Helen Beneš

Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease

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 Parkinsons 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.

Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class.

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.

Chromosomal Mapping of Osteopenia-Associated Quantitative Trait Loci Using Closely Related Mouse Strains

Peak bone mineral density (BMD) is a highly heritable trait in humans and is currently the best predictor of skeletal fragility underlying osteoporosis. The SAMP6 mouse strain displays unusually low BMD at maturity, and age‐dependent osteopenia associated with defective osteoblastogenesis. To identify quantitative trait loci (QTLs) influencing bone density, we constructed crosses between SAMP6 and either AKR/J or SAMP6, two related mouse strains of higher peak BMD. Due to common ancestry of these strains, intercross parents differed at only 39–40% of 227 highly‐polymorphic genotyping markers, thus restricting our search to this informative portion of the genome and reducing the number of mice required for QTL significance. Using dual energy X‐ray absorptiometry (DEXA), we measured spinal BMD in F2 cross progeny at 4 months of age, and selectively genotyped those in the highest and lowest quartiles for BMD. Based on linear regression of bone density on genotype, including Composite Interval Mapping to enhance mapping precision while adjusting for effects of distal markers, we identified multiple QTLs significantly affecting spinal BMD; these were mapped to regions of chromosomes 2 (two sites, one confirmed in both crosses), 7, 11, 13 and 16. One of these loci had been previously identified as a significant bone‐density QTL, while 3 substantiate QTLs suggested by a low‐power study of 24 recombinant‐inbred mouse lines. Such recurrent appearance of QTLs, especially in crosses involving distantly‐related strains, implies that polymorphism at these loci may be favored by evolution and might underlie variation in peak bone density among humans.

The Evolution of Hexamerins and the Phylogeny of Insects

Abstract. The evolutionary relationships among arthropod hemocyanins and insect hexamerins were investigated. A multiple sequence alignment of 12 hemocyanin and 31 hexamerin subunits was constructed and used for studying sequence conservation and protein phylogeny. Although hexamerins and hemocyanins belong to a highly divergent protein superfamily and only 18 amino acid positions are identical in all the sequences, the core structures of the three protein domains are well conserved. Under the assumption of maximum parsimony, a phylogenetic tree was obtained that matches perfectly the assumed phylogeny of the insect orders. An interesting common clade of the hymenopteran and coleopteran hexamerins was observed. In most insect orders, several paralogous hexamerin subclasses were identified that diversified after the splitting of the major insect orders. The dipteran arylphorin/LSP-1-like hexamerins were subject to closer examination, demonstrating hexamerin gene amplification and gene loss in the brachyceran Diptera. The hexamerin receptors, which belong to the hexamerin/hemocyanin superfamily, diverged early in insect evolution, before the radiation of the winged insects. After the elimination of some rapidly or slowly evolving sequences, a linearized phylogenetic tree of the hexamerins was constructed under the assumption of a molecular clock. The inferred time scale of hexamerin evolution, which dates back to the Carboniferous, agrees with the available paleontological data and reveals some previously unknown divergence times among and within the insect orders.

Lifespan and stress resistance of Caenorhabditis elegans are increased by expression of glutathione transferases capable of metabolizing the lipid peroxidation product 4-hydroxynonenal

Caenorhabditis 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.

Molecular cloning of cDNAs for two pro-phenol oxidase subunits from the malaria vector, Anopheles gambiae

Phenol oxidase exists in insect hemolymph as a zymogen, pro-phenol oxidase (pro-PO), which is activated by specific proteolysis in response to infection or wounding. Phenol oxidase catalyses the synthesis of quinones that polymerize to form melanin deposits, which encapsulate parasites and help to seal wounds. Antibodies to pro-PO from Manduca sexta bound to 76, 72, and 71 kDa polypeptide bands from hemolymph of Anopheles gambiae larvae. This antiserum was used to screen a cDNA library from A. gambiae fourth-instar larvae. Full-length clones were isolated for two different pro-POs, designated A. gambiae proPO-p1 and proPO-p2, which are 67% identical in nucleotide sequence and 66% identical in deduced amino acid sequence. The A. gambiae pro-PO sequences are more similar to pro-PO from Drosophila melanogaster than to lepidopteran or crustacean pro-PO sequences in the GenBank database. Like the other arthropod pro-POs, the A. gambiae pro-PO sequences lack a signal peptide and have two conserved regions predicted to bind two copper atoms in the active site of the enzyme. The availability of these pro-PO cDNAs should be useful in examining the biochemical differences between A. gambiae strains that are refractory or susceptible to Plasmodium infection, and differ in their ability to encapsulate the parasites.

Characterization of an EcR/USP heterodimer target site that mediates ecdysone responsiveness of the Drosophila Lsp-2 gene

The Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is uniquely expressed in the fat body tissue from the beginning of the third instar to the end of adult life. Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone. To study the molecular basis for ecdysone regulated Lsp-2 activity, deletion mutants of the Lsp-2 5′-flanking region were constructed by fusion to either the Escherichia coli chloramphenicol acetyltransferase (CAT) gene or to an hsp70-lacZ hybrid gene encoding β-galactosidase. Constructs transfected into Drosophila S2/M3 cells were shown to confer transient ecdysone inducibility on the reporter genes. A single functional ecdysone response element (EIRE) was localized at position — 75 relative to the Lsp-2 transcription initiation site. In gel mobility shift assays using fat body nuclear extracts or nuclear receptors synthesized in vitro, a 27-bp sequence harboring the EcRE bound both the Drosophila ecdysone receptor and the Drosophila retinoid-X homologue, Ultraspiracle, in a cooperative manner. Competition experiments indicate that the affinity of the Lsp-2 EcRE for the ecdysone receptor complex is comparable to that of the canonical EcRE of the hsp27 gene and is at least 4-fold greater than that of Fbp1, another fat body-specific Drosophila gene. Our results suggest that structural features of this EcRE determine its ability to induce ecdysone responsiveness at a lower ligand concentration and may form the basis for differential hormone responsiveness within the fat body.

Lifespan extension in hypomorphic daf‐2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2‐2

Electrophilic stress caused by lipid peroxidation products such as 4‐hydroxynonenal (4‐HNE) and/or related compounds may contribute to aging. The major mode of 4‐HNE metabolism involves glutathione conjugation catalyzed by specialized glutathione transferases. We have previously shown that glutathione transferase CeGSTP2‐2, the product of the Caenorhabditis elegans gst‐10 gene, has the ability to conjugate 4‐HNE, and that its overexpression extends lifespan of C. elegans. We now demonstrate that the expression level of CeGSTP2‐2 correlates highly with lifespan in a series of hypomorphic daf‐2 mutants of C. elegans. The overexpression of CeGSTP2‐2 in daf‐2 is abrogated in daf‐16; daf‐2 mutants, indicating that expression of the gst‐10 gene is modulated by insulin‐like growth factor signaling. To determine whether the relationship between CeGSTP2‐2 and lifespan is causal, we used RNAi to knock down CeGSTP2‐2. Treatment with gst‐10‐specific dsRNA decreased CeGSTP2‐2 protein in wild‐type N2 and in daf‐2 strains to an approximately equal level. The ability to conjugate 4‐HNE was similarly decreased by RNAi, suggesting that the increment of that activity in daf‐2 over N2 is due largely to the overexpression of CeGSTP2‐2. RNAi‐mediated knock‐down of CeGSTP2‐2 led to an increased susceptibility to 4‐HNE, paraquat, and heat shock, and to a shortening of lifespan by 13% in both N2 and daf‐2 strains. These results indicate that CeGSTP2‐2 significantly contributes to the maintenance of the soma, and that this function is augmented in daf‐2 mutants concordantly with other longevity assurance genes, probably via insulin‐like growth factor signaling.

The Somatotrope as a Metabolic Sensor: Deletion of Leptin Receptors Causes Obesity

Leptin, the product of the Lep gene, reports levels of adiposity to the hypothalamus and other regulatory cells, including pituitary somatotropes, which secrete GH. Leptin deficiency is associated with a decline in somatotrope numbers and function, suggesting that leptin may be important in their maintenance. This hypothesis was tested in a new animal model in which exon 17 of the leptin receptor (Lepr) protein was selectively deleted in somatotropes by Cre-loxP technology. Organ genotyping confirmed the recombination of the floxed LepR allele only in the pituitary. Deletion mutant mice showed a 72% reduction in pituitary cells bearing leptin receptor (LEPR)-b, a 43% reduction in LEPR proteins and a 60% reduction in percentages of immunopositive GH cells, which correlated with reduced serum GH. In mutants, LEPR expression by other pituitary cells was like that of normal animals. Leptin stimulated phosphorylated Signal transducer and activator of transcription 3 expression in somatotropes from normal animals but not from mutants. Pituitary weights, cell numbers, IGF-I, and the timing of puberty were not different from control values. Growth curves were normal during the first 3 months. Deletion mutant mice became approximately 30-46% heavier than controls with age, which was attributed to an increase in fat mass. Serum leptin levels were either normal in younger animals or reflected the level of obesity in older animals. The specific ablation of the Lepr exon 17 gene in somatotropes resulted in GH deficiency with a consequential reduction in lipolytic activity normally maintained by GH and increased adiposity.

Studies on the molecular-genetic basis of replicative senescence in Werner syndrome and normal fibroblasts

Based on evidence that human diploid fibroblasts (HDF) from the Werner syndrome (WS) of premature aging might overexpress an inhibitor of DNA synthesis (IDS), we prepared a eukaryotic cDNA expression library from WS mRNA and tested it for IDS activity in a transient assay. Two of six WS cDNA pools tested gave IDS activity, then on plus/minus screening revealed several differentially expressed cDNA clones. By slot blot and Northern analysis, one cDNA clone was found to be overexpressed in WS and normal senescent HDF, but not in quiescent normal HDF, indicating that it is senescence-specific. Further studies are needed to clarify: a) whether this cDNA truly acts as an IDS; b) if so, whether it acts alone or in concert with other cDNAs; and c) whether it is involved in the degenerative and malignant sequelae of WS and normal aging.