AbstractParkinson’s disease (PD) is a neurodegenerative disease characterised by accumulation of α-synuclein protein and loss of midbrain dopaminergic (mDA) neurons of the Substantia nigra (SN), resulting in motor dysfunction. Mutations and multiplications of the SNCA locus, which encodes α-synuclein, have been associated with familial Parkinson’s Disease. Using lentiviruses to overexpress wild-type and mutant α-synuclein in mDA neuron models, the transcriptomic and functional changes associated with increased α-synuclein burden were investigated.
The SH-SY5Y cell line was employed to study gene expression, by profiling actively translated mRNAs isolated by translating ribosome affinity purification (TRAP). Differential expression analysis in α-synuclein overexpressing cells identified actin cytoskeleton, mitochondrial and oxidative stress response proteins, as well as a key protein involved in miRNA biogenesis, DGCR8, to be key pathways affected by α-synuclein overexpression. Validation using molecular biology techniques was able to partially validate some of these targets.
mDA neurons derived from human induced pluripotent stem cells (iPSC) were employed to study functional phenotypes, differentiation capacity and neurite dynamics, in response to overexpression of wild-type and mutant α-synuclein. Whilst the capacity for iPSC cells to differentiate into mDA neurons expressing mature neuronal and dopaminergic markers was not affected, both neurite extension and axonal branching was seen to be reduced in α-synuclein overexpressing cultures. Furthermore, these models were used to interrogate alterations in expression of microRNAs (miRNA) associated with mDA function and PD pathogenesis. Intracellular miRNA analysis resulted in the identification of differential expression of miRs-34b, -34c, -155 and -494, known to be associated with regulation of neuroinflammation, oxidative stress response and post-transcriptional regulation of α-synuclein mRNA.
The data presented implicates several pathways, genes and miRNA that are perturbed in response to α-synuclein overexpression. Further study of these pathways will contribute to our understanding of mechanisms underlying α-synuclein pathology and may represent potential therapeutic targets for PD.
|Date of Award||7 May 2019|
|Supervisor||James B Uney (Supervisor) & Liang-Fong Wong (Supervisor)|
Investigating synucleinopathies using translating ribosome affinity purification in cellular Parkinson’s disease models
Khazim, M. (Author). 7 May 2019
Student thesis: Doctoral Thesis › Doctor of Philosophy (PhD)