Active neuronal death

Take away their supply of nerve growth factor (NGF), and neurons lose the will to live. In 1988, the conventional wisdom held that the cells wasted away because NGF helped keep metabolism churning. After all, NGF is a trophic factor, meaning that it literally “nourishes” the cell. But neuroscientist Eugene Johnson (Washington University, St. Louis) and colleagues suspected that the molecule instead prevented neurons from committing suicide—a controversial assertion that they confirmed with some simple experiments. The issue was important, Johnson says, because droves of surplus neurons perish during development, and uncovering the requirements for cell survival might clarify how this attrition helps wire the nervous system. The knowledge might also reveal measures for sparing neurons jeopardized by stroke and neurodegenerative diseases.

Johnson and colleagues reasoned that if NGF were essential for metabolism, shutting down protein or messenger RNA synthesis would speed the cells' demise. But if NGF stalled a cell-killing mechanism that required fresh mRNA and proteins, halting their production would delay death. To test their hypothesis, the researchers cultured neurons and then added antibodies that neutralized the NGF (Martin et al., 1988). After about a day of deprivation, the cells started to shrink and their membranes began to seethe. The medium showed a surge in adenylate kinase, a protein normally confined to the cytoplasm, demonstrating that the neurons were spilling their guts. But a dose of cycloheximide, a protein synthesis inhibitor, proved to be a balm for the neurons. “Cells without cycloheximide were toast, and cells with cycloheximide were beautiful,” recalls Johnson. Moreover, little adenylate cyclase leaked from treated cells, further evidence that they remained whole. Treating NGF-deprived cultures with actinomycin-D, an RNA synthesis blocker, also derailed cell death. The results “changed how people thought about what trophic factors do and how cells die,” says Johnson. Neurons weren't pining away; they were killing themselves.

Although other researchers had performed similar experiments, their results had been unclear, Johnson recalls. His group, however, made a fortunate choice. Other workers had studied neurons that required a constant supply of new proteins, and the cells died swiftly after translation or transcription halted. But the sympathetic neurons Johnson and colleagues examined could endure a four-day hiatus from protein making, possibly because they were post-mitotic. His group went on to map the sequence of events in a neuron's death, showing that the cell-slaying program eventually reaches a point of no return (Deckwerth and Martin, 1993). The invention of PCR allowed his lab to identify some of the genes that orchestrate neuron suicide (Estus et al., 1994).