We examined the role of lysosomes in a well defined model of necrotic cell death in the nematode. We show that the alkali- zation of endosomal and lysosomal compartments protects against necrotic cell death that is induced by mutations in several ion channels, as well as by prolonged hypoxia. We investigated the effect of mutations that alter lysosome biogenesis in necrotic cell death and found that mutations resulting in the accumula- tion of large lysosomes exacerbate necrosis, whereas mutations that impair lysosome biogenesis are protective. Conditions that counterbalance intracellular acidification enhance suppression of neurodegeneration by aspartyl protease deficiency, indicating that aspartyl proteases are activated by low pH conditions, which develop during necrosis. By monitoring lysosomes during ne- crosis in vivo, we show that lysosomes coalesce around the nu- cleus and dramatically enlarge during the early and intermediate stages of necrosis, although, ultimately, lysosomal definition is lost. Together, these results point to a decisive role for lysosomes in the execution of necrotic cell death. Results Alkalization of endosomal and lysosomal compartments protects against necrotic cell death Recent data suggests that vacuolar H+-ATPase–mediated intra- cellular acidification is required downstream of cytoplasmic calcium overload to promote necrotic cell death, plausibly by enhancing the activity of the low pH–dependent proteases that dismantle the cell (Syntichaki et al., 2005). To emulate impaired lysosomal acidification in degenerating neurons, we treated animals expressing a neurotoxic gain-of-function (gf ) mec-4(d) allele encoding a hyperactive ion channel subunit that is nor- mally required for mechanosensation with NH4Cl and acridine orange. These lysotropic weak bases are known to accumulate in lysosomes and other acidic subcellular compartments, neu- tralizing their pH (Oka and Futai, 2000). Treatment ameliorated degeneration of the six touch receptor neurons of mec-4(d) mutant animals (Fig. 1 A). Similarly, cell death inflicted by the toxic deg-3(d) allele, encoding a hyperactive acetylcholine re- ceptor calcium ion channel, or by overexpression of the hyper- activated Gαs(Q227L) variant (αs(gf)) was also suppressed after treatment with NH4Cl and acridine orange (Fig. 1 A). We as- sessed cell survival by scoring for expression of a touch receptor– specific mec-4::GFP reporter fusion in adult animal neurons. Fluorescent neuron number increased after treatment with alka- lizing agents in adult mec-4(d) mutant animals (309 ± 19 NH4Cl and 192 ± 13 acridine orange vs. 176 ± 11 fluorescent neurons in untreated mec-4(d) mutants; n = 100; P < 0.001, unpaired t test). Prolonged hypoxia, which is a condition of low oxygen availability that emerges during ischemia and stroke, induces necrotic cell death in the nematode (Scott et al., 2002). We ex- amined the effect of alkalization in hypoxia-induced cell death. Nematodes were treated with sodium azide, which inhibits complex IV (cytochrome c oxidase) of the respiratory chain and simulates hypoxia. Treatment with NH4Cl and acridine orange reduced the hypoxic death of wild-type animals (Fig. 1 B). 232 JCB • VOLUME 173 • NUMBER 2 • 2006 Figure 1. Alkalization of endosomal compartments by weak bases pro- tects against necrotic cell death. (A) Degenerating touch receptor neurons in mec-4(d) and PVC interneurons in deg-3(d) and αs(gf) in untreated animals or animals treated with NH4Cl and acridine orange. n > 150. P < 0.001, unpaired t test. (B) The effects of alkalizing agents on hypoxic death. The graph shows the percentage of animals that survived near-lethal treatment with sodium azide. n > 200. P < 0.001; unpaired t test. Error bars represent the SD of the mean. We considered whether suppression of necrosis is an indi- rect effect of probable alterations in animal growth and develop- ment caused by alkalizing agents. We assayed developmental timing after egg hatching and past the L1 stage, which is where we assayed for cell death. We also assayed for animal locomo- tion, pharyngeal pumping, and defecation. Treatment with NH4Cl and acridine orange, at the concentrations and under the conditions used, does not result in any discernible defects in animal growth and development that could influence the course of necrotic cell death. We conclude that dependence on acidi- fied intracellular compartments is a common denominator of necrotic cell death triggered by diverse stimuli. Altered lysosomal biogenesis affects necrosis To further evaluate the lysosomal role in necrotic cell death, we examined mutants defective in lysosomal biogenesis. We exam- ined necrosis in cup-5 loss-of-function (lf; ar465) mutant animals. Cells of cup-5(lf) mutants contain increased numbers of enlarged acidic lysosomes. cup-5 encodes the C. elegans mucolipin-1 homologue that is implicated in mucolipidosis type IV, which is a lysosomal storage disease that results in severe devel- opmental neuropathology in humans (Fares and Greenwald, 2001; Hersh et al., 2002; Treusch et al., 2004). Neurodegeneration