Functionally Validating Evolutionary Conserved Risk Genes for Parkinson's Disease in Drosophila melanogaster

Abstract

Parkinson's disease (PD) is a heterogeneous and complex neurodegenerative disorder and large-scale genetic studies have identified >130 genes associated with PD. Although genomic studies have been decisive for our understanding of the genetic contributions underlying PD, these associations remain as statistical associations. Lack of functional validation limits the biological interpretation; however, it is labour extensive, expensive, and time consuming. Therefore, the ideal biological system for functionally validating genetic findings must be simple. The study aim was to assess systematically evolutionary conserved PD-associated genes using Drosophila melanogaster. From a literature review, a total of 136 genes have found to be associated with PD in GWAS studies, of which 11 are strongly evolutionary conserved between Homo sapiens and D. melanogaster. By ubiquitous gene expression knockdown of the PD-genes in D. melanogaster, the flies' escape response was investigated by assessing their negative geotaxis response, a phenotype that has previously been used to investigate PD in D. melanogaster. Gene expression knockdown was successful in 9/11 lines, and phenotypic consequences were observed in 8/9 lines. The results provide evidence that genetically modifying expression levels of PD genes in D. melanogaster caused reduced climbing ability of the flies, potentially supporting their role in dysfunctional locomotion, a hallmark of PD.

Keywords: RING assay; climbing ability; model organism; neurodegenerative disease.

Figure 1

Overview of gene selection from the four large PD genome-wide association studies (GWAS). A total of 136 unique genes were identified, of which 25 had an orthology score above 12. Among the well-conserved PD genes, 11 were available as homozygous viable from Vienna Drosophila Stock Centre (VDRC) [33]. Supplementary Table S1 contains a complete list of all 136 human PD associated genes [9,12,13,14].

Figure 2

Effect of gene expression knockdown of the 11 selected genes. Each bar represents the log₂ of the ratio of the TMM normalised gene expression of the target gene for the RNAi line and the control line (w₁₁₁₈). Purple colours indicate successful reduction in gene expression of the target gene, whereas the green colour indicates upregulation of gene expression. Only knockdown lines with indications of reduced gene expression were used for the phenotypic characterisation. As there are no biological replicates for the RNA sequencing experiments, no error bars can be obtained.

Figure 3

Mean distance climbed per 3 s as function of age for each of the RNAi knockdown lines and the control line (shown in black). Lines highlighted in purple indicate a significant effect of ageing, and their corresponding gene names are listed (together with the control line). Mean values and statistics supporting the figure can be accessed in Supplementary Table S5.

Figure 4

Phenotypic assessment of RNAi lines displaying reduced gene expression of target gene compared with their common control. Each bar represents the absolute difference between a particular RNAi knockdown line and the control line. Error bars represents the standard error of the difference in mean computed as $\sqrt{\frac{S{D}_{UAS-GAL4}}{n_{UAS-GAL4}}+\frac{S{D}_{W1118-GAL4}}{n_{W1118-GAL4}}}$. Purple colour indicates reduced climbing ability of the RNAi line, and orange colour indicates increased climbing ability. Asterisks indicate significant age-specific difference between RNAi knockdown and control line (FDR > 0.05). Mean values and statistics supporting the figure can be accessed in Supplementary Tables S6–S8.