Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

15.1 Introduction

Early studies in the field of cell death described two major forms of cellular demise, apoptosis and necrosis, and contrasted them as being diametrically different in every aspect examined (Walker et al. 1988). In 1972, Kerr, Wyllie, and Currie described apoptosis as a controlled cell death process and proposed that it functions as a tissue homeostatic mechanism that is complementary and opposite to cell division (Kerr et al. 1972). Necrosis was classically contrasted to apoptosis, also referred to as caspase-dependent programmed cell death, not only on grounds of context and mechanistic regulation or lack thereof but also based on notable morphological differences. The apoptotic cell profile is characterized by cell rounding, detachment from the basal membrane or cell culture substrate, chromatin condensation and nuclear fragmentation, blebbing of the plasma membrane, and shedding of vacuoles known as apoptotic bodies (Galluzzi et al. 2007). Necrotic cells were initially characterized in a negative fashion, exhibiting neither an apoptotic morphological profile nor an extensive vacuolization characteristic of autophagic cell death. However, specific morphological features were soon attributed to necrotic cells. These included in particular an increasingly translucent cytoplasm, osmotic swelling of most organelles, increased cell volume, and finally rupture of the plasma membrane (Fig. 15.1). Notably, unlike apoptosis, necrosis does not feature major nuclear modifications but only minor ultrastructural changes. Moreover, necrotic cells do not fragment into distinct corpses as their apoptotic counterparts do (Galluzzi et al. 2007).

Fig. 15.1
figure 1

Morphology of necrotic cell death in Caenorhabditis elegans. Nematode neurons undergoing necrosis as a result of degenerin ion channel hyperactivation show distinct morphological features (Kourtis and Tavernarakis 2007). (a) The degenerating cell (arrow) appears extensively swollen, while the nucleus is distended and has a distorted morphology (arrowhead). (b) Distinctive electron-dense membranous circumvolutions (arrows) observed under the electron microscope (Hall et al. 1997). Similar membranous inclusions are observed in rat neurons undergoing excitotoxic cell death

Full size image

C. elegans has been instrumental in deciphering both apoptotic and necrotic cellular programs. This can be largely attributed to the specific characteristics and well-described developmental stages of this nematode, which make it exceptionally well suited for the study of both normal and aberrant cell death at the cellular, genetic, and molecular level. Due to its transparency, the visualization and tracking of single cells as well as of individual nuclei is readily feasible by differential interference contrast optics, enabling researchers to follow somatic cell divisions from the fertilized egg all the way to the 959 cell adult hermaphrodite (Sulston and Horvitz 1977; Sulston et al. 1983). The resulting cell lineage map indicated early on that in certain lineages, particular divisions generate cells which are destined to die at specific times and locations that remain faithfully invariant from one animal to another. Exactly 131 somatic cells die every time the fertilized egg normally develops into the adult animal, by an apoptotic programmed cell death (PCD) process.

Genetic and molecular studies performed in C. elegans provided a fundamental insight into the mechanisms underlying this cell death process. In the 131 cells destined to die during development, the level of EGL-1, a BH3 domain protein, is increased. EGL-1 interacts with a protein complex composed of CED-9 (similar to the mammalian B-cell lymphoma protein 2, BCL-2) and CED-4 (similar to the mammalian apoptotic protease activating factor 1, Apaf1), releasing CED-4 which in turn activates CED-3 (similar to human caspases) (Hengartner 2000). In C. elegans, four caspase-related genes exist: ced-3, csp-1, csp-2, and csp-3 (Yuan et al. 1993; Shaham 1998); however, only ced-3 seems to be required for programmed cell death (Yuan et al. 1993; Abraham and Shaham 2004), and only ced-3 and csp-1 are proteolytically active (Shaham 1998). The CSP-2 caspase lacks key active-site residues, and csp-3 encodes only a C-terminal caspase domain, entirely lacking the active site (Shaham 1998). As it turns out, the genetic encoding for the regulation and execution of developmental apoptosis has been remarkably conserved between C. elegans and mammals.

15.2 Necrotic Cell Paradigms During C. elegans Development

The identification of the caspase CED-3 as a key regulator of apoptosis has been a key contribution of C. elegans to the cell death field, as caspases also play crucial roles in the execution of programmed cell death across many species. However, is all developmental cell death in C. elegans dependent on caspases? As it turns out, not quite all cell death events during C. elegans development follow the typical apoptotic pathway that involves CED-4 and CED-3.

15.2.1 Death of the Linker Cell

The C. elegans linker cell has been identified as such an exemption (Horvitz et al. 1983). The linker cell is born during the second larval stage (L2) in the central region of the animal and follows a stereotypical path of migration. As the cell migrates, it leads the extension of the male gonad behind it (Kimble and Hirsh 1979; Sulston et al. 1980), and upon completion of its migratory route, it is positioned between the gonad (vas deferens) and the cloacal tube, serving as an exit channel for sperm in the adult. It is generally thought that the death and removal of the linker cell around the L4/adult transition facilitates the fusion between the vas deferens and cloaca, to connect the male reproductive system to the exterior.

Following up on early observations that the programmed death of the linker cell persists even in ced-3 mutant animals, Abraham and colleagues thoroughly studied the death of this cell by following the fate of a GFP-marked linker cell in animals harboring mutations in core genes of the apoptotic machinery, such as ced-3 and ced-4 , as well as in engulfment genes. They convincingly demonstrated that the linker cell dies in a cell autonomous manner that, unlike what was postulated by previous reports (Sulston et al. 1980), does not require extrinsic signals from engulfing or other cells. Moreover, they showed that this death event is independent of any known apoptotic genes, in line with the lack of apoptotic morphological features, such as chromatin condensation. Instead, there was a noted presence of swollen and degraded mitochondria within large multilayered membrane-bound structures, as well as small electron-translucent “empty” membrane-bound cytoplasmic structures that resembled vacuoles typically seen during necrotic cell death in C. elegans (Hall et al. 1997) (Fig. 15.1). Although linker cell death does not satisfy all classical criteria of necrotic death, it is even further away from classical apoptotic paradigms, perhaps falling under the characteristics of more recently described programmed necrosis processes, also known as necroptosis. More experiments are however needed to further characterize the precise mode of death of the linker cell.

15.2.2 Death of Mis-specified Uterine–Vulval (uv1) Cells

Yet, a more robust example of a necrotic event during development is the demise of mis-specified uterine–vulval (uv1) cells that have an important role in egg laying. Egg laying in C. elegans requires a connection between the lumens of the uterus in the somatic gonad and the vulva in the extra-gonadal epithelium, facilitated by cell–cell interactions between gonadal and vulval cells. Two specialized cell types of the ventral uterine π lineage are integral components of the uterine–vulval connection. These are the syncytial uterine seam (utse) cell, which overlies the vulval lumen, and the four uterine–vulval (uv1) cells, which directly contact the most dorsal vulval cell vulF (Newman et al. 1996). The temporal and spatial specification of both these cell types largely relies on a specific signaling axis, where an inductive LIN-3 epidermal growth factor (EGF) signal derived from a single gonadal cell called the anchor cell (AC) activates the LET-23 EGF receptor on the receiving vulval precursor cells (Aroian et al. 1990; Hill and Sternberg 1992). Mutations in genes of the LIN-3/LET-23/Ras signaling pathway compromise uv1 fate specification. Work from the laboratory of Hanna-Rose (Huang and Hanna-Rose 2006) described the isolation of the cog-3(ku212) mutant, which uncouples gonadogenesis from its normal progression relative to the development of the vulva and shares phenotypes with heterochronic mutations that disturb the temporal coordination of vulval and uterine development. In cog-3(ku212) mutants, the entire uterus, including the pre-uv1 cells, is generated at a later stage of vulval development than is normal. Notably, the delayed pre-uv1 cells subsequently die by necrosis leading to the absence of uv1 cells in the adult stage. Moreover, the study investigated if a LIN-3/LET-23/Ras signaling defect underlies the necrosis of uv1 defect in cog-3 (ku212) mutants, by analyzing cog-3(ku212) double mutants with a gain-of-function allele of let-23. The results indicated that the let-23(gf) mutation rescued the mis-specification and death phenotype of uv1 cells, suggesting that the necrotic program is recruited during development in response to uncoordinated spatiotemporal development.

A recent study revealed the involvement of the ku212 allele in uv1 cell necrosis, which maps to the pnc-1 gene locus, encoding a nicotinamidase (van der Horst et al. 2007; Vrablik et al. 2009). Nicotinamidases are the first enzymes of the NAD+ salvage pathway in invertebrates, using nicotinamide (NAM) as a substrate (Magni et al. 1999). Administration of high levels of nicotinamide causes uv1 cells to die by necrosis at high frequency in wild-type animals. Thus, instead of compromised EGF signaling, the necrotic death of uv1 cells in pnc-1 mutants may result from accumulation of the substrate nicotinamide. In addition, the gonad-defective and uv1 cell death phenotypes are separable in pnc-1 mutants. Constitutively active LET-23/EGF receptor prevents NAM-induced uv1 necrotic cell death, suggesting that EGF signaling may provide a survival cue that rescues uv1 cells from NAM-induced necrosis (reviewed in Vlachos and Tavernarakis 2010).

15.3 Nondevelopmental Necrotic Death

In the adult nematode, necrotic cell death can be triggered by a wide variety of both extrinsic and intrinsic signals (Walker et al. 1988). Several well-defined conditions are known to trigger necrotic cell death in C. elegans. The best-characterized case is that of unusual gain-of-function mutations, in several ion channel genes, which inflict a necrotic pattern of death on the neurons that express their protein products. Cell demise in these paradigms is accompanied by characteristic morphological features of necrosis, starting with the appearance of a distorted nucleus and cell body during the early phase of death. Gradually, the cell swells to several times its normal diameter, and small, tightly wrapped membrane whorls form, originating from the plasma membrane and coalescing into large, electron-dense membranous structures (Hall et al. 1997). Interestingly enough, these membranous inclusions also represent characteristic hallmarks in mammalian neurodegenerative disorders, such as in neuronal ceroid lipofuscinosis (Batten’s disease, the mnd mouse) as well as in the wobbler mouse, a model of amyotrophic lateral sclerosis (ALS) (Cooper et al. 1999; Blondet et al. 2002).

15.3.1 Cell Death Induced by Ionic Imbalance

The most extensively characterized paradigm of nonprogrammed cell death in adult C. elegans animals is the necrosis of cells expressing aberrant ion channels harboring unusual gain-of-function mutations (Syntichaki and Tavernarakis 2003). For example, dominant mutations in deg-1 [degenerin; deg-1(d)] induce death of a group of interneurons of the nematode posterior touch sensory circuit (Chalfie and Wolinsky 1990). Similarly, dominant mutations in the mec-4 gene [mechanosensory; mec-4(d)] induce degeneration of six touch receptor neurons required for the sensation of gentle touch to the body (Syntichaki and Tavernarakis 2004).

deg-1 and mec-4 encode proteins that are very similar in sequence and were the first identified members of the C. elegans “degenerin” family, so named because several members can mutate to forms that induce cell degeneration (Chalfie et al. 1993). Degenerins bear sequence similarity to mammalian epithelial sodium channels (ENaCs). The time of degeneration onset correlates with the initiation of degenerin gene expression, and the severity of cell death is analogous to the dose of the toxic allele (Hall et al. 1997). Expression of mammalian homologous proteins, carrying amino acid substitutions analogous to those of toxic degenerins, leads to degeneration of cells in a manner reminiscent of necrotic cell death in C. elegans. Additional members of the degenerin family are mec-10, which can be engineered to encode toxic degeneration-inducing substitutions; unc-8, which can mutate to a semidominant form that induces swelling and dysfunction of ventral nerve cord; and unc-105, which appears to be expressed in muscle and can mutate to a semidominant form that induces muscle hyper-contraction (Syntichaki and Tavernarakis 2004). Thus, a unifying feature of degenerin family members is that specific gain-of-function mutations have deleterious consequences for the cells in which they are expressed, which, at least in neurons, culminate into a necrotic cell death event.

C. elegans degenerins share sequence similarity with Drosophila Ripped Pocket (RPK) and Pickpocket (PPK), with subunits of the vertebrate amiloride-sensitive epithelial sodium channel (ENaC), and with other neuronally expressed ion channels. Together, these proteins define the DEG/ENaC protein superfamily (Tavernarakis and Driscoll 2001). Although mutant degenerins can kill different groups of neurons depending on their expression patterns, the morphological features of the cell death that they induce are the same and resemble those of mammalian cells undergoing necrotic cell death. The pattern of necrotic cell death inflicted by degenerins is not a peculiarity of this gene class. For example, C. elegans deg-3, whose product is related to the vertebrate α-7 nicotinic acetylcholine receptor and together with the related protein DES-2 forms a very efficient calcium channel, can mutate to induce necrotic cell death similar to that induced by degenerins (Treinin et al. 1998). In addition, mutant-activated forms of the heterotrimeric G protein α-subunit (Gαs Q208L), from both C. elegans and rat, cause swelling and degeneration of many cell types when expressed in C. elegans (Korswagen et al. 1997; Berger et al. 1998).

In addition to degenerins, gain-of-function mutations in other ion channel genes, such as deg-3, lead to vacuolar degeneration of various types of C. elegans neurons. deg-3 encodes an acetylcholine receptor ion channel, related to the vertebrate nicotinic acetylcholine receptor (nAChr) that participates in the formation of a channel highly permeable to Ca2+ (Treinin and Chalfie 1995). Moreover, expression of a constitutively active form of a heterotrimeric G protein subunit Gαs results in degeneration of a specific subset of neurons. Genetic suppressor analysis identified an adenylyl cyclase as a downstream effector of Gαs-induced neurodegeneration, indicating that cAMP signaling is critical for degeneration (Berger et al. 1998; Korswagen et al. 1998).

Ionic imbalance and subsequent necrotic cell death induced by aberrant ion channel function in C. elegans are mechanistically and morphologically similar to excitotoxicity in vertebrates. Excitotoxic cell death is prevalent during stroke, where the energy required for sustaining ionic gradients and the resting potential of neurons is lost. Because membrane potential collapses, massive amounts of the excitatory neurotransmitter glutamate are released at synaptic clefts (Kauppinen et al. 1988a, b). Energy depletion also prevents reuptake of glutamate by dedicated transporters leading to accumulation of glutamate at synapses, hyperexcitation, and eventually necrotic death of downstream synaptic target neurons. Excitotoxicity is critically dependent on Ca2+ influx through glutamate-gated receptor ion channels (reviewed in Kourtis and Tavernarakis 2007).

Malfunction of glutamate transporters and the resulting accumulation of glutamate are known to trigger excitotoxicity in several neurodegenerative diseases (Cleveland and Rothstein 2001). However, the details on the cascade of events leading to neurodegeneration remain unclear. The molecular components of glutamatergic synapses assembled in C. elegans are highly conserved from nematodes to humans. A recent study describes a novel paradigm for nematode excitotoxicity, by investigating the in vivo effects of multiple mediators of glutamate-induced neuronal necrosis (Mano and Driscoll 2009). Combined Δglt-3 glutamate transporter-null mutations and expression of a constitutively active form of the alpha subunit of the G protein Gs induce extensive neurodegeneration in head interneurons. Δglt-3-dependent neurodegeneration acts through Ca2+-permeable Glu receptors of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype, requires calreticulin function, and is modulated by calcineurin and type-9 adenylyl cyclase (AC9). This glutamate-dependent toxicity defines a novel necrotic death paradigm in C. elegans that shares many basic features with excitotoxicity in mammalian neurons and may potentially be operative also in higher organisms.

15.3.2 Heat-Induced Necrotic Death

Climate change has brought about a dramatic increase in the cases of heatstroke and related pathologies in humans. With core body temperature reaching over 40 °C, heatstroke causes immediate devastating tissue damage and inflammatory response that can be fatal, as well as long-term defects. To gain insight into the molecular mechanisms of heat cytotoxicity and to circumvent the confounding influence of secondary physiological and inflammatory responses, our laboratory developed and characterized a genetically tractable model of heatstroke in C. elegans. Widespread cell death across several tissues could be observed in animals exposed to hyperthermia, which in the nematode was simulated by a short exposure to 39 °C (Kourtis et al. 2012). Dying cells displayed morphological features characteristic of necrosis, expressed markers of necrotic death, and became permeable to propidium iodide. Moreover, depletion of proteins required for necrosis strongly facilitated survival after heatstroke. In contrast, loss of key mediators and core components of the apoptotic or autophagic machineries did not suppress heatstroke-induced cell death. Thus, heatstroke compromises viability by triggering extensive necrotic cell death and represents a newly added necrotic cell paradigm in the nematode.

Notably, we also observed that preconditioning animals at an intermediate, nonlethal temperature markedly enhanced their capacity to withstand a subsequent heatstroke. This protective effect is in line with the previously described phenomenon of hormesis (Calabrese 2004), where preexposure to mild stress elicits increased resistance to subsequent severe stress. It is also worth noting that in addition to heatstroke, heat preconditioning conferred resistance against a wide range of necrotic death insults, including in particular ionic imbalance paradigms (discussed earlier), overexpression of aggregation-prone proteins (such as α-synuclein), and hypoxic conditions. In the case of hormesis by heat preconditioning, we found that cytoprotection is orchestrated at the molecular level by the hermetic induction of a single sHSP, HSP-16.1. sHSPs assemble into oligomeric complexes and serve as molecular chaperones, efficiently binding denatured proteins and/or preventing irreversible protein aggregation and insolubilization (Van Montfort et al. 2001). HSP-16.1 localizes in the Golgi, where it functions together with the PMR-1 pump to prevent cytoplasmic Ca2+ overload under extreme stress. We propose that HSP-16.1 contributes to stabilize and protect the stress-labile PMR-1 pump, allowing for efficient clearance of Ca2+ from the cytoplasm, after necrotic insult (see Fig. 15.2).

Fig. 15.2
figure 2

A cytoprotective mechanism against necrotic cell death engaged by heat preconditioning. The contribution of the medial Golgi, shown here by brown cisternae, is depicted (a) in healthy cells under normal conditions, (b) upon exposure to heatstroke or other necrotic stimuli, (c) upon heat preconditioning and subsequent heatstroke, and (d) upon heat preconditioning and subsequent heatstroke in the absence of PMR-1. Under normal conditions (a) the Golgi P-type ATPase Ca2+/Mn2+ pump PMR-1 and SCA-1 maintain Ca2+ homeostasis by sequestering Ca2+ inside the Golgi. (b) Heatstroke perturbs the normal function of PMR-1 leading to aberrant release of Ca2+ into the cytoplasm and causing necrosis. Heat preconditioning (c) increases the levels of HSP-16.1, which restores PMR-1 function upon subsequent heatstroke, therefore preventing necrosis. The effect of HSP-16.1 is entirely dependent on PMR-1; as in the absence of PMR-1 (b), heat preconditioning cannot confer cytoprotection against subsequent heatstroke (Adapted from Kourtis et al. 2012)

Full size image

Importantly, mammalian PMR1 is selectively impaired during ischemic or reperfusion brain injury (Lehotsky et al. 2002; Gidday 2006; Pavlikova et al. 2009). Given the strong evolutionary conservation of the proteins involved, this mechanism is probably relevant to related human pathologies. Relevant to that, we also demonstrated that heatstroke induces widespread necrotic death in mammalian neurons, which can be largely prevented by heat preconditioning. Moreover, hormesis in mammalian neurons in response to heat preconditioning also requires the function of PMR1 and is mediated by the same molecular players as in the nematode.

15.3.3 Bacterial Infection-Induced Necrosis

Infection of C. elegans with different bacterial pathogens has been shown to induce necrotic death of intestinal cells as part of a pathogen-shared response to infection (Wong et al. 2007). At later stages of infection, necrotic vacuoles are also observed in epidermal and gonadal cells. Mutations in genes required for necrosis ameliorate the consequences of infection, suggesting that necrosis is an integral part of host–pathogen interaction that contributes to the pathology associated with infection in C. elegans.

15.3.4 Hypoosmotic Shock-Induced Cell Death

Lysosomal integrity and lysosomal proteolytic mechanisms are key factors modulating necrotic cell death in the nematode. Serpins are extracellular or intracellular regulators of proteolytic pathways and inhibitors of multiple peptidases (Silverman et al. 2001). One of the functions of intracellular serpins is the inhibition of lysosomal cysteine peptidases. SRP-6 is such an intracellular serpin in C. elegans. srp-6-null mutants experiencing hypoosmotic conditions die rapidly and display marked increase of necrotic cell death of the intestinal epithelium (Luke et al. 2007). Ca2+ release from endoplasmic reticulum (ER) stores, together with other factors, induces calpain-mediated lysosomal rupture and massive release of lysosomal peptidases into the cytoplasm that mediate necrotic cell death. In addition to hypoosmotic conditions, srp-6-null mutants are susceptible to other stressors such as thermal and oxidative stress, hypoxia, and channel hyperactivity. SRP-6 appears to protect cells from lysosomal rupture and also ameliorate the deleterious consequences of lysosomal rupture triggered by various stressors. The protective function of SRP-6 may be adaptive by enhancing the degradation of misfolded proteins or by aiding cytoskeletal rearrangements through altering lysosomal membrane permeability and allowing the leakage of small amounts of peptidases. In the absence of SRP-6, the uncontrolled release of these peptidases leads to necrotic cell death.

15.4 Execution of Necrosis

Intracellular calcium overload through different sources is considered as one of the leading steps in the necrotic pathway. Calcium may enter the cell through voltage-gated channels, and the Na+/Ca2+ exchanger and mutations that increase sodium influx augment calcium entry through these paths. The main intracellular compartment for calcium storage is the endoplasmic reticulum (ER) (Mattson et al. 2000; Paschen 2001; Paschen and Frandsen 2001), where calcium is sequestered by the sarco–endoplasmic reticulum Ca2+-ATPase (SERCA) and is released back to the cytoplasm by ryanodine (RyR) and inositol 1,4,5-trisphosphate receptors [Ins(1,4,5)P3PR]. In C. elegans, extensive genetic screens for suppressors of mec-4(d)-induced necrosis have identified genes required for the execution of necrotic cell death. Two of these genes encode the calcium-binding chaperones calreticulin and calnexin, which were found to regulate intracellular calcium levels and to be required for necrotic cell death (Xu et al. 2001). Moreover, treatment of animals with thapsigargin, a drug that induces release of calcium from the ER to the cytoplasm, triggers necrotic cell death, whereas pharmacological treatments or genetic mutations that inhibit calcium release from the ER have a strong protective effect against necrotic cell death.

Genetic studies in C. elegans have also shown that in addition to calcium homeostasis, intracellular pH is also an important modulator of necrotic cell death. Cytoplasmic acidification occurs during necrosis, whereas the vacuolar H+-ATPase, which is a pump that acidifies lysosomes and other intracellular organelles, is required downstream of cytoplasmic calcium overload to promote necrotic cell death (Syntichaki et al. 2005). In line with this, reduced vacuolar H+-ATPase activity or alkalization of acidic endosomal/lysosomal compartments by weak bases has a neuroprotective role against necrosis. Acidic conditions are required for full activity of cathepsins, aspartyl proteases that are primarily confined to lysosomes and other acidic endosomal compartments (Ishidoh and Kominami 2002).

Lysosomal as well as cytoplasmic proteases have been implicated as downstream effectors of cellular destruction in necrosis. Calpains are cytoplasmic, papain-like cysteine proteases that depend on calcium for their activity. Under normal conditions, calpains function to mediate essential signaling and metabolic processes. However, during the course of necrotic cell death, these proteases localize onto lysosomal membranes and may compromise lysosomal integrity, thereby causing leakage of their acidic contents, including lysosomal proteases, into the cytoplasm (Yamashima 2004). In primates, calpains rapidly localize to lysosomal membranes after the onset of ischemic episodes (Yamashima 2000). In C. elegans, two specific calpains, TRA-3 and CLP-1, and two lysosomal cathepsin proteases, ASP-3 and ASP-4, are required for neurodegeneration (Syntichaki et al. 2002). It is likely that ensuing cytoplasmic acidification, activation of the lysosomal, low-pH-dependent cathepsins and hydrolases contributes to cell demise. Mutations that interfere with lysosomal biogenesis and function influence necrotic cell death. For example, necrosis is exacerbated in mutants that accumulate abnormally large lysosomes, whereas impairment of lysosomal biogenesis protects from cell death (Artal-Sanz et al. 2006). Interestingly, lysosomes appear to coalesce around the nucleus and dramatically enlarge during early and intermediate stages of necrosis. In advanced stages of cell death, GFP-labeled lysosomal membranes fade, as lysosomes rupture.

In a recent study from our laboratory, we utilized well-characterized necrosis models in C. elegans to dissect the involvement of clathrin-mediated endocytosis and intracellular trafficking by kinesin motor proteins in cellular destruction during necrotic death (Troulinaki and Tavernarakis 2012a). Our findings revealed for the first time that both clathrin-mediated endocytosis and intracellular trafficking are required for the execution of necrosis in the nematode. Downregulation of endocytosis or kinesin-mediated trafficking by interfering with key proteins regulating these processes, including SNT-1, endophilin (UNC-57), AP180 (UNC-11), synaptojanin (UNC-26), heavy chain of kinesin 1 (UNC-116), and the monomeric kinesin UNC-104, significantly suppresses neurodegeneration induced by hyperactive ion channels without affecting the expression, the localization, or the function of the toxic insults.

Moreover, using the same well-defined necrotic cell paradigm, we assayed animals that were deficient for both autophagy and endocytosis and observed significant synergistic protection against degeneration. These results suggest that autophagy and endocytosis function in parallel to contribute to necrotic cell death (Troulinaki and Tavernarakis 2012a). A graphical representation of the crosstalk of the different mechanisms that cooperate in the execution of necrosis is depicted in Fig. 15.3.

Fig. 15.3
figure 3

Crosstalk between necrotic cell death mechanisms in C. elegans. Necrotic insults converge to increase intracellular Ca2+ levels by calcium influx from extracellular pools through various plasma membrane channels, such as voltage-gated receptors or sodium/calcium exchangers (NCX), or by calcium efflux from subcellular organelles with substantial Ca2+ stores, such as the endoplasmic reticulum via the ryanodine (RyR) and the inositol 1,4,5-trisphosphate receptors [Ins(1,4,5)P3R]. Ca2+ ions then activate cytoplasmic calpain proteases that attack lysosomal membrane proteins, compromising lysosomal integrity and causing the release of hydrolytic enzymes, such as cathepsin proteases. Vacuolar H+ ATPase (V-ATPase)-mediated lysosomal acidification is important for subsequent acidification of the cytoplasm and enhancement of cathepsin activity. In addition, autophagy is induced during necrosis, either directly or through calpain activation, and synergizes with lysosomal cathepsin proteases to facilitate cellular destruction. Moreover, both clathrin-mediated endocytosis and intracellular trafficking are required for cell death and become upregulated by necrosis-triggering insults. [Ca 2+] i cytoplasmic calcium, InsP3R inositol trisphosphate receptor, RyR ryanodine receptor, ER endoplasmic reticulum, AP adaptor proteins for clathrin-mediated endocytosis, V-ATPase vacuolar H+ ATPase (Adapted from Troulinaki and Tavernarakis 2012b)

Full size image

15.5 C. elegans as a Model for Human Diseases Entailing Necrosis

Nematode genes and major signaling pathways show significant conservation during evolution, and more than 50 % of the C. elegans genes have counterparts in humans. In addition to its contribution in elucidating developmental processes, the worm has also served as a platform to model many human pathological conditions such as neurodegenerative disorders, cancer, aging, and associated diseases (Lee et al. 2001; Baumeister and Ge 2002; Poulin et al. 2004). Systematic mapping of gene interactions and signaling pathways implicated in human disease using C. elegans has provided better understanding of complex pathologies (Bussey et al. 2006). The ability to produce “humanized” worms, which express human genes not present in the C. elegans genome, has further enhanced the experimental value of the nematode by allowing the dissection of molecular mechanisms relevant to human disorders. In addition, the ease of drug testing coupled with the efficiency of genetic screens in worms has made C. elegans a favorable tool for the identification and validation of novel drugs and drug targets, aiming to battle human pathological conditions (Kaletta and Hengartner 2006). Here, we overview C. elegans models of human diseases that entail necrosis, focusing on hypoxia, Parkinson’s disease, and tauopathies. Clearly, this list is only indicative of the applications of C. elegans in understanding complex human pathologies that involve necrotic death, and many more such diseases that are not mentioned here have been usefully modeled in the nematode.

15.5.1 Hypoxia

In humans oxygen deprivation induces cell death in pathological conditions such as stroke and heart attack. In C. elegans, hypoxia inflicts necrotic death in a variety of cell types (Scott et al. 2002). Interestingly, mutations in the daf-2 gene, which encodes the C. elegans insulin/IGF receptor tyrosine kinase, confer resistance against hypoxic cell death. DAF-2 is also known to regulate aging and dauer formation in C. elegans (Libina et al. 2003). Related to this, many human neurodegenerative disorders show a late-onset pathogenesis, indicating that aging may alter the vulnerability of cells to various insults. However, while hypoxia resistance in C. elegans appears to be modulated by insulin signaling, other daf-2 mutations that affect longevity and stress resistance do not affect hypoxic death. Selective expression of wild-type daf-2 in neurons and muscles restores hypoxic death in daf-2 hypoxia-resistant mutants, demonstrating a role of the insulin/IGF receptor in the protection of myocytes and neurons from hypoxic injury. Na+-activated potassium channels (KNa) have been identified in cardiomyocytes and neurons as mediators of the protective mechanisms against hypoxic death (Kameyama et al. 1984; Bader et al. 1985). In C. elegans, a KNa ion channel is encoded by the slo-2 gene. slo-2 mutants are hypersensitive to hypoxic death, suggesting that SLO-2 protects against hypoxia effects. Thus, molecular characterization of KNa channels may allow the development of specific agonists and antagonists, in an effort to combat hypoxia-caused pathologies (Yuan et al. 2003).

15.5.2 Parkinson’s Disease

Inclusions of α-synuclein represent a hallmark feature of pathology in both sporadic and familial cases of Parkinson’s disease. α-synuclein is the main component of Lewy bodies found in degenerating dopamine neurons (Spillantini et al. 1997). Mutations in the α-synuclein gene or multiplications of the α-synuclein locus have also been associated with some autosomal dominant familial cases of Parkinson’s disease (Polymeropoulos et al. 1997; Singleton et al. 2003; Chartier-Harlin et al. 2004). C. elegans models of wild-type or mutated human α-synuclein overexpression have been established, either pan-neuronally or specifically in dopaminergic neurons (Lakso et al. 2003; Cao et al. 2005; Cooper et al. 2006; Kuwahara et al. 2006; Qiao et al. 2008), and result in significant motor deficits. No inclusion bodies or α-synuclein aggregation is observed, and intracellular inclusions are rarely observed in these transgenic animals. Overexpression of wild-type or mutant human α-synuclein specifically in worm dopaminergic neurons causes their degeneration, which becomes more pronounced as the animal ages (Cao et al. 2005; Cooper et al. 2006; Kuwahara et al. 2006).

One of the mechanisms implicated in the pathogenesis of Parkinson’s disease is mitochondrial dysfunction (Schapira 2008). Autosomal dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) have been associated with both familial and late-onset cases of PD, with G2019S being a prominent such mutation. C. elegans engineered to express the human LRRK2 (G2019S) mutant form show extensive loss of dopaminergic neurons (Saha et al. 2009), by increasing their vulnerability to mitochondrial stress. Expression of the wild-type LRRK2 has a milder effect on neuron loss. Similarly, loss-of-function mutations in the lrk-1 gene, encoding the worm orthologue of LRRK2, also sensitize dopaminergic neurons to mitochondrial stress.

C. elegans models of α-synuclein-induced dopaminergic neurodegeneration have been used as a platform to identify suppressors of dopaminergic neuron loss with some success. For example, specific overexpression of human torsinA or the worm homologue TOR-2 protects dopamine neurons in these models (Cao et al. 2005). In addition, overexpression of the human lysosomal enzyme cathepsin D has a similar neuroprotective effect (Qiao et al. 2008). Several other molecules involved in autophagy, lysosomal function, trafficking, and G protein signaling have also been identified in RNAi suppressor screenings (Hamamichi et al. 2008).

15.5.3 Tau Toxicity

A significant number of neurodegenerative diseases (including in particular Alzheimer’s disease, frontotemporal dementia and Parkinsonism linked to chromosome 17) is characterized by neurofibrillary tangles consisting of hyperphosphorylated forms of the microtubule-associated protein Tau, encoded by the mapt gene (Lee et al. 2001). Although the exact role of tau in the pathogenesis of these diseases is not clear, the identification of autosomal dominant mutations in the mapt gene indicates a crucial role for the altered tau protein in the neurodegenerative process (Hutton et al. 1998; Poorkaj et al. 1998; Spillantini et al. 1998).

Two studies have reported the expression of human tau (wild-type tau or tau carrying FTDP-17 mutations) either pan-neuronally, under the control of the aex-3 promoter (Kraemer et al. 2003), or specifically in touch receptor neurons of C. elegans, under the control of the mec-7 promoter (Miyasaka et al. 2005). In the first model, expression of the human tau results in reduced lifespan, behavioral abnormalities, progressive uncoordinated movement, and accumulation of insoluble phosphorylated tau, defective cholinergic neurotransmission, and age-dependent axonal and neuronal degeneration. Among the morphological features of neurodegeneration are axonal vacuolar clearing, collapsed membrane structure, and membranous infoldings and whorls (which are characteristic of necrotic cell death), with associated amorphous tau accumulations and abnormal tau-positive aggregates. Axonal degeneration and uncoordinated movement are more severe in lines expressing mutant tau. However, no tau filaments are observed.

15.6 Concluding Remarks

In this chapter, we have attempted to provide a comprehensive overview of the necrotic cell death paradigms that have been established in C. elegans (see Table 15.1) and to also convey our current understanding of the molecular mechanisms involved. The rich repertoire of necrotic cell death events that occur in C. elegans both during development and in the adult renders the nematode a particularly attractive platform for dissecting the mechanisms of pathological cell death in humans, which is typically mediated by necrotic processes.

Table 15.1 Triggers and paradigms of necrotic death in C. elegans. Necrotic stimuli and cell populations affected
Full size table

The similarity of necrotic cell death triggered by hyperactive ion channels in C. elegans to excitotoxic cell death and neurodegeneration in mammals, both in terms of morphological characteristics and mechanistic aspects, reflects the extensive evolutionary conservation of necrosis-relevant genes between C. elegans and mammals. Moreover, conservation of the mechanisms that protect C. elegans and mammalian cells from necrotic death inflicted by diverse stimuli, as exhibited, for example, by the hormetic induction of HSF16.1 upon heat preconditioning, provides new prospects for employing the nematode in the battle against degeneration. Concomitantly, modeling of human degenerative disorders, such as Parkinson’s disease and others, in C. elegans has already accelerated the pace of the molecular dissection of the underlying mechanisms and holds promise for the development and testing of innovative intervention strategies.