Investigation of Compensatory Mechanisms of Dopamine Deficiency in a Drosophila Model
Cichewicz, Karol, Biology - Graduate School of Arts and Sciences, University of Virginia
Hirsh, Jay, As-Biology, University of Virginia
Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting one million people in the United States. The disease is caused by death of neurons synthesizing the neurotransmitter dopamine (DA) in the brain region substantia nigra pars compacta. Death of these DA neurons causes severe impairment of the locomotor functions, including tremor, rigidity and bradykinesia. The locomotor symptoms do not appear until at least 50% of DA neurons die, suggesting that compensatory mechanisms play a significant role in maintaining normal brain function during the presymptomatic phase. Current therapies are mostly limited to DA restoration with the DA precursor L-DOPA. A peripheral DOPA decarboxylase inhibitor carbidopa is used to increase L DOPA transport through the blood-brain-barrier. Currently, there is no therapy that slows down or reverses the neuropathology of PD. Understanding and utilizing the compensatory mechanisms may provide new targets for developing more effective therapies.
Drosophila melanogaster is the most genetically tractable model organism. It expresses complex behaviors, and has DA system that is conserved with human system. We developed a new Drosophila DA-deficient model, which replicates the locomotor hypoactivity of PD. These flies rescue a null mutation of tyrosine hydroxylase, the rate limiting enzyme in DA biosynthesis, in the hypoderm, but maintain DA-deficiency in the brain. DA rescue in the hypoderm is necessary for these flies to develop into adulthood. We developed a transgene allowing precise restoration of DA biosynthesis with a binary expression GAL4/UAS system and demonstrated that DA biosynthesis can be restricted to selected neurons in the brain. Pharmacological restoration of DA biosynthesis with L-DOPA and carbidopa demonstrates that the selectivity of blood-brain-barrier is conserved in Drosophila and humans.
In the population of our DA-deficient flies we identified individuals exhibiting almost normal level of locomotor activity. We isolated two sublines differing in their locomotor activity by about two-fold. We named the high activity subline DA-Bypass because it circumvents the need for DA in the brain to express locomotion. We confirmed that both sublines do not synthesize any DA in the brain by high performance liquid chromatography, and by testing the effects of drugs modulating DA synthesis and release on the behavior. We think that the DA-Bypass subline may be rescuing its locomotor activity using a mechanism relevant to compensatory mechanisms in PD.
Using genetic, transcriptomic, and genomic techniques, we are trying to elucidate the molecular mechanism and the genomic locus/loci responsible for this phenotype. We found that the locus/loci responsible for the DA-Bypass is/are encoded on the X chromosome, and the phenotype manifests stronger in males than females. In an RNA-Seq experiment we identified a number of differentially expressed candidate genes, which can be further tested for modulating locomotor activity in a reverse genetic screen. The chromosomal location of these genes overlaps with our genomic mapping indicating that they are good candidates. Further work is necessary to validate these targets and increase the precision of the genomic mapping.
Our laboratory assays locomotor activity using the Drosophila Activity Monitor (DAM) system developed by TriKinetics (Waltham, MA). We developed a web application ShinyR-DAM for analyzing DAM locomotor activity, sleep and circadian rhythms. Compared to existing programs, ShinyR-DAM greatly decreases the complexity and time required to analyze the data, producing informative and customizable plots, summary tables, and data files for statistical analysis. Our program has an intuitive graphical user interface that enables novice users to quickly perform complex analyses. ShinyR-DAM greatly accelerated our work and has been widely adopted in the community of DAM system users.
PHD (Doctor of Philosophy)
dopamine, Parkinson's disease, Drosophila, L-DOPA, carbidopa, dopamine deficiency, compensatory mechanisms of dopamine deficiency, ShinyR-DAM, R programming language, Shiny application
National Institutes of Health