Saunders, Lisa D., Annika F.M. Haywood, 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

Corresponding Author: Dr. Brian E. Staveley, Department of Biology, Science Building, Rm. SN-3017, Memorial University of Newfoundland, St. John's, Newfoundland & Labrador, Canada, A1B 3X9
e-mail address: bestave@mun.ca
telephone number: (709) 737-4317; telefax number (709) 737-3018

Abstract

Parkinson's disease (PD) is a prevalent neurodegenerative disease marked by the selective loss of dopaminergic neurons that is accompanied by resting tremors and other symptoms. The study of organismal models of PD, including the well-studied a-synucleinopathic model, in Drosophila melanogaster has lead to a greater understanding of the biological basis of the disease.  In an attempt to establish additional Drosophila models of PD via the manipulation of cell survival signaling, the UAS/GAL4 system was used to overexpress two forms of phosphatidylinositol 3-OH kinase (PI3K) in the dopaminergic neurons of flies. The directed expression of PI3K in this manner dramatically reduces life span and climbing ability while an inhibitory form, a dominant negative version of PI3K, reduces life span in a far less dramatic way.  These novel models should provide the basis for a series of investigations into the role of cell survival signaling in Parkinson's disease.

Introduction

Parkinsonís disease (PD) is a common, age-related neurodegenerative disease characterized by muscle rigidity, resting tremors, and postural instability (Spacey and Wood, 1999; Lansbury and Brice, 2002). Post-mortem analysis of patients reveal that PD appears to be due to the selective loss of dopaminergic neurons in the substantia nigra region of the brain. The underlying cause of this distinctive loss of neurons may be classified as either sporadic or familial in origin.  Although the underlying mechanism is not well understood, defects in several genes as well as a number of environmental toxins have been linked to the cause of this neuronal loss. As it is difficult to research the pathogenesis of PD in living patients, a number of animal models (Dawson, 2000; Hashimoto et al., 2003), including a well established Drosophila model (Feany and Bender, 2000), have been developed to investigate aspects of PD.
A promising series of investigations into the biological basis of PD have been initiated through the generation of a PD model by the conditional expression of human a-synuclein in transgenic Drosophila (Feany and Bender, 2000).  The expression of a-synuclein, in both a pan-neural and dopaminergic neuron-specific manner, produce an age-dependent loss of dopaminergic neurons.  The neuronal loss is accompanied with the premature loss of climbing ability and the formation of cytoplasmic inclusions in the dopaminergic neurons.  In addition, expression of a-synuclein in the developing eye results in an age-dependent degeneration of the retina.  In further experiments, the dopamine precursor levodopa, dopamine receptor agonists, and the anticholinergic agent atropine act to counter the age-dependent loss of climbing ability (Pendleton et al., 2002).  Expression of the molecular chaperone gene hsp70 with a-synuclein prevents dopaminergic neuronal degeneration (Auluck et al., 2002).  The expression of parkin can suppress the loss of dopaminergic neurons (Yang et al., 2003), the premature loss of climbing ability and the age-dependent degeneration of the retina (Haywood and Staveley, in preparation) induced by a-synuclein in Drosophila.  In addition, another model has recently been established with the description of mutants in the parkin gene (Greene et al., 2003).  The Drosophila models of PD are proving to be very effective tools in the investigation of the biological basis of this disease.
 Dopaminergic neurons may die as a result of apoptosis in PD (for review see Lev et al., 2003).  This process may be caused by the accumulation of endogenous toxic proteins or environmental toxins.  Exploration of the role of cell survival signaling in the selective loss of dopaminergic neurons in Drosophila may provide further insight into the basis of PD.  The insulin receptor/ PI3 kinase/ akt anti-apoptotic signaling pathway is highly conserved between mammals and Drosophila (Fernandez et al., 1995, Leevers et al., 1996; Staveley et al., 1998; Datta et al., 1999; Oldham et al., 2000).  To initiate this signal, insulin or insulin-like growth factors bind to receptor tyrosine kinases at the cell membrane and activate the protein phosphatidylinositol 3-OH kinase (PI3K) via phosphorylation (Vanhaesebroeck et al., 2000).  In turn, PI3K phosphorylates inositol lipids on the inner membrane of the cell, which leads to the co-localization of akt and phosphoinosotide-dependent kinase 1 (PDK-1) and, as a result, the activation of akt.  An anti-apoptotic or cell survival signal results from activated akt.  Consequently, manipulation of the InR/PI3K/akt pathway in the dopaminergic neurons of Drosophila melanogaster may produce selective apoptotic death of those cells and produce flies with symptoms similar to other models of PD.  As PI3K is an essential component of this pathway, it is a good candidate for manipulating cell survival signaling.
 The UAS/GAL4 ectopic expression system (Brand and Perrimon, 1993) was used to overexpress wild type and mutant forms of PI3K in the dopaminergic neurons. Climbing and longevity assays were performed and the results demonstrate that overexpression of PI3K dramatically reduces climbing ability and viability of the flies from the time of eclosion.  Overexpression of an inhibitory PI3K also reduces the length of life span when compared to controls but does not prematurely reduce the climbing ability of the flies.

Materials & Methods

Fly stocks and culture: The Ddc-GAL44.3D and Ddc-GAL44.36 transgenic lines (Li et al., 2000) were obtained from Dr. Jay Hirsh at the Department of Biology, University of Virginia.  The UAS-PI3K-dp110 and UAS-PI3K-dp110D954A flies were obtained from Dr. Sally Leevers at the Ludwig Institute for Cancer Research and the Department of Biochemistry and Molecular Biology, University College, London.  The w1118 strain was provided by Dr. Howard D. Lipshitz of the Hospital for Sick Children and the University of Toronto.  All flies were cultured on standard cornmeal/yeast/agar medium at 25oC.
Transgene Expression: The UAS/GAL4 ectopic expression system (Brand and Perrimon, 1993) was used to express wild type and mutant forms of phosphatidylinositol 3-OH kinase (PI3K; Leevers et al., 1996) in the dopaminergic neurons using Ddc-GAL4 transgenes (Li et al., 2000). The progeny of crosses of the Ddc-GAL4 lines to transgenic UAS-PI3K-dp110 flies will express the catalytic subunit of PI3K (dp110) in the dopaminergic neurons. The same Ddc-GAL4 driver lines were crossed to UAS-PI3K-dp110D954A to induce the expression of an inhibitory form of this subunit of PI3K.  The controls were produced by crossing w1118 to the Ddc-GAL4 transgenics.
Aging assay: Adult male flies were collected within 24 hours of eclosion and scored for viability every two to three days to determine the adult life span characteristics as previously described (Staveley et al., 1990). Flies were maintained under non-crowded conditions of approximately 5 to 15 individuals upon standard cornmeal/yeast/agar medium at 250C.
Climbing assay: The climbing ability of male flies of the same age were assayed every four days to determine their locomotor abilities throughout their life span as previously described (Feany and Bender, 2000).  To be precise, the proportion of a cohort of ten (or fewer) flies to climb a distance of 8 centimetres within a period of 18 seconds was determined.  In total, twenty trials were carried out at each time point. From this data, the average number of flies that successfully completed the climb at each time point was calculated.
Data Analysis: Data from the aging and climbing assays were compiled and graphed using Microsoft Excel.

Results and Discussion

Transgenic flies expressing one of the two forms of PI3K in the dopaminergic neurons were tested for viability with an aging assay (Figure 1).  Overexpression of PI3K-dp110 with both of the Ddc-GAL4 transgenes greatly decreased the life span of the flies. The median age of survival (50%) for flies expressing PI3K-dp110 was between 18 and 20 days when expressed by Ddc-GAL44.3D and between 12 and 14 days when expressed by Ddc-GAL44.36.  Expression of the dominant negative form of PI3K (PI3K-dp110D954A) produced a small decrease in survival.  The median age of survival (50%) was between 58 and 60 days under the control of the Ddc-GAL44.36 driver and between 68 and 70 days with the Ddc-GAL44.3D transgene.  The GAL4 heterozygotes, Ddc-GAL44.3D and Ddc-GAL44.36, were tested and the results show a median age of survival between 70 and 72 days for the former and between 82 and 84 for the latter.  The expression of PI3K-dp110D954A resulted in a decrease in median survival of approximately 14 days when compared to the Ddc-GAL4 heterozygote controls while the expression of PI3K-dp110 resulted in a major decrease in life span by between 50 and 70 days.
 To monitor the effects upon locomotion, the climbing ability of these transgenic flies were tested (Figure 2).  Flies that express the wild type version PI3K-dp110 under the control of Ddc-GAL4 climb poorly while those expressing PI3K-dp110D954A appear to climb as well as the controls throughout the duration of the experiment.
In addition to the defects in climbing ability and the greatly reduced life span, flies overexpressing PI3K-dp110 exhibit a blistered wing phenotype shortly after emerging from the pupae cases (data not shown).  Within a day or so, most adult Ddc-GAL4/UAS-PI3K-dp110 flies have shriveled wings.  This defect may be indirectly caused by neuronal loss.
Overexpression of PI3K in the dopaminergic neuron during development may lead to selective apoptotic death of these neurons.  Contrary to the common role of PI3K in supporting cell survival, overexpression of PI3K has been shown to cause apoptosis.  In cultured rat embryo fibroblasts, prolonged activation of PI3K in the absence of other stimuli (serum) results in apoptosis (Klippel et al., 1998).  Prolonged overexpression of PI3K increases in the proportion of cells in G2/M and induces apoptosis in Drosophila (Vanhaesebroeck et al., 2000).  This may be due to deregulation of the cell cycle or the induction of an apoptotic feedback program by the hyperactivation of many signaling pathways.  The selective loss of the dopaminergic neurons via a cell death mechanism could be responsible for the observed poor climbing ability and reduced life span of adult Ddc-GAL4/UAS-PI3K-dp110 flies.
Although active PI3K acts to prevent apoptosis of cells, larvae survive for twenty days without PI3K (Weinkove et al., 1999). In contrast, the inhibitory form of PI3K has been shown to cause cell death when expressed in embryos (Scanga et al., 2000).  In our experiments, the expression level of PI3K-dp110D954A may have been sufficient to induce neuronal loss only in late life.  The small decrease in life span may have resulted from this late loss in neurons in the Ddc-GAL4/UAS-PI3K- dp110D954A flies.
In conclusion, this experiment analyzed the viability and climbing ability of flies expressing two forms of PI3K in an attempt to model characteristics of Parkinson's disease.  Unexpectedly, the ectopic expression of PI3K showed dramatically reduced life span coupled with poor climbing ability.  Unlike Parkinson disease patients, the locomotor dysfunction begins early, rather than arising in a gradual manner, which may be due to the larval expression of PI3K, and subsequent loss of dopaminergic neurons at that stage.  The dominant negative version of PI3K reduced life span by a modest amount but did not seem to influence the ability of these flies to climb.  In summary, our experiments show that the overexpression of PI3K in dopaminergic neurons can produce defects that may recapitulate some aspects of Parkinson's disease in Drosophila melanogaster.

Acknowledgements

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 BES). LDS was funded by an Undergraduate Student Research Award from the Natural Sciences and Engineering Research Council of Canada.  Many thanks to Dr. Jay Hirsh, Dr. Sally J. Leevers and Dr. Howard D. Lipshitz for providing fly stocks. We thank Lloyd Smith for design and production of the climbing assay device.  We thank Justin Moores and Jamie Kramer for a critical reading of the manuscript.
 References

Auluck, P.K., H.Y. Chan, J.Q. Trojanowski, V.M. Lee, and N.M. Bonini 2002, Science 295: 865-8.
Brand, A.H., and N. Perrimon 1993, Development 118: 401-15.
Datta, S.R., A. Brunet, and M.E. Greenburg 1999, Genes Dev. 13: 2905-27.
Dawson, T.M., 2000, Cell 101: 115-8.
Feany, M.B., and W.W. Bender 2000, Nature 404: 394-8.
Fernandez, R., D. Tabarini, N. Azpiazu, M. Frasch, and J. Schlessinger 1995, EMBO J. 14: 3373-84.
Greene, J.C., A.J. Whitworth, I. Kuo, L.A. Andrews, M.B. Feany, and L.J. Pallanck 2003, Proc. Natl. Acad. Sci. USA 100: 4078?83.
Hashimoto, M., E. Rockenstein, and E. Masliah 2003, Ann. N.Y. Acad. Sci. 991: 171-88.
Klippel, A., M.-A. Escobedo, M.S. Wachowicz, G. Apell, T.W. Brown, M.A. Giedlin, W.M. Kavanaugh, and L.T. Williams 1998, Mol. Cell. Biol. 18: 5699?711.
Lansbury, P.T., and A. Brice 2002, Curr. Opin. Cell. Biol. 14: 653-60.
Leevers S.J., D. Weinkove, L.K. MacDougall, E. Hafen, and M.D. Waterfield 1996, EMBO J. 23: 6584-94.
Lev, N., E. Melamed, and D. Offen 2003. Prog. Neuropsychopharmacol. Biol. Psychiatry 27: 245-50.
Li, H., S. Chaney, I.J. Roberts, M. Forte, and J. Hirsh 2000, Curr. Biol. 10: 211-4.
Oldham, S., R. Bohni, H. Stocker, W. Brogiolo, and E. Hafen 2000, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 355: 945-52.
Pendleton, R.G., A. Rasheed, T. Sardina, T. Tully, and R. Hillman 2002, Behav. Genetics 2: 89-94.
Scanga, S.E., L. Ruel, R.C. Binari, B. Snow, V. Stambolic, D. Bouchard, M. Peters, B. Calvieri, T.W. Mak, J.R. Woodgett, and A.S. Manoukian 2000,  Oncogene 19: 3971-7.
Spacey, S.D., and N.W. Wood 1999, Curr. Opin. Neurol. 12: 427-432.
Staveley, B.E., L. Ruel, J. Jin, V. Stambolic, F.G. Mastronardi, P. Heitzler, J.R. Woodgett, and. A.S. Manoukian 1998, Curr. Biol. 8: 599-602.
Staveley, B.E., J.P. Phillips, and A.J. Hilliker 1990, Genome 33: 867-72.
Vanhaesebroeck, B., S.J. Leevers, K. Ahmadi, J. Timms, R. Katso, P.C. Driscoll, R. Woscholski, P.J. Parker, and M.D. Waterfield 2001, Annu. Rev. Biochem. 70: 535?602.
Weinkove, D. T. Twardzik, M.D. Waterfield, and S.J. Leevers 1999, Curr. Biol. 9: 1019-29.
Yang, Y., I. Nishimura, Y. Imai, R. Takahashi, and B. Lu 2003, Neuron 37: 911-24.

Figure legends

Figure 1. Survival of flies expressing wild type (PI3K) and dominant negative PI3K (PI3KDN) in the dopaminergic neurons.  Adult males that express the wild type version of PI3K in the dopaminergic neurons Ddc-GAL44.36/UAS-PI3K-dp110 (large solid triangles) and Ddc-GAL44.3D/UAS-PI3K-dp110 (large solid squares) have a greatly reduced life span when compared to controls.  Expression of the dominant negative PI3K transgene under the same circumstances, Ddc-GAL44.36/UAS-PI3K-dp110D954A (small solid triangles) and Ddc-GAL44.3D/UAS-PI3K-dp110D954A (small solid squares), leads to a slightly reduced life span, when compared to the GAL4-expressing controls, Ddc-GAL44.36/+: (small open triangles) and Ddc-GAL44.3D/+: (small open squares).  The number of individuals aged was as follows: Ddc-GAL44.36/UAS-PI3K-dp110, n = 122; Ddc-GAL44.3D/UAS-PI3K-dp110, n = 129; Ddc-GAL44.36/UAS-PI3K-dp110D954A, n = 107; Ddc-GAL44.3D/UAS-PI3K-dp110D954A, n = 195; Ddc-GAL44.36/+, n = 119; Ddc-GAL44.3D/+, n = 280.

Figure 2.  The measurement of climbing ability of flies expressing wild type (PI3K) and dominant negative PI3K (PI3KDN) in the dopaminergic neurons.  Adult males that express the wild type version of PI3K in the dopaminergic neurons Ddc-GAL44.36 /UAS-PI3K-dp110 (large solid triangles) and Ddc-GAL44.3D/UAS-PI3K-dp110 (large solid squares) have a poor ability to climb when compared to controls.  Expression of the dominant negative PI3K transgene under the same circumstances, Ddc-GAL44.36/UAS-PI3K-dp110D954A (small solid triangles) and Ddc-GAL44.3D/UAS-PI3K-dp110D954A (small solid squares), maintain the ability to climb in a manner similar to the controls, Ddc-GAL44.36/+ (small open triangles) and Ddc-GAL44.3D/+ (small open squares).  The climbing experiments were discontinued when death reduced the number significantly.