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Efficient protein delivery using cationic lipid transfection reagents enables high efficiency protein-based genome editing in vivo and in vitro . Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo , achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Sensory hair cells are mechanoreceptors required for hearing and balance functions. From embryonic development, hair cells acquire apical stereociliary bundles for mechanosensation, basolateral ion channels that shape receptor potential, and synaptic contacts for conveying information centrally. These key maturation steps are sequential and presumed coupled; however, whether hair cells emerging postnatally mature similarly is unknown. Here, we show that in vivo postnatally generated and regenerated hair cells in the utricle, a vestibular organ detecting linear acceleration, acquired some mature somatic features but hair bundles appeared nonfunctional and short. The utricle consists of two hair cell subtypes with distinct morphological, electrophysiological and synaptic features. In both the undamaged and damaged utricle, fate-mapping and electrophysiology experiments showed that Plp1+ supporting cells took on type II hair cell properties based on molecular markers, basolateral conductances and synaptic properties yet stereociliary bundles were absent, or small and nonfunctional. By contrast, Lgr5+ supporting cells regenerated hair cells with type I and II properties, representing a distinct hair cell precursor subtype. Lastly, direct physiological measurements showed that utricular function abolished by damage was partially regained during regeneration. Together, our data reveal a previously unrecognized aberrant maturation program for hair cells generated and regenerated postnatally and may have broad implications for inner ear regenerative therapies.

Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) cause hearing loss without vestibular dysfunction in both mice and humans, we investigated the contribution of Tmc1 and the closely related Tmc2 to mechanotransduction in mice. We found that Tmc1 and Tmc2 were expressed in mouse vestibular and cochlear hair cells and that GFP-tagged TMC proteins localized near stereocilia tips. Tmc2 expression was transient in early postnatal mouse cochlear hair cells but persisted in vestibular hair cells. While mice with a targeted deletion of Tmc1 (Tmc1(Δ) mice) were deaf and those with a deletion of Tmc2 (Tmc2(Δ) mice) were phenotypically normal, Tmc1(Δ)Tmc2(Δ) mice had profound vestibular dysfunction, deafness, and structurally normal hair cells that lacked all mechanotransduction activity. Expression of either exogenous TMC1 or TMC2 rescued mechanotransduction in Tmc1(Δ)Tmc2(Δ) mutant hair cells. Our results indicate that TMC1 and TMC2 are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex. Our data also suggest that persistent TMC2 expression in vestibular hair cells may preserve vestibular function in humans with hearing loss caused by TMC1 mutations.

The cochlear sympathetic system plays a key role in auditory function and susceptibility to noise-induced hearing loss (NIHL). The formation of reactive oxygen species (ROS) is a well-documented process in NIHL. In this study, we aimed at investigating the effects of a superior cervical ganglionectomy (SCGx) on NIHL in Sprague-Dawley rats. We explored the effects of unilateral and bilateral Superior Cervical Ganglion (SCG) ablation in the eight-ten weeks old Sprague-Dawley rats of both sexes on NIHL. Auditory function was evaluated by auditory brainstem response (ABR) testing and Distortion product otoacoustic emissions (DPOAEs). Outer hair cells (OHCs) counts and the expression of [alpha].sub.2A-adrenergic receptor (AR) in the rat cochlea using immunofluorescence analysis. Cells culture and treatment, CCK-8 assay, Flow cytometry staining and analysis, and western blotting were to explore the mechanisms of SCG fibers may have a protective role in NIHL. We found that neither bilateral nor unilateral SCGx protected the cochlea against noise exposure. In HEI-OC1 cells, H.sub.2O.sub.2-induced oxidative damage and cell death were inhibited by the application of norepinephrine (NE). NE may prevent ROS-induced oxidative stress in OHCs and NIHL through the [alpha].sub.2A-AR. These results demonstrated that sympathetic innervation mildly affected cochlear susceptibility to acoustic trauma by reducing oxidative damage in OHCs through the [alpha].sub.2A-AR. NE may be a potential therapeutic strategy for NIHL prevention.

Key Points Hearing benefits from an active process that amplifies acoustic inputs by more than a hundred-fold, sharpens frequency discrimination to facilitate the comprehension of speech and the recognition of sound sources, and compresses responses so that we can resolve sounds over a million-fold range in amplitude. The gating of transduction channels endows a mechanically sensitive hair bundle with negative stiffness, an instability that interacts with the motor protein myosin 1c to produce a mechanical amplifier and oscillator. This active hair-bundle motility constitutes the active process of some non-mammalian tetrapods. An outer hair cell of the mammalian cochlea displays somatic motility, in which changes in the transmembrane voltage alter the membrane area occupied by the piezoelectric protein prestin. Depolarization causes the cell body to contract and hyperpolarization causes it to extend at frequencies that can exceed 100 kHz. Acoustic stimulation evokes on the elastic basilar membrane a travelling wave that progresses from the cochlear base towards the apex, peaking at a specific position determined by the stimulus frequency. As this wave advances, the active process of successive hair cells adds energy to counter viscous dissipation. The active process of the mammalian cochlea combines active hair-bundle motility and somatic motility; the former mechanism probably regulates the phase of responsiveness, whereas the latter provides most of the mechanical power. The characteristics of the active process reflect the operation of hair cells near a dynamical instability, the Hopf bifurcation, the generic properties of which explain various phenomena associated with hearing. When extreme quiet excites the active process sufficiently, hair cells traverse the bifurcation and — even in most individuals with normal hearing — produce spontaneous oscillations that emerge from the ears. The sensitivity, frequency resolution and dynamic range of hearing depend upon the cochlear active process, a mechanical-amplification system within the cochlea. In this Review, Hudspeth summarizes evidence that these features result from the operation of hair cells near a dynamical instability, the Hopf bifurcation. Uniquely among human senses, hearing is not simply a passive response to stimulation. Our auditory system is instead enhanced by an active process in cochlear hair cells that amplifies acoustic signals several hundred-fold, sharpens frequency selectivity and broadens the ear's dynamic range. Active motility of the mechanoreceptive hair bundles underlies the active process in amphibians and some reptiles; in mammals, this mechanism operates in conjunction with prestin-based somatic motility. Both individual hair bundles and the cochlea as a whole operate near a dynamical instability, the Hopf bifurcation, which accounts for the cardinal features of the active process.

Exposure to aminoglycoside antibiotics can lead to the generation of toxic levels of reactive oxygen species (ROS) within mechanosensory hair cells of the inner ear that have been implicated in hearing and balance disorders. Better understanding of the origin of aminoglycoside-induced ROS could focus the development of therapies aimed at preventing this event. In this work, we used the zebrafish lateral line system to monitor the dynamic behavior of mitochondrial and cytoplasmic oxidation occurring within the same dying hair cell following exposure to aminoglycosides. The increased oxidation observed in both mitochondria and cytoplasm of dying hair cells was highly correlated with mitochondrial calcium uptake. Application of the mitochondrial uniporter inhibitor Ru360 reduced mitochondrial and cytoplasmic oxidation, suggesting that mitochondrial calcium drives ROS generation during aminoglycoside-induced hair cell death. Furthermore, targeting mitochondria with free radical scavengers conferred superior protection against aminoglycoside exposure compared with identical, untargeted scavengers. Our findings suggest that targeted therapies aimed at preventing mitochondrial oxidation have therapeutic potential to ameliorate the toxic effects of aminoglycoside exposure.

Two types of human ES-cell-derived otic progenitors are shown to have the ability to differentiate in vitro into hair-cell-like cells and auditory neurons, and to engraft, differentiate and improve auditory-evoked response thresholds when transplanted into an auditory neuropathy model; this indicates that it may be possible to use cell-based therapeutic strategies to recover damaged sensory circuitry in deafness. Stem cells counter hearing loss Auditory neuropathy is a form of hearing loss in which the sensory-hair cells of the inner ear are often relatively unscathed, making cochlear implants alone ineffective as therapy. Rather, it is the next step in the auditory pathway that is impaired by damage sustained by the spiral ganglion neurons, and there are no routine treatments available to counter sensory-neuron loss. This paper reports the generation of ear-cell progenitors from human embryonic stem cells, and shows that these otic progenitor cells can differentiate into functional cells involved in auditory response. Transplant of the otic progenitor cells into chemically damaged gerbil ears restores auditory evoked response in the brainstem, suggesting that this type of procedure, combined with cochlear implants, could form the basis of a cell-based therapy for some types of deafness. Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells 1 , is responsible for a substantial proportion of patients with hearing impairment 2 . Although the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss, as poor innervation limits the prospective performance of an implant 3 . Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair-cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory-evoked response thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

The sensory cells that transduce the signals for hearing and balance are highly specialized mechanoreceptors called hair cells that together with supporting cells comprise the sensory epithelia of the inner ear. Loss of hair cells from toxin exposure and age can cause balance disorders and is essentially irreversible due to the inability of mammalian vestibular organs to regenerate physiologically active hair cells. Here, we show substantial regeneration of hair cells in a mouse model of vestibular damage by treatment with a combination of glycogen synthase kinase 3β and histone deacetylase inhibitors. The drugs stimulated supporting cell proliferation and differentiation into hair cells. The new hair cells were reinnervated by vestibular afferent neurons, rescuing otolith function by restoring head translation-evoked otolith afferent responses and vestibuloocular reflexes. Drugs that regenerate hair cells thus represent a potential therapeutic approach to the treatment of balance disorders.

Outer-hair-cell bundles are sensory organelles required for normal hearing in mammals. These bundles convert sound-induced forces into receptor currents. This conversion depends on the resting receptor current of each bundle, which increases when extracellular calcium is decreased to the physiological level. How extracellular calcium regulates the bundle’s resting state is not well understood. We propose a mechanism explaining how extracellular calcium can regulate the outer-hair-cell bundle’s resting state. Each bundle comprises filamentous stereocilia linked by gating springs that are attached to ion channels. Sound-induced forces deflect stereocilia, increasing and decreasing gating-spring tensions, opening and closing the ion channels, resulting in an oscillating receptor current. We hypothesize that decreasing extracellular calcium, decreases the heights of the shorter stereocilia, increasing resting gating-spring tensions, which increases the resting receptor current and decreases the bundle’s resting deflection. To determine the plausibility of this mechanism, we build a mathematical model of an outer-hair-cell bundle and calibrate the model using seven independent experimental observations. The calibrated model shows that the mechanism is quantitatively plausible and predicts that a decrease of only 10 nm in the heights of the shorter stereocilia when extracellular calcium is lowered is sufficient to explain the observed increase in the resting receptor current. The model predicts the values of nine parameters and makes several additional predictions.

Transmembrane channel-like protein 1 (TMC1) is a transmembrane protein forming mechano-electrical transduction (MET) channel, which transduces mechanical stimuli into electrical signals at the top of stereocilia of hair cells in the inner ear. As an unexpected phenomenon, we found that the cytosolic N-terminal (Nt) region of heterologously-expressed mouse TMC1 (mTMC1) was localized in nuclei of a small population of the transfected HEK293 cells. This raised the possibility that the Nt region of heterologously-expressed mTMC1 was cleaved and transported into the nucleus. To confirm the cleavage, we performed western blot analyses. The results revealed that at least a fragment of the Nt region was produced from heterologously-expressed mTMC1. Site-directed mutagenesis experiments identified amino acid residues which were required to produce the fragment. The accumulation of the heterologously-expressed Nt fragment into the nuclei depended on nuclear localization signals within the Nt region. Furthermore, a structural comparison showed a similarity between the Nt region of mTMC1 and basic region leucine zipper (bZIP) transcription factors. However, transcriptome analyses using a next-generation sequencer showed that the heterologously-expression of the Nt fragment of mTMC1 hardly altered expression levels of genes. Although it is still unknown what is the precise mechanism and the physiological significance of this cleavage, these results showed that the cytosolic Nt region of heterologously-expressed mTMC1 could be cleaved in HEK293 cells. Therefore, it should be taken into account that the cleavage of Nt region might influence the functional analysis of TMC1 by the heterologous-expression system using HEK293 cells.