Receptor Linked to Brain Development, May Play Role in Alzheimer’s

Defects in Astrocyte Formation Could Contribute to Disease

In its earliest moments, the developing brain is a formless array of precursor cells, each facing an ultimate decision. Either become a -message-bearing neuron or join the ranks of that broadly defined class of helper cells, the glia. The majority will follow the latter course—glial cells are thought to outnumber neurons by as much as 10 to one. Of these, most will become star-shaped astrocytes. Yet precursor cells develop into glia only after the neurons have started forming, which has led to speculation. Are the precursors simply receiving their astrocyte-determining signal after the neurons receive their direction? Or does the fate-determining message arrive promptly but get repressed, perhaps by another signal?

(clockwise from left) Gabriel Corfas, Samir Koirala, Joshua Murtie, and S. Pablo Sardi
Photo by Graham Ramsay

Astrocyte formation entails a novel and surprising pathway. The findings by (clockwise from left) Gabriel Corfas, Samir Koirala, Joshua Murtie, and S. Pablo Sardi could have implications for Alzheimer’s and other neurodegenerative diseases.

The results of a four-year study suggest the latter may be the case. Gabriel Corfas, S. Pablo Sardi, and colleagues report that a well-known messenger, neuregulin, represses a growth factor known to stimulate astrocyte formation. And it does so by setting in motion a lively sequence of events, beginning with the activation of a quirky receptor protein, erbB4. Most receptors relay messages by a phosphorylation cascade, but erbB4 can take a less conventional route. Once activated, it breaks off just inside the membrane. The Corfas team found that the cleaved bit teams up with at least two other proteins and travels directly to the nucleus, where it turns off a key astrocyte-determining gene.

This sequence appears to play out in mammals. Mice lacking the erbB4 gene produced astrocytes earlier in embryonic development than did controls. Astrocyte development could be put back on course by introducing the cleaved portion of erbB4. The findings appear in the Oct. 6 Cell.

Making the Cut
What makes the scenario especially intriguing is that the protein responsible for cleaving erbB4 is none other than presenilin, long implicated in Alzheimer’s disease. “Alterations in presenilin activity cause Alzheimer’s disease, but it is believed that this is primarily through the processing of amyloid precursor protein,” said Corfas, HMS associate professor of neurology at Children’s Hospital Boston. The new findings suggest that defects in presenilin activity could also work through the processing of erbB4.

This is not the first time erbB4 has been linked to Alzheimer’s. Plaques found in the brains of patients have already been shown to exhibit an abundance of the protein. The new findings suggest that presenilin-mediated defects in the processing of erbB4 could act early in a patient’s life. “This study raises the possibility that there is a developmental contribution to Alzheimer’s disease, that people who develop Alzheimer’s may have some essential weakness in the brain that makes it more susceptible to the accumulation of stress and insult during life, which can bring about neurodegeneration,” Corfas said. “Perhaps alterations in types or locations of astrocytes affect the susceptibility of neurons to insults.”

A Receptor with Legs
Astrocytes do more than protect neurons. They regulate the ionic balance in the extracellular environment, clean up excess neurotransmitters, induce the formation of synapses, and make the blood–brain barrier. An imbalance in the ratio of astrocytes to neurons, caused perhaps by defects in the erbB4 gene itself, could be devastating not just in early development but throughout life. Variations in the erbB4 gene have been linked to schizophrenia, a disease characterized by an overgrowth of synaptic connections in certain regions of the brain.

Illustration by Rachel Eastwood, based on original courtesy of S. Pablo Sardi

Twist of fate. Upon activation by neuregulin, erbB4 is cleaved extracellularly by TACE and intracellularly by presenilin. The erbB4 intracellular domain, E4ICD, pairs with TAB2, which in turn binds N-CoR. The trio travels to the nucleus and, with an unknown DNA-binding partner, turns off key astrocyte-determining genes.

Corfas; Sardi, an HMS research fellow in neurology; and their colleagues had no idea that erbB4 played a role in astrocyte differentiation back in 2002. What they did know, thanks in part to studies in the Corfas lab, is that erbB4 was a peculiar protein with the strange habit of splitting and translocating to the nucleus when activated. “That suggested that the intracellular domain of the receptor could have new and different ways of signaling, but it was not clear what, how, or where,” Corfas said.

Embarking on what would be an arduous quest, he and Sardi began looking for possible partners of the erbB4 intracellular domain (E4ICD). Using a classic yeast two-hybrid system, they netted several candidates, including TAB2. The protein looked promising—it was known to shuttle between the cytoplasm and nucleus, and it could bind to a known transcription repressor, N-CoR. They decided to test the pair in cultured mammalian cells and found that E4ICD did team up with TAB2, and that TAB2 bound N-CoR. The trio then moved to the nucleus.

Yet it was still not clear where—in which cells—the interaction was occurring. The researchers tested a variety of cells before focusing on neural precursors. They then reran all the previous experiments. The same erbB4–TAB2–N-CoR scenario played out in the neural precursor cells. They wanted to find out what effect the activation and subsequent translocation of erbB4 was having. “Generally when you activate a receptor tyrosine kinase like erbB4, something happens—cells proliferate, they die, they differentiate, they move,” said Corfas. He and Sardi exposed the neural precursor cells to erbB4’s preferred ligand, neuregulin. “Nothing happened,” he said. “It was a big disappointment.”

“This study raises the possibility that there is a developmental contribution to Alzheimer’s disease, that people who develop Alzheimer’s may have some essential weakness in the brain that makes it more susceptible to the accumulation of stress and insult during life.”

Reasoning that the situation might be more complex—that activation of erbB4 by neuregulin might be affecting how the neural precursor cells were responding to other signals—they exposed neural precursors to platelet-derived growth factor (PDGF), which is known to stimulate neurons to differentiate. They then added neuregulin. “Still nothing happened. That was another depressing point,” said Corfas. It was only when they added ciliary neurotrophic factor (CNTF), which promotes the differentiation of astrocytes, that they began to see a repressive effect.

Three years into their series of experiments, the researchers had pieced together a compelling tale, but it was still not clear if erbB4 activation was repressing astrocyte formation in living animals. It turns out, a viable erbB4-knockout mouse had been generated. Corfas and colleagues found that the brains of the animals exhibited precocious astrocyte development. Clearly erbB4 was important, but the protein is also capable of signaling by conventional phosphorylation. They wanted to show that erbB4 was carrying out its repressive effects by breaking off and traveling to the nucleus. “To do that, we had to do another series of tough experiments,” Sardi said.

Working with Joshua Murtie and Samir Koirala, HMS research fellows in neurology, they introduced two different forms of erbB4, normal and noncleavable, into the knockout embryos and waited to see which would be rescued. Only the cleavable form of erbB4 was able to rescue the mice and put astrocyte differentiation on a normal timetable. A final experiment showed that it does so single-handedly, without any phosphorylation cascade.

It is still not clear how defects in presenilin might affect the cleaving and translocation of erbB4. Making matters more interesting, erbB4 is likely to interact with proteins other than TAB2 and N-CoR. “Most probably this is quite a large complex, and we are working on identifying all the components,” said Corfas.