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. 2012 Feb 1;31(3):654-66.
doi: 10.1038/emboj.2011.447. Epub 2011 Dec 9.

Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans

Kostoula Troulinaki  1 , Nektarios Tavernarakis
Affiliations

Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans

Kostoula Troulinaki et al. EMBO J. .

Abstract

Unlike apoptosis, necrotic cell death is characterized by marked loss of plasma membrane integrity. Leakage of cytoplasmic material to the extracellular space contributes to cell demise, and is the cause of acute inflammatory responses, which typically accompany necrosis. The mechanisms underlying plasma membrane damage during necrotic cell death are not well understood. We report that endocytosis is critically required for the execution of necrosis. Depletion of the key endocytic machinery components dynamin, synaptotagmin and endophilin suppresses necrotic neurodegeneration induced by diverse genetic and environmental insults in C. elegans. We used genetically encoded fluorescent markers to monitor the formation and fate of specific types of endosomes during cell death in vivo. Strikingly, we find that the number of early and recycling endosomes increases sharply and transiently upon initiation of necrosis. Endosomes subsequently coalesce around the nucleus and disintegrate during the final stage of necrosis. Interfering with kinesin-mediated endosome trafficking impedes cell death. Endocytosis synergizes with autophagy and lysosomal proteolytic mechanisms to facilitate necrotic neurodegeneration. These findings demonstrate a prominent role for endocytosis in cellular destruction during neurodegeneration, which is likely conserved in metazoans.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Clathrin-mediated endocytosis is required for necrotic cell death in C. elegans. (A) Number of cell corpses at the L1 stage of development per 100 animals carrying the neurotoxic mec-4(d) or deg-3(d) alleles in SNT-1-deficient mutants. For mec-4(d) mutant animals, bars denote touch receptor neuron corpses. For deg-3(d) mutants, bars denote inner labial neuron 1 (L1) sensory neuron and PVC interneuron corpses. (B) Expression of a full-length MEC-4::GFP reporter fusion under the control of the mec-4 promoter in touch receptor neurons of wild-type, snt-1 and unc-57 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P<0.05 compared with wild type; t-test). (C) Percentage of SNT-1-deficient animals that survive near-lethal treatment with sodium azide. This chemical inhibits the activity of the respiratory electron transport complex IV (cytochrome C oxidase) and simulates hypoxia. (D) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec-4(d) or deg-3(d) alleles in UNC-57-deficient animals. (E) Percentage of UNC-57-deficient animals that survive after the hypoxia-inducing treatment with sodium azide. (F) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec-4(d) or deg-3(d) alleles together with lesions in genes encoding key proteins that participate in all four steps of clathrin-mediated endocytosis. UNC-11 and DPY-23 are adaptor proteins required for the formation of clathrin-coated pits, DYN-1 is a GTPase necessary for fission of clathrin-coated vesicles and UNC-26 participates in the uncoating of vesicles. Error bars denote s.e.m. values (n>250 for all populations examined; P<0.001, compared with wild-type animals, unpaired t-test).
Figure 2
Figure 2
Endosome formation is induced early during necrosis. (A) Confocal images of touch receptor neurons expressing a pmec-17APS-2::GFP reporter transgene. During early necrosis, the number of APS-2::GFP puncta, corresponding to clathrin-coated pits and vesicles, in touch receptor neurons of mec-4(d) animals does not change significantly, whereas at later stages it decreases. A similar decrease is also observed in unc-57(e1190) mutants, defective for clathrin-mediated endocytosis. (B) Confocal images of touch receptor neurons expressing a pmec-7GFP::RAB-5 reporter transgene. The number of fluorescent puncta that correspond to early endosomes in touch receptor neurons of mec-4(d) animals significantly increases during early necrosis, whereas it declines later. (C) Confocal images of touch receptor neurons expressing a pmec-7GFP::RAB-11 reporter transgene. The number of GFP::RAB-11 puncta that correspond to recycling endosomes in touch receptor neurons of mec-4(d) animals increases early during cell death, while it declines as degeneration proceeds. Both early and recycling endosomes tend to accumulate around a swollen structure that is probably the nucleus. Bars denote 4 μm. Error bars denote s.e.m. values (n>250 for all populations examined; P<0.001, compared with control animals, unpaired t-test).
Figure 3
Figure 3
Endocytosis functions together with lysosomal proteolysis to facilitate necrotic cell death. (A) Depletion of synaptotagmin (SNT-1) or endophilin (UNC-57) does not further suppress MEC-4(d)-induced necrosis in cad-1 mutants with reduced cathepsin activity or with V-ATPase dysfunction (B, C). Similarly, no synthetic reduction of necrotic cell death is observed in calpain-deficient animals that also carry lesions in the synaptotagmin (D) or endophilin (E) genes. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t-test).
Figure 4
Figure 4
Endocytosis synergizes with autophagy to mediate necrotic cell death. (A) Synaptotagmin (SNT-1) deficiency further suppresses MEC-4(d)-induced necrosis in animals with impaired autophagy. (B) Endophilin dysfunction enhances suppression of necrosis in mec-4(d);lgg-1(RNAi) animals, where autophagy is impaired. Error bars denote s.e.m. values (n>250 for all populations examined; P<0.01, compared with single mutant control animals, unpaired t-test).
Figure 5
Figure 5
Intracellular trafficking by kinesins UNC-116 and UNC-104 is required for necrotic cell death. (A) Depletion of the kinesin 1 heavy chain UNC-116 suppresses necrosis induced by both mec-4(d) and deg-3(d) toxic alleles. (B) Expression of a full-length MEC-4::GFP reporter fusion under the control of the mec-4 promoter in touch receptor neurons of wild-type, unc-116 and unc-104 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P<0.05 compared with wild type; t-test). (C) Percentage of unc-116 animals that survive after treatment with sodium azide. (D) Depletion of the monomeric kinesin UNC-104 protects neurons against MEC-4(d)-induced degeneration. (E) unc-104 mutant animals are more resistant to hypoxic death induced by sodium azide. (F) Combined inactivation of endocytosis and kinesin-mediated intracellular trafficking does not enhance protection of touch receptor neurons against MEC-4(d)-induced degeneration. Error bars denote s.e.m. values (n>250 for all populations examined; P<0.001, compared with control animals, unpaired t-test).
Figure 6
Figure 6
Genetic interaction between kinesin-mediated intracellular trafficking and cellular proteolytic pathways mediating necrotic cell death. Dysfunction of either kinesin 1 heavy chain (UNC-116) or the monomeric kinesin UNC-104 does not significantly enhance suppression of neurodegeneration in aspartyl protease-deficient mutant animals (A, B), in animals with compromised lysosomal acidification (C, D), or in calpain protease-deficient mutants animals (E, F). Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t-test).
Figure 7
Figure 7
Kinesin-mediated intracellular trafficking functions together with autophagy to facilitate necrotic cell death. Number of neuron corpses, at the L1 larval stage of development, per 100 animals carrying the neurotoxic mec-4(d) allele in genetic backgrounds deficient for both intracellular trafficking and autophagy. (A) Kinesin 1 heavy chain (UNC-116) deficiency. (B) Monomeric kinesin UNC-104 deficiency. In both cases, no significant synthetic protection of neurons is observed in LGG-1/LC3-depleted animals with impaired autophagy. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t-test).
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