Zebrafish as a translational model of Parkinson’s disease – a study of microRNAs

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, currently there are no disease modifying treatments. It is thought that microRNAs may be implicated in pathogenesis of PD. In this thesis the function of three microRNAs, which may be relevant to PD, were investigated in zebrafish. These miRNAs are: miR-133b, thought to be involved in development of dopaminergic neurons through its target PITX3; miR-205, which has been shown to regulate the PD-associated gene LRRK2; and miR-155, a key regulator of immune and inflammatory processes. Despite being more evolutionarily distant to humans than rodent models, the zebrafish has emerged as an effective model of human disease due it its low cost, fast development and transparency. A further aim of this thesis was to develop a high throughput imaging strategy to assess dopaminergic neuron number in the developing zebrafish brain, which can be used as a screening tool to identify potentially therapeutic compounds.
The effect of both miR-133b and miR-205 knockdown on the number of dopaminergic neurons was investigated. In wild type zebrafish miR-205 knockdown causes a slight but significant decrease in dopaminergic neurons whereas miR-133b knockdown causes a significant increase in dopaminergic neuron number. In a previously established zebrafish model of PD, miR-133b knockdown completely rescued a 15% decrease in dopaminergic neuron number. The expression of miR-155 was assessed in three different zebrafish models of PD. While no change was observed in pink1- or parkin-deficient zebrafish, an increase in this miRNA and related inflammatory markers was observed in both larvae and juvenile brains of gba1-deficient zebrafish. As this miRNA and inflammatory markers are shown to be upregulated before any neuron loss is detectable in these zebrafish, it is possible that miR 155-related inflammation contributes to neurodegeneration. A stable miR-155 mutant line was also generated which will be used to determine whether genetic ablation of this miRNA has a protective effect in gba1-deficient zebrafish.
A fluorescent reporter line expressing GFP in monoaminergic neurons has been validated for study of dopaminergic neurons. A fluorescence-based method developed using this line was able to reproduce data using a previously established in situ hybridisation-based method for neuron counting. To allow for more time and cost effective mounting of embryos for imaging, a mould has been developed. Use of this mould to mount embryos allows for high throughput imaging of the developing brain.
The miRNAs presented in this study may have therapeutic potential in PD through modulation of dopaminergic neuron survival or by regulating the expression of PD-associated genes. The development of a high throughput screening process may allow a PD-related drug or toxin screen to be performed in our group in the near future.