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Front Page

Blocking Protein Might Reverse Hearing Loss

Well-known Cell-cycle Regulator Prevents Regrowth of Inner Ear Hair Cells in Mammals

In the inner ear, bundles of tiny hairs on specialized cells are essential for converting sound vibrations into electrical signals the brain can understand. Like neurons, these cells are terminally differentiated, and once lost, they cannot be renewed. At least not yet. Now HMS researchers led by Zheng-Yi Chen, assistant professor of neurology at Massachusetts General Hospital, have discovered the signal that prevents these cells from dividing. It turns out to be a single and very familiar cell-cycle regulator, the retinoblastoma protein. The findings, described in the Jan. 13 online Science (doi:10.1126/science.1106642), could someday lead to new approaches for treating deafness.

Retinoblastoma prevents hair cell division. In the mouse embryo at left, the cell division marker PCNA (light green) is absent in the three outer hair cells (OHC), one inner hair cell (IHC), and support cells (SC) of the inner ear, indicating that these cells are not dividing. Ablating the retinoblastoma gene, however, causes rampant cell cycling and a surge in PCNA, shown at right. (Image courtesy of Zheng-Yi Chen)

Every person is born with about 17,000 delicate, easily damaged inner ear hair cells. Over the years, attrition of both the cells and their hair bundles leads to hearing loss in about 30 percent of those over 65 and about 50 percent of those over 75. For this reason, finding out how to repair and replenish these cells has been the focus of a core group of researchers at HMS including David Corey—co-author on the Science paper—who studies how hair cells convert mechanical into electrical energy (see Focus, Oct. 15, 2004), and Stefan Heller, who has found that the inner ear is a rich source of progenitor cells (see Focus, Sept. 26, 2003). While fixing damaged cells or implanting new ones may turn out to be a viable means of restoring hearing in some patients, an alternative may be to coax the cells already there to divide.

“In lower vertebrates like fish, amphibians, and chicks, inner ear hair cells spontaneously regenerate if they are damaged, so we wondered what might prevent this from happening in mammals,” said Chen.

Hair bundles function without retinoblastoma. Hair bundles in the inner ear, imaged by differential interference contrast microscopy (left), take up the the dye FM1-43 (right) through the mechanotransduction channel. (Image courtesy of Zheng-Yi Chen)

The Inside Story
To figure this out, research fellows Cyrille Sage, Mingqian Huang, and colleagues turned to the developing mouse inner ear. There, hair cells are derived from the sensory progenitor cells at around day 12 of embryonic development. To find the switch that regulates this process, they used oligonucleotide microarrays to determine exactly when thousands of genes are switched on and off in the embryonic mouse ear. “We found a pattern of expression for the retinoblastoma family members that correlated very nicely with development and maturation of the hair cells,” explained Chen. More specifically, they found that retinoblastoma 1 (Rb1) was very poorly expressed up to the 12th day of the embryo, but was upregulated from embryonic day 14, just when the full complement of inner ear hair cells has been established. The finding suggested that the retinoblastoma protein, well known for its ability to block progression of the cell cycle, may be the switch that prevents the growth and renewal of inner ear hair cells.

Once lost, hair cells in the inner ear are not naturally replenished, a major cause of deafness. But Cyrille Sage, Mingqian Huang, and Zheng-Yi Chen (left to right) have found a way to make these cells reproduce. (Photo by Steve Gilbert)

To find out where the extra cells were coming from, the researchers let the embryos develop in the presence of BrdU, a nucleotide analog that is incorporated into the DNA of dividing cells. Two days later, on examining the embryo inner ear, they found that the hair cells themselves, in addition to support cells, had taken up the label. Hair cells were, in fact, dividing. This was confirmed when the authors labeled inner ear cells with an antibody to PCNA, or proliferating cell nuclear antigen, a protein that is associated with dividing cells (see figure above). “This was very exciting for us, to see that the hair cells had the capacity to keep dividing,” said Chen.

Getting to Work
Of course, having cells divide is one thing; having the progeny be useful is another. Neurons, for example, can be forced to re-enter the cell cycle, but instead of making new working neurons, they usually enter an apoptotic spiral. In the case of inner ear hair cells, however, this does not seem to be the case because Chen and colleagues failed to find any signs of apoptosis, such as activation of pro-apoptotic proteases. What’s more, the cells appeared to express the right hair cell markers at the right time during development, suggesting that they were maturing normally and forming synapses with neurons. “But while you can measure differentiation of these cells whatever way you like, ultimately, you want to know whether they are really functional or not,” explained Chen.

“This was very exciting for us, to see that the hair cells had the capacity to keep dividing.”

The function of a cochlear hair cell is to convert mechanical into electrical energy. As the cells sway back and forth in the sound wave, mechanical stress forces open a channel in the hair bundles that allows an influx of calcium (see Focus, Oct. 15, 2004). The resulting current activates adjacent neurons. The central questions for the researchers were, would the channel work in Rb1-negative hair cells and would the cells mount a current in response? The answer to both seems to be yes. Rb1-negative hair cells took up the dye FM1-43, for example, which enters through the functional channel (see figure below), and they also generated currents, though significantly smaller than those in control cells. This weakness, however, might be explained by the known lag between cell division and hair bundle development.

What are the chances that shutting off the retinoblastoma protein could help generate new inner ear hair cells in adults? To answer this question, Chen and colleagues turned to an in vitro system, growing utricles from postnatal mice. “All the hair cells in these utricles are postmitotic and are both morphologically and functionally mature,” said Chen. To turn off the retinoblastoma gene, the researchers infected the utricles with adenoviruses carrying DNA engineered to ablate the Rb1 gene in infected cells. These cells then began taking up the DNA marker, BrdU, indicating that they had re-entered the cell cycle and had started to divide. “The only problem was that we couldn’t measure if these cells are functional because adenovirus is actually toxic to these cells and damages the hair bundles,” said Chen.

Because mouse and human inner ears are almost identical, the next step will be to try regenerating hair cells in deaf mice to see if hearing can be restored. “If we can manage that, then we will have a good chance to achieve recovery in humans,” Chen said.

— Tom Fagan

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