Jeffery Molkentin says that death comes for cells in three main guises. Because our bodies are all cells (or things made by cells), we should pay close attention. Cells can eat themselves, says Molkentin. That's autophagy. Cells can organize their own suicide. That's apoptosis. Cells can puff up and explode. That's necrosis, the most common, the most dangerous, and, surprisingly, the least understood pathway to cell death.
Most biologists consider necrosis to be the cellular equivalent of a five-alarm fire in a fireworks factory—that is, violent, dangerous, and unpredictable. In contrast, the well-studied apoptotic pathway is an orderly evacuation, with the suicidal cell carefully packing up its organelles and waste products for tidy disposal.
Yet Molkentin believes that necrosis is anything but chaotic. He is building the case that necrosis is a highly regulated process, just like apoptosis. Molkentin argues that if scientists understood more about the molecular chain of events that signals the beginning of necrosis, they could manipulate its onset or blunt its impact. They could also, he notes, make real progress against an array of progressive cell-killing diseases—from congestive heart failure to muscular dystrophy to Parkinson's.
Molkentin stumbled on the starting point for orderly necrosis in a protein that controls permeability in mitochondria, the cell's energy-generating power plants. He used a time-honored scientific technique: he ran a carefully prepared experiment that produced "weird results." At the time, Molkentin was studying calcineurin, a calcium-signaling protein that plays a key role in cardiac hypertrophy, the enlargement of individual heart muscle cells (myocytes) that is a classic response of an injured or failing heart. Helping to trace the calcineurin connection to cardiac hypertrophy was one of Molkentin's early triumphs as a postdoctoral fellow working under Eric Olson at the University of Texas Southwestern Medical Center at Dallas.
When he started his own lab at Cincinnati Children's Hospital Medical Center, Molkentin wanted to probe calcineurin signaling further by blocking it with cyclosporin, the famous immunosuppressive drug that made organ transplantation a practical reality. Cyclosporin was also known to inhibit calcineurin, and Molkentin was curious to see how mice with cardiac hypertrophy fared under stress when their calcineurin signaling was blocked.
The results of those experiments left him baffled. The cyclosporin-treated cells survived in good order, especially their mitochondria, which typically swell up under stress. They sailed through, unruffled. Whatever protection the mitochondria were getting was not coming from the blocked calcineurin, which acts in the cell nucleus. Scrambling to identify the unknown protein inhibited by cyclosporin, Molkentin created a series of transgenic mouse tissue cultures in which key proteins in muscle cells were knocked out. Even mouse tissue completely null for calcineurin was protected against cell death by cyclosporin. Molkentin worked patiently through his other knockouts, narrowing it down to a mitochondrial protein, cyclophilin-D, which is associated with the mitochondrial permeability transition (MPT) pore, the organelle's safety valve.
Molkentin, however, saw cyclophilin-D as something more than a vulnerable protein. He recognized it as the first falling domino in an ordered sequence leading to necrosis. Under sufficient stress, cyclophilin-D somehow opens the MPT pore, allowing water to flood into the mitochondrial interior and flush out the solutes that drive the generation of energy in the form of ATP (adenosine triphosphate). As the mitochondria lose ATP power, the MPT pores open wider, causing their intricately folded membranes and matrix layers to swell and finally rupture. In Molkentin's view, necrosis is a sort of rolling power blackout, crashing one mitochondrial power plant after another, leaving the rest of the cellular machinery powerless to resist breakup and death. But when cyclosporin blocked cyclophilin-D, none of this catastrophe happened because the MPT pores stayed shut.
Molkentin's transgenic mice engineered to be totally null for cyclophilin-D were even more impervious to cell death. Brain and heart cells without cyclophilin-D shrugged off the laboratory-induced equivalent of an ischemic heart attack. The cyclophilin-D–null mice were not, however, super mice. They remained vulnerable to other cell-death signals associated with apoptosis. Clearly cell death could run on more than one track.
In 2005, Molkentin published the results of his cyclophilin-D–null mice experiments in Nature. "That's when people started to get excited," Molkentin remembers, "because here was this whole other scientific vantage point from which to go after almost any disease where progressive cell death is an issue." That includes heart failure and stroke but also wasting and degenerative diseases like muscular dystrophy, Alzheimer's, or multiple sclerosis. Molkentin says that research trials are in progress on a number of these disorders, evaluating the veteran drug cyclosporine (and its analogs) in its new role of mitochondrial protector.
But for Molkentin, the most significant discoveries will come from the careful elucidation of the complete necrosis pathway. He says that being named an HHMI investigator will give him the long-term, flexible resources necessary for an open-ended, three-track proteomic and genomic screening to identify all the upstream and downstream proteins associated with cyclophilin-D in controlling necrosis.
"We couldn't propose something this risky to the NIH," says Molkentin. "I mean, a lot of what we want to do is screening-based, and one of the risks of screening is that you may not find anything interesting." Molkentin will also reexamine the structure of the MPT pore. The old model for its molecular architecture is clearly wrong, he says, so the precise role of cyclophilin-D at the MPT pore is yet another mystery for his HHMI lab.