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Improved TMC1 Gene Therapy Restores Hearing and Balance in Mice

By Christopher Geissler, Ph.D.

Half of all inner ear disorders, which have a negative impact on hearing and/or balance, are caused by genetic mutations. A study published in January 2019 in Nature Communications demonstrates the effectiveness of a gene therapy targeting one specific gene mutation, TMC1 (transmembrane channel-like 1). The research was conducted by Carl A. Nist-Lund in the Harvard Medical School lab of Gwenaëlle S. Géléoc, Ph.D., and Jeffrey R. Holt, Ph.D., with contributions from colleagues including 2017 Emerging Research Grants (ERG) recipient Jennifer Resnik, Ph.D., and her ERG co-principal investigator Daniel B. Polley, Ph.D., both also of Harvard Medical School.

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So far, 35 TMC1 mutations have been identified in humans, including several that are responsible for moderate to severe hearing loss, representing between 3 to 8 percent of cases of genetic hearing loss. This TMC1 gene therapy has had an encouraging level of success in mice and may prove capable of addressing similar genetic mutations in humans in the future.

Previous studies targeting this gene were only moderately successful in restoring function in inner hair cells, with little or no success in outer hair cells. Both types of hair cell are necessary for hearing.

The team decided to look at improving the mechanism that encodes TCM1 in affected mice, using a synthetic delivery vehicle they hoped would be more effective than the conventional one used in previous studies. In mice with this TCM1 mutation, hair cells begin to die when the mouse reaches 4 weeks of age. The treated mice in this study showed improved rates of survival in both inner and outer hair cells.

Most importantly, the improvement in hearing in the mice that received this intervention occurred primarily in the lower frequencies. Human speech is at the low to mid frequency range of the auditory spectrum, so if future human trials are able to replicate the success of this study, speech perception may improve.

The study additionally provided evidence of improved responses in the brain of the treated mice. This indicates that treatment of the cochlea by injection had knock-on effects in the auditory cortex, the part of the brain that plays an important role in hearing.

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Finally, the team recorded improved balance function in the mice that received the gene therapy. While only very young mice experienced better hearing, even older mice showed improvement in balance. The team writes that this improvement in balance function in mature mice may contribute to eventually developing a way to treat balance disorders in humans.

Jennifer Resnik, Ph.D., is a postdoctoral fellow in the Polley Lab, part of the Eaton Peabody Laboratories, Massachusetts Eye and Ear/Harvard Medical School. Her 2017 Emerging Research Grant was generously funded by Hyperacusis Research Ltd. Christopher Geissler, Ph.D., is HHF’s director of program and research support.

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Single-Cell RNA Sequencing Reveals More Clues for Hair Cell Regeneration

By Mark E. Lush, Ph.D., and Daniel C. Diaz

Sensorineural hearing loss in mammals can often be attributed to damage or destruction of the delicate hair cells located within the inner ear. The microscopic hairlike projections on the surface of these cells are the key structure responsible for converting sound waves to electrical signals that travel to the brain through the auditory nerve. Unlike mammals, other vertebrates such as fish, birds, and reptiles routinely regenerate sensory hair cells during homeostasis and following injury. By studying the genetic program of hair cell regeneration in nonmammalian vertebrate organisms, researchers may discover therapeutic targets for treating hearing loss in humans.

The lateral line is a sensory system that allows aquatic vertebrates to orient themselves by detecting water motion. The lateral line organs (neuromasts), distributed on the head and along the body, contain approximately 60 cells, composed of central sensory hair cells surrounded by support cells and an outer ring of mantle cells. Using single-cell RNA sequencing, we combined some of the less well-defined clusters and identified major neuromast cell types, shown in this illustration, ranging from support cells to mature sensory hair cells. Credit: The lab of Tatiana Piotrowski, Ph.D., Stowers Institute for Medical Research, Kansas City

The lateral line is a sensory system that allows aquatic vertebrates to orient themselves by detecting water motion. The lateral line organs (neuromasts), distributed on the head and along the body, contain approximately 60 cells, composed of central sensory hair cells surrounded by support cells and an outer ring of mantle cells. Using single-cell RNA sequencing, we combined some of the less well-defined clusters and identified major neuromast cell types, shown in this illustration, ranging from support cells to mature sensory hair cells. Credit: The lab of Tatiana Piotrowski, Ph.D., Stowers Institute for Medical Research, Kansas City

One such organism, the zebrafish, has emerged as a powerful model for studying sensory hair cell regeneration. Like other fish, zebrafish contain a network of sensory hair cells throughout their body to detect changes in water movement. The hair cells are located in small organs in the skin called neuromasts, which also contains cell types that are remarkably similar to those found in the mammalian inner ear. To study the genetic program of hair cell regeneration in zebrafish, we sequenced the RNA of individual cells within neuromasts, allowing us to classify cell types based on their gene expression signature. This included cells transitioning from support cells to fully mature sensory hair cells, thereby identifying new genes that are expressed during hair cell development. In addition, we characterized the role of the growth factor fgf3, and found that it acts to inhibit hair cell progenitor proliferation. Our results were published in the journal eLife on Jan. 25, 2019. Future work will examine the function of these genes in sensory hair cell regeneration.

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Mark E. Lush, Ph.D., and Daniel C. Diaz both work in the lab of Tatjana Piotrowski, Ph.D., at Stowers Institute for Medical Research in Kansas City. Piotrowski is a member of the Hearing Restoration Project, which helped fund this study.

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New Insights into the Development of the Hair Cell Bundle

By Yishane Lee

Recent genetic studies have identified that the protein Ripor2 (formerly known as Fam65b) is an important molecule for hearing. It localizes to the stereocilia of auditory hair cells and causes deafness when mutations disrupt its function.

In a study published in the Journal of Molecular Medicine in November 2018, Oscar Diaz-Horta, Ph.D., a 2017 Emerging Research Grants (ERG) scientist, and colleagues further show the role the protein plays by demonstrating how it interacts with other proteins during the development of the hair cell bundle. The team found that the absence of Ripor2 changes the orientation of the hair cell bundle, which in turn affects hearing ability.

Ripor2 interacts with Myh9, a protein encoded by a known deafness gene, and Myh9 is expressed in the hair cell bundle stereocilia as well as its kinocilia (apices). The team found that the absence of Ripor2 means that Myh9 is low in abundance. In the study, Ripor2-deficient mice developed hair cell bundles with atypically localized kinocilia and reduced abundance of a phosphorylated form of Myh9. (Phosphorylation is a cellular process critical for protein function.)

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Another specific kinociliary protein, acetylated alpha tubulin, helps stabilize cell structures. The researchers found it is also reduced in the absence of Ripor2.

The study concludes that Ripor2 deficiency affects the abundance and/or role of proteins in stereocilia and kinocilia, which negatively affects the structure and function of the auditory hair cell bundle. These newly detailed molecular aspects of hearing will help to better understand how, when these molecular actions are disrupted, hearing loss occurs.

A 2017 ERG scientist funded by the Children’s Hearing Institute (CHI), Oscar Diaz-Horta, Ph.D., was an assistant scientist in the department of human genetics at the University of Miami. He passed away suddenly in August 2018, while this paper was in production. HHF and CHI both send our deepest condolences to Diaz-Horta’s family and colleagues.

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Headlines in Hearing Restoration

By Yishane Lee

The cornerstone of Hearing Health Foundation for six decades has been funding early-career hearing and balance researchers through its Emerging Research Grants (ERG) program. Many ERG scientists have gone on to obtain prestigious National Institutes of Health (NIH) funding to continue their HHF-funded research; since 1958, each dollar awarded to ERG scientists by HHF has been matched by NIH investments of more than $90. Within the scientific community, ERG is a competitive grant awarded to the most promising investigators, and we’re always especially pleased when our ERG alumni who are now also members of or affiliated with our Hearing Restoration Project consortium make headlines in the mainstream news for their scientific breakthroughs.

Hair cells in the mouse cochlea courtesy of the lab of Hearing Restoration Project (HRP) member Andy Groves, Ph.D., Baylor College of Medicine.

Hair cells in the mouse cochlea courtesy of the lab of Hearing Restoration Project (HRP) member Andy Groves, Ph.D., Baylor College of Medicine.

Ronna Hertzano, M.D., Ph.D. (2009–10): Hearing Restoration Project consortium member Hertzano, an associate professor at the University of Maryland School of Medicine, and colleagues identified a gene, Ikzf2, that acts as a key regulator for outer hair cells whose loss is a major cause of age-related hearing loss. The Ikzf2 gene encodes helios, a transcription factor (a protein that controls the expression of other genes). The mutation of the gene in mice impairs the activity of helios in the mice, leading to an outer hair cell deficit.

Reporting in the Nov. 21, 2018, issue of Nature, the team tested whether the opposite effect could be created—if an abundance of helios could boost the population of outer hair cells. They introduced a virus engineered to overexpress helios into the inner ear hair cells of newborn mice, and found that some mature inner hair cells became more like outer hair cells by exhibiting electromotility, a property limited to outer hair cells. The finding that helios can drive inner hair cells to adopt critical outer hair cell characteristics holds promise for future treatments of age-related hearing loss.

Patricia White, Ph.D. (2009, 2011), with Hearing Restoration Project member Albert Edge, Ph.D.: White, a research associate professor at the University of Rochester Medical Center, Edge, a professor of otolaryngology at Massachusetts Eye and Ear and Harvard Medical School, and team have been able to regrow the sensory hair cells found in the mouse cochlea. The study, published in the European Journal of Neuroscience on Sep. 30, 2018, builds on White’s prior research that identified a family of receptors called epidermal growth factor (EGF) that is responsible for activating supporting cells in the auditory organs of birds. When triggered, these cells proliferate and foster the generation of new sensory hair cells. In mice, EGF receptors are expressed but do not drive regeneration of hair cells, so it could be that as mammals evolved, the signaling pathway was altered.

The new study aimed to unblock the regeneration of hair cells and also integrate them with nerve cells, so they are functional, by switching the EGF signaling pathway to act as it does in birds. The team focused on a specific receptor called ERBB2, found in supporting cells. They used a number of methods to activate the EGF signaling pathway: a virus targeting ERBB2 receptors; mice genetically altered to overexpress activated ERBB2; and two drugs developed to stimulate stem cell activity in the eye and pancreas that are already known to activate ERBB2 signaling. The researchers found that activating the ERBB2 pathway triggered a cascading series of cellular events: Supporting cells began to proliferate and started the process of activating other neighboring stem cells to lead to “apparent supernumerary hair cell formation,” and these hair cells’ integration with the network of neurons was also supported.

This was prepared using press materials from the University of Maryland and the University of Rochester. For more, see hhf.org/hrp.

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Uncovering a Signaling Molecule That Modulates Avian Hair Cell Regeneration

By Rebecca M. Lewis, Au.D., Ph.D., and Jennifer Stone, Ph.D.

Mammals including humans cannot regenerate hair cells, but other species such as birds and fish readily regenerate hair cells after damage to restore auditory function. The gene ATOH1 produces a protein that pushes supporting cells—cells that neighbor hair cells—to either directly convert into a hair cell or to divide and form a new hair cell. However, ATOH1 expression (when the gene is turned on) does not guarantee that hair cells develop in birds or mammals, which suggests that there are factors that prevent supporting cells from changing into hair cells. Identifying these factors in birds may help us better understand the lack of hair cell regeneration in mammals.

This schematic depicts our current ideas for how BMP4 regulates ATOH1 expression and therefore hair cell regeneration in the avian hearing organ. It shows (from left) typical hair cells, hair cell damage, and hair cell regeneration. Typical hair cells secrete BMP4. When hair cells die, BMP4 signaling is reduced, which allows ATOH1 to be expressed in supporting cells and pushes supporting cells to turn into hair cells. The newly regenerated hair cells secrete BMP4, suppressing ATOH1 in supporting cells and restoring the normal condition.

This schematic depicts our current ideas for how BMP4 regulates ATOH1 expression and therefore hair cell regeneration in the avian hearing organ. It shows (from left) typical hair cells, hair cell damage, and hair cell regeneration. Typical hair cells secrete BMP4. When hair cells die, BMP4 signaling is reduced, which allows ATOH1 to be expressed in supporting cells and pushes supporting cells to turn into hair cells. The newly regenerated hair cells secrete BMP4, suppressing ATOH1 in supporting cells and restoring the normal condition.

We examined the avian auditory system to characterize a potential inhibitor to ATOH1 during hair cell regeneration: bone morphogenetic protein 4 (BMP4). Bone morphogenetic proteins are secreted signaling molecules that regulate cellular processes in many regions of the body, including the nervous system. We found that BMP4 localizes to hair cells of the mature avian hearing organ and disappears when hair cells die or sustain damage. From this, we hypothesized that BMP4 may prevent ATOH1 expression in supporting cells and loss of BMP4 when hair cells die may enable ATOH1 to be expressed in supporting cells, driving them to convert into hair cells.

When we exposed avian auditory organs to BMP4 after selectively killing hair cells, this prevented ATOH1 expression and hair cell regeneration. When we antagonized BMP4 using an inhibitor, we found a generally opposite result: an increase in the number of regenerated hair cells.

We conclude that BMP4 is a potent inhibitor of ATOH1 and therefore suppresses hair cell regeneration. We recommend that BMP4 be explored further in studies of mammalian hair cell regeneration.

Published in Hearing Research on May 2, 2018, this study detailing BMP4’s negative effect on ATOH1 expands our knowledge of signaling molecules that suppress hair cell regeneration in birds and may also modulate hair cell regeneration in humans.

Rebecca M. Lewis, Au.D., Ph.D., is a clinical audiologist and auditory neuroscientist at Massachusetts Eye and Ear/Harvard Medical School in Boston. HRP researcher Jennifer Stone, Ph.D., is the director of research in the department of otolaryngology–head and neck surgery at the Virginia Merrill Bloedel Hearing Research Center at the University of Washington.

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Researchers Fighting the Effects of Noise

By Yishane Lee

The cornerstone of Hearing Health Foundation, ever since its founding in 1958 as the Deafness Research Foundation, has been funding early-career researchers who bring innovative thinking to hearing and balance research. HHF’s Emerging Research Grants (ERG) are awarded to the most promising scientists in the field, with many going on to earn prestigious National Institutes of Health backing.

HHF is always proud to see ERG grantees thrive in their careers and research. Most recently, two ERG scientists funded in the mid-1990s have made headlines, each for treatments for noise-induced hearing loss (NIHL).

1996 and 1997 ERG scientist John Oghalai, M.D., of the University of Southern California, coauthored a study showing promise for preventing NIHL. Published May 7, 2018, in the Proceedings of the National Academy of Sciences, Oghalai and team used miniature optics to examine the mouse cochlea after exposure to extremely loud noise, and found that in addition to immediate hair cell death, a fluid buildup in the inner ear over several hours eventually led to nerve cell loss. The fluid buildup, or endolymph hydrops, contributes to synaptopathy, or damage to the auditory nerve cell synapse. In a USC News press release, Oghalai described the excess fluid as a feeling of fullness and ringing in the ear that a person may experience after attending a loud concert.

Because the extra fluid showed a high concentration of potassium, the team saw a method to re-balance the fluids that naturally occur in the inner ear by injecting a salt (sodium) and sugar solution into the middle ear three hours after exposure. Nerve cell loss was reduced by 45 to 64 percent, which may help preserve hearing. The researchers see applications for this treatment for military service members who experience blast trauma as well as for people who have Ménière’s disease, the hearing and balance condition that is associated with inner ear fluid buildup.

Images from the cochleae of guinea pigs show the presence of more hair cells in animals treated with a short interfering RNA that interrupts a gene upregulated after damage (right; control on left). Inner and outer hair cells (IHC and OHC) are labeled in green, stereocilia in yellow, and nuclei in blue. Arrowheads indicate ectopic hair cells. Credit:    The Scientist    via    Molecular Therapy   .

Images from the cochleae of guinea pigs show the presence of more hair cells in animals treated with a short interfering RNA that interrupts a gene upregulated after damage (right; control on left). Inner and outer hair cells (IHC and OHC) are labeled in green, stereocilia in yellow, and nuclei in blue. Arrowheads indicate ectopic hair cells. Credit: The Scientist via Molecular Therapy.

1996 ERG scientist Richard Kopke, M.D., FACS, of the Hough Ear Institute in Oklahoma, spent more than 20 years serving with the U.S. Army, becoming well aware of the dangers of NIHL for service members. In a paper in Molecular Therapy, published online in March 2018, Kopke and colleagues used “small interfering RNAs” (siRNAs) to block the activity of the Notch signaling pathway gene Hes1 that itself blocks hair cell differentiation in developing supporting cells and may contribute to the failure of hair cells to regenerate after injury.

These siRNAs were delivered using nanoparticles directly injected to the cochleae of live, adult guinea pigs. Kopke’s team had previously shown using siRNAs to block Hes1 to be effective in regenerating hair cells in cultured mouse cochlea. In the current study, the 24-hour, sustained-release of siRNAs through nanoparticles three days after deafening resulted in the recovery of some hearing ability, measured using auditory brainstem responses, at three weeks and continuing to nine weeks, when the study ended. Compared with the control mice, the RNA-injected mice showed less overall hair cell loss and early signs of immature hair cell development, which the authors say may signal hair cell regeneration. Hearing loss caused by noise, chemotherapy drugs, or aging that damages or kills hair cells are all targets for this potential treatment.

In an article in The Scientist, HHF’s Hearing Restoration Project consortium member Jennifer Stone, Ph.D., who was not involved in the paper, echoed the study authors in saying further research should work to determine which cells are turning into hair cells, and whether the observed hair cell development is truly new hair cells and not the repair of damaged hair cells. Kopke and team plan to test the treatment using longer periods between deafening and injection, while also modifying dose and delivery.

We need your help supporting innovative hearing and balance science through our Emerging Research Grants program. Please make a contribution today.

 
 
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The Countdown to Operation Regrow

By Gina Russo

Hearing Health Foundation (HHF) is counting down the days until the start of Operation Regrow, a two-week movement when you can help us to further progress toward better treatments and cures for hearing loss.

Beginning Tuesday, June 5, at 8:00 AM EDT, you can support the team of scientists conducting life-changing research to restore lost hearing, and more importantly, your generosity will have double the impact! All contributions received by 11:59 PM EDT on Tuesday, June 19 will be matched by an anonymous donor.

Transverse section through the embryonic day 20 chicken utricle (inner ear organ) at 20X magnification. Photo by Amanda Janesick, Ph.D., of the lab of Stefan Heller, Ph.D., a Hearing Restoration Project consortium member

Transverse section through the embryonic day 20 chicken utricle (inner ear organ) at 20X magnification. Photo by Amanda Janesick, Ph.D., of the lab of Stefan Heller, Ph.D., a Hearing Restoration Project consortium member

With just five days remaining until launch, you can share the five most important facts about Operation Regrow with friends and family:

  1. The Hearing Restoration Project (HRP) is the HHF-funded scientific consortium dedicated to finding biological cures for hearing loss.

  2. Damage to the sensory cells in the human inner ear causes irreversible hearing loss.

  3. The HRP members know that the key to hearing loss cures is the human ability to regrow cells in the inner ear. This phenomenon is already possible in certain species. The HRP has observed cell regrowth in chickens, fish, and young mice.

  4. The HRP is comprised of 15 senior scientists who work collaboratively by openly sharing data and ideas, and this collaboration helps to speed up the research process.

  5. HHF maintains stellar charity ratings from Better Business Bureau Wise Giving Alliance, Guidestar, Charity Navigator, and CharityWatch for using 100% of donations to support critical research, ensuring that all Operation Regrow contributions will directly help the HRP.

If you are able to make a gift to Operation Regrow, please visit www.hhf.org/regrow between June 5 and June 19. Gifts may also be made by phone during business hours, 9:00 AM to 5:30 PM EDT, at 212-257-6140. We’ll be sure to keep you updated on our progress. Thank you for supporting HRP and hearing health!

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New Method Enables Systematic Study of Hair Cell Loss and Regeneration in Chickens

By Carol Stoll

Most forms of hearing loss are permanent because damage to inner ear sensory hair cells is irreversible in mammals, including humans. Mammalian vestibular hair cells have the potential to regenerate albeit at a low rate, but the hair cells of the adult mammalian cochlea are not regenerated. Birds, however, have a robust regenerative response to hair cell damage and are able to restore structure and function in inner ear organs. Consequently, the study of the molecular mechanisms that trigger the onset of avian hair cell regeneration in the balance organs as well as in the cochlea is important and may lead to therapies for hearing loss in humans.

This image shows the undamaged and damaged utricle, an inner ear balance organ, in a chicken. HRP researchers have devised a new method to study the precise timing of hair cell regeneration in chickens using a single surgical application of an ototoxic drug. Photo by Amanda Janesick, Ph.D.

This image shows the undamaged and damaged utricle, an inner ear balance organ, in a chicken. HRP researchers have devised a new method to study the precise timing of hair cell regeneration in chickens using a single surgical application of an ototoxic drug. Photo by Amanda Janesick, Ph.D.

Past experiments that investigate these regeneration mechanisms in living chickens required multiple injections of a drug to induce hair cell loss, making it difficult to determine the exact timing of the regeneration response. A collaboration of two Hearing Restoration Project researchers, Stefan Heller, Ph.D. and Jennifer Stone, Ph.D., and two talented postdoctoral fellows from their laboratories was recently published in Journal of the Association for Research in Otolaryngology identifying a potential solution to this problem. They developed an experimental framework that uses a single ototoxic drug application, enabling them to study the precise onset and timing of hair cell regeneration in vivo.

Heller, Stone, and colleagues performed their experiments on a total of 75 chickens. At seven days of age, the chickens were anesthetized and underwent surgery to eliminate hair cells in the inner ear organs. During the surgery, streptomycin (an ototoxic antibiotic) was delivered to the chicken’s inner ear. At various time points after the surgery, two sensory organs—the utricle, a vestibular organ; and the basilar papilla, the hearing organ—were dissected, labeled for various cellular markers, and analyzed under a microscope. Hair cells and their surrounding supporting cells were counted and observed for damage. EdU, a marker of cell division, was administered to the chickens to determine whether or not new hair cells were generated by cell division. These techniques enabled the researchers to quantitatively characterize the regenerative response of the utricle after damage.

The results of the study demonstrate that surgical application of a single streptomycin dose is a feasible approach to elicit hair cell loss and regeneration in the chicken utricle and basilar papilla. Just hours after streptomycin delivery, hair cell numbers significantly declined and DNA replication was activated. The team was then able to record specific events of the regeneration process, which get initiated around 12 hours after streptomycin-induced hair cell loss, and continue over the course of several days.

Supporting cells produce new hair cells either by converting into a hair cell (direct transdifferentiation), or by dividing, usually asymmetrically, into a supporting cell and a hair cell.  Throughout this regenerative response, supporting cell numbers and density in the utricle remain relatively constant, suggesting that there is a mechanism that responds to specific levels of damage and coordinates the individual events of the regeneration process.

The study establishes a framework for the refined study of the two modes of hair cell regeneration in the chicken utricle. The next steps of the work will focus on understanding the exact timing and mechanism of coordination of the regeneration response. With only a single application of streptomycin necessary to induce near-complete hair cell loss in hearing and balance organs, the new animal model allows for study of the entire process including initiation, realization, and termination. The fundamental understanding of the avian regenerative mechanisms may lead to future development of therapies for loss of hearing and balance in humans.

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