Brain Development Is Guided by Junk DNA that Isn’t Really Junk

Specific DNA once dismissed as junk plays an important role in brain development and might be involved in several devastating neurological diseases, UC San Francisco scientists have found.

Their discovery in mice is likely to further fuel a recent scramble by researchers to identify roles for long-neglected bits of DNA within the genomes of mice and humans alike.

While researchers have been busy exploring the roles of proteins encoded by the genes identified in various genome projects, most DNA is not in genes. This so-called junk DNA has largely been pushed aside and neglected in the wake of genomic gene discoveries, the UCSF scientists said.

In their own research, the UCSF team studies molecules called long noncoding RNA (lncRNA, often pronounced as “link” RNA), which are made from DNA templates in the same way as RNA from genes.

“The function of these mysterious RNA molecules in the brain is only beginning to be discovered,” said Daniel Lim, MD, PhD, assistant professor of neurological surgery, a member of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF, and the senior author of the study, published online April 11 in the journal Cell Stem Cell.

Alexander Ramos, a student enrolled in the MD/PhD program at UCSF and first author of the study, conducted extensive computational analysis to establish guilt by association, linking lncRNAs within cells to the activation of genes.

Ramos looked specifically at patterns associated with particular developmental pathways or with the progression of certain diseases. He found an association between a set of 88 long noncoding RNAs and Huntington’s disease, a deadly neurodegenerative disorder. He also found weaker associations between specific groups of long noncoding RNAs and Alzheimer’s disease, convulsive seizures, major depressive disorder and various cancers.

“Alex was the team member who developed this new research direction, did most of the experiments, and connected results to the lab’s ongoing work,” Lim said. The study was mostly funded through Lim’s grant – a National Institutes of Health (NIH) Director’s New Innovator Award, a competitive award for innovative projects that have the potential for unusually high impact.

LncRNA versus Messenger RNA

Unlike messenger RNA, which is transcribed from the DNA in genes and guides the production of proteins, lncRNA molecules do not carry the blueprints for proteins. Because of this fact, they were long thought to not influence a cell’s fate or actions.

Nonetheless, lncRNAs also are transcribed from DNA in the same way as messenger RNA, and they, too, consist of unique sequences of nucleic acid building blocks.

Evidence indicates that lncRNAs can tether structural proteins to the DNA-containing chromosomes, and in so doing indirectly affect gene activation and cellular physiology without altering the genetic code. In other words, within the cell, lncRNA molecules act “epigenetically” — beyond genes — not through changes in DNA.

The brain cells that the scientists focused on the most give rise to various cell types of the central nervous system. They are found in a region of the brain called the subventricular zone, which directly overlies the striatum. This is the part of the brain where neurons are destroyed in Huntington’s disease, a condition triggered by a single genetic defect.

Ramos combined several advanced techniques for sequencing and analyzing DNA and RNA to identify where certain chemical changes happen to the chromosomes, and to identify lncRNAs on specific cell types found within the central nervous system. The research revealed roughly 2,000 such molecules that had not previously been described, out of about 9,000 thought to exist in mammals ranging from mice to humans.

In fact, the researchers generated far too much data to explore on their own. The UCSF scientists created a website through which their data can be used by others who want to study the role of lncRNAs in development and disease.

“There’s enough here for several labs to work on,” said Ramos, who has training grants from the California Institute for Regenerative Medicine (CIRM) and the NIH.

“It should be of interest to scientists who study long noncoding RNA, the generation of new nerve cells in the adult brain, neural stem cells and brain development, and embryonic stem cells,” he said.

Babies’ brains to be mapped in the womb and after birth

UK scientists have embarked on a six-year project to map how nerve connections develop in babies’ brains while still in the womb and after birth.

By the time a baby takes its first breath many of the key pathways between nerves have already been made. And some of these will help determine how a baby thinks or sees the world, and may have a role to play in the development of conditions such as autism, scientists say.

But how this rich neural network assembles in the baby before birth is relatively unchartered territory.

Researchers from Guy’s and St Thomas’ Hospital, King’s College London, Imperial College and Oxford University aim to produce a dynamic wiring diagram of how the brain grows, at a level of detail that they say has been impossible until now.

They hope that by charting the journeys of bundles of nerves in the final three months of pregnancy, doctors will be able to understand more about how they can help in situations when this process goes wrong.

Prof David Edwards, director of the Centre for the Developing Brain, who is leading the research, says: “There is a distressing number of children in our society who grow up with problems because of things that happen to them around the time of birth or just before birth.

"It is very important to be able to scan babies before they are born, because we can capture a period when an awful lot is changing inside the brain, and it is a time when a great many of the things that might be going wrong do seem to be going wrong."

'Neural networks'

The study - known as the Developing Human Connectome Project - hopes to look at more than 1,500 babies, studying many aspects of their neurological development.

By examining the brains of babies while they are still growing in the womb, as well as those born prematurely and at full term, the scientists will try to define baselines of normal development and investigate how these may be affected by problems around birth.

And they plan to share their map with the wider research community.

Central to this project are advanced MRI scanning techniques, which the scientists say are able to pick up on details of the growing brain that have been difficult to capture until now.

While in the womb, foetuses are free to somersault in their amniotic sacs, and this constant movement has so far hindered clear images of growing brains.

But researchers at the Centre for the Developing Brain have found ways to counter the effects of these movements, building up full three-dimensional pictures while the foetus is in motion.

And by placing the MRI machine in the neonatal intensive care unit at Evelina Children’s Hospital in London they are one of the few centres in the world to have a scanner in such close proximity to the babies who often need it most, Prof Edwards says.

This means the same scanning system can be used to find out more about the brains of the sickest and smallest newborn babies, he says.

'Macro level'

Daniel Rueckert, professor of visual information processing at Imperial College London, who is also involved in the research, says: “We are trying to look at brain connectivity in two ways: firstly, from a structural perspective, to find out which parts of the brain are wired to other parts. And secondly we are looking at functional connectivity - how strongly two brain regions are linked across time and activity.”

But Prof Partha Mitra, a neuroscientist at Cold Spring Harbor Laboratory, New York state, says we need to be aware of the limitations of the technology in use.

"It would obviously be a very good thing to know more about the circuits in the developing human brain. Much of what we know hasn’t changed in a hundred years and has come from dissection studies.

"But we need to keep in mind the imaging techniques we have are indirect - we can’t open up a human brain and look at the connections while someone is alive so we rely on these non-invasive methods. But there is a big gap between the real circuits in the brain and what images can show us."

Prof Rueckert acknowledges that this map will provide a “macro-level” view of the developing brain and not be the “final answer”.

But he points to early results from the adult version of this project - the Human Connectome Project, based in the US: “There is so much evidence already from the adult project that there are significant changes in the brain that can be mapped with the technology we have now.

"It will be incredibly useful to be able to do this with the still growing and developing brain - perhaps giving us more time to intervene when things go wrong."

Sleep study reveals how the adolescent brain makes the transition to mature thinking

A new study conducted by monitoring the brain waves of sleeping adolescents has found that remarkable changes occur in the brain as it prunes away neuronal connections and makes the major transition from childhood to adulthood.

“We’ve provided the first long-term, longitudinal description of developmental changes that take place in the brains of youngsters as they sleep,” said Irwin Feinberg, professor emeritus of psychiatry and behavioral sciences and director of the UC Davis Sleep Laboratory. “Our outcome confirms that the brain goes through a remarkable amount of reorganization during puberty that is necessary for complex thinking.”

The research, published in the February 15 issue of American Journal of Physiology: Regulatory, Integrative and Comparative Physiology, also confirms that electroencephalogram, or EEG, is a powerful tool for tracking brain changes during different phases of life, and that it could potentially be used to help diagnose age-related mental illnesses. It is the final component in a three-part series of studies carried out over 10 years and involving more than 3,500 all-night EEG recordings. The data provide an overall picture of the brain’s electrical behavior during the first two decades of life.

Feinberg explained that scientists have generally assumed that a vast number of synapses are needed early in life to recover from injury and adapt to changing environments. These multiple connections, however, impair the efficient problem solving and logical thinking required later in life. His study is the first to show how this shift can be detected by measuring the brain’s electrical activity in the same children over the course of time.

Two earlier studies by Feinberg and his colleagues showed that EEG fluctuations during the deepest (delta or slow wave) phase of sleep, when the brain is most recuperative, consistently declined for 9- to 18-year-olds. The most rapid decline occurred between the ages of 12 and 16-1/2. This led the team to conclude that the streamlining of brain activity — or “neuronal pruning” — required for adult cognition occurs together with the timing of reproductive maturity.

Questions remained, though, about electrical activity patterns in the brains of younger children.

For the current study, Feinberg and his research team monitored 28 healthy, sleeping children between the ages of 6 and 10 for two nights every six months. The new findings show that synaptic density in the cerebral cortex reaches its peak at age 8 and then begins a slow decline. The recent findings also confirm that the period of greatest and most accelerated decline occurs between the ages of 12 and 16-1/2 years, at which point the drop markedly slows.

“Discovering that such extensive neuronal remodeling occurs within this 4-1/2 year timeframe during late adolescence and the early teen years confirms our view that the sleep EEG indexes a crucial aspect of the timing of brain development,” said Feinberg.

The latest study also confirms that EEG sleep analysis is a powerful approach for evaluating adolescent brain maturation, according to Feinberg. Besides being a relatively simple, accessible technology for measuring the brain’s electrical activity, it is more accurate than more cumbersome and expensive options.

“Structural MRI, for instance, has not been able to identify the adolescent accelerations and decelerations that are easily and reliably captured by sleep EEG,” said Feinberg. “We hope our data can aid the search for the unknown genetic and hormonal biomarkers that drive those fluctuations. Our data also provide a baseline for seeking errors in brain development that signify the onset of diseases such as schizophrenia, which typically first become apparent during adolescence. Once these underlying processes have been identified, it may become possible to influence adolescent brain changes in ways that promote normal development and correct emerging abnormalities.”

(Image: iStockphoto)

New Early Warning System for the Brain Development of Babies

A new research technique, pioneered by Dr. Maria Angela Franceschini, was published in JoVE (Journal of Visualized Experiments) on March 14th. Researchers at Massachusetts General Hospital and Harvard Medical School have developed a non-invasive optical measurement system to monitor neonatal brain activity via cerebral metabolism and blood flow.

Of the nearly four million children born in the United States each year, 12% are born preterm, 8% are born with low birth weight, and 1-2% of infants are at risk for death associated with respiratory distress. The result is an average infant mortality rate of 6 deaths per 1,000 live births. These statistics, though low compared to those of 50 or even 20 years ago, are troubling both to parents and to clinicians. Until recently there were no effective bedside methods to screen for brain injury or monitor injury progression that can contribute to developmental abnormalities or infant mortality. Dr. Franceschini’s new system does both.

“We want to measure cerebral vascular development and brain health in babies,” Dr. Franceschini tells us. Because neuronal metabolism is hard to measure directly, scientists instead evaluate cerebral oxygen metabolism, which highly corresponds to neuronal metabolism. Dr. Franceschini and her team have developed a near infrared optical system to quantify cerebral oxygen metabolism by measuring blood oxygen saturation and blood flow.

The technology is an improvement on continuous-wave near-infrared spectroscopy (CWNIRS), which measures oxygen saturation but does not provide long-term or real time brain monitoring. Instead, frequency-domain near-infrared spectroscopy (FDNIRS) is used in conjunction with diffuse correlation spectroscopy (DCS) to get a more robust evaluation of infant health. Dr. Franceschini explains, “CWNIRS has been used for many years but it only provides relative measurements of blood oxygen saturation. Our technology allows quantification of multiple vascular parameters and evaluation of oxygen metabolism which gives a more direct picture of infant distress.”

“This technology will let us monitor babies who may be having seizures, cerebral hemorrhages, or other cerebral distresses and may allow us to expedite treatment,” says Dr. Franceschini, who plans to develop and streamline this technology to one that nurses can use clinically. “We chose to publish in JoVE because it is important to show how these measurements can be done and this publication lets us reach early adopters.”

Pig brain models provide insights into human cognitive development

A mutual curiosity about patterns of growth and development in pig brains has brought two University of Illinois research groups together. Animal scientists Rod Johnson and Ryan Dilger have developed a model of the pig brain that they plan to use to answer important questions about human brain development.

“It is important to characterize the normal brain growth trajectory from the neonatal period to sexual maturity,” said Johnson.

“Until we know how the brain grows, we don’t know what is going to change,” added Dilger.

In cooperation with the Beckman Institute, they performed MRI scans on the brains of 16 piglets, starting at the age of 2 weeks, then at 4 weeks, and then at 4-week intervals up to 24 weeks.

“We have world-class people at the Beckman Institute who are pushing and developing the next generation of neuroimaging technology, so we’re able to connect with them and take advantage of their expertise,” said Johnson.

Matt Conrad, a student in Johnson’s lab, used three-dimensional visualization software on over 200 images to manually segment each region on three planes. The software put the information together into a three-dimensional image of the pig brain. This is used to determine the volume of the different structures.

When the piglets were at Beckman for their imaging sessions, Dilger performed other tests, including diffusion tensor imaging (DTI), which shows how neural tracks develop, allowing the exploration of brain complexity and of how neurons form. It was also possible to measure neurochemicals, including creatine and acetylcholine, in the brain, which provides a unique insight into brain metabolism.

The end result of this work is what they call the deformable pig brain atlas.

“We are taking 16 pigs and averaging them, so it’s more representative of all pigs,” said Dilger. “You can then apply it to any individual pig to see how it’s different.”

“It’s called a deformable brain atlas because the software takes information from an individual and deforms it until it fits the template, and then you know how much it had to be deformed to fit,” Johnson explained. “So from that, you can tell whether a brain region is larger or smaller compared to the average.”

Johnson and Dilger said that the goal is to develop a tool for pigs that is equivalent to what is available for the mouse brain and make it publicly available. But they don’t want to stop with tool development.

“We want to use this to address important questions,” Johnson said.

One research direction being pursued in Johnson’s lab is to induce viral pneumonia in piglets at the point in the post-natal period when the brain is undergoing massive growth to see how it alters brain growth and development. They are also looking at effects of prenatal infections in the mother to see if that alters the trajectory of normal brain growth in the offspring. The risk for behavioral disorders and reduced stress resilience is increased by pre- and post-natal infection, but the developmental origins are poorly understood.

Dilger’s group is interested in the effects of early-life nutrition on the brain. They are looking at the effects of specific fatty acids as primary structural components of the human brain and cerebral cortex, and at choline, a nutrient that is important for DNA production and normal functioning of neurons.

“Choline deficiency has been tied to cognitive deficits in the mouse and human, and we’re developing a pig model to study the direct effects choline deficiency has on brain structure and function,” Dilger said. “Many women of child-bearing age may not be receiving enough choline in their diets, and recent evidence suggests this may ultimately affect learning and memory ability in their children. Luckily, choline can be found in common foods, especially eggs and meat products, including bacon.”

“Seq-ing” Insights into the Epigenetics of Neuronal Gene Regulation

The epigenetic control of neuronal gene expression patterns has emerged as an underlying regulatory mechanism for neuronal function, identity, and plasticity, in which short- to long-lasting adaptation is required to dynamically respond and process external stimuli. To achieve a comprehensive understanding of the physiology and pathology of the brain, it becomes essential to understand the mechanisms that regulate the epigenome and transcriptome in neurons. Here, we review recent advances in the study of regulated neuronal gene expression, which are dramatically expanding as a result of the development of new and powerful contemporary methodologies, based on next-generation sequencing. This flood of new information has already transformed our understanding of many biological processes and is now driving discoveries elucidating the molecular mechanisms of brain function in cognition, behavior, and disease and may also inform the study of neuronal identity, diversity, and neuronal reprogramming.

Microglia controls neuron production as brain develops

In a surprise breakthrough, researchers at the UC Davis MIND Institute and their colleagues have found that microglia remove healthy neural progenitor cells (NPCs) through phagocytosis to control neuron production during brain development. This newly discovered mechanism keeps neuron numbers in check, preventing brain overgrowth.

The discovery could open up new avenues for brain research and lead to therapies for a variety of neurological conditions.

The study was published online in the The Journal of Neuroscience.

Microglia are the immune component cell of the central nervous system. Similar to macrophages, microglia provide the brain’s primary defense against pathogens and foreign bodies, clear away dying cells and help repair neural damage. When inactive, they act as sentinels. When a problem is located, they activate and eliminate it. However, until recently, no one had realized the important roles they play in brain development.

"We have known for some time that neurons can undergo apoptosis, a form of cell death, and ultimately be removed by microglia," said Stephen Noctor, assistant professor in the Department of Psychiatry and Behavioral Sciences and the study’s lead author. "But this is new. Microglia are actually eating healthy progenitor cells, thereby regulating the number of neurons produced in the developing brain."

During development, NPCs produce neurons in the brain’s proliferative zones. However, creating too many or too few neurons can have dire consequences.

"If you have too many cells, there’s only so much trophic support (brain infrastructure for cell growth and survival) to keep neurons alive," Noctor said. "All these cells competing for resources could easily throw off connectional properties, altering the way surviving neurons interact. Likewise, having too few cortical cells would have profoundly negative consequences."

(Image: Antoine Triller, Alain Bessis & Serge Marty - Département de Biologie, ENS)