ABSTRACT: Presbycusis, or age-related hearing loss (ARHL) [1], represents a major healthcare and social challenge in rapidly aging populations. ARHL significantly impairs communication, quality of life, and psychological well-being among older adults [2]. It typically manifests as a progressive, irreversible, bilateral, and symmetric sensorineural hearing loss [3]. Multiple intrinsic and extrinsic factors, including genetic predisposition, chronic noise exposure, and ototoxic insults, contribute to its onset and severity [4-6]. Reactive oxygen species (ROS) accumulation and mitochondrial dysfunction are recognized as critical mechanisms driving cochlear degeneration during aging [7-9]. Excess D-galactose (D-gal) metabolism produces ROS, initiating a cascade of oxidative stress, inflammation, mitochondrial impairment, and apoptosis, consistent with age-associated pathology [10, 11]. The D-gal-induced aging model is widely established and serves as a reliable experimental paradigm to investigate aging-related biological processes and auditory pathophysiology mechanisms. Forkhead box G1 (FOXG1) is a nuclear transcription factor indispensable for the proper development of the nervous system [12]. In the auditory system, FOXG1 is integral to cochlear morphogenesis and the differentiation of hair cells (HCs), supporting cells (SCs), and spiral ganglion neurons (SGNs) [13-15]. In addition, FOXG1 regulates cellular redox balance by maintaining mitochondrial integrity and controlling ROS production [9, 16]. Our previous work demonstrated that upregulation of FOXG1 expression activates autophagy, enabling HCs to resist oxidative stress-induced injury; however, the underlying mechanisms governing this protective interaction remains elusive. Therefore, this study sought to elucidate the role of FOXG1 in age-related cochlear HCs damage using a D-gal-induced aging model. Among age-associated molecular alterations, autophagy dysregulation has emerged as a key hallmark of aging [17]. Autophagy, a conserved catabolic process, is fundamental to maintaining cellular equilibrium through the breakdown of defective organelles and misfolded polypeptides [18]. Under physiological and pathological states, autophagy acts as a cytoprotective mechanism that eliminates defective mitochondria and mitigates oxidative stress-induced cellular injury [19, 20]. In the auditory system, autophagy is indispensable for normal cochlear development and the preservation of inner ear cellular functions, particularly in HCs [9, 21, 22]. Increasing evidence indicates that autophagic activity declines with age, leading to the accumulation of depolarized mitochondria and excessive ROS in HCs [23]. Insufficient clearance of these impaired mitochondria can trigger apoptotic cascades and contribute to HC degeneration. Conversely, activation of the autophagy pathway promotes HC survival by sequestering damaged mitochondria into autophagosomes, which subsequently fuse with lysosomes for degradation [24-26]. Although our previous studies suggested a link between FOXG1 expression and autophagy during HC degeneration in ARHL, the mechanistic interplay between these processes remains to be elucidated. Beyond autophagy, growing evidence underscores the pivotal contribution of epigenetic alterations in the aging process. Epigenetic mechanisms orchestrate gene expression programs that regulate development, differentiation, and tissue maintenance, while their dysregulation contributes to diverse age-related diseases, including cancer [27], neurodegeneration [28, 29] and metabolic disorders [30]. These mechanisms include DNA methylation, histone modifications, and non-coding RNA regulation, which collectively modulate chromatin accessibility and transcriptional activity [31]. Among these, histone lysine methylation, particularly histone H3 lysine 9 dimethylation (H3K9me2), is tightly connected with chromatin condensation and transcriptional repression. Aberrant H3K9me2 accumulation has been implicated in various aging-related pathologies, including neurodegenerative diseases and sensory dysfunctions [32]. For instance, elevated H3K9me2 levels have been detected in the occipital cortex of Alzheimer’s disease (AD) patients relative to age-matched healthy controls [33]. Similarly, transgenic mouse models harboring familial AD (FAD) mutations exhibit significantly increased H3K9me2 expression in the prefrontal cortex and hippocampus. This aberrant epigenetic modification contributes to the transcriptional repression of glutamate receptor genes, leading to impaired synaptic transmission and AD-like cognitive deficits. Pharmacological inhibition of H3K9me2 restores glutamate receptor expression, improves synaptic function, and alleviates cognitive impairment in aged FAD mice [34]. Likewise, in models of noise-induced hearing loss, increased H3K9me2 levels in the basal turn outer HCs (OHCs) of the cochlea have been associated with enhanced cell vulnerability, whereas inhibition of H3K9me2 mitigates OHC loss [35]. Although epigenetic regulation is increasingly recognized as essential in cochlear homeostasis and auditory pathology, the specific H3K9me2-dependent modulation of FOXG1 in ARHL remains unexplored. In addition to histone modifications, miRNAs serve as key post-transcriptional epigenetic regulators of gene activity. These small non-coding RNAs are engaged in diverse cell processes, including growth, differentiation, apoptosis, tumorigenesis, and embryogenic development [36-38]. Emerging evidence indicates that several miRNAs contribute to HC maturation, SC plasticity, and neural differentiation within the auditory system [39]. Our previous study demonstrated that miR-34a, miR-96, miR-182, and miR-183 mediated cisplatin-induced HC degeneration via the autophagy pathway [16]. However, the role and regulatory mechanisms of miRNAs in ARHL remain poorly understood
Zuhong He