Examination of gene expression using translating ribosome affinity purification (TRAP) in genetic models of Parkinson's disease

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Parkinson's disease (PD) is the second most common neurodegenerative condition, affecting approximately 1% of individuals over the age of 60. Development of disease-modifying treatments is required to target early stages of PD pathology and prevent or delay progression. This development requires characterisation of the early cellular mechanisms that precede and are associated with subsequent neurodegeneration.PD involves the selective death of dopaminergic neurons projecting to the dorsal striatum. Dopaminergic neurons projecting to the ventral striatum are largely spared. The cellular and genetic processes responsible for this differential sensitivity are not understood. The current hypothesis proposes that there is a differential regulation of gene expression at the axon level in DA neurons between dorsal and ventral striata.This project will use transgenic rodent models of PD to compare gene expression between dopaminergic neurons of the dorsal and ventral striatum. We will use a newly developed method ('TRAP') to purify translating ribosomes selectively from dopaminergic neurons. TRAP involves expression of enhanced green fluorescent protein (eGFP) tagged ribosomal protein L10a. eGFP-L10a tags translating ribosomes, enabling purification by immunoprecipitation. eGFP-L10a will be selectively expressed in dopaminergic neurons by placing it under the control of a dopamine transporter promoter. This will give a more accurate readout of expression compared to other transcriptomic methods.The project aims to demonstrate whether there is local mRNA translation at the axon level of dopaminergic neurons, whether the signature of expression differs between dorsal and ventral striata, whether this expression is altered in genetic models of PD and whether this changes with aging. Results from investigating gene expression can then be used to guide functional studies of dopaminergic neuron function. The results derived from this project will also provide a neurological precedent for application of TRAP gene expression profiling to explore origins of other neurodegenerative conditions, including Alzheimer's disease, amyotrophic lateral sclerosis and Huntington's disease.