Sensory Hair Cell Homeostasis

National Institute On Deafness And Other Communication Disorders

Investigators

Linked publications, trials & patents

Abstract

Mechanical cues are crucial for survival, adaptation, and normal homeostasis in almost every cell type across organisms from bacteria to vertebrates. Cells perceive mechanical stimuli through diverse mechanosensitive molecules at the cell membrane, including mechanosensitive ion channels, integrins, or G protein-coupled receptors, which activate different mechanotransduction pathways. The transduction of mechanical messages into intracellular biochemical or electrical signals is termed mechanotransduction or mechanoelectrical transduction (MET). The inner ear MET apparatus, consisting of specialized mechanosensitive ion channels and associated molecules, is integral to the process of transducing sound vibrations and head movements into neural signals, and thus is essential for hearing and balance. Genetic alterations, aging, and noise exposure that disrupt this MET complex can lead to hearing loss. The MET system is also vulnerable to ototoxic agents, which can damage hair cells and result in sensory deficits. Understanding the intricate mechanisms underlying MET is crucial for grasping how sensory cells function and survive. This report summarizes the research progress made during the third year for the Section on Sensory Cell Physiology and Biophysics (SSPB). Our primary focus has been on training a strong team of scientists and initiating meaningful, innovative, and high-quality research projects in the lab that align with our research objectives. Our first research aim is to elucidate the structural and functional relationships within the MET apparatus. We will explore how the different components of the MET complex work together and contribute to its overall function. This investigation is crucial for understanding how sensory cells detect and respond to mechanical stimuli and will shed light on the foundational processes that support our hearing and balance. We have initially focused on two projects: 1. Characterization of the TMC Proteins as Lipid Scramblases Transmembrane Channel-like (TMC) proteins TMC1 and TMC2 were initially identified as the putative pore-forming subunits of the inner ear’s mechanotransduction (MET) channel complex. Previous work demonstrated that TMC1 and TMC2 exhibit ion channel activity when reconstituted into liposomes, suggesting a mechanosensitive channel function. However, emerging evidence, particularly the structural similarities to TMEM16 lipid scramblases and our recent discovery of a novel role for TMC1 in regulating the apical hair cell membrane, has raised the possibility that TMC proteins themselves may also function as lipid scramblases. Over the past year, we investigated this hypothesis. Through a combination of structural modeling and fluorescent-based in vitro scramblase translocation assays, we found that TMC1 and TMC2 from several organisms exhibit robust lipid scramblase activity that is tuned by cholesterol. Furthermore, deafness-causing mutations in TMC1 enhance its scramblase activity at physiological cholesterol levels, indicating a pathological gain-of-function mechanism. These findings suggest that cholesterol acts as a key modulator of TMC activity and that dysregulation of lipid scrambling may contribute to auditory dysfunction. These studies provide important new insights into the functional versatility of the TMC protein family and the MET apparatus, and they may help uncover molecular mechanisms underlying hearing loss and other TMC-associated diseases. This project is now complete and has been submitted for publication. 2. Investigation of Interactions Within the MET Complex Components Several membrane and soluble proteins have been identified as components of the MET complex. While many of their interactions have been previously examined using immunoprecipitation or pull-down assays in heterologous systems, methods that limit resolution of direct interactions and binding kinetics. We have now established an in vitro biolayer interferometry (BLI) platform using purified murine TMC1/2 cytoplasmic domains, TMIE, and CIB1/2/3. This real-time approach allows us to identify and quantify protein-protein interactions with kinetic information. Our BLI studies revealed direct binding with singular affinities between some of these protein fragments. Collectively, these efforts position us to dissect the cooperativity, specificity, and structural logic of protein-protein interactions within the MET complex. The second SSPB aim focuses on assessing the impact of the MET complex on hair cell homeostasis. Hair cells are essential for hearing and balance, and their health and stability are closely linked to the MET apparatus. By examining how the MET complex affects the maintenance of these cells, we aim to understand its broader implications for sensory cell health. This will help us grasp how disruptions in the MET complex can lead to sensory dysfunction and cell loss. 3. The Effect of TMC1 Deafness Mutations on Sensory Hair Cell Loss Auditory hair cells from mice carrying TMC1 deafness-causing mutations show signs of degeneration at different postnatal developmental stages and present hearing loss. However, the role of TMC1 in hair cell survival and the molecular mechanisms of TMC1 mutations in hair cell degeneration remain unclear. We are investigating the impact of different TMC1 mutations on hair cell degeneration, MET channel function, and dysregulation of membrane homeostasis. These data suggest that TMC1 could play an essential role in hair cell survival by regulating MET function or plasma membrane asymmetry, which could have significant implications for our understanding of hearing loss and potential therapeutic interventions. Our third aim is to examine the effects of membrane remodeling on auditory and vestibular functions. We will investigate how these alterations affect hair cell physiology and contribute to conditions like hearing loss and balance disorders. Understanding these effects is crucial for identifying how membrane dynamics impact sensory physiology. 4. Investigating the Role of XKR Lipid Scramblases in Inner Ear Function The XKR8 gene has recently been implicated in hereditary auditory neuropathy, with the W237X variant providing the first evidence linking XKR8 to late-onset hearing loss in humans and mice. XKR8 encodes a caspase-activated lipid scramblase involved in apoptotic cell clearance and neural development. We are investigating Xkr8 expression, localization, and function in the cochlea using knockout models and molecular profiling. Preliminary data indicate that Xkr8 is one of the primary scramblases transcribed in the inner ear, although its precise cellular localization remains under investigation. Functional assays suggest that this hearing-associated variant disrupts scramblase activity, consistent with a loss-of-function effect. While the exact role of XKR8 in normal auditory function remains unclear, ongoing work aims to further elucidate the role of XKR proteins in the inner ear.