Abstract
Aggregation of α-synuclein is associated with neurodegeneration and a hallmark pathology in synucleinopathies. These aggregates are thought to function as prion-like particles where the conformation of misfolded α-synuclein determines the traits of the induced pathology, similar to prion diseases. Still, little is known about the molecular targets facilitating the conformation-specific biological effects, but their identification could form the basis for new therapeutic interventions. High-throughput screening of annotated compound libraries could facilitate mechanistic investigation by identifying targets with impact on α-synuclein aggregation. To this end, we developed a FRET-based cellular reporter in HEK293T cells, with sensitivity down to 6.5 nM α-synuclein seeds. Using this model system, we identified GF109203X, SB202190, and SB203580 as inhibitors capable of preventing induction of α-synuclein aggregation via inhibition of p38 MAPK and PKC, respectively. We further investigated the mechanisms underlying the protective effects and found alterations in the endo-lysosomal system to be likely candidates of the protection. We found the changes did not stem from a reduction in uptake but rather alteration of lysosomal abundance and degradative capacity. Our findings highlight the value high-throughput screening brings to the mechanistic investigation of α-synuclein aggregation while simultaneously identifying novel therapeutic compounds.
Highlights
Alpha-synuclein (α-syn) aggregation is a common feature of synucleinopathies, a group of neurodegenerative diseases including Parkinson’s disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB)
The final reporter cell line was selected based on normalized fluorescence resonance energy transfer (FRET) intensity (FRET means fluorescence intensity multiplied by %FRET positive) and signal-to-noise ratio of the FRET signal as detected by flow cytometry
We focused our initial screen on kinase inhibitors, as kinases have been implicated in various aspects of PD pathology
Summary
Alpha-synuclein (α-syn) aggregation is a common feature of synucleinopathies, a group of neurodegenerative diseases including Parkinson’s disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB). Α-syn has been shown to take on various conformations upon misfolding, which in turn could alter the disease etiology similar to bona fide prion diseases Such structurally distinct fibrils have been produced in cell-free conditions and were shown to possess a structure-dependent spread of pathology and detrimental effects [10]. Differences in biological activity are observed for aggregated α-syn isolated from PD and MSA patient brains, where MSA-derived aggregates display a higher seeding efficacy [11] These conformational and function differences of such pathological aggregates may form as a consequence of the local environment of the affected cell types, as recently reported [12]
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