Superparamagnetic iron oxide nanoparticle regulates microbiota-gut-inner ear axis for hearing protection.

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.

Ultrasound-Responsive Aligned Piezoelectric Nanofibers Derived Hydrogel Conduits for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) have been considered as promising treatment strategy and frontier trend for peripheral nerve regeneration; while their therapeutic outcomes are limited by the lack of controllable drug delivery and available physicochemical cues. Herein, we propose novel aligned piezoelectric nanofibers derived hydrogel NGCs with ultrasound (US)-triggered electrical stimulation (ES) and controllable drug release for repairing peripheral nerve injury. The inner layer of the NGCs was the barium titanate piezoelectric nanoparticles (BTNPs)-doped polyvinylidene fluoride-trifluoroethylene [BTNPs/P(VDF-TrFE)] electrospinning nanofibers with improved piezoelectricity and aligned orientation. The outer side of the NGCs was the thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) hybrid hydrogel with bioactive drug encapsulation. Such NGCs could not only induce neuronal oriented extension and promote neurite outgrowth with US-triggered wireless ES, but also realize the controllable nerve growth factor (NGF) release with the hydrogel shrinkage under US-triggered heating. Thus, the NGC could positively accelerate the functional recovery and nerve axonal regeneration of rat models with long sciatic nerve defects. We believe that the proposed US-responsive aligned piezoelectric nanofibers derived hydrogel NGCs will find important applications in clinic neural tissue engineering. This article is protected by copyright. All rights reserved.

Pcolce2 overexpression promotes supporting cell reprogramming in the neonatal mouse cochlea.

Hair cell (HC) damage is a leading cause of sensorineural hearing loss, and in mammals supporting cells (SCs) are unable to divide and regenerate HCs after birth spontaneously. Procollagen C-endopeptidase enhancer 2 (Pcolce2), which encodes a glycoprotein that acts as a functional procollagen C protease enhancer, was screened as a candidate regulator of SC plasticity in our previous study. In the current study, we used adeno-associated virus (AAV)-ie (a newly developed adeno-associated virus that targets SCs) to overexpress Pcolce2 in SCs. AAV-Pcolce2 facilitated SC re-entry into the cell cycle both in cultured cochlear organoids and in the postnatal cochlea. In the neomycin-damaged model, regenerated HCs were detected after overexpression of Pcolce2, and these were derived from SCs that had re-entered the cell cycle. These findings reveal that Pcolce2 may serve as a therapeutic target for the regeneration of HCs to treat hearing loss.

Electroacoustic Responsive Cochlea-on-a-Chip.

Organ-on-chips can highly simulate the complex physiological functions of organs, exhibiting broad application prospects in developmental research, disease simulation, as well as new drug research and development. However, there is still less concern about effectively constructing cochlea-on-chips. Here, a novel cochlear organoids-integrated conductive hydrogel biohybrid system with cochlear implant electroacoustic stimulation (EAS) for cochlea-on-a-chip construction and high-throughput drug screening, is presented. Benefiting from the superior biocompatibility and electrical property of conductive hydrogel, together with cochlear implant EAS, the inner ear progenitor cells can proliferate and spontaneously shape into spheres, finally forming cochlear organoids with good cell viability and structurally mature hair cells. By incorporating these progenitor cells-encapsulated hydrogels into a microfluidic-based cochlea-on-a-chip with culture chambers and a concentration gradient generator, a dynamic and high-throughput evaluation of inner ear disease-related drugs is demonstrated. These results indicate that the proposed cochlea-on-a-chip platform has great application potential in organoid cultivation and deafness drug evaluation.

Inhibition of Gpx4-mediated ferroptosis alleviates cisplatin-induced hearing loss in C57BL/6 mice.

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 sequencing, implying the induction of ferroptosis. Ferroptosis-related Gpx4 and Fsp1 knockout mice were established to investigate the specific mechanisms associated with ferroptosis in cochleae. Severe outer hair cell loss and progressive damage of synapses in inner hair cells 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.

Development of Chinese herbal medicine for sensorineural hearing loss.

According to the World Health Organization's world report on hearing, nearly 2.5 billion people worldwide will suffer from hearing loss by 2050, which may contribute to a severe impact on individual life quality and national economies. Sensorineural hearing loss (SNHL) occurs commonly as a result of noise exposure, aging, and ototoxic drugs, and is pathologically characterized by the impairment of mechanosensory hair cells of the inner ear, which is mainly triggered by reactive oxygen species accumulation, inflammation, and mitochondrial dysfunction. Though recent advances have been made in understanding the ability of cochlear repair and regeneration, there are still no effective therapeutic drugs for SNHL. Chinese herbal medicine which is widely distributed and easily accessible in China has demonstrated a unique curative effect against SNHL with higher safety and lower cost compared with Western medicine. Herein we present trends in research for Chinese herbal medicine for the treatment of SNHL, and elucidate their molecular mechanisms of action, to pave the way for further research and development of novel effective drugs in this field.

AAV-mediated Gpm6b expression supports hair cell reprogramming.

Irreversible damage to hair cells (HCs) in the cochlea leads to hearing loss. Cochlear supporting cells (SCs) in the murine cochlea have the potential to differentiate into HCs. Neuron membrane glycoprotein M6B (Gpm6b) as a four-transmembrane protein is a potential regulator of HC regeneration according to our previous research. In this study, we found that AAV-ie-mediated Gpm6b overexpression promoted SC-derived organoid expansion. Enhanced Gpm6b prevented the normal decrease in SC plasticity as the cochlea develops by supporting cells re-entry cell cycle and facilitating the SC-to-HC transformation. Also, overexpression of Gpm6b in the organ of Corti through the round window membrane injection facilitated the trans-differentiation of Lgr5+ SCs into HCs. In conclusion, our results suggest that Gpm6b overexpression promotes HC regeneration and highlights a promising target for hearing repair using the inner ear stem cells combined with AAV.

AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness.

Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno-associated virus (AAV)-mediated OTOF gene replacement therapy is extensively validated in Otof-deficient mice. However, the clinical safety and efficacy of AAV-OTOF is not reported. Here, AAV-OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non-human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV-OTOF is delivered into one cochlea of a 5-year-old deaf patient and into the bilateral cochleae of an 8-year-old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure-tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5-year-old patient can be restored to the normal range at 1 month after AAV-OTOF injection, while the 8-year-old patient can hear the conversational sounds. Most importantly, the 5-year-old patient can hear and recognize speech only through the AAV-OTOF-injected ear. This study is the first to demonstrate the safety and efficacy of AAV-OTOF in patients, expands and optimizes current OTOF-related gene therapy and provides valuable information for further application of gene therapies for deafness.

Gut microbial metabolite facilitates colorectal cancer development via ferroptosis inhibition.

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 ALDH1A3 largely abrogates IDA-promoted tumour development both in vitro and in vivo. It is interesting that P. anaerobius is significantly enriched in patients with colorectal cancer (CRC). IDA treatment or implantation of P. anaerobius promotes CRC progression in both xenograft model and ApcMin/+ mice. Together, our findings demonstrate that targeting the IDA-AHR-ALDH1A3 axis should be promising for ferroptosis-related CRC treatment.

AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial.

BACKGROUND: Autosomal recessive deafness 9, caused by mutations of the OTOF gene, 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 OTOF transgene (AAV1-hOTOF) as a treatment for children with autosomal recessive deafness 9. METHODS: This single-arm, single-centre trial enrolled children (aged 1-18 years) with severe-to-complete hearing loss and confirmed mutations in both alleles of OTOF, and without bilateral cochlear implants. A single injection of AAV1-hOTOF was administered into the cochlea through the round window. The primary endpoint was dose-limiting toxicity at 6 weeks after injection. Auditory function and speech were assessed by appropriate auditory perception evaluation tools. All analyses were done according to the intention-to-treat principle. This trial is registered with Chinese Clinical Trial Registry, ChiCTR2200063181, and is ongoing. FINDINGS: Between Oct 19, 2022, and June 9, 2023, we screened 425 participants for eligibility and enrolled six children for AAV1-hOTOF gene therapy (one received a dose of 9 × 1011 vector genomes [vg] and five received 1·5 × 1012 vg). All participants completed follow-up visits up to week 26. No dose-limiting toxicity or serious adverse events occurred. In total, 48 adverse events were observed; 46 (96%) were grade 1-2 and two (4%) were grade 3 (decreased neutrophil count in one participant). Five children had hearing recovery, shown by a 40-57 dB reduction in the average auditory brainstem response (ABR) thresholds at 0·5-4·0 kHz. In the participant who received the 9 × 1011 vg dose, the average ABR threshold was improved from greater than 95 dB at baseline to 68 dB at 4 weeks, 53 dB at 13 weeks, and 45 dB at 26 weeks. In those who received 1·5 × 1012 AAV1-hOTOF, the average ABR thresholds changed from greater than 95 dB at baseline to 48 dB, 38 dB, 40 dB, and 55 dB in four children with hearing recovery at 26 weeks. Speech perception was improved in participants who had hearing recovery. INTERPRETATION: AAV1-hOTOF gene therapy is safe and efficacious as a novel treatment for children with autosomal recessive deafness 9. FUNDING: National Natural Science Foundation of China, National Key R&D Program of China, Science and Technology Commission of Shanghai Municipality, and Shanghai Refreshgene Therapeutics.

Critical role of TPRN rings in the stereocilia for hearing.

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 Tprn knockout 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-Tprn effectively repairs damaged hearing, which lays the foundation for the clinical application of AAV-mediated gene therapy in patients with TPRN mutation.

Stem Cell-Based Hair Cell Regeneration and Therapy in the Inner Ear.

Hearing loss has become increasingly prevalent and causes considerable disability, thus gravely burdening the global economy. Irreversible loss of hair cells is a main cause of sensorineural hearing loss, and currently, the only relatively effective clinical treatments are limited to digital hearing equipment like cochlear implants and hearing aids, but these are of limited benefit in patients. It is therefore urgent to understand the mechanisms of damage repair in order to develop new neuroprotective strategies. At present, how to promote the regeneration of functional hair cells is a key scientific question in the field of hearing research. Multiple signaling pathways and transcriptional factors trigger the activation of hair cell progenitors and ensure the maturation of newborn hair cells, and in this article, we first review the principal mechanisms underlying hair cell reproduction. We then further discuss therapeutic strategies involving the co-regulation of multiple signaling pathways in order to induce effective functional hair cell regeneration after degeneration, and we summarize current achievements in hair cell regeneration. Lastly, we discuss potential future approaches, such as small molecule drugs and gene therapy, which might be applied for regenerating functional hair cells in the clinic.

Preclinical Efficacy And Safety Evaluation of AAV-OTOF in DFNB9 Mouse Model And Nonhuman Primate.

OTOF mutations are the principal causes of auditory neuropathy. There are reports on Otof-related gene therapy in mice, but there is no preclinical research on the drug evaluations. Here, Anc80L65 and the mouse hair cell-specific Myo15 promoter (mMyo15) are used to selectively and effectively deliver human OTOF to hair cells in mice and nonhuman primates to evaluate the efficacy and safety of OTOF gene therapy drugs. A new dual-AAV-OTOF-hybrid strategy to transfer full-length OTOF is generated, which can stably restore hearing in adult OTOFp.Q939*/Q939* mice with profound deafness, with the longest duration being at least 150 days, and the best therapeutic effect without difference in hearing from wild-type mice. An AAV microinjection method into the cochlea of cynomolgus monkeys without hearing impairment is further established and found the OTOF can be safely and effectively driven by the mMyo15 promoter in hair cells. In addition, the therapeutic dose of AAV drugs has no impact on normal hearing and does not cause significant systemic toxicity both in mouse and nonhuman primates. In summary, this study develops a potential gene therapy strategy for DFNB9 patients in the clinic and provides complete, standardized, and systematic research data for clinical research and application.

Natural Multifunctional Silk Microcarriers for Noise-Induced Hearing Loss Therapy.

Noise-induced hearing loss (NIHL) is a common outcome of excessive reactive oxygen species in the cochlea, and the targeted delivery of antioxidants to the inner ear is a potential therapeutic strategy. In this paper, a novel natural biomaterials-derived multifunctional delivery system using silk fibroin-polydopamine (PDA)-composited inverse opal microcarriers (PDA@SFMCs) is presented for inner ear drug delivery and NIHL therapy. Due to their large specific surface area and interpenetrating nanochannels, PDA@SFMCs can rapidly load active biomolecules making them a convenient medium for the storage and delivery of such molecules. In addition, surface modification of PDA enables the microcarriers to remain in the round window niche, thus facilitating the precise local and directed delivery of loaded drugs. Based on these features, it is demonstrated here that n-acetylcysteine-loaded silk microcarriers have satisfactory antioxidant properties on cells and can successfully prevent NIHL in guinea pigs. These results indicate that the natural multifunctional silk microcarriers are promising agents for local inner ear drug delivery in the clinic.

Advance and Application of Single-cell Transcriptomics in Auditory Research.

Hearing loss and deafness, as a worldwide disability disease, have been troubling human beings. However, the auditory organ of the inner ear is highly heterogeneous and has a very limited number of cells, which are largely uncharacterized in depth. Recently, with the development and utilization of single-cell RNA sequencing (scRNA-seq), researchers have been able to unveil the complex and sophisticated biological mechanisms of various types of cells in the auditory organ at the single-cell level and address the challenges of cellular heterogeneity that are not resolved through by conventional bulk RNA sequencing (bulk RNA-seq). Herein, we reviewed the application of scRNA-seq technology in auditory research, with the aim of providing a reference for the development of auditory organs, the pathogenesis of hearing loss, and regenerative therapy. Prospects about spatial transcriptomic scRNA-seq, single-cell based genome, and Live-seq technology will also be discussed.

Orthosteric ligand selectivity and allosteric probe dependence at Hydroxycarboxylic acid receptor HCAR2.

Hydroxycarboxylic acid receptor 2 (HCAR2), a member of Class A G-protein-coupled receptor (GPCR) family, plays a pivotal role in anti-lipolytic and anti-inflammatory effects, establishing it as a significant therapeutic target for treating dyslipidemia and inflammatory diseases. However, the mechanism underlying the signaling of HCAR2 induced by various types of ligands remains elusive. In this study, we elucidate the cryo-electron microscopy (cryo-EM) structure of Gi-coupled HCAR2 in complex with a selective agonist, MK-6892, resolved to a resolution of 2.60 Å. Our structural analysis reveals that MK-6892 occupies not only the orthosteric binding pocket (OBP) but also an extended binding pocket (EBP) within HCAR2. Pharmacological assays conducted in this study demonstrate that the OBP is a critical determinant for ligand selectivity among the HCARs subfamily. Moreover, we investigate the pharmacological properties of the allosteric modulator compound 9n, revealing its probe-dependent behavior on HCAR2 in response to varying orthosteric agonists. Collectively, our findings provide invaluable structural insights that contribute to a deeper understanding of the regulatory mechanisms governing HCAR2 signaling transduction mediated by both orthosteric and allosteric ligands.

Progress on mechanisms of age-related hearing loss.

Age-related hearing loss, or presbycusis, is a common cause of hearing loss in elderly people worldwide. It typically presents as progressive, irreversible, and usually affects the high frequencies of hearing, with a tremendous impact on the quality of life. Presbycusis is a complex multidimensional disorder, in addition to aging, multiple factors including exposure to noise, or ototoxic agents, genetic susceptibility, metabolic diseases and lifestyle can influence the onset and severity of presbycusis. With the aging of the body, its ability to clean up deleterious substances produced in the metabolic process is weakened, and the self-protection and repair function of the body is reduced, which in turn leads to irreversible damage to the cochlear tissue, resulting in the occurrence of presbycusis. Presently, oxidative stress (OS), mitochondrial DNA damage, low-grade inflammation, decreased immune function and stem cell depletion have been demonstrated to play a critical role in developing presbycusis. The purpose of this review is to illuminate the various mechanisms underlying this age-related hearing loss, with the goal of advancing our understanding, prevention, and treatment of presbycusis.

P2X7 receptor is required for the ototoxicity caused by aminoglycoside in developing cochlear hair cells.

Aminoglycoside antibiotics (AGAs) are widely used in life-threatening infections, but they accumulate in cochlear hair cells (HCs) and result in hearing loss. Increases in adenosine triphosphate (ATP) concentrations and P2X7 receptor expression were observed after neomycin treatment. Here, we demonstrated that P2X7 receptor, which is a non-selective cation channel that is activated by high ATP concentrations, may participate in the process through which AGAs enter hair cells. Using transgenic knockout mice, we found that P2X7 receptor deficiency protects HCs against neomycin-induced injury in vitro and in vivo. Subsequently, we used fluorescent gentamicin-Fluor 594 to study the uptake of AGAs and found fluorescence labeling in wild-type mice but not in P2rx7-/- mice in vitro. In addition, knocking-out P2rx7 did not significantly alter the HC count and auditory signal transduction, but it did inhibit mitochondria-dependent oxidative stress and apoptosis in the cochlea after neomycin exposure. We thus conclude that the P2X7 receptor may be linked to the entry of AGAs into HCs and is likely to be a therapeutic target for auditory HC protection.