Department of Biological Sciences, University of Alabama
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Berkowitz, L. A., Hamamichi, S., Knight, A. L., Harrington, A. J., Caldwell, G. A., Caldwell, K. A. Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes. J. Vis. Exp. (17), e835, doi:10.3791/835 (2008).
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 1. 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 2. 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 3, 4.
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.
A. Expression Plasmid Construction
Two plasmids are required: one for tissue-specific expression of the gene of interest and a second as selectable transformation marker (though the marker plasmid is usually available from within the research community).
Selectable Marker Plasmid
A transgenic marker plasmid consists of a vector with a promoter driving the expression of a fluorescent protein in an obvious tissue. In this particular procedure, the unc-54 promoter drives cherry protein expression in body wall muscle (Punc-54::cherry).
B. Generation of Transgenic C. elegans via Microinjection
See related JoVE article: http://www.jove.com/index/details.stp?ID=833
C. Genetic crosses for DA neurodegeneration Analysis
D. Dopaminergic Neuron Analysis
The age-dependent loss of dopamine neurons is a clinical hallmark of Parkinson's disease and has been associated with the accumulation or misfolding of a protein called alpha-synuclein. Here we demonstrate how to label, via a fluorescent transgene, the dopaminergic neurons of C. elegans and mimic the neurodegeneration seen in Parkinson's disease by coexpressing human alpha-synuclein in these cells.
This video depicts the methodology for growth, genetic crossing, mounting, and scoring of transgenic nematodes to evaluate genetic factors that either enhance neurodegeneration or provide neuroprotection over the course of aging. Care is taken to use markers for transgene maintenance appropriately staged male and hermaphrodites to ensure successful genetic crosses, and consistency in scoring neuron loss or protection. In this manner, C. elegans facilitates rapid evaluation of genetic factors that may either contribute to neurodegeneration or represent therapeutic targets for enhancing neuron survival.
We wish to acknowledge the cooperative spirit of all Caldwell Lab members. Movement disorders research in the lab has been supported by the Bachmann-Strauss Dystonia & Parkinson Foundation, United Parkinson Foundation, American Parkinson Disease Association, Parkinson's Disease Association of Alabama, the Michael J. Fox Foundation for Parkinson's Research, and an Undergraduate Research
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2. Hamamichi, S., Rivas, R.N., Knight, A.L., Cao, S., Caldwell, K.A. and Caldwell, G.A. Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson’s disease model. PNAS 105, 728-733 (2008).
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