Small bits of RNA called microRNAs, which can influence the way a cell’s genes are turned on and off, can single-handedly cause a connective tissue cell from human skin to transform into a nerve cell, new research shows.
The study was conducted by Howard Hughes Medical Institute (HHMI) scientists who first showed two years ago that microRNAs help immature neural stem cells develop into mature neurons. The new work demonstrates that in developing neurons, microRNAs aren’t just handmaidens to change; they’re actually running the show.
“In all the ways we could test them, they were normally functioning neurons.”
Gerald R. Crabtree
“This is the first time microRNAs have been shown to have this instructive role in mammalian or human cells,” said HHMI investigator Gerald R. Crabtree of Stanford University, whose work uncovering this premier role for two particular microRNAs was published July 13, 2011, in the journal Nature. “MicroRNAs were thought not to be capable of accomplishing fate transformation,” Crabtree said.
The discovery introduces a new method of converting adult cells of one type into cells of another, without first dialing them back to the embryonic stage – which was previously the only way to change the identity of a fully differentiated cell. Although the late Hal Weintraub, who was an HHMI investigator at the Fred Hutchinson Cancer Research Center, showed in the early 1990s that fibroblasts -- connective tissue cells that secrete collagen and other molecules -- could be converted to muscle cells, this trick is new for neurons.
The ability to change a cell’s identity offers promise for one day replacing damaged neurons in patients who have a neurodegenerative disease, such as Parkinson’s disease. More immediately, the ability to create neurons from adult cells could lead to new experimental models for diseases in which neuronal function is impaired, such as Down syndrome and Alzheimer’s, Crabtree said.
“We’ve had to contend with mouse models of those diseases, which have been very helpful, but inadequate,” he said. “Those are diseases that affect cognition and intellectual characteristics. It’s quite clear the degree of learning impairment one sees in humans would probably never be noticed in a mouse. For example, Alzheimer’s disease affects learning and memory -- and mice are not nearly as good at these tasks as humans.”
Researchers would like to be able to study in the lab the basic processes that drive human nerve cell function in both healthy individuals and in those with neurodegenerative diseases. But it’s nearly impossible to harvest neurons from living humans. “Now we’ll be able to take a fibroblast cell from someone with Down syndrome and turn it into a neuron,” he said. Because neurons created in this way would carry the patient’s own DNA, they are expected to behave similarly to the patient’s neurons and be a powerful model for studying that individual’s disease.
Crabtree pointed out that he and his colleagues were able to generate the specific type of neuron that is often damaged in many neurodegenerative diseases – those that are found in the frontal cortex region of the brain. “These are the [neurons] that have appeared most recently in human evolution, and are the neurons that team up with other neurons to carry out complex associative and synthetic thought,” he explained. “That is the region most compromised in Down syndrome, Alzheimer’s and many other human neurologic diseases. Mice have few of these neurons, making mice poor models for many human neurologic diseases.”
Crabtree’s lab has long been involved in tracing the pathway neuronal stem cells take to adult differentiation. Neuronal stem cells are constantly dividing cells that can not only reproduce exact copies of themselves, but – when they receive the right signals -- can develop into a variety of fully differentiated cells that play distinct roles in the central nervous system.
In 2007, Crabtree’s lab revealed a clue about the signals that drive neuronal stem cells’ development into specialized nerve cells. His team focused on a soccer ball-like complex of interchangeable proteins called a chromatin regulatory complex. Chromatin regulatory complexes are the master packers and unpackers of a cell’s long ribbon of DNA.
In the nucleus of every human cell, there is a little over two yards of DNA, Crabtree explained. If it were untangled and expanded proportionally so that it was the width of spaghetti, that DNA would stretch from San Francisco to Los Angeles. “Imagine packing all that into a suitcase and having to unpack it and find exactly the right spot in a few minutes,” he said. The chromatin assembly complex is in charge of packing yards of DNA into a space about 1/100,000 of an inch. The complexes also play a critical role in unpacking, revealing specific sections of the genetic material to the machinery of gene expression for transcription into RNA.
Crabtree’s team found that a change in the structure of the chromatin assembly complex coincided with the moment the neuronal stem cells became mature neurons, and identified two specific microRNAs that directed that change: miR-9* and miR-124.
Crabtree said he realized the potency of microRNA not long after he and Andrew S. Yoo, a postdoctoral researcher in his lab, made that discovery. To test the broader effects of put miR-9* and miR-124, Yoo put into a plate of human fibroblast cells and they learned that microRNA wasn’t just a member of the band in differentiation. It waved the baton.
“Andrew came to me and asked me to look through the microscope. I saw cells that, to my eye, looked indistinguishable from neurons. He told me, ‘Those cells have just gotten the microRNA and nothing else, and they were fibroblast cultures.’
“That was our 'Eureka!' moment,” Crabtree said. “At that point we realized there was, to at least some degree, an instructive role for these microRNAs. They were more than just required for the formation of neurons, they were really instructing the formation of neurons.”
He left the lab a very happy – but cautious -- man. “They look just too good to be true,” he remembers thinking. The fibroblast-derived neurons looked like the real thing, but did they behave like them as well? The lab quickly embarked on further experiments to find out.
Crabtree tested the cells for a score of proteins that neurons are known to express. All were present. They checked to see if the new neurons formed synapses with other neurons. They did. They tested the cells’ ability to propagate electrical nerve signals. The new neurons behaved exactly like native neurons. “In all the ways we could test them, they were normally functioning neurons,” Crabtree said.
Although the microRNAs all by themselves are able to turn fibroblasts into neurons, only a few percent of the cells on a laboratory plate make the transition, Crabtree said. He was able to boost the purity of the final population to well over 50 percent with a few tweaks, such as adding regulatory proteins that activate certain neuron-specific genes. With that success, Crabtree’s team is particularly optimistic that their conversion process will be valuable in allowing researchers to create the models they need to study nerve function and disease.