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Introduction
While mechanisms of cell death such as apoptosis are well characterized [1], less is known about the mechanisms of organismal death, particularly in invertebrate model organisms. Here we investigate organismal death in the nematode C. elegans, using a newly discovered, endogenous fluorescent marker of death.
介绍
虽然如细胞凋亡的细胞死亡机制很好的特点[1]，了解较少有机体死亡的机制，特别是在无脊椎动物模式生物。在这里，我们调查有机体死亡线虫，使用一种新发现的，内源性荧光标记死亡。
One possibility is that organismal death results from a cascade of cell death. As first defined by Kerr et al. in 1972 [1], cell death has been viewed as taking two forms: controlled (apoptotic) or uncontrolled (necrotic). However, more recent elucidation of the mechanisms underlying necrotic cell death reveals that it too can be a regulated process [2]–[5]. Biochemical hallmarks of necrosis include calcium-mediated initiation, lysosomal membrane permeabilization (LMP), and activation of noncaspase proteases (calpains and cathepsins) .
一种可能性是，从细胞死亡的级联的有机体死亡结果。作为第一个定义的由Kerr等。于1972年[1]，细胞死亡已经被看作是采取两种形式：控制（凋亡）或失控（坏死）。然而，最近的坏死性细胞死亡的机制的阐明，揭示了，它也可以是一个调节过程[2] - [5]。坏死的生化标志，包括钙离子介导的起始，溶酶体膜通透性（LMP），激活noncaspase蛋白酶（钙蛋白酶和组织蛋白酶).
Necrosis as a regulated process has been characterized mainly in mammalian neuronal models. Excitotoxic neuronal cell death occurs in response to overstimulation with the excitatory neurotransmitter glutamate (e.g., under conditions of ischemia or stroke) [7]. Sustained activation of glutamate receptors causes a cytosolic influx of extracellular Ca2+ [8]. Increased Ca2+ levels lead to cell death, largely through activation of associated proteases [9]. Moreover, Ca2+ may spread between cells via connecting gap junctions, and gap junction inhibition reduces ischemia-induced neurodegeneration .
作为一个稳定的进程已经坏死的特点主要在哺乳动物的神经元模型。兴奋毒性神经细胞死亡发生过度刺激的兴奋性神经递质谷氨酸盐（例如，局部缺血或中风的条件下）[7]。谷氨酸受体持续激活导致胞质涌入的细胞外Ca2 + [8]。增加钙离子浓度导致细胞死亡，主要是通过激活相关的蛋白酶[9]。此外，Ca2 +的可能蔓延细胞之间通过连接缝隙连接，缝隙连接的抑制作用降低缺血诱导的神经退行性疾病.
Through the study of ischemia-induced death in mammalian CA1 hippocampal neurons, Yamashima and co-workers identified the calpain-cathepsin cascade as an effector of necrotic cell death. Ischemia increases intracellular Ca2+ levels, which activate Ca2+-dependent cysteine proteases (calpains) [12]. These calpains cause lysosomal lysis, leading to cytosolic acidosis and the destructive release of lysosomal cathepsin proteases [13].
Many components of the calpain-cathepsin cascade are present in C. elegans, where necrotic cell death can be induced in neurons by mutations such as mec-4(u231) [14]. For example, mec-4-induced neurodegeneration requires the calcium-dependent calpains TRA-3 and CLP-1 and the cathepsins ASP-3 and ASP-4 [15].
LMP is a central event in the necrotic cascade, and the degree of LMP can influence the cellular decision to live or to die via necrosis or apoptosis [3],[5],[16]. In C. elegans, lysosomes are required for osmotic stress-induced necrotic death [17] and interventions that increase lysosomal pH can ameliorate mec-4(d)-induced neurodegeneration [18]. http://www.ukassignment.org/dxtermpaper/
C. elegans intestinal cells contain both lysosomes and gut granules, which are large, melanosome-like lysosome-related organelles [19]. Under ultraviolet light, gut granules emit blue fluorescence, with maximal intensity at λex/λem 340/430 nm (Figure 1A–B) [20]. This fluorescence has been attributed to lipofuscin [21],[22], a heterogeneous, cross-linked aggregate of oxidatively damaged lipids and proteins. Lipofuscin accumulates with age in postmitotic mammalian cells and so has frequently been used as a biomarker of aging [23]–[25]. Lipofuscin composition is highly variable but can be identified by virtue of its autofluorescence [24]. If excited by UV light in vitro it emits blue fluorescence, which may reflect formation of fluorescent Schiff bases between carbonyl and amino groups [26],[27]. However, UV excitation of lipofuscin in vivo results in peak fluorescence in the 540–640 nm (orange-yellow) range.
Several observations have led to the suggestion that the fluorescent material in the C. elegans intestine is lipofuscin. Its fluorescence peak at λex/λem 340/430 nm is similar to that of lipofuscin in vitro, it is localized to the lysosome-like gut granules, and its levels increase in aging populations [20]–[22],[29]. It is often used as a biomarker of aging—for example, to verify that treatments that shorten worm lifespan do so by accelerating aging. The presence of lipofuscin in C. elegans would support the view that aging is caused by accumulation of molecular damage. Yet it remains possible that the fluorescent substance in gut granules is not lipofuscin. For example, studies of flu mutations causing altered gut granule fluorescence suggest that it corresponds to fluorescent tryptophan metabolites [30].
In this study, we describe how a reassessment of blue fluorescence in C. elegans led to the discovery of the phenomenon of death fluorescence (DF), a burst of blue fluorescence that accompanies death in C. elegans. We establish that both DF and gut granule fluorescence originate not from lipofuscin, but from tryptophan-derived anthranilic acid glucosyl esters. We then show that DF is generated by the calpain-cathepsin necrotic cell death pathway, and requires calcium signaling for organismal propagation. Finally, we show that inhibition of this pathway can protect animals against stress-induced death, supporting a role of systemic necrotic cell death in organismal death.
Results
Oxidative and Thermal Stress Do Not Increase Blue Fluorescence
Lipofuscin is formed through accumulation of oxidatively damaged proteins and lipids [24]. For example, raised oxygen level (40% O2) increases lipofuscin levels in human fibroblasts [25]. To probe whether the blue fluorescent material in C. elegans gut granules (Figure 1A–B) is lipofuscin, we exposed them to normobaric hyperoxia (90% O2), and elevated iron levels. Both treatments significantly increased protein oxidative damage but neither increased blue fluorescence levels (Figure 1C–F). Elevated expression of hsp-4::gfp is indicative of the unfolded protein response [31], symptomatic of protein damage. Heat shock increased hsp-4::gfp expression but not blue fluorescence (Figure S1). These results imply that C. elegans blue fluorescence is not generated by oxidative damage, suggesting that it is not lipofuscin.
A Burst of Blue Fluorescence Occurs When C. elegans Die
Like lipofuscin in mammals, mean fluorescence levels rise gradually with age in C. elegans population cohorts [20],[29]. However, population mean data do not address heterogeneity in the fluorescence of individual worms. This concern was raised by a previous study [20], as follows. Aging worms can be classed according to their degree of motility: class A animals move normally, class B animals move more slowly, and class C animals do not move away when touched, and are near to death [32]. Notably, blue fluorescence levels did not differ significantly between class A and B, and only increased in class C worms [20]. This suggests that blue fluorescence levels in worms increase only as they approach death.
To test this directly, fluorescence levels of individually cultured, wild-type C. elegans in situ on nematode growth medium (NGM) agar plates were examined at intervals throughout life (DAPI filter; λex/λem 350/460 nm). As animals approached death (as indicated by reduced movement), time-lapse imaging was used to capture fluorescence changes during death. This revealed that fluorescence levels in individual animals change little until immediately prior to death. A striking and sudden ~400% increase in fluorescence level then occurs, coinciding with cessation of movement (i.e., death) (Figure 2; Video S1). This rise begins at ~2 h prior to death, and then fades by ~6 h after death.
Evidence presented here implies that during death in C. elegans, the intestine, the largest somatic organ, undergoes a stereotyped process of self-destruction involving an intra- and intercellular cascade of cellular necrosis. The mechanisms involved are similar to those active in the propagation of cellular necrosis in mammals. In worms, necrotic propagation requires the innexin INX-16, while in mammals connexin (mammalian gap junction proteins) inactivation reduces ischemia-induced neurodegeneration [10]. Thus, the C. elegans intestine is a potential new model for understanding the propagation of necrotic cell death, and its prevention.
Previous studies of the cellular necrosis pathway have largely focused on neurodegeneration, in mammals and C. elegans. Our findings imply similar action of this pathway in the worm intestine. However, generation of DF appears to be restricted to the intestine, and is not detectable in necrotic mec-4(d) neurons (unpublished data).
Our results imply that intestinal self-destruction by systemic necrosis occurs during both stress- and aging-induced death. However, only in stress-induced death did inhibition of systemic necrosis prevent death. This suggests that while lethal stress causes death by inducing systemic necrosis, aging causes death by a number of processes acting in parallel, likely including systemic necrosis (given that it destroys a major organ). Here there are potential parallels in human aging: estimations of the likely upper limits of human longevity have calculated that removal of a major age-related disease (e.g., cardiovascular disease, cancer) would cause only small increases in lifespan [49]. This is because multiple pathologies act in parallel to increase age-related mortality.
A feature of intestinal necrosis is its origin in the anterior int1 cells. This suggests that the unusual vulnerability of these cells to necrotic death might represent a breaking point within organismal homeostasis; analogously, in humans localized failure (e.g., in the heart or kidneys) can cause rapid organismal death. The existence of an anterior to posterior (A-P) Ca2+ wave is unexpected, given that the defecation-associated Ca2+ wave previously characterized in the intestine flows in the opposite direction, from posterior to anterior [44]. How the A-P Ca2+ wave is specified is unknown. One possibility is that extracellular Ca2+ levels are elevated near the anterior intestine, creating vulnerability to necrosis.
The presence of a mechanism, systemic necrosis, that brings about organismal death in C. elegans raises questions about its evolutionary origin. Could such an organismal self-destruct mechanism serve as an adaptation? When food is limiting, gravid hermaphrodites typically die with multiple embryos in their uterus, which hatch internally and consume their mother's corpse (“bagging”). Potentially, this improves the mother's fitness by increasing survival of her genetically identical offspring [57]. One possibility, then, is that systemic necrosis enhances fitness by aiding efficient transfer of nutrients from mother to offspring during bagging. Alternatively, systemic necrosis may be the nonadaptive product of antagonist pleiotropy, or a quasi-program [58],[59]. By this view, elements of the necrosis cascade contribute to early life fitness, while systemic necrosis is an unselected, deleterious consequence of their action under lethal stress or as a result of aging.
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