ATOH1

A deafened adult cannot recover the ability to hear, because the sensory hearing cells of the inner ear don't regenerate after damage. In two new studies scientists explain why this is the case and how we might be able to change it. A deafened adult cannot recover the ability to hear, because the sensory hearing cells of the inner ear don't regenerate after damage. In two new studies, partially funded by the National Institutes of Health and published in the Proceedings of the National Academy of the Sciences (PNAS), USC Stem Cell scientists explain why this is the case and how we might be able to change it. "In the non-sensory supporting cells of the inner ear, key genes required for conversion to sensory cells are shut off through a process known as 'epigenetic silencing.' By studying how the genes are shut off, we begin to understand how we might turn them back on to regenerate hearing," said John Duc Nguyen, the first author of one of the papers. Nguyen now works at the biotech company Genentech and earned his PhD in the USC Stem Cell laboratory of Neil Segil, who passed away from pancreatic cancer in 2022. The second paper explored when and how the ability to form sensory hearing cells is gained in the inner ear in the first place and describes two specific genes that could be useful for regenerating hearing in adults. "We focused on the genes Sox4 and Sox11 because we found that they are necessary for forming sensory hearing cells during development," said the paper's first author Emily Xizi Wang, who also conducted her research as a PhD student in the Segil Lab and works at the biotech company Atara Biotherapeutics. Gage Crump, a co-author on both papers and the interim chair of USC's Department of Stem Cell Biology and Regenerative Medicine at the Keck School of Medicine of USC, added: "These two papers are not only great science, but also a clear example of Neil Segil's enduring legacy as an exceptional mentor to the next generation of stem cell researchers." Silencing isn't golden One important way that genes are shut off or "silenced" involves chemical compounds called methyl groups that bind to DNA and make it inaccessible -- the focus of Nguyen's paper. When the DNA that instructs a cell to become a sensory hearing cell is methylated, the cell cannot access these instructions. Through their experiments with non-sensory supporting cells extracted from the inner ears of mice, Nguyen and his colleagues confirmed that DNA methylation silences genes that promote conversion into sensory hearing cells, including the gene Atoh1 that is known to be a master regulator of inner ear development. An enzyme called TET can remove methyl groups from the DNA, thus reversing the gene silencing and restoring the capacity of supporting cells to convert into sensory hair cells. Accordingly, when the scientists blocked the activity of TET, the supporting cells retained their DNA methylation and therefore could not convert into sensory hair cells in the Petri dish. Intriguingly, in a separate experiment, the researchers tested the extent of gene silencing in supporting cells from a chronically deafened mouse. They found that gene silencing was partially reversed, meaning that the supporting cells had the capacity to respond to signals to transform into sensory hearing cells. This finding has important implications: the loss of sensory hearing cells itself might partially reverse gene silencing in supporting cells in chronically deaf individuals. If so, the supporting cells of chronically deaf individuals might already be naturally primed to convert into sensory hearing cells. Segil's longtime collaborator Andrew K. Groves from the Baylor College of Medicine served as the paper's corresponding author. Knocking their Sox off In the second paper, Wang and her colleagues explored when and how the progenitor cells of the inner ear gain the ability to form sensory hearing cells. The scientists pinpointed when progenitor cells acquire this ability: between days 12 and 13.5 of embryonic development in mice. During this window, the progenitor cells acquire the capacity to respond to signals from the master regulator gene Atoh1 that triggers the formation of sensory hearing cells later during development. What primes the progenitor cells to respond to Atoh1 are two additional genes, Sox4 and Sox11, that change the state of these cells. In embryonic mice lacking Sox4 and Sox11, progenitor cells in the inner ear failed to develop into sensory hearing cells. Specifically, a loss of Sox4 and Sox11 made the cells' DNA inaccessible -- an effect similar to DNA methylation. With their DNA inaccessible, the progenitor cells couldn't respond to signals from Atoh1. On the flip side, high levels of Sox4 and Sox11 activitystimulated mouse progenitor cells and supporting cells to form sensory hearing cells in a Petri dish. More promising still, in mice with damaged sensory cells in the inner ear, high levels of Sox4 and Sox11 activity increased the percentage of vestibular supporting cells that converted into sensory receptor cells -- from 6 percent to 40 percent. "We're excited to continue exploring the mechanisms by which cells in the inner ear gain the ability to differentiate as sensory cells during development and how these can be used to promote the recovery of sensory hearing cells in the mature inner ear," said the paper's corresponding author Ksenia Gnedeva, who completed her postdoctoral training in the Segil Lab and is now an assistant professor in the USC Tina and Rick Caruso Department of Otolaryngology -- Head and Neck Surgery, and the Department of Stem Cell Biology and Regenerative Medicine. About the studies Juan Llamas from the Segil lab and Tuo Shi from the Crump lab are co-authors of both studies. For Wang and Gnedeva's paper, additional co-authors include Talon Trecek, Litao Tao, and Welly Makmura from the Segil lab. Both studies were supported by the National Institutes of Health (NIH grant RO1 DC015829), as well as a Hearing Restoration ProjectConsortium award from the Hearing Health Foundation. Nguyen and Groves' study received support from three additional NIH grants (F31 DC018703, T32 HD060549, and RO1 DC014832), and Wang and Gnedeva's study received support from two additional NIH grants (R21 DC016984 and T32DC009975).

The researchers looked closer into the possibility of reprograming inner ear cells to repair long-term hearing loss. About 430 million people around the world experience disabling hearing loss. In the United States, approximately 37.5 million adults report some trouble hearing. Hearing loss can happen when any part of the ear or the nerves that carry information on sounds to the brain do not work in the usual way. For instance, damaged hair cells in the inner ear can lead to hearing loss. "These cells allow the brain to detect sounds," said Dr. Amrita Iyer, first author of a new paper published in eLife. Iyer was a graduate student in the lab of Dr. Andrew Groves, professor and Vivian L. Smith Endowed Chair in Neuroscience and of molecular and human genetics at Baylor College of Medicine, while she was working on this project. Hair cells are generated during normal development but this ability is progressively lost after birth as mammals mature. "When hair cells are lost in mature animals, the cells cannot be naturally regenerated, which can lead to permanent hearing loss," Iyer explained. "In the current study, we looked closer into the possibility of promoting hair cell regeneration in mature animals using cell reprograming. Our approach involved the overexpression of various transcription factor combinations." Transcription factors promote the expression of certain genes and prevent the expression of others. By changing the pattern of gene expression, the researchers hoped to lead cells to a state in which they would regenerate hair cells in mature animals similarly to what happens during development. "We compared the reprogramming efficiency of the hair cell transcription factor ATOH1 alone or in combination with two other hair cell transcription factors, GFI1 and POU4F3, in mouse non-sensory cells in the cochlea, the part of the inner ear that supports hearing," Iyer said. "We did this at two timepoints -- eight days after birth and 15 days after birth, assessing the extent of hair cell regeneration in mice." To study the structure of the hair cell bundles generated by reprogramming, Iyer collaborated with Dr. Yeohash Raphael's lab at the University of Michigan to perform scanning electron microscopy imaging on the cochleae of mice conditionally overexpressing these transcription factors. The images clearly showed that the hair cell bundles were in accordance to what is observed on inner hair cells during development. Further studies showed that these cells also had some characteristics that suggested that they were capable of sensing sound. "We found that although expressing ATOH1 with hair cell transcription factors GFI1 and POU4F3 can increase the efficiency of hair cell reprogramming in older animals compared to ATOH1 alone or GFI1 plus ATOH1, the hair cells generated by reprogramming at eight days of age -- even with three hair cell transcription factors -- are significantly less mature than those generated by reprogramming at postnatal day one," Iyer said. "We suggest that reprogramming with multiple transcription factors is better able to access the hair cell differentiation gene regulatory network, but that additional interventions may be necessary to produce mature and fully functional hair cells." These findings are key to advancing the existing understanding of the mammalian inner ear hair cell regeneration process. From a therapeutic standpoint, transcription factor-mediated reprogramming and the underlying biology associated with its function may enable fine-tuning of current gene therapy approaches for long-term hearing loss treatment. Other contributors to this work include Ishwar Hosamani, John D. Nguyen, Tiantian Cai, Sunita Singh, Melissa M. McGovern, Lisa Beyer, Hongyuan Zhang, Hsin-I Jen, Rizwan Yousaf, Onur Birol, Jenny J. Sun, Russell S. Ray, Yehoash Raphael and Neil Segil. The authors are affiliated with one or more of the following institutions: Baylor College of Medicine, the University of Southern California and the University of Michigan. The project was supported by the following grants: RO1 DC014832, R21 OD025327, DC015829 and a Hearing Restoration Project Consortium award from the Hearing Health Foundation. The project was also supported by funding from a CPRIT Core Facility Support Award (CPRIT-RP180672), NIH grants (P30 CA125123, S10 RR024574, S10OD018033, S10OD023469, S10OD025240 and P30EY002520), the R. Jamison and Betty Williams Professorship, the University of Michigan College of Engineering and NSF grant #DMR-1625671.