Testing for apoptosis

I’m trying to test for neurodegeneration due to apoptosis, but am having problems finding a viable procedure. I found some articles on Annexin V (a stain that can detect apoptosis in various cell cultures). Can this be used to test for apoptosis in C. elegans dopamine neurons? If so, what procedure should I use? (I don’t have the proper equipment to slice the worms into sections.)

I’m not sure what you mean when you say “by apoptosis”. If you really mean that the death should be occurring in a manner similar to those seen in normal programmed deaths in C. elegans, there are a number of established approaches that might work; if it is occurring differently, some might not work.

I hope you’ve already read the Wormbook chapter on cell death, and the separate WormMethods section on cell death. I’d also point you to a methods review I wrote awhile back in case it might be helpful, though a number of more recently reported methods, including Annexin V staining, are not mentioned there.

I’ve also written a list of methods you might consider, but don’t take this list to be comprehensive:

  1. The first and most important tool for studying cell death has always been Nomarski microscopy. “Apoptotic” cell deaths in C. elegans have a characteristic Nomarski morphology. It may help to include a mutation in a gene required for the engulfment and removal of cell corpses (ced-1, for example) so the dying cell will persist. Note that mutations blocking cell corpse engulfment also cause the persistence of cells dying by other mechanisms (mec-4(d), for example) and interfere with other non-apoptotic cell deaths (lin-24, for example); cell deaths that occur by other mechanisms can often also be observed using Nomarski microscopy, and have very different morphologies from the “apoptotic” deaths. If you are observing the disappearance of dopaminergic cells, it should be feasible to directly observe what happens to the PDE neurons using Nomarski microscopy in a living animal, as these dopaminergic cells are generated during larval development and ar elocated in a discrete and isolated structure. I’d recommend you only use the right PDE neurons, to avoid confusion caused by cells from the migrating QL neuroblast in the vicinity of the left PDE neurons.

  2. The most common tool used in neurodegeneration models in C. elegans where the neurodegeneration is caused by overexpression of a disease gene is to look for disappearance of a gfp marker expressed in those cells. This doesn’t necessarily tell you much about the process by which the cells disappear, but if you believe your mechanism is apoptotic, you can test for effects of a mutation known to affect apoptotic cell death, such as ced-3(lf), and see whether the kinetics of cell disappearance are altered.

  3. Depending on what you mean by testing “for apoptosis in C. elegans dopamine neurons”, there are good cell-fate reporters for dopaminergic neurons. The eight dopaminergic neurons present in the wild type don’t normally undergo apoptosis, but if you block apoptosis roughly 50% of undead PVD sister cells adopt a dopaminergic cell fate, and the undead sisters of the CEPV neurons also can adopt a dopaminergic cell fate.

  4. A number of reporter transgenes and histological methods have been established. The older and/or more established ones include staining with DAPI or a SYTO dye to examine the nuclear morphology of the dying cells; using Acridine Orange to visualize the acidification of the cell corpse (not reliable outside of the germline in C. elegans, though it’s a standard method in Drosophila); TUNEL staining (not developed for use outside of embryos in C. elegans, though it’s a standard technique in other systems);and ced-1::gfp, which accumulates around apoptotic cell corpses.

  5. A number of recent papers have reported methods for using Annexin V to detect apoptotic corpses in C. elegans (1, 2). In addition, more recent papers from the Hengartner and Zhou labs have described a number of additional markers they used to characterize apoptotic cell death.

Are you sure that you mean apoptosis, or do you mean neuronal cell death in general? These dopamine neurons are also subject to another mode of cell death caused by exposure to 6-OHDA entering through specific membrane channels (DAT, dopamine transporter) found only in dopaminergic neurons. You might not be able to detect those “necrotic cell deaths” by standard markers for apoptosis, although you should be able to detect something by DIC microscopy. Richard Nass has marked those few neurons by GFP in order to view them by fluorescence during exposure to toxins. This would not require TEM to know if the cells were still present.

For examples, see papers by Nass and Blakeley, including
Nass, R., Hall, D.H., Miller, D.M. and Blakeley, R.D. (2002) Neurotoxin-induced degeneration of dopamine neurons in C. elegans. PNAS 99: 3264-3269.

Thank you so much for replying.
I’m a high school student, and my school doesn’t have the equipment to perform procedures such as the Nomarski microscopy. Last year, I conducted an experiment that resulted in loss of dopamine neurons in C. elegans at a slower rate than MPTP. I’m hoping to further develop this as a potential model for Parkinson’s disease. This year, I’d like to see if the loss of neurons was due to apoptosis. I have been looking for staining techniques for C. elegans , but haven’t found any procedures to do so in live/fresh worms. (I do not have the capability to section the worms).
My other option is to do DNA fragmentation (my school does have the ability to do electrophoresis).

If you want to stain for the presence of dopaminergic neurons, there are a few options:

  1. Use fluorescence optics to detect the dopaminergic neurons in animals expressing gfp in dopaminergic neurons. This is by far the easiest way, and strains containing integrated reporters that express in dopaminergic neurons are published and available. If you don’t have a microscope equipped with fluorescence optics, it might be possible to modify an existing scope to detect GFP: You can now buy UV LEDs cheaply, and use them in combination with a filter. I know this can be done, but from what I’ve heard sensitivity is a real problem, I don’t know the details of just what you would need, and I don’t really know that this would be practical - though it might be fun to try.

  2. Formaldehyde-Induced-Fluorescence. The original reference is, I think, Sulston, J., Dew, M., and Brenner, S. (1975). Dopaminergic neurons in the nematode Caenorhabditis elegans. J. Comp. Neurol 163, 215-226, or for a more recent reference, and one available online see Lints and Emmons 1999. Note however that this also requires fluorescence optics.

  3. Obtain a strain expressing gfp in dopaminergic neurons and stain using a commercially available anti-GFP antibody. It should be possible to detect the stained neurons using a secondary antibody coupled to a detection mechanism that does not require fluorescence optics.