Figure 6. Perturbation of lysosomal bio- genesis, function, and neurodegeneration. Lysosomal morphology and distribution after treatment with alkalizing agents and in genetic backgrounds affecting lysosomal biogenesis and function. (A and B) Confocal images of PLM touch receptor neurons of mec-4(d) animals expressing a pmec-17LMP-1::GFP reporter fusion, after alkalization of lysosomal compartments. (A) Images of degenerating neurons after treat- ment with 5 mM NH4Cl. (B) Degenerating neu- rons after treatment with 40 μM acridine orange. Nuclei can be seen because, at the concentration used, acridine orange stains DNA. (C–E) Confocal images of PLM touch receptor neurons of mec-4(d) animals under different genetic backgrounds that either sup- press or enhance necrosis. Double mutants express the same pmec-17LMP-1::GFP transgene. (C) Degenerating neurons of cup-5(lf);mec-4(d) mutants. Note that vacuoles appear larger. (D) Degenerating neurons of glo-1(zu391) mec-4(d) animals. (E) Images of degenerat- ing neurons in cad-1(j1);mec-4(d) mutants. LMP-1::GFP expression, differential interfer- ence contrast (DIC), and merged images are shown. (left) Early to middle stages of neuro- degeneration. (right) Middle to late stages of neurodegeneration. Bars, 5 μm. We investigated whether alkalizing treatments result in altered lysosomal fate. We examined the effects of acridine orange and NH4Cl treatment on LMP-1::GFP distribution in mec-4(d) mutants. Although the number of unvacuolated cells with a wild-type pattern of lysosomal distribution increases, vacuolated neurons show the same pattern of lysosomal dis- tribution as mec-4(d) animals (Fig. 6, A and B). Thus, reduced acidification does not affect lysosome distribution. Our ob- servations are consistent with findings in animals deficient for the ATP-binding cassette transporter P-glycoprotein-2, which is also expressed in neurons. Acridine orange and Lysotracker red staining is reduced in animals lacking P-glycoprotein 2, indicating defective acidification. Neverthe- less, LMP-1::GFP distribution remains unchanged (Nunes et al., 2005). We further examined lysosomes in the different mutant genetic backgrounds that either enhance or suppress necrosis. We generated transgenic animals harboring LMP-1::GFP in cup-5, glo-1, and the aspartyl protease– deficient cad-1 mutant background. The number of vacuo- lated neurons is decreased in glo-1 and cad-1 mutants and increased in cup-5 animals (Figs. 2 and 3). We find that lyso- somal morphology and distribution is similar in neurons that do vacuolate and die (Fig. 6, C–E). Discussion In this study, we have examined the involvement of lysosomes in necrotic cell death using a well characterized model of neurodegeneration in C. elegans. We show that both genetic and pharmacological manipulations that affect lysosomal biogene- sis and function modulate necrosis in the nematode. By follow- ing lysosome fate during neurodegeneration in vivo, we found that lysosomes fuse and localize exclusively around a swollen nucleus. In the advanced stages of cell death, GFP-labeled lyso- somal membranes fade, indicating lysosomal rupture. What is the cause of lysosomal rupture during necrosis? Interestingly, calcium, which is one of the major upstream death-initiating signals, has been implicated in this process (Zhao et al., 2005). Activated calcium-dependent calpain prote- ases have been found to localize to disrupted lysosomal mem- branes in hippocampal neurons of primates after acute ischemia (Yamashima, 2000, 2004), leading to the hypothesis that cal- pains compromise the integrity of lysosomal membranes and cause leakage of their acidic contents into the cytoplasm. Calpains become activated after the abrupt increase of intracel- lular calcium concentration that signals the initiation of necrosis (Syntichaki and Tavernarakis, 2002). Excessive calcium influx 236 JCB • VOLUME 173 • NUMBER 2 • 2006