Letters to Nature

Nature 404, 394-398 (23 March 2000) | doi:10.1038/35006074; Received 14 February 2000; Accepted 2 March 2000

A Drosophila model of Parkinson's disease

Mel B. Feany1 & Welcome W. Bender2

  1. Department of Pathology, Division of Neuropathology, Brigham and Women's Hospital and Harvard Medical School, and
  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

Correspondence to: Mel B. Feany1 Correspondence and requests for materials should be addressed to M.B.F. (e-mail: Email: mel_feany@hms.harvard.edu).

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Parkinson's disease is a common neurodegenerative syndrome characterized by loss of dopaminergic neurons in the substantia nigra, formation of filamentous intraneuronal inclusions (Lewy bodies) and an extrapyramidal movement disorder. Mutations in the alpha-synuclein gene are linked to familial Parkinson's disease1, 2 and alpha-synuclein accumulates in Lewy bodies and Lewy neurites3, 4, 5. Here we express normal and mutant forms of alpha-synuclein in Drosophila and produce adult-onset loss of dopaminergic neurons, filamentous intraneuronal inclusions containing alpha-synuclein and locomotor dysfunction. Our Drosophila model thus recapitulates the essential features of the human disorder, and makes possible a powerful genetic approach to Parkinson's disease.

It is unclear how mutations in alpha-synuclein, an abundant neuronal protein of unknown function, produce neurodegeneration in familial cases of Parkinson's disease. However, the dominant inheritance pattern and production of insoluble protein aggregates indicate a toxic dominant mechanism, perhaps relating to abnormal protein accumulation. Expression of human alpha-synuclein in Drosophila might therefore model Parkinson's disease. We have produced transgenic fly lines that produce normal human alpha-synuclein and separate lines with each of the two mutant proteins linked to familial Parkinson's disease, A30P and A53T alpha-synuclein.

We use a bipartite expression system that relies on transcriptional activation by the yeast protein GAL4 (ref. 6) to express normal and mutant alpha-synuclein in flies. Normal and mutant human alpha-synuclein complementary DNA constructs are placed downstream of multiple binding sites for GAL4. Transgenic animals carrying the GAL4-responsive constructs are then crossed to a number of well characterized lines that express the yeast activator in a variety of tissue- and cell-type-specific patterns (the 'drivers'). Development of neuronal and non-neuronal tissues proceeds normally in the presence of human alpha-synuclein, as indicated by appropriate external morphology, histological appearance (Fig. 1a, b), viability and behaviour of newly eclosed flies (Table 1). To confirm appropriate activity of the drivers, we expressed an unrelated toxic protein, mutant ataxin-1, in the same developmental and tissue-specific patterns. In contrast to alpha-synuclein, mutant ataxin-1 produces marked defects during development (Table 1). Other laboratories have also induced phenotypes with these driver lines7.

Figure 1: Histological and immunocytochemical analysis of alpha-synuclein transgenic flies.
Figure 1 : Histological and immunocytochemical analysis of |[alpha]|-synuclein transgenic
flies. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a,b, Frontal sections of 60-day-old control fly (a, elav– GAL4/+) and 60-day-old A30P alpha-synuclein transgenic fly (b, UAS–A30P alpha-synuclein/elav–GAL4) stained with haematoxylin and eosin. Overall brain volume, including the outer cellular cortex layer containing neuronal and glial cell bodies (arrows) and central neuropil areas, and overall architecture are preserved. c, 30-day-old control fly (elav–GAL4/+) shows immunostaining for tyrosine hydroxylase in 4–5 cells in the dorsomedial cluster. d, 30-day-old alpha-synuclein-expressing fly (elav/+; UAS–wild-type alpha-synuclein/+) shows no cell-body-associated immunostaining in the same area. e, 10-day-old control fly expressing alpha-galactosidase in both cell cortex and neuronal processes (arrows) of dopaminergic neurons (UAS–lacZ/Ddc– GAL4). f, 10-day-old fly carrying in addition an alpha-synuclein transgene (UAS–A30P alpha-synuclein/+; UAS–lacZ/ Ddc–GAL4) shows no alpha-galactosidase expression in the outer cellular cortex or central neuropil. g, Immunostaining of alpha-synuclein inclusions in the brain from a 30-day-old transgenic fly (UAS–A30P alpha-synuclein /elav–GAL4) in the area of the suboesophageal ganglia with an antibody against alpha-synuclein. h, Human cortical Lewy body (arrow) from the cingulate cortex of a patient with diffuse Lewy body disease, stained with an antibody against ubiquitin (same scale as g). i , Immunostaining of three alpha-synuclein inclusions in the brain of a young adult fly (1 day post-eclosion; Ddc–GAL4/UAS–wild-type alpha-synuclein ) showing irregularity of selected inclusions (arrow) and diffuse immunoreactivity in a larger neuron (arrowhead). j, Neuritic pathology consisting of alpha-synuclein immunoreactive thread- (arrowhead) and grain-like structures. 60-day-old fly (UAS–A30P alpha-synuclein/elav– GAL4). Compare with abnormal neurite in the cingulate cortex of a Lewy body disease patient (h, arrowhead). k, Immunofluoresence staining of eye imaginal disc from wandering third instar larva (UAS–A53T alpha-synuclein /+; gmr–GAL4/+) with antibody against alpha-synuclein showing no inclusions. j, Diffuse cytoplasmic alpha-synuclein immunoreactivity in the adult gut from a 30-day-old fly (UAS–A53T alpha-synuclein /+; e29c–GAL4/+). Scale bars: a,b,e, f, 30 microm; c,d,g,h, 10 microm; i,j, 5 microm.

High resolution image and legend (27K)


The nervous system is appropriately formed in alpha-synuclein transgenic flies, and the aging brain shows no widespread degenerative changes (Fig. 1a, b). Overall brain volume is likewise preserved in many patients with Parkinson's disease, but marked degeneration occurs in specific groups of neurons. Dopaminergic neurons are preferentially lost in Parkinson's disease; we therefore examined dopaminergic neurons in alpha-synuclein transgenic flies. We used multiple independent markers to identify dopaminergic cells.

First, we stained serial tissue sections through the entire brain of the adult fly with an antibody against tyrosine hydroxylase, which specifically identifies dopaminergic neurons. The position and arrangement of dopaminergic neurons in the developing and adult Drosophila nervous system has been documented in detail8, 9, 10. Tyrosine hydroxylase immunostaining shows a prominent cluster of dopaminergic neurons (the dorsomedial group) that is always represented in well oriented sections by four or five robustly staining cells (Fig. 1c). These cells are present in one-day-old control flies and are not lost with age, being readily identified in 60-day-old flies. The lifespan of control flies and alpha-synuclein transgenic animals is about 60 days under our 25 °C culture conditions.

In contrast, Drosophila that express alpha-synuclein in a pan-neural distribution (elav–GAL4 driver) show a marked, age-dependent loss of dorsomedial dopaminergic neurons (Fig. 1d). In young adult flies expressing wild-type and mutant forms of alpha-synuclein, the dorsomedial cluster consists of the normal complement of four or five neurons. In 30–60-day-old alpha-synuclein transgenic flies, however, the cluster is either absent or consists of a single tyrosine-hydroxylase-positive cell (Fig. 1d). These dopaminergic neurons disappear in flies expressing wild-type, A30P or A53T alpha-synuclein.

To confirm that dopaminergic neurons degenerate in alpha-synuclein transgenic animals, we used a driver line containing the promotor for the DOPA decarboxylase gene (Ddc–GAL4), another marker of dopaminergic neurons. The Ddc line drives reporter-gene expression specifically in dopaminergic cells, allowing us to identify them. Transgenic flies expressing A30P alpha-synuclein under the control of the Ddc driver line show robust immunostaining for alpha-synuclein at one day of age. However, after 30 days, there is no alpha-synuclein immunostaining associated with cell bodies, consistent with degeneration of dopaminergic neurons. To ensure that the Ddc driver line remains active at 30 days, the marker protein beta-galactosidase was expressed instead of alpha-synuclein. beta-galactosidase immunoreactivity is present at 30 days when the Ddc line is used to drive beta-galactosidase expression. We also confirmed that dorsomedial cluster neurons are depleted at 30 days by tyrosine hydroxylase immunostaining brains from flies expressing wild-type, A30P or A53T alpha-synuclein under the control of the Ddc –GAL4 driver.

As a final test for degeneration of dopaminergic neurons, both beta-galactosidase and A30P alpha-synuclein were expressed at the same time, in the same dopaminergic neurons, using the Ddc driver line. In one-day-old transgenic flies, dopaminergic neurons are readily identified by beta-galactosidase immunostaining. In contrast, at 10 days of age, beta-galactosidase expression is undetectable (Fig. 1f, compare with 10-day-old controls in Fig. 1e).

Not all dopaminergic neurons degenerate in alpha-synuclein transgenic flies. Substantial numbers of tyrosine-hydroxylase-positive cells remain in aged flies that express alpha-synuclein in a pan-neural distribution ( elav–GAL4 driver) or specifically in dopaminergic neurons (Ddc –GAL4 driver). Preserved neurons may reflect variation in the amount of GAL4 activator protein available to drive alpha-synuclein expression in particular cells. Alternatively, certain subsets of Drosophila dopaminergic neurons may be particularly sensitive to alpha-synuclein toxicity. In patients with Parkinson's disease, preferential degeneration of specific dopaminergic neurons occurs even within the same neuronal nucleus11.

Neurodegeneration induced by alpha-synuclein shows specificity for dopaminergic neurons. Pan-neural expression of alpha-synuclein in the brain produces no demonstrable loss of volume in the outer cellular cortex or in the central neuropil area (Fig. 1b). The excess vacuolization characteristic of other, more generalized, neurodegenerative mutations in Drosophila is not present12, 13. In addition, no excess of degenerating neurons is detected by toluidine blue staining or ultrastructural examination. Thus, most neurons are preserved in flies expressing alpha-synuclein. Of course, such anatomical investigations do not exclude degeneration of a minor neuronal subpopulation.

To address the possibility that subsets of non-dopaminergic neurons are vulnerable to alpha-synuclein toxicity, we examined a second major amine transmitter in Drosophila, serotonin. Serotonergic neurons can degenerate in patients with Parkinson's disease14. Whole-mount preparations of adult brains stained with an antibody against serotonin revealed no differences between 30-day-old flies expressing A30P alpha-synuclein in a pan-neural pattern and controls. The anatomical arrangement of serotonergic cells has been described in detail in Drosophila15. We can identify all the major serotonergic cell groups in experimental animals and controls. However, we cannot exclude an effect of alpha-synuclein expression on a small subgroup of serotonergic cells.

Expression of human alpha-synuclein in flies thus replicates three key features of the pathology of Parkinson's disease: adult onset, involvement restricted to the nervous system and anatomical specificity within the nervous system.

The most specific and diagnostic feature of Parkinson's disease is an alpha-synuclein-rich cytoplasmic filamentous aggregate called the Lewy body. Lewy bodies in the cerebral cortex are best visualised by immunostaining16, 17. When we immunostain brains from aged flies expressing normal and mutant alpha-synuclein in a pan-neural pattern with antibodies against human alpha-synuclein, we observe a distinct punctate pattern of staining suggestive of aggregate formation (Fig. 1g). The fly alpha-synuclein inclusions strongly resemble cortical Lewy bodies from patients with diffuse Lewy body disease, a disorder closely related to Parkinson's disease ( Fig. 1h). Most neuronal cytoplasmic inclusions are single, round and regular, as in Fig. 1g. Occasional inclusions are more irregular (Fig. 1i). Neuritic alpha-synuclein pathology is also present in transgenic fly brains, and consists of both thread- and grain-like inclusions (Fig. 1j). The light microscopic morphology of inclusions produced by wild-type, A30P or A53T alpha-synuclein transgenic flies is indistinguishable.

Electron microscopy reveals cytoplasmic inclusions in brains from flies expressing alpha-synuclein (pan-neural elav–GAL4 driver) that have a relatively homogenous core and are edged by radiating filaments projecting into a surrounding halo (Fig. 2a). The filaments are 7–10 nm in diameter and are sometimes associated with cellular organelles (Fig. 2b, arrow). Granular material is admixed with loosely packed filaments in less compact inclusions ( Fig. 2b, arrowhead), as in cortical Lewy bodies16, 17, 18. The overall morphology of the inclusions, the filamentous and granular nature of the aggregates and the size and disposition of the component filaments are all reminiscent of human Lewy bodies18. We never saw similar inclusions in aged control flies. Immunoelectron microscopy reveals Hrp labelling concentrated over the inclusions (Fig. 2c). Faint radiating filaments are visible peripherally in the unstained preparation ( Fig. 2c, arrow). No immunoreactivity was present in identically treated specimens from control flies of the same age.

Figure 2: Electron microscopy of alpha-synuclein inclusions in flies.
Figure 2 : Electron microscopy of |[alpha]|-synuclein inclusions in flies. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, Neuronal cytoplasmic inclusion with relatively homogenous core and peripheral radiating filaments (arrow) from a 25-day-old alpha-synuclein transgenic fly (UAS–A30P alpha-synuclein/elav–GAL4). nu, nucleus. b, Higher magnification of a more diffuse inclusion from a fly of the same age and genotype showing loosely arranged filaments, sometimes associated with cellular organelles (arrow), intermixed with granular material (arrowhead). c, Immunoelectron microscopy showing central electron-dense Hrp immunolabelling in an inclusion from a 60-day-old fly of the same genotype. Faint radiating filaments (arrow) are visible peripherally in this unstained specimen. Scale bars: a, 0.5 microm; b,c, 0.1 microm.

High resolution image and legend (28K)

The time of appearance of alpha-synuclein inclusions in the fly brain depends on the level of alpha-synuclein expression. The inclusions appear at 20–30 days of age when alpha-synuclein expression is pan-neural. In younger brains, the immunostaining pattern is diffuse and cytoplasmic (Fig. 1i, arrowhead). Immunoreactivity is also diffuse and cytoplasmic in developing tissues (Fig. 1k, eye imaginal disc) and non-neuronal tissues (Fig. 1l, adult gut) when the GAL4 drivers induce expression in these tissues. Inclusion formation thus parallels alpha-synuclein toxicity in both its restriction to the nervous system and its timing. Inclusions are present in 2–10% of neurons throughout the central body complex at 30 days of age (pan-neural elav–GAL4 driver), and are not restricted to dopaminergic neurons. Lewy bodies in classic Parkinson's disease and in diffuse Lewy body disease are also present in a variety of nuclei and neuronal subtypes, not all of which show obvious degeneration.

Given the neuronal degeneration and widespread inclusion formation present in alpha-synuclein transgenic flies, we searched for behavioural manifestations of nervous system dysfunction. Locomotor behaviour is grossly preserved in young flies. However, as transgenic flies with pan-neural expression of alpha-synuclein age, they develop locomotor dysfunction (Fig. 3). Normal Drosophila display a strong negative geotactic response. When tapped to the bottom of a vial they rapidly climb to the top of the vial, and most flies remain there. As they get older, normal flies no longer climb to the top of the vial, but instead make short abortive climbs and fall back to the bottom of the vial. The loss of the climbing response has been used to monitor aging-related changes in Drosophila19, 20. Flies transgenic for alpha-synuclein initially climb as well as control flies. However, over time they decline in performance more rapidly than controls (Fig. 3). The progressive, accelerated decline in climbing ability in alpha-synuclein transgenic flies demonstrates a functional deficit produced by alpha-synuclein expression in the nervous system. The time course of locomotor dysfunction parallels the degeneration of dopaminergic neurons and the appearance of alpha-synuclein inclusions.

Figure 3: Premature loss of climbing ability in alpha-synuclein transgenic flies.
Figure 3 : Premature loss of climbing ability in |[alpha]|-synuclein transgenic
flies. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

Flies expressing wild-type, A30P and A53T alpha-synuclein are significantly different from control flies from days 23 to 45 (P < 0.01, one-way analysis of variance with supplementary Newman–Keuls test). Asterisk, climbing scores for A30P transgenic flies that are significantly different from the wild-type and A53T transgenic scores. The s.e.m.s of 20 trials are within the symbols. Control genotype: elav–GAL4/+. Experimental genotypes: (1) elav/+; UAS–wild-type alpha -synuclein/+; (2) UAS–A30P alpha-synuclein/elav–GAL4; (3) UAS–A53T alpha-synuclein/elav–GAL4.

High resolution image and legend (12K)

Drosophila expressing A30P alpha-synuclein lose their climbing ability earlier than flies expressing wild-type or A53T alpha-synuclein. This small but statistically significant effect may reflect either enhanced biological toxicity of the A30P mutant protein or small variations in the amount of alpha-synuclein produced in the transgenic lines. We examined alpha-synuclein expression in multiple independent transgenic lines for each alpha-synuclein variant by western blotting, by immunofluorescence on eye imaginal discs and by immunohistochemical staining on sections of adult Drosophila, and we have compared transgenic lines with similar levels of alpha-synuclein as determined by all three assays. However, small differences in alpha-synuclein protein levels may have escaped our detection.

Degenerative changes are not restricted to the brain. Retinal degeneration occurs when alpha-synuclein is expressed specifically in the eye (gmr –GAL4 driver). Expression of wild-type or mutant alpha-synuclein during development of the eye produces no effect. However, continued expression of alpha-synuclein in the adult eye produces retinal degeneration that is detectable by ten days, and marked at 30 days in transgenic flies expressing wild-type (Fig. 4), A30P or A53T alpha-synuclein. Retinal degeneration can be readily monitored under the dissecting microscope in live flies by examining an optical effect termed the retinal pseudopupil21. The pseudopupil is quite sensitive to disruptions in the normal architecture of the retina, and becomes abnormal as the retinas of the alpha-synuclein transgenic flies degenerate. The presence of an easily assayed, nervous-system-specific, degenerative phenotype will facilitate generation of second site modifiers and other pharmacological and transgenic manipulations designed to modify neurodegeneration.

Figure 4: Retinal degeneration in alpha-synuclein transgenic flies.
Figure 4 : Retinal degeneration in |[alpha]|-synuclein transgenic flies. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, One-day-old control fly. b, Normal retina in one-day-old alpha-synuclein transgenic fly. c, Well preserved retina in 30-day-old control fly. d, 30-day-old alpha-synuclein-expressing fly showing retinal degeneration with vacuolization and architectural distortion. Scale bar, 15 microm. Genotypes include control: gmr–GAL4/+; transgenic: UAS–wild-type alpha-synuclein /gmr–GAL4 flies.

High resolution image and legend (50K)

Retinal degeneration in alpha-synuclein transgenic flies shows that in flies, as in people, alpha-synuclein-related degenerative changes show relative rather than absolute specificity for dopaminergic neurons. In fact, the Drosophila retina may be resistant to alpha-synuclein toxicity because more transgenic alpha-synuclein is produced in the eye (gmr–GAL4) than the brain (elav–GAL4).

The modest difference between the toxicity of wild-type and A30P mutant alpha-synuclein in the locomotor assay, and the similar effects of all three alpha-synuclein variants on dopaminergic neuronal loss and retinal degeneration, are consistent with the fact that most patients with Parkinson's disease have the wild-type form of the protein, which does become incorporated into Lewy bodies. We cannot show that A53T mutant alpha-synuclein is more toxic than the wild-type protein in any of our assays. A53T mutant alpha-synuclein shows accelerated aggregation in vitro22, 23 and enhanced toxicity in some assays24, 25. The mouse, however, carries the A53T allele as its normal sequence26. A53T mutant alpha-synuclein may be better tolerated in animals than is suggested by in vitro and cellular assays.

We have created a model of Parkinson's disease by expressing human alpha-synuclein in Drosophila. The parallel time course of dopaminergic cell degeneration, inclusion formation and locomotor dysfunction indicates that the three abnormalities may be causally related. We will now be able to delineate underlying pathogenetic mechanisms and identify novel proteins mediating alpha-synuclein toxicity in a genetically tractable organism. Our transgenic fruit flies provide a genetic complement to alpha-synuclein transgenic mice27.

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Methods

Transgenic Drosophila

We cloned normal and mutant alpha-synuclein and SCA1 cDNAs into the GAL4-responsive pUAST expression vector, and created transgenic strains by embryo injection. At least four independent transgenic lines were derived for wild-type alpha-synuclein and each alpha-synuclein mutant, and all were tested for protein expression. Western blots with two monoclonal antibodies (LB509 and clone 42, see below) revealed an appropriately sized band (Mr 19K) in heads of transheterozygous flies of the following six genotypes: (1) elav– GAL4/+; UAS–wild-type alpha-synuclein/+; (2) UAS–A30P alpha-synuclein /elav–GAL4; (3) UAS–A53T alpha-synuclein/ elav–GAL4; (4) UAS–wild-type alpha-synuclein/ gmr–GAL4; (5) UAS–A30P alpha-synuclein/+; gmr– GAL4/+; (6) UAS–A53T alpha-synuclein/+; gmr–GAL4/+. Wild-type and mutant lines expressed 30% or less alpha-synuclein per mg total brain protein compared with rat and human controls. For wild-type alpha-synuclein and each mutant, we analysed 2–5 independent transgenic lines with varying alpha-synuclein expression in the assays described.

The GAL4 driver lines used include: 24B–GAL4 (ref. 6), 32B–GAL4 (ref. 6), dpp–GAL4, Ddc–GAL4 (ref. 28), e29c–GAL4, elav–GAL4 (ref. 29) and gmr–GAL4 (ref. 30). Flies expressing GAL4 from the Ddc, elav or gmr promotor, but without a UAS–alpha-synuclein target, were used as controls, and had the following genotypes: Ddc–GAL4/+, elav–GAL/+ and gmr–GAL4/+. The wild-type chromosome from the control heterozygotes was derived from the w- background strain used for embryo injections. The GAL4-responsive lacZ reporter construct was UAS–lacZ Bg4-1-2 (ref. 6).

We used a Ddc–GAL4 line to express alpha-synuclein and beta-galactosidase in dopaminergic cells28. At the time points reported, in flies of the genotype Ddc–GAL4/UAS–lacZBg4-1-2 , all cells clearly positive for beta-galactosidase also expressed tyrosine hydroxylase by double-label immunohistochemistry. Only a subset of tyrosine-hydroxylase-positive cells expressed beta-galactosidase at our level of detection. We thus restrict our conclusions to a subset of dopaminergic cells in experiments with the Ddc–GAL4 line.

Sectioning, immunostaining and electron microscopy

Adult flies were fixed in formalin at 1, 10, 30 and 60 days, and embedded in paraffin. Whole-mount preparations fixed in formalin were also used. Immunostaining on paraffin sections was performed using an avidin-biotin-peroxidase complex (ABC) method. Antibodies, sources and dilutions include: anti-tyrosine hydroxylase, Chemicon, 1:500; LB509, Zymed Labouratories, 1:500; clone 42, Transduction Laboratories, 1:200; anti-serotonin, Sigma, 1:500; anti-beta-galactosidase, Promega, 1:500; anti-ubiquitin polyclonal serum, Chemicon, 1:1,000. No endogenous immunoreactivity was revealed in tissue sections from nontransgenic control Drosophila stained with the anti-alpha-synuclein antibodies LB509 and clone 42.

To assess brain morphology, serial 4-microm sections including the entire brain were prepared from formalin-fixed, paraffin-embedded heads from flies of the following three genotypes: (1) elav–GAL4/+; UAS–wild-type alpha-synuclein /+; (2) UAS–A30P alpha-synuclein/elav–GAL4; and (3) UAS–A53T alpha-synuclein/elav–GAL4. Sections were stained with haematoxylin and eosin. Time points monitored were 1, 10, 30 and 60 days. In addition, serial 1-microm sections of glutaraldehyde-fixed heads from flies of the same genotypes prepared at 1, 30 and 60 days were stained with toluidine blue to highlight degenerating cells. No evidence of excess neurodegeneration was detected using either technique.

To evaluate dopaminergic cells of the dorsomedial cluster by tyrosine hydroxylase immunostaining, serial 4-microm sections were cut to include the entire brain. Immunopositive cells at the level of the giant interneuron commissure, posterior to the fan-shaped body, were counted in well oriented frontal sections at 1, 10, 30 and 60 days. At 1 day all control and experimental sections contained four or five cells in the delineated region. At 30 and 60 days all controls showed four or five cells. At 30 and 60 days all alpha-synuclein-expressing animals (alpha-synuclein, elav–GAL4 and alpha-synuclein, Ddc–GAL4 transheterozygotes) showed 0 or 1 tyrosine-hydroxylase-positive cell in the defined region. Tyrosine-hydroxylase-positive cells outside the dorsomedial cluster were present, and served as internal controls for the immunostaining procedure. At least four, and usually between six and ten brains were examined for wild-type alpha-synuclein and each mutant alpha-synuclein. Controls included young and aged flies of the genotypes elav–GAL4/+ and Ddc–GAL4/+. We evaluated expression of alpha-synuclein and beta-galactosidase on similar serial section preparations. Quantification was simplified in these experiments because no clear cell-body-associated alpha-synuclein or beta-galactosidase immunoreactivity was observed in the aged alpha-synuclein transgenic flies at the times reported.

For histological examination of retinas, heads were fixed in glutaraldehyde and embedded in epon. Tangential retinal sections were prepared at a thickness of 1 microm and stained with toluidine blue (Fig 4).

Standard electron microscopy was performed on brains from 25-day-old experimental (UAS–A30P alpha-synuclein/elav–GAL4) and control (elav–GAL4/+) flies. For immunoelectron microscopy, pre-embedding immunohistochemistry with an Hrp-congugated secondary antibody was performed on 60-day adult brains from experimental (UAS–A30P alpha-synuclein /elav–GAL4) and control (elav–GAL4/+) flies fixed in 4% paraformaldehyde with 0.5% glutaraldehyde. Tissue was post-fixed in osmium and embedded in epon. Unstained ultrathin sections and ultrathin sections stained with uranyl acetate and lead citrate were examined.

Climbing assay

The climbing assay was performed as described19, 20. Forty flies were placed in a plastic vial, and gently tapped to the bottom of the vial. The number of flies at the top of the vial was counted after 18 s of climbing. Twenty trials were performed for each time point. The data shown represent results from a cohort of flies tested serially over 55 days. The experiment was repeated three times, with independently derived transgenic lines. Similar results were obtained from each experiment. The experiment was carried out under red light (Kodak Safelight Filter 1A). Control flies were of the genotype elav–GAL4/+. Experimental animals were of the following genotypes: (1) elav–GAL4/+; UAS–wild-type alpha-synuclein/+; (2) UAS–A30P alpha-synuclein /elav–GAL4; and (3) UAS–A53T alpha-synuclein/ elav–GAL4.

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Acknowledgements

We thank J. Hirsh, J. Hardy, M. Farrer and H. Orr for flies and DNAs; J. Hirsh, D. Dickson, M. Frosch, K. Buckley, W. Quinn and D. Morisato for discussions; and H. Shing, L. Trakimas, A. Merola, C. Ridolfi and M. Ericsson for technical assistance. M.B.F. thanks J. Gusella and the American Parkinson Disease Foundation for encouragement. Support was provided by a Howard Hughes Physician Postdoctoral Fellowship and a grant from the N.I.A. to M.B.F, and by a grant from the N.I.H. to W.B.