Seed amplification assays for diagnosing synucleinopathies: the issue of influencing factors

Bellomo, G.; Paciotti, Silvia; Gatticchi, Leonardo; Rizzo, Domenico; Paoletti, Federico Paolini; Fragai, Marco; Parnetti, Lucilla

doi:10.52586/5010

Cited by 12 publications

(9 citation statements)

References 62 publications

(106 reference statements)

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“…The inflection point of the second elongation phase (t2) was estimated using a double-sigmoidal fitting and correlated with the log mass of synthetic seeds (Supplementary MaterialFig. S 3 ), in agreement with previous literature [ 1 , 12 ]. To further characterize our experimental conditions in PBS, we analysed samples from both plateaus of the aggregation curve by TEM.…”

Section: Resultssupporting

confidence: 84%

“…CSF inhibition of α-syn aggregation in SAAs remains poorly characterized, although it has been already reported in the literature [ 1 , 2 , 11 , 12 ]. We spiked 20 fg of synthetic α-syn aggregates (synthetic seeds) in CSF of healthy control subjects (HC) to determine if the CSF inhibition is similar and intrinsic to all CSFs or if it is subject dependent.…”

Section: Resultsmentioning

confidence: 99%

“…After the addition of 20 fg α-syn seeds, all the NC CSF samples tested SAA-positive. Considering the fact that SAA time variables (TTT and T50) should be theoretically linearly related to the logarithm of α-syn seed mass [ 12 ], we preferred to use Log2-transfomed total protein, ApoA1 and ApoE levels to calculate correlation coefficients and to perform linear regressions. However, similar results were found when logarithm transformation was not applied.…”

Section: Resultsmentioning

confidence: 99%

“…It remains unknown if disease progression correlates with the amount of seeds in a CSF sample, but if it does, estimation of α-syn seed levels by kinetic parameters will need models to control for CSF components such as apolipoproteins or total protein as a surrogate measure. Indeed, in the absence of matrix effects, TTT and T50 should be linearly associated with the logarithm of the aggregated α-syn mass [ 12 ]. The addition of CSF lipoprotein/total protein content as a covariate can restore this linear relationship, thus allowing the quantification of aggregated α-syn mass.…”

Section: Discussionmentioning

confidence: 99%

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Cerebrospinal fluid lipoproteins inhibit α-synuclein aggregation by interacting with oligomeric species in seed amplification assays

Bellomo

Paciotti

Concha‐Marambio

et al. 2023

Mol Neurodegeneration

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Background Aggregation of α-synuclein (α-syn) is a prominent feature of Parkinson’s disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification. Methods In this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn. Results We found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates. Conclusions Our results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts.

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Cerebrospinal fluid lipoproteins inhibit α-synuclein aggregation by interacting with oligomeric species in seed amplification assays

Bellomo

Paciotti

Concha‐Marambio

et al. 2023

Mol Neurodegeneration

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“…Under our experimental condition, higher diluted (10 −5 ) skin samples of PD cases seem to induce more positive reactions in α-Syn RT-QuIC than lower diluted (10 −3 and 10 −4 ) ones. Higher concentration of samples but lower capacity in the aggregation of α-Syn may reflect that some unidentified components in the tested samples are not conducive to the nucleation and growth of α-Syn aggregates (Bellomo et al, 2021). Less seeding activity of larger quantity of CSF of sCJD cases is also observed in prion RT-QuIC (Xiao et al, 2018).…”

Section: Discussionmentioning

confidence: 95%

Application of α-Syn Real-Time Quaking-Induced Conversion for Brain and Skin Specimens of the Chinese Patients With Parkinson’s Disease

Chen

Jiao²,

Huang³

et al. 2022

Front. Aging Neurosci.

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The real-time quaking-induced conversion (RT-QuIC) assay has been developed and used as an in vitro diagnostic tool for Parkinson’s disease (PD). In this study, we established α-Syn RT-QuIC using recombinant human α-Syn as the substrate. All 5 brain homogenates of neuropathological PD cases and 13 skin homogenates of clinical PD cases showed positive results, whereas all the samples of negative controls remain negative. Meantime, randomly selected 6 skin samples of PD cases and 6 skin samples of sCJD cases showed negative in opposite prion RT-QuIC and α-Syn RT-QuIC. Our α-Syn RT-QuIC showed dose-dependent manner between the lag times and peak ThT fluorescent values. Additionally, the detecting limitation was about 10–7 dilution for brain tissues and 10–6 for skins. Those data indicate a reliable specificity and good sensitivity of the established α-Syn RT-QuIC in identifying and amplifying the misfolded α-Syn in brain and skin tissues of patients with PD.

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Investigating alpha‐synuclein co‐pathology in Alzheimer's disease by means of cerebrospinal fluid alpha‐synuclein seed amplification assay

Bellomo,

Toja,

Paolini Paoletti

et al. 2024

Alzheimer's & Dementia

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INTRODUCTIONLewy body disease, a frequently observed co‐pathology in Alzheimer's disease (AD), can be identified antemortem in cerebrospinal fluid (CSF) by α‐synuclein seed amplification assay (αS‐SAA). The prevalence and clinical impact of CSF αS‐SAA positivity in AD are still unknown.METHODSαS‐SAA was performed on CSF samples from 240 AD patients (preclinical, prodromal, and dementia stages), 85 controls, 84 patients with Parkinson's disease (PD), and 21 patients with PD with dementia or dementia with Lewy bodies. In AD patients, associations between αS‐SAA positivity and cognitive changes were also evaluated.RESULTSIn agreement with available neuropathological studies, αS‐SAA positivity was observed in 30% of AD patients (vs 9% in controls), and was associated with cognitive decline, visuospatial impairment, and behavioral disturbances.DISCUSSIONαS‐SAA positivity in AD patients reflects the prevalence observed in neuropathological series and is associated with a worse clinical outcome. These data confirm the validity of CSF αS‐SAA positivity as biomarker of synucleinopathy.

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Seed amplification assays for diagnosing synucleinopathies: the issue of influencing factors

Cited by 12 publications

References 62 publications

Cerebrospinal fluid lipoproteins inhibit α-synuclein aggregation by interacting with oligomeric species in seed amplification assays

Cerebrospinal fluid lipoproteins inhibit α-synuclein aggregation by interacting with oligomeric species in seed amplification assays

Application of α-Syn Real-Time Quaking-Induced Conversion for Brain and Skin Specimens of the Chinese Patients With Parkinson’s Disease

Investigating alpha‐synuclein co‐pathology in Alzheimer's disease by means of cerebrospinal fluid alpha‐synuclein seed amplification assay

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