Research Collaboration Remains

Collaboration Remains Key

A year into the working groups reorganization, the Hearing Restoration Project remains focused on cross-species analyses and is meeting with regeneration experts in the other research fields. By Lisa Goodrich, Ph.D.

We have our first glimpses of genes expressed across datasets and are well on our way to having lists of markers that will benefit the entire inner ear research community. As ever larger and more complex data sets are generated, centralizing some of this work is critical to finding genes and pathways relevant to hair cell regeneration and moving the HRP into its next phase.

As February drew to a close, Hearing Health Foundation’s Hearing Restoration Project (HRP) gathered over two days for its annual meeting, again by Zoom, to assess progress over the past year and prioritize for the future, near and longer term. The 2022 format differed somewhat from that of prior years in dividing each day into two parts, the first half featuring talks and presentations, and the second half dedicated to taking stock of progress since our last annual meeting and strategizing next steps. One important advantage in structuring the meeting this way is the ability to include HRP members’ postdocs in the first half. These early career researchers are instrumental in completing the day-to-day work of HRP projects, and we take seriously the consortium’s role in training the next generation of research scientists.

One of the presentations this year was by Seth Ament, Ph.D., co-chair of the Integrative Analysis working group, and Mahashweta Basu, Ph.D., HRP’s new full-time data analyst (see “Like a Master Class in Consensus,” opposite page). The Integrative Analysis working group has made outstanding progress toward focusing the cross-species analysis, starting with defining commonalities and differences among our target cells—the hair cells—which regenerate in some species and tissues and not in others. Having a dedicated data analyst is a welcome addition to the analysis that is conducted in every HRP member’s lab.

Due to Dr. Basu’s efforts, we have our first glimpses of genes expressed across datasets and are well on our way to having lists of markers that will benefit the entire inner ear research community. As ever larger and more complex data sets are generated, centralizing some of this work is critical to finding genes and pathways relevant to hair cell regeneration and moving the HRP into its next phase.

I was particularly excited to include this year an outside speaker—Freda Miller, Ph.D., professor of medical genetics at the University of British Columbia in Canada and an expert in stem cells and developmental neurobiology. Over the past year, the HRP has made a concerted effort to include perspectives from experts in regeneration in other fields. The spirit of collaboration that motivates the HRP itself similarly recommends that we learn from exchange with colleagues in other fields who grapple with challenges similar to the ones we face in the inner ear. Fittingly, Dr. Miller’s talk was followed by an update from consortium member Albert Edge, Ph.D., on stem cells in the inner ear and their ability to generate hair cells in cochlear organoids, a platform that will be increasingly important for screening genes and drugs in the future.

The second half of each day was dedicated to more fine-grained discussions about assessing the mechanics of HRP collaboration and clarifying goals and

priorities for the coming year. Limiting the second half of each day’s meeting to the HRP members encourages frank, open discussion among the group, which is critical for the type of team science the HRP embodies.

The group dove into details on specific initiatives, such as a joint HRP publication, and undertook broader evaluations of the working group structure and activities. Everyone agreed that we are already seeing the benefits of the changes we decided to make at the March 2021 annual meeting—that is, clustering research projects into three working groups: Cross-Species Epigenetics, Integrative Analysis, and Reprogramming and Gene Delivery. There is much excitement and enthusiasm for what we are able to accomplish together.

As well as setting goals for the coming year and reaching consensus on priorities, individual working groups began planning for proposals for the next round of funding. Given the working group structure, projects are now even more pronouncedly multiyear in nature, which means work plans are revised progressively in response to results.

Holding regular working group meetings in between our annual meetings is key to our ability to pivot as we learn new information, address unexpected roadblocks, and encourage brainstorming among members. Additionally, the consortium members genuinely enjoy coming together on a regular basis to talk about science and brainstorming new and creative ways to move the field forward.

As always, all of us in the HRP appreciate the opportunity to gather annually to take a bird’s eye view of the consortium’s work as a whole. Now that this year’s annual meeting is behind us and the working groups have begun drafting proposals, we are already looking forward to next year’s meeting, which—fingers crossed—will see us finally able to gather in person again.

HRP scientific director Lisa Goodrich, Ph.D., is a professor of neurobiology at Harvard Medical School. For more, see hhf.org/hrp.

Mahashweta Basu, Ph.D., joins HHF’s Hearing Restoration Project consortium as a bioinformatics analyst.

Like a Master Class

in Consensus By Mahashweta Basu, Ph.D.

I had been at the University of Maryland’s Institute of Genome Sciences for about two years when HRP member Seth Ament, Ph.D., mentioned that the HRP was looking to add a full-time data analyst to the consortium. Having honed my skills as a bioinformatician over the prior five years, I was excited by this opportunity to work with a team of researchers on data coming from and being compared across a number of labs spread across North America.

The opportunity to work with so many principal investigators and analyze data across species— fresh data, well in advance of its being published— is especially attractive. I am intrigued by the patterns they have in common, which is specifically what we are looking for.

Originally trained in physics at the University of Calcutta in India, I began to apply my training in statistical modeling to biological research as a doctoral researcher at the Saha Institute of Nuclear Physics, working on protein interaction and microRNA network projects. This was followed by postdoctoral research at the University of Maryland and then two Oak Ridge Institute for Science and Education fellowships at the U.S. Food and Drug Administration. This work included transcriptomic analysis in different disease conditions and a variety of tissue types throughout the human body, and machine learning projects including prediction models and module identification in complex biological networks.

After I moved to the Institute of Genome Sciences, my focus shifted primarily to the brain, and so closer to the HRP’s work. I have continued to gain experience in working with large datasets and I am especially interested in prediction models, so the chance to join HRP was particularly exciting. Having previously worked on studies led by a single principal investigator, I am keen to contribute to this kind of multidisciplinary, collaborative effort.

The opportunity to work with so many principal investigators and analyze data across species—fresh data, well in advance of its being published—is especially attractive. I am intrigued by the patterns they have in common, which is specifically what we are looking for. Of course, working with multiple large datasets and varied timelines like we have in the HRP can be a challenge, too, but one I welcome, and the HRP is a master class in how groups of researchers can come to a consensus about aims, timelines, and focuses.

Joining the HRP has been eye-opening, too, in learning specifically about hearing and the ear. People tend not to think about sensory organs like the ear until a problem arises. I continue to be fascinated with what I learn about the mechanisms whereby sound is converted to electrical impulses, and how certain genes can play a role in regeneration in some species, but not in others. With each step we learn more, which is what I find most exciting about the research sciences: This is a process, and each new bit of information gives us some answers and also new questions.

HRP data analyst Mahashweta Basu, Ph.D., is at the Institute for Genome Sciences at the University of Maryland School of Medicine.

Recent Research by Hearing Health Foundation Scientists, Explained

Advancing Discoveries via Biologist-Friendly Access to Multi-Omic Data

Data processing that analyzes a large amount of data about individual cells and measures them through multiple “omics” (such as genomics, transcriptomics, proteomics, epigenomics, and metabolomics) has advanced our understanding of biological sciences and medicine in an unprecedented way. This process is termed high-throughput, cell type-specific multi-omic analyses.

The full benefit of this data, however, is achieved through its reuse. Successful reuse of data requires identifying users and ensuring data democratization (accessible to average users) and federation, meaning users’ databases can be connected through a virtual centralized meta-database so their access to others’ data is meaningful. In our paper published in Human Genetics in March 2022, we discuss challenges in access and reuse of multi-omic data and possible solutions, including the gEAR (the gene Expression Analysis Resource).

Omics data generation and analysis has undergone rapid expansion since the publication of the human and mouse genomes two decades ago. Since then, technological advances have improved the speed, throughput (data processing), accuracy, and affordability of these technologies. Also, advancements in the past few years enable many of these interrogations to be performed at the resolution of a single cell, allowing us to understand spatial and temporal dynamics in extreme detail.

Multi-omic data serves as the basis for discovery and is usually published in conjunction with peer-reviewed manuscripts. While the manuscripts highlight key findings, and may offer pertinent gene lists as attached tables, by convention all the data, raw as well as processed, is deposited in repositories.

The value of the data increases when it is made available and subsequently reused by other users for new discoveries. Standardized computational approaches are needed that allow for the data’s findability and reusability. Size of files, access to data, appropriate form of data storage, data annotation, and lack of sufficient experimental metadata are a few of the challenges for sharing and reuse of data. In parallel, we need to continue developing solutions to provide meaningful access to multi-omic data for biologists who are not trained specifically in informatics.

Having progressed from initial seed funding from HHF’s Hearing Restoration Project to now receiving significant National Institute on Deafness and Other Communication Disorders funding, the gEAR portal is an important example of this approach of democratizing data for a specific research community, that of auditory science.

Hearing loss, which affects 1 in 1,000 newborns and over 50 percent of the population older than age 70, may result from mutations in more than 150 genes distributed across the different cell types of the mammalian inner ear. Cell type-specific omics have advanced our understanding of the inner ear cell types, identified critical regulators of cell fate, and uncovered some of the challenges in hair cell regeneration in mammals.

As a primary resource for data sharing within the ear field, the gEAR is cited for data validation, hypothesis generation, and data dissemination. The code, which is open source, has now been used to support research initiatives in other fields beyond the ear. However, such efforts require extensive investment. Funding agencies could propel discovery via the broad use and reuse of multi-omic data across disciplines. —Ronna Hertzano, M.D., Ph.D., and Anup Mahurkar

A 2009–2010 Emerging Research Grants (ERG) scientist and a member of HHF’s Hearing Restoration Project, Ronna Hertzano, M.D., Ph.D. (left), is a professor in the department of otorhinolaryngology–head & neck surgery and an affiliate member of the Institute for Genome Sciences, University of Maryland School of Medicine at the University of Maryland School of Medicine. For more, see page 14. Anup Mahurkar is the executive director of software engineering & information technology at the Institute for Genome Sciences, University of Maryland School of Medicine.

Evidence of Brain Tissue Damage From Blast Overpressure

Blast overpressure is responsible for 86 percent of U.S. service members wounded in action in Iraq and Afghanistan between 2001 and 2017. Many report postconcussive symptoms such as hearing loss, cochlear synaptopathy, tinnitus, hyperacusis, poor speech perception in noise, and cognitive deficits (such as learning, memory, attention, and emotional problems). Concerns have emerged about the long-term effects of mild traumatic brain injury (mTBI), a signature injury in blast explosions, to which an accelerated incidence of lateonset neurodegenerative disorders has been attributed.

Specifically, the positive pressure phase of briefduration blast overpressure inflicts mechanical damage to the cochlea and its central pathways, initiating a cascade of pathological cellular processes, primarily activation of oxidative mechanism and neuroinflammation, that result in neurodegeneration and cell death in the cochlea and higher auditory centers of the brain, such as the cochlear nucleus, inferior colliculus, and auditory cortex.

Evidence demonstrates that even a single-blast TBI can cause lasting neurological changes—without clinical symptoms—that are considered a risk factor for late-onset neurodegenerative disease, such as chronic traumatic encephalopathy (brain degeneration as a result of repeated head trauma). Researchers are investigating a potential association between tinnitus and dementia, for which there is some preliminary evidence.

Blast overpressure-induced conditions often overlap with post-traumatic stress disorder and chronic traumatic encephalopathy. In veterans, hearing loss and tinnitus are consistently rated as the top two serviceconnected disabilities, according to the Veterans Benefits Administration Annual Benefits Report for Fiscal Year 2020. However, there are to date no Food and Drug Administration-approved medications for tinnitus.

In addition, there are no evidence-based guidelines available for the definitive diagnosis, or directed treatment, of blast-induced mTBI. This too is due to a poor understanding of the underlying pathology, as few studies have focused on the unique lesions associated with blast overpressure. As a result it is critical to characterize and consolidate our understanding of blast-induced pathology in the central auditory neuraxis, or the auditory pathway in the higher brain centers.

For our study published in the Journal of Neurotrauma in November 2021, my team and I characterized blastinduced auditory neurodegeneration in the chinchilla, a widely adopted animal model in auditory neuroscience. Before delving into the molecular pathways underlying blast-induced pathology, we studied the structural changes using highly powerful 9.4T small animal diffusion tensor imaging (DTI) at the Center for Biomedical Imaging at the University at Buffalo.

Our investigation is the first of its kind to perform DTI in the chinchilla. DTI is a powerful technique for monitoring the response of the brain to trauma. In this study, the tissue microstructure of the auditory pathway was probed using a quantitative and noninvasive magnetic resonance DTI approach to collect images of water diffusion properties in auditory higher centers.

The water diffusion in tissues is highly sensitive to differences in the microstructural architecture of cellular membranes, making it a powerful method for detecting microscopic differences in tissue properties. Thus, the quantitative scalar DTI metrics are directly linked to the anatomic organization and microstructural features of white matter fiber tracts in the central auditory neuraxis.

This study’s findings are more generalizable to humans for two reasons: 1) The chinchilla has a hearing range and size of tissue microstructure similar to humans; and 2) the measures used to probe the pathophysiological changes (such as auditory brainstem response, or ABR, and neuroimaging) are commonly applied to humans as well.

Our results indicate that a single unilateral blast significantly impairs the structural and functional integrity at all levels of the central auditory neuraxis. Overall, it is evident that the cytoarchitecture, the structural integrity of brain tissue, is compromised at all levels, particularly at the cochlear nucleus and auditory cortex. —Vijaya Prakash Krishnan Muthaiah, Ph.D.

A 2019 2019 ERG scientist generously funded by the General Grand Chapter Royal Arch Masons International, Vijaya Prakash Krishnan Muthaiah, Ph.D., is an assistant professor in the department of rehabilitation sciences at the University at Buffalo, the State University of New York.

Schematics of noise stimuli presented to mice.

With or Without Significant Hearing Loss, Older Mice Show Difficulty With Brain Processing

Many older adults notice changes in their ability to understand speech, which can affect quality of life. These types of changes in auditory processing affect individuals with or without measurable hearing loss. To better distinguish between the effects of hearing loss and aging, my graduate student compared cortical responses in two strains of mice with differing levels of age-related hearing loss, reporting our results in Hearing Research in October 2021.

Previous studies had already demonstrated in animal models that noise-induced hearing loss results in increased central gain, which compensates for the lower levels of input, without any concomitant improvement in temporal processing. Until our study, it remained unclear whether the same was true in the case of presbycusis (age-related hearing loss), which develops over longer time frames. We tested temporal processing and central gain in mice with severe or mild hearing loss with age using electrophysiological responses from awake and freely moving mice.

We show that severe presbycusis leads to increased gain in the auditory cortex, but with reduced temporal fidelity. Data from the mice with more moderate hearing loss demonstrated age-related changes in temporal processing without concomitant increase in cortical gain. However, in the mice with moderate hearing loss, cortical temporal processing deficits were seen only when tested with more challenging sounds (shorter gaps and shallower modulation). This indicates that even mild hearing loss with aging may result in a decline in temporal processing under challenging conditions, such as environments with increased noise.

This has implications for treating age-related changes in auditory processing in humans. Sound amplification via hearing aids or cochlear implants may be insufficient; interventions may have to be multifaceted, involving amplification devices, behavioral training, and pharmacological approaches, as well as more detailed treatment plans that identify the most appropriate time points for each type of intervention.

Our data from mice is largely consistent with EEG studies of aging humans, suggesting that the EEG measures in mice can be used as translation-relevant biomarkers to test pharmacological intervention and behavioral training studies aimed at reducing agerelated auditory processing deficits in humans. —Khaleel Razak, Ph.D.

A 2009 and 2018 ERG scientist whose 2018 grant was generously funded by the General Grand Chapter Royal Arch Masons International, Khaleel Razak, Ph.D., is a psychology professor at the University of California, Riverside.

A Historical Perspective on Surgery to Treat Ménière’s Disease

Ménière’s disease is a disorder of the inner ear consisting of intermittent, spontaneous episodes of vertigo (dizziness) in combination with other fluctuating ear symptoms including low-frequency sensorineural hearing loss, aural fullness, and tinnitus. The condition is associated with Prosper Ménière who in 1861 identified the inner ear labyrinth as a likely source for symptoms of a syndrome involving episodic vertigo and hearing loss. The cause of Ménière’s disease remains unclear.

Endolymphatic hydrops is a swelling of the endolymph (fluid) spaces that has been observed consistently in postmortem studies of patients with a history of Ménière’s disease, by examining their tissues and cells (known as histology). But hydrops can also occur in asymptomatic individuals and in association with other diseases, suggesting that while hydrops is associated with Ménière’s disease, it may not cause the disorder.

Since it was first discovered, Ménière’s disease has been a disorder managed primarily by otolaryngologists, also known as ear, nose, and throat specialists, or ENTs. As a result, surgical treatments have accompanied attempts at medical management. Inspired by patients’ sensations of ear fullness and later by the histologic findings of hydrops, surgeons began manipulating the membranous labyrinth to relieve episodes of vertigo while attempting to preserve hearing. The membranous labyrinth lies within the bony labyrinth and is filled with endolymph fluid.

Published in Frontiers in Neurology in December 2021, our review highlights this history of manipulation of the membranous labyrinth. The studies involving patients are uniformly retrospective, with some procedures performed first in animal models of endolymphatic hydrops.

These procedures indicate a rich history of innovation that parallels developments in otologic surgery. Lateral semicircular canal fenestration, the introduction of the operating microscope, the development of stapes surgery, and now increasing experience with semicircular canal plugging have each influenced procedures that aim to address the finding of endolymphatic hydrops.

The broad goals of these procedures are eliminating episodic vertigo while preserving hearing. Assessing the success of interventions is difficult due to the natural history of Ménière’s disease in which cessation of vertigo episodes over time is common. Also, whether the procedures improved endolymphatic hydrops specifically was unknown, since historically this could be assessed only by histology on postmortem specimens.

While hearing loss remains frequent following these procedures, many individuals who underwent procedures

Surgical manipulations of the membranous labyrinth for treatment of Ménière’s disease: (A) This depiction of Femenic’s shunt in the lateral semicircular canal to relieve endolymphatic hydrops and vertigo episodes. Adapted with permission from Femenic. (B) This histological representation of endolymphatic hydrops in the cochlear duct and saccule, with a Fick sacculotomy needle placed through a fenestration in the stapes to drain excess endolymph. (C) This permanent tack placement in the stapes bone was intended to enable repeated decompression of the hydropic saccule in the Cody tack operation. (D) Two views of the cochleosacculotomy procedure, where a 90-degree pick is driven through the round window to rupture the cochlear duct and saccule and create a permanent fistula in the osseous spiral lamina. Adapted with permission from Schuknecht and Kinney et al. (E) Depiction of the Otic-Perotic shunt procedure, where a platinum tube is placed in the basilar membrane of the basal cochlea to enable decompression of the scala media. Adapted with permission from Pulec.

experienced relief from vertigo without hearing loss. Again, this relief might have occurred despite the intervention, but other variables such as technical differences among surgeons and other patient-specific factors like the extent of hydrops may underlie undesirable complications and variable outcomes.

Advances in surgical technology—such as preoperative imaging, operative robotics, and improved magnification— will lead to new surgical approaches on the membranous labyrinth. Our hope is that this also will improve outcomes for patients with inner ear disorders like Ménière’s disease. —Calvin J. Kersbergen and Bryan K. Ward, M.D.

Calvin J. Kersbergen is an M.D./Ph.D. candidate at Johns Hopkins University School of Medicine, where 2020 ERG scientist Bryan K. Ward, M.D. (left), is an assistant professor of otolaryngology–head and neck surgery. For more, see page 17.

When You Have to Think and Walk, What Happens to Your Balance?

Most activities of daily living require us to do two or more things at the same time, especially motor tasks (walking, standing, moving) with some form of a cognitive task (navigating, talking, decision-making). But it is not yet entirely clear what happens to balance performance in healthy individuals when they are also performing a cognitive task.

Several studies found that balance performance deteriorated (e.g., people are less stable and sway more) when attention needs to be divided. This is supported by an attentional resource-competition theory suggesting that our brain has some limited capacity and when we need to divide it—between control of movement and thinking—neither activity receives adequate attention, or one needs to be prioritized over the other.

Other studies found that balance improved when people allocated their attention to a cognitive task. The theory here is that balance control is largely controlled at lower, automatic brain levels while cognition requires high cortical function. As a result, attempting to overly control balance is not ideal. If we use higher brain resources for cognition, then we let balance happen automatically, as it should.

In our study published in the Journal of Motor Behavior in December 2021, we investigated how these theories may work together. We hypothesized that some of the apparent discrepancy between these theories is related to how we measure balance. Twenty-three healthy young adults each stood in a heel-to-toe position while wearing an HTC Vive head-mounted display, via which they observed a display of stars on three walls that was static or dynamic. On half of the trials, participants also counted backward in intervals of three from a three-digit number.

We found that the amount of sway—particularly a side-to-side directional path and velocity—significantly increased when participants were counting, particularly with dynamic visuals, while side-to-side variance (how concentrated was the sway around its mean) decreased. When we analyzed the frequency components of the sway, we found that the increase in path was explained by an increase in high frequency (fast) movements and the decrease in variance was explained by a decrease in low frequency (slow) movements. Fast corrective movements are typically attributed to relying on somatosensory input for balance (joint position sense). Slower movements are associated with cortical control of movement.

We conclude that when adding a cognitive task (counting backward) to a challenging balance task (standing in a heel-to-toe position and observing a dynamic visual movement via the head-mounted display), young adults responded by increasing small corrective movements, and thus the overall path, while also reducing slow frequency movements, and thus the overall variance.

This suggests that both attentional resourcecompetition (the increased challenge to maintain position with the cognitive task) as well as a switch to a more automatic, somatosensory-based control of the stance took place simultaneously. —Anat V. Lubetzky, Ph.D.

A 2019 ERG scientist, Anat V. Lubetzky, Ph.D., is an associate professor at New York University’s department of physical therapy.

For references to all the papers cited, see hhf.org/spring2022-references.