Stunted neuron branching restored in mice

In a new study in Neuron, Brown University researchers report that mutation of a gene associated with some autism forms in humans can hinder the proper growth and connectivity of brain cells in mice. They also show how that understanding allowed them to restore proper cell growth in the lab.

Brown University researchers have traced a genetic deficiency implicated in autism in humans to specific molecular and cellular consequences that cause clear deficits in mice in how well neurons can grow the intricate branches that allow them to connect to brain circuits. The researchers also show in their study (online Sep. 12, 2013, in Neuron) that they could restore proper neuronal growth by compensating for the errant molecular mechanisms they identified.

The study involves the gene that produces a protein called NHE6. Mutation of the gene is directly associated with a rare and severe autism-related condition known as Christianson syndrome. But scientists, including senior author Dr. Eric Morrow, have also associated the protein with more general autism.

“In generalized autism this protein is downregulated,” said Morrow, assistant professor of biology in the Department of Molecular Biology, Cellular Biology, and Biochemistry at Brown and a psychiatrist who sees autism patients at the Bradley Hospital in East Providence. “That meant to us that downregulation of NHE6 is relevant to a sizeable subset of autism.”

The NHE6 protein helps to regulate acidity in the endosomes of cells. These endosomes are responsible for transporting material around cells and for degrading proteins including ones that signal neurons to grow the elaborately branched axons and dendrites that form neural connections.

In their experiments the researchers measured acidity in the endosomes of brain cells of normal mice and in mice with mutations in the NHE6 gene. They found that the mutant mice had significantly higher endosome acidity. The mutant mice with the higher endosome acidity also had more degradation of a receptor protein, called TrkB, that responds a neurotrophic factor called BDNF. Together they signal axon and dendrite growth and branching.

Did the higher acidity and lower levels of TrkB signaling affect the neurons? Morrow and his colleagues were able to show directly in the mouse brain that the neuronal branching was diminished as were the number and maturity of connections between neurons, called synapses. Further still, working with co-author Julie Kauer, professor of medical science in the Department of Molecular Pharmacology, Physiology, and Biotechnology, they looked at synaptic and circuit function in the mice, and they found deficits corresponding to those anatomical findings.

“One of the overriding problems in disorders like autism, we think, is that it’s a problem of communication between different areas of the brain and neurons communicating with each other in networks,” said Morrow, who is affiliated with the Brown Institute for Brain Science.

Searching for a rescue

Having discovered a specific chain of events by which NHE6 mutations undermine neural branching and connectivity, Morrow and lead authors Qing Ouyang and Sofia Lizarraga sought to find out why and whether they could fix it.

Sometimes acidity in the endosome can activate protein-degrading enzymes called proteases. The team hypothesized that perhaps the acidity resulting from the absence of NHE6 was leading proteases to degrade TrkB, reducing its levels in mutant neurons compared to normal ones. When they treated mutant cells with a protease inhibitor called leupeptin, they found that the TrkB levels and signaling returned to levels close to those found in the normal cells.

Given that TrkB’s job is to bind with BDNF, the researchers also hypothesized that if the problem of NHE6 mutation was a reduction of TrkB, perhaps a suitable end-run around the problem would be to administer BDNF to cells directly. Indeed they found that NHE6 mutant cells, if given extra BDNF, produced axon and dendrite growth and branching that was more like normal neurons.

“In this paper we show that BDNF signaling is attenuated in the mutant mice, but it’s not blocked,” Morrow said. “You can rescue the [neuronal growth] by turning up the signaling.”

There are already drugs developed to deliver doses of chemicals that increase or mimic BDNF in the body, Morrow said, but many more tests beyond this study would have to be done before scientists and doctors could know whether a BDNF-related drug could have a therapeutic effect for patients with Christianson syndrome or any related form of autism.

“We don’t think that this is everything about the condition,” Morrow said. “But if we were able to treat this one mechanism by adding exogenous drug, would it repair enough or some element of it?”

Christianson syndrome and perhaps only a subset of autism appears to relate to deficits in neural branching. Some forms of autism, in fact, may result from too much branch growth. Moreover, doctors have no precise ways to tell whether a child diagnosed with autism has too much or too little neural branching.

But given the study results suggesting that NHE6 may play a role in some autism forms perhaps by hindering neural branching, the new research suggests a target for addressing it.

How neural stem cells create new and varied neurons

A new study examining the brains of fruit flies reveals a novel stem cell mechanism that may help explain how neurons form in humans. A paper on the study by researchers at the University of Oregon appeared in the online version of the journal Nature in advance of the June 27 publication date.

"The question we confronted was ‘How does a single kind of stem cell, like a neural stem cell, make all different kinds of neurons?’" said Chris Doe, a biology professor and co-author on the paper "Combinatorial temporal patterning in progenitors expands neural diversity."

Researchers have known for some time that stem cells are capable of producing new cells, but the new study shows how a select group of stem cells can create progenitors that then generate numerous subtypes of cells.

"Instead of just making 100 copies of the same neuron to expand the pool, these progenitors make a whole bunch of different neurons in a particular way, a sequence," Doe said. "Not only are you bulking up the numbers but you’re creating more neural diversity."

The study, funded by the Howard Hughes Medical Institute and the NIH National Institute of Child Health and Human Development, builds on previous research from the Doe Lab published in 2008. That study identified a special set of stem cells that generated neural progenitors. These so-called intermediate neural progenitors (INPs) were shown to blow up into dozens of new cells. The research accounted for the number of cells generated, but did not explain the diversity of new cells.

"While it’s been known that individual neural stem cells or progenitors could change over time to make different types of neurons and other types of cells in the nervous system, the full extent of this temporal patterning had not been described for large neural stem cell lineages, which contain several different kinds of neural progenitors," said lead author Omer Bayraktar, a doctoral student in developmental neurobiology who recently defended his dissertation.

The cell types in the study, Bayraktar said, have comparable analogs in the developing human brain and the research has potential applications for human biologists seeking to understand how neurons form.

The Nature paper appears alongside another study on neural diversity by researchers from New York University. Together the two papers provide new insight into the processes involved in producing the wide range of nerve cells found in the brains of flies.

For their study, Bayraktar and Doe zeroed in on the stem cells in drosophila (fruit flies) known as type II neuroblasts. The neuroblasts, which had previously been shown to generate INPs, were shown in this study to be responsible for a more complex patterning of cells. The INPs were shown to sequentially generate distinct neural subtypes. The research accounted for additional neural diversity by revealing a second axis in the mechanism. Instead of making 100 neurons, as had been previously thought, a stem cell may be responsible for generating some 400 or 500 neurons.

The study concludes that neuroblasts and INP patterning act together to generate increased neural diversity within the central complex of the fruit fly and that progenitors in the human cerebral cortex may use similar mechanisms to increase neural diversity in the human brain. One long-term application of the research may be to eventually pinpoint stem cell treatments to target specific diseases and disorders.

"If human biologists understand how the different types of neurons are made, if we can tell them ‘This is the pathway by which x, y and z neurons are made,’ then they may be able to reprogram and redirect stem cells to make these precise neurons," Doe said.

The mechanism described in the paper has its limits. Eventually the process of generating new cells stops. One of the next questions to answer will be what makes the mechanism turn off, Doe said.

"This vital research will no doubt capture the attention of human biologists," said Kimberly Andrews Espy, vice president for research and innovation and dean of the UO graduate school. "Researchers at the University of Oregon continue to further our understanding of the processes that undergird development to improve the health and well-being of people throughout the world."