CRISPR Caspase 9 strategies to explore parkin loss‐of‐function Drosophila development

Abstract

Parkinson’s disease (PD) is a neurodegenerative disease in which dopaminergic (DA) neurons selectively degenerate within the substantia nigra pars compacta. Parkin loss‐of‐function Drosophila melanogaster, a model for familial PD, have neurodegeneration in structures homologous to those of the human substantia nigra. Mitochondrial dysfunction and reactive oxygen species (ROS) are implicated in PD‐associated neurodegeneration and in the parkin‐null fly phenotype. In healthy cells, ROS are involved in signaling and are regulated by antioxidant activity. Glutathione (GSH) is a major antioxidant that combats the effects of excess damaging ROS (oxidative stress) and may be dysregulated in parkin mutant Drosophila. Preliminary data from our lab has shown that parkin loss‐of‐function Drosophila (park25/25; Greene et al., 2003) have elevated hydrogen peroxide levels that are sustained as the fly ages, a transient increase in glutathione activity, and increased levels of protein oxidation. Whether oxidative stress persists because of low GSH levels, dysfunction of GSH, or excessive ROS is unclear. Our study aims to address the possibility that low levels of GSH contribute to the parkin loss‐of‐function fly phenotype. To this end, we utilized a modified CRISPR/Cas9 system to overexpress cystathionine beta synthase (CBS), a key, rate‐limiting‐limiting enzyme involved in the production of GSH. To overexpress CBS, we have used a modified Cas9 construct in which the enzymatic function of Cas9 has been deactivated (“dead Cas9” or “dCas9”). Transcriptional activators VPR have been fused to dCas9, resulting in overexpression of CBS. Since parkin–null Drosophila have developmental deficits, we have measured pupariation and eclosion efficiencies in flies overexpressing CBS to shed light on the potential use of this intervention for future studies. Parent flies are placed in a vial and allowed to lay embryos. First and second instar larva are collected and transferred to a secondary vial. The larva are then monitored, and the number of larva that pupate are recorded (pupariation efficiency). The number of flies that eclose (emerge from the pupa case) are also recorded and compared to the pupa count (eclosion efficiency). Additionally, the distance between pupa and the food level is measured to indicate larval mobility. Preliminary data indicate the overexpression of CBS increases pupariation efficiency and decreases pupariation times in the heterozygous parkin mutants. Parkin‐null larva CBS overexpression also appear to have increased climbing. This initial data implicates overexpression of CBS in neuroprotection of the park loss‐of‐function model. Additional experiments on neuron degeneration, mitochondrial functionality, and antioxidant activity can provide further insight into why parkin‐null dopamine neurons are susceptible to degradation. By incorporating Cas9 and dCas9 into our parkin‐null flies, we can modify expression of hundreds of genes in order to elucidate the pathways implicated in oxidative stress in this model.