Person:

Stankovic, Konstantina

Fibroblast growth factor 23 (FGF23) is a circulating hormone important in phosphate homeostasis. Abnormal serum levels of FGF23 result in systemic pathologies in humans and mice, including renal phosphate wasting diseases and hyperphosphatemia. We sought to uncover the role FGF23 plays in the auditory system due to shared molecular mechanisms and genetic pathways between ear and kidney development, the critical roles multiple FGFs play in auditory development and the known hearing phenotype in mice deficient in klotho (KL), a critical co-factor for FGF23 signaling. Using functional assessments of hearing, we demonstrate that Fgf mice are profoundly deaf. Fgf mice have moderate hearing loss above 20 kHz, consistent with mixed conductive and sensorineural pathology of both middle and inner ear origin. Histology and high-voltage X-ray computed tomography of Fgf mice demonstrate dysplastic bulla and ossicles; Fgf mice have near-normal morphology. The cochleae of mutant mice appear nearly normal on gross and microscopic inspection. In wild type mice, FGF23 is ubiquitously expressed throughout the cochlea. Measurements from Fgf mice do not match the auditory phenotype of Kl−/− mice, suggesting that loss of FGF23 activity impacts the auditory system via mechanisms at least partially independent of KL. Given the extensive middle ear malformations and the overlap of initiation of FGF23 activity and Eustachian tube development, this work suggests a possible role for FGF23 in otitis media.

Vestibular schwannoma (VS) is an intracranial tumor that causes significant morbidity, including hearing loss, tinnitus, dizziness, and possibly even death from brainstem compression. However, FDA-approved pharmacologic treatments for VS do not exist. Sulforaphane (SFN) is a naturally occurring isothiocyanate found in cruciferous vegetables, such as broccoli, with potent chemoprotective effects in several cell types. Our objective was to determine whether SFN is effective against VS in vitro and in vivo. Human primary VS cells, HEI-193 schwannoma cells, and SC4 Nf2−/− Schwann cells were used to investigate the inhibitory effects of SFN in vitro. Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation, and cell viability and metabolic activity was calculated by MTT assay. Apoptosis was measured by flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and Western blot for cleaved caspases. A mouse model with a murine schwannoma allograft was also used to examine the antitumor activity of SFN. SFN exhibited significant antiproliferative activity in schwannoma cells in vitro, via the inhibition of HDAC activity and the activation of ERK. SFN treatment induced apoptosis and cell cycle arrest at the G2/M phase. SFN also significantly inhibited schwannoma growth in vivo. Our preclinical studies motivate a future prospective clinical study of SFN for the treatment of VS.

Osteoprotegerin (OPG) is a key regulator of bone remodeling. Mutations in OPG are involved in a variety of human diseases. We have shown that cochlear spiral ganglion cells secrete OPG at high levels and lack of OPG causes sensorineural hearing loss in addition to the previously described conductive hearing loss. In order to study the regulation of OPG expression, we conducted a database search on regulatory elements in the promoter region of the OPG gene, and identified two potential GATA-3 binding sites. Using luciferase assays and site directed mutagenesis, we demonstrate that these two elements are GATA-3 responsive and support GATA-3 transactivation in human HEK and HeLa cells. The expression of wild type GATA-3 activated OPG mRNA and protein expression, while the expression of a dominant negative mutant of GATA-3 or a GATA-3 shRNA construct reduced OPG mRNA and protein levels. GATA-3 deficient cells generated by expressing a GATA-3 shRNA construct were sensitive to apoptosis induced by etoposide and TNF-α. This apoptotic effect could be partly prevented by the co-treatment with exogenous OPG. Our results suggest new approaches to rescue diseases due to GATA-3 deficiency – such as in hypoparathyroidism, sensorineural deafness, and renal (HDR) syndrome – by OPG therapy.

Summary Tumor necrosis factor (TNF) family cytokines are important mediators of inflammation. Elevated levels of serum TNF‐α are associated with human sensorineural hearing loss via poorly understood mechanisms. We demonstrate, for the first time, expression of TNF‐related apoptosis‐inducing ligand (TRAIL) and its signaling death receptor 5 (DR5) in the murine inner ear and show that exogenous TRAIL can trigger hair cell and neuronal degeneration, which can be partly prevented with DR5‐blocking antibodies.

Vestibular schwannomas (VSs) are the most common tumours of the cerebellopontine angle. Ninety-five percent of people with VS present with sensorineural hearing loss (SNHL); the mechanism of this SNHL is currently unknown. To establish the first model to study the role of VS-secreted factors in causing SNHL, murine cochlear explant cultures were treated with human tumour secretions from thirteen different unilateral, sporadic VSs of subjects demonstrating varied degrees of ipsilateral SNHL. The extent of cochlear explant damage due to secretion application roughly correlated with the subjects’ degree of SNHL. Secretions from tumours associated with most substantial SNHL resulted in most significant hair cell loss and neuronal fibre disorganization. Secretions from VSs associated with good hearing or from healthy human nerves led to either no effect or solely fibre disorganization. Our results are the first to demonstrate that secreted factors from VSs can lead to cochlear damage. Further, we identified tumour necrosis factor alpha (TNFα) as an ototoxic molecule and fibroblast growth factor 2 (FGF2) as an otoprotective molecule in VS secretions. Antibody-mediated TNFα neutralization in VS secretions partially prevented hair cell loss due to the secretions. Taken together, we have identified a new mechanism responsible for SNHL due to VSs.

The mammalian cochlea has historically resisted attempts at high-resolution, non-invasive imaging due to its small size, complex three-dimensional structure, and embedded location within the temporal bone. As a result, little is known about the relationship between an individual’s cochlear pathology and hearing function, and otologists must rely on physiological testing and imaging methods that offer limited resolution to obtain information about the inner ear prior to performing surgery. Micro-optical coherence tomography (μOCT) is a non-invasive, low-coherence interferometric imaging technique capable of resolving cellular-level anatomic structures. To determine whether μOCT is capable of resolving mammalian intracochlear anatomy, fixed guinea pig inner ears were imaged as whole temporal bones with cochlea in situ. Anatomical structures such as the tunnel of Corti, space of Nuel, modiolus, scalae, and cell groupings were visualized, in addition to individual cell types such as neuronal fibers, hair cells, and supporting cells. Visualization of these structures, via volumetrically-reconstructed image stacks and endoscopic perspective videos, represents an improvement over previous efforts using conventional OCT. These are the first μOCT images of mammalian cochlear anatomy, and they demonstrate μOCT’s potential utility as an imaging tool in otology research.

Moderate acoustic overexposure in adult rodents is known to cause acute loss of synapses on sensory inner hair cells (IHCs) and delayed degeneration of the auditory nerve, despite the completely reversible temporary threshold shift (TTS) and morphologically intact hair cells. Our objective was to determine whether a cochlear synaptopathy followed by neuropathy occurs after noise exposure in pubescence, and to define neuropathic versus non-neuropathic noise levels for pubescent mice. While exposing 6 week old CBA/CaJ mice to 8-16 kHz bandpass noise for 2 hrs, we defined 97 dB sound pressure level (SPL) as the threshold for this particular type of neuropathic exposure associated with TTS, and 94 dB SPL as the highest non-neuropathic noise level associated with TTS. Exposure to 100 dB SPL caused permanent threshold shift although exposure of 16 week old mice to the same noise is reported to cause only TTS. Amplitude of wave I of the auditory brainstem response, which reflects the summed activity of the cochlear nerve, was complemented by synaptic ribbon counts in IHCs using confocal microscopy, and by stereological counts of peripheral axons and cell bodies of the cochlear nerve from 24 hours to 16 months post exposure. Mice exposed to neuropathic noise demonstrated immediate cochlear synaptopathy by 24 hours post exposure, and delayed neurodegeneration characterized by axonal retraction at 8 months, and spiral ganglion cell loss at 8-16 months post exposure. Although the damage was initially limited to the cochlear base, it progressed to also involve the cochlear apex by 8 months post exposure. Our data demonstrate a fine line between neuropathic and non-neuropathic noise levels associated with TTS in the pubescent cochlea.

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.

The cochlear implant (CI) is the most successful neural prosthesis, restoring the sensation of sound in people with severe-to-profound hearing loss by electrically stimulating the cochlear nerve. Existing CIs have an external, visible unit, and an internal, surgically-placed unit. There are significant challenges associated with the external unit, as it has limited utility and CI users often report a social stigma associated with prosthesis visibility. A fully-implantable CI (FICI) would address these issues. However, the volume constraint imposed on the FICI requires less power consumption compared to today’s CI. Because neural stimulation by CI electrodes accounts for up to 90% of power consumption, reduction in stimulation power will result directly in CI power savings. To determine an energy-efficient waveform for cochlear nerve stimulation, we used a genetic algorithm approach, incorporating a computational model of a single mammalian myelinated cochlear nerve fiber coupled to a stimulator-electrode-tissue interface. The algorithm’s prediction was tested in vivo in human CI subjects. We find that implementation of a non-rectangular biphasic neural stimulation waveform may result in up to 25% charge savings and energy savings within the comfortable range of hearing for CI users. The alternative waveform may enable future development of a FICI.

Vestibular schwannoma (VS) is the most common tumor of the cerebellopontine angle, and it typically presents with sensorineural hearing loss. The genomic landscape of schwannoma is complex and many of the molecules implicated in VS pathogenesis represent targets not amenable to antibody-based or small molecule therapeutics. Tumor-targeted delivery of small interfering RNA (siRNA) therapeutics provides a direct and effective means to interrogate targets while minimizing off-target effects. To establish a preclinical model for therapeutic inhibition of putative targets in VS, archived tumor specimens, fresh tumor cells derived from patients with sporadic VS, and an established schwannoma cell line were screened. Nanoparticles directed by the tumor-homing peptide iRGD were selectively taken up by primary VS cultures in vitro via interactions with αvβ3/β5 integrins and neuropilin-1 (NRP-1). Cellular uptake was inhibited by a neutralizing antibody against αv integrin in a dose-dependent manner. When applied to primary VS cultures, iRGD-targeted nanoparticles delivered siRNA directed against TNFα in a receptor-specific fashion to potently silence gene expression and protein secretion. Taken together, our results provide a proof of principle for tumor-targeted, nanoparticle-mediated delivery of siRNA to VS and establish a novel platform for the development and pre-clinical screening of molecular therapeutics against VS.