Hair Cell Mechanotransduction Research Articles

Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

Elasticity and Thermal Stability are Key Determinants of Hearing Rescue by Mini-Protocadherin-15 Proteins.

Protocadherin-15 is a core protein component of inner-ear hair-cell tip links pulling on transduction channels essential for hearing and balance. Protocadherin-15 defects can result in non-syndromic deafness or Usher syndrome type 1F (USH1F) with hearing loss, balance deficits, and progressive blindness. Three rationally engineered shortened versions of protocadherin-15 (mini-PCDH15s) amenable for gene therapy have been used to rescue function in USH1F mouse models. Two can successfully or partially rescue hearing, while another one fails. Here we show that despite varying levels of hearing rescue, all three mini-PCDH15 versions can rescue hair-cell mechanotransduction. Negative-stain electron microscopy shows that all three versions form dimers like the wild-type protein, while crystal structures of some engineered fragments show that these can properly fold and bind calcium ions essential for function. In contrast, simulations predict distinct elasticities and nano differential scanning fluorimetry shows differences in melting temperature measurements. Our data suggest that elasticity and thermal stability are key determinants of sustained hearing rescue by mini-PCDH15s.

In vivo screening for toxicity-modulating drug interactions identifies antagonism that protects against ototoxicity in zebrafish.

Introduction: Ototoxicity is a debilitating side effect of over 150 medications with diverse mechanisms of action, many of which could be taken concurrently to treat multiple conditions. Approaches for preclinical evaluation of drug-drug interactions that might impact ototoxicity would facilitate design of safer multi-drug regimens and mitigate unsafe polypharmacy by flagging combinations that potentially cause adverse interactions for monitoring. They may also identify protective agents that antagonize ototoxic injury. Methods: To address this need, we have developed a novel workflow that we call Parallelized Evaluation of Protection and Injury for Toxicity Assessment (PEPITA), which empowers high-throughput, semi-automated quantification of ototoxicity and otoprotection in zebrafish larvae via microscopy. We used PEPITA and confocal microscopy to characterize in vivo the consequences of drug-drug interactions on ototoxic drug uptake and cellular damage of zebrafish lateral line hair cells. Results and discussion: By applying PEPITA to measure ototoxic drug interaction outcomes, we discovered antagonistic interactions between macrolide and aminoglycoside antibiotics that confer protection against aminoglycoside-induced damage to lateral line hair cells in zebrafish larvae. Co-administration of either azithromycin or erythromycin in zebrafish protected against damage from a broad panel of aminoglycosides, at least in part via inhibiting drug uptake into hair cells via a mechanism independent from hair cell mechanotransduction. Conversely, combining macrolides with aminoglycosides in bacterial inhibition assays does not show antagonism of antimicrobial efficacy. The proof-of-concept otoprotective antagonism suggests that combinatorial interventions can potentially be developed to protect against other forms of toxicity without hindering on-target drug efficacy.

Differential expression of mechanotransduction complex genes in auditory/vestibular hair cells in zebrafish.

Ciliated sensory cells such as photo- and olfactory receptors employ multiple types of opsins or hundreds of unique olfactory G-protein coupled receptors to respond to various wavelengths of light or odorants. With respect to hearing and balance, the mechanotransduction machinery involves fewer variants; however, emerging evidence suggests that specialization occurs at the molecular level. To address how the mechanotransduction complex varies in the inner ear, we characterized the expression of paralogous genes that encode components required for mechanotransduction in zebrafish hair cells using RNA-FISH and bioinformatic analysis. Our data indicate striking zonal differences in the expression of two components of the mechanotransduction complex which are known to physically interact, the transmembrane channel-like 1 and 2 (tmc1/2) family members and the calcium and integrin binding 2 and 3 (cib2/3) paralogues. tmc1, tmc2b, and cib3 are largely expressed in peripheral or extrastriolar hair cells, whereas tmc2a and cib2 are enriched in central or striolar hair cells. In addition, a gene implicated in deaf-blindness, ush1c, is highly enriched in a subset of extrastriolar hair cells. These results indicate that specific combinations of these components may optimize responses to mechanical stimuli in subtypes of sensory receptors within the inner ear.

Novel heterozygous USH1C mutation impacts hair cell mechanotransduction and causes progressive hearing loss

Novel heterozygous USH1C mutation impacts hair cell mechanotransduction and causes progressive hearing loss

Influence of the vestibular system on the neonatal motor behaviors in the gray short-tailed opossum (Monodelphis domestica)

Marsupials are born very immature yet must be sufficiently autonomous to crawl on the mother’s belly, find a teat and attach to it to pursue their development. Sensory inputs are necessary to guide the newborn to a teat and induce attachment. The vestibular system, which perceives gravity and head movements, is one of the senses proposed to guide newborns towards the teats but there are conflicting observations about its functionality at birth (postnatal day (P) 0). To test if the vestibular system of opossum newborns is functional and can influence locomotion, we used two approaches. First, we stimulated the vestibular apparatus in in vitro preparations from opossums aged from P1 to P12 and recorded motor responses: at all ages studied, mechanical pressures applied on the vestibular organs induced spinal roots activity whereas head tilts did not induce forelimb muscle contractions. Second, using immunofluorescence, we assessed the presence of Piezo2, a protein involved in mechanotransduction in vestibular hair cells. Piezo2 labeling was scant in the utricular macula at birth, but observed in all vestibular organs at P7, its intensity increasing up to P14; it seemed to stay the same at P21. Our results indicate that neural pathways from the labyrinth to the spinal cord are already in place around birth but that the vestibular organs are too immature to influence motor activity before the end of the second postnatal week in the opossum. It may be the rule in marsupial species that the vestibular system becomes functional only after birth.

Rab11a Is Essential for the Development and Integrity of the Stereocilia and Kinocilia in the Mammalian Organ of Corti.

The cochlea hair cells transform mechanic sounds to neural signals with a remarkable sensitivity and resolution. This is achieved via the precisely sculpted mechanotransduction apparatus of the hair cells and the supporting structure of the cochlea. The shaping of the mechanotransduction apparatus, the staircased stereocilia bundles on the apical surface of the hair cells, requires an intricate regulatory network including planar cell polarity (PCP) and primary cilia genes in orienting stereocilia bundles and building molecular machinery of the apical protrusions. The mechanism linking these regulatory components is unknown. Here, we show that a small GTPase known for its role in protein trafficking, Rab11a, is required for ciliogenesis in hair cells during development in mice. In addition, in the absence of Rab11a, stereocilia bundles lost their cohesion and integrity, and mice are deaf. These data indicate an essential role of protein trafficking in the formation of hair cell mechanotransduction apparatus, implicating a role of Rab11a or protein trafficking in linking the cilia and polarity regulatory components with the molecular machinery in building the cohesive and precisely shaped stereocilia bundles.

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining.

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

Cholesterol as a tool to probe the role of membrane in cochlear hair cell mechanotransduction.

Most mechanically gated channels are sensitive to force translated through the membrane. The lipid bilayer can modulate channel function directly through lipid/protein interactions or indirectly based on membrane mechanical properties. Cholesterol is an important component of the membrane and a major modulator of membrane mechanical properties. While other ion channels and mechanically gated channels can be activated or modulated by changes to the membrane cholesterol, the functional role of membrane cholesterol in regulation of mammalian cochlear mechanotransduction (MET) channels has not been closely investigated. Using whole-cell patch clamping and live-cell fluorescence lifetime imaging (FLIM) of a viscosity-sensitive molecular rotor BODIPY 1c for the first time in the inner ear, we examined the role of membrane cholesterol in modulating the MET response properties of rat cochlear hair cells. Molecular rotors are fluorophores for which the fluorescence lifetime (the average time a fluorophore remains in the excited state) increase with increasing viscosity of their immediate environment. We confirmed extraction of cholesterol, using methyl β cyclodextrin (MβCD), with reduced filipin staining in both inner and outer hair bundles. MβCD results in reversible reduction in fluorescence lifetime in hair bundles, suggesting initial reduction followed by gradual recovery in the stereocilia membrane viscosity. MβCD reversibly increases the channel resting open probability, suggesting that cholesterol depletion increases force transfer to the MET channel. Together this data suggests that the cell membrane is part of the force relay machinery to the MET channel and could possibly interact directly with components of the MET machinery. Further studies are needed to generate causal link between MET channel gating and membrane mechanics.

Structure of a non-mammalian cadherin heterodimer suggests a general binding mechanism essential for hair-cell mechanotransduction.

Vertebrate hair cells have evolved over 500 million years to function as exquisitely precise and robust mechanosensors that transform mechanical stimuli into electrochemical signals for brain processing. Critical for this process of mechanotransduction is the tip link, a fine protein filament conveying force to gate ion channels and trigger sensory perception. In various mammals, cadherin-23 and protocadherin-15 interact to form tip links. Crystal structures of mouse protein fragments have shown that this interaction is mediated by the protein tips (EC1-2), which engage in an antiparallel “handshake complex” essential for hair-cell mechanotransduction that has been validated in vitro and in vivo. However, less is known about non-mammalian tip links. There is evidence supporting the existence of tip links made of cadherin-23 and protocadherin-15 in birds and fish, and our surface plasmon resonance experiments suggest that reptile tip-link proteins also form handshake complexes. Here we report the X-ray crystal structure of the non-mammalian catfish (Pangasianodon hypophthalmus) cadherin-23 and protocadherin-15b EC1-2 heterodimer complex at 2.6-Å resolution. The catfish protein fragments arrange in an antiparallel dimer that strongly resembles that of the mammalian handshake complex, suggesting a general binding mechanism for cadherin-23 and protocadherin-15 throughout vertebrates and paralogues.

In-silico insights into aminoglycoside intake by TMC1.

Aminoglycosides are small-molecule antibiotics that are widely used as treatment against a variety of bacterial infections. A side-effect of aminoglycosides use is dose-dependent hearing loss, often referred to as cochleotoxicity. This is hypothesized to be due to the inability of the inner-ear sensory hair cells to clear these molecules out leading to permanent hair-cell death. Multiple experiments involving manipulations of components of the hair-cell mechanotransduction machinery, including tip links and transmembrane channel-like 1 (TMC1) and TMC2 proteins, have shown that an important pathway for aminoglycoside intake by hair-cells is their transduction channel. Recent studies have also shown that TMC1 and TMC2 proteins are the pore forming components of the hair-cell mechanotransduction machinery. Although experiments suggest that these positively charged aminoglycoside molecules can enter hair cells through TMCs, the mechanism and the energetics of this process have not been studied. Here we use AlphaFold2 models of the human TMC1 protein with and without calcium- and integrin-binding protein 2 (CIB2) in the presence of Kanamycin A (KanA) along with microsecond-long voltage simulations to study the pore opening mechanism and the interactions between KanA and pore residues. We also present the free-energy surface of the process obtained using metadynamics simulations (a biased sampling method) which quantifies the energetics of this pore-crossing event. The open-pore conformations that were generated because of the KanA crossing event were further simulated with voltage to calculate the conductance of the channel in its putative open state and to assess the role of CIB2 in its conduction. These results give qualitative and a quantitative insights into the mechanism of pore opening and KanA crossing through the inner-ear transduction channel.

Structural characterization of mini-PCDH15 proteins for Usher syndrome type 1F therapy.

Hair cells, the sensory cells of the inner ear, carry actin-filled microvillus-like projections called stereocilia, which are interconnected by long protein filaments called “tip links”. Upon sound stimulation, tip links enable hair-cell mechanotransduction, the critical conversion of mechanical deflection into an electrical signal our brain can understand. Tip links are formed by two pairs of large Ca2+-binding proteins, cadherin-23 (CDH23) and protocadherin-15 (PCDH15), interacting tip-to-tip in a Ca2+-dependent manner. The ectodomain of PCDH15 is made of 11 extracellular cadherin (EC) repeats, with EC linkers stabilized by Ca2+ ions bound to acidic residues. Mutations in PCDH15 cause Usher Syndrome Type 1F, characterized by congenital hearing loss, balance deficit, and progressive blindness. Adeno-associated virus (AAV) vectors have been found to be efficient and effective for gene therapy in the inner ear. However, the PCDH15 coding sequence is too large to fit in AAV. We have developed novel “mini-PCDH15” constructs that retain critical domains but lack 3-5 EC repeats, and that consequently fit in AAV vectors. Some of the mini-PCDH15s are functional and rescue hearing and balance in Pcdh15-knockout mice despite having engineered Ca2+-binding linkers that are not found in nature. Here, we present structural and computational studies of mini-PCDH15s, including an X-ray crystal structure of an artificial EC3-EC7 bent linker of a mini-PCDH15 that partially rescues hearing. In addition, we report on biochemical assays, low-resolution cryo-EM, and molecular dynamics simulations used to compare the flexibility, rigidity, and Ca2+-binding ability of wild-type and mini-PCDH15 ectodomains. Our mini-PCDH15s show different stability and behave as dimers in solution, showing diverse conformational arrangements that might modulate binding to CDH23. Our results provide an atomistic view of engineered mini-PCDH15s and promote the use of rational, iterative, structure-based mini-gene approach to develop gene therapies for large proteins.

Mutation-agnostic RNA interference with engineered replacement rescues Tmc1-related hearing loss.

Hearing loss is the most common sensory deficit, of which genetic etiologies are a frequent cause. Dominant and recessive mutations in TMC1, a gene encoding the pore-forming subunit of the hair cell mechanotransduction channel, cause DFNA36 and DFNB7/11, respectively, accounting for ∼2% of genetic hearing loss. Previous work has established the efficacy of mutation-targeted RNAi in treatment of murine models of autosomal dominant non-syndromic deafness. However, application of such approaches is limited by the infeasibility of development and validation of novel constructs for each variant. We developed an allele-non-specific approach consisting of mutation-agnostic RNAi suppression of both mutant and WT alleles, co-delivered with a knockdown-resistant engineered WT allele with or without the use of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to augment transgene expression. This therapeutic construct was delivered into the mature murine model of DFNA36 with an AAV vector and achieved robust hair cell and auditory brainstem response preservation. However, WPRE-enhanced Tmc1 expression resulted in inferior outcomes, suggesting a role for gene dosage optimization in future TMC1 gene therapy development.

Prolonged Dexamethasone Exposure Enhances Zebrafish Lateral-Line Regeneration But Disrupts Mitochondrial Homeostasis and Hair Cell Function

The synthetic glucocorticoid dexamethasone is commonly used to treat inner ear disorders. Previous work in larval zebrafish has shown that dexamethasone treatment enhances hair cell regeneration, yet dexamethasone has also been shown to inhibit regeneration of peripheral nerves after lesion. We therefore used the zebrafish model to determine the impact of dexamethasone treatment on lateral-line hair cells and primary afferents. To explore dexamethasone in the context of regeneration, we used copper sulfate (CuSO4) to induce hair cell loss and retraction of nerve terminals, and then allowed animals to recover in dexamethasone for 48 h. Consistent with previous work, we observed significantly more regenerated hair cells in dexamethasone-treated larvae. Importantly, we found that the afferent processes beneath neuromasts also regenerated in the presence of dexamethasone and formed an appropriate number of synapses, indicating that innervation of hair cells was not inhibited by dexamethasone. In addition to regeneration, we also explored the effects of prolonged dexamethasone exposure on lateral-line homeostasis and function. Following dexamethasone treatment, we observed hyperpolarized mitochondrial membrane potentials (ΔΨm) in neuromast hair cells and supporting cells. Hair cells exposed to dexamethasone were also more vulnerable to neomycin-induced cell death. In response to a fluid-jet delivered saturating stimulus, calcium influx through hair cell mechanotransduction channels was significantly reduced, yet presynaptic calcium influx was unchanged. Cumulatively, these observations indicate that dexamethasone enhances hair cell regeneration in lateral-line neuromasts, yet also disrupts mitochondrial homeostasis, making hair cells more vulnerable to ototoxic insults and possibly impacting hair cell function.

Considering gene therapy to protect from X-linked deafness DFNX2 and associated neurodevelopmental disorders.

Mutations and deletions in the gene or upstream of the gene encoding the POU3F4 transcription factor cause X-linked progressive deafness DFNX2 and additional neurodevelopmental disorders in humans. Hearing loss can be purely sensorineural or mixed, that is, with both conductive and sensorineural components. Affected males show anatomical abnormalities of the inner ear, which are jointly defined as incomplete partition type III. Current approaches to improve hearing and speech skills of DFNX2 patients do not seem to be fully effective. Owing to inner ear malformations, cochlear implantation is surgically difficult and may predispose towards severe complications. Even in cases where implantation is safely performed, hearing and speech outcomes remain highly variable among patients. Mouse models for DFNX2 deafness revealed that sensorineural loss could arise from a dysfunction of spiral ligament fibrocytes in the lateral wall of the cochlea, which leads to reduced endocochlear potential. Highly positive endocochlear potential is critical for sensory hair cell mechanotransduction and hearing. In this context, here, we propose to develop a therapeutic approach in male Pou3f4 -/y mice based on an adeno-associated viral (AAV) vector-mediated gene transfer in cochlear spiral ligament fibrocytes. Among a broad range of AAV vectors, AAV7 was found to show a strong tropism for the spiral ligament. Thus, we suggest that an AAV7-mediated delivery of Pou3f4 complementary DNA in the spiral ligament of Pou3f4 -/y mice could represent an attractive strategy to prevent fibrocyte degeneration and to restore normal cochlear functions and properties, including a positive endocochlear potential, before hearing loss progresses to profound deafness.

High-Frequency Hearing Is Required to Compute a Topographic Map of Auditory Space in the Mouse Superior Colliculus.

A topographic map of auditory space is a feature found in the superior colliculus (SC) of many species, including CBA/CaJ mice. In this genetic background, high-frequency monaural spectral cues and interaural level differences (ILDs) are used to compute spatial receptive fields (RFs) that form a topographic map along the azimuth. Unfortunately, C57BL/6 mice, a strain widely used for transgenic manipulation, display age-related hearing loss (AHL) because of an inbred mutation in the Cadherin 23 gene (Cdh23) that affects hair cell mechanotransduction. To overcome this problem, researchers have used young C57BL/6 mice in their studies, as they have been shown to have normal hearing thresholds. However, important details of the auditory response characteristics of the SC such as spectral responses and spatial localization, have not been characterized in young C57BL/6 mice. Here, we show that two- to four-month C57BL/6 mice lack neurons with frontal auditory RFs and therefore lack a topographic representation of auditory space in the SC. Analysis of the spectrotemporal RFs (STRFs) of the SC auditory neurons shows that C57BL/6 mouse SC neurons lack the ability to detect the high-frequency (>40 kHz) spectral cues that are needed to compute frontal RFs. We also show that crossing C57BL/6 mice with CBA/CaJ mice or introducing one copy of the wild-type Cdh23 to C57BL/6 mice rescues the high-frequency hearing deficit and improves the topographic map of auditory space. Taken together, these results demonstrate the importance of high-frequency hearing in computing a topographic map of auditory space.

Two kinematic gains underlying the organ of Corti mechanotransduction

The organ of Corti (OoC) is the sensory epithelium in the mammalian hearing organ where vibrations are encoded into neural signals. The OoC vibrates due to external and internal agitations. Externally, differential fluid pressures across the OoC cause vibrations. Internally, actuator cells (outer hair cells) are known to boost the vibrations. These two agitations result in distinct vibration patterns. Eventually, these vibrations deflect the stereocilia bundle to activate mechanotransduction channels in them. How OoC vibrations result in the hair cell mechanotransduction is critical to hearing research, but a fundamental information (kinematic gain) has not been quantified clearly. Using the optical coherence tomography, we measured 2-D vibrations of the OoC in cochlear turns acutely excised from young Mongolian gerbils. The excised tissues were stimulated either mechanically or electrically, which are comparable to external or internal agitations, respectively. The stereocilia deflection was estimated from relative motion between overlying and underlying structures. Two kinematic gains were defined. Passive kinematic gain represents the amplitude ratio between the stereocilia deflection and the basilar membrane transverse vibration. Active kinematic gain represents the amplitude ratio between the stereocilia deflection and the outer hair cell length change. (Work supported by NIH NIDCD R01 DC014685.)

Fast adaptation of cooperative channels engenders Hopf bifurcations in auditory hair cells

Since the pioneering work of Thomas Gold, published in 1948, it has been known that we owe our sensitive sense of hearing to a process in the inner ear that can amplify incident sounds on a cycle-by-cycle basis. Called the active process, it uses energy to counteract the viscous dissipation associated with sound-evoked vibrations of the ear’s mechanotransduction apparatus. Despite its importance, the mechanism of the active process and the proximate source of energy that powers it have remained elusive, especially at the high frequencies characteristic of amniote hearing. This is partly due to our insufficient understanding of the mechanotransduction process in hair cells, the sensory receptors and amplifiers of the inner ear. It has been proposed previously that cyclical binding of Ca2+ ions to individual mechanotransduction channels could power the active process. That model, however, relied on tailored reaction rates that structurally forced the direction of the cycle. Here we ground our study on our previous model of hair-cell mechanotransduction, which relied on cooperative gating of pairs of channels, and incorporate into it the cyclical binding of Ca2+ ions. With a single binding site per channel and reaction rates drawn from thermodynamic principles, the current model shows that hair cells behave as nonlinear oscillators that exhibit Hopf bifurcations, dynamical instabilities long understood to be signatures of the active process. Using realistic parameter values, we find bifurcations at frequencies in the kilohertz range with physiological Ca2+ concentrations. The current model relies on the electrochemical gradient of Ca2+ as the only energy source for the active process and on the relative motion of cooperative channels within the stereociliary membrane as the sole mechanical driver. Equipped with these two mechanisms, a hair bundle proves capable of operating at frequencies in the kilohertz range, characteristic of amniote hearing.

Ca2+ entry through mechanotransduction channels localizes BAIAP2L2 to stereocilia tips.

Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 (BAIAP2L2), a membrane-binding protein required for the maintenance of mechanotransduction in hair cells, is selectively retained at the tips of transducing stereocilia. BAIAP2L2 trafficked to stereocilia tips in the absence of EPS8, but EPS8 increased the efficiency of localization. A tripartite complex of BAIAP2L2, EPS8, and MYO15A formed efficiently in vitro, and these three proteins robustly targeted to filopodia tips when coexpressed in cultured cells. Mice lacking functional transduction channels no longer concentrated BAIAP2L2 at row 2 stereocilia tips, a result that was phenocopied by blocking channels with tubocurarine in cochlear explants. Transduction channels permit Ca2+ entry into stereocilia, and we found that membrane localization of BAIAP2L2 was enhanced in the presence of Ca2+. Finally, reduction of intracellular Ca2+ in hair cells using BAPTA-AM led to a loss of BAIAP2L2 at stereocilia tips. Taken together, our results show that a MYO15A-EPS8 complex transports BAIAP2L2 to stereocilia tips, and Ca2+ entry through open channels at row 2 tips retains BAIAP2L2 there.

Single-Cell RNA-Seq of Cisplatin-Treated Adult Stria Vascularis Identifies Cell Type-Specific Regulatory Networks and Novel Therapeutic Gene Targets.

The endocochlear potential (EP) generated by the stria vascularis (SV) is necessary for hair cell mechanotransduction in the mammalian cochlea. We sought to create a model of EP dysfunction for the purposes of transcriptional analysis and treatment testing. By administering a single dose of cisplatin, a commonly prescribed cancer treatment drug with ototoxic side effects, to the adult mouse, we acutely disrupt EP generation. By combining these data with single cell RNA-sequencing findings, we identify transcriptional changes induced by cisplatin exposure, and by extension transcriptional changes accompanying EP reduction, in the major cell types of the SV. We use these data to identify gene regulatory networks unique to cisplatin treated SV, as well as the differentially expressed and druggable gene targets within those networks. Our results reconstruct transcriptional responses that occur in gene expression on the cellular level while identifying possible targets for interventions not only in cisplatin ototoxicity but also in EP dysfunction.