Pathophysiological processes underlying hidden hearing loss revealed in Kcnt1/2 double knockout mice

Abstract

Presbycusis is a prevalent condition in older adults characterized by the progressive loss of hearing due to age-related changes in the cochlea, the auditory portion of the inner ear. Many adults also struggle with understanding speech in noise despite having normal auditory thresholds, a condition termed "hidden" hearing loss because it evades standard audiological assessments. Examination of animal models and postmortem human tissue suggests that hidden hearing loss is also associated with age-related changes in the cochlea and may, therefore, precede overt age-related hearing loss. Nevertheless, the pathological mechanisms underlying hidden hearing loss are not understood, which hinders the development of diagnostic biomarkers and effective treatments for age-related hearing loss. To fill these gaps in knowledge, we leveraged a combination of tools, including transcriptomic profiling and morphological and functional assessments, to identify these processes and examine the transition from hidden to overt hearing loss. As a novel approach, we took advantage of a recently characterized model of hidden hearing loss: Kcnt1/2 double knockout mice. Using this model, we find that even before observable morphological pathology, hidden hearing loss is associated with significant alteration in several processes, notably proteostasis, in the cochlear sensorineural structures, and increased susceptibility to overt hearing loss in response to noise exposure and aging. Our findings provide the first insight into the pathophysiology associated with the earliest and, therefore, most treatable stages of hearing loss and provide critical insight directing future investigation of pharmaceutical strategies to slow and possibly prevent overt age-related hearing loss.

Keywords: aging; cochlea; hidden hearing loss; mouse; presbycusis; proteostasis.

FIGURE 1

DKO mice show greater transcriptomic changes in the sensorineural compared to metabolic structures of the cochlea. (a) Schematic of the inner ear illustrates the cochlea and cochlear structures, including the sensorineural structures (organ of Corti and spiral ganglion neurons, OC/SGN, blue) and metabolic structures (stria vascularis and spiral ligament, SV/SL, red), isolated for transcriptomic analysis. (b) Correlation heat map with hierarchical clustering analysis shows distinct clustering by structure and genotype (WT and DKO). (c) Principal component analysis (PCA) shows the largest variance is attributed to differences in cochlear structure (PC1). Additional variance among the cochlear sensorineural structures is attributed to genotype (PC4). (d) Differential expression analysis identifies both up‐ and downregulated genes in the cochlear sensorineural (OC/SGN) and metabolic (SV/SL) structures, some of which are shared.

FIGURE 2

DKO mice show differential expression of genes specifically enriched in type I SGNs. (a) Schematic illustrates the synaptic connectivity between the type I (cyan) and type II (yellow) spiral ganglion neurons (SGNs) and inner and outer hair cells (IHCs and OHCs). (b) Venn diagram shows the overlap in genes differentially expressed in the cochlear sensorineural structures (organ of Corti and spiral ganglion neuron, OC/SGN, differentially expressed genes, DEGs) in DKO mice (blue) compared to previously identified enriched genes (EG) in either the type I (cyan) or type II (yellow) SGNs. (c) Heat map analysis of standardized r‐log transformed expression levels of the overlapping genes show distinct clustering by genotype and SGN subtype.

FIGURE 3

DKO mice show changes in gene expression associated with several mechanisms, including altered proteostasis, in the cochlear sensorineural structures of DKO mice. (a) Gene ontology (GO) enrichment analysis identifies several cellular components, molecular functions, and biological processes that are differentially enriched in the cochlear sensorineural structures (organ of Corti and spiral ganglion neurons, OC/SGN) isolated from DKO mice. GO terms were categorized into five main categories: (1) Proteasome, (2) Actin and myosin, (3) axon, synapse, and cell junction, (4) mitochondria, and (5) hormone and lipid. For many categories, different terms were identified by the same gene list. In this case, only the term with the most significant p value is listed. Only terms identified by ≥3 genes are listed. Complete lists are available in Table S3. (b) Z‐projections of PROTEOSTAT‐labeled SGNs isolated from 6‐week‐old WT and DKO mice reveals the greater abundance of aggresomes (red aggregates, left panel) in SGNs from DKO compared to WT mice (right panel). Scale bar equals 10 μm. Significant difference is indicated with an asterisk (*).

FIGURE 4

DKO mice show changes in gene expression associated with aging and deafness in the cochlear sensorineural structures of DKO mice (a) Venn diagram shows the overlap in differentially expressed genes (DEGs) in the cochlear sensorineural structures (OC/SGN) in DKO mice (blue) compared to previously identified DEGs identified in the cochlear sensorineural structures of aged (two‐year‐old) mice with overt hearing loss (Aged WT, light blue). (b) There is a significant positive correlation in the fold changes observed in these overlapping DEGs. (c) Venn diagram shows the overlap in differentially expressed genes in the cochlear sensorineural structures (OC/SGN DEGs) in DKO mice (blue) compared to genes previously associated with aging and deafness (purple).

FIGURE 5

DKO mice show reduced recovery of cochlear function but no differences in auditory synapse loss following noise exposure. (a) Auditory brainstem response (ABR) wave I absolute thresholds (upper panels) and wave I amplitude input/output (I/O) responses (lower panels) before (black line) and 1 day (red line) and 1 week (grey line) after noise exposure are shown as a function of stimulus frequency for both WT (left panels) and DKO (right panels) mice. (b) Z‐projections of the organs of Corti from WT (left panels) and DKO (right panels) mice show CTBP2‐immunostained presynaptic ribbons (green) and GluA2‐immunolabled postsynaptic glutamate receptors (red) in control mice (upper panels) and mice 1 week after noise exposure (lower panels). 32 kHz region is shown. Scale bar equals 10 μm. (c) Synapses (paired presynaptic ribbons and postsynaptic glutamate receptor patches) per inner hair cell (IHC) in control mice (black line) and mice 1 week after noise exposure (grey line) are shown as a function of tonotopic region for WT (filled circles, left panel) and DKO (open circles, right panel) mice. Significant differences are indicated with an asterisk (*).

FIGURE 6

DKO mice show accelerated age‐related loss of cochlear function and auditory synapses. (a) Auditory brainstem response (ABR) wave I absolute thresholds are shown as a function of age for WT (filled circles) and DKO (open circles) mice for the indicated auditory stimuli. (b) The rates of age‐related hearing loss (ARHL) are shown as a function of auditory stimuli for WT (filled bars) and DKO mice (open bars). (c) ABR wave I amplitude input/output (I/O) responses are shown as a function of age for WT (filled circles) and DKO mice (open circles) for click stimuli. (d) ABR wave I latency I/O responses are shown as a function of age for WT (filled circles) and DKO mice (open circles) for click stimuli. (e) Z‐projections of the organs of Corti from WT (upper panels) and DKO (lower panels) mice show CTBP2‐immunostained presynaptic ribbons (green) and GluA2‐immunolabled postsynaptic glutamate receptors (red) at the ages indicated. 32 kHz region is shown. Scale bar equals 10 μm. (f) Synapses (paired presynaptic ribbons and postsynaptic glutamate receptor patches) per IHC are shown as a function of age for WT (filled circles) and DKO mice (open circles) for the indicated tonotopic regions. Significant differences are indicated with an asterisk (*).