Effects of pharmacological agents on the lifespan phenotype of Drosophila DJ-1β mutants

Abstract

Mutations in the DJ-1 gene cause autosomal recessive, early-onset Parkinsonism. The DJ-1 protein exerts a protective role against oxidative stress damage, working as a cellular oxidative stress sensor, and it seems to regulate gene expression at different levels. In Drosophila, two DJ-1 orthologs have been identified: DJ-1α and DJ-1β. Several studies have shown that loss of DJ-1β function causes Parkinson's disease (PD)-like phenotypes in flies such as age-dependent locomotor defects, reduced lifespan, and enhanced sensitivity to toxins that induce oxidative stress, like the herbicide paraquat. However, no dopaminergic neurodegeneration is observed. These results suggested that both locomotor and lifespan phenotypes could be either related to defects in oxidative stress response, or in dopaminergic physiology as proposed in mice models. In this study, we have employed pharmacological approaches to modify the lifespan phenotype of DJ-1β mutant flies. We have assessed the effects of chronic treatments with antiparkinsonian drugs as well as with antioxidant compounds on such phenotype finding that only antioxidants show statistically significant beneficial effects on DJ-1β mutants' lifespan. These results strongly suggest that oxidative stress plays a causal role in the lifespan phenotype of DJ-1β mutants. Consistent with this, we find that loss of DJ-1β function results in cellular accumulation of reactive oxygen species (ROS) in adult brains, elevated levels of lipid peroxidation and an increased catalase enzymatic activity, thus indicating the existence of high oxidative stress levels in DJ-1β mutants and confirming the essential function of the DJ-1β protein in protecting the organism against oxidative insults. Our study further shows that the lifespan phenotype of DJ-1β mutant flies is amenable to pharmacological intervention, and validates Drosophila as a valuable model for testing and identifying new drugs with therapeutic potential for PD.

Introduction

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Although most PD cases are sporadic, several genes are associated with familial disease providing important insights into PD pathogenesis (Schultz, 2008). One of them is PARK7 whose mutations cause autosomal recessive, early-onset Parkinsonism (Bonifati et al., 2003). PARK7 (or DJ-1) encodes DJ-1, a small protein that was originally identified as an oncogenic factor (Nagakubo et al., 1997). However, studies in cell culture and animal models have demonstrated that the DJ-1 protein exerts a protective role against oxidative stress damage, being modified in several cysteine residues under these conditions (Canet-Aviles et al., 2004, Betarbet et al., 2006, Choi et al., 2006, Meulener et al., 2006). This modification increases with age and exposure to oxidative toxins, probably leading to DJ-1 inactivation and eventually predisposing dopaminergic (DA) neurons to stress-induced degeneration. Although the molecular function of DJ-1 is unclear, it seems that it can regulate gene expression either at the transcriptional or translational level and, interestingly, both functions are impaired by oxidation (Van der Brug et al., 2008, Zhong and Xu, 2008, Blackinton et al., 2009). It has been proposed a model whereby DJ-1 interacts with mRNAs and dissociates under conditions of oxidative stress, thus the apparent pleiotropic effects of DJ-1 may be related to the single function of binding to multiple transcripts (van der Brug et al., 2008).

In Drosophila, two DJ-1 orthologs have been identified, DJ-1α and DJ-1β. Loss of function of these genes causes defects in oxidative stress response and locomotion as well as shortened lifespan, but only inactivation of DJ-1α expression via RNAi results in DA neurodegeneration (Meulener et al., 2005, Menzies et al., 2005, Park et al., 2005, Yang et al., 2005a, Lavara-Culebras and Paricio, 2007). DJ-1β^A286 mutant flies were characterized in our laboratory and contain a transposable element insertion in the DJ-1β gene, thus encoding a truncated and probably inactive DJ-1β protein. Despite this, they are viable and fertile but show motor impairments and a significantly shorter lifespan than wild-type flies. Moreover, they are highly sensitive to paraquat exposure (Lavara-Culebras and Paricio, 2007). Since loss of DJ-1β function does not affect DA neuron survival (Menzies et al., 2005, Meulener et al., 2005, Park et al., 2005, Lavara-Culebras and Paricio, 2007), one possibility is that the observed motor and lifespan phenotypes are due to defects in dopamine neurotransmission or DA neuron malfunction, as suggested in mice models (Chen et al., 2005, Goldberg et al., 2005). Alternatively, they could be caused by oxidative damage. Supporting this, it was previously shown that age-dependent locomotor defects exhibited by DJ-1β mutants carrying a different allele were substantially enhanced by paraquat treatment (Park et al., 2005). Furthermore, a recent study has shown that two compounds with antioxidant and anti-inflammatory properties partially suppress DA neuron loss and locomotor dysfunction of DJ-1α RNAi flies (Faust et al., 2009). In such a scenario, we have investigated the molecular mechanisms involved in the lifespan phenotype of DJ-1β^A286 mutant flies, by testing the effects on this phenotype of several compounds that were added to the flies' diet. In this study, DJ-1β mutant flies were chronically treated with representatives of the major classes of drugs used to mitigate the effects of dopaminergic neuron loss in PD patients, like l-DOPA or pergolide, and the experimental compound SK&F 38393, as well as with compounds that exhibit antioxidant activity, including vitamin C, melatonin or α-tocopherol. We find that lifespan of DJ-1β^A286 mutants is significantly extended after chronic treatments with antioxidant agents but it is unaffected by exposure to antiparkinsonian drugs. Although lifespan values are not restored to wild-type in the DJ-1β mutant flies, our results indicate that the lifespan phenotype is mediated, at least in part, by oxidative damage and not by defects in DA neuron physiology as previously suggested (Lavara-Culebras and Paricio, 2007). Confirming these results, we have observed an increase in several markers of oxidative stress in DJ-1β^A286 mutants when compared to control flies.