Figure 3. Suppression of necrosis by aspartyl protease deﬁciency is enhanced by conditions that impede intracellular acidiﬁcation. The number of vacuolated touch receptor neurons per 100 L1 stage mec-4(d) animals. Bars represent the mean of three independent experiments. n > 300. Knockdown of both vacuolar H+-ATPase and aspartyl protease genes resulted in signiﬁcantly more extended quenching of neurodegeneration than for any single gene. P < 0.001, unpaired t test. Efﬁcacy of RNAi was assessed as described in Materials and methods. Error bars represent the SD of the mean. mutants (Fig. 3). In contrast, reduced V-ATPase activity did not further enhance suppression of necrosis by calpain protease deficiency (Table I). We conclude that suppression of necrosis by aspartyl protease deficiency is enhanced by conditions that impede intracellular acidification. We considered the contribution of additional cellular pH homeostasis mechanisms in necrosis. Two other major mecha- nisms have been implicated in cytoplasmic and subcellular or- ganelle pH regulation; first, the sodium–hydrogen exchanger (NHX), and second, the cation transporter P-type ATPase. These mechanisms operate both on the plasma membrane and at the membranes of subcellular organelles, such as mitochondria, to facilitate proton trafficking and pH homeostasis. Multiple nhx genes are encoded in the C. elegans genome (more than 9 nhx isoforms; Nehrke and Melvin, 2002; http://www.wormbase.org). pmr-1 is the gene encoding the nematode P-type ATPase homologue. Knockdown of nhx-4, -5, and -9, which are three nhx isoforms expressed in neurons, did not alter the extent of neurodegeneration induced by mec-4(d) (137 ± 19 nhx-4(RNAi)mec-4(d), 134 ± 16 nhx-5(RNAi)mec-4(d), and 134 ± 21 nhx-9(RNAi);mec-4(d) versus 139 ± 14 vacuolated neurons in mec-4(d) mutants; n = 100). To address the potentially Table I. Calpain and V-ATPase activity in necrotic cell death Strain mec-4(d) gfp(RNAi);mec-4(d) clp-1(RNAi);mec-4(d) vha-12(n2915)mec-4(d) gfp(RNAi);vha-12(n2915)mec-4(d) clp-1(RNAi);vha-12(n2915)mec-4(d) Corpses 205 ± 9 193 ± 11 96 ± 18 112 ± 9 101 ± 13 89 ± 16 Suppression of necrosis by calpain protease deﬁciency is not further enhanced by reduced vacuolar H+-ATPase activity. Values represent vacuolated touch receptor neurons (± SD) per 100 animals at the L1 stage. n > 250. 234 JCB • VOLUME 173 • NUMBER 2 • 2006 redundant function of these genes in the nervous system, we as- sayed cell death in animals in which all three isoforms were knocked down by RNAi. We did not observe significant sup- pression of necrosis in these animals (131 ± 28 nhx-9(RNAi); nhx-5(RNAi) nhx-4(RNAi)mec-4(d) versus 139 ± 14 vacuolated neurons in mec-4(d) mutants; n = 100.). Similarly knockdown of pmr-1 did not affect necrotic cell death (pmr-1(RNAi); mec-4(d): 141 ± 17, versus 139 ± 14 vacuolated neurons in mec-4(d) mutants; n = 100). Therefore, neurodegeneration is not affected by compromising these other, nonlysosomal pH homeostasis mechanisms. Lysosomal fate during necrosis The importance of lysosomal membrane permeabilization in cell death has previously been established (Kroemer and Jaattela, 2005). Approaches combining electron microscopy and immunodetection show that calpains concentrate on lysosomal membranes during ischemic stroke in primates (Yamashima, 2000; Yamashima et al., 2003). However, information on lyso- some fate and lysosomal system alterations during necrosis in vivo is lacking. We monitored the distribution and morphology of lysosomes in vivo during necrotic cell death in C. elegans. To visualize lysosomes and late endosomes, we fused GFP at the COOH terminus of LMP-1, which is the only C. elegans protein bearing a lysosomal targeting sequence (GYXXΦ; Φ, large hy- drophobic amino acid residue) at its COOH terminus (Kostich et al., 2000). LMP-1 shows similarity to the vertebrate lysosome- associated membrane protein LAMP/CD68 (Kostich et al., 2000; Eskelinen et al., 2003), and it is widely used as a lysosomal marker (Treusch et al., 2004; Hermann et al., 2005; Nunes et al., 2005). We examined lysosomal distribution and morphology in touch receptor neurons of wild-type and mec-4(d) animals expressing an LMP-1::GFP fusion. In the neurons of wild-type animals, lysosomes appear scattered throughout the cytoplasm (Fig. 4 A). In contrast, dur- ing the early stages of neurodegeneration, lysosomes enlarge and localize close to the nucleus (Fig. 4 B, i). As neurodegen- eration progresses, lysosomes fuse to surround an internally vacuolated structure (Fig. 4 B, ii–viii). This encapsulated vacu- ole is likely the swollen nucleus of the dying neuron (Hall et al., 1997). To confirm the nuclear origin of the internal vacu- ole, we performed DAPI staining in mec-4(d) animals express- ing the LMP-1::GFP fusion protein. As shown in Fig. 5, LMP-1–labeled internal membranes are positive for DAPI staining. In agreement with our observation, elevation of cyto- solic Ca2+ concentration can induce lysosomal fusion (Bakker et al., 1997). Consistent with previous studies (Hughes and August, 1982; Lippincott-Schwartz and Fambrough, 1986; Hermann et al., 2005), a portion of LMP-1 localizes to the plasma membrane (Fig. 4 B, ii–viii). Lysosomes remain confined around the distended nucleus during necrotic cell death (Fig. 4 B, ii–v). As neurodegeneration proceeds, the nucleus migrates to the periphery of the cell and condenses (Fig. 4 B, iv–vi). In the advanced stages of neurode- generation, GFP intensity decreases and lysosomal definition is ultimately lost (Fig. 4 B, vii and viii). Interestingly, a decrease in LMP-1::GFP immunoreactivity is associated with neuronal