Key Signaling Pathway That Makes Young Neurons Connect Discovered By Scientists

In the new study, the researchers identified a molecular program that controls an essential step in the fast-growing brains of young mammals. The researchers found that this signaling pathway spurs the growth of neuronal output connections by a mechanism called "mitochondrial capture," which has never been described before.

"Mutations that may affect this signaling pathway already have been found in some autism cases," said TSRI Professor Franck Polleux, who led the research, published June 20, 2013 in the journal Cell.

Branching Out

Polleux's laboratory is focused on identifying the signaling pathways that drive neural development, with special attention to the neocortex?a recently evolved structure that handles the "higher" cognitive functions in the mammalian brain and is highly developed in humans.

In a widely cited study published in 2007, Polleux's team identified a trigger of an early step in the development of the most important class of neocortical neurons. As these neurons develop following asymmetric division of neural stem cells, they migrate to their proper place in the developing brain. Meanwhile they start to sprout a root-like mesh of input branches called dendrites from one end, and, from the other end, a long output stalk called an axon. Polleux and his colleagues found that the kinase LKB1 provides a key signal for the initiation of axon growth in these immature cortical neurons.

In the new study, Polleux's team followed up this discovery and found that LKB1 also is crucially important for a later stage of these neurons' development: the branching of the end of the axon onto the dendrites of other neurons.

"In experiments with mice, we knocked the LKB1 gene out of immature cortical neurons that had already begun growing an axon, and the most striking effect was a drastic reduction in terminal branching," said Julien Courchet, a research associate in the Polleux laboratory who was a lead co-author of the study. "We saw this also in lab dish experiments, and when we overexpressed the LKB1 gene, the result was a dramatic increase in axon branching."

Further experiments by Courchet showed that LKB1 drives axonal branching by activating another kinase, NUAK1. The next step was to try to understand how this newly identified LKB1-NUAK1 signaling pathway induced the growth of new axon branches.

Stopping the Train in Its Tracks

Following a thin trail of clues, the researchers decided to look at the dynamics of microtubules. These tiny railway-like tracks are laid down within axons for the efficient transport of molecular cargoes and are altered and extended during axonal branching. Although they could find no major change in microtubule dynamics within immature axons lacking LKB1 or NUAK1, the team did discover one striking abnormality in the transport of cargoes along these microtubules. Tiny oxygen-reactors called mitochondria, which are the principal sources of chemical energy in cells, were transported along axons much more actively?and by contrast, became almost immobile when LKB1 and NUAK1 were overexpressed.

But the LKB1-NUAK1 signals weren't just immobilizing mitochondria randomly. They were effectively inducing their capture at points on the axons where axons form synaptic connections with other neurons.

"When we removed LKB1 or NUAK1 in cortical neurons, the mitochondria were no longer captured at these points," said Tommy Lewis, Jr., a research associate in the Polleux Laboratory who was co-lead author of the study.

"We argue that there must be an active 'homing factor' that specifies where these mitochondria stop moving," said Polleux. "And we think that this is essentially what the LKB1-NAUK1 signaling pathway does here."Looking Ahead

Precisely how the capture of mitochondria at nascent synapses promotes axonal branching is the object of a further line of investigation in the Polleux laboratory. "We think that we have uncovered something very interesting about mitochondrial function at synapses," Polleux said.

In addition to its basic scientific importance, the work is likely to be highly relevant medically. Developmentally related brain disorders such as epilepsy, autism and schizophrenia typically involve abnormalities in neuronal connectivity. Recent genetic surveys have found NUAK1-related gene mutations in some children with autism, for example. "Our study is the first one to identify that NUAK1 plays a crucial role during the establishment of cortical connectivity and therefore suggests why this gene might play a role in autistic disorder," Polleux says.

He notes, too, that declines in normal mitochondrial transport within axons have been observed in neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. "In the light of our findings, we wonder if the decreased mitochondrial mobility observed in these cases might be due not to a transport defect, but instead to a defect in mitochondrial capture in aging neurons," he said. "We're eager to start doing experiments to test such possibilities."

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2-step Mechanism of Inner Ear Tip Link Regrowth Discovered By Researchers

The discovery offers a possible mechanism for potential interventions that could preserve hearing in people whose hearing loss is caused by genetic disorders related to tip link dysfunction. The work was supported by the National Institute on Deafness and Other Communication Disorders (NIDCD), a component of the National Institutes of Health.

The findings appear in the June 11, 2013 online edition of PLOS Biology. The senior author of this study is Gregory I. Frolenkov, an associate professor in the College of Medicine at the University of Kentucky, Lexington, and his fellow, Artur A. Indzhykulian, Ph.D., is the lead author.

Stereocilia are bundles of bristly projections that extend from the tops of sensory cells, called hair cells, in the inner ear. Each stereocilia bundle is arranged in three neat rows that rise from lowest to highest like stair steps. Tip links are tiny thread-like strands that link the tip of a shorter stereocilium to the side of the taller one behind it. When sound vibrations enter the inner ear, the stereocilia, connected by the tip links, all lean to the same side and open special channels, called mechanotransduction channels. These pore-like openings allow potassium and calcium ions to enter the hair cell and kick off an electrical signal that eventually travels to the brain where it is interpreted as sound.

The findings build on a number of recent discoveries in laboratories at NIDCD and elsewhere that have carefully plotted the structure and function of tip links and the proteins that comprise them. Earlier studies had shown that tip links are made up of two proteins -- cadherin-23 (CDH23) and protocadherin-15 (PCDH15) - that join to make the link, with PCDH15 at the bottom of the tip link at the site of the mechanotransduction channel, and CDH23 on the upper end. Scientists assumed that the assembly was static and stable once the two proteins bonded.

Tip links break easily with exposure to noise. But unlike hair cells, which can't regenerate in humans, tip links repair themselves, mostly within a matter of hours. The breaking of tip links, and their regeneration, has been known about for many years, and is seen as one of the causes of the temporary hearing loss you might experience after a loud blast of sound (or a loud concert). Once the tip links regenerate, hair cell function returns usually to normal levels. What scientists didn't know was how the tip link reassembled.

To study tip link assembly, the researchers treated young, postnatal (5-7 days) mouse sensory hair cells with BAPTA - a substance that, like loud noise, damages and disrupts tip links. To image the proteins, the group pioneered an improved scanning electron microscopy (SEM) technique of immunogold labeling that uses antibodies bound to gold particles that attach to the proteins. Then using SEM they imaged the cells at high resolution to determine the positions of the proteins before, during, and after BAPTA treatment.

What the researchers found was that after a tip link is chemically disrupted, a new tip link forms, but instead of the normal combination of CDH23 and PCDH15, the link is made up of PCDH15 proteins at both ends. Over the next 24 hours, the PCDH15 protein at the upper end is replaced by CDH23 and the tip link is back to normal.

Why tip links regenerate using a two-step instead of a neat one-step process is not known. For reasons that are still unclear, CDH23 disappears from stereocilia after noise damage while PDCH15 stays around. Looking to regenerate quickly, the lower PDCH15 latches onto another PDCH15, forming a shorter and functionally slightly weaker tip link. Later, at some time during the 36 hours after the damage, when CDH23 returns, PDCH15 gives up its provisional partner and latches onto its much stronger mate in CDH23. In other words, PDCH15 prefers to be with CDH23, but in a pinch it will bond weakly with another bit of PDCH15 until CDH23 shows up.

The researchers coupled the SEM observations with electrophysiology studies to show how the functional properties of the tip links changed throughout this two-step process. The temporary PCDH15/PCDH15 tip link has a slightly different functional response than the permanent PDCH15/CDH23 combination. Researchers were able to correlate the differences in function with the protein combinations that make up the tip link.

Additional experiments revealed that when hair cells develop, the tip links use the same two-step process.

Previous research has shown that both CDH23 and PCDH15 are required for normal hearing and vision. In fact, NIDCD scientists in earlier studies have shown that mutations in either of these genes can cause the hearing loss or deaf-blindness found in Usher Syndrome types 1D and 1F.

"In the case of deaf individuals who are unable to make functional CDH23, knowledge of this new temporary alliance of PCDH15 proteins to form a weaker, but still functional, tip link could inform treatments that would encourage the double PCDH15 bond to become permanent and maintain at least limited hearing," said Tom Friedman, Ph.D., chief of the Laboratory of Molecular Genetics at NIDCD where the research began.

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Gene That Controls Aggressiveness in Breast Cancer Cells Discovered

The researchers, whose findings are published this week in the journal Cell, report that the ZEB1 gene is held in a poised state in basal non-CSCs, such that it can readily respond to environmental cues that consequently drive those non-CSCs into the dangerous CSC state. Basal-type breast carcinoma is a highly aggressive form of breast cancer. According to a 2011 epidemiological study, the 5-year survival rate for patients with basal breast cancer is 76%, compared with a roughly 90% 5-year survival rate among patients with other forms of breast cancer.

"We may have found a root source, maybe the root source, of what ultimately determines the destiny of breast cancer cells-their future benign or aggressive clinical behavior," says Whitehead Founding Member Robert Weinberg, who is also a professor of biology at MIT and Director of the MIT/Ludwig Center for Molecular Oncology.

Transcription factors are genes that control the expression of other genes, and therefore have a significant impact on cell activities. In the case of ZEB1, it has an important role in the so-called epithelial-to-mesenchymal transition (EMT), during which epithelial cells acquire the traits of mesenchymal cells. Unlike the tightly-packed epithelial cells that stick to one another, mesenchymal cells are loose and free to move around a tissue. Previous work in the Weinberg lab showed that adult cancer cells passing through an EMT are able to self-renew and to seed new tumors with high efficiency, hallmark traits of CSCs.

Other earlier work led by Christine Chaffer, a postdoctoral researcher in the Weinberg lab, demonstrated that cancer cells are able to spontaneously become CSCs. Now Chaffer and Nemanja Marjanovic have pinpointed ZEB1, a key player in the EMT, as a gene critical for this conversion in breast cancer cells.

Breast cancers are categorized into at least five different subgroups based on their molecular profiles. More broadly these groups can be subdivided into the less aggressive ‘luminal'' subgroup or more aggressive ‘basal'' subgroup. The aggressive basal-type breast cancers often metastasize, seeding new tumors in distant parts of the body. Patients with basal breast cancer generally have a poorer prognosis than those with the less aggressive luminal-type breast cancer.

Chaffer and Marjanovic, a former research assistant in the Weinberg lab, studied non-CSCs from luminal- and basal-type cancers and determined that cells from basal cancers are able to switch relatively easily into CSC state, unlike luminal breast cancer cells, which tend to remain in the non-CSC state.

The scientists determined that the difference in ZEB1''s effects is due to the way the gene is marked in the two types of cancers. In luminal breast cancer cells, the ZEB1 gene is occupied with modifications that shut it down. But in basal breast cancer cells, ZEB1''s state is more tenuous, with repressing and activating markers coexisting on the gene. When these cells are exposed to certain signals, including those from TGFs, the repressive marks are removed and ZEB1 is expressed, thereby converting the basal non-CSCs into CSCs.

So what does this new insight mean for treating basal breast cancer?

"Well, we know that these basal breast cancer cells are very plastic and we need to incorporate that kind of thinking into treatment regimes," says Chaffer. "As well as targeting cancer stem cells, we also need to think about how we can prevent the non-cancer stem cells from continually replenishing the pool of cancer stem cells. For example, adjuvant therapies that inhibit this type of cell plasticity may be a very effective way to keep metastasis at bay."

Marjnaovic agrees but cautions that the model may not be applicable for every cancer.

"This is an example of how adaptable cancer cells can be,," says Marjanovic, who is currently a research assistant at the Broad Institute. "We have yet to determine if ZEB1 plays a similar role in all cancer types, but the idea that cancer cells reside in a poised state that enables them to adapt to changing environments may be a mechanism used by many cancers to increase their aggressiveness."

This work is supported Vertex Scholars Program, the National Science Foundation (NSF), Jerome and Florence Brill Fellowship, Croucher and Ludwig Research Fellowship, the National Institutes of Health (NIH) (1 F32 GM099153-01A1, HD 045022 and R37CA084198).

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