The role of SM-20 in neuronal cell death

The withdrawal of nerve growth factor (NGF) from sympathetic neurons results in cell death. Since the death of these neurons is blocked by inhibitors of RNA and protein synthesis, it is hypothesized that genes specifically expressed following NGF withdrawal are likely to be important in the apoptotic pathway. Using differential display, a gene called SM-20 was found to be preferentially expressed in NGF-deprived versus NGF-maintained sympathetic neurons. SM-20 expression is also elevated in NGF-maintained sympathetic neurons during apoptosis induced by two differently acting pharmacological agents indicating that the increase in SM-20 mRNA is associated with the death of these cells in response to various stimuli. In addition, synthesis of SM-20 protein increases after NGF withdrawal prior to the onset of cell death. Importantly, expression of SM-20 in sympathetic neurons and in a tetracycline-inducible PC12 cell line can promote cell death in the presence of survival factors. SM-20 protein is localized to mitochondria, an organelle with a prominent role in apoptosis. In dual-immunofluorescence experiments, SM-20 co-localizes with cytochrome oxidase I and mitochondria-selective dyes in nonneuronal and neuronal cells. Consistent with its mitochondrial localization, the first 25 amino acids of SM-20 are sufficient to target a normally cytosolic protein to mitochondria demonstrating that SM-20 contains an amino-terminal mitochondrial targeting sequence. In cell fractionation experiments, a 33-kD processed form of SM-20 was present predominately in the mitochondria-enriched fraction suggesting that the mitochondrial targeting sequence of SM-20 is enzymatically removed following import into mitochondria. A form of SM-20 lacking 59 amino-terminal residues (SM-20(60–355)) is not targeted to mitochondria but instead localizes uniformly throughout the cytosol and nucleus. Surprisingly, this truncated form of SM-20 still elicits cell death in the presence of NGF indicating that the death promoting activity of SM-20 may be independent of its mitochondrial localization. SM-20 induced neuronal death can be inhibited by a general caspase inhibitor. Furthermore, expression of SM-20 or SM-20 (60–355) in sympathetic neurons leads to caspase-3 activation but does not promote cytochrome c release from mitochondria. Taken together, these data identify SM-20 as a novel mitochondrial protein that can cause caspase-dependent cell death in NGF-dependent sympathetic neurons.