Figure 2. Effect of lysosomal biogenesis mutants on necrotic cell death. (A) Neurodegeneration is enhanced in a cup-5(lf) genetic background, where the lysosomal system is expanded. This enhancement is suppressed by vacuolar H+-ATPase deﬁciency. Degenerating neurons in mec-4(d), cup-5(lf);mec-4(d), outcrossed cup-5(lf);mec-4(d), cup-5(RNAi);mec-4(d), deg-3(d), and cup-5(lf);deg-3(d) animals. Knockdown of cup-5 by RNAi also enhances mec-4(d)–induced neurodegeneration. n > 250. P < 0.005; unpaired t test. (B) Neurodegeneration is suppressed by three different glo-1 mutant alleles, where lysosomal biogenesis is defective. Degenerating neurons in mec-4(d), glo-1(zu391)mec-4(d), glo-1(kx92)mec-4(d), and glo-1(zu437)mec-4(d) animals. n > 350. P < 0.005, unpaired t test. Error bars represent the SD of the mean. inflicted by mec-4(d) and deg-3(d) is exacerbated in cup-5(lf) mutants (Fig. 2 A). We confirmed the reduced cell survival by scoring for expression of a touch receptor–specific mec-4::GFP reporter fusion in adult animal neurons. Fluorescent neuron number decreased in cup-5(lf);mec-4(d) double mutants, com- pared with mec-4(d) mutant animals (109 ± 16 cup-5(lf);mec-4(d) versus 176 ± 11 fluorescent neurons in mec-4(d) mutants; n = 150; P < 0.001, unpaired t test). In addition, cup-5(lf) mutants showed increased sensitiv- ity to hypoxia compared with wild type (16.5 ± 5.3% wild-type survival versus 5.3 ± 4.1% cup-5(lf) animal survival. n > 200; P < 0.005, unpaired t test). Knockdown of vha-2, which en- codes for a subunit of the vacuolar H+-ATPase, by RNAi in cup-5(lf);mec-4(d) double mutants abolished cup-5(lf)–mediated enhancement of cell death (Fig. 2 A). This suggests that the enhanced cell death observed in cup-5(lf);mec-4(d) double mu- tants is caused by increased lysosome-mediated acidification. To confirm the effect of cup-5 deficiency on cell death, we out- crossed cup-5(lf) mutant animals in an effort to minimize the possibility that the effects on neurodegeneration we observed were caused by unlinked spurious mutations in the genetic background of the cup-5(lf) allele. Furthermore, we used RNAi to specifically target cup-5. Both outcrossed cup-5(lf) deri- vatives and RNAi-mediated knockdown of cup-5 resulted in enhanced mec-4(d)–induced neurodegeneration (Fig. 2 A). In a reciprocal approach, we examined necrosis in glo-1(lf) mutants. The glo-1 gene encodes a predicted Rab GTPase that is similar to proteins implicated in the biogenesis of special- ized lysosome-related organelles (Hermann et al., 2005). glo-1 mutant alleles were recovered in a screen aimed at identifying genes involved in the formation of birefringent gut granules, which are lysosome-related organelles (Hermann et al., 2005). glo-1(lf) mutants are defective in the biogenesis of lysosome- related gut granules, show little or no staining with lysosomal markers, and lack detectable expression of the vacuolar H+-ATPase subunits VHA-17 and VHA-11 in intestinal pre- cursor cells (Hermann et al., 2005). We found that all three glo-1(lf) alleles ameliorate necrotic cell death triggered by mec-4(d) (Fig. 2 B). This suggests that the reduced number of lysosomes in touch receptor neurons of glo-1(lf)mec-4(d) double mutants results in reduced intracellular acidification and, consequently, in reduced necrotic cell death. Suppression of necrosis by aspartyl protease deﬁciency is enhanced by conditions that impede lysosome- mediated intracellular acidiﬁcation Specific calpain and aspartyl proteases are implicated in the ex- ecution of necrotic cell death in both nematodes and mammals (Syntichaki et al., 2002; Yoshida et al., 2002), and the impor- tance of calpain and aspartyl protease activation in acute cell injury and necrotic cell death triggered by calcium influx has been previously established (for review see Artal-Sanz and Tavernarakis, 2005). Calpain proteases become activated upon the abrupt increase of intracellular calcium that occurs in re- sponse to diverse necrosis-initiating stimuli, whereas aspartyl proteases function optimally under the highly acidic conditions present in the lumen of lysosomes and other acidic endosomal compartments (Ishidoh and Kominami, 2002; Goll et al., 2003). We assessed the effect of lysosome-mediated intracellu- lar acidification on the requirement for aspartyl proteases in necrosis. cad-1(j1) mutants maintain aspartyl protease activity that is 90% lower than in wild-type animals (Jacobson et al., 1988). mec-4(d)–induced neurodegeneration is attenuated in cad-1(j1);mec-4(d) double-mutant strains (Syntichaki et al., 2002). RNAi-mediated knockdown of vha-2 diminishes cell death inflicted by mec-4(d) (Fig. 2 B and Fig. 3; Syntichaki et al., 2005). Cell death was further reduced in cad-1(j1);mec-4(d) mutant animals by RNAi-mediated knockdown of vha-2 (Fig. 3). Two aspartyl proteases, ASP-3 and -4, contribute the bulk of protease activity required for neurodegeneration in- flicted by diverse genetic insults in C. elegans (Syntichaki et al., 2002). Similarly, knockdown of asp-3 or asp-4 by RNAi in vha-12(n2915)mec-4(d) double mutants augmented survival of the six receptor neurons, compared with single mec-4(d) LYSOSOMAL FATE IN NECROTIC CELL DEATH • ARTAL-SANZ ET AL. 233