Recombinant human Neuritin protects cochlear ribbon synapses and hearing function via ERK1/2 activation post noise-induced injury

Abstract

The preservation of synaptic integrity and physiological activity is pivotal for post-traumatic auditory rehabilitation following acoustic overexposure. Neuritin, a neurotrophic factor that facilitates synapse formation, maturation, and enhanced synaptic transmission, is essential for synapse development. In this study, we established a noise-induced cochlear synaptopathy model in CBA/CaJ mice, revealing a temporal association between endogenous Neuritin expression and synaptic density. Furthermore, administration of recombinant Human Neuritin (rhNeuritin) effectively preserves synaptic density in the cochlear basal turn at 7 days and 14 days following noise exposure. Importantly, it preserves the density of functional synapses (represented by overlapping CtBP2 and GluA2 puncta) and synapse function (indicated by ABR I wave amplitudes), thus diminishing the impairment of auditory function. In addition, rhNeuritin reverses the decrease in phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) levels resulting from noise exposure. By primarily preserving both the number and functionality of synapses in the basal turn, potentially via the induction of ERK1/2 phosphorylation, rhNeuritin mitigated hearing loss. These findings underscore the protective efficacy of rhNeuritin against noise-induced synaptic injury.

Graphical abstract

Highlights

• •Recombinant human Neuritin (rhNeuritin) protects hearing function after noise exposure.
• •RhNeuritin maintains synapse density and function in the cochlear basal turn.
• •RhNeuritin protects cochlear synapses by upregulating ERK1/2 phosphorylation.

1. Introduction

World Health Organization (WHO) data indicate Noise Induced Hearing Loss (NIHL) affects over 6 % of the global population, posing a substantial socioeconomic burden [[1], [2], [3]]. Moderate noise exposure in occupational and recreational settings represents a primary etiology of NIHL. Emerging evidence reveals that such exposure induces degeneration of Inner Hair Cell (IHC)-Spiral Ganglion Neuron (SGN) ribbon synapses, a pathological process often unaccompanied by persistent hearing threshold shifts [4,5]. The clinical significance of this cochlear injury is frequently underestimated, partially due to non-perceived loudness of causative sound levels. Notably, moderate noise can acutely and irreversibly disrupt these synapses without immediate or delayed Hair Cell (HC) loss, ultimately leading to SGN degeneration and permanent hearing loss [6].

Cochlear ribbon synapses consist of a presynaptic ribbon structure at the IHC basal pole and a postsynaptic domain within SGN terminals. These synapses harbor neurotransmitter vesicles in the presynaptic active zone and concentrate AMPA receptors in the postsynaptic density [7]. This specialized architecture enables efficient encoding and transmission of auditory signals from IHCs to SGNs. Given their susceptibility to acoustic trauma and limited regenerative capacity [8], preserving synaptic density and functionality is critical to prevent noise-induced auditory dysfunction.

Neuritin is a neurotrophic factor that is highly expressed during the development and maturation of synapses [9,10]. Genetic ablation of Neuritin in KO mice leads to impaired synaptic development, characterized by reduced spine density and immature synaptic connections [11]. Conversely, Neuritin overexpression enhances synaptic maturation through the augmentation of functional AMPA receptor clustering, expansion of presynaptic and postsynaptic compartments, and stabilization of synaptic junctions [12]. Neuritin also contributes to neuronal viability and morphological plasticity by triggering ERK1/2-dependent neurite elongation [13,14].

Our group previously reported that Neuritin overexpression in supporting cells ameliorates kanamycin-induced cochlear damage, including HC loss and hearing impairment, by blocking the Notch signaling cascade [15]. In a pharmacological model of SGN injury, rhNeuritin preserved the density and organization of SGN nerve fibers while safeguarding gerbils' high-frequency auditory capabilities [16]. Despite these findings, the expression patterns and functional roles of Neuritin in cochlear synapses remain largely uncharacterized.

Thus, there is an urgent need to explore the role of Neuritin in noise-induced cochlear synaptopathy. In this study, we established a murine model of noise-induced cochlear synaptopathy using CBA/CaJ mice. Our objectives were twofold: first, to analyze the relationship between Neuritin expression levels and ribbon synapse density; second, to evaluate rhNeuritin's potential to protect auditory function and synaptic density, as well as its involvement in ERK1/2 signaling modulation during acoustic trauma. Addressing these questions may shed new light on the mechanisms underlying noise-induced hearing loss and identify potential therapeutic targets for intervention.

2. Materials and methods

2.1. Animals and ethics statement

4-week-old male CBA/CaJ mice (Shanghai Jihui Laboratory Animal Co., Ltd., certificate no. 20170012005154) were acclimated in the Laboratory Animal Center of Hangzhou Normal University under controlled conditions (12L:12D photoperiod, ad libitum access to food and water). Animals with pre-existing outer/middle ear pathologies were excluded. All experimental procedures complied with protocols approved by the Institutional Animal Care and Use Committee of Hangzhou Normal University (HSD20200101).

2.2. Noise exposure and model establishment

For model establishment, a total of 39 mice were used and divided into groups based on post-noise exposure time points for different analyses.

30 mice were randomly allocated into 5 groups of 6 for auditory function detection and histological analysis, with time points set at pre-noise exposure (baseline control), 1, 3, 7, and 14 days post-exposure. Each group endured a 2-h exposure to octave-band noise (8–16 kHz, 91 dB SPL). Following exposure, at their designated time points, auditory function was measured, and then cochleae were harvested for histological and WB examination.

9 additional mice were split into 3 groups of 3 to elucidate the expression trend of Neuritin after noise injury. These groups corresponded to 1.5, 3, and 6 h post-exposure. Like the other groups, they underwent the same 2-h noise exposure, after which cochleae were collected for WB analysis to evaluate Neuritin expression.

All mice were placed in mesh-enclosed compartments within a reverberant noise chamber. Acoustic stimuli were generated using a Tucker-Davis Technologies RP2 signal processor (TDT System III), amplified by a P9500S power amplifier, and transmitted through four TW67 tweeters. Sound pressure levels were calibrated before each session to ensure a variation of less than 1 dB across chambers.

2.3. Preparation of rhNeuritin

Recombinant human Neuritin (rhNeuritin) was generated using a previously described protocol [17]. Briefly, the Pichia pastoris expression system (Invitrogen) was employed, involving strain GS115 and vector pPIC9K. The human Neuritin cDNA was amplified via polymerase chain reaction and subcloned into the pPIC9K vector under the control of the alcohol oxidase promoter. Sequence-verified pPIC9K-hNeuritin plasmids were introduced into GS115 cells via electroporation, generating stable transformants. Positive clones were expanded in buffered glycerol complex medium and induced with methanol in buffered methanol complex medium to induce rhNeuritin expression. Culture supernatants were subjected to hydrophobic interaction chromatography, followed by dialysis and lyophilization. Protein identity and bioactivity were confirmed via Western blot and neurite outgrowth assays, respectively, prior to in vivo administration.

2.4. rhNeuritin intervention

30 mice were used for the rhNeuritin intervention. Under anesthesia induced by intraperitoneal injection of a ketamine-xylazine cocktail (100 mg/kg and 20 mg/kg, respectively), each mouse was placed on a thermoregulated heated pad to maintain a core body temperature of 38 ± 0.5 °C. A retroauricular approach was employed to access the cochlear bulla, creating a 2 mm fenestration to expose the round window niche. The left ear received 32 μL of rhNeuritin (1 mg/mL in sterile NS) absorbed onto a gelatine sponge pledget, while the right ear received an NS-soaked gelatine sponge via round window delivery. NS served as a negative control to isolate the specific effects of rhNeuritin.

After a 30-min interval, all mice were exposed to the same octave-band noise (8–16 kHz, 91 dB SPL) for 2 h as detailed in 2.2. Subsequently, they were divided into 5 groups of 6, corresponding to pre-noise exposure, 1, 3, 7, and 14 days post-exposure time points. At these designated time points, auditory function was detected, and cochleae were collected for histological or WB analysis. The bulla fenestration was sealed with muscle tissue, and the skin incision was closed using absorbable sutures [18,19].

2.5. ABR measurements

Auditory brainstem response (ABR) assessments [20] were conducted in all subjects prior to surgery and at 1 day, 3 days, 7 and 14 days post-operation. Anesthesia was induced via intraperitoneal injection of ketamine (100 mg/kg) and xylazine (20 mg/kg). Animals were positioned on a thermostatic heating pad (maintaining 38 ± 0.5 °C) to stabilize electrophysiological parameters. Stainless steel needle electrodes were subcutaneously implanted at the vertex and ventrolateral regions of both auricles. Unilateral ABR recordings were performed using open-field configurations, with left/right ears analyzed independently. Customized earplugs (3 M, MN, USA) were fabricated to occlude non-tested ear canals, with placement verified prior to each session. TDT hardware/software systems were utilized for stimulus generation and biosignal acquisition. Tone burst stimuli (8, 16, 32 kHz) were delivered via a broadband speaker (MF1; TDT) positioned 10 cm from the external auditory meatus. Threshold determination utilized ABR wave II morphology. Starting from the maximum intensity, sound pressure level (SPL) was decremented in 10 dB (pre-noise) or 5 dB (post-noise) steps until waveform disappearance. Amplitude-SPL curves were constructed by visually identifying wave I peaks at each intensity level and calculating peak-to-peak amplitudes. Data analysis was performed using GraphPad Prism 9.0.1.

2.6. Histological preparation, confocal imaging, and synaptic counts

For immunostaining and quantitative analysis of ribbon synapses, cochlear samples were perfusion-fixed with 4 % paraformaldehyde in phosphate-buffered saline (PBS) (4 °C, 2 h), followed by decalcification in 0.12 M EDTA (room temperature, 2 days). Microdissected cochleae (apical, middle, basal turns) were permeabilized with 1 % Triton X-100/PBS (1 h) and blocked with 5 % goat serum/PBS (1 h). Samples were incubated with primary antibodies: mouse anti-CtBP2 (IgG1; BD Biosciences, 612044, 1:200) and mouse anti-GluA2 (IgG2a, Millipore, MABN1189, 1:2000). Alexa Fluor-conjugated secondary antibodies (Invitrogen) enabled fluorescent labeling. Confocal images were acquired using a Zeiss LSM 710 META microscope (63 × water immersion objective). Image stacks were processed in ImageJ (NIH). Synaptic ribbons were quantified and normalized to the total number of IHC nuclei per field.

Synapse counting criteria: CtBP2 (presynaptic marker) and GluA2 (postsynaptic marker) co-localization defined functional synapses. Only CtBP2⁺/GluA2⁺ double-positive synapses were counted.

2.7. Western blotting

Protein expression of Neuritin, total ERK1/2 (t-ERK1/2), and phosphorylated ERK1/2 (p-ERK1/2) in murine cochleae was evaluated via Western blot analysis. Cochlear tissues were dissected and snap-frozen in liquid nitrogen immediately post-euthanasia. Cold RIPA buffer containing 1 % protease inhibitor cocktail facilitated protein extraction. Lysates were centrifuged at 14,000 rpm for 10 min at 4 °C, and supernatants were collected. Total protein was separated by 10 % SDS-PAGE and transferred to PVDF membranes at 23 V for 43 min. Membranes were blocked with 5 % non-fat milk in Tris-buffered saline (TBS) for 2 h at room temperature (RT) and incubated overnight at 4 °C with primary antibodies in TBST (TBS with 0.1 % Tween-20)/3 % non-fat milk. Primary antibodies included: anti-Neuritin (1:1000; ab64186, Abcam, UK), anti-t-ERK1/2 (#8544, Cell Signaling Technology [CST], USA, 1:1000), anti-p-ERK1/2 (#4370S, CST, USA, 1:1000), and anti-β-actin (1:2000; ZSGB-BIO, China). On the following day, membranes were incubated with HRP-conjugated secondary antibody (1:2500; ZB-2301, ZSGB-BIO, China) for 2 h at RT. Protein signals were visualized using an ECL kit (Millipore, USA) and analyzed with Adobe Photoshop CC 2015.

2.8. Statistical analysis

All experiments included a minimum of three biological replicates per condition. Data were analyzed with GraphPad Prism 9 (GraphPad Software, CA, USA) and reported as mean ± standard error of the mean (SEM). Normality and variance homogeneity were verified prior to statistical testing. One-way or two-way analysis of variance (ANOVA) was applied based on experimental design, followed by appropriate post hoc test for multiple comparisons. Statistical significance was defined as p < 0.05.

3. Results

3.1. Characterization of noise-induced hearing loss in adult CBA/CaJ mice

To establish a robust noise-induced synaptic loss model and define a functional baseline for evaluating the protective effects of rhNeuritin, we measured ABR thresholds for clicks and tone bursts (8, 16, 32 kHz) before and 1, 3, 7, 14 days after noise exposure. This time-course design aimed to capture acute injury (1 day), potential recovery (3–14 days), and frequency-specific vulnerabilities relevant to cochlear synaptic pathology.

A one-way ANOVA followed by Bonferroni's multiple comparison test revealed a significant threshold shift in click responses 1 day post-exposure, indicating successful induction of acute auditory impairment. By 14 days, click thresholds nearly normalized (p < 0.0001, Fig. 1A).Tone test analysis using two-way ANOVA showed that while 8 and 16 kHz thresholds recovered to baseline by 14 days, the 32 kHz threshold remained significantly elevated (p < 0.0001, Fig. 1B). This frequency-specific pattern aligns with the known vulnerability of cochlear basal turns (processing 32 kHz) to noise-induced ribbon synapse loss [21].

Fig. 1.

Characterization of synaptic loss model in CBA/CaJ mice.

A- B. Auditory Brainstem Response(ABR) thresholds for click (A) and high-frequency tone (B) stimuli were measured pre-noise and at 1-, 3-, 7-, and 14 days post-noise insult. Data are presented as mean ± SEM (N = 6 mice/time point). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (one-way ANOVA [A], two-way ANOVA [B]; Bonferroni's post hoc test).

C. Confocal microscopy of cochlear basal turns per Inner Hair Cell(IHC): CtBP2 (presynaptic ribbons, green) and GluA2 (postsynaptic AMPARs, red). Representative images at pre-noise exposure, 1 day and 14 days post-noise exposure (scale bar = 10 μm). CtBP2⁺/GluA2⁺ co-localized puncta were indicated by arrows.

D. Synapse density across cochlear turns (apical/middle/basal). CtBP2⁺/GluA2⁺ puncta counted (N = 6 cochleae/group). ∗∗∗∗p < 0.0001 (two-way ANOVA with Bonferroni's multiple comparisons).

Immunofluorescent labeling of CtBP2 (presynaptic ribbons) and GluA2 (postsynaptic AMPA receptor subunits) was used to quantify noise-induced synaptic alterations, with functional synapses defined as CtBP2⁺/GluA2⁺ co-localized puncta. Representative confocal micrographs from the cochlear basal turn(pre-noise, 1 day, 14 days post-noise), which is corresponding to the high-frequency 32 kHz region in tone tests, are presented in Fig. 1C. Two-way ANOVA with Bonferroni's multiple comparisons test revealed that the noise induced significant synaptic loss across all cochlear regions (apical/middle/basal) at 1 day post-exposure (p < 0.0001). Notably, this synaptic loss persisted at 14 days, with no recovery observed (Fig. 1D). These structural findings align with the functional ABR results, while click thresholds showed partial recovery by 14 days, synaptic damage remained permanent.

Together, these results establish a robust noise-induced synaptic damage model, this integrated functional-structural validation provides a critical baseline for evaluating rhNeuriitn protection against synaptic damage in subsequent experiments.

3.2. Expression of Neuritin after noise exposure

To determine whether Neuritin is involved in noise-induced synaptic damage, we characterized its temporal expression profile in cochlear tissues using Western blot. Unlike the 1–14 day time points for ABR and synaptic density, which focus on functional recovery and persistent structural damage, Neuritin expression was evaluated at acute time points (1.5, 3, 6 h; 1, 3 days post exposure). This timing was informed by our preliminary experiments, which revealed peak expression changes within 6 h post-exposure, allowing us to capture the early molecular responses to noise trauma.

Protein quantification (normalized to β-actin) revealed a biphasic expression pattern: transient upregulation at 1.5–3 h post-exposure, followed by sustained downregulation from 6 h to 3 days (p < 0.001, Fig. 2A–B). This early stress-induced surge likely represents an endogenous attempt to mitigate acute synaptic stress, while the subsequent decline coincides with the onset of synaptic loss observed at 1 day (Fig. 1D), when Neuritin levels drop below baseline. This deficit highlights a potential therapeutic window for exogenous Neuritin supplementation.

Fig. 2.

Expression of Neuritin following synapse loss elicited by noise exposure. A. Western blot analysis was conducted to examine the expression levels of Neuritin in cochlear tissues. The samples were taken from the cochlear tissues before noise exposure (serving as the baseline control) and at 1.5 h, 3 h, 6 h, 1 day, and 3 days after noise exposure. B. Quantitative analysis was carried out on the expression levels of Neuritin. For each time point, the cochleae from 3 mice (a total of 6 cochleae) were used. Statistical significance was determined by one - way ANOVA followed by Bonferroni's multiple comparisons test, ∗∗∗p < 0.001.

3.3. RhNeuritin maintained the function and number of synapses in adult mice after noise exposure

To test whether exogenous recombinant Neuritin (rhNeuritin) protects against noise-induced synaptic injury, particularly in the high-frequency basal turn identified as vulnerable in Section 3.1, we evaluated both functional (ABR P1 amplitude) and structural (synaptic density) endpoints. ABR P1 amplitude, a proxy for auditory nerve synaptic transmission strength, was measured before and 1, 3, 7, 14 days post-noise exposure, with cochleae harvested immediately after each time point for CtBP2/GluA2 immunostaining to quantify functional synapses (CtBP2⁺/GluA2⁺ puncta).

In the cochlear basal turn, corresponding to the 32 kHz high-frequency vulnerable to noise-induced synaptic loss, rhNeuritin significantly enhanced ABR P1 amplitude at 14 days post-exposure (p < 0.05, Fig. 3A). This functional improvement reflects restored synaptic transmission efficiency under rhNeuritin treatment. Structural analysis showed that while both groups exhibited acute synaptic loss at 1 day, the rhNeuritin group retained significantly more synapses in the basal turn at 7 and 14 days (p < 0.05, Fig. 3C), as visualized by representative confocal micrographs at 14 days (Fig. 3B). This time- and region-dependent preservation of CtBP2⁺/GluA2⁺ puncta demonstrates that rhNeuritin specifically mitigates progressive synaptic degeneration in the basal turn, the primary site of noise-induced structural damage.These results showed that exogenous rhNeuritin compensates for endogenous downregulation, protecting vulnerable high-frequency synapses from persistent loss.

Fig. 3.

RhNeuritin preserves synaptic density and function in noise-damaged cochleae.

A. ABR P1 amplitudes (high-frequency stimuli) were measured in Normal Saline (NS,negative control) and rhNeuritin treated ears before and 1/14 days post-noise exposure. Data represent N = 6 mice/time point. p < 0.05 (two-way ANOVA with Bonferroni's post-hoc test. B. Representative immunohistochemical images of cochlear synapses in the basal turn 14 days post-exposure. Presynaptic ribbons (CtBP2, green) and postsynaptic (GluA2, red) are shown (scale bar = 10 μm). CtBP2⁺/GluA2⁺ co-localized puncta were indicated by arrows. C. Quantification of cochlear synapses (CtBP2⁺/GluA2⁺ colocalization) in apical/middle/basal turns, corresponding to 8 kHz, 16 kHz, and 32 kHz frequency regions, respectively. N = 6 cochleae (6 mice)/time point. ∗p < 0.05 (two-way ANOVA with Bonferroni's post-hoc test).

3.4. RhNeuritin protects hearing function against noise-induced injury

Building on the synaptic protective effects of rhNeuritin, we tested whether this structural preservation translates to functional hearing protection, particularly in the high-frequency region prone to persistent damage. ABR thresholds for click stimuli (global hearing function) and 32 kHz tone bursts (high-frequency specificity) were measured in rhNeuritin and NS treated mice before and 1, 3, 7, 14 days post-noise exposure.

RhNeuritin significantly attenuated noise-induced click threshold shifts at 7 days (p < 0.01, Fig. 4A), an effect consistent with the preserved basal turn synaptic density in Fig. 3C. More critically, in the high-frequency 32 kHz range, rhNeuritin promoted sustained recovery at both 7 days(p < 0.05, Figs. 4B) and 14 days(p < 0.01, Fig. 4B). This frequency-specific functional protection mirrors the regional synaptic preservation in the basal turn (Fig. 3C), establishing a direct link between Neuritin-mediated synaptic integrity and hearing threshold maintenance.

Fig. 4.

RhNeuritin preserves hearing thresholds in noise-exposed adult mice. A. ABR thresholds following click and high-frequency 32 kHz. B. Stimulation were measured before and at 1, 3, 7, and 14 days post-noise exposure in NS and rhNeuritin treated cochleae. N = 6 mice/time point. ∗p < 0.05, ∗∗p < 0.01; two-way ANOVA with Bonferroni's post-hoc test.

These results demonstrate that rhNeuritin not only protects cochlear synapses but also mitigates long-term functional deficits in the most noise-vulnerable frequency region.

3.5. RhNeuritin promoted the phosphorylation of ERK1/2 after noise exposure

ERK1/2, a mitogen-activated protein kinase (MAPK) family member, orchestrates diverse cellular processes. Given that genetic deletion of ERK2 leads to profound cochlear ribbon synapse loss and hearing deficits [22] and that Neuritin exerts its neurotrophic effects by activating ERK1/2 signaling in various cellular contexts [13,14], we investigated whether the protective effects of rhNeuritin in noise-exposed mice are mediated through ERK1/2 pathway activation.

Western blot analysis was performed on cochlear tissues collected at the same time points as synaptic density and ABR measurements (before and 1, 3, 7 days post-exposure), focusing on phosphorylated ERK1/2 (p-ERK1/2)-a key marker of ERK pathway activation. Compared to NS controls, rhNeuritin treatment significantly upregulated p-ERK1/2 levels at 1, 3, and 7 days post-exposure (p < 0.01, Fig. 5A–B). This phosphorylation increase was specific to the activated (p-ERK) form, as total ERK (t-ERK) levels remained unchanged, confirming pathway-specific activation.

Fig. 5.

RhNeuritin modulates the levels of p-ERK1/2 in adult mice following noise exposure. A. Western blot analysis was conducted to assess the expression levels of p - ERK1/2 and t - ERK1/2 in the cochleae of mice treated with either NS or rhNeuritin at 1, 3, and 7 days after noise exposure. B.Quantitative analysis was performed to determine the relative levels of p - ERK1/2:t - ERK1/2 in the cochleae at different time - points. For each time - point, cochleae from 6 mice (a total of 6 cochleae) were used. Significance was evaluated by two - way ANOVA followed by Bonferroni's multiple comparisons test. ∗∗p < 0.01, ∗∗∗p < 0.001; two-way ANOVA, Bonferroni's multiple comparisons test.

These findings establish a mechanistic link between rhNeuritin and ERK1/2 signaling: by restoring p-ERK1/2 in noise-exposed cochleae, Neuritin likely promotes synaptic survival pathways, consistent with its role in protecting basal turn synapses (Fig. 3C) and preserving high-frequency hearing function (Fig. 4B). The coincidence of ERK activation with synaptic and functional recovery provides a molecular basis for the protective effects observed, positioning the Neuritin-ERK axis as a critical target for interventions against noise-induced synaptic degeneration.

4. Discussion

Neurotrophins (NTs) serve as critical regulators of synaptic development and plasticity in the central nervous system [22]. In this study, we unveiled a newly discovered role for rhNeuritin in the inner ear, demonstrating its capacity to attenuate noise-induced synaptic loss and preserve cochlear synaptic function. Mechanistically, these effects are linked to ERK1/2 phosphorylation, as demonstrated by elevated p-ERK1/2 levels in rhNeuritin-treated cochleae.

In this investigation, a CBA/CaJ mouse model of noise-induced cochlear synaptopathy was established. CBA/CaJ mice, renowned for maintaining robust auditory function even at 39 weeks of age, represent the gold standard in NIHL research [23]. These animals have been instrumental in developing diverse synaptic damage models, including those induced by noise, aging, and ototoxic drugs. Based on cochlear structural damage characteristics, these models are categorized into synaptopathy-restricted models and combined synaptopathy-cochleopathy models [24]. Given that the current focus of this study is to clarify the effect of rhNeuritin on ribbon synapses and its potential mechanism, a synaptopathy-restricted model was selected to isolate variables and minimize confounding cochlear pathologies [25].

Neuritin, a neurotrophic factor, plays a pivotal role in nervous system development and synaptic plasticity [9,10] RhNeuritin has been shown to facilitate functional recovery in sciatic nerve [26] and spinal cord injuries [27]. However, its role in the auditory system remains underexplored. This study initially characterized Neuritin expression dynamics in noise-exposed cochleae, revealing a temporal correlation between Neuritin levels, hearing thresholds, and synaptic density (Fig. 2A and B).

The ABR wave I amplitude (P1) reflects synaptic integrity between auditory nerve fibers and inner hair cells (IHCs), correlating with both synaptic density and function [28]. In the high-frequency (32 kHz) basal cochlear turn, rhNeuritin-treated ears exhibited significantly higher P1 amplitudes than NS controls at 14 days post-exposure. Concomitant preservation of synaptic density in this region, which is critical for high-frequency processing, demonstrates rhNeuritin's regional specificity in maintaining synaptic density and function (Fig. 3A–C).

Notably, cochlear synapses exhibit limited intrinsic regenerative potential following noise trauma. However, regenerated or residual synaptic ribbons fail to restore auditory function, likely due to incomplete formation of functional connections with AMPA receptor-expressing neurons, thereby constraining auditory nerve recovery [29]. Importantly, our results showed that rhNeuritin treatment preserved a significantly higher density of functional synapses (defined by overlapping GluA2 and CtBP2 immunoreactive puncta) compared to NS controls.

Glutamate-mediated excitotoxicity is speculated to initiate noise-induced synaptic damage by inducing swelling of SGN terminals contacting IHCs [30,31]. Phosphorylated ERK1/2 (p-ERK1/2), the active form of this kinas, acts as a prosurvival signaling hub, promoting SGN viability and neurite outgrowth [32,33]. Previous studies have linked Neuritin to ERK1/2 phosphorylation-dependent neuritogenesis [13,14]. In the present work, Western blot analysis revealed noise exposure downregulated cochlear p-ERK1/2 levels, an effect antagonized by rhNeuritin treatment (Fig. 5A–B). This suggests a protective cascade: rhNeuritin-elicited ERK1/2 phosphorylation enhances SGN survival and terminal arborization, enabling residual synaptic ribbons to form functional connections with AMPA receptor-expressing SGNs. Consequently, rhNeuritin preserves basal turn synaptic integrity, a key determinant of high-frequency hearing, by restoring p-ERK1/2 levels, which correlates with improved ABR I-wave amplitudes and thresholds.

While this study demonstrates rhNeuritin's potential for NIHL therapy, several critical limitations require addressing. Large-scale longitudinal studies are essential to validate its long-term therapeutic efficacy in NIHL models. Elucidating the precise molecular mechanisms specifically how rhNeuritin orchestrates ERK1/2 phosphorylation to preserve synaptic integrity will be pivotal for developing targeted interventions. Additionally, exploring combinatorial therapies with complementary neuroprotectants may uncover synergistic interactions that enhance auditory restoration.

Collectively, this work establishes rhNeuritin as a novel synaptic protectant against noise trauma, operating via ERK1/2-dependent preservation of cochlear synapse density and function. These findings underscore rhNeuritin's translational potential for synaptic NIHL and advocate for mechanistic studies to guide clinical translation.

CRediT authorship contribution statement

Haiyan Wang: Writing – original draft, Project administration, Funding acquisition. Jinchi Hu: Methodology, Formal analysis. Shuangyan Liu: Software, Data curation. Fei Gui: Data curation. Xiaopin Sun: Investigation. Rong Chen: Resources. Guanwu Yin: Writing – review & editing. Xiaoming Song: Supervision, Resources, Conceptualization. Yi Yang: Writing – review & editing, Supervision, Project administration. Yu Hong: Writing – review & editing, Funding acquisition, Conceptualization.

Ethics approval

This study was carried out by the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996. All experimental protocols were approved by the Animal Care and Use Committee of Hangzhou Normal University (permit no. HSD20200101). All efforts were made to minimize the number of animals used and their suffering.

Funding

This work was supported by the Zhejiang Provincial Natural Science Foundation of China (No. LTGY23H130001 and No. LY18H260002).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.