Auditory Research publications

Aminoglycoside antibiotics are essential in managing many life‐threatening diseases. However, their derivatives, such as neomycin, are associated with severe side effects such as persistent sensorineural hearing loss. Therefore, it is essential to elucidate the molecular and biochemical mechanisms of aminoglycoside‐induced ototoxicity and identify targets for alleviating ototoxic injury. Here, we provide a detailed cochlear cell atlas of neomycin‐induced acute and chronic ototoxicity‐related changes through single‐nucleus RNA sequencing profiling. Utilising this cochlear cell atlas, we used the Augur and scDist algorithms to evaluate cell‐type‐specific susceptibility to neomycin injury. We observed aberrant expression of X‐linked inhibitor of apoptosis (Xiap)–associated factor 1 (Xaf1) in neomycin‐exposed cochleae using the cochlear cell atlas, and we identified a novel role for Xaf1 in facilitating PANoptosis through overexpression and knockdown assays in vitro. Finally, we assessed the protective role of Xaf1 against neomycin‐induced ototoxicity by Xaf1 knockdown in cochlear hair cells using adeno‐associated virus‐based gene delivery. Mechanistically, Xaf1 orchestrates PANoptosis activation through direct interaction with and transcriptional regulation of ZBP1, establishing its hierarchical position upstream in the signalling cascade. This study presents detailed cochlear cellular maps of neomycin‐induced ototoxicity and serves as a valuable resource for identifying transcriptome‐wide disease‐driving perturbations at the single‐cell level. More importantly, we identified Xaf1 as a critical target for modulating the PANoptosis pathway, offering a promising treatment strategy for aminoglycoside‐induced ototoxicity.

Noise-induced hearing loss (NIHL) is a widespread auditory disorder in industrialized nations, posing significant global and domestic health concerns. Despite its prevalence, the mechanisms underlying NIHL remain poorly understood, hindering the development of effective therapeutic interventions. Emerging evidence from clinical and animal studies suggests a potential relationship between altered gut microbiota and hearing loss, although causality remains unclear. In this study, we investigated the role of postnatal microbial signals in NIHL pathogenesis using acute noise exposure models in C57BL/6 mice. Our findings indicate that acute noise exposure does not significantly alter gut microbiota composition in this strain and that microbiota depletion does not confer protection against hearing loss following acute acoustic trauma. These results suggest no link between gut microbiota and acute NIHL in C57BL/6 mice.

The "cocktail party problem" challenges our ability to understand speech in noisy environments and often includes background music. Here, we explored the role of background music in speech-in-noise listening. Participants listened to an audiobook in familiar and unfamiliar music while tracking keywords in either speech or song lyrics. We used EEG to measure neural tracking of the audiobook. When speech was masked by music, the modeled temporal response function (TRF) peak latency at 50 ms (P1TRF) was prolonged compared to unmasked. Additionally, P1TRF amplitude was larger in unfamiliar background music, suggesting improved speech tracking. We observed prolonged latencies at 100 ms (N1TRF) when speech was not the attended stimulus, though only in less musical listeners. Our results suggest early neural representations of speech are stronger with both attention and concurrent unfamiliar music, indicating familiar music is more distracting. One's ability to perceptually filter "musical noise" at the cocktail party also depends on objective musical listening abilities.

Age-related hearing loss (ARHL) is one of the most prevalent and complex disorders. Our previous study demonstrated that abnormal activation of mammalian target of rapamycin complex 1 (mTORC1) signaling in the cochlear neurosensory epithelium causes auditory hair cell (HC) damage and contributes to ARHL. However, the underlying mechanism of mTORC1 activation remains unclear. In this study, we identified tumor necrosis factor-alpha-induced protein 8-like 2 (TNFAIP8L2), an immune regulatory gene, as a potential candidate. To elucidate the effect of TNFAIP8L2 on mTORC1 signaling in the neurosensory epithelium and on hearing function, we generated a Tnfaip8l2-deficient (Tnfaip8l2-/-) mouse model. We discovered that Tnfaip8l2 deficiency led to features of oxidative stress in cochlear HCs and age-related hearing degeneration, exhibiting a similar phenotype to the mTORC1-over-activated Tsc1-cKO mice described previously. Furthermore, rapamycin, a well-known mTORC1 inhibitor, significantly mitigated the hearing dysfunction caused by Tnfaip8l2-deficiency. Mechanistically, we found that TNFAIP8L2 regulates mTORC1 signaling by simultaneously inhibiting the GTPase activity of Ras homolog enriched in brain (RHEB) and Ras-related C3 botulinum toxin substrate 1 (RAC1). Notably, both RHEB and RAC1 inhibitors alleviated the hearing phenotype observed in Tnfaip8l2-/- mice by inhibiting mTORC1 signaling. Collectively, our results provide insights into the activation of the mTORC1 pathway in aged mouse cochleae and positions TNFAIP8L2 as a valuable theoretical strategy.

Fear retrieval is fundamental for animals to rapidly enter a self-defense state, which enhances their survival rate. Although the cellular mechanisms and neural circuits underlying fear retrieval have been relatively well studied, particularly in classic fear-related nuclei such as the amygdala, the neural circuits and intrinsic microcircuits of some non-classical fear-related nuclei remain poorly understood. A disinhibitory microcircuit in the temporal association cortex (TeA) was identified in this study, demonstrating a critical role in fear retrieval. Additionally, the response of the TeA to fear retrieval is largely driven by acetylcholine (Ach), which then relay information to the tail of striatum (TS) to mediate freezing behavior. Together, these findings demonstrate the essential role of the TeA and its microcircuit in auditory fear retrieval in response to pure tones, providing new insights into the mechanisms of fear retrieval in the cortex.

Auditory processing in the cerebral cortex is considered to begin with thalamocortical inputs to layer 4 (L4) of the primary auditory cortex (A1). In this canonical model, A1 L4 inputs initiate a hierarchical cascade that propagates to higher-order cortices for slower integration of complex sounds. Here, we identify parallel ascending pathways in mice that bypass A1 and directly reach the secondary auditory cortex (A2), alongside the canonical hierarchical route. We found that layer 6 (L6) of both A1 and A2 receive short-latency (<10 ms) sound inputs via higher-order thalamic nuclei. Additionally, A2 L4 is innervated by a caudal subdivision of the traditionally defined primary thalamus, which we now re-classify as non-primary. Notably, both identified thalamic regions receive projections from distinct subdivisions of the higher-order inferior colliculus, which in turn receive direct projections from cochlear nucleus neurons. Thus, higher-order auditory cortex integrates both slower, pre-processed information and rapid, direct sensory inputs, enabling parallel processing of fast sensory information across cortical areas.

Atypical sensory processing, particularly in the auditory domain, is one of the most common and quality-of-life affecting symptoms seen in autism spectrum disorders (ASD). Fragile X Syndrome (FXS) is a leading inherited cause of ASD and a majority of FXS individuals present with auditory processing alterations. While auditory hypersensitivity is a common phenotype observed in FXS and Fmr1 knockout (KO) rodent models, it is important to consider other auditory coding impairments that could contribute to sound processing difficulties and disrupted language comprehension in FXS. We have shown previously that a Fmr1 KO rat model of FXS exhibits heightened sound sensitivity that coincided with abnormal perceptual integration of stimulus bandwidth, indicative of altered spectral processing. Frequency discrimination is a fundamental aspect of sound encoding that is important for a range of auditory processes, such as source segregation and speech comprehension, and disrupted frequency coding could thus contribute to a range of auditory issues in FXS and ASD. Here we explicitly characterized spectral processing deficits in male Fmr1 KO rats using an operant conditioning tone discrimination assay and in vivo electrophysiological recordings from the auditory cortex and inferior colliculus. We found that Fmr1 KO rats exhibited poorer frequency resolution, which corresponded with neuronal hyperactivity and broader frequency tuning in auditory cortical but not collicular neurons. Using an experimentally informed population model, we show that these cortical physiological differences can recapitulate the observed behavior discrimination deficits, with decoder performance being tightly linked to differences in cortical tuning width and signal-to-noise ratios. Together, these findings indicate that cortical hyperexcitability in Fmr1 KO rats may act to preserve signal-to-noise ratios and signal detection threshold at the expense of sound sensitivity and fine feature discrimination, highlighting a potential mechanistic locus for a range of auditory behavioral phenotypes in FXS.

The activity of neurotransmitter-based cell types in the cuneiform and pedunculopontine nuclei during locomotion, non-locomotor behaviors, and following sensory stimulation is not fully understood. Using fiber photometry in mice, we found cell-type specific responses to sensory stimuli. Glutamatergic and GABAergic cells responded to sound, visual looming, and air puffs, except for pedunculopontine GABAergic cells, which did not respond to visual looming. Cholinergic cells responded to air puffs. Air puffs triggered high-speed locomotion, whereas visual looming and sound stimuli evoked low-speed locomotion. During air puff–evoked locomotion, cuneiform glutamatergic neuron activity was higher than in trials without locomotion. In contrast, during locomotion evoked by visual looming or sound, activity in pedunculopontine glutamatergic neurons was higher than when no locomotion occurred. In the open-field arena, mice exhibited spontaneous low-speed locomotion during which activity increased in pedunculopontine glutamatergic cells. Activity also increased in a cell type-specific manner during grooming or rearing. Our study shows cell type-specific activity in the cuneiform or pedunculopontine nuclei during locomotion, non-locomotor behaviors, and following sensory stimulation. Sensory responsiveness likely has relevance in Parkinson’s disease, where sensory circuits are increasingly targeted to improve walking.

Identifying neural signatures of loss of consciousness is a major goal of neuroscience. The local/global auditory novelty paradigm has been useful in characterizing sensory processing across arousal states. Propofol suppresses responses to long‐term novelty (global deviance, GD) at subhypnotic doses; suppression of responses to short‐term novelty (local deviance, LD) outside auditory cortex may represent a biomarker of loss of consciousness. Dexmedetomidine is an alpha‐2 adrenergic agonist that induces sleep‐like sedation. This study examined whether the changes in auditory novelty processing observed with propofol, a GABA‐ergic agent, also occur with dexmedetomidine and during sleep. Intracranial recordings were obtained in neurosurgical patients undergoing monitoring for refractory epilepsy. Stimuli were vowel sequences incorporating LD and GD. Neural activity was recorded during wakefulness, administration of dexmedetomidine, and sleep and was examined as the averaged evoked potential (AEP) and high gamma (70–150 Hz) power. AEP responses were more broadly distributed than high gamma activity. Results previously observed with propofol were replicated with dexmedetomidine. Subhypnotic doses led to decreased LD effects and a precipitous decline in GD effects. Loss of responsiveness was associated with loss of LD effects outside the auditory cortex. Likewise, daytime sleep was associated with cessation of GD effects and confinement of LD effects to the auditory cortex. Results support the generalizability of changes in auditory novelty processing to dexmedetomidine and sleep. Preservation of LD effects in the auditory cortex indicates that the auditory cortex continues to monitor the environment following loss of responsiveness.

Analyzing the functional connectivity of the brain is an enormous challenge, as deciphering functional connectivity requires knowledge of functional responses and connections. One promising strategy is analyzing the spatial pattern of activity correlations across cell populations. In the primary auditory cortex (A1), cells respond to different sound features. On the large scale, there exists a tonotopic map, which is fractured at the small scale, raising the question of whether functional connections are spatially ordered or disordered. To test whether functional connectivity on a local and a global scale is also disordered, we first designed a robust statistical model to estimate parameters and test for the significance of the estimated correlation maps. We developed an inference method that allows efficient model fitting and statistical testing to project the correlation maps to 2D space. We then performed in vivo two-photon calcium imaging in layer 2/3 of A1 with pure tones (PT) or a combination of two tones (TT; harmonically related or not). We found that the spatial patterns of signal correlations (SCs) depend on the type of sound stimuli that were presented. The functional 2D maps of PT-driven SCs are more restricted to local neurons than TT signal correlations which showed more global textures. 2D SC patterns for harmonic stimuli showed spatially distinct relationships. TT SCs revealed spatially precise functional connectivity between harmonically related neurons. Thus, even though the frequency preference of neighboring neurons in A1 is functionally diverse, the functional connection pattern of these neurons is functionally precise and harmonically related.

Insects’ sound and vibration detection including proprioception rely on the scolopidium—a mechanosensory unit enclosing the sensory cilium of chordotonal organ neurons. The cilium, enclosed by a scolopale cell, contains mechanosensitive ion channels with the ciliary tip embedded in a cap. Despite knowledge of the scolopidial structure in multiple insects, the mechanism by which mechanical force elicits transduction remains speculative. We examined scolopidia in the auditory Müller’s organ of the desert locust and present a comprehensive three-dimensional (3D) ultrastructure of a scolopidium using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). Next, we characterised sound-evoked motions of Müller’s organ and the scolopidium using Optical Coherence Tomography (OCT) and high-speed light microscopy. Finally, we measured transduction currents via patch clamp electrophysiology during mechanical stimulation of individual scolopidia. By combining ultrastructure, sound-evoked motions, and transduction current recordings, our finding suggests that the scolopidium is activated best by stretch along the ciliary axis.

Most patients with sensorineural hearing loss (SNHL) may initially experience high‐frequency hearing loss linked to greater vulnerability of outer hair cells (OHCs) in the cochlear basal turn. However, the molecular mechanism underlying this susceptibility in the high‐frequency region remains unclear. Here, NOX2 is first identified as a differentially expressed gene related to oxidative damage in the apical and basal turns through single‐cell RNA sequencing. In mouse cochleae damaged by neomycin and noise, Nox2‐positive OHCs increase progressively from the apex to base, indicating that NOX2 expression clearly differed significantly between the apical and basal OHCs. In NOX2 knockout mice, OHC damage caused by neomycin and noise exposure is significantly reduced. Furthermore, knocking out NOX2 increases the expression of Nrf2 in the cytoplasm and Nrf2 nuclear translocation in OHCs. In addition, Nrf2 inhibitors counteract the protective effect of NOX2 knockout against neomycin. Finally, when Nox2 is used as the target, ginsenoside Rg1 is identified as a compound with protective effects against neomycin‐induced hearing loss. In summary, these results indicate NOX2 is highly correlated with high‐frequency OHCs vulnerability in the cochlea and is a potential intervention target for the clinical treatment of SNHL.

Hearing loss is a pervasive global health challenge with profound impacts on communication, cognitive function, and quality of life. Recent studies have established age-related hearing loss as a significant risk factor for dementia, highlighting the importance of hearing loss research. Auditory brainstem responses (ABRs), which are electrophysiological recordings of synchronized neural activity from the auditory nerve and brainstem, serve as in vivo readouts for sensory hair cell, synaptic integrity, hearing sensitivity, and other key features of auditory pathway functionality, making them highly valuable for both basic neuroscience research and clinical diagnostics. Despite their utility, traditional ABR analyses rely heavily on subjective manual interpretation, leading to considerable variability and limiting reproducibility across studies. Here, we introduce Auditory Brainstem Response Analyzer (ABRA), a novel open-source graphical user interface powered by deep learning, which automates and standardizes ABR waveform analysis. ABRA employs convolutional neural networks trained on diverse datasets collected from multiple experimental settings, achieving rapid and unbiased extraction of key ABR metrics, including peak amplitude, latency, and auditory threshold estimates. We demonstrate that ABRA’s deep learning models provide performance comparable to expert human annotators while dramatically reducing analysis time and enhancing reproducibility across datasets from different laboratories. By bridging hearing research, sensory neuroscience, and advanced computational techniques, ABRA facilitates broader interdisciplinary insights into auditory function. An online version of the tool is available for use at no cost at https://abra.ucsd.edu.

Negative feedback of the cochlear efferent system plays a critical role in control of hearing sensitivity and protection from noise trauma. Type II auditory nerves (ANs) innervate outer hair cells (OHCs) in the cochlea and provide an input to the cochlear efferent system to achieve hearing sensitivity controlling and protection; in particular, medial olivocochlear efferent nerves innervate OHCs to control OHC electromotility, which is an active cochlear amplifier in mammals. However, little is known about channel information underlying type II AN activity and consequent function. Here, we report that ATP-gated P2x7 receptor had a predominant expression at type II spiral ganglion (SG) neurons and the synaptic areas under inner hair cells and OHCs with lateral and medial olivocochlear efferent nerves. Knockout (KO) of P2x7 increased hearing sensitivity with enhanced acoustic startle response, auditory brainstem response, and cochlear microphonics by increasing OHC electromotility. P2x7 KO also increased susceptibility to noise and exacerbated ribbon synapse degeneration. Middle-level noise exposure could impair active cochlear mechanics resulting in hearing loss in P2x7 KO mice. These data demonstrate that P2x7 receptors have a critical role in type II SG neuron’s function and the cochlear efferent system to control hearing sensitivity; deficiency of P2x7 receptors can impair type II SG neuron’s function and the cochlear efferent suppression leading to increase of active cochlear amplification and hearing oversensitivity, i.e., hyperacusis, and susceptibility to noise, which may also associate with other hearing disorders, such as tinnitus.

Background: Acoustic trauma is a common cause of acute sensorineural hearing loss associated with tinnitus; however, the underlying molecular mechanisms remain unclear. Extracellular signal-regulated kinase 2 (ERK2), a member of the mitogen-activated protein kinase family, is crucial in cellular signaling, especially in the nervous system, where it helps regulate neuroprotection, neurogenesis, and neuronal plasticity. ERK2 is activated in the cochlea by acoustic stimuli and plays a protective role in cochlear hair cells (HCs), which are the primary sensory receptors for hearing. However, the significance of ERK2 expression in HCs associated with development and etiology of tinnitus is largely unexplored. Methods: To investigate the role of ERK2 in tinnitus development, the gap detection test (GAP) was used to evaluate HC-specific ERK2-conditional knockout mice (HC-E2CKO) exposed to moderate acoustic stimuli. Results: Both control and HC-E2CKO mice showed normal prepulse inhibition levels (<0.6) before and after auditory damage, indicating normal functioning of sensorimotor gating pathways, excluding gross sensorimotor deficits. This confirmed that the animals were eligible for the GAP. HC-E2CKO mice showed a transient increase in the GAP ratio, indicating tinnitus development, 1 week after noise exposure, although the hearing threshold was not significantly elevated. The GAP ratio returned to normal after 2 weeks. In contrast, control mice did not exhibit elevation in hearing threshold and the GAP ratio remained normal. Conclusion: Our findings suggest that ERK2 in the inner ear plays a role in tinnitus onset or perception after acoustic stress, potentially through inhibitory mechanisms.

Introduction Prenatal exposure to valproic acid (VPA) is a common environmental cause of autism spectrum disorder (ASD) and often leads to expressive and receptive language impairments. Similar communication difficulties among individuals with ASD are often linked to abnormal subcortical and cortical sound processing. Rodents prenatally exposed to VPA exhibit degraded cortical responses to speech and an impaired ability to behaviorally discriminate speech sounds. Methods We sought to determine whether sound processing could be restored with paired vagus nerve stimulation (VNS). In a first experiment, we evaluated whether sound-paired VNS would alter in vivo extracellular multi-unit responses to tones, noise burst trains, and speech sounds from the anterior auditory field. We next sought to evaluate whether improvements to neural sound processing led to improvements in sound discrimination ability. In a second experiment, rats underwent go/no-go sound discrimination testing where VNS was paired with successful trials. Results We found that VPA-exposed rats had degraded spectral, temporal, and speech sound processing compared to saline-exposed control rats. VPA-exposed rats which received sound-paired VNS exhibited a partial or full restoration of processing across sound types. However, across several sound discrimination tasks, we did not observe changes in behavioral performance in response to prenatal exposure to VPA or VNS. Discussion Our study is the first to show that speech-paired VNS leads to a generalized improvement in cortical sound processing across sound types, rescuing neural processing among VPA-exposed rats. These results provide a framework for future studies to develop VNS-based interventions for communication disorders.

Hearing loss is common in conditions caused by ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency, such as generalized arterial calcification of infancy and autosomal recessive hypophosphatemic rickets type 2. Mechanistically, it is hypothesized that poor mineralization of the auditory ossicles leads to impaired sound transmission in the middle ear. Here we investigated whether enzyme replacement therapy (ERT) improves hearing loss in an Enpp1-deficient mouse model and whether this is associated with corrected bone properties in the ossicles. For this purpose, male Enpp1asj/asj mice were subjected to either a soluble or bone-targeted ERT of ENPP1-Fc. Hearing function and bone properties of the malleus, the first bone of the ossicular chain, were evaluated and compared to untreated Enpp1asj/asj and WT mice at the age of 17 wk. In untreated Enpp1-deficient mice, we found elevated hearing thresholds at stimulus frequencies of 8, 16, and 32 kHz, suggesting a generalized impairment of hearing. The hearing deficits observed in Enpp1asj/asj mice were partially or fully corrected by both soluble and bone-targeted ERT, as evidenced by the restoration of auditory brainstem response thresholds, with dose dependence and generally stronger effect of bone-targeted ERT. This was not only associated with corrected blood plasma markers of mineral metabolism but also improved matrix mineralization and restored osteocyte properties in the malleus. In addition, for the first time in mice, we were able to demonstrate the occurrence of mineralized osteocyte lacunae in Enpp1asj/asj mice, a phenomenon that was corrected by bone-targeted but not soluble ERT, underscoring the need for targeted delivery for effective treatment. In contrast to osteocyte lacunar mineralization in humans (ie, micropetrosis), the intra-lacunar mineral was overall less mineralized than the adjacent bone. In conclusion, our results point toward the potential benefit of correcting ossicular mineralization to prevent hearing loss in ENPP1 deficiency.

Many elderly listeners have difficulties with speech-in-noise perception, even if auditory thresholds in quiet are normal. The mechanisms underlying this compromised speech perception with age are still not understood. For identifying the physiological causes of these age-related speech perception difficulties, an appropriate animal model is needed enabling the use of invasive methods. In a comparative behavioral study, we used young-adult and quiet-aged Mongolian gerbils as well as young and elderly human subjects to investigate age-related changes in the discrimination of speech sounds in background noise, evaluating whether gerbils are an appropriate animal model for the age-related decline in speech-in-noise processing of human listeners. Gerbils and human subjects had to report a deviant consonant-vowel-consonant combination (CVC) or vowel-consonant-vowel combination (VCV) in a sequence of CVC or VCV standards, respectively. The logatomes were spoken by different speakers and masked by a steady-state speech-shaped noise. Response latencies were measured to generate perceptual maps employing multidimensional scaling, visualizing the subjects’ internal representation of the sounds. By analyzing response latencies for different types of vowels and consonants, we investigated whether aging had similar effects on the discrimination of speech sounds in background noise in gerbils compared to humans. For evaluating peripheral auditory function, auditory brainstem responses and audiograms were measured in gerbils and human subjects, respectively. We found that the overall phoneme discriminability in gerbils was independent of age, whereas consonant discriminability was declined in humans with age. Response latencies were generally longer in aged than in young gerbils and humans, respectively. Response latency patterns for the discrimination of different vowel or consonant types were different between species, but both gerbils and humans made use of the same articulatory features for phoneme discrimination. The species-specific response latency patterns were mostly unaffected by age across vowel types, while there were differential aging effects on the species-specific response latency patterns of different consonant types.

The mediodorsal thalamic nucleus (MD) forms neural circuits with various brain regions, including the prefrontal cortex (PFC), the reticular thalamic nucleus (TRN), and the midbrain reticular nucleus (MRN). However, the specific roles and underlying mechanisms in auditory information processing remain unclear. Here, we perform multi-channel electrophysiological recordings in awake mice to investigate the response patterns of the MD to auditory stimuli, as well as the regulatory effects of PFC, MRN, and TRN inputs. We identify two distinct types of sound-evoked responses. The Phasic-response features a transient burst firing to the stimulus with short latency, rapidly adapting to baseline and corresponding to the onset fluctuation of the local field potential. The Sustained-response is marked by prolonged firing with longer latency and is accompanied by persistent enhancement of oscillatory power following stimulus offset. The response patterns of MD neurons remain consistent across different types of auditory stimuli. Optogenetic inactivation of the MRN suppresses both response types in the MD. The Sustained-response is attenuated by PFC inactivation but enhanced by TRN inactivation, while the Phasic-response remains unaffected by inactivation of either the PFC or TRN. Our findings expand the understanding of the MD's role in sound information integration and auditory cognitive regulation.

Stereocilia are rod-like mechanosensory projections consisting of unidirectionally oriented actin filaments that extend into the inner ear hair cell cytoskeleton, forming dense rootlets. Taperin (TPRN) localizes to the narrowed-down base of stereocilia, where they pivot in response to sound and gravity. We show that TPRN-deficient mice have progressive deafness characterized by gradual asynchronous retraction and fusion of outer and inner hair cell stereocilia, followed by synaptic abnormalities. Stereocilia that lack TPRN develop warped rootlets with gradual loss of TRIOBP-5 and ANKRD24 from mechanosensory rows starting postnatally. In contrast, TPRN overexpression causes excessive F-actin bundling, extra rows, and over-elongation of stereocilia during development. Purified full-length mouse TPRN cross-links F-actin into bendable bundles reflecting in vivo data. This F-actin–bundling ability is attributed to the TPRN N-terminal region. TPRN interacts with the membrane receptor PTPRQ, connecting the F-actin core to the plasma membrane, stabilizing stereocilia. Thus, TPRN is a specialized F-actin bundler strategically located to augment stereocilia rootlet formation and their pivot point flexibility for sustained sound-induced deflections.