Abstract
Mutations in the SNCA gene cause autosomal dominant Parkinson's disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small β-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34-41 post differentiation. Once midbrain identity fully developed, at day 48-62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.
| Original language | English |
|---|---|
| Article number | 162 |
| Number of pages | 22 |
| Journal | npj Parkinson's Disease |
| Volume | 8 |
| Issue number | 1 |
| Early online date | 24 Nov 2022 |
| DOIs | |
| Publication status | Published - 24 Nov 2022 |
Bibliographical note
FundingOpen Access funding provided by The Francis Crick Institute.
Acknowledgements
We would wish to thank the patients for the fibroblast donation. We would also like to thank the Francis Crick Institute Flow Cytometry, Advanced Light Microscopy, Advanced Sequencing, and Bioinformatics and Biostatistics STPs for their help and equipment in conducting and analysing the flow cytometry, fluorescence microscopy, and single-cell RNA-seq experiments. This research was funded in whole or in part by Aligning Science Across Parkinson’s [ASAP-000509 and ASAP-000463] through the Michael J. Fox Foundation for Parkinson’s Research (MJFF). For the purpose of open-access, the author has applied a CC public copyright license to all Author Accepted Manuscripts arising from this submission. G.S.V. acknowledges funding from the UCL-Birkbeck MRC DTP. S.G. acknowledges funding from the i2i G.S. Virdi et al. 21 Published in partnership with the Parkinson’s Foundation npj Parkinson’s Disease (2022) 162 grant (The Francis Crick Institute), MJFox foundation, the Wellcome Trust, and is an MRC Senior Clinical Fellow [MR/T008199/1]. D.A. is funded by the National Institute for Health Research. R.P. holds an MRC Senior Clinical Fellowship [MR/S006591/1] and a Lister Research Prize Fellowship. H.A. acknowledges funding from the Kuwait University, Kuwait. M.H. acknowledges funding from UCB Biopharma, and Dr. Jim Love. N.P. acknowledges funding from Medical Research Scotland [PHD-50193–2020]. This work is supported by the Francis Crick Institute which receives funding from the UK Medical Research Council, Cancer Research UK, and the Wellcome Trust. Research at UCL Great Ormond Street Institute of Child Health benefits from funding from the NIHR Biomedical Research Centre at Great Ormond Street Hospital.
Data Availability Statement
Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41531-022-00423-7.
Access to Document
- Virdi_etal_NPJPD_Protein_Aggregation_And_VoR
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