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 JoVE Biology

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

1, 1, 1, 1, 1, 1

1Department of Biological Sciences, University of Alabama

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    Summary

    This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

    Date Published: 7/18/2008, Issue 17; doi: 10.3791/835

    Cite this Article

    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).

    Abstract

    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.

    Protocol

    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).

    Experimental Plasmid

    1. Select a promoter that is expressed in the tissue/cell type of interest; in this case, the DA transporter (Pdat-1) promoter is used. This expression plasmid was created as an Invitrogen Gateway system-compatible destination vector (pDEST-DAT-1) to allow the insertion of any gene of interest downstream of the promoter by recombinational cloning 1.
    2. The cDNA of the gene of interest is PCR amplified using primers that contain the Gateway att B recombinational sequence. The amplified cDNA is first recombined into the donor vector pDONR201 to create the entry vector and then transformed into E. coli strain DH5α. Following selection of successful recombinants and mini-prep isolation of DNA, the entry vector is recombined into the pDEST-DAT-1 destination vector to create the expression plasmid. Details on recombinational cloning are available from either the Invitrogen Gateway manual or in Caldwell et al. 5.

    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

    1. Worms are grown using standard procedures 6.
    2. Place 10 Pdat-1::α-syn; Pdat-1::GFP males 1, 2 onto a mating plate with 3-4 L4 stage transgenic hermaphrodites (expressing Pdat-1::gene X and Punc-54::cherry). This should be performed individually for each of the three separate stable lines created. After two days, remove the males.
    3. Inspect the F1 generation; if there are several male progeny, the mating was successful.
    4. Clone out 5 hermaphrodite L4 animals from each cross that exhibit both the Pdat-1::GFP fluorescent marker (inherited from the male parent) and the Punc-54::cherry fluorescent body wall muscle marker (inherited from the hermaphrodite parent). The cloned animals need to be at the L4 stage to ensure that they have not yet mated with male animals present on the plate. Allow them to self-cross and produce F2 progeny.
    5. F2 animals produced in step 3 are cloned out. Specifically, transfer ~5-10 animals that exhibit both fluorescent markers to their own individual plates. Screen the F3 generation for plates where 100% of the animals express GFP in DA neurons and some of the animals are express cherry in body wall muscle cells. These animals will be homozygous for the Pdat-1::α-syn; Pdat-1::GFP gene while stably transmitting the newly created transgene (gene X).

    D. Dopaminergic Neuron Analysis

    1. Prepare a fresh plate for each of the three lines created above, as well as the Pdat-1::α-syn; Pdat-1::GFP alone strain. Allow them to grow to adulthood.
    2. Transfer 30-40 transgenic adults from each line onto a fresh plate. Allow them to lay embryos for 4 hours at 20ºC.
    3. Remove the adults. Allow the embryos to develop for 3-4 days at 20ºC until they reach the L4 stage.
    4. Pick ~100 transgenic L4 animals to a plate containing 0.04 mg/ml 5-fluoro-2'-deoxyuridine (FUDR). This nucleotide analog blocks development of the next generation via inhibition of DNA synthesis, thus preventing the offspring from overwhelming the experimental animals 7. Protect the FUDR plates from the light since it is light sensitive.
    5. Adult animals will be analyzed at various days following egg laying The appropriate days of analysis are determined empirically. For example, we have determined that 77%, 87%, and 90% of the Pdat-1::α-syn; Pdat-1::GFP animals exhibit degenerated DA neurons at days 6, 7 and 10 post development, respectively. Therefore, if we predict that the transgene of interest might enhance neurodegeneration, we will analyze at day 6 and/or 7. Likewise, transgenes that might suppress neurodegeneration will be analyzed at days 7 and 10.
    6. Make 4 fresh agarose pads (unlike the microinjection agar pads, these are used fresh and are not allowed to dry out). Set out two microscope slides that have a piece of tape across them; the tape serves as spacer for equivalently thick pads. Lay a third slide between them on the bench (they are positioned three-abreast). Place a drop of molten agarose on the center slide and quickly lay another slide on top of the agarose, perpendicular to and across all three slides. Make several additional pads, leaving the two slides together until the pad is ready for use.
    7. To analyze worm DA neurons, place a 6 μl drop of 3 mM levamisole (an anesthetic) on a 22 x 30 mm cover glass. Pick 40 adult animals (10 extra beyond the 30 to be scored) into the drop, then invert the cover glass onto the agarose pad. Repeat for each of the other lines.
    8. Score 30 animals per line for the presence of each CEP and ADE neuron using a compound microscope with epifluorescence and a filter cube that allows visualization of FITC or GFP. We use an Endow GFP HYQ filter cube (Chroma Technology). Record data on the attached scoring sheet. Wild-type animals will retain complete GFP fluorescence in all 4 CEP and 2 ADE neurons. Individual animals exhibiting loss of DA neurons are scored as non-WT or "degenerating". Likewise, the extent of degeneration can be scored by counting the total neurons lost in each animal as well as the population.
    9. Repeat the analysis 2 more times (30 more animals per line per trial). The total number of worms exhibiting wild-type DA neurons from the three rounds of analyses is averaged. Statistical analysis for neuroprotection is then performed using the student’s t-test (p < 0.05) to compare control worms (Pdat-1::α-syn; Pdat-1::GFP) with strains that over-express candidate genes in DA neurons.

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    Discussion

    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.

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    Disclosures

    Acknowledgements

    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

    Materials

    Name Type Company Catalog Number Comments
    Agarose Ultrapure Invitrogen 15510-027
    Coverglass 20x30 mm Fisher Scientific 12-548-5A
    Microscope Slides Plain, 3x1" Fisher Scientific 12-549
    Dissecting with Fluorescence Microscope Nikon Instruments SMZ800
    Dissecting Microscope Nikon Instruments SMZ645
    Epifluorescent Microscope Nikon Instruments Model E-800
    Filter Cube, GFP HYQ Endow Bandpass Chroma Technology Corp.

    References

    1. Cao, S., Gelwix, C.C., Caldwell, K.A., Caldwell, G.A. Torsin-mediated neuroprotection from cellular stresses to dopaminergic neurons of C. elegans. J Neurosci 25, 3801-3812 (2005).

    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).

    3. Cooper, A.A., Gitler, A.D., Cashikar, A., Haynes, C.M., Hill, K.J., Bhullar, B., Liu, K., Xu, K., Strathearn, K.E., Liu, F., Cao, S., Caldwell, K.A., Caldwell, G.A., Marsischky, G., Kolodner, R.D., Labaer, J., Rochet, J.C., Bonini, N.M., and Lindquist, S. alpha-synuclein blocks ER-golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science 313, 324-328 (2006).

    4. Gitler, A.D., Bevis, B.J., Shorter, J., Strathearn, K.E., Hamamichi, S., Su, L.J., Caldwell, K.A., Caldwell, G.A., Rochet, J-C., McCaffery, J.M., Barlowe, C., and Lindquist, S. The Parkinson’s disease protein alpha-synuclein disrupts cellular Rab homeostasis. PNAS 105, 145-150 (2008).

    5. Caldwell, G. Integrated Genomics: A Discovery-Based Laboratory Course. : John Wiley and Sons. West Sussex, England, (2006).

    6. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71-94 (1974).

    7. Gandhi, S., Santelli, J., Mitchell, J.D.H., Stiles, J.W., and Sanadi, D.R. A simple method for maintaining large, aging populations of Caenorhabditis elegans. Mechanisms of Ageing and Development 12, 137-150 (1980).

    Comments

    7 Comments

    Thank you for your research. My husband has Parkinson's. It's so good to know that young people are doing research which may ultimately help stem the onslaught of this disease, not in our lifetime, but perhaps in the near future. Where dŒs your study go from here? Good luck with your work. (reader from Vancouver, Canada)
    Reply

    Posted by: AnonymousOctober 10, 2008, 2:08 PM

    Hi I would like to know when you can view the GFP::Alpha Synuclein C. elegans? Also, dŒs the Alpha-synuclein slowly degenerate the neurons or dŒs it automatically degenerate them when the C. elegans are born? Do they have to be in the adult stage when viewed under the microscope?
    Reply

    Posted by: Hunter C.September 27, 2010, 9:57 PM

    Oh wow, this is awesome.
    Reply

    Posted by: Dmitriy K.August 12, 2013, 12:35 AM

    Hello! How does the GFP get viewed in the C. elegans? Is there a specific type of microscope needed or does the promoter of the gene need to be activated?
    Reply

    Posted by: Marilyn Z.January 25, 2014, 1:25 AM

    Hi. 1) To view GFP you need a microscope equipped with special (UV) light and fluorescent filters to excite the GFP molecule and capture the emissions. 2) This is a biological question, not so much an equipment question. The power of GFP and similar fluorescent molecules is that as long as they are expressed in the cell that can be visualized by the proper type of microscope. The type of promoter they are fused to just directs the tissue and/or developmental time of expression in the animal. Depending on the type of promoter used it may or may not need to be activated. For example heat-shock promoters need to have the animal heated above normal temp to induce expression. Other promoters are constitutive, so they are active all the time.
    Laura
    Reply

    Posted by: Laura B.February 3, 2014, 3:14 PM

    Oh, I see. Thank you!

    How fast is the GFP produced? (for example, if I have GFP expression in DAergic neurons and do something to increase DAergic activity, can I see the increase in GFP right after, or how long will I have to wait?) Thanks.
    Reply

    Posted by: Marilyn Z.February 18, 2014, 4:34 PM

    Again, how "fast" the GFP is produced depends on the type of transcriptional promoter it is attached to. The dat-1 promoter we use is expressed in the dopaminergic neurons. Once those cells have differentiated, the promoter is functional and the GFP is then expressed. So there is no need to activate these neurons to see the GFP. However, in other systems, a promoter may need to be activated (like the heat-shock example described above) to allow it to express the GFP.
    Laura
    Reply

    Posted by: Laura B.February 19, 2014, 11:49 AM

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