Loss-of-function mutations of the parkin gene are a major cause of early-onset parkinsonism. To explore the mechanism by which loss of parkin function results in neurodegeneration, we are using a genetic approach in Drosophila. Here, we show that Drosophila parkin mutants display degeneration of a subset of dopaminergic (DA) neurons in the brain. The neurodegenerative phenotype of parkin mutants is enhanced by loss-of-function mutations of the glutathione S-transferase S1 (GstS1) gene, which were identified in an unbiased genetic screen for genes that modify parkin phenotypes. Furthermore, overexpression of GstS1 in DA neurons suppresses neurodegeneration in parkin mutants. Given the previous evidence for altered glutathione metabolism and oxidative stress in sporadic Parkinson's disease (PD), these data suggest that the mechanism of DA neuron loss in Drosophila parkin mutants is similar to the mechanisms underlying sporadic PD. Moreover, these findings identify a potential therapeutic approach in treating PD.genetic modifier ͉ neurodegeneration ͉ parkin P arkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra and the accumulation of proteinaceous intraneuronal inclusions known as Lewy bodies. The mechanisms responsible for neurodegeneration in PD are largely unknown, although previous work suggests that mitochondrial complex I dysfunction, oxidative stress, and aberrant proteolytic degradation may contribute to pathogenesis (1). The recent identification of genes responsible for rare inherited forms of parkinsonism presents an opportunity to establish neurodegenerative mechanisms that may be relevant to sporadic forms of PD.Loss-of-function mutations of parkin are a common cause of autosomal recessive juvenile parkinsonism (ARJP), and parkin dysfunction may also contribute to late-onset sporadic PD (2-6). Patients with parkin mutations display many of the typical features of idiopathic PD, including locomotor dysfunction, reduced mitochondrial complex I activity, and degeneration of DA neurons in the substantia nigra. However, most ARJP cases have a significantly earlier age of onset and lack Lewy body pathology. Parkin has been shown to possess ubiquitin-protein ligase activity (7-9), which acts to confer substrate target specificity in the ubiquitin͞proteasome protein degradation pathway. This finding has led to the model that toxic accumulation of parkin substrates may be responsible for DA neuron death. A number of putative substrates of parkin have been identified (10). Several of these parkin substrates, including the Lewy body component ␣-synuclein (11) and the putative G protein-coupled receptor Pael-R (12), have received considerable attention, in part because they implicate specific cellular pathways in parkinmediated pathogenesis. However, the involvement of many of the identified parkin substrates in the etiology of ARJP remains controversial.To identify pathways relevant to parkin pathogenesis, we are using a gen...

Drosophila melanogaster glutathione S‐transferase DmGSTS1‐1 (earlier designated as GST‐2) is related to sigma class GSTs and was previously described as an indirect flight muscle‐associated protein with no known catalytic properties. We now report that DmGSTS1‐1 isolated from Drosophila or expressed in Escherichia coli is essentially inactive toward the commonly used synthetic substrate 1‐chloro‐2,4‐dinitrobenzene (CDNB), but has relatively high glutathione‐conjugating activity for 4‐hydroxynonenal (4‐HNE), an electrophilic aldehyde derived from lipid peroxidation. 4‐HNE is thought to have signaling functions and, at higher concentrations, has been shown to be cytotoxic and involved in the etiology of various degenerative diseases. Drosophila strains carrying P‐element insertions in the GstS1 gene have a reduced capacity for glutathione conjugation of 4‐HNE. In flies with both, one, or none of the GstS1 alleles disrupted by P‐element insertion, there is a linear correlation between DmGSTS1‐1 protein content and 4‐HNE‐conjugating activity. This correlation indicates that in adult Drosophila 70 ± 6% of the capacity to conjugate 4‐HNE is attributable to DmGSTS1‐1. The high abundance of DmGSTS1‐1 (approximately 2% of the soluble protein in adult flies) and its previously reported localization in tissues that are either highly aerobic (indirect flight muscle) or especially sensitive to oxidative damage (neuronal tissue) suggest that the enzyme may have a protective role against deleterious effects of oxidative stress. Such function in insects would be analogous to that carried out in mammals by specialized alpha class glutathione S‐transferases (e.g. GSTA4‐4). The independent emergence of 4‐HNE‐conjugating activity in more than one branch of the glutathione S‐transferase superfamily suggests that 4‐HNE catabolism may be essential for aerobic life.

From the fruitfly, Drosophila melanogaster, ten members of the cluster of Delta-class glutathione S-transferases (GSTs; formerly denoted as Class I GSTs) and one member of the Epsilon-class cluster (formerly GST-3) have been cloned, expressed in Escherichia coli, and their catalytic properties have been determined. In addition, nine more members of the Epsilon cluster have been identified through bioinformatic analysis but not further characterized. Of the 11 expressed enzymes, seven accepted the lipid peroxidation product 4-hydroxynonenal as substrate, and nine were active in glutathione conjugation of 1-chloro-2,4-dinitrobenzene. Since the enzymically active proteins included the gene products of DmGSTD3 and DmGSTD7 which were previously deemed to be pseudogenes, we investigated them further and determined that both genes are transcribed in Drosophila. Thus our present results indicate that DmGSTD3 and DmGSTD7 are probably functional genes. The existence and multiplicity of insect GSTs capable of conjugating 4-hydroxynonenal, in some cases with catalytic efficiencies approaching those of mammalian GSTs highly specialized for this function, indicates that metabolism of products of lipid peroxidation is a highly conserved biochemical pathway with probable detoxification as well as regulatory functions.

SummaryCaenorhabditis elegans expresses a glutathione transferase (GST) belonging to the Pi class, for which we propose the name CeGSTP2-2. CeGSTP2-2 (the product of the gst-10 gene) has the ability to conjugate the lipid peroxidation product 4-hydroxynonenal (4-HNE). Transgenic C. elegans strains were generated in which the 5′ ′ ′ ′ -flanking region and promoter of gst-10 were placed upstream of gst-10 and mGsta4 cDNAs, respectively. mGsta4 encodes the murine mGSTA4-4, an enzyme with particularly high catalytic efficiency for 4-HNE. The localization of both transgenes was similar to that of native CeGSTP2-2. The 4-HNE-conjugating activity in worm lysates increased in the order: control < mGsta4 transgenic < gst-10 transgenic; and the amount of 4-HNE-protein adducts decreased in the same order, indicating that the transgenic enzymes were active and effective in limiting electrophilic damage by 4-HNE. Stress resistance and lifespan were measured in transgenic animals (five independent lines each) and were compared with two independent control lines. Resistance to paraquat, heat shock, ultraviolet irradiation and hydrogen peroxide was greater in transgenic strains. Median lifespan of mGsta4 and gst-10 transgenic strains vs. control strains was increased by 13% and 22%, respectively. In addition to the cause-effect relationship between GST expression and lifespan observed in the transgenic lines, correlative evidence was also obtained in a series of congenic lines of C. elegans in which lifespan paralleled the 4-HNE-conjugating activity in whole-animal lysates. We conclude that electrophilic damage by 4-HNE may contribute to organismal aging.

Cardiotoxicity is one of the major side effects encountered during cancer chemotherapy with doxorubicin (DOX) and other anthracyclines. Previous studies have shown that oxidative stress caused by DOX is one of the primary mechanisms for its toxic effects on the heart. Since the redox-sensitive transcription factor, Nrf2, plays a major role in protecting cells from the toxic metabolites generated during oxidative stress, we examined the effects of the phytochemical sulforaphane (SFN), a potent Nrf2-activating agent, on DOX-induced cardiotoxicity. These studies were carried out both in vitro and in vivo using rat H9c2 cardiomyoblast cells and wild type 129/sv mice, and involved SFN pretreatment followed by SFN administration during DOX exposure. SFN treatment protected H9c2 cells from DOX cytotoxicity and also resulted in restored cardiac function and a significant reduction in DOX-induced cardiomyopathy and mortality in mice. Specificity of SFN induction of Nrf2 and protection of H9c2 cells was demonstrated in Nrf2 knockdown experiments. Cardiac accumulation of 4-hydroxynonenal (4-HNE) protein adducts, due to lipid peroxidation following DOX-induced oxidative stress, was significantly attenuated by SFN treatment. The respiratory function of cardiac mitochondria isolated from mice exposed to DOX alone was repressed, while SFN treatment with DOX significantly elevated mitochondrial respiratory complex activities. Co-administration of SFN reversed the DOX-associated reduction in nuclear Nrf2 binding activity and restored cardiac expression of Nrf2-regulated genes, at both the RNA and protein levels. Together, our results demonstrate for the first time that the Nrf2 inducer, SFN, has the potential to provide protection against DOX-mediated cardiotoxicity.