Age-related hearing loss (ARHL), commonly referred to as presbycusis, is a gradual, symmetrical, sensorineural deafness that occurs with advancing age. In the United States, two-thirds of people older than 70 years suffer from ARHL (Lin et al., 2011). Typically, ARHL initiates with an impact on high-frequency hearing and progressively extends to affect mid- and low-frequency auditory perception (Humes et al., 2009). The cause and pathogenesis of ARHL are complex and largely unknown.

The cochlear sensory epithelia, comprises sensory cells (hair cells) and nonsensory cells (supporting cells). Hair cells play a pivotal role in detecting and transmitting sound while supporting cells contribute to maintaining the structural integrity of sensory organs, regulating ion homeostasis, and modulating the extracellular matrix in the cochlea, all of which are critical for maintaining normal hearing (Babola et al., 2020; Hayashi et al., 2020; Kim et al., 2016; Monzack et al., 2015).

The major pathological changes in the cochlea in ARHL are loss of hair cells, atrophy of the stria vascularis (SV), and degeneration of spiral ganglion neurons (SGNs) (Ishiyama et al., 2007; Nelson and Hinojosa, 2006; Rizk and Linthicum, 2012; Wu et al., 2020a). Age-related hair cell loss and dysfunction of supporting cells are closely associated with ARHL (Sun et al., 2023; Tajima et al., 2020; Wu et al., 2020a; Wu et al., 2020b). Several mechanisms contributing to cochlear aging have been identified. A single-cell transcriptomic atlas of mouse cochlear aging revealed that the hallmarks of cochlear aging include the loss of proteostasis and elevated apoptosis (Sun et al., 2023). Furthermore, various studies have indicated that age-related cochlear pathology is linked to increased oxidative stress, impaired ion channels, mitochondrial dysfunction, immune senescence, and gap junction dysfunction in supporting cells (Bermúdez-Muñoz et al., 2020; Cheng et al., 2023; Peixoto Pinheiro et al., 2021; Xu et al., 2023; Yamasoba et al., 2007).

The cochlear duct exhibits tonotopic organization, with basal hair cells responsible for detecting and transducing high-frequency sounds, while apical hair cells perform this function for low-frequency sounds. ARHL is characterized by cochlear cell loss or dysfunction with a base-to-apex gradient, suggesting that cells at the basal cochlear turn are more sensitive to age-induced damage than those at the apical turn (Kusunoki et al., 2004; Li and Borg, 1991). Although recent research has explored the correlation between cochlear spatial differences and aging at the transcriptional level, proteomic-based studies have not been reported (Liu et al., 2022; Liu et al., 2014; Sun et al., 2023; Tang et al., 2019; Yoshimura et al., 2014).

Proteomics serves as a crucial approach for investigating global changes in protein expression and cellular functions (Gonçalves et al., 2022; Tyers and Mann, 2003). In the context of the cochlea, proteomic techniques have been employed to investigate hearing loss induced by cisplatin, noise, and ototoxicity, leading to the identification of potential treatment targets (Jamesdaniel et al., 2012; Jung et al., 2009; Miao et al., 2021). However, due to the small size of cochlear samples, pooling samples from various animals is often necessary to achieve the required sample size for conventional proteomics (Chen et al., 2020; Maeda et al., 2015; Miao et al., 2021; Waissbluth et al., 2017). Thus, changes in each cochlear sample could not be identified. Additionally, distinguishing the sensory epithelia from other cochlear structures, such as the SV, spiral ligament (SL), and SGNs, in adult mice poses a challenge for detecting protein expression in each sensory epithelium sample.

Unlike conventional proteomics, which typically involves sample materials sufficient for analyzing micrograms of extracted proteins, microscale proteomics focuses on analyzing trace samples containing hundreds of nanograms or sub-micrograms of proteins. This approach has been successfully applied to various samples, including single pancreatic islets with 2,000-4,000 cells or micro-dissected tissue samples (Waanders et al., 2009; Zhu et al., 2018).

This study represents the first application of microscale proteomics to investigate spatially differential protein profiles in the cochlear sensory epithelia of ARHL mice.