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Keywords:

  • α-synuclein;
  • model organism;
  • motor neuron;
  • neurodegeneration;
  • worm transgenic

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid constructs
  5. Transgenics
  6. Measurement of dopaminergic neurons
  7. Thrashing assay
  8. Immunohistochemistry and immunoblotting
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

Overexpression of human α-synuclein in model systems, including cultured neurons, drosophila and mice, leads to biochemical and pathological changes that mimic synucleopathies including Parkinson's disease. We have overexpressed both wild-type (WT) and mutant alanine53[RIGHTWARDS ARROW]threonine (A53T) human α-synuclein by transgenic injection into Caenorhabditis elegans. Motor deficits were observed when either WT or A53T α-synuclein was overexpressed with a pan-neuronal or motor neuron promoter. Neuronal and dendritic loss were accelerated in all three sets of C. elegans dopaminergic neurons when human α-synuclein was overexpressed under the control of a dopaminergic neuron or pan-neuronal promoter, but not with a motor neuron promoter. There were no significant differences in neuronal loss between overexpressed WT and A53T forms or between worms of different ages (4 days, 10 days or 2 weeks). These results demonstrate neuronal and behavioral perturbations elicited by human α-synuclein in C. elegans that are dependent upon expression in specific neuron subtypes. This transgenic model in C. elegans, an invertebrate organism with excellent experimental resources for further genetic manipulation, may help facilitate dissection of pathophysiologic mechanisms of various synucleopathies.

Abbreviations used
A53T

mutant alanine53[RIGHTWARDS ARROW]threonine

ADE

anterior deirid

CEP

cephalic neurons

GFP

green fluorescent protein

PD

Parkinson's disease

PDE

posterior deirid

UCHL1

ubiquitin carboxyl-terminal hydrolase L1

TBSB

Tris-buffered saline with 0.5% bovine serum albumin

WT

wild type

Synucleopathies represent a large range of neuropathologically defined conditions that include Parkinson's disease (PD), dementia with Lewy bodies, Pick's disease and multiple system atrophy (Spillantini et al. 1997, 1998; Baba et al. 1998; Takeda et al. 1998). PD is a neurodegenerative disorder that affects 1% of the population over the age of 50 years. PD neurodegeneration is found predominantly in dopaminergic neurons of the substantia nigra where the pathological hallmark is the appearance of intracellular inclusions termed Lewy bodies. These bodies consist of protein complexes that include neurofilaments, ubiquitin and α-synuclein (Forno 1996; Spillantini et al. 1997). Rare familial forms of PD have provided an opportunity to understand the pathophysiologic mechanisms of this disease and at least eight PD loci have been identified (Lansbury and Brice 2002). Autosomal dominant forms of PD have been linked to mutations in α-synuclein. Two mutations, alanine to threonine at position 53 (A53T) and alanine to proline at position 30 (A30P), have been identified that are highly penetrant (Polymeropoulos et al. 1997; Kruger et al. 1998). Another autosomal dominant form of PD is caused by a mutation in ubiquitin carboxy-terminal hydrolase (UCH-L1) (Leroy et al. 1998). Recently, it was discovered that UCH-L1 also contains dimerization-dependent, ubiquityl ligase activity (Liu et al. 2002). Mutations in parkin represent a gene defect that underlies PD with an autosomal recessive mode of inheritance (Lucking et al. 2000). Parkin, like numerous other RING finger-containing proteins, has E3 ubiquitin ligase activity and autosomal recessive juvenile-linked Parkin mutants are defective in E3 activity.

A model that describes the interactions of mutant forms of α-synuclein, Parkin and UCH-L1 has been proposed (Shimura et al. 2001), whereby mutation leads to over-accummulation of α-synuclein and formation of Lewy bodies. Most patients with Parkin mutations lack Lewy bodies. Nonetheless, sporadic cases constitute the substantial bulk of PD cases in which the wild-type (WT) form of α-synuclein is present. A mechanism by which dopaminergic neurons are selectively lost in PD has been proposed by Conway et al. (2001) and Xu et al. (2002). It is suggested that oxidative ligation of α-synuclein to dopamine leads to accumulation of neurotoxic adducts.

The best studied rodent and primate models of PD use the administration of neurotoxic agents such as 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which target dopaminergic neurons. Cenorhabditis elegans contain well defined dopaminergic neurons that are visible under fluorescence microscopy (Sulston et al. 1975; Nass et al. 2002) and have a range of locomotor activities (Thomas and Lockery 1999). 6-Hydroxydopamine has been administered to C. elegans whereupon dopaminergic neuron degeneration and membrane blebbing of axons and dendrites have been demonstrated (Nass et al. 2002).

Several different α-synuclein-overproducing transgenic mouse models and a rat virally transduced model have been produced (Kahle et al. 2000; Masliah et al. 2000; van der Putten et al. 2000; Kahle et al. 2002; Kirik et al. 2002; Lee et al. 2002; Richfield et al. 2002). Although many of the results obtained in these models indicate neuronal abnormalities, the studies report differences in time course, progression of pathologic effects, effects elicited by WT or mutant forms, and localization of pathologic changes. In order to explore the effects of α-synuclein in a less complex genetic environment, with a better defined neuronal system, and to obtain better control of the transgene expression, we have overexpressed human WT and a mutant form of α-synuclein in C. elegans. We have taken advantage of different promoters to direct expression of the transgenes into specific subsets of neurons. This transgenic C. elegans model that overexpresses α-synuclein might help to elucidate the pathophysiology of synucleopathies and facilitate the development of novel therapeutic strategies.

Plasmid constructs

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid constructs
  5. Transgenics
  6. Measurement of dopaminergic neurons
  7. Thrashing assay
  8. Immunohistochemistry and immunoblotting
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

The transgenic and neuronal markers Pdat-1::green fluorescent protein (GFP) (Nass et al. 2001; Nass et al. 2002) and Paex3::GFP (Iwasaki et al. 1997) were constructed as described previously. Human α-synuclein was amplified by RT–PCR (Finnzymes, Helsinki, Finland) from human brain RNA using primers (5′-AAAGGAATTCATTAGCCATG; 3′-GGGAGCAAAGATATTTCTTA). The cDNA was cloned into pGEM-TEasy (Promega, Madison, WI, USA) and the sequence matched precisely the coding region for GenBank entry XM_003494. The insert was cloned into the SmaI site of TJ644 (Iwasaki et al. 1997) to create Paex-3::WT. The A53T mutant form of α-synuclein was prepared by the overlapping PCR-mutagenesis strategy (Ausubel et al. 1997) and cloned into the MscI site of TJ1078 (Iwasaki et al. 1997) to create Paex-3::A53T. The acr-2 promoter (Hallam et al. 2000) was cloned into the HindIII–BamHI site of pPD49.26 (Professor Andy Fire, Carnegie Institute of Washington, Baltimore, MD, USA) and the cDNA for wild-type α-synuclein was cloned to the NcoI site, whereas mutant α-synuclein was cloned to the NcoI–SacI site, to create Pacr-2::WT and Pacr-2::A53T respectively. The unc-30 promoter (Allyson McCormick, University of Washington, Seattle, WA, USA) was cloned into the HindIII–XbaI site of pPD49.26, and the cDNA for wild-type α-synuclein was cloned to the NcoI site and that for A53T mutant α-synuclein to the NcoI–SacI site to create Punc-30::WT and Punc-30::A53T respectively. The DAT-1 promoter was amplified and cloned into the HindIII–BamHI site of pPD95.73 (Andy Fire). This promoter was exchanged with the unc-30 promoter of Punc-30::WT and Punc-30::A53T by HindIII–SmaI digestion and ligation to create Pdat-1::WT and Pdat-1::A53T. All plasmids were sequenced to verify cDNA identity, cloning sites and correct orientation.

Transgenics

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid constructs
  5. Transgenics
  6. Measurement of dopaminergic neurons
  7. Thrashing assay
  8. Immunohistochemistry and immunoblotting
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

Transgenic animals were generated using microinjection techniques as described previously for C. elegans (Mello and Fire 1995). The standard DNA concentration injected was 70 ng/µL per construct. pBSK plasmid (Stratagene, La Jolla, CA, USA) was used as carrier DNA for controls. Co-injection of either Paex-3::GFP or Pdat-1::GFP was used as a marker for transgenic animals, and simultaneously to help visualize and locate neurons. A Nikon (Tokyo, Japan) SMZ800 microscope equipped with a P-FLAP fluorescence attachment was used for routine selection of transgenic animals. Transgenic animals collected for the assays were generally from F2–F5 generations.

Measurement of dopaminergic neurons

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid constructs
  5. Transgenics
  6. Measurement of dopaminergic neurons
  7. Thrashing assay
  8. Immunohistochemistry and immunoblotting
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

L4 transgenic animal lines produced by co-injection with Pdat-1::GFP as a marker were selected by fluorescence microscopy a day before the assay and transferred to a fresh plate. On the day of the assay, 10–15 worms at a time were transferred to an agar pad and examined using an Olympus AX70 fluorescence microscope with an Olympus (Tokyo, Japan) UPlan Apo 60X/0.90 objective. The different classes of dopamine neurons [cephalic neurons (CEP), anterior deirid (ADE) and posterior deirid (PDE)] were clearly visible, and the locations and dendritic patterns agreed with those described previously (White et al. 1986; Nass et al. 2002). Presence of a cell body, dendritic morphology and positions were scored individually for each worm. Photographs were taken with a Sensys camera (Photometrics, Roper Scientific, Tucson, AZ, USA) that was connected to the microscope. Cell bodies and dendrites were scored as present if fluorescence could be seen. Dendrites were scored as abnormal if they had breaks or were barely visible.

Thrashing assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid constructs
  5. Transgenics
  6. Measurement of dopaminergic neurons
  7. Thrashing assay
  8. Immunohistochemistry and immunoblotting
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

L4 transgenic animals were selected by fluorescence microscopy a day before the assay and transferred to a fresh plate. On the day of the assay, animals were placed on to a 10-µL drop of M9 buffer on a standard microscope slide and allowed to equilibrate for ∼ 30 s. Animals were scored for the number of times the head crossed an axis drawn across the length of the body in 30 s (Miller et al. 1996; Thomas and Lockery 1999).

Immunohistochemistry and immunoblotting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid constructs
  5. Transgenics
  6. Measurement of dopaminergic neurons
  7. Thrashing assay
  8. Immunohistochemistry and immunoblotting
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

The whole-mount freeze-cracking method was carried out according to Crittenden and Kimble (1999) with a few modifications. Worms were collected into 0.5 mL of M9 medium from starved agar plate and transferred to a centrifuge tube. Worms were spun for 2 min at 225 g and extra medium was removed. Worm suspension (8 µL volume) was transferred on to an object glass that was covered with subbing solution. No paraformaldehyde was added. A coverslip was placed on top of the animals and extra liquid was removed with Whatman #1 paper. After freeze-cracking, worms were blocked in Tris-buffered saline with 0.5% bovine serum albumin (TBSB) for 30 min at 22°C, incubated in 50 µL primary antibody (1 : 300 dilution, mouse IgG1 anti-α-synuclein; BD Transduction Laboratories, San Jose, CA, USA) in phosphate-buffered saline overnight at 4°C or 2 h at room temperature. Worms were washed three times each for 15 min with 200 µL TBSB and incubated with 100 µL secondary antibody (1 : 200 dilution, donkey anti-mouse IgG-RRX; Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA) in TBSB for 1 h at room temperature under a light cover. After washing again as described above, mounting medium was added (FluorSave™ Reagent; CalBiochem, LaJolla, CA, USA) and a coverslip applied. Worms were viewed under epifluorescence with either an Olympus AX70 microscope or Nikon Eclipse/Ultra VIEW confocal microscope.

For immunoblot analysis, 20-µg samples of cell extracts were fractionated on polyacrylamide gels under denaturing conditions and transferred to nitrocellulose membrane (Protran; Schleicher & Schuell, Keene, NH, USA). α-Synuclein was detected with α-synuclein antibody (1 : 250; BD Transduction Laboratories), and immunocomplexes were visualized by using rabbit anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1 : 1000; Amersham Biotech, Amersham, UK) and chemiluminescent detection (Luminol; Sigma, St Louis, MO, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid constructs
  5. Transgenics
  6. Measurement of dopaminergic neurons
  7. Thrashing assay
  8. Immunohistochemistry and immunoblotting
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

Transgenic worms were generated to provide a model with which to investigate the impact of α-synuclein expression on dopaminergic neuron integrity and motor performance. Promoters that direct pan-neuronal (aex-3), dopaminergic (dat-1), or motor neuron-specific expression (acr-2, unc-30) were fused to WT and A53T mutant α-synuclein cDNAs and injected. Transgenic worms carrying constructs of promoters fused to GFP or a stop codon at the second amino acid position of α-synuclein had thrashing values similar to N2 wildtype worms (Table 1). Transgenic worms overexpressing WT and A53T forms of α-synuclein under control of a dopaminergic promoter (dat-1) also displayed thrashing values similar to N2 worms. Transgenic worms overexpressing WT and A53T α-synuclein under control of a pan-neuronal (aex-3) or motor neuron promoter (acr-2, unc-30) displayed significantly lower thrashing values in comparison to controls. An example of transgenic worms carrying a GFP marker only or GFP marker with Paex-3::WT is shown in Figs 1(a) and (b).

Table 1.   Transgenic overexpression of α-synuclein causes impairment in a thrashing assay which is a test of motoric movement
ConstructLocation of expressionNo. of linesThrashes per 30 sn
  1. Adult worms (∼ 4 days old) were transferred from Petri dishes to microscope slides containing isotonic buffer. Each time the worm moved across its body axis a thrash was counted (Thomas and Lockery 1999). Thrashes per 30 s are shown as the mean ±SD. The total number of worms scored is shown (n). STOP, a stop codon was placed at amino acid position 2 of human α-synuclein WT cDNA. *p < 0.05, p < 0.001 versus Paex-3::GFP (Student t-test).

Paex-3::GFPPan-neuronal396 ± 686
Pdat-1::GFPDopaminergic491 ± 10105
Paex-3::STOPPan-neuronal691 ± 796
Pdat-1::WTDopaminergic1598 ± 14144
Pdat-1::A53TDopaminergic896 ± 590
Paex-3::WTPan-neuronal556 ± 19*127
Paex-3::A53TPan-neuronal348 ± 5116
Punc-30::WTMotor neurons777 ± 18135
Punc-30::A53TMotor neurons665 ± 19*86
Pacr-2::WTMotor neurons736 ± 17128
Pacr-2::A53TMotor neurons548 ± 12131
image

Figure 1.  Transgenic C. elegans overexpressing GFP markers and human α-synuclein. Worms were injected with Paex-3::GFP, Paex-3::WT and Pdat-1::GFP. (a) Transmitted light microscope image of an adult carrying the transgenic marker Paex-3::GFP only. (b) Image of an adult carrying the transgenic marker and Paex-3::WT. (c) Epifluorescence image of a transgenic worm injected with Pdat-1::GFP. The two pairs (four neurons) of CEP and one pair (two neurons) of ADE are indicated by arrows. Magnification 400 ×. (d) Image of a PDE neuron pair (two neurons). Magnification was 200 ×. (e) Image of Pdat-1::GFP; Paex-3::WT-injected worm showing CEP but missing ADE neurons. (f) Image of Pdat-1::GFP; Paex-3::WT worm showing only one of two PDE neurons.

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The C. elegans dopamine transporter promoter (Pdat-1) was fused to green fluorescent protein (GFP) and injected to mark dopaminergic neurons in worms under epifluoresence microscopy. The same promoter was fused to α-synuclein WT and α-synuclein A53T, and co-injected with the marker plasmid. Hermaphrodite worms have four pairs of dopaminergic neurons: two pairs of CEP, one pair of ADE and one pair of PDE. These neurons were visible in worms injected with the marker plasmid (Figs 1b–e).

Immunohistochemical analysis demonstrated that α-synuclein was produced in dopaminergic cells when expression was driven by the dat-1 promoter (Fig. 2b) and in many unspecified neurons within the nerve ring when the expression was driven by the aex-3 promoter (Fig. 2a). Confocal microscopy demonstrated that, in some dopaminergic neurons, the dat-1 promoter-driven expression appeared as intracellular inclusions (Fig. 2c). This event was rare and was observed in four of ∼ 200 neurons screened. Immunoblot analysis revealed anti-α-synuclein immunoreactivity in α-synuclein-expressing transgenic worms (Fig. 2d). Immunoreactivity varied between the lines carrying non-integrated WT and A53T α-synuclein constructs. Transmission of transgene expression to each generation was ∼ 20–50% and lines were maintained by regular selection. Immunoreactivity was observed in a human cerebrum lysate (BD Transduction Laboratories) but not in worms carrying the marker construct Pdat-1::GFP only (Fig. 2e).

image

Figure 2.  Immunofluorescence images of C. elegans carrying Paex-3::WT or Pdat-1::WT constructs. Worms (L2–L4 stage) were reacted with mouse anti-human-α-synuclein antibody and donkey anti-mouse IgG secondary antibody. (a) Reactivity can be seen with neurons in nerve ring and other head neurons. (b) Reactivity can be seen in CEP and ADE neurons. (c) Confocal image of transgenic C. elegans worm carrying Pdat-1::WT. Expression of α-synuclein as discrete intracellular inclusions can be seen. (d) Western blot of human α-synuclein proteins. Samples containing 20 μg protein per lane from three independently injected Paex-3::WT or Paex-3::A53T non-integrated transgenic worm lines were blotted. Sizes in kilodaltons (kd) are as indicated. (e) western blot of α-synuclein proteins. Protein (20 µg) from N2 worms carrying only a transgenic marker (dat-1::GFP) was compared with human brain cerebrum proteins (HB; 5 µg protein) as a control. No visible band can be observed from N2 worms. Size markers are shown.

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Dopaminergic neurons were scored after injection of marker (Pdat-1::GFP plasmid) plus carrier, marker plasmid plus α-synuclein WT or marker plasmid plus α-synuclein A53T. A significant reduction in the number of dopaminergic neurons was observed in all sets after co-injection of the α-synuclein constructs (Fig. 3a). Neurons were scored from 30 worms each from four independently injected lines (120 worms) for each construct set. Data shown are mean ± SEM from the four lines. The percentage of CEP neurons present was 93 ± 1.9, 72 ± 2.0 and 66 ± 5.2% for control, WT and A53T respectively. Similar reductions were observed in ADE neurons (94 ± 1.4, 75 ± 1.8 and 82 ± 3.0% respectively) and PDE neurons (85 ± 7.0, 57.5 ± 4.0 and 76 ± 4.8% respectively). There were no observable differences between worms injected with WT or A53T forms of α-synuclein (two-way anova). There were no apparent effects of age as similar results were obtained when worms were scored at 4, 10 or 14/15 days (Fig. 3c and d). Different worms were used for each time point. Loss of dendritic processes from dopaminergic neurons mirrored cell loss (Fig. 3b). Processes present in CEP neurons were (mean ± SEM): 91 ± 2.9, 68 ± 2.6 and 62 ± 6.4% for control, WT and A53T respectively. Similar values were observed in ADE processes (94 ± 1.7, 66 ± 3.8 and 78 ± 3.7% respectively) and PDE processes (83 ± 8.2, 40 ± 4.6 and 58 ± 9.7% respectively). Older worms showed similar results to those scored at 4 days (Fig. 3d).

image

Figure 3.  Quantitation of dopaminergic neurons and dendritic processes in transgenic C. elegans carrying marker (Pdat-1::GFP) and carrier DNA (control), marker and Pdat-1::WT, or marker and Pdat-1::A53T. Neurons were quantitated under epifluorescence microscopy. (a, b) Analysis of dopaminergic neurons and processes from CEP, ADE and PDE sets. (c, d) Presence of dopaminergic neurons and processes at different ages. Values are mean ± SEM from four independently injected lines, 30 worms per line. The maximum score for each line is 120, 60 and 60 for CEP, ADE and PDE neurons respectively. *p < 0.05, **p < 0.001 versus controls from same neuron set and age; #p < 0.05 versus control from same neuron set at 4 days (two-way anova followed by Dunnett's post-hoc test).

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Significant reductions in dopaminergic neurons were also observed when worms were transfected with α-synuclein constructs driven by the aex-3 promoter but not by the motor neuron promoter acr-2. The aex3::WT and aex-3::A53T transgenic worms had mean ± SEM values of 71 ± 6.7 and 73 ± 4.3% for CEP neurons, 78 ± 4.0 and 74 ± 5.0% for ADE neurons, and 57 ± 11 and 56 ± 8.7% for PDE neurons respectively (Fig. 4a). Similar reductions were observed for the existence of dendrites in CEP and ADE dopaminergic neurons (Figs 4 b and c).

image

Figure 4.  Quantitation of dopaminergic neurons and dendritic processes in transgenic C. elegans carrying Pdat-1::GFP (marker) only, or marker with Pacr-2::WT, Pacr-2::A53T, Paex-3::WT or P-aex-3::A53T. (a) Neuron sets CEP, ADE and PDE were quantitated under epifluorescence microscopy. (b,c) Percentage processes existing and percentage abnormal processes were quantitated as described in the Methods section. Values are mean ± SEM from 3–6 independently injected lines, 30 worms per line. *p < 0.05, **p < 0.01 versus controls from same neuron set and age (one-way anova followed by Dunnet's post-hoc test).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid constructs
  5. Transgenics
  6. Measurement of dopaminergic neurons
  7. Thrashing assay
  8. Immunohistochemistry and immunoblotting
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

In the present study, we produced transgenic C. elegans overexpressing human α-synuclein directed to different sets of neurons. Motor deficits, as well as loss of dopaminergic neurons, loss of dendrites and increases in neuronal process breaks, were observed in transgenic worms expressing both WT and A53T forms of human α-synuclein.

Synucleopathies are age-related neurodegenerative conditions, whereas in this C. elegans transgenic model overexpressing α-synuclein, synucleopathy appeared to be a developmental process; no increase in dopaminergic neuronal loss was observed between young and old adults. Dopamine and dopamine transporters, which have been implicated in the neurotoxicity of α-synuclein (Conway et al. 2001; Lee et al. 2001; Xu et al. 2002), are present early on and are apparently sufficient for the observed dopaminergic neuron losses. As C. elegans has a short lifespan, approximately 21 days under laboratory conditions, longer-lived animals may have an increase in neurotoxicity. The results in C. elegans support the hypothesis that complex, accumulative, age-related processes that occur during etiology and pathogenesis are important for full development and progression of human synucleopathies. Mechanisms such as oxidative stress, mitochondrial dysfunction and environmental influences (Olanow and Tatton 1999) occurs in both humans and nematodes, but varies in C. elegans due to growth under controlled laboratory conditions.

α-Synuclein expression as inclusion bodies was rare and aggregation of α-synuclein was not observed in western blots (data not shown). This contrasts to Lewy bodies which are known to contain fibrils of α-synuclein (Spillantini et al. 1997, 1998). It is possible that C. elegans does not contain sufficient cellular components, such as polyamines, that promote fibrillization (Antony et al. 2003) or modifying enzymes (Junn et al. 2003). Alternatively, C. elegans may be more efficient at suppressing aggregation by expressing kinases (Negro et al. 2002; Seo et al. 2002), torsinA or heat-shock proteins (McLean et al. 2002). Finally, it has been reported that concentration greatly affects fibril formation (Uversky et al. 2001) and surviving dopaminergic neurons may not express sufficient α-synuclein for aggregation to take place.

Motor deficits, as measured by a thrashing assay, were dependent on the location of transgene expression. Deficits were observed when α-synuclein was expressed in motor neurons driven by acr-2, unc-30 or aex-3 promoters, but not when expression was limited to dopaminergic neurons. The acr-2 gene encodes a non-α-subunit nicotinic acetylcholine receptor and the promoter directs expression to ventral cholinergic motor neurons (Hallam et al. 2000). The unc-30 gene encodes a homeodomain transcription factor that is expressed in GABAergic D type motor neurons (Jin et al. 1994). Although several studies in mice and one in rat have demonstrated motor deficits following α-synuclein overexpression, it is not clear whether this was due to significant loss of dopaminergic terminals or motor neuron pathology. Motor neurons have been shown to be especially vulnerable to α-synuclein in transgenic mice (Sommer et al. 2000; van der Putten et al. 2000). A general neuronal promoter (Thy-1) was used to direct expression in these studies, so it remains to be determined whether axonal damage or denervation of neuromuscular junctions occurs by direct expression of α-syncuclein in motor neurons. The results presented here suggest that perturbations elicited by α-synuclein depend directly upon the subset of neurons in which the proteins are expressed. Thus, motor deficits were observed in this C. elegans model when α-synuclein was expressed in motor neurons.

Motor deficits were not observed in transgenic α-synuclein worms when the expression was directed to dopaminergic neurons. In C. elegans, dopaminergic neurons are thought to be mechanosensory and to modulate, but do not control motor responses (reviewed in Nass and Blakely 2003). Previous studies have indicated that ablation of all dopaminergic neurons is required for a differential locomotor response to bacteria (Sawin 1996). Moreover, cat-1 mutants that are largely devoid of dopamine have similar thrashing rates to N2 WT worms (Duerr et al. 1999). Taken together with our data that show ∼ 30% loss of GFP-positive dopaminergic neurons in worms that overexpress α-synuclein, and a paucity of α-synuclein expression as cellular inclusions, the lack of correlation between dopaminergic cell loss and motor deficits may be expected.

Significant dopaminergic neuron loss and dendritic breaks were seen in worms injected with α-synuclein WT and A53T when dopaminergic (dat-1) or pan-neuronal (aex-3) promoters drove the expression, but not with a motor neuron promoter (acr-2). The promoters used, dat-1 and aex-3, drive expression that can be detected in early larval stages (Iwasaki et al. 1997; Nass et al. 2002; data not shown) and both neuronal loss and motor deficits were observed at the young adult stage. These findings suggest that the cellular machinery needed to mediate the effects of α-synuclein is already present at an early stage. Moreover, because loss of dopaminergic neurons has been observed in transgenic overexpressing models in nematodes in this study, and flies, mice and human cell cultures in other studies (Feany and Bender 2000; Zhou et al. 2000, 2002; Xu et al. 2002), a common cellular mechanism may ultimately be involved in α-synuclein toxicity.

Similar effects were seen with WT and mutant A53T forms of α-synuclein in both motoric and neuronal measures. To date, it has been unclear in model systems whether the effects of WT and A53T forms can be differentiated. The expression of the A53T form leads to a highly penetrant PD phenotype in humans, whereas in rats the A53T form is the WT form, apparently with no unusual pathology. Neuronal pathology has been observed in transgenic mouse models expressing both WT and mutant forms of α-synuclein in some (Kahle et al. 2000; Kirik et al. 2002) but not all (Richfield et al. 2002) studies.

To our knowledge, a worm synuclein ortholog in C. elegans has not been identified; however, some other proteins known to be involved in synucleopathies, such as ubiquitin and 14-3-3, have been previously described in C. elegans (Ostrerova et al. 1999; Wang and Shakes, 1997; Graham et al. 1989). It has already been suggested that oxidative stress and mitochondrial dysfunction may ultimately underly neurodegeneration in PD (Olanow and Tatton 1999; Hsu et al. 2000) and this might be further investigated in this model (Nass et al. 2001, 2002; Nass and Blakely 2003).

C. elegans has been proposed as a model for various neurodegenerative diseases (Culetto and Sattelle 2000; Link 2001; Nass et al. 2001; Baumeister and Ge 2002). Although the cardinal features of human PD (bradykinesia, rigidy, resting tremor) cannot be recapitulated realistically in nematodes, loss of dopaminergic neurons suggests that C. elegans may provide some insights into the cellular pathology of various synucleopathies. To our knowledge, this is the first published example of an overexpressing α-synuclein C. elegans model. Genetic crosses with null-mutant alleles and forward genetic screens (Jorgensen and Mango 2002) of this model should further help us to understand the role of dopamine and other molecules involved in α-synuclein interactions and ultimately in evolution of synucleopathies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid constructs
  5. Transgenics
  6. Measurement of dopaminergic neurons
  7. Thrashing assay
  8. Immunohistochemistry and immunoblotting
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References

The basic GFP vectors were provided by Andy Fire (Carnegie Institute of Washington). The acr-2 promoter was a gift from Yishi Jin (University California, Santa, Cruz). The unc-30 promoter was from Allyson McCormick (University Washington, Seattle). Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center which is funded the NIH National Center for Research Resources. The technical assistance of Anne Lehtelä and the microscopy expertise of Riitta Miettinen are gratefully acknowledged. This study was supported by a National Parkinson's Foundation Award (RB), the National Institute of Environmental Health grant P30 ES00267 (RN), the Academy of Finland (ML and GW) and the Finnish Ministry of Education (SV).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Plasmid constructs
  5. Transgenics
  6. Measurement of dopaminergic neurons
  7. Thrashing assay
  8. Immunohistochemistry and immunoblotting
  9. Results
  10. Discussion
  11. Acknowledgements
  12. References
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