Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Department of Biological Sciences, University of Alabama, USA.
Journal of Visualized Experiments (Impact Factor: 1.33). 02/2008; DOI: 10.3791/835
Source: PubMed


Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration. Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets. Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

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    • "We investigated whether overexpression of LRRK2 WT and PD-linked mutants R1441C and G2019S induces neurodegeneration in C. elegans DA neurons. Previous studies have established that DA neurodegeneration can be reliably assayed in live worms by monitoring the morphological changes of DA neurons co-expressing GFP reporter (Berkowitz et al., 2008; Nass et al., 2002). We scored transgenic worms throughout their life span for the survival of each of the four cephalic neurons (CEPs), as these DA neurons are relatively large in size and are free from interference by autofluorescence that can develop in old animals. "
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    • "Toxicity of various types of toxicants, including metals and pesticides, has been studied in C. elegans and has been evaluated as a model for various neurodegenerative disorders (Berkowitz et al., 2008; Silverman et al., 2008). The extensive advantages of using a C. elegans model system have given researchers the capability of examining various endpoints, many of which assess nervous system functioning. "
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