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International Parkinson and Movement Disorder Society
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        VOLUME 27, ISSUE 2 • JUNE 2023  Full issue »

α-Synuclein seed amplification assay shows potential to unmask pathology and enable biologically-based therapeutic development 

Biomarkers for α-synucleinopathies that reflect underlying pathology are sorely needed to improve the accuracy of early diagnosis and identify subtypes based on molecular factors and thus accelerate clinical trials.1  Clinical entities including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB) are the most common neuronal α-synucleinopathies. The pathological hallmark of these disorders is the accumulation of misfolded, aggregated α-synuclein in brainstem nuclei and neocortex into Lewy Bodies and Lewy Neurites.2,3   

In the past 5 years, misfolded-protein amplification techniques, originally developed for the detection of self-propagating PrPSc in prion diseases,4 have been applied to detect pathological α-synuclein in PD and other synucleinopathies, and represent a major breakthrough in biomarker development.5,6 These assays have been reported under the names real-time quaking-induced conversion (RT-QuIC),7 protein misfolding cyclic amplification (PMCA),8 and most recently, the consensus name, seed amplification assay (SAA).9,10  Tiny amounts self-replicating misfolded synuclein aggregates (seeds) from biological samples are multiplied during SAA to reach quantities easily detectable by standard fluorescence measurements. Driven by stringent SAA conditions, these seeds multiply by imprinting their pathological conformation into the monomeric recombinant synuclein used in the assay as substrate, and generate more and more misfolded synuclein (for more information please see Concha-Marambio et al. 2023)

Prior studies have shown that α-synuclein SAAs performed on cerebrospinal fluid (CSF) distinguish PD patients from healthy controls (HC) with high sensitivity and specificity.10,11 Preliminary studies have also shown αSyn-SAA positive results in a high proportion of patients from at-risk groups, such as patients with isolated rapid eye movement (REM) sleep behavior disorder (RBD)12,13 A recent study showed excellent inter-laboratory agreement when samples from the same participants were run on three different assay platforms.10  

In our study, reported in Lancet Neurology,14 we describe αSyn-SAA results on over 1,100 participants in the Parkinson’s Progression Markers Initiative (PPMI) study using assay conditions developed by Amprion. The study included PD patients with and without PD-associated genetic variants, healthy controls (HCs), and patients at risk for PD (either because of prodromal features or non-manifesting carriers [NMCs] of genetic variants). The goals of this analysis were to determine assay sensitivity and specificity using a large number of samples, to test the ability of αSyn-SAA to detect early signs of PD pathophysiology in at-risk individuals, and to leverage clinical and biomarker data within PPMI to examine clinical and genetic heterogeneity among PD patients based on αSyn-SAA status. 

The main result of the analysis was that overall diagnostic accuracy was high, with sensitivity of 87.7% (95% CI 84.9, 90.5) across all PD patients and specificity of 96.3% (95% CI 93.4, 99.2) in healthy controls.  Somewhat surprisingly, there was marked heterogeneity in the proportion with positive α-synuclein SAA among PD subgroups.  In sporadic PD patients who also had a typical smell deficit, sensitivity was 99%. In other subgroups, including those with preserved olfaction (63.0%) and carriers of the LRRK2 G1920S variant (67.5%), the proportion with a positive α-synuclein SAA was much lower. In LRRK2 variant carriers, this proportion mirrors the frequency of typical Lewy pathology seen at autopsy that has been reported.15  Taken together, these results suggest that the α-synuclein SAA is not only highly accurate, but also reveals molecular heterogeneity among patients who (with the exception of olfactory impairment) are clinically indistinguishable. 

The last important finding in the paper was that α-synuclein SAA was positive in a high proportion of so-called “prodromal” individuals with either isolated hyposmia or REM sleep behavior disorder. A number of NMCs of either LRRK2 or GBA were also positive, including those who had no physiological evidence of neurodegeneration (ie. normal dopamine transporter imaging). Combined, these findings suggest that presence of α-synuclein seeds in CSF detected by SAA is a very early event in pathophysiological progression and precedes symptoms and dopaminergic neurodegeneration.  

How will the results of this study affect therapeutic development and clinical care? Tools like α-synuclein seed amplification assay will allow researchers seeking to test interventions that target the pathology that underlies the vast majority of PD and DLB cases to do so in patients that actually have that pathology. In a future where therapies that target specific pathology are offered to patients as part of standard treatment, molecular characterization may become a foundational component of clinical care. 



1. Parnetti L, Gaetani L, Eusebi P, et al. CSF and blood biomarkers for Parkinson's disease. The Lancet Neurology 2019; 18(6): 573-86. 

2. Braak H, Del Tredici K, Rüb U, De Vos RA, Steur ENJ, Braak E. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiology of aging 2003; 24(2): 197-211. 

3. Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M. Alpha-synuclein in Lewy bodies. Nature 388(6645):839-40, 1997. 

4. Saborio GP, Permanne B, Soto C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 2001; 411(6839): 810-3. 

5. Shahnawaz M, Tokuda T, Waragai M, et al. Development of a Biochemical Diagnosis of Parkinson Disease by Detection of α-Synuclein Misfolded Aggregates in Cerebrospinal Fluid. JAMA Neurology 2017; 74(2): 163-72. 

6. Fairfoul G, McGuire LI, Pal S, et al. Alpha-synuclein RT-QuIC in the CSF of patients with alpha-synucleinopathies. Annals of Clinical and Translational Neurology 2016; 3(10): 812-8. 

7. Groveman BR, Orrù CD, Hughson AG, et al. Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC. Acta Neuropathologica Communications 2018; 6(1): 7. 

8. Shahnawaz M, Mukherjee A, Pritzkow S, et al. Discriminating α-synuclein strains in Parkinson’s disease and multiple system atrophy. Nature 2020; 578(7794): 273-7. 

9. Concha-Marambio L, Farris CM, Holguin B, et al. Seed Amplification Assay to Diagnose Early Parkinson's and Predict Dopaminergic Deficit Progression. Movement Disorders 2021; 36(10): 2444-6. 

10. Russo MJ, Orru CD, Concha-Marambio L, et al. High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson’s disease. Acta Neuropathologica Communications 2021; 9(1): 179. 

11. Rossi M, Candelise N, Baiardi S, et al. Ultrasensitive RT-QuIC assay with high sensitivity and specificity for Lewy body-associated synucleinopathies. Acta Neuropathol 2020; 140(1): 49-62. 

12. Iranzo A, Fairfoul G, Ayudhaya ACN, et al. Detection of α-synuclein in CSF by RT-QuIC in patients with isolated rapid-eye-movement sleep behaviour disorder: a longitudinal observational study. Lancet Neurol 2021; 20(3): 203-12. 

13. Concha-Marambio L, Weber S, Farris CM, et al. Accurate Detection of α-Synuclein Seeds in Cerebrospinal Fluid from Isolated Rapid Eye Movement Sleep Behavior Disorder and Patients with Parkinson's Disease in the DeNovo Parkinson (DeNoPa) Cohort. Mov Disord 2023; 38(4): 567-78. 

14. Siderowf A, Concha-Marambio L, Lafontant D-E, et al. Assessment of heterogeneity among participants in the Parkinson's Progression Markers Initiative cohort using α-synuclein seed amplification: a cross-sectional study. The Lancet Neurology 2023; 22(5): 407-17. 

15. Kalia LV, Lang AE, Hazrati LN, et al. Clinical correlations with Lewy body pathology in LRRK2-related Parkinson disease. JAMA Neurol 2015; 72(1): 100-5. 


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