Dopa decarboxylase(Ddc)-GAL4 dramatically reduces life span.

Haywood, Annika F.M., Lisa D. Saunders, and Brian E. Staveley.  Department of Biology, Memorial University of Newfoundland, St. John's, Newfoundland & Labrador, Canada A1B 3X9; telephone: (709) 737-4317, telefax: (709) 737-3018, bestave@mun.ca

Abstract

     The GAL4 system is extensively used for the directed expression of genes of interest in Drosophila melanogaster.  One main property of this system is that GAL4, the yeast transcription factor, is believed to be inactive in Drosophila and that its expression under the control of D. melanogaster-specific promoters will have little effect on its own cells.  We have shown that expression of GAL4 under the control of the dopa decarboxylase (Ddc) promoter element does have an effect upon adult viability.  Although Ddc-GAL4 heterozygotes appear normal when raised at 25°C, the homozygotes have a have a greatly reduced life span.  While the homozygotes simply do not survive, Ddc-GAL4 heterozygotes demonstrate normal locomotion over their life span.

Introduction

     The GAL4 ectopic expression system (Brand and Perrimon, 1993; reviewed in Phelps and Brand, 1998) has become an extremely useful approach for the study of specific genes in Drosophila melanogaster.  This expression system relies upon GAL4 to bind the upstream activation sequence (UAS) in order to activate transcription of the target gene.  A variety of transgenic Drosophila lines are readily available that express GAL4 in specific tissues or cell types.  Our laboratory is interested in models of neurodegenerative diseases and we have initiated work with the dopa decarboxylase (Ddc)-GAL4 transgenic expression lines to model Parkinson’s disease in Drosophila.

     The Drosophila melanogaster model of Parkinson’s disease was developed by the generation of transgenic lines bearing wild type and mutant forms of the human a-synuclein gene cloned downstream of the UAS yeast promoter (Feany and Bender, 2000).  There is no apparent Drosophila homologue of a-synuclein, but expression of the human a-synuclein protein in the Drosophila nervous system recapitulated some features of Parkinson’s disease.  Expression of a-synuclein in the dopaminergic neurons (Feany and Bender, 2000) was driven by a transgene comprised of the dopa decarboxylase (Ddc) gene promoter cloned upstream of GAL4.  Originally, this transgene was developed to examine a Drosophila model of cocaine addiction (Li et al., 2000).  The Parkinsonian flies, apparently normal at a young age, demonstrated a premature loss of locomotor (climbing) ability, loss of the dopaminergic neurons and accumulation of a-synuclein-containing inclusions.  In addition, GMR-GAL4 driven expression in the developing eye resulted in age-dependent retinal neurodegeneration.  Subsequent treatment of the transgenic a-synuclein expressing flies with a number of pharmacological agents such as the dopamine precursor L-DOPA, dopamine receptor agonists (bromocriptine, pergolide and SK&F38393) and the anticholinergic atropine, all restored or partially restored the age-dependent loss climbing ability (Pendleton et al., 2002).  Further, this model has been used to examine the suppression of the a-synuclein toxicity by the molecular chaperone, hsp70 (Auluck et al., 2002).  A good understanding of the effects of Ddc-GAL4 expression in Drosophila melanogaster is essential to properly interpreting this model system.

     As a prelude to our exploration of models of Parkinson's disease in Drosophila, we began to investigate the biological properties of the Ddc-GAL4 driver lines.  We have shown that Ddc-GAL4 causes reduced viability and is therefore not inactive in D. melanogaster.

Materials and Methods

     Fly stocks and culture: Ddc-GAL4 ^4.3D and Ddc-GAL4 ^4.36 flies (Li et al., 2000) were obtained from Jay Hirsh at the Department of Biology, University of Virginia and w¹¹¹⁸ was obtained from Dr. Howard Lipshitz at the Hospital for Sick Children in Toronto.  To obtain heterozygotes, Ddc-GAL4 homozygous males were crossed to w¹¹¹⁸ females.  All flies were cultured on standard cornmeal/yeast/molasses/agar media at 25°C.

     Aging analysis: Adult males were aged in small groups upon standard media at 25°C and scored for viability every two to three days as described previously (Staveley et al., 1990).  The number of individuals aged are as follows:  Ddc-GAL4 ^4.3D homozygotes n = 102; Ddc-GAL4 ^4.36 homozygotes n = 133; Ddc-GAL4 ^4.3D heterozygotes n = 280; Ddc-GAL4 ^4.36 heterozygotes n = 119; w¹¹¹⁸  n =  83).

     Locomotion assay: The flies were assayed for their ability to climb in a manner similar to that described by Feany and Bender (2000).  Every 4 to 5 days, 10 male flies of a cohort of aged flies were assayed for their ability to climb to the top of the vial within a period of 18 seconds.  Twenty trials were carried out for each time point.

Results and Discussion

     An investigation of the baseline biological consequences of GAL4 expression as directed by the dopa decarboxy-lase(Ddc) promoter is essential to our studies of Parkinson's disease models in Drosophila.  We began by crossing the Ddc-GAL4 ^4.3D and Ddc-GAL4^4.36 driver lines to w¹¹¹⁸ to conduct climbing assays. It quickly became apparent that the stocks of both Ddc-GAL4 insertion lines required extra care to maintain and that the flies were apparently short-lived in both cases.  As a result we decided to conduct longevity trials.

Homozygotes of both insertions of the Ddc-GAL4 trans-gene display a greatly reduced life span (Figure 1). For example, by the 6^th day, Ddc-GAL4^4.3D and Ddc-GAL4^4.36 homozygous males showed only 43% and 44% survival, respectively.       While 50% of Ddc-GAL4^4.3D male het-erozygotes survive past the age of 88 days and Ddc-GAL4^4.36 male heter-ozygotes past 70 days of age.  We assigned w¹¹¹⁸ as the control strain in these experiments, 50% of which survived between 58 and 60 days.  The shorter median life span of the control may reflect an insufficiency of the w¹¹¹⁸ stock rather than an increase in viability the heterozygotes.  It is important to note that the very similar longevity profile of the two independent insertions of Ddc-GAL4 suggest that the reduction in life span is due to the expression of the transgene and not the site of insertion.

     The reduced viability of the Ddc-GAL4 homozygotes forced us to reexamine the locomotor activity of the Ddc-GAL4 heterozygotes (Figure 2).  However, the Ddc-GAL4 ^4.3D and Ddc-GAL4 ^4.36 heterozygotes retain their ability to climb with age in a manner similar to the w¹¹¹⁸ controls.  Due to greatly reduced viability, the locomotion of homozygotes was not measured.

     The UAS/GAL4 ectopic expression system has made it possible to express genes and test the effects of overexpression.  The Ddc-GAL4 driver has been used for expression of transgenes in the dopaminergic neurons including expressing the a-synuclein gene to model Parkinson’s disease (Feany and Bender, 2000).  Although no UAS (cggagtactgtcctcc) promoter sequences are found in D. melanogaster (Berkeley Drosophila Genome Project, pers. comm.), our laboratory has demonstrated that expression of GAL4 in the eye with the GMR-GAL4 transgene leads to increased levels of apoptosis and morphological defects (Kramer and Staveley, submitted).   Although the mechanism by which GAL4 induces cell death is unclear, death of the dopaminergic neurons could certainly result in premature lethality in these flies.

     Acknowledgments:  This work was funded by the Natural Sciences and Engineering Research Council of Canada and the Dean of Science of Memorial University of Newfoundland (start-up funds) to B.E.S.  L.D.S. was funded by an Undergraduate Student Research Award from the Natural Sciences and Engineering Research Council of Canada.  We thank Lloyd Smith for design and production of the climbing assay device.  We thank Dr. Jay Hirsh at the Department of Biology, University of Virginia, and Dr. Howard Lipshitz at the Hospital for Sick Children in Toronto for providing fly stocks.

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