Virdi, Gurvir Singh; (2021) Using induced pluripotent stem cells to delineate the role of midbrain dopaminergic neurons in Parkinson's disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterised by the loss of midbrain dopaminergic neurons (mDA) in the substantia nigra. To date, there have been no successful disease modulating therapies, likely due to the lack of robust relevant human models to study the pathogenesis of the disease. Directed differentiation of hiPSCs into region-specific cell subtypes is critical to understanding disease mechanisms. However, the generation of pure midbrain dopaminergic (mDA) neurons to model PD has remained a significant challenge, and protocols are variable in enrichment, cost and time efficiency. I developed rapid and efficient specification of highly enriched mDA neurons (>80%) utilising small molecules. Single-cell RNA-sequencing reconstructed the temporal transcriptomic sequence of cellular fates, confirming the development trajectory of neural precursor cells (NPC) into mDA neurons. Within 4 weeks of terminal differentiation the mDA neurons synthesise and secrete dopamine, exhibit electrical activity, and form functional synapses and networks in vitro. The protocol was used to model PD, by using two sets of familial PD mutations. Mutations in SNCA cause autosomal dominant PD, through poorly defined mechanisms. I investigated the temporal sequence of pathophysiological events in mDA synucleinopathies. Adopting super resolution approaches, I discovered the earliest accumulation of abnormal protein assemblies, in the form of beta-sheet rich oligomeric aggregates of alpha-synuclein. Toxic aggregates of alpha-synuclein subsequently lead to early calcium dysregulation and calcium buffering. This is then followed by abnormalities in mitochondrial calcium, impaired respiration, oxidative stress, and at later stages, up-regulation of autophagy, and ultimately cell death. Similarly, mutations in PINK1 cause autosomal recessive PD. Kinase activity of PINK1 is abolished in these neurons, and as a consequence they displayed hallmark mitochondrial dysfunction, characterised by complex I dysregulation, an increase in mitochondrial ROS production and an abnormal mitochondrial PTP opening. Super resolution microscopy revealed that the neurons have a fragmented mitochondrial network. Finally, regardless of the abundant mitochondrial dysfunction, PINK1 PD neurons are still able to degrade their mitochondria via mitophagy. The rapid and efficient differentiation paradigm to generate enriched mDA neurons from hiPSCs provides a robust human cellular model for PD.

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