Exploring receptors involved in the intracellular uptake of fibrillated α–synuclein

Abstract

Neurodegeneration of Parkinson disease (PD) is thought to be caused by intracellular accumulation of misfolded, toxic α–synuclein (αS). Recently, prionoid hypothesis, which proposes that aggregated αS can transfer from cell–to–cell and spread pathological changes to surrounding tissues, has become widely accepted. Although the molecular mechanisms involved in the transcellular spreading of aggregated αS remain unclear, uptake via endocytosis has been postulated. Several molecules have been reported as endocytic receptors for αS, but most of them are not comprehensively explored, and most of them are poorly expressed in neurons. In addition, surfactants used in the isolation and purification process of receptor proteins may affect the screening results by interfering with the physiological structure of the receptors and their ligand–binding ability. To overcome these situations, we prepared a whole mouse brain–derived membrane protein library without surfactant, and combined affinity column chromatography and mass spectrometry to comprehensively search for fibrillar αS receptors. After secondary screening of the identified molecules by co–immunoprecipitation using cultured cells, sortilin, which is highly expressed in neurons, was found to strongly bind fibrillar forms of αS. The internalization of fibrillar αS into cells depends on the expression level of sortilin on the cell surface, and time–lapse imaging confirmed the internalization of fibrillar αS and sortilin and their transport into endosomes. Furthermore, pretreatment with sortilin–specific antibody significantly inhibited the uptalke of fibrillar αS. Finally, immunohistochemistry confirmed the presence of sortilin in the core structure of Lewy bodies and GCI in the patient's brain. We hope that the discovery of a novel function of sortilin in the formation of αS pathology will lead to further understanding of the pathogenesis of PD and provide hints for the development of disease–modifying therapy.