Tlr2/4 Double Knockout Attenuates the Degeneration of Primary Auditory Neurons: Potential Mechanisms From Transcriptomic Perspectives

The transcriptomic landscape of mice with primary auditory neurons degeneration (PAND) indicates key pathways in its pathogenesis, including complement cascades, immune responses, tumor necrosis factor (TNF) signaling pathway, and cytokine-cytokine receptor interaction. Toll-like receptors (TLRs) are important immune and inflammatory molecules that have been shown to disrupt the disease network of PAND. In a PAND model involving administration of kanamycin combined with furosemide to destroy cochlear hair cells, Tlr 2/4 double knockout (DKO) mice had auditory preservation advantages, which were mainly manifested at 4–16 kHz. DKO mice and wild type (WT) mice had completely damaged cochlear hair cells on the 30th day, but the density of spiral ganglion neurons (SGN) in the Rosenthal canal was significantly higher in the DKO group than in the WT group. The results of immunohistochemistry for p38 and p65 showed that the attenuation of SGN degeneration in DKO mice may not be mediated by canonical Tlr signaling pathways. The SGN transcriptome of DKO and WT mice indicated that there was an inverted gene set enrichment relationship between their different transcriptomes and the SGN degeneration transcriptome, which is consistent with the morphology results. Core module analysis suggested that DKO mice may modulate SGN degeneration by activating two clusters, and the involved molecules include EGF, STAT3, CALB2, LOX, SNAP25, CAV2, SDC4, MYL1, NCS1, PVALB, TPM4, and TMOD4.

Researchers have confirmed that many environmental insults immediately and directly damage hair cells, but the resulting SGN degeneration is chronic because of a lack of neurotrophic factors and peripheral stimuli (Kujawa and Liberman, 2009;Makary et al., 2011;Wu et al., 2021). The degeneration of the SGN leads to the loss and/or distortion of auditory information in the brain. Even as little as 10% of neural tissue degeneration can lead to a disproportionate change in the stimulation profile of the auditory nerve (Sriperumbudur et al., 2020). Spiral ganglion neurons would mainly have Ic subtype loss if the loss of peripheral stimulus occurs, which is similar to SGN aging (Shrestha et al., 2018). The degeneration of SGNs could induce distinct forms of plasticity in cortical excitatory and inhibitory neurons that culminate in net hyperactivity, increased neural gain, and reduce the adaptation to background noise (Resnik and Polley, 2021). On the other hand, once SGN degeneration occurs, cochlear implants will inevitably have poor performance. Histology of 12 temporal bones from 6 subjects indicated that higher residual SGNs could predict better performance after implantation in a given patient (Riss et al., 2008;Cusumano et al., 2017).
To elucidate the mechanism of SGN degeneration, a mouse model of rapid hair cell ablation with a single dose injection of kanamycin sulfate and furosemide (Taylor et al., 2008;Hu et al., 2017, p. 3;Ye et al., 2019), has frequently been used. In this SGN degeneration model, a single dose injection of kanamycin sulfate and furosemide immediately damaged hair cells but not SGNs (Gao et al., 2017). As auditory epithelia ablated, SGN, lacking neurotrophic factors and stimulus, will degenerate with increased lipofuscin area, damaged autophagic flux, and reduced density (Hu et al., 2017;Ye et al., 2019).
Tlr polymorphisms were correlated with hearing preservation in bacterial meningitis survivors (van Well et al., 2012). Koo et al. (2015) assumed that this kind of hearing preservation difference is associated with Tlr-mediated BLB permeability. Researchers have also found that Tlr4 knockout mice had better hearing function following noise trauma without affecting sensory cell viability under physiological conditions (Vethanayagam et al., 2016). De Paola et al. (2012) found that Tlr4 inhibition attenuates motor neuron degeneration both in vitro and in vivo. Tlr4 deficiency could also protect mice against ischemia and axotomy-induced retinal ganglion cells degeneration, and they assumed that better neuron preservation was associated with reduced parenchymal stress responses via the ERK, JNK, and P38 signaling pathways (Kilic et al., 2008). The innate immune response of the nerve microenvironment is involved in functional recovery during Wallerian degeneration, and Tlr2 and Tlr4 can regulate synaptic stability in the spinal cord after peripheral nerve injury (Freria et al., 2016). Researchers have found that systemic microbial TLR2 agonists could induce neuron degeneration in Alzheimer's disease mice and direct CNS delivery of a selective TLR2 antagonist blocked the neurotoxicity of systemically administered zymosan, indicating that CNS TLR2 mediates this neuron degeneration process (Lax et al., 2020). Previous studies have also demonstrated that administration of anti-TLR2 alleviated α-synuclein accumulation in neuronal, neuroinflammation, neurodegeneration, and behavioral deficits in an α-synuclein tg mouse model of PD/DLB and proposed TLR2 immunotherapy as a novel therapeutic strategy for neuron degeneration (Kim et al., 2018). These studies suggest that Tlr molecules may regulate the degeneration pathogenesis of auditory systems, especially spiral ganglion neurons.
Although SGN degeneration is characterized by increased lipofuscin area, damaged autophagic flux, and reduced density, a comprehensive understanding of the SGN degeneration transcriptome landscape is still lacking, and the role of molecules such as Tlr that may regulate the pathogenesis remain unknown. Here, we analyzed the transcriptome landscape of SGN degeneration and identified that Tlr2 and Tlr4 (Tlr2/4) might regulate the pathogenesis of degeneration. Based on these findings, we analyzed the role of Tlr2 and Tlr4 in SGN degeneration and found that genetic deletion of Tlr2/4 in combination attenuates SGN degeneration in mice without interfering with classic Tlr signaling pathways. Furthermore, we used transcriptome analysis to identify the key modules and pathways. Identification of these specific molecules and pathways could potently facilitate the protection of the auditory system.

Animals
B6.B10ScN-Tlr4lps-del/JthJ and C.129(B6)-Tlr2 t m1K ir /J mice (The Jackson Laboratory, Bar Harbor, ME, United States) were used for Tlr2/4 double knockout. Only 8-week-old male mice without a history of ototoxic damage or noise exposure were used for model establishment. The animals were housed under a standard 12-h light/dark cycle and were allowed free access to water and a regular mouse diet. The cochleae of each animal were collected and assigned to different assays to reduce animal usage and maintain sufficient sample sizes. The following genotyping primers were used to examine the Tlr2 knockout allele: 5 -CTTCCTGAATTTGTCCAGTACA-3 ; 5 -GGGCCAGCTCATTCCTCCCAC-3 ; 5 -ACGAGCAAGATC AACAGGAGA-3 (mutation allele 334 bp, WT allele 499 bp). The following genotyping primers were used to examine the Tlr4 knockout allele: 5 -GCAAGTTTCTATATGCATTCTC-3 ; 5 -CCTCCATTTCCAATAGGTAG-3 ; 5 -ATATGCATGATCAA CACCACAG-3 ; 5 -TTTCCATTGCTGCCCTATAG-3 (mutated allele 140 bp, WT allele 390 bp). The procedures were approved by the Ethics Committee of Xinhua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine (Shanghai, China).

Spiral Ganglion Neurons Degeneration Model
Mice of the SGN degeneration group were subcutaneously injected with 1 g/kg kanamycin sulfate (Sigma-Aldrich, E004000), and intraperitoneally injected with 0.4 g/kg furosemide (Tianjin Pharmaceutical Group, H12020527; 10 mg/ml) after 30 min (Hu et al., 2017;Ye et al., 2019). Mice in the control group were simultaneously and intraperitoneally treated with equal doses of saline. The mice were sacrificed 7, 15, and 30 days after drug administration, and the temporal bones were removed and dissected in PBS at 4 • C.

Measurement of Auditory Brainstem Response
The mice were anesthetized with a ketamine (0.1 mg/g)/xylazine (0.01 mg/g) mixture. Body temperature was maintained at 37 • C with a warming blanket. An active needle electrode was placed in the midline of the vertex of the skull, a reference electrode at the mastoid areas, and a ground electrode in the low back area. The ABRs were provoked with tone bursts of 4,8,11,16,22, and 32 kHz, generated by a D/A converter (RP2.1; TDT) and relayed to an attenuator (PA5; TDT), an amplifier (SA1; TDT), and a magnetic speaker (MF1; TDT). Mouse auditory brainstem responses were filtered (100-3,000 Hz), amplified, and averaged using TDT hardware and software. Responses were recorded from 90 dB SPL to 10 dB below the threshold level in 5 dB descending steps. The ABR threshold was defined as the lowest intensity that reliably elicited a detectable response. The average ABR threshold shifts between wild-type and DKO mice were compared using two-way ANOVA with the two factors group × frequency. If significant group effects were identified, the Welch two-sample t-test was used to evaluate each frequency between the two groups.

High Throughput Sequencing
Total RNA from Cochlear modiolus tissues was extracted using the QIAGEN RNA Extraction Kit (Cat. No.74104). Agarose gels (1.5%) were used to detect RNA degradation and contamination. Illumina Hiseq 2,500 and Hiseq 4,000 were used to characterize the transcriptome landscape of SGN following the manufacturer's recommendations. The following procedures were performed using Hisat2 (version 2.1.0) to align the clean reads with the reference genome of mice (Kim et al., 2015) and StringTie (version 1.3.3b) to evaluate gene expression (Pertea et al., 2015).

Functional Annotation
Gene set enrichment analysis (GSEA) (Subramanian et al., 2005;Yu et al., 2012) was used to extract biological insight from RNAseq data. GSEA aims to determine whether members of gene set S tend to occur in the extremes (top or bottom) of a given list from RNA-seq results. The enrichment score (ES) indicates the degree of a set S overrepresentation at the extremes of the gene ranked list L. ES was calculated as follows: P-value was calculated by estimating the statistical significance of the ES by empirical phenotype-based permutation test procedures.
Terms used for functional annotation include Gene Ontology (Ashburner et al., 2000) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa and Goto, 2000). Functional annotation of GO, including biological processes, cellular components, and molecular functions, and KEGG is a common omnibus to systematically interpret gene functions, facilitating an intensive understanding of genomic information and higherorder functional information.

Protein-Protein Interaction and Module Analysis
The construction of PPI networks imported the Search Tool for the Retrieval of Interacting Genes database (STRING) (Szklarczyk et al., 2021), and was analyzed using Cytoscape (version 3.6.1) software (Shannon et al., 2003). STRING (version 11.0) covers 2,000 million interactions of 24.6 million proteins from 5,090 organisms. When fed with differentially expressed genes, STRING was able to identify the most significantly interactive associations, in which a criterion of a combined score > 0.4, was set as the significance level. Cytoscape plugin molecular complex detection (MCODE) can illustrate the core modules in the PPI network. The false degree cut-off, node score cut-off, haircut, false K-core, and maximum depth from seed were set at 2, 0.2, true, 2, and 100, respectively Yan et al., 2019).

Statistical Analysis
Computations and data visualization were performed using R 4.0.2. Distribution of the data was assumed to be normal, but this was not formally tested. No statistical methods were used predetermine sample sizes, but our sample sizes are like those reported in our previous publications (Hu et al., 2017;Ye et al., 2019). Statistical differences in auditory physiology, SGN counts were analyzed using two-way ANOVA, followed by Welch t-test. All statistical analysis results were expressed as the mean ± standard error of the mean (SEM). Statistical significance was defined as P < 0.05.

Spiral Ganglion Neurons Degeneration Established by Cochlear Sensory Epithelial Ablation
SGN degeneration was successfully induced by the destruction of cochlear hair cells, which is consistent with the results of previous studies (Hu et al., 2017;Ye et al., 2019). Apoptotic activities in hair cells were significantly upregulated within the following 7 days, whereas no change was seen in SGNs ( Figure 1A). The structure of the organ of Corti was almost completely collapsed and destroyed, and nearly no sensory epithelial cells remained in the middle turn on the 30th day after kanamycin and furosemide injection, while almost only a continuous layer of flattened cubic epithelial cells remained on the basilar membrane ( Figure 2B). A time-series evaluation of SGN density was conducted on the 7th, 15th, and 30th day after kanamycin and furosemide injection ( Figure 1B, auditory physiology data in Supplementary Figure 1 and Supplementary Table 1). On the 30th day after kanamycin and furosemide injection, SGN density has significantly decreased in the apical, middle, and basal region of the cochlea (Welch two sample t-test, apical: t = 8.4, P = 0.00009; middle: t = 7.53, P = 0.0005; basal: t = 10.33, P = 0.00003). Thus, this time point was used to decipher the transcriptome changes during SGN degeneration.
GO analysis showed that the dysregulated functions included positive regulation of peptidyl-serine phosphorylation of STAT protein, type I interferon receptor binding, T cell activation involved in immune response, B cell proliferation, natural killer cell activation involved in the immune response, membrane disruption, negative regulation of B cell apoptosis, and embryonic brain development ( Figure 4B). The analysis of PPI interactions indicated six key modules (Table 1), and 10 molecules were the most significant, including COMP, COL1A1, STAT3, HAPLN1, HBEGF, CCL2, MMP13, SMAD4, CXCL1, and CTSK. We noticed that these enriched activities of immune responses, TNF signaling pathway, and cytokine-cytokine receptor interaction could be regulated by Tlr molecules (Peek et al., 2020). At the same time, Tlr2 and Tlr4 disrupted the SGN degeneration signaling network (Figure 3C). Based on these results, we hypothesized that Tlr might contribute to the regulation of SGN degeneration.

Tlr2/4 Double Knockout Mice Has Auditory Advantage in Spiral Ganglion Neurons Degeneration
Given that Tlr may play an important role in SGN degeneration, we established Tlr2 and Tlr4 knockout mice. We detected the On the 30th day, SGN density significantly decreased at the apical, middle, and basal turn of the cochlea (apical: N = 9, t = 8.14, P < 0.01, middle: N = 9, t = 7.53, P < 0.01; basal: N = 9, t = 10.33, P < 0.01). Statistical significance of other time points was also denoted with the order of apical, middle and basal, respectively (ns: not significant; *P < 0.05; **P < 0.01.). Gray area indicated the standard error range calculated by loess method. Right panel shows the representative images of SGN before and 30 days after the injection of kanamycin and furosemide. Scale bar: 10 µm.
We also observed morphological changes in the Organ of Corti and spiral ganglion neurons. The Tlr2/4 DKO mice and their wild-type littermates treated with normal saline (NS) maintained relatively intact inner and outer hair cells, while the Tlr DKO mice and their wild-type littermates under the SGN degeneration model lost most inner and outer hair cells, and the tunnel of Corti collapsed ( Figure 2B).
The Tlr DKO mice and their wild-type littermates treated with NS maintained a relatively normal density, with no significant difference in neuron density (P = 0.675, t = 0.43, N = 24, Welch Two Sample t-test). SGN density significantly decreased in Tlr DKO mice and wild-type mice under the SGN degeneration model (Figure 2C), but the SGN density was significantly higher in the DKO group mice (P < 0.001, t = 11.71, N = 32, Welch Two Sample t-test) (Figure 2D). These results suggest that the hearing preservation advantage of Tlr2/4 DKO mice mainly relies on the protection of spiral ganglion neurons.

Inverted Gene Set Enrichment of Double Knockout Mice During Spiral Ganglion Neurons Degeneration
The p38-mediated MAPK and p65-mediated NF-κB signaling pathways are canonical signaling pathways of Tlr molecules (Li et al., 2010). We used immunohistochemistry to detect the expression of these genes in SGNs. SGN p38 expression was found in both DKO and WT mice, mainly in the cytoplasm and nuclei. There was no significant difference in p38 expression between the two groups on the 30th day of degeneration induction ( Figure 2E, neuron density evaluation in Supplementary Figure 2). The SGNs of DKO and WT mice express p65 (RelA), which is mainly located in the cytoplasm and nuclei. P65 expression decreased 30 days after SGN degeneration, but no significant differences in expression were identified between the two groups ( Figure 2F, neuron density evaluation in Supplementary Figure 2).
In general, Tlr2/4 DKO mice showed downregulated biological activities during SGN degeneration compared to WT mice. The top 30 enriched KEGG pathways included 1 upregulated and 29 downregulated pathways ( Figure 6A). Oxidative phosphorylation was downregulated during SGN degeneration, but upregulated in DKO mice compared with  WT mice, which is called inverted gene set enrichment. The top 10 KEGG terms with inverted gene set enrichments included arachidonic acid metabolism, complement and coagulation cascades, coronavirus disease-COVID-19, cytokine-cytokine receptor interaction, hepatitis C, inflammatory mediator regulation of TRP channels, JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, TNF signaling pathway, and oxidative phosphorylation ( Figure 6B). Similarly, the top 30 enriched GO terms included one upregulated and 29 downregulated activities ( Figure 6C). The top 10 KEGG terms with inverted gene set enrichments included iron ion binding, monooxygenase activity, negative regulation of endopeptidase activity, platelet aggregation, positive regulation of myoblast differentiation, protein polymerization, response to cytokines, response to peptide hormones, serine-type endopeptidase activity, and serine-type endopeptidase inhibitor activity ( Figure 6D).

Suspectable Core Modules That Attenuate Spiral Ganglion Neurons Degeneration in Double Knockout Mice
In order to detect whether DKO mice had consistent morphology across the entire signaling network, i.e., whether there is an inverted gene set enrichment relationship with the primary auditory neurons degeneration (PAND)-specific gene set, we analyzed the different genes in the PAND-specific gene set. We found that there was an inverted GSEA relationship between the PAND process and the PAND-specific gene set in DKO mice, and the PAND-associated DEGs were relatively reverse regulated in the progression of PAND in DKO mice (NES = -1.84, P < 0.001).
We further analyzed the attenuation of SGN degeneration in DKO mice from the perspective of the entire signaling network (Supplementary Figure 3). We found that 130 of the 661 genes specifically upregulated during the degeneration of SGN in WT mice were downregulated mice during the degeneration of spiral neurons in DKO mice, and 380 genes were specifically downregulated during the degeneration of SGN in WT mice; 116 genes were downregulated during the degeneration of spiral neurons in DKO mice.
Protein interaction analysis of the proteins encoded by these genes showed that DKO mice had a specific anti-degenerative signal network compared to WT mice. The disease module analysis showed that there were two key modules ( Table 2). The score of the first module was 3.667, including seven genes, namely EGF, STAT3, CALB2, LOX, SNAP25, CAV2, and SDC4. The score of the second module was 3.5, which included five genes: MYL1, NCS1, PVALB, TPM4, and TMOD4. In addition, EGF, STAT3, CALB2, SNAP25, SDC4, TPM4, and TMOD4 are key molecules involved in SGN degeneration. CALB2 is a specific marker of the SGN Ia and Ib subtypes in mice (Shrestha et al., 2018), PVALB is a neuron-specific marker (Petitpré et al., 2018), and the interaction of these molecules may contribute to the attenuation of SGN degeneration in DKO mice.

DISCUSSION
Studies have shown that primary and secondary SGN degeneration may account for half of the SGN degeneration (Makary et al., 2011;Waqas et al., 2017;Guo et al., 2021). Morphological studies of the human cochlea show that with the growth of age, even if there is no loss of inner and outer hair cells, SGN will be lost at a rate of about 100 cells per year. This rate can increase to approximately 185 cells per year when there is simultaneous loss of inner and outer hair cells (Makary et al., 2011). Unlike auditory hair cells, which are easily damaged by noise and ototoxic drugs such as aminoglycosides and cisplatin, spiral ganglion neurons are relatively robust. In a study of animal models and human temporal bone, histopathological analysis of acquired sensorineural hearing loss showed that severe degeneration of cochlear spiral neurons is often accompanied by a significant loss of hair cells (Miller et al., 1997;McFadden et al., 2004). The degeneration of SGNs seems to be related to the loss of inner hair cells, because 95% of spiral neurons only have synaptic connections with inner hair cells. However, the causal relationship based on histopathological correlation is often frustrated (Zilberstein et al., 2012), which may be because the loss of inner hair cells is usually accompanied by the loss or complete destruction of the organ of Corti after cochlear injury, and both hair cells and supporting cells contribute to the support of SGN in the cochlea (Sugawara et al., 2005). Temporal bone studies from 54 to 89 years of age without ear diseases suggest that the number of hair cells is almost normal, but the pattern of axonal degeneration of the cochlear nerve may be an important form of human presbycusis (Viana et al., 2015).
Transcriptome analysis of age-related cochlear degeneration indicated that no immune or inflammatory activity was enriched (Supplementary Figure 4). Therefore, the present study utilized the secondary SGN degeneration model of a single dose injection of kanamycin and furosemide, and the SGN degeneration model is suitable for the study of Tlr regulation during SGN degeneration. In the present SGN degeneration model, almost all outer hair cells were destroyed within 7 days after the injection of   (Hu et al., 2017;Ye et al., 2019). These results were also in consistent with the study of Taylor (Taylor et al., 2008). Another important question is when this transcriptome changes happen in DKO mice. We consider that the key points might be the opposite regulated direction of DKO mice compared with a normal SGN degeneration transcriptome change. In other words, the overlapping and opposite terms would be highlighted that might play key role in the alleviation of SGN degeneration in DKO mice whenever it happened. But we should acknowledge that it is preferred if these changes occurred after the degeneration induction, otherwise a systematic influence and specific delivery of these molecules should be considered. Tlr-regulated innate immune signaling regulates neuron-glia interactions (McLaughlin et al., 2019). McLaughlin et al. demonstrated that glia non-canonical Toll-like receptor signaling could be non-autonomously activated by neuronal apoptosis, priming their capacity to engulf apoptotic neurons and regulate the maintenance of a healthy brain (Kashio and Miura, 2019). Glia continuously surveys neuronal health during development, providing trophic support to healthy neurons, while rapidly engulfing dying ones. With neuronal health being surveyed, glia could provide trophic support to healthy neurons and rapidly engulf dying neurons. Glia necessitates a foolproof mechanism to unambiguously identify those neurons to support vs. engulf. To ensure specificity, glia is proposed to interact with dying neurons via a series of carefully choreographed steps. Dying neurons and glia communicate via toll-receptorregulated innate immune signaling, while neuronal apoptosis drives the processing and activation of the Toll-6 ligand and activates the dSARM-mediated Toll-6 transcriptional pathway, which controls the expression of the Draper engulfment receptor. Pathway loss drives early onset neurodegeneration, underscoring its functional importance. Zhang et al. (2019) demonstrated that in the cochleae (lateral wall and spiral ganglion neurons), TLR-4 and the downstream signaling molecule MyD88 were significantly upregulated 3 d after noise exposure. It has also been reported that Tlr4 knockout mice could inhibit the expression of major histocompatibility complex class II and participate in the antigen-presenting function of macrophages after acoustic trauma (Vethanayagam et al., 2016). Their results suggested that Tlr4 regulates multiple aspects of the immune response in the cochlea and contributes to cochlear pathogenesis after acoustic injury. According to the single-cell transcriptome of Shrestha et al. (2018) and our IHC results (data not shown), SGN may rarely be Tlr2/4 positive. Tlr2/4 double knockout may attenuate SGN degeneration by disturbing the local microenvironment of circular macrophage cells or Schwann cells and detecting Tlr downstream in SGN fails to signify the attenuation of SGN degeneration.
We identified some relatively novel molecules that may play an essential role in attenuating SGN degeneration in Tlr2/4 DKO mice. Researchers have found that the interaction between Tlr and these molecules may regulate the pathogenesis of many diseases. For example, myofibroblast Tlr signaling promotes colitis-associated carcinogenesis by mediating macrophage M2 polarization and STAT3 activation via intracellular communication (Yuan Q. et al., 2021, p. 88). Hsu et al. (2010) found that the differentially regulated expression of Tlr-associated epidermal growth factor influences mucosal healing.
DFNA44, an autosomal dominant deafness, is tightly associated with the effector of EGF-mediated cell signaling (Modamio-Hoybjor et al., 2007). Rats administered oral epigallocatechin-3-gallate demonstrated reduced cisplatininduced hearing loss, experienced reduced loss of OHCs in the basal region of the cochlea and reduced oxidative stress and apoptotic markers. The preservation of STAT3 and Bcl-xL activation and increased STAT3/STAT1 ratio may contribute to protection (Borse et al., 2017). STAT3 and its activated form are specifically expressed in hair cells during cochlear development (Chen et al., 2017). CALB2 is a candidate gene for human dominant optic atrophy (Carelli et al., 2011, p. 8). Calb2 is highly expressed in the Ia and Ib subtypes of SGN (Shrestha et al., 2018;Sun et al., 2018). In noise-induced hearing loss (NIHL), noise exposure damages cochlear sensory hair cells, which lack the capacity to regenerate. Following noise insult, intense metabolic activity occurs, resulting in a free-radical imbalance in the cochlea. Oxidative stress and antioxidant enzyme alterations, including lipoxygenase (LOX) upregulation, have been linked to chronic inflammation, which contributes to hearing impairment. Inhibition of LOX showed greater efficacy in the treatment of NIHL (Rodriguez et al., 2017). LOX products may contribute to the hypersensitivity of SGN to hair cell inputs in a variety of pathological conditions (Balaban et al., 2003). Snap25 is a key marker of neuronal identity. Noda et al. (2018) used the neurogenic pioneer transcription factor Ascl1 and the auditory neuron differentiation factor NeuroD1 to reprogram spiral ganglion non-neuronal cells into induced neurons.
Their transcriptome results indicated that induced neurons maintained some key markers of neuronal identity, such as Tubb3, Map2, Prph, Snap25, and Prox1 (Noda et al., 2018). The mutation of the otoferlin C2 domain, which causes deafness in humans, impairs the ability of otoferlin to bind syntaxin, SNAP-25, and the Cav1.3 calcium channel, which may mediate regulation by otoferlin of hair cell synaptic exocytosis, which is critical to inner ear hair cell function (Ramakrishnan et al., 2009). The damaged interaction between SDC4 and GIPCs also contributes to the progression of non-syndromic hearing loss (Katoh, 2013).

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI Gene Expression Omnibus (GEO) under accession number GSE182978.

ETHICS STATEMENT
The animal study was reviewed and approved by the Ethics Committee of Xinhua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine.

AUTHOR CONTRIBUTIONS
MX and QW were responsible for the initiation and conduction, respectively. QW was responsible for writing the manuscript. QW, YS, YP, TZ, AZ, DG, CF, HH, LM, KC, and BY performed the experiments. BY and QW processed and analyzed the data. All authors contributed to the article and approved the submitted version.