Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.802345
Title: Phenotypic analysis of the CRISPR-engineered G51D α-synuclein rat
Author: West, Stephen Annan
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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Abstract:
Parkinson’s disease (PD) is a common neurodegenerative disease that is most often idiopathic, but a significant proportion (5-10%) of cases are caused by highly penetrant monogenic mutations. Point mutations and multiplications of the SNCA gene, encoding α-synuclein (αSyn), are known to cause early onset aggressive familial PD. The recently described G51D mutation is particularly aggressive resulting in symptoms and pathologies that span PD, dementia with Lewy bodies, and multiple system atrophy (MSA). The average age of onset for these patients is around 37 years of age and they rapidly develop motor symptoms and cognitive impairments. Post-mortem analysis revealed atrophy and neuronal loss in the frontal cortices, caudate and putamen and complete loss of tyrosine hydroxylase positive cells in the substantia nigra. aSyn inclusions are widespread and present both in neurons, as expected in PD, and oligodendrocytes, more reminiscent of MSA pathology. In order to model this familial PD mutation our lab we used CRISPR/Cas9 technology to introduce the G51D mutation into the endogenous rat SNCA gene. A 2-nucleotide mutation was introduced to codon-51 to change GGA (glycine) to GAT (aspartic acid). This created a new BspHI restriction site (TCATGG → TCATGA) that facilitated genotyping. αSyn is an intrinsically disordered protein, however the N-terminal two-thirds of the protein has been demonstrated to form an amphipathic α-helical structure upon interaction with membranes. The G51 residue lies within this N-terminal region, therefore the introduction of a charged aspartic acid residue is hypothesized to disrupt the ability of αSyn to form a helix upon membrane interaction. Previous in vitro work has supported this and shown that the introduction of the G51D mutation does result in a reduction in the helicity of αSyn when bound to membranes. This work has led to our hypothesis that the αSynG651D protein will no longer be able to interact with membranes at synapses in the rat brain and this could lead to pathogenic effects. To investigate this, confocal microscopy was used to compare the localisation of αSyn with proteins which localise to synapses in wild-type, SNCAG51D/+ and SNCAG51D/G51D rats. I observed that αSynG651D was significantly depleted from synapses in SNCAG51D/G51D rat brain at 12 months of age in the cortex and pons and there was a trend to mislocalisation from the synapse in the striatum and olfactory bulb. Total αSyn protein levels did not change in SNCAG51D/G51D rats, so I then investigated whether αSynG51D was ectopically localised to other intracellular organelles, namely the lysosome and mitochondria as both are implicated in PD pathology. αSyn was colocalised with the lysosome of all genotypes and there was no change between genotypes. The localisation of αSyn was negatively correlated with TOMM20, a mitochondria marker, in wild-type rats, and this changed to being positively correlated in SNCAG51D/G51D rat brain. However, the differences in mitochondrial localisation of αSyn in the different genotypes were not statistically significant. The density of tyrosine hydroxylase positive terminals in the striatum and cell bodies in the substantia nigra were investigated for all genotypes, and no evidence of cell loss was found in rats with the SNCAG51D mutation up to 12 months of age suggesting no overt neurodegeneration. In the absence of degeneration, early pathological changes were investigated. Human sporadic and G51D Parkinson’s disease shows changes to the inflammatory environment of the brain and so this was investigated in the G51D rats using FACS to isolate different immune cell types. There were no significant changes in immune cell numbers (microglia, T cells, monocyte-derived macrophages or neutrophils) at 12 months of age, however an increased number of monocyte-derived macrophages was observed in male, but not female, heterozygous SNCAG51D/+ rats compared to wild-type rats at 18 months in the ventral midbrain and striatum. To investigate any changes in gene expression due to the G51D mutation RNA-seq and quantitative mass spectrometry experiments were performed. The RNA-seq data revealed genes involved in long-term potentiation were dysregulated, suggesting the mislocalisation of αSyn was affecting synaptic function. To investigate this further, synaptosomes were isolated from the cortex and the proteomes of both the synapse and cortex of different genotypes were compared by Tandem Mass Tag (TMT) mass spectrometry. In the cortex, Kyoto Encyclopedia of genes and genomes (KEGG) pathway analysis showed there was a dysregulation of proteins involved in Parkinson’s disease, synaptic and mitochondrial function in αSynG51D/+ mutants. The synaptosome data revealed there was a dysregulation of proteins involved in synaptic and mitochondrial function and an increase in complement proteins suggesting that dysfunctional synapses could be targeted for degradation. Together these results demonstrate the αSynG51D/+ mutant rats exhibit molecular changes that may represent some of the earliest pathological events during synucleinopathy prior to the onset of neurodegeneration.
Supervisor: Kunath, Tilo ; Sieger, Dirk Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.802345  DOI:
Keywords: Parkinson’s disease ; a-synuclein ; SNCA gene ; G51D mutation ; CRISPR/Cas9
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