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. Author manuscript; available in PMC: 2015 Jun 30.
Published in final edited form as: Methods Mol Biol. 2013;1004:171–182. doi: 10.1007/978-1-62703-383-1_13

Necrosis in C. elegans

Matt Crook, Avni Upadhyay, Wendy Hanna-Rose
PMCID: PMC4485986  NIHMSID: NIHMS700861  PMID: 23733577

Abstract

To use Caenorhabditis elegans to study the mechanisms for initiation and execution of necrosis, the experimentalist should be familiar with the established models of necrosis in C. elegans and the genetic and molecular tools available. We present a summary of two contrasting models for studying necrosis in C. elegans and outline the methods for scoring necrosis in each. These methods are useful for the study of necrosis under other conditions in C. elegans and for comparative studies both between established and new necrosis models. We also present a list of the genetic and drug tools available for perturbing pathways known to be important for initiation or execution of necrosis and describe their use in C. elegans. Specifically, we outline methods to inhibit autophagy, to perturb calcium homeostasis, and to disrupt lysosomal function in the C. elegans system.

Keywords: Cell death, Degenerins, Nicotinamide, TOR, Vitamin B3 metabolism, Mechanosensory cells, Uv1 cells, Necrosis, C. elegans

1 Introduction

Cell death occurs via multiple genetic programs and studies in Caenorhabditis elegans have been key to both the recognition of specific death programs and the elucidation of their underlying genetic control. Cell death programs can be distinguished based on the morphological and molecular changes that occur during dismantling of the cell as well as on the signals for initiation of the program. Necrosis is one such cell death program. It proceeds via a stereotypical series of morphological changes, excessive cell swelling being one of the most dramatic (reviewed in refs. 1–3). While the morphology of necrotic cells has been described in detail, we are just beginning to decipher the genetic and molecular events that underlie necrotic death, and it is becoming clear that distinct pathways for both initiation and execution of morphologically similar death programs may exist. These differences highlight the importance of multiple models for studying necrotic death and demonstrate that the C. elegans system will continue to be valuable to the advancement of the field of cell death.

Necrotic death in C. elegans is not programmed from a developmental standpoint as in some other systems, but C. elegans cells do respond to various insults with one or more genetically controlled necrotic-type death programs. There are several models for studying necrosis in C. elegans and these models are proving useful for understanding how cells die in response to insult, a crucial first step to learning to prevent such deaths in clinical settings where necrosis plays a role in disability (e.g., after stroke or ischemia). We briefly describe two specific models for studying necrosis in C. elegans and then describe methods for using these systems to analyze mechanisms of necrosis.

1.1 Touch Cell Death Induced by Degenerins

Gain-of-function mutations in certain ion channel subunits cause neurons expressing these “degenerins” to necrose with predictable spatial and temporal patterns [4–6]. The best-studied degenerin gene is mec-4. Dominant, gain-of-function mutations in mec-4 cause necrosis of six specific touch receptor cells (PVM, AVM, a pair of PLMs, and a pair of ALMs) (Fig. 1a, see wormatlas.org or ref. 7 for detailed information about cell morphology and location). Necrotic cells appear in the mec-4(d) mutants shortly before hatching and the corpses are absorbed by mid-L1 [8]. Genetic analysis of degenerin-induced necrosis has revealed an important role for calcium influx in induction of the necrotic program and for calcium-dependent proteases in its execution [9, 10]. Lysosomal proteases are also involved in executing necrosis downstream of calcium signaling in this model [11, 12]. Finally, efficient autophagy is required for the progression of necrosis [13, 14]. We explain how to use tools to probe each of these processes in mec-4d-induced touch cell necrosis.

Fig. 1.

Fig. 1

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Scoring touch cell necrosis. (a) Schematic diagram indicating approximate positions of the six touch cells in the animal. The shaded Xs are left/right pairs of cells and may not be visible in the same focal plane, particularly in the mid-region of the body. (b) mec-4d(e1611) L1 larva with two necrotic cells (arrows) in the tail

1.2 Uv1 Cell Death Induced by Nicotinamide or Loss of PNC-1 Nicotinamidase Function

The four uterine-vulva 1 cells that lie between the uterine seam cell and the vulF cell in C. elegans (Fig. 2a) are sensitive to perturbations in vitamin B3 metabolism. Supplementation of wild-type animals with nicotinamide or mutation of the nicotinamidase PNC-1, which converts nicotinamide to nicotinic acid, causes these cells to necrose [15, 16]. While the morphological changes associated with uv1 and degenerin-induced necrosis are similar, studies suggest that the triggers and the signals that activate the death program in these two models are distinct, highlighting the value of further comparative analyses.

Fig. 2.

Fig. 2

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Staging animals for scoring uv1 necrosis. (a) Schematic diagram indicating approximate positions of the four uv1 cells in the animal. There are two uv1 cells on each side, flanking the vulva. Only two uv1 cells are visible in a single focal plane. (b–d) A temporal series of wild-type, mid to late L4 hermaphrodites, showing the morphology of the vulva. (b ) The “Christmas tree” stage: uv1 cells begin to die shortly after this stage and animals should be scored when slightly older. (c) A slightly older animal. The first subtle signs of eversion are evident. The two large medial cells (arrows ) are moving towards one another and will eventually make contact, as in the older animal in (d). The protrusions into the vulval lumen in the ventral vulva (arrowheads) are beginning to “droop” ventrally and this feature becomes more evident in (d). Asterisks mark the open uterine lumen in all animals. (c′ and c″) Fluorescent images in two different focal planes from the same animal as in (c). This animal carries the Pida-1::GFP transgene. Arrows indicate the two uv1 cells visible in the same focal plane. The uv1 cells appear to have a convex surface on the ventral side and a concave surface on the dorsal side. The HSN cell (arrowhead ) is in a slightly different focal plane and has a distinct morphology with a fl at ventral surface. (e) pnc-1(pk9605) mutant with a necrotic uv1 cell (arrow)

2 Materials

2.1 Mounting Worms for Microscopy

  1. Compound microscope with 100× differential interference contrast (DIC) oil immersion objective, preferably with a UV light source for fluorescence microscopy, but scoring can be accomplished without fluorescence microscopy.

  2. Stereoscope for manipulating animals on culture plates.

  3. 25×75×1 mm microscope slides and 22×22 mm no.1 coverslips.

  4. Anesthetic.

    1. 100 mM stock solution of tricaine in water (store aliquots at −20 °C).

    2. 100 mM stock solution of tetramisole hydrochloride in water (store aliquots at −20 °C).

    3. Working solution of 1 mM each tricaine and tetramisole hydrochloride in M9: 22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl, 1 mM MgSO4.

  5. 5 5 % agarose in water for making pads.

2.2 Strains for Scoring Necrosis

In touch cells

  1. N2 wild type, available from C. elegans Genetics Center.

  2. CB1611 mec-4d(e1611) X: [8], available from C. elegans Genetics Center.

  3. ZB1656 tdIs5 [Pmec-4::GFP] I; mec-4d(u231) X [10], available by request from M. Driscoll lab.

In uv1 cells

  1. N2 wild type, available from C. elegans Genetics Center.

  2. HV727 pnc-1(pk9605) IV: [16], available by request from W. Hanna-Rose lab.

    3. HV560 inIs179 [Pida-1::GFP] II; pnc-1(pk9605) IV: available by request from W. Hanna-Rose lab.

  3. Wild type or other animals carrying the inIs179[Pida-1::GFP] transgene cultured on NGM plates with 25 mM nicotinamide, which induces uv1 cell death.

2.3 RNAi

  1. LB broth and agar.

  2. NGM agar culture plates. Add 3 g NaCl, 17 g agar, and 2.5 g bacto peptone to a 2 l flask and add ddH2O to 975 ml. Autoclave for 50 min on liquid cycle, allow to cool to 55 °C, and then add 0.5 ml 1 % cholesterol (dissolved in 95 % ethanol and filter sterilized), 1 ml each of 1 M CaCl2 and M MgSO4, and 25 ml of 1 M KHPO4. Swirl to mix and pour into 10 cm plates.

  3. 1 M isopropyl beta-d-1-thiogalactopyranoside (IPTG).

  4. 12.5 mg/ml tetracycline.

  5. 50 mg/ml ampicillin.

  6. 100 mg/ml carbenicillin.

  7. NGM plates with 1 mM IPTG and 25 μg/ml carbenicillin that are 2–3 days old.

  8. LB agar plates with 50 μg/ml ampicillin and 12.5 μg/ml tetracycline.

  9. Clones from RNAi feeding library for genes listed in Table 1.

Table 1.

Genes and drugs for manipulating processes that affect initiation and/or execution of necrosis in various systems

Autophagy
Loss of function of these genes will block autophagy and rescue mec-4d-induced touch cell necrosis [13, 14], but not uv1 necrosis
unc-51(e369) Atg1p ortholog, S/T kinase
bec-1(ok700) Atg6/Vps30/Beclin1 ortholog
lgg-1 Atg8p ortholog, member of preautophagosome complex
Ca2+
Loss of function of these genes or application of these drugs will dampen an increase in cytosolic Ca 2+ and rescue mec-4d-induced touch cell necrosis [10], but not uv1 necrosis
crt-1(bz29) ER Ca 2+-binding protein/chaperone
cnx-1 ER Ca 2+-binding protein
50 mM EGTA Ca 2+ chelator
10 μM dantrolene Blocks ER Ca 2+ release
TOR-mediated nutrient sensing
Loss of function of these genes will block TOR-mediated nutrient sensing and exacerbate mec-4d-induced touch cell necrosis [14]. This may be via a negative effect of Tor on autophagy
CeTor/let-363 Tor1/2p PIK ortholog
Lysosomal proteases
Loss of function of these genes or application of this drug will disrupt the function of lysosomal proteases and will rescue mec-4d-induced touch cell necrosis [11] and uv1 necrosis [15]
50 μM Bafilomycin-A1 Inhibits processing of cathepsin D, a lysosomal protease [19]
vha-2, vha-10, vha-12 Subunits of V-ATPase that cause cytosolic acidification and activation of lysosomal proteases [11]
asp-3, asp-4 Aspartyl protease, asp-3 and asp-4 act in parallel and downstream of clp-1 and tra-3 [9]
clp-1 Calpain homologue, acts in parallel with tra-3
tra-3 Atypical calpain regulatory protease
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Allele names are provided for genes where traditional genetic experiments have been done. Where no allele name is provided, RNAi is recommended as a method to block gene function. Additional information about any C. elegans gene is available at Wormbase.org

2.4 Drug Treatments

  1. Compound microscope equipped for microinjection of C. elegans.

  2. 1 M ethylene glycol tetraacetic acid (EGTA).

  3. 10 mM dantrolene in water.

  4. 100 μM Bafilomycin-A1 stock solution: Add 160 μl of 98 % ethanol to 10 μg bafilomycin-A1 (Sigma-Aldrich). Mix well to dissolve. Prepare aliquots. Cover the tubes with aluminum foil and parafilm the cap; store at −20 °C. Avoid frequent freeze-thawing of stock solution. To prepare the 50 μM working solution, add 10 μl of stock solution to 10 μl of 98 % ethanol. Keep the solution on ice and covered in foil during use.

3 Methods

General procedures for maintenance and manipulation of C. elegans including culturing conditions, preparing animals for microscopy, and performing genetics and microinjection are available from several excellent sources including wormbook.org [17, 18]. We recommend consultation with experienced C. elegans researchers for experimenters who are novices with the system.

3.1 Mounting Worms for Microscopy

  1. Prepare two support slides by applying a strip of lab tape to the top and place a clean glass slide between them.

  2. Melt the 5 % agarose and place a small drop on the slide using a plastic Pasteur pipette.

  3. Squash the agarose drop by placing a third slide, perpendicular to the other slides, over the drop and the support slides, applying firm pressure with the thumbs over the tape, creating a round agarose pad of uniform thickness.

  4. Carefully slide the top slide off of the agarose pad (see Note 1).

  5. Add 2–5 μl of anesthetic solution to the agarose pad.

  6. Add 10–20 animals of the appropriate stage to the drop on the pad.

  7. Place a coverslip over the drop, add immersion oil to the cov-erslip, and view with 100× DIC objective on the compound microscope.

3.2 Scoring Necrotic Phenotypes

Necrotic cells can be visualized using DIC microscopy based on two obvious morphological characteristics: their swollen cytoplasm and their disintegrating nuclear membrane. The swollen cytoplasm expands the dying cell to several times its original size and appears empty of any organelles. The nuclear membrane maintains its original size but appears fragmented, similar to the islands in a coral atoll chain (Figs. 1b and 2e). Because necrotic cells can be cleared from the animal after a period of time, a less ambiguous and recommended method for scoring is to use transgenic strains with GFP expressed in the cells of interest (e.g., Fig. 2c). However, the choice between scoring with or without GFP will be governed by the ease of genetic crosses to put markers in the desired genetic background and the availability of a microscope with the appropriate UV light source and filters for visualizing GFP. We describe and discuss procedures for scoring using both methods.

  1. Place animals on the slide for microscopy.

    1. mec-4d-induced touch cell necrosis for DIC: Select first larval stage (L1) animals from a mec-4d strain (see Note 2).

    2. mec-4d-induced touch cell necrosis for fluorescence microscopy: Select post-L3-stage animals from a mec-4d strain car- rying a Pmec-4::GFP transgene that is expressed in the touch cells (see Note 3).

    3. Uv1 cell necrosis for DIC: Select animals that are in the second half of the fourth larval stage (L4), just after vulva eversion begins and before the uterine lumen closes (see Notes 4 and 5) from a pnc-1 mutant strain or a wild-type strain cultured on 25 mM nicotinamide.

    4. Uv1 cell necrosis for fluorescence microscopy: Select late-L4 animals, after vulva eversion has begun, or early adults, before oocytes are present, from a strain carrying a Pida-1::GFP transgene that is expressed in the uv1 cells.

  2. Scan the slide, and for each animal at the appropriate stage, determine the presence or the absence of necrotic cells (see Notes 6 and 7) or the presence or the absence of GFP+ cells (see Notes 8 and 9), as appropriate.

  3. Record the number of animals at the appropriate stage that were scored as well as the necrotic phenotype (see Notes 10 and 11).

3.3 RNAi Protocol for Genes Listed in Table 1 (See Note 12)

Reagents, including drugs and genes, for perturbing known pathways involved in initiation or execution of necrosis in C. elegans are listed in Table 1. Allele names are provided for genes where traditional genetic experiments have been done. While either traditional genetics or RNAi could be applied with most of the genes, where no allele name is provided, RNAi has been the method applied to block gene function. RNAi can also be used to knock down the genes that are listed with alleles.

  1. Streak for single colonies on an LB Amp Tet plate with an inoculum from a glycerol stock of a bacterial strain carrying the desired RNAi clone (e.g., from the C. elegans RNAi library (http://www.gurdon.cam.ac.uk/~ahringerlab/pages/rnai.html)).

  2. Incubate at 37 °C overnight.

  3. Pick a single colony and inoculate a 2–3 ml LB Amp broth culture.

  4. Incubate at 37 °C overnight with agitation.

  5. Prepare a backup glycerol stock from the overnight culture (1:2 culture:50 % glycerol).

  6. Prepare plasmid DNA from the overnight culture.

  7. Verify the identity of RNAi insert by sequencing with an M13 forward primer for the C. elegans RNAi library clones (other RNAi clones may differ).

  8. Once verified, inoculate another 2–3 ml LB Amp broth culture using the glycerol stock from step 5.

  9. Incubate at 37 °C overnight with agitation.

  10. Add approximately 200 μl of culture to the top of NGM IPTG Carb plates. Incubate at 20 °C for 3–4 days to allow the plates to dry and the bacteria to express the double-stranded RNA. Five plates per RNAi treatment usually provides enough worms of the right stage to score.

  11. Add animals to the prepared RNAi plates (see Note 13).

    1. For mec-4d-induced necrosis, pick five to ten L2 larvae to each RNAi plate.

    2. For uv1 necrosis, pick five L4 larvae to each RNAi plate (see Note 14).

  12. Incubate RNAi plates at 20 °C until the progenies are ready to score as described (see Note 15).

3.4 Drug Treatments to Perturb Calcium Homeostasis (Table 1)

  1. Add EGTA, a Ca2+ chelator, or dantrolene, a chemical that inhibits Ca2+ release from the ER (see Note 16), to NGM agar before pouring the culture plates to a final concentration of 50 mM and 10 μM, respectively.

  2. Spot plates with E. coli OP50 and incubate at 20 °C for 3–4 days.

  3. For uv1 necrosis, pick five L4 larvae to each plate. For mec-4d-induced touch cell necrosis, pick five to ten L2 larvae to each plate (see Note 14). Five plates per treatment usually provides enough worms of the right stage to score (see Note 17).

  4. Incubate plates at 20 °C until progenies are at the appropriate age to score, which will take approximately 3–4 days, depending on strain and treatment.

3.5 Drug Treatment to Disrupt Lysosome Function (Table 1)

  1. Inject the body cavity of gravid hermaphrodites with a working solution of Bafilomycin-A1 (see Note 18).

  2. As a control inject a matched set of animals with 98 % ethanol.

  3. Transfer injected animals to an NGM plate with OP50 food (see Note 19).

  4. Culture the animals in the dark at 20 °C overnight.

  5. Transfer surviving animals to fresh NGM plates with OP50.

  6. Score the progeny of injected animals at the appropriate stage as described above.

Acknowledgments

This work was supported by NIH grant GM086786 to W.H.R.

Footnotes

1

Use a razor blade to remove the edges of the round agar pad until it is square. The square pad is easier to scan under the microscope without losing track of your position during scoring.

2

Select animals immediately after hatching and before any darkening of the intestine because necrotic touch cells are broken down and absorbed before the end of the L1 stage. Do not score animals that are older than mid-L1. www.Wormatlas.org is helpful if additional information about staging animals is required.

3

Score older animals when using the Pmec-4 ::GFP transgene to avoid accidental inclusion of corpses with persistence of faint GFP signal in counts of surviving GFP+ cells.

4

Staging worms this precisely is not easy to accomplish as the animals are chosen for placement on the slide. However, as the slide is being scored, it is simple to include only animals of the appropriate stage. Figure 2 has additional information about staging.

5

Uv1 cells are not specified until early in the L4 stage. Necrotic uv1 cells do not appear until shortly after the vulva reaches the “Christmas tree” stage when the vulva begins to evert (Fig. 2b–d). The ideal time to score is just after vulva eversion begins and before the uterine lumen closes at the late-L4 stage. While the window for scoring uv1 cell necrosis is broader than that of touch cell necrosis because necrotic uv1 cells are not cleared quickly, scoring too early is still a risk. Scoring for the presence or the absence of cell corpses after L4 is possible, but the ability to count exactly how many corpses are present becomes difficult after oocytes are fertilized and deposited in the uterus.

6

Touch cell necrosis: mec-4d animals have a maximum of six necrotic touch cell corpses (Fig. 1a), but on average will have two to three by DIC. In addition, the majority of easily visible necrotic cells are in the tail (Fig. 1b). Precisely counting the number of touch cells that have necrosed is challenging due to the short scoring window during which necrotic touch cells are visible before they are absorbed and disappear. This factor can result in high variance in the number of necrotic cells per animal. Uv1 necrosis: pnc-1 mutants have a maximum of four necrotic uv1 cells (Fig. 2a) and 100 % of animals will have at least one necrotic uv1 cell. Most animals will have four necrotic uv1 cells [15].

7

Occasionally you will see large empty vacuole-like structures lacking a disintegrating nucleus in various areas of the animal; these are most likely the result of worms being on the slide too long before scoring.

8

One disadvantage to using Pida-1::GFP as a uv1 marker is its expression in the nearby HSN cell body, which, although medial to the four uv1 cells and of clearly distinct morphology (Fig. 2c), can be mistaken for a surviving uv1 cell by the novice. Alternative GFP markers more specific for the uv1 cells are available. However, markers that are expressed earlier than Pida-1::GFP, which is only expressed after the point when the cells die, can lead to inaccuracies in scoring if an animal that is too young is accidentally included.

9

Intervening cells can obscure both necrotic uv1 cells and GFP+ uv1 cells on the side of the animal opposite the objective, especially after the uterine lumen has begun to close. If the observer has difficulty scoring both sides of every animal, the side nearest the objective can be exclusively scored in each animal.

10
Touch cell necrosis is reported in a number of ways:
  1. By the presence/absence of necrosis per animal, determined by observation of one or more corpses (presence) or no corpses (absence) or by observation of less than six GFP+ cells (presence) or exactly six GFP+ cells (absence).
  2. By the number of necrotic touch cells in the tail of each animal because these are the most readily visible corpses.
  3. By the total number of necrotic touch cells per animal, often reported as the number of necrotic touch cells per 100 animals. This is determined by the number of corpses observed or by subtracting the number of GFP+ cells observed from total number of touch cells expected, which is six.
11
Uv1 necrosis can be reported in a number of ways:
  1. By the total number of necrotic uv1 cells per animal (or per side of the animal nearest the objective—see Note 8). This number is determined by either the number of corpses observed or subtracting the number of GFP+ cells observed from the total number of cells expected, which is four (two per side).
  2. As the proportion of uv1 cells that survive out of the total number of possible uv1 cells.
  3. By the presence/absence of necrotic uv1 cells per animal or per side of the animal nearest the objective.
12

For a comprehensive guide to RNAi in C. elegans, see the Reverse Genetics chapter from www.Wormbook.org (http://www.wormbook.org/chapters/www_introreversegenetics/introreversegenetics.html). We carry out RNAi by feeding, both for ease of use and because of the availability of the C. elegans RNAi feeding library (http://www.gurdon.cam.ac.uk/~ahringerlab/pages/rnai.html).

13

Washing worms three times in M9 before placement on the RNAi plates may reduce some of the variability of the RNAi effect caused by carryover of OP50 from stock plates.

14

It is important to take the time to transfer the younger larvae when planning to score touch cell necrosis because gene expression must be knocked down before the embryos form. When planning to score uv1 necrosis, it is not necessary to select such young animals and using older larva can avoid potential problems that gene knockdown may cause in late-stage larval development.

15

Incubate approximately 3–4 days, depending on strain and RNAi clone.

16

Solubility of dantrolene in water at this concentration can be problematic. Mix the stock solution thoroughly by vortexing to ensure that the final concentration is as close as possible to the desired concentration.

17

Altering Ca2+ availability affects ovulation in C. elegans. As a result, animals on EGTA or dantrolene may become sterile or produce fewer progeny than normal; therefore the number of plates per treatment may need to be increased.

18

We recommend microinjection of this drug. Because Bafilomycin-A1 is not soluble in water, it is difficult to add to culture plates as with the drugs to manipulate calcium. Moreover, the drug is expensive and larger quantities are needed when used as a supplement in culture plates as compared to when administered by injection. Acute exposure to Bafilomycin-A1 via soaking in drug solution is a possible substitute for injection but this treatment is associated with higher lethality.

19

Animals are transferred to new plates to eliminate the progeny that were embryos in the uterus during microinjection and, thus, were not exposed to drug.

References

  • 1.Golstein P, Kroemer G. Cell death by necrosis: towards a molecular definition. Trends Biochem Sci. 2007;32:37–43. doi: 10.1016/j.tibs.2006.11.001. [DOI] [PubMed] [Google Scholar]
  • 2.McCall K. Genetic control of necrosis—another type of programmed cell death. Curr Opin Cell Biol. 2010;22:882–888. doi: 10.1016/j.ceb.2010.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Syntichaki P, Tavernarakis N. Death by necrosis. Uncontrollable catastrophe, or is there order behind the chaos? EMBO Rep. 2002;3:604–609. doi: 10.1093/embo-reports/kvf138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Chalfie M, Wolinsky E. The identification and suppression of inherited neurodegeneration in Caenorhabditis elegans. Nature. 1990;345:410–416. doi: 10.1038/345410a0. [DOI] [PubMed] [Google Scholar]
  • 5.Hall DH, Gu G, Garcia-Anoveros J, et al. Neuropathology of degenerative cell death in Caenorhabditis elegans. J Neurosci. 1997;17:1033–1045. doi: 10.1523/JNEUROSCI.17-03-01033.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Driscoll M, Chalfie M. The mec-4 gene is a member of a family of Caenorhabditis ele-gans genes that can mutate to induce neuronal degeneration. Nature. 1991;349:588–593. doi: 10.1038/349588a0. [DOI] [PubMed] [Google Scholar]
  • 7.Hall DH, Altun ZF. C. elegans atlas. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY: 2008. [Google Scholar]
  • 8.Chalfie M, Sulston J. Developmental genetics of the mechanosensory neurons of Caenorhabditis elegans. Dev Biol. 1981;82:358–370. doi: 10.1016/0012-1606(81)90459-0. [DOI] [PubMed] [Google Scholar]
  • 9.Syntichaki P, Xu K, Driscoll M, et al. Specific aspartyl and calpain proteases are required for neurodegeneration in C. elegans. Nature. 2002;419:939–944. doi: 10.1038/nature01108. [DOI] [PubMed] [Google Scholar]
  • 10.Xu K, Tavernarakis N, Driscoll M. Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca(2+) release from the endoplasmic reticulum. Neuron. 2001;31:957–971. doi: 10.1016/s0896-6273(01)00432-9. [DOI] [PubMed] [Google Scholar]
  • 11.Syntichaki P, Samara C, Tavernarakis N. The vacuolar H+-ATPase mediates intracellular acidification required for neurodegeneration in C. elegans. Curr Biol. 2005;15:1249–1254. doi: 10.1016/j.cub.2005.05.057. [DOI] [PubMed] [Google Scholar]
  • 12.Artal-Sanz M, Samara C, Syntichaki P, et al. Lysosomal biogenesis and function is critical for necrotic cell death in Caenorhabditis elegans. J Cell Biol. 2006;173:231–239. doi: 10.1083/jcb.200511103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Samara C, Syntichaki P, Tavernarakis N. Autophagy is required for necrotic cell death in Caenorhabditis elegans. Cell Death Differ. 2008;15:105–112. doi: 10.1038/sj.cdd.4402231. [DOI] [PubMed] [Google Scholar]
  • 14.Toth ML, Simon P, Kovacs AL, et al. Influence of autophagy genes on ion-channel-dependent neuronal degeneration in Caenorhabditis elegans. J Cell Sci. 2007;120:1134–1141. doi: 10.1242/jcs.03401. [DOI] [PubMed] [Google Scholar]
  • 15.Huang L, Hanna-Rose W. EGF signaling overcomes a uterine cell death associated with temporal mis-coordination of organogenesis within the C. elegans egg-laying apparatus. Dev Biol. 2006;300:599–611. doi: 10.1016/j.ydbio.2006.08.024. [DOI] [PubMed] [Google Scholar]
  • 16.Vrablik TL, Huang L, Lange SE, et al. Nicotinamidase modulation of NAD+ biosynthesis and nicotinamide levels separately affect reproductive development and cell survival in C. elegans. Development. 2009;136:3637–3646. doi: 10.1242/dev.028431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Strange K. C. elegans: methods and applications. Humana; Totowa, NJ: 2006. [Google Scholar]
  • 18.Hope IA. C. elegans: a practical approach. Oxford University Press; Oxford, NY: 1999. [Google Scholar]
  • 19.Pivtoraiko VN, Harrington AJ, Mader BJ, et al. Low-dose bafilomycin attenuates neuronal cell death associated with autophagy-lysosome pathway dysfunction. J Neurochem. 2010;114:1193–1204. doi: 10.1111/j.1471-4159.2010.06838.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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