Selective Vulnerability of GABAergic Inhibitory Interneurons to Bilirubin Neurotoxicity in the Neonatal Brain.

Hyperbilirubinemia (HB) is a key risk factor for hearing loss in neonates, particularly premature infants. Here we report that bilirubin (BIL)-dependent cell death in auditory brainstem of neonatal mice of both sexes is significantly attenuated by ZD7288, a blocker for hyperpolarization-activated cyclic nucleotide-gated (HCN) channel mediated current (Ih), or by genetic deletion of HCN1. GABAergic inhibitory interneurons predominantly express HCN1, on which BIL selectively acts to increase their intrinsic excitability and mortality by enhancing HCN1 activity and Ca2+-dependent membrane targeting. Chronic BIL elevation in neonatal mice in vivo increases the fraction of spontaneously active interneurons and their firing frequency, Ih and death, compromising audition at young adult stage in HCN1+/+, but not in HCN1-/- genotype. We conclude that HB preferentially targets HCN1 to injure inhibitory interneurons, fueling a feedforward loop in which lessening inhibition cascades hyperexcitability, Ca2+ overload, neuronal death and auditory impairments. These findings rationalize HCN1 as a potential target for managing HB encephalopathy.Significance Statement This study demonstrated that bilirubin preferentially targets GABAergic interneurons where it facilitates not only gating of HCN1 channels but also targeting of intracellular HCN1 to plasma membrane in calcium-dependent manner, resulting in neuronal hyperexcitability, injury and sensory dysfunction. These findings implicate HCN1 channel not only as a potential driver for auditory abnormalities in neonatal patients with bilirubin encephalopathy, but also potential intervention target for clinical management of neurological impairments associated with severe jaundice. Selective vulnerability of interneurons to neurotoxicity may be of general significance for understanding other forms of brain injury.

Glutamate acts on acid-sensing ion channels to worsen ischaemic brain injury.

Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-D-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3-7. Here we show that glutamate and its structural analogues, including NMDAR antagonist L-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4-7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.

Measuring endogenous levels of unconjugated bilirubin released from isolated murine brain tissue during oxygen-glucose deprivation.

Quantitative assessment of endogenously synthesized and released bilirubin from brain tissue remains a challenge. Here, we present a sensitive and reproducible experimental paradigm to quantify, in real time, unconjugated bilirubin (UCB) from isolated murine brain tissue during oxygen-glucose deprivation (OGD). We describe steps for perfusion, brain dissection, brain slice preparation and incubation, glucose depletion, and OGD processing. We then detail procedures for standard calibration plotting and sample UCB measurement. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Surface Functional Modification by Ti3 C2 Tx MXene on PLLA Nanofibers for Optimizing Neural Stem Cell Engineering.

Optimizing cell substrates by surface modification of neural stem cells (NSCs), for efficient and oriented neurogenesis, represents a promising strategy for treating neurological diseases. However, developing substrates with the advanced surface functionality, conductivity, and biocompatibility required for practical application is still challenging. Here, Ti3 C2 Tx MXene is introduced as a coating nanomaterial for aligned poly(l-lactide) (PLLA) nanofibers (M-ANF) to enhance NSC neurogenesis and simultaneously tailor the cell growth direction. Ti3 C2 Tx MXene treatment provides a superior conductivity substrate with a surface rich in functional groups, hydrophilicity, and roughness, which can provide biochemical and physical cues to support NSC adhesion and proliferation. Moreover, Ti3 C2 Tx MXene coating significantly promotes NSC differentiation into both neurons and astrocytes. Interestingly, Ti3 C2 Tx MXene acts synergistically with the alignment of nanofibers to promote the growth of neurites, indicating enhanced maturation of these neurons. RNA sequencing analysis further reveals the molecular mechanism by which Ti3 C2 Tx MXene modulates the fate of NSCs. Notably, surface modification by Ti3 C2 Tx MXene mitigates the in vivo foreign body response to implanted PLLA nanofibers. This study confirms that Ti3 C2 Tx MXene provides multiple advantages for decorating the aligned PLLA nanofibers to cooperatively improve neural regeneration.

Bilirubin gates the TRPM2 channel as a direct agonist to exacerbate ischemic brain damage.

Stroke prognosis is negatively associated with an elevation of serum bilirubin, but how bilirubin worsens outcomes remains mysterious. We report that post-, but not pre-, stroke bilirubin levels among inpatients scale with infarct volume. In mouse models, bilirubin increases neuronal excitability and ischemic infarct, whereas ischemic insults induce the release of endogenous bilirubin, all of which are attenuated by knockout of the TRPM2 channel or its antagonist A23. Independent of canonical TRPM2 intracellular agonists, bilirubin and its metabolic derivatives gate the channel opening, whereas A23 antagonizes it by binding to the same cavity. Knocking in a loss of binding point mutation for bilirubin, TRPM2-D1066A, effectively antagonizes ischemic neurotoxicity in mice. These findings suggest a vicious cycle of stroke injury in which initial ischemic insults trigger the release of endogenous bilirubin from injured cells, which potentially acts as a volume neurotransmitter to activate TRPM2 channels, aggravating Ca2+-dependent brain injury.

Electroencephalography Microstate Alterations in Otogenic Vertigo: A Potential Disease Marker.

Objectives: A huge population, especially the elderly, suffers from otogenic vertigo. However, the multi-modal vestibular network changes, secondary to periphery vestibular dysfunction, have not been fully elucidated. We aim to identify potential microstate electroencephalography (EEG) signatures for otogenic vertigo in this study. Materials and Methods: Patients with recurrent otogenic vertigo and age-matched healthy adults were recruited. We performed 256-channel EEG recording of all participants at resting state. Neuropsychological questionnaires and vestibular function tests were taken as a measurement of patients' symptoms and severity. We clustered microstates into four classes (A, B, C, and D) and identified their dynamic and syntax alterations of them. These features were further fed into a support vector machine (SVM) classifier to identify microstate signatures for vertigo. Results: We compared 40 patients to 45 healthy adults, finding an increase in the duration of Microstate A, and both the occurrence and time coverage of Microstate D. The coverage and occurrence of Microstate C decreased significantly, and the probabilities of non-random transitions between Microstate A and D, as well as Microstate B and C, also changed. To distinguish the patients, the SVM classifier, which is built based on these features, got a balanced accuracy of 0.79 with a sensitivity of 0.78 and a specificity of 0.8. Conclusion: There are several temporal dynamic alterations of EEG microstates in patients with otogenic vertigo, especially in Microstate D, reflecting the underlying process of visual-vestibular reorganization and attention redistribution. This neurophysiological signature of microstates could be used to identify patients with vertigo in the future.

Multisensory Exercise Improves Balance in People with Balance Disorders: A Systematic Review.

OBJECTIVE: To examine the effect of multisensory exercise on balance disorders. METHODS: PubMed, Scopus and Web of Science were searched to identify eligible studies published before January 1, 2020. Eligible studies included randomized control trials (RCTs), non-randomized studies, case-control studies, and cohort studies. The methodological quality of the included studies was evaluated using JBI Critical Appraisal Checklists for RCTs and for Quasi-Experimental Studies by two researchers independently. A narrative synthesis of intervention characteristics and health-related outcomes was performed. RESULTS: A total of 11 non-randomized studies and 9 RCTs were eligible, including 667 participants. The results supported our assumption that multisensory exercise improved balance in people with balance disorders. All of the 20 studies were believed to be of high or moderate quality. CONCLUSION: Our study confirmed that multisensory exercise was effective in improving balance in people with balance disorders. Multisensory exercises could lower the risk of fall and enhance confidence level to improve the quality of life. Further research is needed to investigate the optimal strategy of multisensory exercises and explore the underlying neural and molecular mechanisms of balance improvement brought by multisensory exercises.

Malate-Aspartate Shuttle Plays an Important Role in LPS-Induced Neuroinflammation of Mice Due to its Effect on STAT3 Phosphorylation.

Neuroinflammation is a key pathological factor in numerous neurological disorders. Cumulating evidence has indicated critical roles of NAD+/NADH metabolism in multiple major diseases, while the role of malate-aspartate shuttle (MAS) - a major NADH shuttle - in inflammation has remained unclear. In this study we investigated the roles of MAS in LPS-induced neuroinflammation both in vivo and in vitro. Immunofluorescence staining, Western blot assay and Real-time PCR assays were conducted to determine the activation of Iba-1, the protein levels of iNOS and COX2 and the mRNA levels of IL-1ß, IL-6, and TNF-α in vivo, showing that both pre-treatment and post-treatment of aminooxyacetic acid (AOAA) - an MAS inhibitor - profoundly decreased the LPS-induced neuroinflammation in mice. BV2 microglia was also used as a cellular model to investigate the mechanisms of this finding, in which such assays as Western blot assay and nitrite assay. Our study further indicated that AOAA produced its effects on LPS-induced microglial activation by its effects on MAS: Pyruvate treatment reversed the effects of AOAA on the cytosolic NAD+/NADH ratio, which also restored the LPS-induced activation of the AOAA-treated microglia. Moreover, the lactate dehydrogenase (LDH) inhibitor GSK2837808A blocked the effects of pyruvate on the AOAA-produced decreases in both the cytosolic NAD+/NADH ratio and LPS-induced microglial activation. Our study has further suggested that AOAA produced inhibition of LPS-induced microglial activation at least partially by decreasing STAT3 phosphorylation. Collectively, our findings have indicated AOAA as a new and effective drug for inhibiting LPS-induced neuroinflammation. Our study has also indicated that MAS is a novel mediator of LPS-induced neuroinflammation due to its capacity to modulate LPS-induced STAT3 phosphorylation, which has further highlighted a critical role of NAD+/NADH metabolism in inflammation.

Stepwise Induction of Inner Ear Hair Cells From Mouse Embryonic Fibroblasts via Mesenchymal- to-Epithelial Transition and Formation of Otic Epithelial Cells.

Although embryonic stem cells or induced pluripotent stem cells are able to differentiate into inner ear hair cells (HCs), they have drawbacks limiting their clinical application, including a potential risk of tumourigenicity. Direct reprogramming of fibroblasts to inner ear HCs could offer an alternative solution to this problem. Here, we present a stepwise guidance protocol to induce mouse embryonic fibroblasts to differentiate into inner ear HC-like cells (HCLs) via mesenchymal-to-epithelial transition and then acquisition of otic sensory epithelial cell traits by overexpression of three key transcription factors. These induced HCLs express multiple HC-specific proteins, display protrusions reminiscent of ciliary bundle structures, respond to voltage stimulation, form functional mechanotransduction channels, and exhibit a transcriptional profile of HC signature. Together, our work provides a new method to produce functional HCLs in vitro, which may have important implications for studies of HC development, drug discovery, and cell replacement therapy for hearing loss.

Bilirubin enhances the activity of ASIC channels to exacerbate neurotoxicity in neonatal hyperbilirubinemia in mice.

Neonatal hyperbilirubinemia is a common clinical condition that can lead to brain encephalopathy, particularly when concurrent with acidosis due to infection, ischemia, and hypoxia. The prevailing view is that acidosis increases the permeability of the blood-brain barrier to bilirubin and exacerbates its neurotoxicity. In this study, we found that the concentration of the cell death marker, lactate dehydrogenase (LDH) in cerebrospinal fluid (CSF), is elevated in infants with both hyperbilirubinemia and acidosis and showed stronger correlation with the severity of acidosis rather than increased bilirubin concentration. In mouse neonatal neurons, bilirubin exhibits limited toxicity but robustly potentiates the activity of acid-sensing ion channels (ASICs), resulting in increases in intracellular Ca2+ concentration, spike firings, and cell death. Furthermore, neonatal conditioning with concurrent hyperbilirubinemia and hypoxia-induced acidosis promoted long-term impairments in learning and memory and complex sensorimotor functions in vivo, which are largely attenuated in ASIC1a null mice. These findings suggest that targeting acidosis and ASICs may attenuate neonatal hyperbilirubinemia complications.

Ca2+-dependent recruitment of voltage-gated sodium channels underlies bilirubin-induced overexcitation and neurotoxicity.

Neonatal jaundice is prevalent among newborns and can lead to severe neurological deficits, particularly sensorimotor dysfunction. Previous studies have shown that bilirubin (BIL) enhances the intrinsic excitability of central neurons and this can potentially contribute to their overexcitation, Ca2+ overload, and neurotoxicity. However, the cellular mechanisms underlying elevated neuronal excitability remain unknown. By performing patch-clamp recordings from neonatal neurons in the rat medial vestibular nucleus (MVN), a crucial relay station for locomotor and balance control, we found that BIL (3 µM) drastically increases the spontaneous firing rates by upregulating the current-mediated voltage-gated sodium channels (VGSCs), while shifting their voltage-dependent activation toward more hyperpolarized potentials. Immunofluorescence labeling and western immunoblotting with an anti-NaV1.1 antibody, revealed that BIL elevates the expression of VGSCs by promoting their recruitment to the membrane. Furthermore, we found that this VGSC-trafficking process is Ca2+ dependent because preloading MVN neurons with the Ca2+ buffer BAPTA-AM, or exocytosis inhibitor TAT-NSF700, prevents the effects of BIL, indicating the upregulated activity and density of functional VGSCs as the core mechanism accountable for the BIL-induced overexcitation of neonatal neurons. Most importantly, rectification of such overexcitation with a low dose of VGSC blocker lidocaine significantly attenuates BIL-induced cell death. We suggest that this enhancement of VGSC currents directly contributes to the vulnerability of neonatal brain to hyperbilirubinemia, implicating the activity and trafficking of NaV1.1 channels as a potential target for neuroprotection in cases of severe jaundice.

Changes in tinnitus after vestibular schwannoma surgery.

We designed a prospective study to evaluate changes in tinnitus after vestibular schwannoma (VS) surgery. Subjects included 41 patients who were diagnosed with a VS and underwent translabyrinthine microsurgery (TLM) between January 2015 and May 2016. All patients underwent related examinations and were asked to answer the Tinnitus Handicap Inventory (THI) scale and a visual analog scale (VAS) of tinnitus severity both pre- and postoperatively. Of the 41 patients, 31 (75.6%) suffered from tinnitus before surgery. Microsurgery was associated with an overall decrease in tinnitus (p < 0.001). There was a significant improvement in THI and VAS scores after surgery (p = 0.001 and p = 0.005, respectively). The decrease in THI scores in the low-frequency group was significantly larger than that of the mid- and high-frequency groups after surgery (p = 0.034 and p = 0.001, respectively). The loudness of tinnitus decreased significantly after surgery (p = 0.031). Tinnitus in patients with VS improved after TLM. Patients with mid-/high-frequency tinnitus and louder tinnitus preoperatively seemed to have a worse prognosis than those with low-frequency and quieter tinnitus.

Relationships between cardiometabolic disorders and obstructive sleep apnea: Implications for cardiovascular disease risk.

Previous studies have reported the effects of obstructive sleep apnea (OSA) and cardiometabolic disorders on cardiovascular disease (CVD), but associations between cardiometabolic biomarkers and two cardinal features of OSA (chronic intermittent hypoxia and sleep fragmentation) and their interactions on CVD in OSA populations remain unclear. A total of 1727 subjects were included in this observational study. Data on overnight polysomnography parameters, biochemical biomarkers, and anthropometric measurements were collected. Metabolic syndrome (MS), including blood pressure, waist circumference (WC), fasting glucose, triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C), was diagnosed based on modified criteria of the Adult Treatment Panel III. WC, mean arterial pressure, TG and low-density lipoprotein cholesterol (LDL-C) were independently associated with apnea-hypopnea index (AHI) after adjustment for confounding factors (ß = 0.578, P = 0.000; ß = 0.157, P = 0.001; ß = 1.003, P = 0.019; and ß = 4.067, P = 0.0005, respectively). Furthermore, the interaction analysis revealed joint effects between hypertension, obesity, hyperglycemia, and LDL-C dyslipidemia and AHI on CVD. The relative excess risks of CVD due to the interactions with OSA were 2.06, 1.02, 0.48, and 1.42, respectively (all P < 0.05). In contrast, we found no independent effect of the microarousal index (MAI) on CVD. However, LDL-C level and some MS components (WC, TG) were associated with MAI. Our findings indicate that hypoxemia and cardiometabolic disorders in OSA may potentiate their unfavorable effects on CVD. Sleep fragmentation may indirectly predispose patients with OSA to an increased risk of CVD. Thus, cardiometabolic disorders and OSA synergistically influence cardiometabolic risk patterns.

Accelerated Development of the First-Order Central Auditory Neurons With Spontaneous Activity.

In developing sensory systems, elaborate morphological connectivity between peripheral cells and first-order central neurons emerges via genetic programming before the onset of sensory activities. However, how the first-order central neurons acquire the capacity to interface with peripheral cells remains elusive. By making patch-clamp recordings from mouse brainstem slices, we found that a subset of neurons in the cochlear nuclei, the first central station to receive peripheral acoustic impulses, exhibits spontaneous firings (SFs) as early as at birth, and the fraction of such neurons increases during the prehearing period. SFs are reduced but not eliminated by a cocktail of blockers for excitatory and inhibitory synaptic inputs, implicating the involvement of intrinsic pacemaker channels. Furthermore, we demonstrate that these intrinsic firings (IFs) are largely driven by hyperpolarization- and cyclic nucleotide-gated channel (HCN) mediated currents (Ih), as evidenced by their attenuation in the presence of HCN blockers or in neurons from HCN1 knockout mice. Interestingly, genetic deletion of HCN1 cannot be fully compensated by other pacemaker conductances and precludes age-dependent up regulation in the fraction of spontaneous active neurons and their firing rate. Surprisingly, neurons with SFs show accelerated development in excitability, spike waveform and firing pattern as well as synaptic pruning towards mature phenotypes compared to those without SFs. Our results imply that SFs of the first-order central neurons may reciprocally promote their wiring and firing with peripheral inputs, potentially enabling the correlated activity and crosstalk between the developing brain and external environment.

Treatment choice by patients with obstructive sleep apnea: data from two centers in China.

BACKGROUND: Standard management has been recommended for obstructive sleep apnea (OSA) by several guidelines, but patient choice in the practical setting is unclear. METHODS: A survey nested in two prospective cohort studies of OSA (enrollment: 2001-2010) in China. The last interview was conducted between July 2014 and May 2015, using a comprehensive 10-point questionnaire administered in a face-to-face or telephone interview, and assessed (I) whether the participant had received any OSA treatment; (II) why he or she had decided for or against treatment; (III) what treatment was received; (IV) whether the participant used continuous positive airway pressure (CPAP) or OA daily; and (V) the perceived efficacy of therapy. RESULTS: A total of 4,097 subjects with a mean age of 45 years [37-55] responded to this survey, with a response rate of 79.4% (4,097/5,160); 2,779 subjects (67.8%) did not receive any treatment: 1,485 (53.4%) believed that their condition was not serious, despite severe OSA in 53.7% of the patients. A multivariate regression showed that the decision to receive treatment was associated with: age between 45-59 years [odds ratio (OR) 0.805, 95% CI: 0.691-0.936; P<0.001], female gender (OR 0.492, 95% CI: 0.383-0.631; P<0.001), severe OSA (OR 1.92, 95% CI: 1.01-3.64; P<0.001), hypertension (OR 1.414, 95% CI: 1.209-1.654; P<0.001) and diabetes (OR 1.760, 95% CI: 1.043-2.972; P=0.034). In subjects receiving treatment (n=1,318), 50.9% reported negative perceptions about the treatments. CONCLUSIONS: Nearly two thirds of Chinese patients choose not to receive treatment after OSA diagnosis, and nearly half are negative about their treatments for OSA. This requires clinical attention, and warrants further study in different geographic settings.

Bilirubin augments Ca2+ load of developing bushy neurons by targeting specific subtype of voltage-gated calcium channels.

Neonatal brain is particularly vulnerable to pathological levels of bilirubin which elevates and overloads intracellular Ca2+, leading to neurotoxicity. However, how voltage-gated calcium channels (VGCCs) are functionally involved in excess calcium influx remains unknown. By performing voltage-clamp recordings from bushy cells in the ventral cochlear nucleus (VCN) in postnatal rat pups (P4-17), we found the total calcium current density was more than doubled over P4-17, but the relative weight of VGCC subtypes changed dramatically, being relatively equal among T, L, N, P/Q and R-type at P4-6 to predominantly L, N, R over T and P/Q at P15-17. Surprisingly, acute administration of bilirubin augmented the VGCC currents specifically mediated by high voltage-activated (HVA) P/Q-type calcium currents. This augment was attenuated by intracellular loading of Ca2+ buffer EGTA or calmodulin inhibitory peptide. Our findings indicate that acute exposure to bilirubin increases VGCC currents, primarily by targeting P/Q-type calcium channels via Ca2+ and calmodulin dependent mechanisms to overwhelm neurons with excessive Ca2+. Since P/Q-subtype calcium channels are more prominent in neonatal neurons (e.g. P4-6) than later stages, we suggest this subtype-specific enhancement of P/Q-type Ca2+ currents likely contributes to the early neuronal vulnerability to hyperbilirubinemia in auditory and other brain regions.

NAD+ Attenuates Bilirubin-Induced Hyperexcitation in the Ventral Cochlear Nucleus by Inhibiting Excitatory Neurotransmission and Neuronal Excitability.

Nicotinamide adenine dinucleotide (NAD+) is an important molecule with extensive biological functions in various cellular processes, including protection against cell injuries. However, little is known regarding the roles of NAD+ in neuronal excitation and excitotoxicity associated with many neurodegenerative disorders and diseases. Using patch-clamp recordings, we studied its potential effects on principal neurons in the ventral cochlear nucleus (VCN), which is particularly vulnerable to bilirubin excitotoxicity. We found that NAD+ effectively decreased the size of evoked excitatory postsynaptic currents (eEPSCs), increased paired-pulse ratio (PPR) and reversed the effect of bilirubin on eEPSCs, implicating its inhibitory effects on the presynaptic release probability (Pr). Moreover, NAD+ not only decreased the basal frequency of miniature EPSCs (mEPSCs), but also reversed bilirubin-induced increases in the frequency of mEPSCs without affecting their amplitude under either condition. Furthermore, we found that NAD+ decreased the frequency of spontaneous firing of VCN neurons as well as bilirubin-induced increases in firing frequency. Whole-cell current-clamp recordings showed that NAD+ could directly decrease the intrinsic excitability of VCN neurons in the presence of synaptic blockers, suggesting NAD+ exerts its actions in both presynaptic and postsynaptic loci. Consistent with these observations, we found that the latency of the first postsynaptic spike triggered by high-frequency train stimulation of presynaptic afferents (i.e., the auditory nerve) was prolonged by NAD+. These results collectively indicate that NAD+ suppresses presynaptic transmitter release and postsynaptic excitability, jointly weakening excitatory neurotransmission. Our findings provide a basis for the exploration of NAD+ for the prevention and treatment of bilirubin encephalopathy and excitotoxicity associated with other neurological disorders.

Genetic Correction of Induced Pluripotent Stem Cells From a Deaf Patient With MYO7A Mutation Results in Morphologic and Functional Recovery of the Derived Hair Cell-Like Cells.

UNLABELLED: The genetic correction of induced pluripotent stem cells (iPSCs) induced from somatic cells of patients with sensorineural hearing loss (caused by hereditary factors) is a promising method for its treatment. The correction of gene mutations in iPSCs could restore the normal function of cells and provide a rich source of cells for transplantation. In the present study, iPSCs were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T; P-iPSCs), the asymptomatic father of the patient (MYO7A c.1184G>A mutation; CF-iPSCs), and a normal donor (MYO7A(WT/WT); C-iPSCs). One of MYO7A mutation sites (c.4118C>T) in the P-iPSCs was corrected using CRISPR/Cas9. The corrected iPSCs (CP-iPSCs) retained cell pluripotency and normal karyotypes. Hair cell-like cells induced from CP-iPSCs showed restored organization of stereocilia-like protrusions; moreover, the electrophysiological function of these cells was similar to that of cells induced from C-iPSCs and CF-iPSCs. These results might facilitate the development of iPSC-based gene therapy for genetic disorders. SIGNIFICANCE: Induced pluripotent stem cells (iPSCs) were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T). One of the MYO7A mutation sites (c.4118C>T) in the iPSCs was corrected using CRISPR/Cas9. The genetic correction of MYO7A mutation resulted in morphologic and functional recovery of hair cell-like cells derived from iPSCs. These findings confirm the hypothesis that MYO7A plays an important role in the assembly of stereocilia into stereociliary bundles. Thus, the present study might provide further insight into the pathogenesis of sensorineural hearing loss and facilitate the development of therapeutic strategies against monogenic disease through the genetic repair of patient-specific iPSCs.

The role of gamma-aminobutyric acid/glycinergic synaptic transmission in mediating bilirubin-induced hyperexcitation in developing auditory neurons.

Hyperbilirubinemia is a common clinical phenomenon observed in human newborns. A high level of bilirubin can result in severe jaundice and bilirubin encephalopathy. However, the cellular mechanisms underlying bilirubin excitotoxicity are unclear. Our previous studies showed the action of gamma-aminobutyric acid (GABA)/glycine switches from excitatory to inhibitory during development in the ventral cochlear nucleus (VCN), one of the most sensitive auditory nuclei to bilirubin toxicity. In the present study, we investigated the roles of GABAA/glycine receptors in the induction of bilirubin hyperexcitation in early developing neurons. Using the patch clamp technique, GABAA/glycine receptor-mediated spontaneous inhibitory synaptic currents (sIPSCs) were recorded from bushy and stellate cells in acute brainstem slices from young mice (postnatal day 2-6). Bilirubin significantly increased the frequency of sIPSCs, and this effect was prevented by pretreatments of slices with either fast or slow Ca(2+) chelators BAPTA-AM and EGTA-AM suggesting that bilirubin can increase the release of GABA/glycine via Ca(2+)-dependent mechanisms. Using cell-attached recording configuration, we found that antagonists of GABAA and glycine receptors strongly attenuated spontaneous spiking firings in P2-6 neurons but produced opposite effect in P15-19 neurons. Furthermore, these antagonists reversed bilirubin-evoked hyperexcitability in P2-6 neurons, indicating that excitatory action of GABA/glycinergic transmission specifically contribute to bilirubin-induced hyperexcitability in the early stage of development. Our results suggest that bilirubin-induced enhancement of presynaptic release GABA/Glycine via Ca(2+)-dependent mechanisms may play a critical role in mediating neuronal hyperexcitation associated with jaundice, implicating potential new strategies for predicting, preventing, and treating bilirubin neurotoxicity.