Jan. 20, 2013 — A new finding by Harvard stem cell biologists turns one of the basics of neurobiology on its head — demonstrating that it is possible to turn one type of already differentiated neuron into another within the brain.
The discovery by Paola Arlotta and Caroline Rouaux “tells you that maybe the brain is not as immutable as we always thought, because at least during an early window of time one can reprogram the identity of one neuronal class into another,” said Arlotta, an Associate Professor in Harvard’s Department of Stem Cell and Regenerative Biology (SCRB).
The principle of direct lineage reprogramming of differentiated cells within the body was first proven by SCRB co-chair and Harvard Stem Cell Institute (HSCI) co-director Doug Melton and colleagues five years ago, when they reprogrammed exocrine pancreatic cells directly into insulin producing beta cells.
Arlotta and Rouaux now have proven that neurons too can change their mind. The work is being published on-line Jan. 20 by the journal Nature Cell Biology.
In their experiments, Arlotta targeted callosal projection neurons, which connect the two hemispheres of the brain, and turned them into neurons similar to corticospinal motor neurons, one of two populations of neurons destroyed in Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease. To achieve such reprogramming of neuronal identity, the researchers used a transcription factor called Fezf2, which long as been known for playing a central role in the development of corticospinal neurons in the embryo.
What makes the finding even more significant is that the work was done in the brains of living mice, rather than in collections of cells in laboratory dishes. The mice were young, so researchers still do not know if neuronal reprogramming will be possible in older laboratory animals — and humans. If it is possible, this has enormous implications for the treatment of neurodegenerative diseases.
“Neurodegenerative diseases typically effect a specific population of neurons, leaving many others untouched. For example, in ALS it is corticospinal motor neurons in the brain and motor neurons in the spinal cord, among the many neurons of the nervous system, that selectively die,” Arlotta said. “What if one could take neurons that are spared in a given disease and turn them directly into the neurons that die off? In ALS, if you could generate even a small percentage of corticospinal motor neurons, it would likely be sufficient to recover basic functioning,” she said.
The experiments that led to the new finding began five years ago, when “we wondered: in nature you never seen a neuron change identity; are we just not seeing it, or is this the reality? Can we take one type of neuron and turn it into another?” Arlotta and Rouaux asked themselves.
Over the course of the five years, the researchers analyzed “thousands and thousands of neurons, looking for many molecular markers as well as new connectivity that would indicate that reprogramming was occurring,” Arlotta said. “We could have had this two years ago, but while this was a conceptually very simple set of experiments, it was technically difficult. The work was meant to test important dogmas on the irreversible nature of neurons in vivo. We had to prove, without a shadow of a doubt, that this was happening.”
The work in Arlotta’s lab is focused on the cerebral cortex, but “it opens the door to reprogramming in other areas of the central nervous system,” she said.
Arlotta, an HSCI principal faculty member, is now working with colleague Takao Hensch, of Harvard’s Department of Molecular and Cellular Biology, to explicate the physiology of the reprogrammed neurons, and learn how they communicate within pre-existing neuronal networks.
“My hope is that this will facilitate work in a new field of neurobiology that explores the boundaries and power of neuronal reprogramming to re-engineer circuits relevant to disease,” said Paola Arlotta.
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- Caroline Rouaux, Paola Arlotta. Direct lineage reprogramming of post-mitotic callosal neurons into corticofugal neurons in vivo. Nature Cell Biology, 2013; DOI: 10.1038/ncb2660
ScienceDaily (Aug. 7, 2012) — Scientists from The Scripps Research Institute have identified a new stem cell population that may be responsible for giving birth to the neurons responsible for higher thinking. The finding also paves the way for scientists to produce these neurons in culture — a first step in developing better treatments for cognitive disorders, such as schizophrenia and autism, which result from disrupted connections among these brain cells.
Scientists from The Scripps Research Institute have identified a new stem cell population that may be responsible for giving birth to the neurons responsible for higher thinking. (Credit: © rolffimages / Fotolia)
Published in the August 10, 2012 issue of the journal Science, the new research reveals how neurons in the uppermost layers of the cerebral cortex form during embryonic brain development.
“The cerebral cortex is the seat of higher brain function, where information gets integrated and where we form memories and consciousness,” said the study’s senior author Ulrich Mueller, a professor and director of the Dorris Neuroscience Center at Scripps Research. “If we want to understand who we are, we need to understand this area where everything comes together and forms our impression of the world.”
In the new study, Mueller’s team identified a neural stem cell in mice that specifically gives rise to the neurons that make up the upper layers of the cerebral cortex. Previously, it was thought that all cortical neurons — those making up both the lower and upper layers — came from the same type of stem cell, called a radial glial cell, or RGC. A neuron’s fate was thought to be determined by the timing of its birth date. The Scripps Research team, however, showed that there is a distinct stem cell progenitor that gives rise to upper layer neurons, regardless of birth date or place.
“Advanced functions like consciousness, thought, and creativity require a lot of different neuronal cell types and a central question has been how all this diversity is produced in the cortex,” said Santos Franco, a senior research associate in Mueller’s laboratory and first author of the paper. “Our study shows this diversity already exists in the progenitor cells.”
Peeling Back the Onion Layers
In mammals, the cortex is made up of six distinct anatomic layers holding different types of excitatory neurons. They are not the uniform layers of a cake, but rather, they are more like the layers wrapped around an onion. The smaller lower layers, on the inside, host neurons that connect to the brain stem and spinal cord to help regulate essential functions such as breathing and movement. The larger upper layers, closer to the outer surface of the brain, contain neurons that integrate information coming in from the senses and connect across the two halves of the brain.
The upper layers are a “relatively young invention,” evolutionarily speaking, having been greatly expanded during primate evolution, said Mueller. They give humans in particular the unique abilities to think abstractly, plan for the future and problem-solve.
For the last two decades, scientists have believed that the fate of cerebral cortex neurons was determined by their birth date because each layer is formed in a time-dependent manner. The lower layer neurons form in the center of the “ball” first, and then the cells that will become the upper layers form last, migrating through the lower layers.
“So the model was that there is a stem cell in the center of the ball that generates the different types of neurons in successive waves,” said Mueller. “What we now show is that there are at least two different populations of RGCs and potentially more.”
Franco first created a line of mice in which he could track upper-layer neurons as they were born and migrated. The team followed a marker gene called Cux2, which was previously reported to be expressed only by upper-layer neurons. By linking a gene for an enzyme called Cre to the Cux2 gene, the scientists could watch any cell expressing Cux2 under the microscope, because the Cre enzyme flips on another gene that glows fluorescent red.
Surprisingly, the team observed Cux2 already turned on in some of the RGCs, even at the earliest points in brain development — embryonic day nine or ten — before any upper-layer neurons exist. Following this population of glowing stem cells through development, the team showed that the cells almost exclusively generated upper-layer neurons. In contrast, the subgroup of RGCs not expressing Cux2 became lower-layer neurons.
Next, the team removed these Cux2-positive precursor cells from their niche in the embryonic brain to see how they would develop in a lab dish. When they cultured both types of RGCs, again only Cux2-expressing RGCs developed into upper-layer neurons.
In developing brains, these Cux2-positive stem cells first self-renew and proliferate before differentiating later into neurons. So, the team wanted to know if a neuron’s birth date determined its fate. To test this, the researchers delivered a TCF4 molecule in utero that forced the Cux2-positive RGCs to prematurely differentiate. Even though it was too early in normal development, the Cux2-positive RGCs still produced upper-layer neurons.
In other words, regardless of position or timing, the Cux2-positive RGCs are destined to become upper-layer neurons. Mueller and colleagues concluded that these stem cells have some intrinsic property that determines their fate from the start.
The work also shows that this RGC subset is responsible for the huge proliferation of cells necessary to create the larger upper-layer cortex found in primate brains. “If we want to understand how the human brain evolved, how we are different from an amphibian, then this one precursor cell may have been important,” said Mueller.
But, bigger brains came with a risk, making humans more prone to disorders when upper-layer neurons don’t form connections properly. Up until now, researchers trying to reproduce human cortical neurons in the lab from stem cells have only generated lower-layer-type neurons. “This opens a door now to try to make the upper-layer neurons, which are frequently affected in psychiatric disorders,” said Mueller.
In addition to Mueller and Franco, authors of the paper, “Fate-restricted neural progenitors in the mammalian cerebral cortex,” were Cristina Gil-Sanz, Isabel Martinez-Garay, Ana Espinosa, Sarah R. Harkins-Perry, and Cynthia Ramos of Scripps Research. Martinez-Garay is now at the University of Oxford.
This research was supported by the Dorris Neuroscience Center, US National Institutes of Health (grant award numbers NS060355, NS046456, MH078833), and California Institute for Regenerative Medicine, and conducted in affiliation with the NIH Blueprint-funded Cre Driver Network.
- Santos J. Franco, Cristina Gil-Sanz, Isabel Martinez-Garay, Ana Espinosa, Sarah R. Harkins-Perry, Cynthia Ramos, and Ulrich Müller. Fate-Restricted Neural Progenitors in the Mammalian Cerebral Cortex. Science, 10 August 2012: 746-749 DOI: 10.1126/science.1223616
ScienceDaily (July 31, 2012) — Speech, sensory perception, thought formation, decision-making processes and movement are complex tasks that the brain only masters when individual nerve cells (neurons) are well connected. Berlin neuroscientists have now discovered a molecular switch that regulates this networking of nerve cells.
The scientists from Charité — Universitätsmedizin Berlin, the NeuroCure Cluster of Excellence and the Max Delbrück Center for Molecular Medicine (MDC) have published their work in the journal Genes and Development.
The dendritic tree, a highly branched structure of neurons, plays an important role in these brain functions. The dendrites act like antennae to receive signals from other cells and send them on to the nerve cell body. Congenital neurological conditions, like mental retardation, are often associated with errors in dendritic tree development.
Marta Rosário’s research team, in cooperation with Victor Tarabykin from Charité and Walter Birchmeier from MDC, has now discovered how this branching process is controlled during development. In living mice, it could be shown that the NOMA-GAP protein serves as a switch in this process. Maturing neurons produce this switch protein, which then starts a chain of signals in cells that leads to dendritic branching. A central element of this signal chain is a protein, called Cdc42. It plays an important role in the first developmental stages of neurons, but inhibits the branching of the dendritic tree in later developmental stages. When NOMA-GAP becomes active, it turns off Cdc42 allowing maturing neurons to form complex dendritic trees. The correct deployment of the switch protein and control of the signal chain regulated by Cdc42 are thus essential for the proper dendritic branching of neurons and thus for the development of the neocortex (the cerebral cortex) that steers sensory perception, memory, speech and movement, among other functions.
“Errors in this signal cascade lead to an incompletely developed dendritic tree. The result is a risk of mental limitations as signals in the brain cannot be adequately processed,” explains Marta Rosário. “For us the study forms an important foundation for researching various conditions, like mental retardation, schizophrenia or depression, that will hopefully point out new therapeutic avenues.”
- M. Rosario, S. Schuster, R. Juttner, S. Parthasarathy, V. Tarabykin, W. Birchmeier. Neocortical dendritic complexity is controlled during development by NOMA-GAP-dependent inhibition of Cdc42 and activation of cofilin. Genes & Development, 2012; DOI: 10.1101/gad.191593.112
ScienceDaily (July 19, 2012) — By decoding brain activity, scientists were able to “see” that two monkeys were planning to approach the same reaching task differently — even before they moved a muscle.
Anyone who has looked at the jagged recording of the electrical activity of a single neuron in the brain must have wondered how any useful information could be extracted from such a frazzled signal.
But over the past 30 years, researchers have discovered that clear information can be obtained by decoding the activity of large populations of neurons.
Now, scientists at Washington University in St. Louis, who were decoding brain activity while monkeys reached around an obstacle to touch a target, have come up with two remarkable results.
Their first result was one they had designed their experiment to achieve: they demonstrated that multiple parameters can be embedded in the firing rate of a single neuron and that certain types of parameters are encoded only if they are needed to solve the task at hand.
Their second result, however, was a complete surprise. They discovered that the population vectors could reveal different planning strategies, allowing the scientists, in effect, to read the monkeys’ minds.
By chance, the two monkeys chosen for the study had completely different cognitive styles. One, the scientists said, was a hyperactive type, who kept jumping the gun, and the other was a smooth operator, who waited for the entire setup to be revealed before planning his next move. The difference is clearly visible in their decoded brain activity.
The study was published in the July 19th advance online edition of the journal Science.
All in the task
The standard task for studying voluntary motor control is the “center-out task,” in which a monkey or other subject must move its hand from a central location to targets placed on a circle surrounding the starting position.
To plan the movement, says Daniel Moran, PhD, associate professor of biomedical engineering in the School of Engineering & Applied Science and of neurobiology in the School of Medicine at Washington University in St. Louis, the monkey needs three pieces of information: current hand and target position and the velocity vector that the hand will follow.
In other words, the monkey needs to know where his hand is, what direction it is headed and where he eventually wants it to go.
A variation of the center-out task with multiple starting positions allows the neural coding for position to be separated from the neural coding for velocity.
By themselves, however, the straight-path, unimpeded reaches in this task don’t let the neural coding for velocity to be distinguished from the neural coding for target position, because these two parameters are always correlated. The initial velocity of the hand and the target are always in the same direction.
To solve this problem and isolate target position from movement direction, doctoral student Thomas Pearce designed a novel obstacle-avoidance task to be done in addition to the center-out task.
Crucially, in one-third of the obstacle-avoidance trials, either no obstacle appeared or the obstacle didn’t block the monkey’s path. In either case, the monkey could move directly to the target once he got the “go” cue.
The population vector corresponding to target position showed up during the third hold of the novel task, but only if there was an obstacle. If an obstacle appeared and the monkey had to move its hand in a curved trajectory to reach the target, the population vector lengthened and pointed at the target. If no obstacle appeared and the monkey could move directly to the target, the population vector was insignificant.
In other words, the monkeys were encoding the position of the target only when it did not lie along a direct path from the starting position and they had to keep its position “in mind” as they initially moved in the “wrong” direction.
“It’s all,” Moran says, “in the design of the task.”
And then some magic happens
Pearce’s initial approach to analyzing the data from the experiment was the standard one of combining the data from the two monkeys to get a cleaner picture.
“It wasn’t working,” Pearce says, “and I was frustrated because I couldn’t figure out why the data looked so inconsistent. So I separated the data by monkey, and then I could see, wow, they’re very different. They’re approaching this task differently and that’s kind of cool.”
The difference between the monkey’s’ styles showed up during the second hold. At this point in the task, the target was visible, but the obstacle had not yet appeared.
The hyperactive monkey, called monkey H, couldn’t wait. His population vector during that hold showed that he was poised for a direct reach to the target. When the obstacle was then revealed, the population vector shortened and rotated to the direction he would need to move to avoid the obstacle.
The smooth operator, monkey G, in the meantime, idled through the second hold, waiting patiently for the obstacle to appear. Only when it was revealed did he begin to plan the direction he would move to avoid the obstacle.
Because he didn’t have to correct course, monkey G’s strategy was faster, so what advantage was it to monkey H to jump the gun? In the minority of trials where no obstacle appeared, monkey H approached the target more accurately than monkey G. Maybe monkey H is just cognitively adapted to a Whac-A-Mole world. And monkey G, when caught without a plan, was at a disadvantage.
Working with the monkeys, the scientists had been aware that they had very different personalities, but they had no idea this difference would show up in their neural recordings.
“That’s what makes this really interesting,” Moran says.
- Thomas M. Pearce and Daniel W. Moran. Strategy-Dependent Encoding of Planned Arm Movements in the Dorsal Premotor Cortex. Science, 2012; DOI: 10.1126/science.1220642
Washington University in St. Louis (2012, July 19). Scientists read monkeys’ inner thoughts: Brain activity decoded while monkeys avoid obstacle to touch target. ScienceDaily. Retrieved July 21, 2012, from http://www.sciencedaily.com /releases/2012/07/120719141804.htm
ScienceDaily (July 16, 2012) — Children quickly learn to avoid negative situations and seek positive ones. But humans are not the only species capable of remembering positive and negative events; even the small brain of a fruit fly has this capacity. Dopamine-containing nerve cells connected with the mushroom body of the fly brain play a role here. Scientists from the Max Planck Institute of Neurobiology in Martinsried have identified four different types of such nerve cells. Three of the nerve cell types assume various functions in mediating negative stimuli, while the fourth enables the fly to form positive memories.
From earliest childhood we learn to avoid things that harm us and seek positive experiences instead. Aversive memory is created by experiences like pricking our finger on a rose thorn, which we remember for a long time. Conversely, the smell of fresh food is positively associated with a feeling of satiety and creates a reward memory.
Hiromu Tanimoto and his colleagues at the Max Planck Institute of Neurobiology recently localised and identified the most important types of nerve cells involved in forming positive and negative memories of a fruit fly. All four nerve cell types they discovered use dopamine to communicate with other nerve cells. The dopamine signals released by these cells are received in the mushroom body, a prominent brain structure in insect brains. “It is really surprising that similar dopamine-releasing nerve cells can play such different roles,” says Tanimoto.
The scientists investigate the functions of the individual nerve cell types in two separate studies. In the process of learning avoidance strategies, the flies were presented with an odour that was associated with a negative stimulus, a foot shock. As a result, the flies learned to avoid this odour in future.
In the next round of experiments, the scientists replaced the shock with artificial activation of defined sets of nerve cells during an odour presentation. They discovered that the transient activation of these nerve cells alone is sufficient to signal aversive stimulus in the fly brain and lead to the formation of an aversive odour memory — even when no real aversive stimulus is present.
The scientists were also able to demonstrate that the three types of nerves cells that are responsible for the memory of punishment fulfil different functions. A major difference here is the stability of the induced memories. One of the cell types is responsible for the long-lasting memory, while memories formed by other dopamine cells are short-lived. “Punishing events induce aversive memories with different stabilities by combining distinct dopamine cells in the fly brain” explains Tanimoto.
The same method was employed to demonstrate that another set of dopamine cells signals reward to form positive odour memory. When these cells were artificially activated, the flies remembered the smell and tried to get to the source of the odour even in the absence of sugar reward. The scientists proved that specific dopamine neurons also play a key role in this process.
The messenger substance dopamine is not only significant for fruit flies and other insects. Particularly, it is also needed for reward-based learning in humans. These new discoveries suggest that functional diversity of dopamine is a highly conserved mechanism in brains.
1. Chang Liu, Pierre-Yves Plaçais, Nobuhiro Yamagata, Barret D. Pfeiffer, Yoshinori Aso, Anja B. Friedrich, Igor Siwanowicz, Gerald M. Rubin, Thomas Preat, Hiromu Tanimoto. A subset of dopamine neurons signals reward for odour memory in Drosophila. Nature, 2012; DOI: 10.1038/nature11304
2. Yoshinori Aso, Andrea Herb, Maite Ogueta, Igor Siwanowicz, Thomas Templier, Anja B. Friedrich, Kei Ito, Henrike Scholz, Hiromu Tanimoto. Three Dopamine Pathways Induce Aversive Odor Memories with Different Stability. PLoS Genetics, 2012; 8 (7): e1002768 DOI: 10.1371/journal.pgen.1002768
Max-Planck-Gesellschaft (2012, July 16). Dopamine: A substance with many messages. ScienceDaily. Retrieved July 19, 2012, from http://www.sciencedaily.com /releases/2012/07/120718131355.htm