Neutrophil Extracellular Traps Mediate in vitro and in vivo Degradation of α-Synuclein Amyloid Fibrils

Thyago R Cardim-Pires,1,2,* Ariele da Silva Martins,1,* Fernanda Verdini Guimarães,3 Thayana Roberta Ferreira De Mattos,1 Eva Lepinay,2 Martine Saint-Pierre,2 Francesca Cicchetti,2,4 Elvira M Saraiva,5 Patrícia Machado Rodrigues Silva,3 Debora Foguel11Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; 2Centre de Recherche du CHU de Québec, Axe Neurosciences, Québec, QC, Canada; 3Laboratório de Inflamação, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brasil; 4Département de Psychiatrie & Neurosciences, Faculté de Médecine, Université Laval, Québec, QC, Canada; 5Departamento de Imunologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, RJ, Brasil*These authors contributed equally to this workCorrespondence: Debora Foguel, Universidade Federal do Rio de Janeiro, Av Carlos Chagas Filho, 373 – Bloco E – sala E42- Cidade Universitária, Rio de Janeiro, RJ, CEP: 21941-902, Brazil, Tel +55 21 39386761, Email [email protected]: Neutrophil extracellular traps (NETs) are chromatin-based structures decorated with neutrophil enzymes such as elastase and myeloperoxidase (MPO). We previously demonstrated that amyloid fibrils (AFs), regardless of their protein composition, induce NET release in human neutrophils in vitro through a mechanism dependent on reactive oxygen species generated by NADPH oxidase 2 (NOX2). In this study, we investigated whether α-synuclein amyloid fibrils (αSF) induce NET formation in vivo and whether NETs modulate amyloid-associated pathology.Methods: αSF were instilled intratracheally into wild-type (WT) and gp91phox knockout (KO) mice, which lack NOX-2 activity and are unable to form NETs. Both male and female mice were analyzed. Eight hours post-instillation, lungs were assessed for neutrophil infiltration, NET formation (citrullinated histones and myeloperoxidase), amyloid fibril persistence (Congo red staining), and lung mechanics (elastance and resistance).Results: αSF induced robust neutrophil recruitment and lung inflammation in both WT and KO mice. However, NET formation was detected exclusively in WT animals. Congo red–positive amyloid-like structures persisted in KO lungs but were largely absent in WT mice, indicating NET-associated proteolysis promotes fibril clearance. Functionally, αSF impaired lung elastance in both genotypes at early time points. As fibrils were cleared in WT mice, lung function recovered. In contrast, KO animals exhibited persistent fibril deposition and sustained elastance impairment. Additionally, NET structures were observed in post-mortem brain tissue from patients with Parkinson’s disease.Conclusion: These findings demonstrate that NETs act as an innate immune mechanism capable of sensing and degrading amyloid fibrils in vivo, thereby promoting their clearance and preserving tissue function. This mechanism may play a relevant role in modulating amyloid-associated pathology, including in human neurodegenerative diseases such as Parkinson’s disease.Keywords: amyloid fibrils, amyloidolysis, neutrophils, NADPH oxidase 2, Parkinson’s disease