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

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

Chronic neurotransmission increases the susceptibility of lateral-line hair cells to ototoxic insults.

Sensory hair cells receive near constant stimulation by omnipresent auditory and vestibular stimuli. To detect and encode these stimuli, hair cells require steady ATP production, which can be accompanied by a buildup of mitochondrial byproducts called reactive oxygen species (ROS). ROS buildup is thought to sensitize hair cells to ototoxic insults, including the antibiotic neomycin. Work in neurons has shown that neurotransmission is a major driver of ATP production and ROS buildup. Therefore, we tested whether neurotransmission is a significant contributor to ROS buildup in hair cells. Using genetics and pharmacology, we disrupted two key aspects of neurotransmission in zebrafish hair cells: presynaptic calcium influx and the fusion of synaptic vesicles. We find that chronic block of neurotransmission enhances hair-cell survival when challenged with the ototoxin neomycin. This reduction in ototoxin susceptibility is accompanied by reduced mitochondrial activity, likely due to a reduced ATP demand. In addition, we show that mitochondrial oxidation and ROS buildup are reduced when neurotransmission is blocked. Mechanistically, we find that it is the synaptic vesicle cycle rather than presynaptic- or mitochondrial-calcium influx that contributes most significantly to this metabolic stress. Our results comprehensively indicate that, over time, neurotransmission causes ROS buildup that increases the susceptibility of hair cells to ototoxins.

EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia.

Hair cells detect sound, head position or water movements when their mechanosensory hair bundle is deflected. Each hair bundle has an asymmetric architecture that restricts stimulus detection to a single axis. Coordinated hair cell orientations within sensory epithelia further tune stimulus detection at the organ level. Here, we identify GPR156, an orphan GPCR of unknown function, as a critical regulator of hair cell orientation. We demonstrate that the transcription factor EMX2 polarizes GPR156 distribution, enabling it to signal through Gαi and trigger a 180° reversal in hair cell orientation. GPR156-Gαi mediated reversal is essential to establish hair cells with mirror-image orientations in mouse otolith organs in the vestibular system and in zebrafish lateral line. Remarkably, GPR156-Gαi also instructs hair cell reversal in the auditory epithelium, despite a lack of mirror-image organization. Overall, our work demonstrates that conserved GPR156-Gαi signaling is integral to the framework that builds directional responses into mechanosensory epithelia.

Retrograde Mitochondrial Transport Is Essential for Organelle Distribution and Health in Zebrafish Neurons.

In neurons, mitochondria are transported by molecular motors throughout the cell to form and maintain functional neural connections. These organelles have many critical functions in neurons and are of high interest as their dysfunction is associated with disease. While the mechanics and impact of anterograde mitochondrial movement toward axon terminals are beginning to be understood, the frequency and function of retrograde (cell body directed) mitochondrial transport in neurons are still largely unexplored. While existing evidence indicates that some mitochondria are retrogradely transported for degradation in the cell body, the precise impact of disrupting retrograde transport on the organelles and the axon was unknown. Using long-term, in vivo imaging, we examined mitochondrial motility in zebrafish sensory and motor axons. We show that retrograde transport of mitochondria from axon terminals allows replacement of the axon terminal population within a day. By tracking these organelles, we show that not all mitochondria that leave the axon terminal are degraded; rather, they persist over several days. Disrupting retrograde mitochondrial flux in neurons leads to accumulation of aged organelles in axon terminals and loss of cell body mitochondria. Assays of neural circuit activity demonstrated that disrupting mitochondrial transport and function has no effect on sensory axon terminal activity but does negatively impact motor neuron axons. Taken together, our work supports a previously unappreciated role for retrograde mitochondrial transport in the maintenance of a homeostatic distribution of mitochondria in neurons and illustrates the downstream effects of disrupting this process on sensory and motor circuits.SIGNIFICANCE STATEMENT Disrupted mitochondrial transport has been linked to neurodegenerative disease. Retrograde transport of this organelle has been implicated in turnover of aged organelles through lysosomal degradation in the cell body. Consistent with this, we provide evidence that retrograde mitochondrial transport is important for removing aged organelles from axons; however, we show that these organelles are not solely degraded, rather they persist in neurons for days. Disrupting retrograde mitochondrial transport impacts the homeostatic distribution of mitochondria throughout the neuron and the function of motor, but not sensory, axon synapses. Together, our work shows the conserved reliance on retrograde mitochondrial transport for maintaining a healthy mitochondrial pool in neurons and illustrates the disparate effects of disrupting this process on sensory versus motor circuits.

Synaptic mitochondria regulate hair-cell synapse size and function.

Sensory hair cells in the ear utilize specialized ribbon synapses. These synapses are defined by electron-dense presynaptic structures called ribbons, composed primarily of the structural protein Ribeye. Previous work has shown that voltage-gated influx of Ca2+ through CaV1.3 channels is critical for hair-cell synapse function and can impede ribbon formation. We show that in mature zebrafish hair cells, evoked presynaptic-Ca2+ influx through CaV1.3 channels initiates mitochondrial-Ca2+ (mito-Ca2+) uptake adjacent to ribbons. Block of mito-Ca2+ uptake in mature cells depresses presynaptic-Ca2+ influx and impacts synapse integrity. In developing zebrafish hair cells, mito-Ca2+ uptake coincides with spontaneous rises in presynaptic-Ca2+ influx. Spontaneous mito-Ca2+ loading lowers cellular NAD+/NADH redox and downregulates ribbon size. Direct application of NAD+ or NADH increases or decreases ribbon size respectively, possibly acting through the NAD(H)-binding domain on Ribeye. Our results present a mechanism where presynaptic- and mito-Ca2+ couple to confer proper presynaptic function and formation.

Corrigendum: Transcriptomic Profiling of Zebrafish Hair Cells Using RiboTag.

[This corrects the article DOI: 10.3389/fcell.2018.00047.].

Transcriptomic Profiling of Zebrafish Hair Cells Using RiboTag.

The zebrafish inner ear organs and lateral line neuromasts are comprised of a variety of cell types, including mechanosensitive hair cells. Zebrafish hair cells are evolutionarily homologous to mammalian hair cells, and have been particularly useful for studying normal hair cell development and function. However, the relative scarcity of hair cells within these complex organs, as well as the difficulty of fine dissection at early developmental time points, makes hair cell-specific gene expression profiling technically challenging. Cell sorting methods, as well as single-cell RNA-Seq, have proved to be very informative in studying hair cell-specific gene expression. However, these methods require that tissues are dissociated, the processing for which can lead to changes in gene expression prior to RNA extraction. To bypass this problem, we have developed a transgenic zebrafish model to evaluate the translatome of the inner ear and lateral line hair cells in their native tissue environment; the Tg(myo6b:RiboTag) zebrafish. This model expresses both GFP and a hemagglutinin (HA) tagged rpl10a gene under control of the myo6b promoter (myo6b:GFP-2A-rpl10a-3xHA), resulting in HA-tagged ribosomes expressed specifically in hair cells. Consequently, intact zebrafish larvae can be used to enrich for actively translated hair cell mRNA via an immunoprecipitation protocol using an antibody for the HA-tag (similar to the RiboTag mice). We demonstrate that this model can be used to reliably enrich for actively translated zebrafish hair cell mRNA. Additionally, we perform a global hair cell translatome analysis using RNA-Seq and show enrichment of known hair cell expressed transcripts and depletion of non-hair cell expressed transcripts in the immunoprecipitated material compared with mRNA extracted from whole fish (input). Our results show that our model can identify novel hair cell expressed genes in intact zebrafish, without inducing changes to gene expression that result from tissue dissociation and delays during cell sorting. Overall, we believe that this model will be highly useful for studying changes in zebrafish hair cell-specific gene expression in response to developmental progression, mutations, as well as hair cell damage by noise or ototoxic drug exposure.

Synaptically silent sensory hair cells in zebrafish are recruited after damage.

Analysis of mechanotransduction among ensembles of sensory hair cells in vivo is challenging in many species. To overcome this challenge, we used optical indicators to investigate mechanotransduction among collections of hair cells in intact zebrafish. Our imaging reveals a previously undiscovered disconnect between hair-cell mechanosensation and synaptic transmission. We show that saturating mechanical stimuli able to open mechanically gated channels are unexpectedly insufficient to evoke vesicle fusion in the majority of hair cells. Although synaptically silent, latent hair cells can be rapidly recruited after damage, demonstrating that they are synaptically competent. Therefore synaptically silent hair cells may be an important reserve that acts to maintain sensory function. Our results demonstrate a previously unidentified level of complexity in sculpting sensory transmission from the periphery.

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.

Enlargement of Ribbons in Zebrafish Hair Cells Increases Calcium Currents But Disrupts Afferent Spontaneous Activity and Timing of Stimulus Onset.

In sensory hair cells of auditory and vestibular organs, the ribbon synapse is required for the precise encoding of a wide range of complex stimuli. Hair cells have a unique presynaptic structure, the synaptic ribbon, which organizes both synaptic vesicles and calcium channels at the active zone. Previous work has shown that hair-cell ribbon size is correlated with differences in postsynaptic activity. However, additional variability in postsynapse size presents a challenge to determining the specific role of ribbon size in sensory encoding. To selectively assess the impact of ribbon size on synapse function, we examined hair cells in transgenic zebrafish that have enlarged ribbons, without postsynaptic alterations. Morphologically, we found that enlarged ribbons had more associated vesicles and reduced presynaptic calcium-channel clustering. Functionally, hair cells with enlarged ribbons had larger global and ribbon-localized calcium currents. Afferent neuron recordings revealed that hair cells with enlarged ribbons resulted in reduced spontaneous spike rates. Additionally, despite larger presynaptic calcium signals, we observed fewer evoked spikes with longer latencies from stimulus onset. Together, our work indicates that hair-cell ribbon size influences the spontaneous spiking and the precise encoding of stimulus onset in afferent neurons.SIGNIFICANCE STATEMENT Numerous studies support that hair-cell ribbon size corresponds with functional sensitivity differences in afferent neurons and, in the case of inner hair cells of the cochlea, vulnerability to damage from noise trauma. Yet it is unclear whether ribbon size directly influences sensory encoding. Our study reveals that ribbon enlargement results in increased ribbon-localized calcium signals, yet reduces afferent spontaneous activity and disrupts the timing of stimulus onset, a distinct aspect of auditory and vestibular encoding. These observations suggest that varying ribbon size alone can influence sensory encoding, and give further insight into how hair cells transduce signals that cover a wide dynamic range of stimuli.

Additive reductions in zebrafish PRPS1 activity result in a spectrum of deficiencies modeling several human PRPS1-associated diseases.

Phosphoribosyl pyrophosphate synthetase-1 (PRPS1) is a key enzyme in nucleotide biosynthesis, and mutations in PRPS1 are found in several human diseases including nonsyndromic sensorineural deafness, Charcot-Marie-Tooth disease-5, and Arts Syndrome. We utilized zebrafish as a model to confirm that mutations in PRPS1 result in phenotypic deficiencies in zebrafish similar to those in the associated human diseases. We found two paralogs in zebrafish, prps1a and prps1b and characterized each paralogous mutant individually as well as the double mutant fish. Zebrafish prps1a mutants and prps1a;prps1b double mutants showed similar morphological phenotypes with increasingly severe phenotypes as the number of mutant alleles increased. Phenotypes included smaller eyes and reduced hair cell numbers, consistent with the optic atrophy and hearing impairment observed in human patients. The double mutant also showed abnormal development of primary motor neurons, hair cell innervation, and reduced leukocytes, consistent with the neuropathy and recurrent infection of the human patients possessing the most severe reductions of PRPS1 activity. Further analyses indicated the phenotypes were associated with a prolonged cell cycle likely resulting from reduced nucleotide synthesis and energy production in the mutant embryos. We further demonstrated the phenotypes were caused by delays in the tissues most highly expressing the prps1 genes.

oca2 Regulation of chromatophore differentiation and number is cell type specific in zebrafish.

We characterized a zebrafish mutant that displays defects in melanin synthesis and in the differentiation of melanophores and iridophores of the skin and retinal pigment epithelium. Positional cloning and candidate gene sequencing link this mutation to a 410-kb region on chromosome 6, containing the oculocutaneous albinism 2 (oca2) gene. Quantification of oca2 mutant melanophores shows a reduction in the number of differentiated melanophores compared with wildtype siblings. Consistent with the analysis of mouse Oca2-deficient melanocytes, zebrafish mutant melanophores have immature melanosomes which are partially rescued following treatment with vacuolar-type ATPase inhibitor/cytoplasmic pH modifier, bafilomycin A1. Melanophore-specific gene expression is detected at the correct time and in anticipated locations. While oca2 zebrafish display unpigmented gaps on the head region of mutants 3 days post-fertilization, melanoblast quantification indicates that oca2 mutants have the correct number of melanoblasts, suggesting a differentiation defect explains the reduced melanophore number. Unlike melanophores, which are reduced in number in oca2 mutants, differentiated iridophores are present at significantly higher numbers. These data suggest distinct mechanisms for oca2 in establishing differentiated chromatophore number in developing zebrafish.

Maintenance of melanophore morphology and survival is cathepsin and vps11 dependent in zebrafish.

Here, we characterize a Danio rerio zebrafish pigment cell mutant (melanophore integrity mutant), which displays a defect in maintenance of melanophore and iridophore number. Mapping and candidate gene analysis links the melanophore integrity mutant mutation to the vacuolar protein sorting 11 (vps11(w66)) gene. Quantification of vps11(w66) chromatophores during larval stages suggests a decrease in number as compared to wildtype siblings. TUNEL analysis and treatment with the caspase inhibitor, zVAD-fmk, indicate that vps11(w66) chromatophore death is caspase independent. Western blot analysis of PARP-1 cleavage patterns in mutant lysates suggests that increases in pH dependent cathepsin activity is involved in the premature chromatophore death observed in vps11(w66) mutants. Consistently, treatment with ALLM and Bafilomycin A1 (cathepsin/calpain and vacuolar-type H+-ATPase inhibitors, respectively), restore normal melanophore morphology and number in vps11(w66) mutants. Last, LC3B western blot analysis indicates an increase in autophagosome marker, LC3B II in vps11(w66) mutants as compared to wildtype control, but not in ALLM or Bafilomycin A1 treated mutants. Taken together, these data suggest that vps11 promotes normal melanophore morphology and survival by inhibiting cathepsin release and/or activity.