2):175-178. doi:10.1038/nm.2294

6. Liu R, Chen Z, Wang Y, Wu L. Application of protein misfolding amplification techniques in prion diseases. Ageing Neur Dis. 2025;5:15. https://www.oaepublish.com/articles/and.2025.18

7. Concha-Marambio L, Pritzkow S, Shahnawaz M, et al. Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid. Nat Protoc. 2023;18(4):1179-1196. doi:10.1038/s41596-022-00787-3

8. Orrú CD, Vaughan DP, Vijiaratnam N, et al. Diagnostic and prognostic value of α-synuclein seed amplification assay kinetic measures in Parkinson's disease: a longitudinal cohort study. Lancet Neurol. 2025;24(7):580-590. doi:10.1016/S1474-4422(25)00157-7

* StressMarq Biosciences’ products are intended for Research Use Only (RUO).