Reshaping the brain: scientists reprogram neurons after birth

The cerebral cortex—the gray matter that forms the outer layers of the mammalian cerebrum and cerebellum—is divided into six different layers based on the presence of specialized neurons, and we’ve known that since the early 1900s. Denis Jabaudon is interested in using the tools of modern biology to understand the genetic mechanisms that establish and maintain those layers. Over the past few years, his lab has published papers implicating various genes in the generation of specific neuronal subtypes.

Now they have gone a step further. They have developed a new electrochemical method to transfer genes into specific types of neurons—they call it iontoporation. Using it, they have transformed one type of neuron in a mature brain into a different type entirely.

Although Jabaudon and others have made some headway in working out how the different neurons arise, they still don’t know how plastic they are—if they can change fates after they started differentiating down one particular path. In the context of brain injury, it would be useful to know if certain neural circuits could be reprogrammed and repaired by having the neurons that are already present change fates to adapt to the damage. But this has been challenging to determine, because changing the fate of specific neurons in the latter stages of differentiation has been technically difficult.

Layer 4 mouse spiny neurons have round bodies, with many short dendrites (connections with other cells) that stay within their layer of the brain. They receive sensory signals from the thalamus. Layer 5B output neurons are pyramidal in shape, with a prominent dendrite that extends all the way to layer 1.

Fezf2 is a transcription factor that regulates the activity of other genes. It is expressed throughout the L5B neuron’s entire life, and it is necessary and sufficient for turning early cortical cells into L5B neurons.

When Jabaudon’s colleagues iontoporated Fezf2 into L4 neurons the day after mice were born, a week after the neurons had established their identity, it completely transformed them. You guessed it: the L4 neurons walked, talked, looked, and quacked just like L5B neurons. They looked like L5B’s and began transmitting signals to other nerves just as these cells did. Most significantly, however, they rearranged their intracortical inputs, meaning that they now received signals from layer 2/3 neurons instead of from the thalamus.

The researchers tried their iontoporation as late as ten days after the mice were born, and found that the neurons become less amenable to reprogramming with time, but some features were still malleable for a week after the mice were born—two weeks after the neurons originated.

The authors hope to further explore the molecular mechanisms responsible for the emergence and patterning of different cortical areas during brain development and their plasticity after injury. They hope that one day, reprogramming existing neurons could be a means of nervous system repair.

Lack of Protein Sp2 Disrupts Neuron Creation in Brain

A protein known as Sp2 is key to the proper creation of neurons from stem cells, according to researchers at North Carolina State University. Understanding how this protein works could enable scientists to “program” stem cells for regeneration, which has implications for neural therapies.

Troy Ghashghaei and Jon Horowitz, both faculty in NC State’s Department of Molecular Biomedical Sciences and researchers in the Center for Comparative Medicine and Translational Research, wanted to know more about the function of Sp2, a cell cycle regulator that helps control how cells divide. Previous research from Horowitz had shown that too much Sp2 in skin-producing stem cells resulted in tumors in experimental mice. Excessive amounts of Sp2 prevented the stem cells from creating normal cell “offspring,” or skin cells. Instead, the stem cells just kept producing more stem cells, which led to tumor formation.

“We believe that Sp2 must play a fundamental role in the lives of normal stem cells,” Horowitz says. “Trouble ensues when the mechanisms that regulate its activity are overwhelmed due to its excess abundance.”

Ghashghaei and his team – led by doctoral candidate Huixuan Liang – took the opposite approach. Using genetic tools, they got rid of Sp2 in certain neural stem cells in mice, specifically those that produce the major neurons of the brain’s cerebral cortex. They found that a lack of Sp2 disrupted normal cell formation in these stem cells, and one important result was similar to Horowitz’s: the abnormal stem cells were unable to produce normal cell “offspring,” or neurons. Instead, the abnormal stem cells just created copies of themselves, which were also abnormal.

“It’s interesting that both an overabundance of this protein and a total lack of it result in similar disruptions in how stem cells divide,” Ghashghaei says. “So while this work confirms that Sp2 is absolutely necessary for stem cell function, a lot of questions still remain about what exactly it is regulating, and whether it is present in all stem cells or just a few. We also need to find out if Sp2 deletion or overabundance can produce brain tumors in our mice as in the skin.

“Finally, we are very interested in understanding how Sp2 regulates a very important decision a stem cell has to make: whether to produce more of itself or to produce offspring that can become neurons or skin cells,” Ghashghaei adds. “We hope to address those questions in our future research, because these cellular mechanisms have implications for cancer research, neurodevelopmental diseases and regenerative medicine.”

The results appear online in Development. NC State graduate students Guanxi Xiao, and Haifeng Yin, as well as Dr. Simon Hippenmeyer, a collaborator with the Ghashghaei lab from Austria’s Institute of Science and Technology, contributed to the work. The work was funded by the National Institutes of Health and the American Federation for Aging Research.

Chimpanzees successfully play the Ultimatum Game

Researchers at the Yerkes National Primate Research Center, Emory University, are the first to show chimpanzees possess a sense of fairness that has previously been attributed as uniquely human. Working with colleagues from Georgia State University, the researchers played the Ultimatum Game with the chimpanzees to determine how sensitive the animals are to the reward distribution between two individuals if both need to agree on the outcome.

The researchers say the findings, available in an early online edition of the Proceedings of the National Academy of Sciences (PNAS) available this week, suggest a long evolutionary history of the human aversion to inequity as well as a shared preference for fair outcomes by the common ancestor of humans and apes.

According to first author Darby Proctor, PhD, “We used the Ultimatum Game because it is the gold standard to determine the human sense of fairness. In the game, one individual needs to propose a reward division to another individual and then have that individual accept the proposition before both can obtain the rewards. Humans typically offer generous portions, such as 50 percent of the reward, to their partners, and that’s exactly what we recorded in our study with chimpanzees.”

Co-author Frans de Waal, PhD, adds, “Until our study, the behavioral economics community assumed the Ultimatum Game could not be played with animals or that animals would choose only the most selfish option while playing. We’ve concluded that chimpanzees not only get very close to the human sense of fairness, but the animals may actually have exactly the same preferences as our own species.” For purposes of direct comparison, the study was also conducted separately with human children.

In the study, researchers tested six adult chimpanzees (Pan troglodytes) and 20 human children (ages 2 – 7 years) on a modified Ultimatum Game. One individual chose between two differently colored tokens that, with his or her partner’s cooperation, could be exchanged for rewards (small food rewards for chimpanzees and stickers for children). One token offered equal rewards to both players, whereas the other token favored the individual making the choice at the expense of his or her partner. The chooser then needed to hand the token to the partner, who needed to exchange it with the experimenter for food. This way, both individuals needed to be in agreement.

Both the chimpanzees and the children responded like adult humans typically do. If the partner’s cooperation was required, the chimpanzees and children split the rewards equally. However, with a passive partner, who had no chance to reject the offer, chimpanzees and children chose the selfish option.

Chimpanzees, who are highly cooperative in the wild, likely need to be sensitive to reward distributions in order to reap the benefits of cooperation. Thus, this study opens the door for further explorations into the mechanisms behind this human-like behavior.

Parkinson’s Treatment Can Trigger Creativity

Parkinson’s experts across the world have been reporting a remarkable phenomenon — many patients treated with drugs to increase the activity of dopamine in the brain as a therapy for motor symptoms such as tremors and muscle rigidity are developing new creative talents, including painting, sculpting, writing, and more.

Prof. Rivka Inzelberg of Tel Aviv University’s Sackler Faculty of Medicinefirst noticed the trend in her own Sheba Medical Center clinic when the usual holiday presents from patients — typically chocolates or similar gifts — took a surprising turn. “Instead, patients starting bringing us art they had made themselves,” she says.

Inspired by the discovery, Prof. Inzelberg sought out evidence of this rise in creativity in current medical literature. Bringing together case studies from around the world, she examined the details of each patient to uncover a common underlying factor — all were being treated with either synthetic precursors of dopamine or dopamine receptor agonists, which increase the amount of dopamine activity in the brain by stimulating receptors. Her report published in the journal Behavioral Neuroscience.

Giving in to artistic impulse

Dopamine is involved in several neurological systems, explains Prof. Inzelberg. Its main purpose is to aid in the transmission of motor commands, which is why a lack of dopamine in Parkinson’s patients is associated with tremors and a difficulty in coordinating their movements.

But it’s also involved in the brain’s “reward system” — the satisfaction or happiness we experience from an accomplishment. This is the system which Prof. Inzelberg predicts is associated with increasing creativity. Dopamine and artistry have long been connected, she points out, citing the example of the Vincent Van Gogh, who suffered from psychosis. It’s possible that his creativity was the result of this psychosis, thought to be caused by a spontaneous spiking of dopamine levels in the brain.

There are seemingly no limits to the types of artistic work for which patients develop talents, observes Prof. Inzelberg. Cases include an architect who began to draw and paint human figures after treatment, and a patient who, after treatment, became a prize-winning poet though he had never been involved in the arts before.

It’s possible that these patients are expressing latent talents they never had the courage to demonstrate before, she suggests. Dopamine-inducing therapies are also connected to a loss of impulse control, and sometimes result in behaviors like excessive gambling or obsessional hobbies. An increase in artistic drive could be linked to this lowering of inhibitions, allowing patients to embrace their creativity. Some patients have even reported a connection between their artistic sensibilities and medication dose, noting that they feel they can create more freely when the dose is higher.

Therapeutic value

Prof. Inzelberg believes that such artistic expressions have promising therapeutic potential, both psychologically and physiologically. Her patients report being happier when they are busy with their art, and have noted that motor handicaps can lessen significantly. One such patient is usually wheelchair-bound or dependent on a walker, but creates intricate wooden sculptures that have been displayed in galleries. External stimuli can sometimes bypass motor issues and foster normal movement, she explains. Similar types of art therapy are already used for dementia and stroke patients to help mitigate the loss of verbal communication skills, for example.

The next step is to try to characterize those patients who become more creative through treatment through comparing them to patients who do not experience a growth in artistic output. “We want to screen patients under treatment for creativity and impulsivity to see if we can identify what is unique in those who do become more creative,” says Prof. Inzelberg. She also believes that such research could provide valuable insights into creativity in healthy populations, too.

Scientists Discover Structure of Protein Essential for Quality Control, Nerve Function

Using an innovative approach, scientists at The Scripps Research Institute (TSRI) have determined the structure of Ltn1, a recently discovered “quality-control” protein that is found in the cells of all plants, fungi and animals.

Ltn1 appears to be essential for keeping cells’ protein-making machinery working smoothly. It may also be relevant to human neurodegenerative diseases, for an Ltn1 mutation in mice leads to a motor-neuron disease resembling amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease).

“To better understand Ltn1’s mechanism of action, we needed to solve its structure, and that’s what we’ve done here,” said TSRI Associate Professor Claudio Joazeiro.

“In addition, this project has brought us a set of structural analysis techniques that we can apply to other exciting problems in biology,” said TSRI Professor Bridget Carragher.

Joazeiro and Carragher, along with Clint Potter, also a TSRI professor, are senior authors of the new report, which appears in the online Early Edition of the Proceedings of the National Academy of Sciences the week of January 14, 2013.

Links to Neurodegenerative Disease

Ltn1 first turned up on biologists’ radar screens several years ago when a joint Novartis-Phenomix research team noted that mice with an unknown gene mutation were born normal but suffered from progressive paralysis. The scientists dubbed the animals lister mice, because they listed to one side as they walked. Collaborating with Joazeiro, the Novartis team reported in a 2009 paper that the mutated gene normally codes for a type of enzyme known as an E3 ubiquitin ligase, and that the mouse phenotype was due to a neurodegenerative syndrome resembling ALS.

In a study published in the journal Nature the following year, Joazeiro and his postdoctoral research associate Mario H. Bengtson found that the enzyme serves as a crucial quality-control manager for the cellular protein-making factories called ribosomes. Occasionally a ribosome receives miscoded genetic instructions and produces certain types of abnormal proteins, known as “nonstop proteins”— jamming the ribosomal machinery like a wrinkled sheet of paper in an office printer. Bengtson and Joazeiro found that Ltn1 fixes jammed ribosomes by tagging nonstop proteins with ubiquitin molecules, thereby marking them for quick destruction by roving cellular garbage-disposers called proteasomes.

“The question for us then was, ‘How does Ltn1 do this?’” said Joazeiro.

Pushing the Boundaries of Electron Microscopy

To help find out, he began a collaboration with Carragher and Potter, who run the National Resource for Automated Molecular Microscopy (NRAMM), an advanced electron microscope facility at TSRI that is funded by the National Institutes of Health’s National Center for Research Resources.

Ltn1 was deemed too large for its structure to be determined by current nuclear magnetic resonance (NMR) technology, and, as the scientists know now, too flexible to allow the highly regular crystalline packing needed by X-ray crystallographers. “It’s a very floppy molecule, so it would be hard to crystallize,” said Potter.

Advanced electron microscopy offered a way, however. Dmitry Lyumkis, a graduate student in the NRAMM laboratory and first author of the study, took high-resolution images of yeast Ltn1 with an electron microscope. He then used sophisticated image and data processing software to align and average individual images. The technique eliminates much of the random “noise” that obscures single images and produces a sharp 3D picture of the protein.

No one has ever used electron microscopy to distinguish so many—more than 20—conformations of such a small protein. “Usually electron microscopists determine no more than two or three conformational states, and they work with protein complexes whose size is in the megadalton range, but Ltn1 is only 180 kilodaltons, an order of magnitude smaller,” Lyumkis said.

An Unusually Flexible Structure

The analysis revealed that Ltn1 has an elongated, double-jointed and extraordinarily flexible structure with two working ends—the N-terminus and C-terminus. “We anticipate that the N-terminus is responsible for association with the ribosome and know that the C-terminus is responsible for the ubiquitylation of nonstop proteins,” said Lyumkis. “We suspect that the high flexibility of this structure is needed for it to work on the variety of nonstop proteins that can get stuck in ribosomes.”

One of the next steps for the team is to evaluate Ltn1’s individual segments, which appear to be more rigid, using X-ray crystallography, in order to develop a piece-by-piece atomic-resolution model of the enzyme. Another is to determine the structure of Ltn1 when it is attached to a ribosome and operating on a nonstop protein. Joazeiro notes that a typical yeast cell has nearly 200,000 ribosomes but requires only 200 Ltn1 copies for adequate quality control under normal growth conditions. “Somehow this enzyme can efficiently sense which ribosomes are jammed, and we expect that by solving the joint structure of Ltn1 and a ribosome, we’ll be able to understand how it does this,” he says.

Lyumkis, Carragher, Potter and their colleagues at NRAMM also plan to use a similar electron microscopy-based approach to find the structures of other important proteins with highly variable “heterogeneous” conformations. “Heterogeneity has been a big challenge,” said Potter, “and being able to collect this large dataset and do all of this data processing successfully has been a critical breakthrough.”

Protein identified that can disrupt embryonic brain development and neuron migration

Interneurons – nerve cells that function as ‘dimmers’ – play an important role in the brain. Their formation and migration to the cerebral cortex during the embryonic stage of development is crucial to normal brain functioning. Abnormal interneuron development and migration can eventually lead to a range of disorders and diseases, from epilepsy to Alzheimer’s. New research by Dr. Eve Seuntjens and Dr. Veronique van den Berghe of the Department of Development and Regeneration (Danny Huylebroeck laboratory, Faculty of Medicine) has identified two proteins, Sip1 and Unc5b, that play an important role in the development and migration of interneurons to the cerebral cortex – a breakthrough in our understanding of early brain development.

Two types of nerve cells are crucial to healthy brain functioning. Projection neurons, the more widely known of the two, make connections between different areas of the brain. Interneurons, a second type, work as dimmers that regulate the signalling processes of projection neurons. A shortage or irregular functioning of interneurons can cause short circuits in the nervous system. This can lead to seizures, a common symptom of many brain disorders. Interneuron dysfunction even appears to play a role in schizophrenia, autism and neurodegenerative diseases such as Alzheimer’s, Parkinson’s and ALS.

Trailblazers

Researchers have only recently understood how different kinds of neuron are formed during embryonic development. During early brain development, stem cells form projection neurons in the cerebral cortex. Interneurons are made elsewhere in the brain. These interneurons then migrate to the cortex to mix with the projection neurons. Dr. Eve Seuntjens of the Celgen laboratory led by Professor Danny Huylebroeck explains: “The journey of interneurons is very complex: their environment changes constantly during growth and there are no existing structures — such as nerve pathways — available for them to follow.”

The question is how young interneurons receive their ‘directions’ to the cerebral cortex. Several proteins play a role, says Dr. Seuntjens. “We changed the gene containing the production code for the protein Sip1 in mice so that this protein was no longer produced during brain development.  In those mice, the interneurons never made it to the cerebral cortex — they couldn’t find the way.

That has to do with the guidance signals – substances that repel or attract interneurons and thus point them in the right direction – encountered by the interneurons on their way to the cerebral cortex. Without Sip1 production, interneurons see things through an overly sharp lens, so to speak. They see too many stop signs and become blocked. That overly sharp lens is Unc5b, a protein. Unc5b is deactivated by Sip1 in healthy mice. There are several known factors that influence the migration of interneurons, but Unc5b is the first protein we’ve isolated that we now know must be switched off in order for interneuron migration to move ahead smoothly.”

The next step is to study this process in the neurons of humans. “Now that there are techniques to create stem cells from skin cells, we can mimic the development of stem cells into interneurons and study what can go wrong. From there, we can test whether certain drugs can reverse the damage. That’s all still on the horizon, but you can see that the focus of research on many brain disorders and diseases is increasingly shifting to early child development because that just might be where a cause can be found.”

New Discovery in Autism-Related Disorder Reveals Key Mechanism in Brain Development and Disease

A new finding in neuroscience for the first time points to a developmental mechanism linking the disease-causing mutation in an autism-related disorder, Timothy syndrome, and observed defects in brain wiring, according to a study led by scientist Ricardo Dolmetsch and published online yesterday in Nature Neuroscience. These findings may be at the heart of the mechanisms underlying intellectual disability and many other brain disorders.

The present study reveals that a mutation of the disease-causing gene throws a key process of neurodevelopment into reverse. That is, the mutation underlying Timothy syndrome causes shrinkage, rather than growth, of the wiring needed for the development of neural circuits that underlie cognition.

“In addition to the implications for autism, what’s really exciting is that we now have a way to get at the core mechanisms tying genes and environmental influences to development and disease processes in the brain,” said Dolmetsch, Senior Director of Molecular Networks at the Allen Institute for Brain Science.

“Imagine what we can learn if we do this hundreds and hundreds of times for many different human genetic variations in a large-scale, systematic way. That’s what we are doing now at the Allen Institute,” Dolmetsch continued.

In normal brain development, brain activity causes branches emanating from neural cells to stretch or expand. In cells with the mutation, these branched extensions, called dendrites, instead retract in response to neural activity, according to this study. This results in abnormal brain circuitry favoring connections with nearby neurons rather than farther-reaching connections. Further, the study identified a previously unknown mode of signaling to uncover the chemical pathway that causes the dendritic retraction.

This finding may have wide-reaching implications in neuroscience, as impaired dendrite formation is a common feature of many neurodevelopmental disorders. Further, the same gene has been implicated in other disorders including bipolar disorder and schizophrenia.

Under Dolmetsch’s leadership, the Molecular Networks program at the Allen Institute, one of three major new initiatives announced by the Institute last March, is using similar methods on a grand scale. The Institute is probing a large number of human genetic variations and many pathways in the brain to untangle the cellular mechanisms of neurodevelopment and disease. In addition to identifying the molecular and environmental rules that shape how the brain is built, the program will create new research tools and data sets that it will share publicly with the global research community.

Timothy syndrome is a neurodevelopmental disorder associated with autism spectrum disorders and caused by a mutation in a single gene. In addition to autism, it is also characterized by cardiac arrhythmias, webbed fingers and toes, and hypoglycemia, and often leads to death in early childhood.

(Image: iStock)