Zhang, Weijia; (2023) Therapeutic opportunities for modulating gene expression in neurodegeneration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Alzheimer's disease (AD) and Parkinson’s disease (PD) are both devastating neurodegenerative conditions with no cure. In this thesis, I want to explore if gene expression can be modulated to rescue neurodegenerative phenotypes. This report is in two parts, looking at gene expression at the level of transcription and then, splicing. First, I demonstrate how effective CRISPR/Cas13d, a RNA guided, RNA targeting system, can be used to modulate alternative splicing of LILRB2, an AD related gene. I first show this in a HeLa Flp-in mini-gene model, then take this further by using antisense oligonucleotide (ASO) technology and apply it to a clinically relevant model using human induced pluripotent stem cell (hiPSC) derived neurons and microglia. Both approaches are examples of direct modulation of gene expression via disruption to the splicing mechanisms. In the second part of the thesis, I ask a more fundamental question - why is gene expression dysregulated in pathogenic mutations? I focus specifically on exploring the transcription machinery (RNAPII) and explore this in the context of SNCA gene and Parkinson’s disease. I first show data demonstrating the presence of nuclear α- synuclein in vitro and in human post-mortem brain tissue. I postulate that the species of nuclear α-synuclein could be dominated by oligomers. I then use hiPSC-derived dopaminergic neurons as a cellular model, coupled with a proteomic approach, using silica particle-assisted chromatin enrichment and mass spectrometry (SPACE) to show how mutations in the SNCA gene affect proteins co-localized with RNAPII. I show perturbed pathways in the mutant cells relating to mitochondrial dysfunction and lipid metabolism. Both are known to be associated with Parkinson’s pathology and more broadly, mechanisms of neurodegeneration. I observed reduction in RNAPII-binding of proteins in mutant cells, including those that are known to be associated with PD pathology. Taken together, these findings postulate how there may be both direct and indirect effects of α-synuclein on the transcriptional machinery, as modulators of gene 3 expression. These observations highlight potential druggable targets and pathways in Parkinson’s disease. Overall, I present therapeutic opportunities for modulating gene expression in neurodegenerative diseases. These new approaches may ultimately lead to new treatment options where it forms a part of a cocktail of targeted therapies, fine-tuning aberrant pathways.

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