scholarly journals Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking

2021 ◽  
pp. mbc.E20-10-0657
Author(s):  
Aayushi Manchanda ◽  
Josephine A. Bonventre ◽  
Sean M. Bugel ◽  
Paroma Chatterjee ◽  
Robyn Tanguay ◽  
...  
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Gaba Receptors ◽  
Transmembrane Domain ◽  
Stop Codon ◽  
Wild Type ◽  
Zebrafish Model ◽  
Sensory Hair Cells ◽  
Early Developmental ◽  
Vestibular Pathways

Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domains of the protein have not been fully characterized. Using a zebrafish model, we investigated a mutant otoferlin with a stop codon at the start of the transmembrane domain. We found that both the phenotype severity and the expression level of mutant otoferlin changed with the age of the zebrafish. At the early developmental timepoint of 72 hours post-fertilization (hpf) low expression of the otoferlin mutant coincided with synaptic ribbon deficiencies, reduced endocytosis, and abnormal transcription of several hair cell genes. As development proceeded, expression of the mutant otoferlin increased, and both synaptic ribbons and hair cell transcript levels resembled wild type. However, hair cell endocytosis deficits and abnormalities in the expression of GABA receptors persisted even after upregulation of mutant otoferlin. Analysis of membrane-reconstituted otoferlin measurements suggest a function for the transmembrane domain in liposome docking. We conclude that that deletion of the transmembrane domain reduces membrane docking, attenuates endocytosis, and results in developmental delay of the hair cell.

scholarly journals Putative pore-forming subunits of the mechano-electrical transduction channel, Tmc1/2b, require Tmie to localize to the site of mechanotransduction in zebrafish sensory hair cells

2018 ◽  
Author(s):  
Itallia V. Pacentine ◽  
Teresa Nicolson
Keyword(s):  
Hair Cell ◽  
Inner Ear ◽  
Hair Cells ◽  
Transmembrane Domain ◽  
Cell Activity ◽  
Sensory Hair ◽  
Transmembrane Channel ◽  
Extracellular Region ◽  
Normal Site ◽  
Sensory Hair Cells

AbstractMutations in transmembrane inner ear (TMIE) cause deafness in humans; previous studies suggest involvement in the mechano-electrical transduction (MET) complex in sensory hair cells, but TMIE’s precise role is unclear. In tmie zebrafish mutants, we observed that GFP-tagged Tmc1 and Tmc2b, which are putative subunits of the MET channel, fail to target to the hair bundle. In contrast, overexpression of Tmie strongly enhances the targeting of Tmc2b-GFP to stereocilia. To identify the motifs of Tmie underlying the regulation of the Tmcs, we systematically deleted or replaced peptide segments. We then assessed localization and functional rescue of each mutated/chimeric form of Tmie in tmie mutants. We determined that the first putative helix was dispensable and identified a novel critical region of Tmie, the extracellular region and transmembrane domain, which mediates both mechanosensitivity and Tmc2b-GFP expression in bundles. Collectively, our results suggest that Tmie’s role in sensory hair cells is to target and stabilize Tmc subunits to the site of MET.Author summaryHair cells mediate hearing and balance through the activity of a pore-forming channel in the cell membrane. The transmembrane inner ear (TMIE) protein is an essential component of the protein complex that gates this so-called mechanotransduction channel. While it is known that loss of TMIE results in deafness, the function of TMIE within the complex is unclear. Using zebrafish as a deafness model, Pacentine and Nicolson demonstrate that Tmie is required for the localization of other essential complex members, the transmembrane channel-like (Tmc) proteins, Tmc1/2b. They then evaluate twelve unique versions of Tmie, each containing mutations to different domains of Tmie. This analysis reveals that some mutations in Tmie cause dysfunctional gating of the channel as demonstrated through reduced hair cell activity, and that these same dysfunctional versions also display reduced Tmc expression at the normal site of the channel. These findings link hair cell activity with the levels of Tmc in the bundle, reinforcing the currently-debated notion that the Tmcs are the pore-forming subunits of the mechanotransduction channel. The authors conclude that Tmie, through distinct regions, is involved in both trafficking and stabilizing the Tmcs at the site of mechanotransduction.

scholarly journals Radixin deficiency causes deafness associated with progressive degeneration of cochlear stereocilia

2004 ◽  
Vol 166 (4) ◽  
pp. 559-570 ◽  
Author(s):  
Shin-ichiro Kitajiri ◽  
Kanehisa Fukumoto ◽  
Masaki Hata ◽  
Hiroyuki Sasaki ◽  
Tatsuya Katsuno ◽  
...  
Keyword(s):  
Hair Cells ◽  
Plasma Membranes ◽  
Wild Type ◽  
Cross Link ◽  
Erm Proteins ◽  
Hearing Ability ◽  
Adult Mice ◽  
Progressive Degeneration ◽  
Sensory Hair Cells

Ezrin/radixin/moesin (ERM) proteins cross-link actin filaments to plasma membranes to integrate the function of cortical layers, especially microvilli. We found that in cochlear and vestibular sensory hair cells of adult wild-type mice, radixin was specifically enriched in stereocilia, specially developed giant microvilli, and that radixin-deficient (Rdx−/−) adult mice exhibited deafness but no obvious vestibular dysfunction. Before the age of hearing onset (∼2 wk), in the cochlea and vestibule of Rdx−/− mice, stereocilia developed normally in which ezrin was concentrated. As these Rdx−/− mice grew, ezrin-based cochlear stereocilia progressively degenerated, causing deafness, whereas ezrin-based vestibular stereocilia were maintained normally in adult Rdx−/− mice. Thus, we concluded that radixin is indispensable for the hearing ability in mice through the maintenance of cochlear stereocilia, once developed. In Rdx−/− mice, ezrin appeared to compensate for radixin deficiency in terms of the development of cochlear stereocilia and the development/maintenance of vestibular stereocilia. These findings indicated the existence of complicate functional redundancy in situ among ERM proteins.

scholarly journals The challenge of hair cell regeneration

2010 ◽  
Vol 235 (4) ◽  
pp. 434-446 ◽  
Author(s):  
Andrew K Groves
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Electrical Energy ◽  
Cell Regeneration ◽  
Hair Cell Regeneration ◽  
Therapeutic Approaches ◽  
Auditory Hair Cells ◽  
Sensory Hair Cells ◽  
Ototoxic Drugs ◽  
Effects Of Aging

Sensory hair cells of the inner ear are responsible for translating auditory or vestibular stimuli into electrical energy that can be perceived by the nervous system. Although hair cells are exquisitely mechanically sensitive, they can be easily damaged by excessive stimulation by ototoxic drugs and by the effects of aging. In mammals, auditory hair cells are never replaced, such that cumulative damage to the ear causes progressive and permanent deafness. In contrast, non-mammalian vertebrates are capable of replacing lost hair cells, which has led to efforts to understand the molecular and cellular basis of regenerative responses in different vertebrate species. In this review, we describe recent progress in understanding the limits to hair cell regeneration in mammals and discuss the obstacles that currently exist for therapeutic approaches to hair cell replacement.

scholarly journals Synaptic mitochondria regulate hair-cell synapse size and function

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Hiu-tung C Wong ◽  
Qiuxiang Zhang ◽  
Alisha J Beirl ◽  
Ronald S Petralia ◽  
Ya-Xian Wang ◽  
...  
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Direct Application ◽  
Structural Protein ◽  
Ribbon Synapses ◽  
Presynaptic Function ◽  
Sensory Hair Cells ◽  
Synaptic Mitochondria ◽  
And Function ◽  
Cell Synapse

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.

scholarly journals Multiple PDZ domain protein maintains patterning of the apical cytoskeleton in sensory hair cells

Development ◽  
2021 ◽  
Author(s):  
Amandine Jarysta ◽  
Basile Tarchini
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Critical Role ◽  
Pdz Domain ◽  
Hair Bundle ◽  
Structural Defects ◽  
Sensory Hair ◽  
High Frequencies ◽  
Sensory Hair Cells ◽  
Sound Transduction

Sound transduction occurs in the hair bundle, the apical compartment of sensory hair cells in the inner ear. The hair bundle is formed of actin-based stereocilia aligned in rows of graded heights. It was previously shown that the GNAI-GPSM2 complex is part of a developmental blueprint that defines the polarized organization of the apical cytoskeleton in hair cells, including stereocilia distribution and elongation. Here we report a novel and critical role for Multiple PDZ domain (MPDZ) protein during apical hair cell morphogenesis. We show that MPDZ is enriched at the hair cell apical membrane along with MAGUK p55 subfamily member 5 (MPP5/PALS1) and the Crumbs protein CRB3. MPDZ is required there to maintain the proper segregation of apical blueprints proteins, including GNAI-GPSM2. Loss of the blueprint coincides with misaligned stereocilia placement in Mpdz mutant hair cells, and results in permanently misshapen hair bundles. Graded molecular and structural defects along the cochlea can explain the profile of hearing loss in Mpdz mutants, where deficits are most severe at high frequencies.

Comparing Sensory Organs to Define the Path for Hair Cell Regeneration

2019 ◽  
Vol 35 (1) ◽  
pp. 567-589 ◽  
Author(s):  
Nicolas Denans ◽  
Sungmin Baek ◽  
Tatjana Piotrowski
Keyword(s):  
Hair Cell ◽  
Olfactory Epithelium ◽  
Current Literature ◽  
Hair Cells ◽  
Sensory Cell ◽  
Cell Regeneration ◽  
Hair Cell Regeneration ◽  
Sensory Hair ◽  
Sensory Hair Cells ◽  
Technical Advances

Deafness or hearing deficits are debilitating conditions. They are often caused by loss of sensory hair cells or defects in their function. In contrast to mammals, nonmammalian vertebrates robustly regenerate hair cells after injury. Studying the molecular and cellular basis of nonmammalian vertebrate hair cell regeneration provides valuable insights into developing cures for human deafness. In this review, we discuss the current literature on hair cell regeneration in the context of other models for sensory cell regeneration, such as the retina and the olfactory epithelium. This comparison reveals commonalities with, as well as differences between, the different regenerating systems, which begin to define a cellular and molecular blueprint of regeneration. In addition, we propose how new technical advances can address outstanding questions in the field.

scholarly journals In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness

2020 ◽  
Vol 12 (546) ◽  
pp. eaay9101 ◽  
Author(s):  
Wei-Hsi Yeh ◽  
Olga Shubina-Oleinik ◽  
Jonathan M. Levy ◽  
Bifeng Pan ◽  
Gregory A. Newby ◽  
...  
Keyword(s):  
Hair Cell ◽  
Point Mutation ◽  
Hair Cells ◽  
Point Mutations ◽  
Sensory Transduction ◽  
Auditory Function ◽  
Sensory Hair ◽  
Base Editing ◽  
Sensory Hair Cells

Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide therapeutic restoration of gene function. Mutations of transmembrane channel-like 1 gene (TMC1) can cause dominant or recessive deafness. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (c.A545G; resulting in the substitution p.Y182C) in Tmc1 that causes deafness. Tmc1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. We found that sensory hair cells of Baringo mice have a complete loss of auditory sensory transduction. To repair the mutation, we tested several optimized cytosine base editors (CBEmax variants) and guide RNAs in Baringo mouse embryonic fibroblasts. We packaged the most promising CBE, derived from an activation-induced cytidine deaminase (AID), into dual adeno-associated viruses (AAVs) using a split-intein delivery system. The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Injected mice showed up to 51% reversion of the Tmc1 c.A545G point mutation to wild-type sequence (c.A545A) in Tmc1 transcripts. Repair of Tmc1 in vivo restored inner hair cell sensory transduction and hair cell morphology and transiently rescued low-frequency hearing 4 weeks after injection. These findings provide a foundation for a potential one-time treatment for recessive hearing loss and support further development of base editing to correct pathogenic point mutations.

Functional Expression of Exogenous Proteins in Mammalian Sensory Hair Cells Infected With Adenoviral Vectors

1999 ◽  
Vol 81 (4) ◽  
pp. 1881-1888 ◽  
Author(s):  
Jeffrey R. Holt ◽  
David C. Johns ◽  
Sam Wang ◽  
Zheng-Yi Chen ◽  
Robert J. Dunn ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Hair Cell ◽  
Hair Cells ◽  
Cell Function ◽  
Functional Expression ◽  
Adenoviral Vectors ◽  
Inward Rectifier ◽  
Sensory Hair ◽  
Sensory Hair Cells ◽  
Infected Cells

Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors. To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell’s exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for β-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 × 107pfu/ml were sufficient for expression in 50% of the ∼1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel–GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by −17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function.

scholarly journals The effects of Tmc1 Beethoven mutation on mechanotransducer channel function in cochlear hair cells

2015 ◽  
Vol 146 (3) ◽  
pp. 233-243 ◽  
Author(s):  
Maryline Beurg ◽  
Adam C. Goldring ◽  
Robert Fettiplace
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Outer Hair Cells ◽  
Wild Type ◽  
Cochlear Hair Cells ◽  
Transmembrane Channel ◽  
Adaptive Shift ◽  
Electrical Transducer ◽  
Protein Isoform ◽  
The Relationship

Sound stimuli are converted into electrical signals via gating of mechano-electrical transducer (MT) channels in the hair cell stereociliary bundle. The molecular composition of the MT channel is still not fully established, although transmembrane channel–like protein isoform 1 (TMC1) may be one component. We found that in outer hair cells of Beethoven mice containing a M412K point mutation in TMC1, MT channels had a similar unitary conductance to that of wild-type channels but a reduced selectivity for Ca2+. The Ca2+-dependent adaptation that adjusts the operating range of the channel was also impaired in Beethoven mutants, with reduced shifts in the relationship between MT current and hair bundle displacement for adapting steps or after lowering extracellular Ca2+; these effects may be attributed to the channel’s reduced Ca2+ permeability. Moreover, the density of stereociliary CaATPase pumps for Ca2+ extrusion was decreased in the mutant. The results suggest that a major component of channel adaptation is regulated by changes in intracellular Ca2+. Consistent with this idea, the adaptive shift in the current–displacement relationship when hair bundles were bathed in endolymph-like Ca2+ saline was usually abolished by raising the intracellular Ca2+ concentration.

scholarly journals Identification of a 275-kD protein associated with the apical surfaces of sensory hair cells in the avian inner ear.

1990 ◽  
Vol 110 (4) ◽  
pp. 1055-1066 ◽  
Author(s):  
G P Richardson ◽  
S Bartolami ◽  
I J Russell
Keyword(s):  
Hair Cell ◽  
Inner Ear ◽  
Hair Cells ◽  
Proximal Region ◽  
Basilar Papilla ◽  
Relative Molecular Mass ◽  
Apical Surface ◽  
Sensory Hair ◽  
Sensory Hair Cells ◽  
Avian Inner Ear

Immunological techniques have been used to generate both polyclonal and monoclonal antibodies specific for the apical ends of sensory hair cells in the avian inner ear. The hair cell antigen recognized by these antibodies is soluble in nonionic detergent, behaves on sucrose gradients primarily as a 16S particle, and, after immunoprecipitation, migrates as a polypeptide with a relative molecular mass of 275 kD on 5% SDS gels under reducing conditions. The antigen can be detected with scanning immunoelectron microscopy on the apical surface of the cell and on the stereocilia bundle but not on the kinocilium. Double label studies indicate that the entire stereocilia bundle is stained in the lagena macula (a vestibular organ), whereas in the basilar papilla (an auditory organ) only the proximal region of the stereocilia bundle nearest to the apical surface is stained. The monoclonal anti-hair cell antibodies do not stain brain, tongue, lung, liver, heart, crop, gizzard, small intestine, skeletal muscle, feather, skin, or eye tissues but do specifically stain renal corpuscles in the kidney. Experiments using organotypic cultures of the embryonic lagena macula indicate that the antibodies cause a significant increase in the steady-state stiffness of the stereocilia bundle but do not inhibit mechanotransduction. The antibodies should provide a suitable marker and/or tool for the purification of the apical sensory membrane of the hair cell.

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