Touch sensation requires the mechanically gated ion channel ELKIN1.

Touch perception is enabled by mechanically activated ion channels, the opening of which excites cutaneous sensory endings to initiate sensation. In this study, we identify ELKIN1 as an ion channel likely gated by mechanical force, necessary for normal touch sensitivity in mice. Touch insensitivity in Elkin1-/- mice was caused by a loss of mechanically activated currents (MA currents) in around half of all sensory neurons activated by light touch (low-threshold mechanoreceptors). Reintroduction of Elkin1 into sensory neurons from Elkin1-/- mice restored MA currents. Additionally, small interfering RNA-mediated knockdown of ELKIN1 from induced human sensory neurons substantially reduced indentation-induced MA currents, supporting a conserved role for ELKIN1 in human touch. Our data identify ELKIN1 as a core component of touch transduction in mice and potentially in humans.

Insights into the Structural Basis of Amyloid Resistance Provided by Cryo-EM Structures of AApoAII Amyloid Fibrils.

Amyloid resistance is the inability or the reduced susceptibility of an organism to develop amyloidosis. In this study we have analysed the molecular basis of the resistance to systemic AApoAII amyloidosis, which arises from the formation of amyloid fibrils from apolipoprotein A-II (ApoA-II). The disease affects humans and animals, including SAMR1C mice that express the C allele of ApoA-II protein, whereas other mouse strains are resistant to development of amyloidosis due to the expression of other ApoA-II alleles, such as ApoA-IIF. Using cryo-electron microscopy, molecular dynamics simulations and other methods, we have determined the structures of pathogenic AApoAII amyloid fibrils from SAMR1C mice and analysed the structural effects of ApoA-IIF-specific mutational changes. Our data show that these changes render ApoA-IIF incompatible with the specific fibril morphologies, with which ApoA-II protein can become pathogenic in vivo.

FADS1-arachidonic acid axis enhances arachidonic acid metabolism by altering intestinal microecology in colorectal cancer.

Colonocyte metabolism shapes the microbiome. Metabolites are the main mediators of information exchange between intestine and microbial communities. Arachidonic acid (AA) is an essential polyunsaturated fatty acid and its role in colorectal cancer (CRC) remains unexplored. In this study, we show that AA feeding promotes tumor growth in AOM/DSS and intestinal specific Apc-/- mice via modulating the intestinal microecology of increased gram-negative bacteria. Delta-5 desaturase (FADS1), a rate-limiting enzyme, is upregulated in CRC and effectively mediates AA synthesis. Functionally, FADS1 regulates CRC tumor growth via high AA microenvironment-induced enriched gram-negative microbes. Elimination of gram-negative microbe abolishes FADS1 effect. Mechanistically, gram-negative microbes activate TLR4/MYD88 pathway in CRC cells that contributes FADS1-AA axis to metabolize to prostaglandin E2 (PGE2). Cumulatively, we report a potential cancer-promoting mechanism of FADS1-AA axis in CRC that converts raising synthesized AA to PGE2 via modulating the intestinal microecology of gram-negative.

Compensatory gene expression potentially rescues impaired brain development in Kit mutant mice.

While loss-of-function mutations in the murine dominant white spotting/Kit (W) locus affect a diverse array of cell lineages and organs, the brain, organ with the highest expression show the least number of defective phenotypes. We performed transcriptome analysis of the brains of KitW embryos and found prominent gene expression changes specifically in the E12.5 KitW/W homozygous mutant. Although other potentially effective changes in gene expression were observed, uniform downregulation of ribosomal protein genes and oxidative phosphorylation pathway genes specifically observed in the E12.5 brain may comprise a genetic compensation system exerting protective metabolic effects against the deleterious effect of KitW/W mutation in the developing brain.

Organ defects of the Usp7K444R mutant mouse strain indicate the essential role of K63-polyubiquitinated Usp7 in organ formation.

BACKGROUND: K63-linked polyubiquitination of proteins have nonproteolytic functions and regulate the activity of many signal transduction pathways. USP7, a HIF1α deubiquitinase, undergoes K63-linked polyubiquitination under hypoxia. K63-polyubiquitinated USP7 serves as a scaffold to anchor HIF1α, CREBBP, the mediator complex, and the super elongation complex to enhance HIF1α-induced gene transcription. However, the physiological role of K63-polyubiquitinated USP7 remains unknown. METHODS: Using a Usp7K444R point mutation knock-in mouse strain, we performed immunohistochemistry and standard molecular biological methods to examine the organ defects of liver and kidney in this knock-in mouse strain. Mechanistic studies were performed by using deubiquitination, immunoprecipitation, and quantitative immunoprecipitations (qChIP) assays. RESULTS: We observed multiple organ defects, including decreased liver and muscle weight, decreased tibia/fibula length, liver glycogen storage defect, and polycystic kidneys. The underlying mechanisms include the regulation of protein stability and/or modulation of transcriptional activation of several key factors, leading to decreased protein levels of Prr5l, Hnf4α, Cebpα, and Hnf1ß. Repression of these crucial factors leads to the organ defects described above. CONCLUSIONS: K63-polyubiquitinated Usp7 plays an essential role in the development of multiple organs and illustrates the importance of the process of K63-linked polyubiquitination in regulating critical protein functions.

SOCS2 regulation of growth hormone signaling requires a canonical interaction with phosphotyrosine.

Suppressor of cytokine signaling (SOCS) 2 is the critical negative regulator of growth hormone (GH) and prolactin signaling. Mice lacking SOCS2 display gigantism with increased body weight and length, and an enhanced response to GH treatment. Here, we characterized mice carrying a germ-line R96C mutation within the SOCS2-SH2 domain, which disrupts the ability of SOCS2 to interact with tyrosine-phosphorylated targets. Socs2R96C/R96C mice displayed a similar increase in growth as previously observed in SOCS2 null (Socs2-/-) mice, with a proportional increase in body and organ weight, and bone length. Embryonic fibroblasts isolated from Socs2R96C/R96C and Socs2-/- mice also showed a comparable increase in phosphorylation of STAT5 following GH stimulation, indicating the critical role of phosphotyrosine binding in SOCS2 function.

Pax6 mutant cerebral organoids partially recapitulate phenotypes of Pax6 mutant mouse strains.

Cerebral organoids show great promise as tools to unravel the complex mechanisms by which the mammalian brain develops during embryogenesis. We generated mouse cerebral organoids harbouring constitutive or conditional mutations in Pax6, which encodes a transcription factor with multiple important roles in brain development. By comparing the phenotypes of mutant organoids with the well-described phenotypes of Pax6 mutant mouse embryos, we evaluated the extent to which cerebral organoids reproduce phenotypes previously described in vivo. Organoids lacking Pax6 showed multiple phenotypes associated with its activity in mice, including precocious neural differentiation, altered cell cycle and an increase in abventricular mitoses. Neural progenitors in both Pax6 mutant and wild type control organoids cycled more slowly than their in vivo counterparts, but nonetheless we were able to identify clear changes to cell cycle attributable to the absence of Pax6. Our findings support the value of cerebral organoids as tools to explore mechanisms of brain development, complementing the use of mouse models.

Endocannabinoid-dependent formation of columnar axonal projection in the mouse cerebral cortex.

Columnar structure is one of the most fundamental morphological features of the cerebral cortex and is thought to be the basis of information processing in higher animals. Yet, how such a topographically precise structure is formed is largely unknown. Formation of columnar projection of layer 4 (L4) axons is preceded by thalamocortical formation, in which type 1 cannabinoid receptors (CB1R) play an important role in shaping barrel-specific targeted projection by operating spike timing-dependent plasticity during development (Itami et al., J. Neurosci. 36, 7039-7054 [2016]; Kimura & Itami, J. Neurosci. 39, 3784-3791 [2019]). Right after the formation of thalamocortical projections, CB1Rs start to function at L4 axon terminals (Itami & Kimura, J. Neurosci. 32, 15000-15011 [2012]), which coincides with the timing of columnar shaping of L4 axons. Here, we show that the endocannabinoid 2-arachidonoylglycerol (2-AG) plays a crucial role in columnar shaping. We found that L4 axon projections were less organized until P12 and then became columnar after CB1Rs became functional. By contrast, the columnar organization of L4 axons was collapsed in mice genetically lacking diacylglycerol lipase α, the major enzyme for 2-AG synthesis. Intraperitoneally administered CB1R agonists shortened axon length, whereas knockout of CB1R in L4 neurons impaired columnar projection of their axons. Our results suggest that endocannabinoid signaling is crucial for shaping columnar axonal projection in the cerebral cortex.

Collagen (I) homotrimer potentiates the osteogenesis imperfecta (oim) mutant allele and reduces survival in male mice.

The osteogenesis imperfecta murine (oim) model with solely homotrimeric (α1)3 type I collagen, owing to a dysfunctional α2(I) collagen chain, has a brittle bone phenotype, implying that the (α1)2(α2)1 heterotrimer is required for physiological bone function. Here, we comprehensively show, for the first time, that mice lacking the α2(I) chain do not have impaired bone biomechanical or structural properties, unlike oim homozygous mice. However, Mendelian inheritance was affected in male mice of both lines, and male mice null for the α2(I) chain exhibited age-related loss of condition. Compound heterozygotes were generated to test whether gene dosage was responsible for the less-severe phenotype of oim heterozygotes, after allelic discrimination showed that the oim mutant allele was not downregulated in heterozygotes. Compound heterozygotes had impaired bone structural properties compared to those of oim heterozygotes, albeit to a lesser extent than those of oim homozygotes. Hence, the presence of heterotrimeric type I collagen in oim heterozygotes alleviates the effect of the oim mutant allele, but a genetic interaction between homotrimeric type I collagen and the oim mutant allele leads to bone fragility.

ADAR1 Prevents Autoinflammatory Processes in the Heart Mediated by IRF7.

BACKGROUND: ADAR1 (adenosine deaminase acting on RNA-1)-mediated adenosine to inosine (A-to-I) RNA editing plays an essential role for distinguishing endogenous from exogenous RNAs, preventing autoinflammatory ADAR1 also regulates cellular processes by recoding specific mRNAs, thereby altering protein functions, but may also act in an editing-independent manner. The specific role of ADAR1 in cardiomyocytes and its mode of action in the heart is not fully understood. To determine the role of ADAR1 in the heart, we used different mutant mouse strains, which allows to distinguish immunogenic, editing-dependent, and editing-independent functions of ADAR1. METHODS: Different Adar1-mutant mouse strains were employed for gene deletion or specific inactivation of ADAR1 enzymatic activity in cardiomyocytes, either alone or in combination with Ifih1 (interferon induced with helicase C domain 1) or Irf7 (interferon regulatory factor 7) gene inactivation. Mutant mice were investigated by immunofluorescence, Western blot, RNAseq, proteomics, and functional MRI analysis. RESULTS: Inactivation of Adar1 in cardiomyocytes resulted in late-onset autoinflammatory myocarditis progressing into dilated cardiomyopathy and heart failure at 6 months of age. Adar1 depletion activated interferon signaling genes but not NFκB (nuclear factor kappa B) signaling or apoptosis and reduced cardiac hypertrophy during pressure overload via induction of Irf7. Additional inactivation of the cytosolic RNA sensor MDA5 (melanoma differentiation-associated gene 5; encoded by the Ifih1 gene) in Adar1 mutant mice prevented activation of interferon signaling gene and delayed heart failure but did not prevent lethality after 8.5 months. In contrast, compound mutants only expressing catalytically inactive ADAR1 in an Ifih1-mutant background were completely normal. Inactivation of Irf7 attenuated the phenotype of Adar1-deficient cardiomyocytes to a similar extent as Ifih1 depletion, identifying IRF7 as the main mediator of autoinflammatory responses caused by the absence of ADAR1 in cardiomyocytes. CONCLUSIONS: Enzymatically active ADAR1 prevents IRF7-mediated autoinflammatory reactions in the heart triggered by endogenous nonedited RNAs. In addition to RNA editing, ADAR1 also serves editing-independent roles in the heart required for long-term cardiac function and survival.

The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

Transient early life growth hormone exposure permanently alters brain, muscle, liver, macrophage, and adipocyte status in long-lived Ames dwarf mice.

The exceptional longevity of Ames dwarf (DF) mice can be abrogated by a brief course of growth hormone (GH) injections started at 2 weeks of age. This transient GH exposure also prevents the increase in cellular stress resistance and decline in hypothalamic inflammation characteristic of DF mice. Here, we show that transient early-life GH treatment leads to permanent alteration of pertinent changes in adipocytes, fat-associated macrophages, liver, muscle, and brain that are seen in DF mice. Ames DF mice, like Snell dwarf and GHRKO mice, show elevation of glycosylphosphatidylinositol specific phospholipase D1 in liver, neurogenesis in brain as indicated by BDNF and DCX proteins, muscle production of fibronectin type III domain-containing protein 5 (a precursor of irisin), uncoupling protein 1 as an index of thermogenic capacity in brown and white fat, and increase in fat-associated anti-inflammatory macrophages. In each case, transient exposure to GH early in life reverts the DF mice to the levels of each protein seen in littermate control animals, in animals evaluated at 15-18 months of age. Thus, many of the traits seen in long-lived mutant mice, pertinent to age-related changes in inflammation, neurogenesis, and metabolic control, are permanently set by early-life GH levels.

Meteorin-like promotes heart repair through endothelial KIT receptor tyrosine kinase.

Effective tissue repair after myocardial infarction entails a vigorous angiogenic response, guided by incompletely defined immune cell-endothelial cell interactions. We identify the monocyte- and macrophage-derived cytokine METRNL (meteorin-like) as a driver of postinfarction angiogenesis and high-affinity ligand for the stem cell factor receptor KIT (KIT receptor tyrosine kinase). METRNL mediated angiogenic effects in cultured human endothelial cells through KIT-dependent signaling pathways. In a mouse model of myocardial infarction, METRNL promoted infarct repair by selectively expanding the KIT-expressing endothelial cell population in the infarct border zone. Metrnl-deficient mice failed to mount this KIT-dependent angiogenic response and developed severe postinfarction heart failure. Our data establish METRNL as a KIT receptor ligand in the context of ischemic tissue repair.

A set point in the selection of the αßTCR T cell repertoire imposed by pre-TCR signaling strength.

Signaling via the T cell receptor (TCR) is critical during the development, maintenance, and activation of T cells. Quantitative aspects of TCR signaling have an important role during positive and negative selection, lineage choice, and ability to respond to small amounts of antigen. By using a mutant mouse line expressing a hypomorphic allele of the CD3ζ chain, we show here that the strength of pre-TCR­mediated signaling during T cell development determines the diversity of the TCRß repertoire available for positive and negative selection, and hence of the final αßTCR repertoire. This finding uncovers an unexpected, pre-TCR signaling­dependent and repertoire­shaping role for ß-selection beyond selection of in-frame rearranged TCRß chains. Our data furthermore support a model of pre-TCR signaling in which the arrangement of this receptor in stable nanoclusters determines its quantitative signaling capacity.

Vagus nerve stimulation promotes resolution of inflammation by a mechanism that involves Alox15 and requires the α7nAChR subunit.

Nonresolving inflammation underlies a range of chronic inflammatory diseases, and therapeutic acceleration of resolution of inflammation may improve outcomes. Neural reflexes regulate the intensity of inflammation (for example, through signals in the vagus nerve), but whether activation of the vagus nerve promotes the resolution of inflammation in vivo has been unknown. To investigate this, mice were subjected to electrical vagus nerve stimulation (VNS) or sham surgery at the cervical level followed by zymosan-induced peritonitis. The duration of inflammation resolution was significantly reduced and efferocytosis was significantly increased in mice treated with VNS as compared with sham. Lipid mediator (LM) metabololipidomics revealed that mice treated with VNS had higher levels of specialized proresolving mediators (SPMs), particularly from the omega-3 docosahexaenoic (DHA) and docosapentaenoic (n-3 DPA) metabolomes, in peritoneal exudates. VNS also shifted the ratio between proinflammatory and proresolving LMs toward a proresolving profile, but this effect by VNS was inverted in mice deficient in 12/15-lipoxgenase (Alox15), a key enzyme in this SPM biosynthesis. The significant VNS-mediated reduction of neutrophil numbers in peritoneal exudates was absent in mice deficient in the cholinergic α7-nicotinic acetylcholine receptor subunit (α7nAChR), an essential component of the inflammatory reflex. Thus, VNS increased local levels of SPM and accelerated resolution of inflammation in zymosan-induced peritonitis by a mechanism that involves Alox15 and requires the α7nAChR.

Thalamocortical axons regulate neurogenesis and laminar fates in the early sensory cortex.

Area-specific axonal projections from the mammalian thalamus shape unique cellular organization in target areas in the adult neocortex. How these axons control neurogenesis and early neuronal fate specification is poorly understood. By using mutant mice lacking the majority of thalamocortical axons, we show that these axons are required for the production and specification of the proper number of layer 4 neurons in primary sensory areas by the neonatal stage. Part of these area-specific roles is played by the thalamus-derived molecule, VGF. Our work reveals that extrinsic cues from sensory thalamic projections have an early role in the formation of cortical cytoarchitecture by enhancing the production and specification of layer 4 neurons.

Repair of surviving hair cells in the damaged mouse utricle.

Sensory hair cells (HCs) in the utricle are mechanoreceptors required to detect linear acceleration. After damage, the mammalian utricle partially restores the HC population and organ function, although regenerated HCs are primarily type II and immature. Whether native, surviving HCs can repair and contribute to this recovery is unclear. Here, we generated the Pou4f3DTR/+; Atoh1CreERTM/+; Rosa26RtdTomato/+ mouse to fate map HCs prior to ablation. After HC ablation, vestibular evoked potentials were abolished in all animals, with ∼57% later recovering responses. Relative to nonrecovery mice, recovery animals harbored more Atoh1-tdTomato+ surviving HCs. In both groups, surviving HCs displayed markers of both type I and type II subtypes and afferent synapses, despite distorted lamination and morphology. Surviving type II HCs remained innervated in both groups, whereas surviving type I HCs first lacked and later regained calyces in the recovery, but not the nonrecovery, group. Finally, surviving HCs initially displayed immature and subsequently mature-appearing bundles in the recovery group. These results demonstrate that surviving HCs are capable of self-repair and may contribute to the recovery of vestibular function.

Patient-Specific TBX5-G125R Variant Induces Profound Transcriptional Deregulation and Atrial Dysfunction.

BACKGROUND: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt-Oram syndrome (also known as hand-heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. METHODS: We analyzed ECGs of an extended family pedigree of Holt-Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome (Tbx5G125R) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). RESULTS: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt-Oram syndrome patients. Tbx5G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. CONCLUSIONS: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.

Improved impedance to maladaptation and enhanced VCAM-1 upregulation with resistance-type training in the long-lived Snell dwarf (Pit1dw/dw) mouse.

Snell dwarf mice with the Pit1dw/dw mutation are deficient in growth hormone, prolactin, and thyroid stimulating hormone and exhibit >40% lifespan extension. This longevity is accompanied by compromised muscular performance. However, research regarding young (3-month-old) Snell dwarf mice demonstrate exceptional responsivity to resistance-type training especially in terms of a shifted fiber type distribution and increased protein levels of vascular cell adhesion molecule-1 (VCAM-1), a possible mediator of such remodeling. In the present study, we investigated whether this responsiveness persists at 12 months of age. Unlike 12-month-old control mice, age-matched Snell dwarf mice remained resistant to training-induced maladaptive decreases in performance and muscle mass. This was accompanied by retainment of the remodeling capacity in muscles of Snell dwarf mice to increase VCAM-1 protein levels and a shift in myosin heavy chain (MHC) isoform distribution with training. Even decreasing training frequency for control mice, an alteration which protected muscles from maladaptation at 12 months of age, did not result in the overt remodeling observed for Snell dwarf mice. The results demonstrate a distinct remodeling response to resistance-type exercise operative in the context of the Pit1dw/dw mutation of long-lived Snell dwarf mice.

The timing of auditory sensory deficits in Norrie disease has implications for therapeutic intervention.

Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease.