Amyloid fibrils trigger the release of neutrophil extracellular traps (NETs), causing fibril fragmentation by NET-associated elastase.

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.

Dissecting the structure, thermodynamic stability, and aggregation properties of the A25T transthyretin (A25T-TTR) variant involved in leptomeningeal amyloidosis: identifying protein partners that co-aggregate during A25T-TTR fibrillogenesis in cerebrospinal fluid.

Deposition of amorphous aggregates and fibrils of transthyretin (TTR) in leptomeninges and subarachnoid vessels is a characteristic of leptomeningeal amyloidosis (LA), a currently untreatable cerebral angiopathy. Herein, we report the X-ray structure of the A25T homotetramer of TTR, a natural mutant described in a patient with LA. The structure of A25T-TTR is indistinguishable from that of wild-type TTR (wt-TTR), indicating that the difference in amyloidogenicity between A25T-TTR and wt-TTR cannot be ascribed to gross structural differences. Using pressure-induced dissociation of the tetramer, we show that A25T-TTR is 3 kcal/mol less stable than L55P-TTR, the most aggressive mutant of TTR described to date. After incubation for 15 days at 37 °C (pH 7.3), A25T-TTR forms mature amyloid fibrils. To mimic the environment in which TTR aggregates, we investigated aggregation in cerebrospinal fluid (CSF). Unlike L55P-TTR, A25T-TTR rapidly forms amyloid aggregates in CSF that incorporated several protein partners. Utilizing a proteomics methodology, we identified 19 proteins that copurified with A25T-TTR amyloid fibrils. We confirmed the presence of proteins previously identified to be associated with TTR aggregates in biopsies of TTR amyloidosis patients, such as clusterin, apolipoprotein E, and complement proteins. Moreover, we identified novel proteins, such as blood coagulation proteins. Overall, our results revealed the in vitro characterization of TTR aggregation in a biologically relevant environment, opening new avenues of investigation into the molecular mechanisms of LA.