through several channels and transporter-mediated routes leads to intracellular calcium overload and concomitant cell death (Lipton and Nicotera, 1998; Nicotera and Bano, 2003). Sodium influx amplifies acute neuronal swelling and facilitates calcium entry through voltage-gated channels and the Na+/Ca2+ exchanger (Sattler and Tymianski, 2000). Cell injury and death can also be induced by disturbances of calcium homeostasis in the ER (Mattson et al., 2000; Paschen, 2001). The ER is the major calcium storage compartment of the cell. Sequestration of calcium into the ER is mediated by the sarcoendoplasmic reticulum Ca2+-ATPase, and release back to the cytoplasm is controlled by ryanodine, and 1,4,5-inositol trisphosphate receptors (Carafoli, 2002). Within the ER, calcium binds to calcium-binding molecular chaperones such as calretic- ulin and calnexin (Michalak et al., 1999; Llewellyn et al., 2000). Under conditions of extreme cellular stress, ER calcium stores are rapidly mobilized, boosting the massive increase of intracel- lular calcium concentration, which signals cell demise (Ferri and Kroemer, 2001). Pharmacological treatments or genetic muta- tions that inhibit calcium release from the ER have a strong pro- tective effect against necrotic cell death (Yu et al., 2000; Xu et al., 2001). In contrast, treatment with chemicals such as thap- sigargin, which promotes the discharge of calcium from intra- cellular stores by specifically inhibiting the sarcoendoplasmic reticulum Ca2+-ATPase calcium pump, induces necrotic cell death (Takemura et al., 1989; Xu et al., 2001). We hypothesize that generalized osmotic destabilization of the cell during necro- sis may also contribute to the bursting of lysosomes. The exclusive confinement of lysosomes around the nu- cleus during neurodegeneration may reflect damaged micro- tubule or actin motors, which mediate the movement of lysosomal organelles (Burkhardt et al., 1997). It is known that calpain proteases contribute to cell death by cleaving essential cytoskeletal proteins of neuronal axons (for review see Artal- Sanz and Tavernarakis, 2005). Therefore, calpains may act on microtubules at early stages of neurodegeneration. However, we cannot rule out the possibility that lysosomes are specifically targeted to the periphery of the nucleus. We observed that reduction of necrotic cell death by a drop in aspartyl protease activity is enhanced by conditions that counterbalance intracellular acidification. Such synergy between aspartyl proteases and pH indicates that aspartyl pro- teases become activated by low pH conditions, which develop during necrosis and facilitate cellular destruction. In addition to aspartyl proteases, other proteases that function optimally at low pH may become activated by acidification during necrosis. Such proteases have been implicated in both apoptotic and necrotic cell death (Ferri and Kroemer, 2001). We suggest that preventing acidification suppresses neurodegeneration, in part, by lowering the activity of these enzymes. Alternatively, aspar- tyl proteases and acidification may independently contribute to cell death. However, to discriminate between these alternatives requires complete elimination of aspartyl protease or vacuolar H+-ATPase activity, which results in embryonic lethality (Oka and Futai, 2000; Pujol et al., 2001; unpublished data). The totality of our observations denotes an essential and general role for lysosomes in necrotic cell death induced by various insults. Our study is the first to monitor lysosomal alter- ations during necrosis in vivo, in any organism. Our findings uncovered novel aspects of the cellular changes that transpire during neurodegeneration in the nematode. Such information could be effectively used toward identifying candidate common intervention targets in an effort to battle numerous pathological conditions in humans. We envision that alterations of lysosomal biogenesis and function by genetic mutations or pharmacologi- cal treatments modify the susceptibility of neurons to necrosis. However, once a threshold is exceeded and cell death com- mences the sequence of events is essentially unaltered. Materials and methods Strains and genetics We used standard procedures for C. elegans strain maintenance, crosses, and other genetic manipulations (Brenner, 1974). Nematodes were grown at 20°C. N2 was used as the wild-type strain. The following mutant alleles were used: mec-4(u231)X, which is referred to in the text as mec-4(d); deg-3(u662)V, which is referred to in the text as deg-3(d); nuIs5[pglr-1Gαs(Q227L)pgrl-1GFP], which is referred to in the text as αs(gf); arIs37[pmyo-3ssGFP]I;cup-5(ar465)III;dpy-20(e1282)IV, which is referred to in the text as cup-5(lf), cad-1(j1)II, glo-1(zu391)X, glo-1(Kx92)X, and glo-1(zu437)X. The glo-1 alleles were provided by G. Hermann (Lewis and Clark College, Portland, OR). The following double and triple mu- tants were used: cad-1(j1)II;mec-4(u231)X, vha-12(n2915)mec-4(u231)X, arIs37[pmyo-3ssGFP]I;cup-5(ar465)III;dpy-20(e1282)IV;mec-4(u231)X, arIs37- [pmyo-3ssGFP]I;cup-5(ar465)III;dpy-20(e1282)IV;deg-3(u662)V, glo-1(zu391)- mec-4(u231)X, glo-1(kx92)mec-4(u231)X, and glo-1(zu437)mec-4(u231)X. Plasmids and RNAi To generate pmec-17LMP-1::GFP, we fused GFP at the COOH terminus of the C. elegans LMP-1 protein. The translational fusion includes the entire LMP-1 coding sequence lacking the stop codon, a Gly-Ser-Ser-Pro-Gly-Leu-Ala- Lys-Gly-Pro-Lys-Gly linker, and GFP. The resulting chimera was expressed in touch receptor neurons under the control of the mec-17 promoter. The plasmid carrying the reporter fusion was constructed in two steps. First, the mec-17 promoter was ampliﬁed from N2 genomic DNA with the primers 5′ CGGGATCCGAATCGTCTCACAACTGATCC 3′ and 5′ AACTGCAGG- TGACTACTTGAGACCTG 3′. A 1,900-bp PstI–BamHI fragment was cloned into the promoterless gfp vector pPD95.77 (Fire et al., 1990). Second, the LMP-1 coding region was ampliﬁed from genomic DNA using the primers 5′ CGGGATCCGACGCTGGCATATCCTTGTCTC 3′ and 5′ CGGGATC- CAATTGAACTATGTTGAAATCG 3′. A BamHI PCR fragment was cloned downstream of the mec-17 promoter on the pPD95.77 plasmid vector. For RNAi experiments, we used HT115(DE3) Escherichia coli bacteria, which were transformed with plasmids that direct the synthesis of double-stranded RNAs corresponding to the genes of interest; they were then fed to animals according to a previously described methodology (Kamath et al., 2001). For cup-5 RNAi, we used a 1.5-kb PCR-generated fragment derived from the cup-5 locus using the primers 5′ GGGGTACCCCATGATTTCAGATGTC- TCGC 3′ and 5′ GGGGTACCCCGAATGCAAAGAATGAGAACG 3′. The primers used for nhx RNAi constructs are as follows: 5′ GCTCTAGACTC- TTCACTGGCCTGTG 3′ and 5′ CCGCTCGAGATCAGTATGACTGCG 3′ for nhx-4, 5′ AACTGCAGTTATGGACGATATCAAC 3′ and 5′ CCGCTCG- AGCCACAAACTTCAGCCAC 3′ for nhx-5, and 5′ GCTCTAGATGGTGT- CCTGACTCTTC 3′ and 5′ CCGCTCGAGCTTCCACTCCAGACATC 3′ for nhx-9. For pmr-1 we used the following PCR primers: 5′ AACTGCAGATTG- AAACACTGACATC 3′ and 5′ CCGCTCGAGTACCTGAAACATTCCG 3′. RNAi plasmids for vha-2, aspartyl proteases asp-3 and asp-4, and calpain clp-1 have been previously described (Syntichaki et al., 2002, 2005). We assayed the effectiveness of RNAi by monitoring the expression of full- length GFP reporter fusions. Plasmid vectors for C. elegans were provided by A. Fire (Stanford University School of Medicine, Stanford, CA). Neurodegeneration assays Degeneration of speciﬁc neuron sets in animals bearing deg-3(d), mec-4(d), and αs(gf) alleles was quantiﬁed as previously described (Syntichaki et al., 2002). For alkalization assays, we treated young adult animals with lysotropic alkalizing agents (5 mM NH4Cl and 40–150 μM acridine orange; Sigma-Aldrich) in liquid cultures supplemented with E. coli bacteria for LYSOSOMAL FATE IN NECROTIC CELL DEATH • ARTAL-SANZ ET AL. 237