Researchers have also applied transgenic approaches to C. elegans (Mello and Fire 1995). We have produced a trans-genic Parkinson disease model in C. elegans using overexpression of a-synuclein (Lakso et al. 2003). The product of this gene is found in Lewy bodies, the pathological hallmark of Parkinson disease (Spillantini et al. 1997). Mutations in this gene lead to familial forms of Parkinson disease (Poly-meropoulus et al. 1997). Some of the biochemical features of Parkinson disease were recapitulated in our model. For example, loss of dopaminergic neurons and processes was observed when a-synuclein was overexpressed under control of a dopaminergic neuron-specific promoter. Cellular inclusions consisting of a-synuclein protein were also observed although these events were rare. With regard to movement deficits, a thrashing assay was used. We observed that transgenic C. elegans overexpressing a-synuclein under a pan-neuronal promoter had thrashing values ~50% less than transgenic controls expressing only the transgenic marker. Both overexpressing wild-type (WT) and mutant a-synuclein (A53T) (Lee et al. 2002) worms displayed similar deficit values. An example of tracks left in bacteria by wildtype and transgenic C. elegans overexpressing a-synuclein appears in Figure 1. These transgenic worms were scored at four days of age, which is a young adult stage. In perspective, C. elegans has a life span of three weeks under standard laboratory conditions. Their movement slows during the last week of life and they can appear sluggish. Thus, the basal level in a thrashing assay changes depending upon the age. Whether overexpression of a-synuclein would accelerate this decline in movement remains to be determined. Transgenic C. elegans overexpessing a-synuclein under a pan neuronal promoter also showed deficits in a radial distance assay (unpublished observation). The score for controls ranges from 4 to 7 cm while overexpressing a-synuclein transgenic worms scored from 0 to 2 cm.
One advantage in C. elegans transgenics is direct expression of transgenes specifically to subsets of cells, and in particular to specific subsets of neurons. The deficits in movement that we observed in transgenic C. elegans overexpressing a-synuclein are likely due to pathology in motor neurons. When a-synuclein is overexpressed in dopaminergic neurons, no deficits in movement, as determined by the thrashing assay, were observed. When a-synuclein was overexpressed by the pan-neuronal promoter of either of two different motor neuron promoters, unc-30 or acr-2, investigators observed movement deficits. Unc-30
encodes a homeodomain transcription factor that is necessary and sufficient to specify cell fate of nineteen type-D GABA-ergic motor neurons (Jin et al. 1994). These motor neurons line the ventral side of C. elegans and inhibition by GABA in these neurons, synchronized with excitation by acetylcholine of C. elegans permits the sine wave movement characteristic of worms. Thus, overexpression of a-synuclein within these neurons appears sufficient to perturb movement and is highly suggestive of motor neuron pathology in a-synuclein pathology. An obvious experiment is to mark these neurons with GFP and determine whether they are still present. ACR-2 encodes a non-alpha acetylcholine receptor subunit and ectopic expression of GFP under this promoter indicates expression in most of the ventral cord motor neurons (Hallam et al. 2000). C. elegans overexpressing a-synuclein under control of acr-2 promoter showed the most pronounced motor deficits with thrashing assay values of 40-50% of the controls. How expression of human a-synuclein can alter these neurons remains a matter of speculation, since C. elegans does not have an obvious ortholog to the human gene. Cells need not die for researchers to observe perturbations in movement. For example, transgenic worms overexpressing polyglutamine, as a model of Huntington disease, display neuronal dysfunction in the form of touch insensitivity without neuronal loss (Parker et al. 2001). General effects on gross movement can be attributed in some cases to perturbations in the neuromuscular junction, which would resemble the origin of pathology in many unc mutants. To address possible interacting proteins or downstream targets that contribute to perturbations in movement, we utilize RNA interference (RNAi) studies (Kamath et al. 2003). In this paradigm, phenotypically affected worms are fed bacteria that produce double-stranded RNA upon induction by isopropyl-b-thiogalactopyranoside. Through a mechanism still under intense investigation, the complementary RNA is degraded, and worms undergo a form of worm gene therapy whereby they lose genes necessary to mediate movement deficits. Identifying such genes should indicate new molecules central to the neuropathology of movement disorders. An example of postures of wild-type N2, transgenic a-
synuclein, and unc-22 RNAi-treated C. elegans appears in Figure 2.
While the focus of pathology in Parkinson disease has been degeneration of dopaminergic neurons of the substantia nigra, other regions of the brain are affected. Investigators have shown that brain stem bulbar nuclei that send projections to premotor and motor neurons are affected in human Parkinson disease brains (Braak et al. 2000). In a transgenic mouse model overexpressing a-synuclein controlled by a general promoter, pathology is seen in brainstem and motor neurons that included axonal damage and dener-vation of the neuromuscular junction (van der Putten 2000). At least one approach could help to resolve whether direct pathology of motor neurons underlie movement deficits in the a-synuclein overexpressing worms: direct evaluation of motor neuron pathology in transgenic worms that would be coupled to an inducible transgene system. Such an experimental set-up would eliminate confounds such as developmental effects of the transgene and indirect effects of perturbed motor neurons.
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