Endogenous Levels of Alpha-Synuclein Modulate Seeding and Aggregation in Cultured Cells

Eftychia Vasili,Leonidas Stefanis,Manuel Flores-León,Mohammed Al-Azzani,Antonio Dominguez-Meijide,Angeliki Kanellidi,Ronald Melki,Tiago Fleming Outeiro

doi:10.1007/s12035-021-02713-2

Abstract

Parkinson’s disease is a progressive neurodegenerative disorder characterized by the accumulation of misfolded alpha-synuclein in intraneuronal inclusions known as Lewy bodies and Lewy neurites. Multiple studies strongly implicate the levels of alpha-synuclein as a major risk factor for the onset and progression of Parkinson’s disease. Alpha-synuclein pathology spreads progressively throughout interconnected brain regions but the precise molecular mechanisms underlying the seeding of alpha-synuclein aggregation are still unclear. Here, using stable cell lines expressing alpha-synuclein, we examined the correlation between endogenous alpha-synuclein levels and the seeding propensity by exogenous alpha-synuclein preformed fibrils. We applied biochemical approaches and imaging methods in stable cell lines expressing alpha-synuclein and in primary neurons to determine the impact of alpha-synuclein levels on seeding and aggregation. Our results indicate that the levels of alpha-synuclein define the pattern and severity of aggregation and the extent of p-alpha-synuclein deposition, likely explaining the selective vulnerability of different cell types in synucleinopathies. The elucidation of the cellular processes involved in the pathological aggregation of alpha-synuclein will enable the identification of novel targets and the development of therapeutic strategies for Parkinson’s disease and other synucleinopathies.

Highlights

Misfolding and aggregation of aSyn into large intraneuronal inclusions called Lewy bodies (LBs) and Lewy neurites (LNs) is a common feature and a defining hallmark of Parkinson’s disease (PD) and other related neurodegenerative diseases known as synucleinopathies [1]
The overall objective of our study was to investigate the effect of extracellular aSyn preformed fibrils (PFFs) on the seeding and aggregation of endogenous aSyn expressed at variable levels in cells
Immunofluorescence analyses of treated cells showed that PFFs induced the formation of aSyn inclusions that were EGFP-positive, confirming the seeding of the endogenously expressed aSynEGFP by the PFFs (Fig. 1B), as this was not observed in PBS-treated cells

Summary

Introduction

Materials and MethodsMisfolding and aggregation of aSyn into large intraneuronal inclusions called Lewy bodies (LBs) and Lewy neurites (LNs) is a common feature and a defining hallmark of Parkinson’s disease (PD) and other related neurodegenerative diseases known as synucleinopathies [1]. ASyn pathology seems to progress throughout interconnected brain regions in a pattern consistent with the “prion hypothesis” for the spreading of protein pathology [2]. Cell-to-cell transmission of aSyn pathology involves the conversion of the endogenous protein into a pathological conformation, propagating the pathology. Mounting evidence suggests that the intrinsically disordered monomeric aSyn assembles into small soluble oligomers, with increased β-sheet content that are prone to further aggregation and that may, along the way, result in the formation of toxic species [3, 4]. The injected animals develop aggregates containing endogenous aSyn phosphorylated on Ser129 in brain regions directly or indirectly connected with the injection site, time-dependent neuronal loss, neuroinflammation, and behavioral alterations [5]

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Conclusion