Person:

Holt, Jeffrey

The hyperpolarization-activated, cyclic nucleotide–sensitive current, Ih, is present in vestibular hair cells and vestibular ganglion neurons, and is required for normal balance function. We sought to identify the molecular correlates and functional relevance of Ih in vestibular ganglion neurons. Ih is carried by channels consisting of homo- or heteromeric assemblies of four protein subunits from the Hcn gene family. The relative expression of Hcn1–4 mRNA was examined using a quantitative reverse transcription PCR (RT-PCR) screen. Hcn2 was the most highly expressed subunit in vestibular neuron cell bodies. Immunolocalization of HCN2 revealed robust expression in cell bodies of all vestibular ganglion neurons. To characterize Ih in vestibular neuron cell bodies and at hair cell–afferent synapses, we developed an intact, ex vivo preparation. We found robust physiological expression of Ih in 89% of cell bodies and 100% of calyx terminals. Ih was significantly larger in calyx terminals than in cell bodies; however, other biophysical characteristics were similar. Ih was absent in calyces lacking Hcn1 and Hcn2, but small Ih was still present in cell bodies, which suggests expression of an additional subunit, perhaps Hcn4. To determine the contributions of hair cell mechanotransduction and Ih to the firing patterns of calyx terminals, we recorded action potentials in current-clamp mode. Mechanotransduction currents were modulated by hair bundle defection and application of calcium chelators to disrupt tip links. Ih activity was modulated using ZD7288 and cAMP. We found that both hair cell transduction and Ih contribute to the rate and regularity of spontaneous action potentials in the vestibular afferent neurons. We propose that modulation of Ih in vestibular ganglion neurons may provide a mechanism for modulation of spontaneous activity in the vestibular periphery.

Information flow through neural circuits is determined by the nature of the synapses linking the subtypes of neurons. How neurons acquire features distinct to each synapse remains unknown. We show that the transcription factor Mafb drives the formation of auditory ribbon synapses, which are specialized for rapid transmission from hair cells to spiral ganglion neurons (SGNs). Mafb acts in SGNs to drive differentiation of the large postsynaptic density (PSD) characteristic of the ribbon synapse. In Mafb mutant mice, SGNs fail to develop normal PSDs, leading to reduced synapse number and impaired auditory responses. Conversely, increased Mafb accelerates synaptogenesis. Moreover, Mafb is responsible for executing one branch of the SGN differentiation program orchestrated by the Gata3 transcriptional network. Remarkably, restoration of Mafb rescues the synapse defect in Gata3 mutants. Hence, Mafb is a powerful regulator of cell-type specific features of auditory synaptogenesis that offers a new entry point for treating hearing loss. DOI: http://dx.doi.org/10.7554/eLife.01341.001

The hyperpolarization-activated current, Ih, is carried by members of the Hcn channel family and contributes to resting potential and firing properties in excitable cells of various systems, including the auditory system. Ih has been identified in spiral ganglion neurons (SGNs); however, its molecular correlates and their functional contributions have not been well characterized. To investigate the molecular composition of the channels that carry Ih in SGNs, we examined Hcn mRNA harvested from spiral ganglia of neonatal and adult mice using quantitative RT-PCR. The data indicate expression of Hcn1, Hcn2, and Hcn4 subunits in SGNs, with Hcn1 being the most highly expressed at both stages. To investigate the functional contributions of HCN subunits, we used the whole-cell, tight-seal technique to record from wild-type SGNs and those deficient in Hcn1, Hcn2, or both. We found that HCN1 is the most prominent subunit contributing to Ih in SGNs. Deletion of Hcn1 resulted in reduced conductance (Gh), slower activation kinetics (τfast), and hyperpolarized half-activation (V1/2) potentials. We demonstrate that Ih contributes to SGN function with depolarized resting potentials, depolarized sag and rebound potentials, accelerated rebound spikes after hyperpolarization, and minimized jitter in spike latency for small depolarizing stimuli. Auditory brainstem responses of Hcn1-deficient mice showed longer latencies, suggesting that HCN1-mediated Ih is critical for synchronized spike timing in SGNs. Together, our data indicate that Ih contributes to SGN membrane properties and plays a role in temporal aspects of signal transmission between the cochlea and the brain, which are critical for normal auditory function.

Inner ear sensory epithelia contain mechanosensitive hair cells that transmit information to the brain through innervation with bipolar neurons. Mammalian hair cells do not regenerate and are limited in number. Here we investigate the potential to generate mechanosensitive hair cells from mouse embryonic stem cells in a three-dimensional (3D) culture system. The system faithfully recapitulates mouse inner ear induction followed by self-guided development into organoids that morphologically resemble inner ear vestibular organs. We find that organoid hair cells acquire mechanosensitivity equivalent to functionally mature hair cells in postnatal mice. The organoid hair cells also progress through a similar dynamic developmental pattern of ion channel expression, reminiscent of two subtypes of native vestibular hair cells. We conclude that our 3D culture system can generate large numbers of fully functional sensory cells which could be used to investigate mechanisms of inner ear development and disease as well as regenerative mechanisms for inner ear repair.

Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities1, 2. Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector3, for transgene delivery to the mouse cochlea. Cochlear explants incubated with Anc80L65 encoding eGFP demonstrated high level transduction of inner and outer hair cells (60–100%). Injection of Anc80L65 through the round window membrane resulted in highly efficient transduction of inner and outer hair cells, a substantial improvement over conventional adeno-associated virus (AAV) vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.

Because there are currently no biological treatments for deafness, we sought to advance gene therapy approaches to treat genetic deafness. We reasoned that gene delivery systems that target auditory and vestibular sensory cells with high efficiency would be required to restore complex auditory and balance function. We focused on Usher Syndrome, a devastating genetic disorder that causes blindness, balance disorders and profound deafness, and used a knock-in mouse model, Ush1c c.216G>A, which carries a cryptic splice site mutation found in French-Acadian patients with Usher Syndrome type IC (USH1C). Following delivery of wild-type Ush1c into the inner ears of neonatal Ush1c c.216G>A mice, we find recovery of gene and protein expression, restoration of sensory cell function, rescue of complex auditory function and recovery of hearing and balance behavior to near wild-type levels. The data represent unprecedented recovery of inner ear function and suggest that biological therapies to treat deafness may be suitable for translation to humans with genetic inner ear disorders.

Although genetic factors contribute to almost half of all deafness cases, treatment options for genetic deafness are limited1–5. We developed a genome editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9:guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated in vitro and in primary fibroblasts genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like 1) Beethoven (Bth) mouse model, even though the mutant Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9:guide RNA:lipid complexes targeting the Bth allele into the cochlea of neonatal Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response (ABR) thresholds in injected ears compared with uninjected ears or ears injected with complexes that target an unrelated gene. Enhanced acoustic reflex responses were observed among injected compared to uninjected Bth/+ animals. These findings suggest protein:RNA complex delivery of target gene-disrupting agents in vivo as a potential strategy for the treatment of some autosomal dominant hearing loss diseases.

Human inner ear tissue derived from pluripotent stem cells could provide a powerful platform for drug discovery or a source of sound- or motion-sensing cells for patients with hearing loss or balance dysfunction. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and Wnt signaling to generate multiple otic vesicle–like structures from a single stem-cell aggregate. Over two months, the vesicles develop into inner ear organoids with sensory epithelia that are innervated by sensory neurons. Additionally, using CRISPR/Cas9, we generate an ATOH1-2A-eGFP cell line to detect hair cell induction and demonstrate that derived hair cells exhibit electrophysiological properties similar to those of native sensory hair cells. Our culture system will be useful for elucidating mechanisms of human inner ear development and testing potential inner ear therapies.

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE–PACE) to improve their editing efficiency and target sequence compatibility. We used BE–PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.