Wnt Signaling Protects against Paclitaxel-Induced Spiral Ganglion Neuron Damage in the Mouse Cochlea In Vitro.
ABSTRACT: 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.
Project description:Mammalian Sperm Associated Antigen 6 (SPAG6) is the orthologue of Chlamydomonas PF16, a protein localized in the axoneme central apparatus. Recent studies showed that Spag6 has a role in brain neuronal proliferation and differentiation. The mammalian spiral ganglion neurons (SGNs) are specialzed bipolar neurons in the inner ear. However, the role of SPAG6 in SGN has not been elucidated. Therefore, We hypothesized that a Spag6 knockout would affect the development and function of SGNs. We utilized Spag6-deficient mice and SGN explants to define the role of SPAG6. On postnatal day 30 (P30) mutant mice had lower SGN density compared to their wild-type littermates, and more apoptosis was evident in the mutants. Increased Bax expression, a disturbed distribution of cytochrome c, and cleaved caspase-3 positive staining indicated that increased apoptosis involved a mitochondrial pathway. Transmission electron microscopy revealed abnormalities in the ultrastructure of mutant SGNs as early as P7. In vitro, lack of SPAG6 affected the growth of neurites and growth cones. Additionally, SPAG6 deficiency decreased synapse density in SGN explants. Finally, Spag6 mutant SGNs were more sensitive to the microtubule stabilizing agent, paclitaxel. These findings suggest that Spag6 plays a crucial role in SGN development and function.
Project description:Application of ouabain to the intact round-window (RW) membrane of the gerbil cochlea induces apoptosis in most spiral ganglion neurons (SGNs), leaving a few neurons intact (Schmiedt et al. 2002). Here, physiological measures and immunostaining were used to examine the process of SGN degeneration at 3, 6, 12, and 24 h, 4 days, and 1 and 5 months after ouabain treatment. The few remaining neurons surviving up to 5 months after ouabain treatment were immunoreactive for peripherin, a type II neuron marker. Peripherin-positive cell counts indicate that about 7% of the SGNs in the gerbil cochlea are type II neurons, and these neurons survive intact after ouabain treatment. Ouabain exposure had little effect on the outer hair cell and lateral wall systems, even after a 5 month loss of auditory-nerve function. The cellular locations of cytochrome c, poly (ADP-ribose) polymerase (PARP), and activated caspase 3 were examined in control and ouabain-treated cochleas. A redistribution of cytochrome c in peripherin-negative (type I) neurons was observed at 3 h after ouabain exposure. Degraded PARP and activated caspase 3 were also detected in peripherin-negative SGNs at 6 and 24 h after treatment, respectively. These results suggest that the redistribution of cytochrome c is an early event during apoptosis in type I SGNs and that activation of PARP and caspase 3 are associated with apoptosis in these cells. Calcineurin and NF-kappaB are two important signaling pathways that may modulate cell survival in the central nervous system. Here, we found that calcineurin and NF-kappaB selectively labeled type II neurons. It is speculated that the high levels of calcineurin and NF-kappaB in type II SGNs, as compared with type I SGNs, may play protective roles in enhancing the survival of type II neurons exposed to ouabain.
Project description:Optogenetic stimulation of type I spiral ganglion neurons (SGNs) promises an alternative to the electrical stimulation by current cochlear implants (CIs) for improved hearing restoration by future optical CIs (oCIs). Most of the efforts in using optogenetic stimulation in the cochlea so far used early postnatal injection of viral vectors carrying blue-light activated channelrhodopsins (ChRs) into the cochlea of mice. However, preparing clinical translation of the oCI requires (<i>i</i>) reliable and safe transduction of mature SGNs of further species and (<i>ii</i>) use of long-wavelength light to avoid phototoxicity. Here, we employed a fast variant of the red-light activated channelrhodopsin Chrimson (f-Chrimson) and different AAV variants to implement optogenetic SGN stimulation in Mongolian gerbils. We compared early postnatal (p8) and adult (>8 weeks) AAV administration, employing different protocols for injection of AAV-PHP.B and AAV2/6 into the adult cochlea. Success of the optogenetic manipulation was analyzed by optically evoked auditory brainstem response (oABR) and immunohistochemistry of mid-modiolar cryosections of the cochlea. In order to most efficiently evaluate the immunohistochemical results a semi-automatic procedure to identify transduced cells in confocal images was developed. Our results indicate that the rate of SGN transduction is significantly lower for AAV administration into the adult cochlea compared to early postnatal injection. SGN transduction upon AAV administration into the adult cochlea was largely independent of the chosen viral vector and injection approach. The higher the rate of SGN transduction, the lower were oABR thresholds and the larger were oABR amplitudes. Our results highlight the need to optimize viral vectors and virus administration for efficient optogenetic manipulation of SGNs in the adult cochlea for successful clinical translation of SGN-targeting gene therapy and of the oCI.
Project description: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 <i>in vitro</i> and <i>in vivo</i>. 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.<b>Abbreviations</b>: 3-MA: 3-methyladenine; AAV : adeno-associated virus; ABR: auditory brainstem responses; AKT/protein kinase B: thymoma viral proto-oncogene; Baf: bafilomycin A<sub>1</sub>; CAP: compound action potential; COX4I1: cytochrome c oxidase subunit 4I1; Cys: cysteine; ER: endoplasmic reticulum; H<sub>2</sub>O<sub>2</sub>: 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.
Project description:Cochlear implants electrically stimulate spiral ganglion neurons (SGNs) in order to provide speech cues to severe-profoundly deaf patients. In normal hearing cochleae the SGNs depend on endogenous neurotrophins secreted by sensory cells in the organ of Corti for survival. SGNs gradually degenerate following deafness and consequently there is considerable interest in developing clinically relevant strategies to provide exogenous neurotrophins to preserve SGN survival. The present study investigated the safety and efficacy of a drug delivery system for the cochlea using nanoengineered silica supraparticles. In the present study we delivered Brain-derived neurotrophic factor (BDNF) over a period of four weeks and evaluated SGN survival as a measure of efficacy. Supraparticles were bilaterally implanted into the basal turn of cochleae in profoundly deafened guinea pigs. One ear received BDNF-loaded supraparticles and the other ear control (unloaded) supraparticles. After one month of treatment the cochleae were examined histologically. There was significantly greater survival of SGNs in cochleae that received BDNF supraparticles compared to the contralateral control cochleae (repeated measures ANOVA, p = 0.009). SGN survival was observed over a wide extent of the cochlea. The supraparticles were well tolerated within the cochlea with a tissue response that was localised to the site of implantation in the cochlear base. Although mild, the tissue response was significantly greater in cochleae treated with BDNF supraparticles compared to the controls (repeated measures ANOVA, p = 0.003). These data support the clinical potential of this technology particularly as the supraparticles can be loaded with a variety of therapeutic drugs.
Project description:During inner ear development, primary auditory neurons named spiral ganglion neurons (SGNs) are surrounded by otic mesenchyme cells, which express the transcription factor Pou3f4. Mutations in Pou3f4 are associated with DFNX2, the most common form of X-linked deafness and typically include developmental malformations of the middle ear and inner ear. It is known that interactions between Pou3f4-expressing mesenchyme cells and SGNs are important for proper axon bundling during development. However, Pou3f4 continues to be expressed through later phases of development, and potential interactions between Pou3f4 and SGNs during this period had not been explored. To address this, we documented Pou3f4 protein expression in the early postnatal mouse cochlea and compared SGNs in Pou3f4 knockout mice and littermate controls. In Pou3f4<sup>y/-</sup> mice, SGN density begins to decline by the end of the first postnatal week, with approximately 25% of SGNs ultimately lost. This period of SGN loss in Pou3f4<sup>y/-</sup> cochleae coincides with significant elevations in SGN apoptosis. Interestingly, this period also coincides with the presence of a transient population of Pou3f4-expressing cells around and within the spiral ganglion. To determine if Pou3f4 is normally required for SGN peripheral axon extension into the sensory domain, we used a genetic sparse labeling approach to track SGNs and found no differences compared with controls. We also found that Pou3f4 loss did not lead to changes in the proportions of Type I SGN subtypes. Overall, these data suggest that otic mesenchyme cells may play a role in maintaining SGN populations during the early postnatal period.
Project description:The mammalian cochlea undergoes a highly dynamic process of growth and innervation during development. This process includes spiral ganglion neuron (SGN) branch refinement, a process whereby Type I SGNs undergo a phase of "debranching" before forming unramified synaptic contacts with inner hair cells. Using Sox2 CreERT2 and R26R tdTomato as a strategy to genetically label individual SGNs in mice of both sexes, we report on both a time course of SGN branch refinement and a role for P2rx3 in this process. P2rx3 is an ionotropic ATP receptor that was recently implicated in outer hair cell spontaneous activity and Type II SGN synapse development (Ceriani et al., 2019), but its function in Type I SGN development is unknown. Here, we demonstrate that P2rx3 is expressed by Type I SGNs and hair cells during developmental periods that coincide with SGN branching refinement. P2rx3 null mice show SGNs with more complex branching patterns on their peripheral synaptic terminals and near their cell bodies around the time of birth. Loss of P2rx3 does not appear to confer general changes in axon outgrowth or hair cell formation, and alterations in branching complexity appear to mostly recover by postnatal day (P)6. However, when we examined the distribution of Type I SGN subtypes using antibodies that bind Calb2, Calb1, and Pou4f1, we found that P2rx3 null mice showed an increased proportion of SGNs that express Calb2. These data suggest P2rx3 may be necessary for normal Type I SGN differentiation in addition to serving a role in branch refinement.
Project description:Spontaneous bursts of activity in developing sensory pathways promote maturation of neurons, refinement of neuronal connections, and assembly of appropriate functional networks. In the developing auditory system, inner hair cells (IHCs) spontaneously fire Ca(2+) spikes, each of which is transformed into a mini-burst of action potentials in spiral ganglion neurons (SGNs). Here we show that NMDARs are expressed in SGN dendritic terminals and play a critical role during transmission of activity from IHCs to SGNs before hearing onset. NMDAR activation enhances glutamate-mediated Ca(2+) influx at dendritic terminals, promotes repetitive firing of individual SGNs in response to each synaptic event, and enhances coincident activity of neighboring SGNs that will eventually encode similar frequencies of sound. Loss of NMDAR signaling from SGNs reduced their survival both in vivo and in vitro, revealing that spontaneous activity in the prehearing cochlea promotes maturation of auditory circuitry through periodic activation of NMDARs in SGNs.
Project description:In the mammalian cochlea, spiral ganglion neurons (SGNs) relay the acoustic information to the central auditory circuits. Degeneration of SGNs is a major cause of sensorineural hearing loss and severely affects the effectiveness of cochlear implant therapy. Cochlear glial cells are able to form spheres and differentiate into neurons <i>in vitro</i>. However, the identity of these progenitor cells is elusive, and it is unclear how to differentiate these cells toward functional SGNs. In this study, we found that Sox2<sup>+</sup> subpopulation of cochlear glial cells preserves high potency of neuronal differentiation. Interestingly, Sox2 expression was downregulated during neuronal differentiation and Sox2 overexpression paradoxically inhibited neuronal differentiation. Our data suggest that Sox2<sup>+</sup> glial cells are potent SGN progenitor cells, a phenotype independent of Sox2 expression. Furthermore, we identified a combination of small molecules that not only promoted neuronal differentiation of Sox2<sup>-</sup> glial cells, but also removed glial cell identity and promoted the maturation of the induced neurons (iNs) toward SGN fate. In summary, we identified Sox2<sup>+</sup> glial subpopulation with high neuronal potency and small molecules inducing neuronal differentiation toward SGNs.
Project description:Macroautophagy/autophagy dysfunction is associated with many neurodegenerative diseases. TFEB (transcription factor EB), an important molecule that regulates lysosomal and autophagy function, is regarded as a potential target for treating some neurodegenerative diseases. However, the relationship between autophagy dysfunction and spiral ganglion neuron (SGN) degeneration and the role of TFEB in SGN degeneration has not yet been established. Here, we showed that in degenerated SGNs, induced by sensory epithelial cell loss in the cochlea of mice following kanamycin and furosemide administration, the lipofuscin area and oxidative stress level were increased, the nuclear-to-cytoplasmic TFEB ratio was decreased, and the late stage of autophagic flux was impaired. After autophagy dysfunction was partially ameliorated with an MTOR inhibitor, which promoted TFEB translocation into the nucleus from the cytoplasm, we found that the lysosomal deficits were significantly relieved, the oxidative stress level was reduced, and the density of surviving SGNs and auditory nerve fibers was increased. The results in the present study reveal that autophagy dysfunction is an important component of SGN degeneration, and TFEB may be a potential target for attenuating SGN degeneration following sensory epithelial cell loss in the cochlea of mice. Abbreviations: 3-NT: 3-nitrotyrosine; 4-HNE: 4-hydroxynonenal; 8-OHdG: 8-hydroxy-2'-deoxyguanosine; ABR: auditory brainstem response; APP: amyloid beta (A4) precursor protein; CLEAR: coordinated lysosomal expression and regulation; CTSB: cathespin B; CTSD: cathespin D; SAMR1: senescence-accelerated mouse/resistance 1; SAMP8: senescence-accelerated mouse/prone 8; MAPK1/ERK2: mitogen-activated protein kinase 1; MTOR: mechanistic target of rapamycin kinase; SGN: spiral ganglion neuron; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscope; TFEB: transcription factor EB.