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Mammalian hair cells (HCs) are arranged spirally along the cochlear axis and correspond to different frequency ranges. Serving as primary sound detectors, HCs spatially segregate component frequencies into a topographical map. HCs display significant diversity in anatomical and physiological characteristics, yet little is known about the organization of the cochleotopic map of HCs or the molecules involved in this process. Using single-cell RNA sequencing, we determined the distinct molecular profiles of inner hair cells and outer hair cells, and we identified numerous position-dependent genes that were expressed as gradients. Newly identified genes such as Ptn, Rxra, and Nfe2l2were found to be associated with tonotopy. We employed the SCENIC algorithm to predict the transcription factors that potentially shape these tonotopic gradients. Furthermore, we confirmed that Nfe2l2, a tonotopy-related transcription factor, is critical in mice for sensing low-to-medium sound frequencies in vivo. the analysis of cell-cell communication revealed potential receptor-ligand networks linking inner hair cells to spiral ganglion neurons, including pathways such as BDNF-Ntrk and PTN-Scd4, which likely play essential roles in tonotopic maintenance. Overall, these findings suggest that molecular gradients serve as the organizing principle for maintaining the selection of sound frequencies by HCs.

Fibroblast growth factor receptor 1 (FGFR1) is emerging as a promising molecular target of lung cancer, and various FGFR1 inhibitors have exhibited significant therapeutic effects on lung cancer in preclinical research. Due to their low targeting ability or bioavailability, direct administration of these inhibitors may cause side effects. Herein, a hydrogelator, Nap-Phe-Phe-Phe-Glu-Thr-Glu-Leu-Tyr-OH (Nap-Y), was rationally designed to coassemble with an FGFR1 inhibitor nintedanib (Nin) to form a peptide hydrogel Gel Y/Ninfor localized administration and FGFR1-triggered release of Nin. Upon specific phosphorylation by FGFR1 overexpressed on lung cancer cells, Nap-Yin Gel Y/Ninis converted to the hydrophilic product Nap-Phe-Phe-Phe-Glu-Thr-Glu-Leu-Tyr(H2PO3)-OH (Nap-Yp), leading to dehydrogelation of the gel and subsequent Ninrelease. In vitroexperiments demonstrate that the release of Ninin a sustained manner from Gel Y/Ninsignificantly suppresses the survival, migration, and invasion of A549 cells by inhibiting FGFR1 expression and its phosphorylation function on downstream signaling molecules. Nude mouse studies show that Gel Y/Ninexhibits enhanced therapeutic efficacy on lung tumor than free Nin. We anticipate that Gel Y/Ninwill be utilized for lung cancer treatment in clinical settings in the near future.

Autosomal recessive deafness 9, caused by mutations of the OTOFgene, is characterised by congenital or prelingual, severe-to-complete, bilateral hearing loss. However, no pharmacological treatment is currently available for congenital deafness. In this Article, we report the safety and efficacy of gene therapy with an adeno-associated virus (AAV) serotype 1 carrying a human OTOFtransgene (AAV1-hOTOF) as a treatment for children with autosomal recessive deafness 9.

Inner ear hair cells detect sound vibration through the deflection of mechanosensory stereocilia. Cytoplasmic protein TPRN has been shown to localize at the taper region of the stereocilia, and mutations in TPRN cause hereditary hearing loss through an unknown mechanism. Here, using biochemistry and dual stimulated emission depletion microscopy imaging, we show that the TPRN, together with its binding proteins CLIC5 and PTPRQ, forms concentric rings in the taper region of stereocilia. The disruption of TPRN rings, triggered by the competitive inhibition of the interaction of TPRN and CLIC5 or exogenous TPRN overexpression, leads to stereocilia degeneration and severe hearing loss. Most importantly, restoration of the TPRN rings can rescue the damaged auditory function of Tprnknockout mice by exogenously expressing TPRN at an appropriate level in HCs via promoter recombinant adeno-associated virus (AAV). In summary, our results reveal highly structured TPRN rings near the taper region of stereocilia that are crucial for stereocilia function and hearing. Also, TPRN ring restoration in stereocilia by AAV-Tprneffectively repairs damaged hearing, which lays the foundation for the clinical application of AAV-mediated gene therapy in patients with TPRNmutation.

The gut microbiota play a pivotal role in human health. Emerging evidence indicates that gut microbes participate in the progression of tumorigenesis through the generation of carcinogenic metabolites. However, the underlying molecular mechanism is largely unknown. In the present study we show that a tryptophan metabolite derived from Peptostreptococcus anaerobius, trans-3-indoleacrylic acid (IDA), facilitates colorectal carcinogenesis. Mechanistically, IDA acts as an endogenous ligand of an aryl hydrocarbon receptor (AHR) to transcriptionally upregulate the expression of ALDH1A3(aldehyde dehydrogenase 1 family member A3), which utilizes retinal as a substrate to generate NADH, essential for ferroptosis-suppressor protein 1(FSP1)-mediated synthesis of reduced coenzyme Q10. Loss of AHR or ALDH1A3largely abrogates IDA-promoted tumour development both in vitro and in vivo. It is interesting that P. anaerobiusis significantly enriched in patients with colorectal cancer (CRC). IDA treatment or implantation of P. anaerobiuspromotes CRC progression in both xenograft model and ApcMin/+mice. Together, our findings demonstrate that targeting the IDA–AHR–ALDH1A3axis should be promising for ferroptosis-related CRC treatment.

Auditory hair cells (HCs) are the mechanosensory receptors of the cochlea, and HC loss or malfunction can result from genetic defects. Dock4, a member of the Dock180-related protein superfamily, is a guanine nucleotide exchange factor for Rac1, and previous reports have shown that Dock4mutations are associated with autism spectrum disorder, myelodysplastic syndromes, and tumorigenesis. Here, we found that Dock4 is highly expressed in the cochlear HCs of mice. However, the role of Dock4 in the inner ear has not yet been investigated. Taking advantage of the piggyBac transposon system, Dock4knockdown (KD) mice were established to explore the role of Dock4 in the cochlea. Compared to wild-type controls, Dock4KD mice showed significant hearing impairment from postnatal day 60. Dock4KD mice showed hair bundle deficits and increased oxidative stress, which eventually led to HC apoptosis, late-onset HC loss, and progressive hearing loss. Furthermore, molecular mechanism studies showed that Rac1/β-catenin signaling was significantly downregulated in Dock4KD cochleae and that this was the cause for the disorganized stereocilia and increased oxidative stress in HCs. Overall, our work demonstrates that the Dock4/Rac1/β-catenin signaling pathway plays a critical role in the maintenance of auditory HCs and hearing function.

ABSTRACTAminoglycosides exhibit ototoxicity by damaging mitochondria, which in turn generate reactive oxygen species that induce hair cell death and subsequent hearing loss. It is well known that damaged mitochondria are degraded by mitophagy, an important mitochondrial quality control system that maintains mitochondrial homeostasis and ensures cell survival. However, it is unclear whether dysregulation of mitophagy contributes to aminoglycoside-induced hair cell injury. In the current study, we found that PINK1-PRKN-mediated mitophagy was impaired in neomycin-treated hair cells. Our data suggested that mitochondrial recruitment of PRKN and phagophore recognition of damaged mitochondria during mitophagy were blocked following neomycin treatment. In addition, the degradation of damaged mitochondria by lysosomes was significantly decreased as indicated by the mitophagic flux reporter mt-mKeima. Moreover, we demonstrated that neomycin disrupted mitophagy through transcriptional inhibition of Pink1expression, the key initiator of mitophagy. Moreover, we found that neomycin impaired mitophagy by inducing ATF3 expression. Importantly, treatment with a mitophagy activator could rescue neomycin-treated hair cells by increasing mitophagy, indicating that genetic modulation or drug intervention in mitophagy may have therapeutic potential for aminoglycoside-induced hearing loss.Abbreviations:AAV: adeno-associated virus; ABR: auditory brainstem response; ATF3: activating transcription factor 3; ATOH1/MATH1: atonal bHLH transcription factor 1; BafA1: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; COX4I1/COXIV: cytochrome c oxidase subunit 4I1; CTBP2/RIBEYE: C-terminal binding protein 2; DFP: deferiprone; EGFP: enhanced green fluorescent protein; FOXO3: forkhead box O3; GRIA2/GLUR2: glutamate receptor, ionotropic, AMPA2 (alpha 2); HC: hair cell; HSPD1/HSP60: heat shock protein 1 (chaperonin); IHC: inner hair cell; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MYO7A: myosin VIIA; OPTN: optineurin; OMM: outer mitochondrial membrane; PRKN: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced putative kinase 1; RT-qPCR: real-time quantitative polymerase chain reaction; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; USP30: ubiquitin specific peptidase 30; XBP1: X-box binding protein 1.

Noise-induced hearing loss (NIHL) is a highly prevalent form of sensorineural hearing damage that has significant negative effects on individuals of all ages and there are no effective drugs approved by the US Food and Drug Administration. In this study, we unveil the potential of superparamagnetic iron oxide nanoparticle assembly (SPIOCA) to reshape the dysbiosis of gut microbiota for treating NIHL. This modulation inhibits intestinal inflammation and oxidative stress responses, protecting the integrity of the intestinal barrier. Consequently, it reduces the transportation of pathogens and inflammatory factors from the bloodstream to the cochlea. Additionally, gut microbiota-modulated SPIOCA-induced metabolic reprogramming in the gut–inner ear axis mainly depends on the regulation of the sphingolipid metabolic pathway, which further contributes to the restoration of hearing function. Our study confirms the role of the microbiota–gut–inner ear axis in NIHL and provides a novel alternative for the treatment of NIHL and other microbiota dysbiosis-related diseases.This study presents a novel strategy to protect against noise-induced hearing loss by regulating the microbiota-gut-inner ear axis with iron oxide nanoparticles.

Myosin VI（MYO6） is an unconventional myosin that is vital for auditory and vestibular function. Pathogenic variants in the human MYO6gene cause autosomal-dominant or -recessive forms of hearing loss. Effective treatments for Myo6mutation causing hearing loss are limited. We studied whether adeno-associated virus (AAV)-PHP.eB vector-mediated in vivodelivery of Staphylococcus aureusCas9 (SaCas9-KKH)-single-guide RNA (sgRNA) complexes could ameliorate hearing loss in a Myo6WT/C442Ymouse model that recapitulated the phenotypes of human patients. The in vivoediting efficiency of the AAV-SaCas9-KKH-Myo6-g2 system on Myo6C442Yis 4.05% on average in Myo6WT/C442Ymice, which was ∼17-fold greater than editing efficiency of Myo6WTalleles. Rescue of auditory function was observed up to 5 months post AAV-SaCas9-KKH-Myo6-g2 injection in Myo6WT/C442Ymice. Meanwhile, shorter latencies of auditory brainstem response (ABR) wave I, lower distortion product otoacoustic emission (DPOAE) thresholds, increased cell survival rates, more regular hair bundle morphology, and recovery of inward calcium levels were also observed in the AAV-SaCas9-KKH-Myo6-g2-treated ears compared to untreated ears. These findings provide further reference for in vivogenome editing as a therapeutic treatment for various semi-dominant forms of hearing loss and other semi-dominant diseases.

ABSTRACTSpiral ganglion neurons (SGNs) are auditory neurons that relay sound signals from the inner ear to the brainstem. The ototoxic drug cisplatin can damage SGNs and thus lead to sensorineural hearing loss (SNHL), and there are currently no methods for preventing or treating this. Macroautophagy/autophagy plays a critical role in SGN development, but the effect of autophagy on cisplatin-induced SGN injury is unclear. Here, we first found that autophagic flux was activated in SGNs after cisplatin damage. The SGN apoptosis and related hearing loss induced by cisplatin were alleviated after co-treatment with the autophagy activator rapamycin, whereas these were exacerbated by the autophagy inhibitor 3-methyladenine, indicating that instead of inducing SGN death, autophagy played a neuroprotective role in SGNs treated with cisplatin both in vitroand in vivo. We further demonstrated that autophagy attenuated reactive oxygen species (ROS) accumulation and alleviated cisplatin-induced oxidative stress in SGNs to mediate its protective effects. Notably, the role of the antioxidant enzyme PRDX1 (peroxiredoxin 1) in modulating autophagy in SGNs was first identified. Deficiency in PRDX1 suppressed autophagy and increased SGN loss after cisplatin exposure, while upregulating PRDX1 pharmacologically or by adeno-associated virus activated autophagy and thus inhibited ROS accumulation and apoptosis and attenuated SGN loss induced by cisplatin. Finally, we showed that the underlying mechanism through which PRDX1 triggers autophagy in SGNs was, at least partially, through activation of the PTEN-AKT signaling pathway. These findings suggest potential therapeutic targets for the amelioration of drug-induced SNHL through autophagy activation.Abbreviations: 3-MA: 3-methyladenine; AAV : adeno-associated virus; ABR: auditory brainstem responses; AKT/protein kinase B: thymoma viral proto-oncogene; Baf: bafilomycin A1; CAP: compound action potential; COX4I1: cytochrome c oxidase subunit 4I1; Cys: cysteine; ER: endoplasmic reticulum; H2O2: hydrogen peroxide; HC: hair cell; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; NAC: N-acetylcysteine; PRDX1: peroxiredoxin 1; PTEN: phosphatase and tensin homolog; RAP: rapamycin; ROS: reactive oxygen species; SGNs: spiral ganglion neurons; SNHL: sensorineural hearing loss; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling; WT: wild type.

Lipopolysaccharide (LPS) plays an important role in metabolic syndrome (MetS) and other gut-derived diseases, and detoxifying LPS is considered to be a fundamental approach to prevent and treat these diseases. Here, inspired by the feeding behaviour of scavenger, novel microfluidic microcapsules with alkaline phosphatase (ALP) encapsulation and the scavenger-like molecular sieve shell are presented for cleaning intestinal LPS. Benefiting from the precisely controlled of the pore size and microfluidic electrospray, the generated microcapsules were imparted with porous molecular-sieve shells and ALP encapsulated active cores. These microcapsules could continuously work as an intestinal scavenger after colonized in intestine. It has been demonstrated that the microcapsules could englobe LPS while inhibit the permeation of digestive enzyme, and this ability contributes to promising ALP's activity, protecting cells at the presence of LPS and reducing inflammation. In addition, this scavenger inspired microcapsule could effectively decrease the LPS in organs, reduce inflammation and regulating fat metabolism in vivo. These features make the ALP encapsulated microcapsules an ideal candidate for treating MetS and other LPS related diseases.

Cochlear implants are currently the most effective treatment for profound sensorineural hearing loss. However, their therapeutic effect is limited by the survival and proper physiological function of spiral ganglion neurons (SGNs), which are targeted by the cochlear implant. It is therefore critical to explore the mechanism behind the effect of electric-acoustic stimulation (EAS) on the targeted SGNs. In this work, a biocompatible cochlear implant/graphene EAS system was created by combining a cochlear implant to provide the electrically transformed sound stimulation with graphene as the conductive neural interface. SGNs were cultured on the graphene and exposed to EAS from the cochlear implant. Neurite extension of SGNs was accelerated with long-term stimulation, which might contribute to the development of growth cones. Our system allows us to study the effects of cochlear implants on SGNs in a low-cost and time-saving way, and this might provide profound insights into the use of cochlear implants and thus be of benefit to the populations suffering from sensorineural hearing loss.

Cisplatin-induced hearing loss is a common side effect of cancer chemotherapy in clinics, however, the mechanism of cisplatin-induced ototoxicity is still not completely clarified. Cisplatin-induced ototoxicity is mainly associated with the production of reactive oxygen species, activation of apoptosis, and accumulation of intracellular lipid peroxidation, which also is involved in ferroptosis induction. In this study, the expression of TfR1, a ferroptosis biomarker, was upregulated in the outer hair cells of cisplatin-treated mice. Moreover, several key ferroptosis regulator genes were altered in cisplatin-damaged cochlear explants based on RNA-Seq, implying the induction of ferroptosis. Ferroptosis-related Gpx4and Fsp1knockout mice were established to investigate the specific mechanisms associated with ferroptosis in cochleae. Severe OHC loss and progressive damage of synapses in IHC were observed in Atoh1-Gpx4-/-mice. However, Fsp1-/-mice showed no significant hearing phenotype, demonstrating that Gpx4, but not Fsp1, may play an important role in the functional maintenance of HCs. Moreover, findings showed that FDA-approved luteolin could specifically inhibit ferroptosis and alleviate cisplatin-induced ototoxicity through decreased expression of Transferrin and intracellular concentration of ferrous ions. This study indicated that ferroptosis inhibition through the reduction of intracellular ferrous ions might be a potential strategy to prevent cisplatin-induced hearing loss.

Iridium(III) complexes have been shown to be promising probes in two-photon imaging to real-time track the transplanted cells in stem-cell-based therapy. Here, we report on polymeric nanocapsules loaded with red phosphorescence dye of bis(2-methyldibenzo[f,h]quinoxaline) (acetylacetonate) iridium(III) (Ir(MDQ)2acac) with excellent stability created by the double emulsion method. The Ir(MDQ)2acac nanocapsules present high biocompatibility and an efficient fluorescent labeling rate when incubated with cultured mouse neural stem cells (NSCs). More importantly, the Ir(MDQ)2acac nanocapsules had both one- and two-photon imaging properties with stable phosphorescence lasting for 72 h. Furthermore, data from in vivotracking in nude mice demonstrated that the photoluminescence from Ir(MDQ)2acac nanocapsules in NSCs could be stably monitored for up to 21 days. Our data shed light on the potential clinical application of iridium complexes encapsulated in polymeric nanospheres for two-photon imaging in real-time tracking of the transplanted stem cells.

Multimodal bioimaging is a powerful tool for visualizing the abnormal state at the target site of the related disease. In this study, we used multimodal imaging techniques such as computed tomography, fluorescence, and magnetic resonance imaging to improve early and precise diagnosis of tumor. Herein, we reported the facile in situ biosynthesis of iridium and iron oxide nanoclusters (NCs) in cancer cells or tumor tissue. These NCs are used as a multimodal bioimaging probe to improve the image sensitivity and specificity toward the tumor. These NCs are applied for the in vivo multimodal imaging in the form of an imaging probe capable of enhancing the sensitivity of the image and specificity toward the tumor tissue. Our observation demonstrates that highly luminescent and magnetic NCs are not only biocompatible but also tumor-targeted because NC formation does not take place in normal cells and tissues. In addition, we isolated exosomes and the biosynthesized NCs internalized within exosomes, and these exosomes can be used as cancer biomarkers.

ABSTRACTAminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.

Copper is a microelement with important physiological functions in the body. However, the excess copper ion (Cu2+) may cause severe health problems, such as hair cell apoptosis and the resultant hearing loss. Therefore, the assay of Cu2+is important. We integrate ionic imprinting technology (IIT) and structurally colored hydrogel beads to prepare chitosan-based ionically imprinted hydrogel beads (IIHBs) as a low-cost and high-specificity platform for Cu2+detection. The IIHBs have a macroporous microstructure, uniform size, vivid structural color, and magnetic responsiveness. When incubated in solution, IIHBs recognize Cu2+and exhibit a reflective peak change, thereby achieving label-free detection. In addition, benefiting from the IIT, the IIHBs display good specificity and selectivity and have an imprinting factor of 19.14 at 100 μmol·L−1. These features indicated that the developed IIHBs are promising candidates for Cu2+detection, particularly for the prevention of hearing loss.

Elastin-like protein (ELP) hydrogel helps maintain the three-dimensional (3-D) cochlear structure in culture.

Peripheral nerve injury (PNI), causing loss of sensory and motor function, is a complex and challenging disease in the clinic due to the restricted regeneration capacity. Nerve guidance conduits (NGCs) have become a promising substitute for peripheral nerve regeneration, but their efficacy is often limited. Here, inspired by the physiological structures of peripheral nerves, we present a conductive topological scaffold for nerve repair by modifying Morphobutterfly wing with reduced graphene oxide (rGO) nanosheets and methacrylated gelatin (GelMA) hydrogel encapsulated brain-derived neurotrophic factor (BDNF). Benefiting from the biocompatibility of GelMA hydrogel, the conductivity of rGO and parallel nanoridge structures of wing scales, PC12 cells, and neural stem cells grown on the modified wing have an increased neurite length with guided cellular orientation. In addition, the NGCs are successfully obtained by manually rolling up the scaffolds and exhibited great performance in repairing 10 mm sciatic nerve defects in rats, and we believe that the NGCs can be applied in reparing longer nerve defects in the future by further optimization. We also demonstrate the feasibility of electrically conductive NGCs based on the rGO/BDNF/GelMA-integrated Morphobutterfly wing as functional nerve regeneration conduits, which may have potential value for application in repairing peripheral nerve injuries.

Cochlear implantation has become the most effective treatment method for patients with profound and total hearing loss. However, its therapeutic efficacy is dependent on the number and normal physiological function of cochlear implant-targeted spiral ganglion neurons (SGNs). Electrical stimulation can be used as an effective cue to regulate the morphology and function of excitatory cells. Therefore, it is important to develop an efficient cochlear implant electroacoustic stimulation (EAS) system to study the behavior of SGNs. In this work, we present an electrical stimulation system constructed by combining a cochlear implant and a conductive Ti3C2TxMXene–matrigel hydrogel. SGNs were cultured in the Ti3C2TxMXene–matrigel hydrogel and exposed to electrical stimulation transduced by the cochlear implant. It was demonstrated that low-frequency stimulation promoted the growth cone development and neurite outgrowth of SGNs as well as signal transmission between cells. This work may have potential value for the clinical application of the Ti3C2TxMXene hydrogel to optimize the postoperative listening effect of cochlear implantation and benefit people with sensorineural hearing loss.

Background: Aging, noise, infection, and ototoxic drugs are the major causes of human acquired sensorineural hearing loss, but treatment options are limited. CRISPR/Cas9 technology has tremendous potential to become a new therapeutic modality for acquired non-inherited sensorineural hearing loss. Here, we develop CRISPR/Cas9 strategies to prevent aminoglycoside-induced deafness, a common type of acquired non-inherited sensorineural hearing loss, via disrupting the Htra2gene in the inner ear which is involved in apoptosis but has not been investigated in cochlear hair cell protection. Results: The results indicate that adeno-associated virus (AAV)-mediated delivery of CRISPR/SpCas9 system ameliorates neomycin-induced apoptosis, promotes hair cell survival, and significantly improves hearing function in neomycin-treated mice. The protective effect of the AAV–CRISPR/Cas9 system in vivo is sustained up to 8 weeks after neomycin exposure. For more efficient delivery of the whole CRISPR/Cas9 system, we also explore the AAV–CRISPR/SaCas9 system to prevent neomycin-induced deafness. The in vivo editing efficiency of the SaCas9 system is 1.73% on average. We observed significant improvement in auditory brainstem response thresholds in the injected ears compared with the non-injected ears. At 4 weeks after neomycin exposure, the protective effect of the AAV–CRISPR/SaCas9 system is still obvious, with the improvement in auditory brainstem response threshold up to 50 dB at 8 kHz. Conclusions: These findings demonstrate the safe and effective prevention of aminoglycoside-induced deafness via Htra2gene editing and support further development of the CRISPR/Cas9 technology in the treatment of non-inherited hearing loss as well as other non-inherited diseases.

The mammalian cochlea cannot regenerate functional hair cells (HCs) spontaneously. Atoh1 overexpression as well as other strategies are unable to generate functional HCs. Here, we simultaneously upregulated the expression of Gfi1, Pou4f3, and Atoh1in postnatal cochlear supporting cells (SCs) in vivo, which efficiently converted SCs into HCs. The newly regenerated HCs expressed HC markers Myo7a, Calbindin, Parvalbumin, and Ctbp2 and were innervated by neurites. Importantly, many new HCs expressed the mature and terminal marker Prestin or vesicular glutamate transporter 3 (vGlut3), depending on the subtypes of the source SCs. Finally, our patch-clamp analysis showed that the new HCs in the medial region acquired a large K+current, fired spikes transiently, and exhibited signature refinement of ribbon synapse functions, in close resemblance to native wild-type inner HCs. We demonstrated that co-upregulating Gfi1, Pou4f3,and Atoh1enhances the efficiency of HC generation and promotes the functional maturation of new HCs.

Several clinical studies have reported that hearing loss is correlated with autism in children. However, little is known about the underlying mechanism between hearing loss and autism. p21-activated kinases (PAKs) are a family of serine/threonine kinases that can be activated by multiple signaling molecules, particularly the Rho family of small GTPases. Previous studies have shown that Pak1mutations are associated with autism. In the present study, we take advantage of Pak1knockout (Pak1−/−) mice to investigate the role of PAK1 in hearing function. We find that PAK1 is highly expressed in the postnatal mouse cochlea and that PAK1 deficiency leads to hair cell (HC) apoptosis and severe hearing loss. Further investigation indicates that PAK1 deficiency down-regulates the phosphorylation of cofilin and ezrin-radixin-moesin and the expression of βII-spectrin, which further decreases the HC synapse density in the basal turn of cochlea and disorganized the HC stereocilia in all three turns of cochlea in Pak1−/−mice. Overall, our work demonstrates that the autism-related gene Pak1plays a crucial role in hearing function. As the first candidate gene linking autism and hearing loss, Pak1may serve as a potential target for the clinical diagnosis of autism-related hearing loss.

Mesenchymal niche cells instruct activity of tissue-resident stem and progenitor cell populations. Epithelial stem cells in hair follicles (HFs) have region-specific activity, which may arise from intrinsic cellular heterogeneity within mesenchymal dermal papilla (DP) cells. Here we show that expression of Hoxcgenes is sufficient to reprogram mesenchymal DP cells and alter the regenerative potential of epithelial stem cells. Hoxcgene expression in adult skin dermis closely correlates with regional HF regeneration patterns. Disrupting the region-specific expression patterns of Hoxcgenes, by either decreasing their epigenetic repression via Bmi1loss or inducing ectopic interactions of the Hoxclocus with an active epigenetic region, leads to precocious HF regeneration. We further show that a single Hoxcgene is sufficient to activate dormant DP niches and promote regional HF regeneration through canonical Wnt signaling. Altogether, these results reveal that Hoxcgenes bestow mesenchymal niches with tissue-level heterogeneity and plasticity.