| Abstract |
Necrotic cell death underlies extensive neuronal loss in acute degenerative episodes,
such as ischemic stroke, and in devastating human pathologies, like neurodegenerative
diseases (Martin, 2001; Syntichaki and Tavernarakis, 2002). In the nematode Caenorhabditis
elegans, hyperactivation of specific ion channels or hypoxia evoke an analogous pattern of
neuronal death (Driscoll and Chalfie, 1991; Scott et al., 2002). Investigations in both
nematodes and mammals implicate specific calpains and aspartyl proteases in the execution
of necrotic cell death (Syntichaki and Tavernarakis, 2002; Yoshida et al., 2002). Aspartyl
proteases achieve full activity under acidic conditions (Goll et al., 2003; Ishidoh and
Kominami, 2002). However, the factors that induce aberrant activation of these otherwise
benign enzymes during necrosis have been largely unknown.
In this thesis we show that the function of the vacuolar H+-ATPase, a pump that
acidifies endosomal and lysosomal compartments, is essential for the execution of necrotic
cell death in the nematode Caenorhabditis elegans. Indeed, impairment of the vacuolar H+-
ATPase function or alkalization of normally acidic intracellular organelles by weak bases
protected against necrosis. The use of pH-sensitive green fluorescent protein (GFP)-
molecules revealed reduction of cytoplasmic pH in dying cells. Intracellular acidification
required the vacuolar H+-ATPase function. Suppression of necrosis by aspartyl protease but
not calpain deficiency was further enhanced by conditions that perturbed intracellular
acidification. Thus, intracellular pH is an important modulator of necrosis in C. elegans. We
propose that the vacuolar H+-ATPase activity is required to establish necrosis-promoting
acidic intracellular conditions that augment the function of executioner aspartyl proteases in
degenerating cells.
However, the origin of cytoplasmic acidification remained obscure. Mutations in
genes that affect lysosomal biogenesis and function had a significant impact on necrotic death
levels. By using a genetically encoded fluorescent marker to monitor lysosomal fate during
necrosis in vivo, we found that lysosomes fuse and localize around a swollen nucleus.
However, in advanced stages of cell death GFP–labeled lysosomal membranes faded,
indicating lysosomal rupture. In conjunction with the effect of endocytic oganelle alkalization
on necrotic death, the sum of our data suggests that lysosomes have a prominent role in
cellular destruction during necrosis and facilitate pH reduction by releasing their acidic
content. Similar mechanisms may contribute to necrotic cell death that follows extreme
acidosis—for example, during stroke—in humans.
The lysosomal system participates via the autophagic process to the degradation of
intracellular proteins and organelles. We checked the involvement of autophagy in necrosis
and found that it is required for necrotic death in C. elegans. Impairment of autophagy by
genetic inactivation of autophagy genes or by pharmacological treatment suppressed necrosis.
Excessive autophagosome formation was induced early during neurodegeneration. At more
pronounced stages autophagosomes accumulated in the nucleus periphery, where they
exhibited partial or complete colocalization with lysosomes and mitochondria. Autophagy
induction was preceded by calpain activation and synergized with lysosomal catabolic
mechanisms to facilitate cell death. These findings demonstrate that autophagy contributes to
cellular destruction during necrosis. Thus, interfering with the autophagic process may protect
neurons against necrotic damage in humans.
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Διερεύνηση των μηχανισμών νεκρωτικού θανάτου στο νηματώδη Caenorhabditis elegans
