Somal and axonal translation in Parkinson's dopamine neurons: Sequencing the translatome

Thesis

Nigrostriatal dopamine neurons have long, highly arborised axons, indicating a requirement for axonal translation. The axonal ‘translatome’ will reflect the neuronal response to local stressors that precedes cell death in Parkinson’s disease (PD) as well as revealing key transcriptional differences between protected and vulnerable dopaminergic populations. The translating ribosome affinity purification (TRAP) technique enables the capture of translating ribosomes specifically in dopaminergic neurons by expressing an enhanced green fluorescence protein (eGFP)-tagged ribosomal subunit driven by a cell type-specific promoter.

We have produced a novel transgenic mouse line by crossing a human alphasynuclein overexpression model, previously generated by our lab, with a dopamine transporter (DAT)-Cre driven TRAP line. This thesis describes the collection and analysis of translating mRNA from dopaminergic cell bodies and terminals of 3 and 18-month control and Parkinsonian mice for sequencing. The expression features that best distinguished dopaminergic neurons from neighbouring cell types were first identified. This information was used to prioritise genes located within linkage regions of PD GWAS loci. Novel candidate genes were identified, including the Calcium Sensing Receptor. Comparison of axonal and somal samples demonstrated compartmental specialisation, with autophagic function, MAPK signalling, and markers of axonal homeostasis preferentially expressed in axons. Comparison between dorsal and ventral striatal axons identified a series of genes with greater dorsal expression and prior evidence of a role in axonal degeneration. These genes were considered putative markers of selective vulnerability. With age, coordinated changes in expression were observed in genes related to autophagy, oxidative stress and endocytic recycling. These changes were specific to the dopaminergic neuron and can be used to better understand the role of each gene in dopaminergic function. Results from this thesis have been collated and summarised in the form of a public dopaminergic expression atlas.

Authors: Kilfeather, P

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English