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

Background Aggregation of alpha-synuclein (alpha-syn) is a prominent feature of Parkinson's disease (PD) and other synucleinopathies. Currently, alpha-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 alpha-syn in a patient-dependent manner, potentially undermining unoptimized alpha-syn SAAs and preventing seed quantification. Methods In this study, we characterized the inhibitory effect of CSF milieu on detection of alpha-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 alpha-syn. Results We found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on a-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric alpha-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-alpha-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary alpha-syn intermediates. We observed significantly slower amplification of alpha-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 alpha-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 alpha-syn aggregates. Conclusions Our results describe a novel interaction between lipoproteins and alpha-syn aggregates that inhibits the formation of alpha-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on alpha-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 alpha-syn quantification efforts.