Establishment of a human induced pluripotent stem cell neuronal model for identification of modulators of A53T αsynuclein levels and aggregation - Archive ouverte HAL Access content directly
Journal Articles PLoS ONE Year : 2021

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

(1) , (2) , (3, 4) , (3, 5) , (1) , (2) , (1)
1
2
3
4
5

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 αsynuclein 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.
Fichier principal
Vignette du fichier
Vajhoj et al Lundbeck Plos One 2021.pdf (2.74 Mo) Télécharger le fichier
Origin : Files produced by the author(s)

Dates and versions

cea-03500527 , version 1 (22-12-2021)

Identifiers

Cite

Charlotte Vajhøj, Benjamin Schmid, Ania Alik, Ronald Melki, Karina Fog, et al.. Establishment of a human induced pluripotent stem cell neuronal model for identification of modulators of A53T αsynuclein levels and aggregation. PLoS ONE, 2021, 16 (12), pp.e0261536. ⟨10.1371/journal. pone.0261536⟩. ⟨cea-03500527⟩
13 View
14 Download

Altmetric

Share

Gmail Facebook Twitter LinkedIn More