Cutting‐Edge Advancements in Physical Stimulation for Spiral Ganglion Neuron Protection and Regeneration

Hearing impairment, the most prevalent sensory deficit, affects more than 466 million people worldwide (WHO). We presently lack causative treatment for the most common form, sensorineural hearing impairment; hearing aids and cochlear implants (CI) remain the only means of hearing restoration. We engaged with CI users to learn about their expectations and their willingness to collaborate with health care professionals on establishing novel therapies. We summarize upcoming CI innovations, gene therapies, and regenerative approaches and evaluate the chances for clinical translation of these novel strategies. We conclude that there remains an unmet medical need for improving hearing restoration and that we are likely to witness the clinical translation of gene therapy and major CI innovations within this decade.

Aminoglycoside-Induced Degeneration of Adult Spiral Ganglion Neurons Involves Differential Modulation of Tyrosine Kinase B and p75 Neurotrophin Receptor Signaling

Neurotrophins and electrical stimulation for protection and repair of spiral ganglion neurons following sensorineural hearing loss

Spiral ganglion neurons (SGNs) are auditory neurons that relay sound signals from the inner ear to the brainstem. The ototoxic drug cisplatin can damage SGNs and thus lead to sensorineural hearing loss (SNHL), and there are currently no methods for preventing or treating this. Macroautophagy/autophagy plays a critical role in SGN development, but the effect of autophagy on cisplatin-induced SGN injury is unclear. Here, we first found that autophagic flux was activated in SGNs after cisplatin damage. The SGN apoptosis and related hearing loss induced by cisplatin were alleviated after co-treatment with the autophagy activator rapamycin, whereas these were exacerbated by the autophagy inhibitor 3-methyladenine, indicating that instead of inducing SGN death, autophagy played a neuroprotective role in SGNs treated with cisplatin both in vitro and in vivo. We further demonstrated that autophagy attenuated reactive oxygen species (ROS) accumulation and alleviated cisplatin-induced oxidative stress in SGNs to mediate its protective effects. Notably, the role of the antioxidant enzyme PRDX1 (peroxiredoxin 1) in modulating autophagy in SGNs was first identified. Deficiency in PRDX1 suppressed autophagy and increased SGN loss after cisplatin exposure, while upregulating PRDX1 pharmacologically or by adeno-associated virus activated autophagy and thus inhibited ROS accumulation and apoptosis and attenuated SGN loss induced by cisplatin. Finally, we showed that the underlying mechanism through which PRDX1 triggers autophagy in SGNs was, at least partially, through activation of the PTEN-AKT signaling pathway. These findings suggest potential therapeutic targets for the amelioration of drug-induced SNHL through autophagy activation. Abbreviations: 3-MA: 3-methyladenine; AAV : adeno-associated virus; ABR: auditory brainstem responses; AKT/protein kinase B: thymoma viral proto-oncogene; Baf: bafilomycin A1; CAP: compound action potential; COX4I1: cytochrome c oxidase subunit 4I1; Cys: cysteine; ER: endoplasmic reticulum; H2O2: hydrogen peroxide; HC: hair cell; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; NAC: N-acetylcysteine; PRDX1: peroxiredoxin 1; PTEN: phosphatase and tensin homolog; RAP: rapamycin; ROS: reactive oxygen species; SGNs: spiral ganglion neurons; SNHL: sensorineural hearing loss; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling; WT: wild type.

Cisplatin can damage spiral ganglion neurons (SGNs) and cause sensorineural hearing loss. Wnt activation protects against neomycin-induced hair cell damage in the mouse cochlea, but the role of Wnt signaling in protecting SGNs from cisplatin treatment has not yet been elucidated. This study was designed to investigate the neuroprotective effects of Wnt signaling against cisplatin-induced SGN damage. First, we found that Wnt signaling was activated in SGNs after cisplatin treatment. Next, we discovered that overexpression (OE) of Wnt signaling in SGNs reduced cisplatin-induced SGN loss by inhibiting caspase-associated apoptosis, thus preventing the loss of SGN function after cisplatin treatment. In contrast, inhibition of Wnt signaling increased apoptosis, made SGNs more vulnerable to cisplatin treatment, and exacerbated hearing loss. TP53-induced glycolysis and apoptosis regulator (TIGAR), which scavenges intracellular reactive oxygen species (ROS), was upregulated in SGNs in response to cisplatin administration. Wnt/β-catenin activation increased TIGAR expression and reduced ROS level, while inhibition of Wnt/β-catenin in SGNs reduced TIGAR expression and increased the ROS level. Moreover, OE of TIGAR reduced ROS and decreased caspase 3 expression, as well as increased the survival of SGNs in Wnt-inhibited SGNs. Finally, antioxidant treatment rescued the more severe SGN loss induced by β-catenin deficiency after cisplatin treatment. Innovation and Conclusion: This study is the first to indicate that Wnt signaling activates TIGAR and protects SGNs against cisplatin-induced damage through the inhibition of oxidative stress and apoptosis in SGNs, and this might offer novel therapeutic targets for the prevention of SGN injury. Antioxid. Redox Signal. 00, 000-000.

Sensorineural hearing loss is irreversible and is associated with the loss of spiral ganglion neurons (SGNs) and sensory hair cells within the inner ear. Improving spiral ganglion neuron (SGN) survival, neurite outgrowth, and synaptogenesis could lead to significant gains for hearing-impaired patients. There has therefore been intense interest in the use of neurotrophic factors in the inner ear to promote both survival of SGNs and re-wiring of sensory hair cells by surviving SGNs. Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) represent the primary neurotrophins in the inner ear during development and throughout adulthood, and have demonstrated potential for SGN survival and neurite outgrowth. We have pioneered a hybrid molecule approach to maximize SGN stimulation in vivo, in which small molecule analogues of neurotrophins are linked to bisphosphonates, which in turn bind to cochlear bone. We have previously shown that a small molecule BDNF analogue coupled to risedronate binds to bone matrix and promotes SGN neurite outgrowth and synaptogenesis in vitro. Because NT-3 has been shown in a variety of contexts to have a greater regenerative capacity in the cochlea than BDNF, we sought to develop a similar approach for NT-3. 1Aa is a small molecule analogue of NT-3 that has been shown to activate cells through TrkC, the NT-3 receptor, although its activity on SGNs has not previously been described. Herein we describe the design and synthesis of 1Aa and a covalent conjugate of 1Aa with risedronate, Ris-1Aa. We demonstrate that both 1Aa and Ris-1Aa stimulate neurite outgrowth in SGN cultures at a significantly higher level compared to controls. Ris-1Aa maintained its neurotrophic activity when bound to hydroxyapatite, the primary mineral component of bone. Both 1Aa and Ris-1Aa promote significant synaptic regeneration in cochlear explant cultures, and both 1Aa and Ris-1Aa appear to act at least partly through TrkC. Our results provide the first evidence that a small molecule analogue of NT-3 can stimulate SGNs and promote regeneration of synapses between SGNs and inner hair cells. Our findings support the promise of hydroxyapatite-targeting bisphosphonate conjugation as a novel strategy to deliver neurotrophic agents to SGNs encased within cochlear bone.

It has been reported that paclitaxel administration could cause sensorineural hearing loss, and Wnt activation is important for the development and cell protection of mouse cochlea. However, the effect of Wnt signaling in spiral ganglion neurons (SGNs) damage induced by paclitaxel has not yet been elucidated. In this study, we explored the effect of paclitaxel on SGNs in the mouse cochlea and the neuroprotective effects of Wnt signaling pathway against paclitaxel-induced SGN damage by using Wnt agonist/antagonists in vitro. We first found that paclitaxel treatment resulted in a degenerative change and reduction of cell numbers in SGNs and induced caspase-mediated apoptosis in SGNs. The expression levels of β-catenin and C-myc were increased, thus indicating Wnt signaling was activated in SGNs after paclitaxel treatment. The activation of Wnt signaling pathway protected against SGN loss after exposure to paclitaxel, whereas the suppression of Wnt signaling in SGNs made them more vulnerable to paclitaxel treatment. We also showed that activation of Wnt signaling in SGNs inhibited caspase-mediated apoptosis. Our findings demonstrated that Wnt signaling had an important role in protecting SGNs against paclitaxel-induced damage and thus might be an effective therapeutic target for the prevention of paclitaxel-induced SGN death.

Release of neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF) from hair cells in the cochlea is essential for the survival of spiral ganglion neurons (SGNs). Loss of hair cells associated with a sensorineural hearing loss therefore results in degeneration of SGNs, potentially reducing the performance of a cochlear implant. Exogenous replacement of either or both neurotrophins protects SGNs from degeneration after deafness. We previously incorporated NT3 into the conducting polymer polypyrrole (Ppy) synthesized with para-toluene sulfonate (pTS) to investigate whether Ppy/pTS/NT3-coated cochlear implant electrodes could provide both neurotrophic support and electrical stimulation for SGNs. Enhanced and controlled release of NT3 was achieved when Ppy/pTS/NT3-coated electrodes were subjected to electrical stimulation. Here we describe the release dynamics and biological properties of Ppy/pTS with incorporated BDNF. Release studies demonstrated slow passive diffusion of BDNF from Ppy/pTS/BDNF, with electrical stimulation significantly enhancing BDNF release over 7 days. A 3-day SGN explant assay found that neurite outgrowth from explants was 12.3-fold greater when polymers contained BDNF (p < 0.001), although electrical stimulation did not increase neurite outgrowth further. The versatility of Ppy to store and release neurotrophins, conduct electrical charge, and act as a substrate for nerve-electrode interactions is discussed for specialized applications such as cochlear implants.

F021 Myographic recording of the electrically elicited stapedius reflex

Does cochlear implantation and electrical stimulation affect residual hair cells and spiral ganglion neurons?

Sensorineural hearing loss (SNHL) is caused by the loss of sensory hair cells (HCs) and/or connected spiral ganglion neurons (SGNs). The current clinical conventional treatment for SNHL is cochlear implantation (CI). The principle of CI is to bypass degenerated auditory HCs and directly electrically stimulate SGNs to restore hearing. However, the effectiveness of CI is limited when SGNs are severely damaged. In the present study, oriented nanofiber scaffolds were fabricated using electrospinning technology to mimic the SGN spatial microenvironment in the inner ear. Meanwhile, different proportions of polyaniline (PANI), poly-l-lactide (PLLA), gelatin (Gel) were composited to mimic the composition and mechanical properties of auditory basement membrane. The effects of oriented PANI/PLLA/Gel biomimetic nanofiber scaffolds for neurite outgrowth were analyzed. The results showed the SGNs grew in an orientation along the fiber direction, and the length of the protrusions increased significantly on PANI/PLLA/Gel scaffold groups. The 2% PANI/PLLA/Gel group showed best effects for promoting SGN adhesion and nerve fiber extension. In conclusion, the biomimetic oriented nanofiber scaffolds can simulate the microenvironment of SGNs as well as promote neurite outgrowth in vitro, which may provide a feasible research idea for SGN regeneration and even therapeutic treatments of SNHL in future.

Sensorineural hearing loss is a major public health problem affecting more than 278 million people worldwide. The primary cause of sensorineural hearing loss is loss or damage of sensory hair cells in the organ of Corti. However, approximately 10-15% of cases with profound hearing loss in children are caused by degeneration of the spiral ganglion neurons (SGNs) or neurons in the auditory brainstem. Moreover, SGNs gradually degenerate after the loss of hair cells due to a lack of excitatory stimulation. Since SGNs do not regenerate to any clinically significant extent, novel therapies for their preservation, regeneration or replacement are being heavily sought. Currently, no treatment option is available for peripheral auditory neuropathy. Cell-based therapies offer a strategy to enhance auditory functions in the deaf patient and improve the benefits of cochlear implantation. There are three major areas for potential clinical applications relevant to this approach. First, for patients who have received cochlear implants, generation or preservation of SGNs via cell replacement therapy could significantly improve the quality of their sound perception. Another group of potential recipients who would benefit from cell replacement therapy are patients suffering from acoustic neuroma or neurofibromatosis. These patients generally exhibit significant loss of auditory or vestibular primary neurons with relatively intact sensory hair cells (Kaga et al., 1997; Evans et al., 2000; Sperfeld et al., 2002). Thus, replacing dead or damaged neurons with stem cells could be critical in restoring their hearing or balance sensation. Finally, auditory neurons generated from stem cells could be used in in vitro assays to test the effectiveness and safety of newly developed drugs before clinical trials. Type I SGNs, comprising 95% of all neural populations in the SG, innervate inner hair cells in the organ of Corti and function as the primary auditory afferent neurons (Berglund and Ryugo, 1987; Liberman et al., 1990; Rusznak and Szucs, 2009). These SGNs predominantly express AMPA receptors (mainly GluR2-4) (Niedzielski and Wenthold, 1995; Parks, 2000; Dulon et al., 2006; Chen et al., 2007; Flores-Otero et al., 2007), which bind the neurotransmitter glutamate released from inner hair cells (Fig. 1). This subsequently triggers action potentials that propagate along the nerve fibers to the cochlear nucleus. The majority, if not all, of type I SGNs are glutamatergic and release glutamate from their presynaptic membrane in the cochlear nucleus (Rebillard et al., 2003; Reyes et al., 2008). Expression of AMPA receptors and glutamate transporters thus is the hallmark of mature type I SGNs. During embryonic development, SGNs arise from the otic placode and

Sensorineural hearing loss (SNHL) seriously affects people's lives, and the degradation of spiral ganglion neurons (SGNs) is the main cause of SNHL. Many studies have revealed that long non-coding RNAs (lncRNAs) are linked to SGNs. However, it is still unclear how lncRNAs affect damage repair in SGNs. Fluorescence in situ hybridization (FISH) and nuclear-cytoplasmic separation (NCS) assays were used to verify the cellular localization of lncRNA-HOTAIR in SGNs. Primary SGNs were dissected from the C57BL/6J male mice. We constructed sh-HOTAIR SGNs to investigate the role of HOTAIR. Target gene levels were analyzed using qRT-PCR and Western blot assays. Additionally, SGN proliferation was assessed using the CCK-8 and flow cytometry assays. Moreover, RIP and dual-luciferase assays were conducted to elucidate the interactions among HOTAIR, miRNA, and mRNA. Our findings illustrated that HOTAIR was primarily expressed in the cytoplasm, and downregulation of HOTAIR increased SGN apoptosis by approximately 30% (P < 0.001). In addition, miR-211-5p was identified as being directly downstream of HOTAIR, which could bind with miR-211-5p to modulate SGNs growt (all P < 0.05). Furthermore, miR-211-5p reduced the proliferation of SGNs (P < 0.001) and increased apoptosis (P < 0.01) by binding to the 3'-UTR of EYA3, whereas overexpression of EYA3 reversed this result (all P < 0.05). Our findings suggest that HOTAIR promotes SGN proliferation by competitively binding to miR-211-5p and regulating EYA3 expression, highlighting its potential as a novel target for sensorineural hearing loss therapies.

Background Cochlear implant-based electrical stimulation may be an important reason to induce the residual hearing loss after cochlear implantation. In our previous study, we found that charge-balanced biphasic electrical stimulation inhibited the neurite growth of spiral ganglion neurons (SGNs) and decreased Schwann cell density in vitro. In this study, we want to know whether cochlear implant-based electrical stimulation can induce the change of electrical activity in cultured SGNs. Methods Spiral ganglion neuron electrical stimulation in vitro model is established using the devices delivering cochlear implant-based electrical stimulation. After 48 h treatment by 50 μA or 100 μA electrical stimulation, the action potential (AP) and voltage depended calcium current (ICa) of SGNs are recorded using whole-cell electrophysiological method. Results The results show that the ICa of SGNs is decreased significantly in 50 μA and 100 μA electrical stimulation groups. The reversal potential of ICa is nearly +80 mV in control SGN, but the reversal potential decreases to +50 mV in 50 μA and 100 μA electrical stimulation groups. Interestingly, the AP amplitude, the AP latency, and the AP duration of SGNs have no statistically significant differences in all three groups. Conclusion Our study suggests cochlear implant-based electrical stimulation only significantly inhibit the ICa of cultured SGNs but has no effect on the firing of AP, and the relation of ICa inhibition and SGN damage induced by electrical stimulation and its mechanism needs to be further studied.

There are millions of people who are affected from sensorineural hearing loss. In this type of deafness, sensory hair cells are typically dysfunctional or lost, and thereby not conveying sound information to brain. The state-of-the-art solution is the cochlear implant (CI) which circumvents the receptor problem via direct electric stimulation of spiral ganglion neurons (SGNs) of that from auditory nerve (AN). Thus, the CI enables hearing and provides open speech comprehension. Yet, for instance in noisy background, in most users, speech comprehension fails likely owing to poor frequency resolution. This results from wide current spread in the the saline-filled cochlea activating large sets of the tonotopically organized SGNs. Here, light can serve as an alternative mode of stimulation since it can be conveniently focused to evoke SGN populations more precisely. Clearly, SGNs are not light sensitive and hence we employ an optogenetic approach. The series of studies presented here gathers under the same roof: How to achieve optogenetic manipulation of the SGNs such that it will suit well to the intrinsic physiological properties of auditory system, be stably maintained for many years, and last but not least be safe? Temporal firing properties of SGNs are uniquely fast allowing fine coding of sound. Therefore, channelrhodopsins (ChRs) used should enable reliable control of light-induced spiking in SGNs. To this end, first, we investigated feasibility of blue-light-gated naturally occurring Chronos with the fastest on-/off-kinetics at the mouse AN with the help of molecular tools, i.e. endoplasmic reticulum exit signal (ES) and membrane trafficking signal (TS), enhancing membrane localization of the ChR. By this way, it was possible to drive the AN up to 1000 Hz stimulation rate. While Chronos suits temporal coding very well, we also sought for red-shifted fast alternatives since chronic exposure to blue light bears risk of phototoxicity in SGNs. Here, the engineered red-shifted very-fast-Chrimson (vf-Chrimson-ES/TS) which has similar fast kinetics as fast as Chronos-ES/TS, evoked fast responses until 500 Hz stimulation rate from the mouse AN. Moreover, we further characterized light intensity encoding of vf-Chrimson-ES/TS in comparison to the slower variant fast-Chrimson (f-Chrimson). Third, we studied the long-term availability and biosafety profile of f-Chrimson, a prominent candidate for use with the optical CI, showing expression in SGNs up to two years following early postnatal AAV-based delivery to the inner ear. There was also a tendency of SGN loss due to combinatorial effect of aging and f-Chrimson expression. Expression spread to the non-injection ear and parts of the brain. The spread was restricted to the vicinity of injection and was not detected in the distal organs such as kidney and spleen. All in all, these three projects that I was involved in at different extents pave the way to future medical optogenetic cochlear implants.