Researchers assumed that cells shut down like lights blinking off one by one. Then they looked closer. (Illustration by Calum Heath)

Death Is Weirder Than You Think

Why do some genes get more active when life ends?

Before we become ourselves, before we even have a brain, our cells are active: cleaving, dividing, differentiating, making the building blocks that will eventually add up to a conscious, cognizant organism.

Our cells precede us—but they also outlive us. Long after it’s lights-out for you, individual cells don’t give up the ghost. In fact, some cells can survive for days and others for weeks in a dead body.

What exactly is going on in those cells that rage against the dying of the light? Until recently, no one bothered to look very closely. But now an evocative line of research is blowing apart some assumptions about what death is.

Not only do cells survive for a while after an organism dies, they may actually fight to live. The activity of some genes increases after death, as cells apparently sense that something has gone horribly wrong. It’s like an astronaut in deep space who suddenly gets silence on the radio and frantically beams signals home to Earth, unaware that a nuclear holocaust has wiped out everything she holds dear.

The researchers uncovering the details of this post-mortem genetic activity think it could have consequences for organ transplantation, genetic research, and forensic science. But first they have to convince their fellow researchers that cellular life after death is even worth studying.

“You cannot get an NIH grant for this,” says one of the researchers, Alexander Pozhitkov, a chemist and geneticist at the City of Hope’s cancer research center in California. “This is too weird, right?”

Yeah, it is

The whole thing got started in about 2009, when Pozhitkov was a postdoctoral researcher at the Max Planck Institute for Evolutionary Biology in Germany. It was there that he got a chance to pursue a project he’d been thinking about for more than a decade.

Pozhitkov acquired about 30 zebrafish from the institute’s colony. (These tropical fish are commonly used in research because, among other things, they have transparent embryos, ideal for observing development.) He killed the animals by shocking them with a quick immersion in a cooler of ice water, then put them back in their regular 82-degree Fahrenheit tank.

Over the course of the next four days, he periodically scooped a few fish out of the tank, froze them in liquid nitrogen, and then analyzed their messenger RNA. These are threadlike molecules that do the work of translating DNA into proteins; each strand of messenger RNA is a transcript of some section of DNA. Later Pozhitkov and his colleagues repeated the same process with mice, although their death was meted out by broken neck rather than cold shock.

When Pozhitkov’s colleague Peter Noble, then a biochemist at the University of Washington, dug into the data on how active the messenger RNA was on each day after death, something amazed him. In both the fish and the mice, the translation of genes into proteins generally declined after death, as would be expected. But the count of messenger RNA indicated that about 1 percent of genes actually increased in transcription after death. Some were chugging along four days after life ceased.

It wasn’t that the researchers had expected a total cessation of activity the moment the zebrafish and mice shuffled off this mortal coil. But to detect increases in transcription rather than just the blinking off of the lights one by one? That was “the most bizarre thing I’ve ever seen,” Noble says.

Not everyone was impressed. Noble and Pozhitkov heard a lot of criticism after the story made the rounds, first on the preprint site bioRxiv in 2016 and then in a paper in Open Biology in 2017. The main critique was that they might have misinterpreted a statistical blip. Because cells die off at different rates, perhaps the transcripts recorded in still-living cells merely made up a greater proportion of all the total transcripts, says Peter Ellis, a lecturer in molecular biology at the University of Kent. Think of the transcripts as socks in a drawer, he says. If you lost some of the red ones, the remaining white socks would make up a larger percentage of your total sock collection, but you wouldn’t have acquired more of them.

“The most bizarre thing I’ve ever seen.”

Since that original publication, though, there are hints that something more is going on in the cells that are still churning after the organism dies. In a study published in February in Nature Communications, other researchers examined human tissue samples and found hundreds of genes that alter their expression after death. Some genes declined in activity, but others increased. A gene that promotes growth, EGR3, began ramping up its expression four hours after death. Some fluctuated back and forth, like the gene CXCL2, which codes for a signaling protein that calls white blood cells to the site of inflammation or infection.

These changes weren’t merely the passive result of transcripts degrading at different rates like red socks being sporadically lost, says the University of Porto’s Pedro Ferreira, who led the study. Something, he says, was going on that actively regulated gene expression “even after the death of the organism.”

Final distress calls

When an organism dies, the most important, energy-intensive cells follow first. Goodbye, neurons. But more peripheral cells keep doing their jobs for days or even weeks, depending on factors like temperature and decay. In one 2015 study, researchers were able to coax live cell cultures from goat ears a whopping 41 days after the goats were slaughtered. They got these cells from fibroblasts, which make up connective tissue and are relatively low-energy. Keeping them alive for 41 days required nothing more than normal refrigeration. “Organismal death has no meaning at the cellular level,” Ellis says.

But death does rock the cells’ world by cutting off oxygen and nutrients, at the least. So what is driving posthumous gene expression? That’s an open question, but a new paper by Noble and Pozhitkov might contain clues.

The research, now on bioRxiv, hasn’t yet been peer reviewed, but using the original zebrafish and mouse data, Noble found that the messenger RNA active after death isn’t like the rest of the messenger RNA in cells. About 99 percent of the RNA transcripts floating around in cells degrade rapidly when the organism dies, Noble says. The remaining 1 percent have something special: certain patterns of individual nucleotides that bind to molecules that regulate messenger RNA after transcription. This appears to be a large part of what keeps things going after death.

Pozhitkov and Noble argue that this mechanism could be part of how cells react to a situation that the organism theoretically could come back from, like a near-drowning. Cells might essentially try to “open all the valves” in their death throes, he says, allowing certain stress-related genes, such as ones that respond to inflammation, to express themselves.

It’s like an astronaut in deep space who gets silence on the radio and frantically beams signals home, unaware that a nuclear holocaust has wiped Earth out.

For Ellis, this kind of finding is a side effect of processes that might be interesting during life but are a mere simulacrum in death. Yet Ferreira sees practical implications. Some genetic research is done on tissue samples that have been removed from a body. It’s important to know how transcription changes after death so the results don’t get skewed by cells’ final distress calls.

Ferreira and his team were also able to pinpoint the time of death of an individual based solely on the postmortem changes in the gene expression. In theory that could be handy in, say, a murder investigation. But Ferreira’s group had the advantage of knowing that their tissue samples were taken and stored using state-of-the-art methods from donors without certain medical conditions. In real life, factors ranging from the pre-existing conditions of the corpse to the temperature of the surrounding environment to the time lapsed before sampling could all affect the RNA timeline, Ferreira says. In other words, the technique is far from ready for the justice system.

Noble and Pozhitkov have other practical considerations in mind. Organs taken from donors for transplantation spend at least some time outside the body, and their RNA may start sending out the same sort of distress signals seen in death. It’s possible that this could have long-term health consequences for the transplant recipient, Pozhitkov says. After all, transplant recipients have a higher rate of cancer than the general population. Perhaps it’s not, as commonly believed, primarily from the immune-suppressing drugs they take, but rather because of “the postmortem process in the organ,” he says.

This is all speculative, though transplant researchers are exploring whether to keep organs warm on life support instead of chilled in coolers to improve transplant outcomes. It’s not clear the extent to which RNA transcription explains any of the benefits of warm transplants.

For now, however, research on the cellular afterlife is itself on life support. Noble is looking for a new academic appointment after leaving his last job at the University of Alabama. Pozhitkov’s funding at the City of Hope center is for unrelated projects. Still, both are firm that their findings shouldn’t be relegated to the bin of quirky science. Both want to revive the work.

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