citations

                    Phenotypic manifestation of a-synuclein strains derived from Parkinson's disease and multiple system atrophy in human dopaminergic neurons
                

Authors:

Tanudjojo B, Shaikh SS, Fenyi A, Bousset L, Agarwal D, Marsh J, Zois C, Heman-Ackah S, Fischer R, Sims D, Melki R, Tofaris GK

In:

Source: Nat Commun.
Publication Date: (2021)
Issue: 12 (1): 3817

Research Area:

Neurobiology

Cells used in publication:

293T: Species: human; Tissue Origin: kidney
Induced Pluripotent Stem Cell (iPS), human: Species: human; Tissue Origin:

Platform:

4D-Nucleofector® X-Unit

Experiment

To knock out DJ-1, the SNCATRIP iPSC clone SFC831-03-03 was nucleofected with the CRISPR complexes formed by the two sgRNAs targeting either exon 2 or exon 4 and the HiFi Cas9 protein. Single-guide RNAs for genome editing were dissolved in DNAse- and RNAse-free buffer to a concentration of 100 mM. One microliter of each sgRNA was mixed with 2 µL of HiFi Cas9 protein (IDT, Cat # 1081067) to form the active CRISPR/Cas9 complex. iPSCs were cultured in six-well plates coated with Matrigel (Corning Bioscience) in E8 medium until they reached a density of 70–90%. iPS cells were detached using Accutase (Gibco) and a total of 1.5×10^6 cells were co-nucleofected with the CRISPR/Cas9 complex. For the nucleofection, the P3 Primary Cell Kit (Lonza) was used and run with the programme CA-167.

Abstract

a-Synuclein is critical in the pathogenesis of Parkinson’s disease and related disorders, yet it remains unclear how its aggregation causes degeneration of human dopaminergic neurons. In this study, we induced a-synuclein aggregation in human iPSC-derived dopaminergic neurons using fibrils generated de novo or amplified in the presence of brain homogenates from Parkinson’s disease or multiple system atrophy. Increased a-synuclein monomer levels promote seeded aggregation in a dose and time-dependent manner, which is associated with a further increase in a-synuclein gene expression. Progressive neuronal death is observed with brain-amplified fibrils and reversed by reduction of intraneuronal a-synuclein abundance. We identified 56 proteins differentially interacting with aggregates triggered by brain-amplified fibrils, including evasion of Parkinson’s disease-associated deglycase DJ-1. Knockout of DJ-1 in iPSC-derived dopaminergic neurons enhance fibril-induced aggregation and neuronal death. Taken together, our results show that the toxicity of a-synuclein strains depends on aggregate burden, which is determined by monomer levels and conformation which dictates differential interactomes. Our study demonstrates how Parkinson’s disease-associated genes influence the phenotypic manifestation of strains in human neurons.

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