Researchers survey protein family that helps the brain form synapses

Neuroscientists and bioengineers at Stanford are working together to solve a mystery: How does nature construct the different types of synapses that connect neurons – the brain cells that monitor nerve impulses, control muscles and form thoughts.

In a paper published in the Proceedings of the National Academy of Sciences, Thomas C. Südhof, M.D., a professor of molecular and cellular physiology, and Stephen R. Quake, a professor of bioengineering, describe the diversity of the neurexin family of proteins.

Neurexins help to create the synapses that connect neurons. Think of synapses as switchboards or control panels that connect specific neurons when these brain cells must work together to perform a given task.

Neurexins play a key role in the formation and functioning of synaptic connections. Past human genetics studies have linked neurexins to a variety of cognitive disorders, such as autism and schizophrenia.

Südhof, the Avram Goldstein Professor in the School of Medicine and a winner of the 2013 Nobel Prize in Medicine, has spent years studying the many different forms, or isoforms, of neurexin proteins. He has postulated that different isoforms of neurexins may help to create different types of synaptic connections with distinct properties and functions, and thus enable neurons to do so many complex tasks.

But Südhof had no way to know exactly how many isoforms of neurexins existed until he sat down last year with Quake, the Lee Otterson Professor in the School of Engineering. Quake has pioneered new ways to sequence DNA – the master blueprint that nature follows when making proteins.

The study being published in PNAS represents the results of a year-long collaboration between neuroscientists and bioengineers to better understand how different neurexin proteins affect the behavior of synapses and, ultimately, normal brain functions and neurological conditions such as autism.

Though this will not be the last word on the subject, the findings help illuminate how the brain works and improve our understanding of neurological disorders.

Inside cells, a molecular machine unzips a double-stranded DNA molecule to create an RNA molecule. The RNA molecule is a copy of all the genetic instructions encoded into the DNA. But only specific regions of this RNA molecule contain instructions for making a specific protein. The cell has ways to remove the unnecessary regions and splice the protein-coding regions into a shorter RNA molecule called messenger RNA or mRNA. Thus, each mRNA contains the full instructions for making a specific protein.

To begin this experiment, Ozgun Gokce, a postdoctoral scholar in molecular and cellular physiology in Südhof’s lab, and Barbara Treutlein, a postdoctoral scholar in Quake’s lab, extracted brain cells from the prefrontal cortex of a mouse, then isolated the RNA contained in this tissue.

From this large pool of RNAs they then identified the mRNAs for neurexins. They ran those messenger molecules through equipment designed to read the entire long sequence of chemical instructions for making a specific isoform in the neurexin family of protein.

Quake’s lab is adept at using new instruments that allow researchers to read the long sequence of chemicals in an mRNA strand, allowing them to ascertain exactly what directions this messenger is carrying to the cell’s protein-making machinery.

“This experiment couldn’t have been done even a few years ago,” Treutlein explained.

The mRNAs for neurexins are very long chains of nucleotides – the chemicals that encode genetic information. Only recently have instruments been capable of reading the exact sequence of such long nucleotide chains.

The ability to read the entire sequence of each mRNA was essential because neurexins have 25 constituent parts. But not all of these parts are used each time neurons produce a copy of the protein. Isoforms of neurexin have different combinations of these 25 possible parts. This experiment was designed to discover how many isoforms of neurexin existed and how prevalent each of these isoforms was.

The researchers analyzed more than 25,000 full-length neurexin mRNAs. They found 450 variants. Each variant omitted one or more of the 25 possible components. Most of these isoforms occurred infrequently. A handful accounted for the predominant isoforms.

Although the Stanford scientists sequenced 25,000 mRNAs to discover 450 variants, they believe that if they were to sequence even more mRNAs they would discover more isoforms – their estimate is that at least 2,500 isoforms of the neurexin family exist.

“The fact that we see so many isoforms supports the theory that these protein variants contribute to the huge diversity of synaptic connections that neuroscientists have observed,” Treutlein said.

The experiment raises many questions for future study. For instance, what functions are performed by the predominant isoforms versus the rare variants; how does the inclusion or exclusion of components affect that isoform and the synapse in which it works?

“This experiment was like a flight over the terrain,” Gokce said. “Now we have to go down and look at the details.”

Human brain development is a symphony in three movements

The human brain develops with an exquisitely timed choreography marked by distinct patterns of gene activity at different stages from the womb to adulthood, Yale researchers report in the Dec. 26 issue of the journal Neuron.

The Yale team conducted a large-scale analysis of gene activity in cerebral neocortex —an area of the brain governing perception, behavior, and cognition — at different stages of development. The analysis shows the general architecture of brain regions is largely formed in the first six months after conception by a burst of genetic activity, which is distinct for specific regions of the neocortex. This rush is followed by a sort of intermission beginning in the third trimester of pregnancy. During this period, most genes that are active in specific brain regions are quieted — except for genes that spur connections between all neocortex regions. Then in late childhood and early adolescence, the genetic orchestra begins again and helps subtly shape neocortex regions that progressively perform more specialized tasks, a process that continues into adulthood.

The analysis is the first to show this “hour glass” sketch of human brain development, with a lull in genetic activity sandwiched between highly complex patterns of gene expression, said Nenad Sestan, professor of neurobiology at Yale’s Kavli Institute for Neuroscience and senior author of the study. Intriguingly, say the researchers, some of the same patterns of genetic activity that define this human “hour glass” sketch were not observed in developing monkeys, indicating that they may play a role in shaping the features specific to human brain development.

The findings emphasize the importance of the proper interplay between genes and environment in the child’s earliest years after birth when the formation of synaptic connections between brain cells becomes synchronized, which shape how brain structures will be used later in life, said Sestan. For instance, disruptions of in synchronization of synaptic connections during child’s earliest years have been implicated in autism.

Sestan says the human brain is more like a neighorhood, which is better defined by the community living within its borders than its buildings.

“The neighborhoods get built quickly and then everything slows down and the neocortex focuses solely on developing connections, almost like an electrical grid,” said Sestan.  “Later when these regions are synchronized, the neighborhoods begin to take on distinct functional identities like Little Italy or Chinatown.”

Flip of a single molecular switch makes an old brain young

The flip of a single molecular switch helps create the mature neuronal connections that allow the brain to bridge the gap between adolescent impressionability and adult stability. Now Yale School of Medicine researchers have reversed the process, recreating a youthful brain that facilitated both learning and healing in the adult mouse.

Scientists have long known that the young and old brains are very different. Adolescent brains are more malleable or plastic, which allows them to learn languages more quickly than adults and speeds recovery from brain injuries. The comparative rigidity of the adult brain results in part from the function of a single gene that slows the rapid change in synaptic connections between neurons.

By monitoring the synapses in living mice over weeks and months, Yale researchers have identified the key genetic switch for brain maturation a study released March 6 in the journal Neuron. The Nogo Receptor 1 gene is required to suppress high levels of plasticity in the adolescent brain and create the relatively quiescent levels of plasticity in adulthood.  In mice without this gene, juvenile levels of brain plasticity persist throughout adulthood. When researchers blocked the function of this gene in old mice, they reset the old brain to adolescent levels of plasticity.

“These are the molecules the brain needs for the transition from adolescence to adulthood,” said Dr. Stephen Strittmatter. Vincent Coates Professor of Neurology, Professor of Neurobiology and senior author of the paper. “It suggests we can turn back the clock in the adult brain and recover from trauma the way kids recover.”

Rehabilitation after brain injuries like strokes requires that patients re-learn tasks such as moving a hand. Researchers found that adult mice lacking Nogo Receptor recovered from injury as quickly as adolescent mice and mastered new, complex motor tasks more quickly than adults with the receptor.

“This raises the potential that manipulating Nogo Receptor in humans might accelerate and magnify rehabilitation after brain injuries like strokes,” said Feras Akbik, Yale doctoral student who is first author of the study.

Researchers also showed that Nogo Receptor slows loss of memories.  Mice without Nogo receptor lost stressful memories more quickly, suggesting that manipulating the receptor could help treat post-traumatic stress disorder.

“We know a lot about the early development of the brain,” Strittmatter said, “But we know amazingly little about what happens in the brain during late adolescence.”

Definitive proof for receptor’s role in synapse development

Jackson Laboratory researchers led by Associate Professor Zhong-wei Zhang, Ph.D., have provided direct evidence that a specific neurotransmitter receptor is vital to the process of pruning synapses in the brains of newborn mammals.

Faulty pruning at this early developmental stage is implicated in autism-spectrum disorders and schizophrenia. The definitive evidence for N-methyl-D-aspartate receptor (NMDAR) in pruning has eluded researchers until now, but in research published in the Proceedings of the National Academy of Sciences, Zhang’s lab had serendipitous help in the form of a mouse model containing brain cells lacking NMDAR side-by-side with cells containing the receptor.

Soon after birth, mammals’ brains undergo significant development and change. Initially, large numbers of synapses form between neurons. Then, in response to stimuli, the synaptic connections are refined—some synapses are strengthened and others eliminated, or pruned.

In most synapses, glutamate serves as the neurotransmitter, and NMDAR, a major type of post-synaptic glutamate receptor, was previously known to play an important role in neural circuit development. Previous research has implicated the importance of NMDARs in pruning, but it remained unclear whether they played a direct or indirect role.

Zhang and colleagues focused on the thalamus, a brain region where synapse pruning and strengthening can be monitored and quantified with relative ease. They got unexpected help when they realized the mouse model they were using had thalamus cells lacking NMDARs right next to cells with normal NMDAR levels.

The researchers showed that the refinement process was disrupted in the absence of NMDARs. At the same time, neighboring neurons with the receptors proceeded through normal synaptic strengthening and pruning, clearly establishing the necessity of NMDARs in postsynaptic neurons for synaptic refinement.

"Whenever I give a talk or meet colleagues," Zhang says, "the first question that comes up is whether the NMDA receptor is important. It’s good that this is now settled definitively."

There has been extensive research into synaptic strengthening, and most of these studies indicate that the presence of NMDARs may support the recruitment of larger numbers of another kind of glutamate receptor to strengthen the synaptic connections. How NMDARs regulate the pruning process remains largely unknown, however.