Establishment of a human induced pluripotent stem cell neuronal model for identification of modulators of A53T α-synuclein levels and aggregation

Charlotte Vajhøj,Ronald Melki,Ania Alik,Karina Fog,Tina Charlotte Stummann,Benjamin Schmid,Bjørn Holst,Stephan N Witt

doi:10.1371/journal.pone.0261536

Abstract

Inhibiting formation or promoting degradation of α-synuclein aggregates are among the therapeutical approaches under investigation as disease-modifying treatment strategies for Parkinson’s disease. To support these developments, several in vitro models based on seeded α-synuclein aggregation have been established in immortalized cell lines and murine primary neurons. Here, we report on a humanized model with a reproducibility and throughput that enables its use in supporting target identification and validation in pharmacological research. A human induced pluripotent stem cell (iPSC) line was genetically modified to express HA-tagged α-synuclein with the point mutation in position 53 from Alanine to Threonine (A53T) under an inducible system and differentiated into cortical neurons expressing neuronal markers and exhibiting spontaneous activity. Intracellular α-synuclein aggregation was triggered by exposure to exogenous added fibrillated recombinant wild-type human α-synuclein fibrils91 and demonstrated by several endpoints; the formation of Triton-insoluble SDS-soluble α-synuclein, biochemically in a fluorescence resonance energy transfer based aggregation assay and by immunocytochemistry of phosphorylated α-synuclein positive puncta. We demonstrate the feasibility of upscaling the iPSC neuron production for drug discovery and that the model has a suitable dynamic range allowing for both detection of increased and decreased α-synuclein aggregation. Moreover, gene modulation is feasible using siRNAs, making the model suitable for genetic screening for modulators of α-synuclein aggregation. Data on effects of USP8, USP13 and USP9X knockdown on α-synuclein expression and aggregation contradicts published data from immortalized cell lines and murine systems. This highlight the importance of including humanized neuronal models in the confirmation of biological mechanisms in specific variations of Parkinson’s disease.

Highlights

Targeting α-synuclein is currently one of the most pursued disease-modifying treatment strategies for Parkinson’s disease
The development of cellular models, where addition of exogenous pathological α-synuclein fibrils to immortalized cell lines or murine primary neurons leads to misfolding, aggregation and phosphorylation of endogenous α-synuclein, has provided valuable model systems capturing key pathological phenotypes of Parkinson’s disease [2,3,4]
We decided for A53T α-synuclein, which is more aggregation prone than wildtype α-synuclein [18], to support the establishment of an α-synuclein seeding with a robust assay window

Summary

Introduction

Targeting α-synuclein is currently one of the most pursued disease-modifying treatment strategies for Parkinson’s disease. Toxic α-synuclein species have been shown to disrupt important cellular functions, and inhibiting formation or promoting degradation of α-synuclein aggregates are among the therapeutical approaches under investigation [1]. The development of cellular models, where addition of exogenous pathological α-synuclein fibrils to immortalized cell lines or murine primary neurons leads to misfolding, aggregation and phosphorylation of endogenous α-synuclein, has provided valuable model systems capturing key pathological phenotypes of Parkinson’s disease [2,3,4]. Models based on dopaminergic neurons derived from Parkinson’s patient iPSCs have demonstrated higher α-synuclein protein levels and increased phosphorylation of α-synuclein at serine residue 129 (pS129) compared to controls [5, 6].

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Results

Conclusion