New Assay Demonstrates Ability to Detect Abnormal α-Synuclein in Neurodegenerative Diseases

Nobutaka Hattori, MD, PhD, FAAN

In a recent study published in Nature Medicine, researchers developed a modified assay system that could efficiently detect pathogenic α-synuclein seeds from serum in patients with synucleinopathies.¹ These findings suggest the system, otherwise known as immunoprecipitation-based real-time quaking-induced conversion (IP/RT-QuIC), can be efficient in the diagnosis and distinguishing of patients with neurodegenerative diseases such as Parkinson disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA).

In a cohort among 270 patients with synucleinopathies, IP/RT-QuIC had a high diagnostic performance in differentiating PD from controls (aura under the curve [AUC], 0.96;95% CI, 0.95–0.99) and MSA versus controls (AUC, 0.64;95% CI, 0.49–0.79). Similarly, in a blinded external cohort of 40 patients with synucleinopathy, the system also demonstrated high diagnostic performance in differentiating those with PD (AUC,0.86;95% CI, 0.74–0.99) and MSA (AUC,0.80; 95% CI 0.65–0.97) from the controls.²

“In this study, we validated the usefulness of our novel assay system, IP/RT-QuIC, as a diagnostic marker of synucleinopathies. We propose that the fibril morphology of serum α-synuclein seeds and aggregates derived by IP/RT-QuIC can discriminate between PD, DLB, and MSA,” senior author Nobutaka Hattori, MD, PhD, FAAN, dean, Faculty of Medicine and Graduate School of Medicine at Juntendo University, and colleagues wrote.¹

In the assay, α-synuclein seeds are separated from the patient’s serum through immunoprecipitation, otherwise known as protein separation utilizing an antibody binding only for targeting protein. This is then followed by rapid amplification by real-tme quaking-induced conversion, or amplification induced by vigorous shaking. All told, this approach of diagnosis is highly sensitive since it has the ability to recognize serum α-synuclein seed concentrations as small as 1000pg/ml.

“At present, a neurologist's consultation is necessary to diagnose synucleinopathies. However, using IP/RTQuIC, a general internist can make the diagnosis. Therefore, more patients with synucleinopathies may be diagnosed with precision and could receive appropriate treatment at an earlier stage,” Hattori and colleagues noted.¹

In an analysis of the structural properties of the amplified seeds using transmission electron microscopy (TEM), the synuclein seed structure varied in the type of synucleinopathy. The seeds in PD and DLB displayed paired filaments in comparison with the seeds in MSA, which had 3 distinct structures that were twisted and straight strands. Therefore, this finding validated that IP/RT-QuIC added with TEM can differentiate between synucleinopathies according to the disease specific seed structure.

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As investigators converted the amplified seeds into the HEK293T cell line stably expressing GFP-fused α-synuclein with p.A53T mutation (in vitro) and injected seeds into mouse brains (in vivo), the seeds retained their aggregate forming capacity and diseases-specific seed structure. Researchers observed that the aggregates showed different morphologies depending on the type of disease which, they noted, explains how specific synucleinopathies can be diagnosed based on the structural differences of the α-synuclein seeds and their aggregates.

“Our new IP/RT-QuIC assay may have many future applications as a biomarker for precise diagnosis and monitoring of treatment of neurodegenerative diseases in clinical trials. This simple diagnostic method will enable establishment of personalized therapy options for synucleinopathies,” Hattori et al noted.¹

Synucleinopathies are cluster of neurodegenerative diseases that result from the anomalous buildup of α-synuclein, which is a protein typically found in the brain and neurons. An erroneous folding process of α-synuclein triggers the creation of 'seeds' that magnetize additional α-synuclein proteins, creating more substantial aggregates.^1,2 Despite the discovery of α-synuclein seeds in diverse tissues and blood among patients affected by synucleinopathies, their viability as biomarkers remains to be further investigated.

REFERENCES
1. A novel blood serum assay to diagnose neurodegenerative diseases. News Release. Juntendo University Research Promotion Center. Published May 29, 2023. Accessed August 14, 2023. https://www.eurekalert.org/news-releases/990594
2. Okuzumi A, Hatano T, Matsumoto G, et al. Propagative α-synuclein seeds as serum biomarkers for synucleinopathies. Nat Med. 2023;29(6):1448-1455. doi:10.1038/s41591-023-02358-9