Altered Fhod3 expression involved in progressive high-frequency hearing loss via dysregulation of actin polymerization stoichiometry in the cuticular plate.

Age-related hearing loss (ARHL) is a common sensory impairment with complex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide association studies (GWAS) in mice and identified a novel locus on chromosome 18 associated with ARHL specifically linked to a 32 kHz tone burst stimulus. Consequently, we investigated the role of Formin Homology 2 Domain Containing 3 (Fhod3), a newly discovered candidate gene for ARHL based on the GWAS results. We observed Fhod3 expression in auditory hair cells (HCs) primarily localized at the cuticular plate (CP). To understand the functional implications of Fhod3 in the cochlea, we generated Fhod3 overexpression mice (Pax2-Cre+/-; Fhod3Tg/+) (TG) and HC-specific conditional knockout mice (Atoh1-Cre+/-; Fhod3fl/fl) (KO). Audiological assessments in TG mice demonstrated progressive high-frequency hearing loss, characterized by predominant loss of outer hair cells, and a decreased phalloidin intensities of CP. Ultrastructural analysis revealed loss of the shortest row of stereocilia in the basal turn of the cochlea, and alterations in the cuticular plate surrounding stereocilia rootlets. Importantly, the hearing and HC phenotype in TG mice phenocopied that of the KO mice. These findings suggest that balanced expression of Fhod3 is critical for proper CP and stereocilia structure and function. Further investigation of Fhod3 related hearing impairment mechanisms may lend new insight towards the myriad mechanisms underlying ARHL, which in turn could facilitate the development of therapeutic strategies for ARHL.

Cochlear transcriptome analysis of an outbred mouse population (CFW).

Age-related hearing loss (ARHL) is the most common cause of hearing loss and one of the most prevalent conditions affecting the elderly worldwide. Despite evidence from our lab and others about its polygenic nature, little is known about the specific genes, cell types, and pathways involved in ARHL, impeding the development of therapeutic interventions. In this manuscript, we describe, for the first time, the complete cell-type specific transcriptome of the aging mouse cochlea using snRNA-seq in an outbred mouse model in relation to auditory threshold variation. Cochlear cell types were identified using unsupervised clustering and annotated via a three-tiered approach-first by linking to expression of known marker genes, then using the NSForest algorithm to select minimum cluster-specific marker genes and reduce dimensional feature space for statistical comparison of our clusters with existing publicly-available data sets on the gEAR website, and finally, by validating and refining the annotations using Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH) and the cluster-specific marker genes as probes. We report on 60 unique cell-types expanding the number of defined cochlear cell types by more than two times. Importantly, we show significant specific cell type increases and decreases associated with loss of hearing acuity implicating specific subsets of hair cell subtypes, ganglion cell subtypes, and cell subtypes within the stria vascularis in this model of ARHL. These results provide a view into the cellular and molecular mechanisms responsible for age-related hearing loss and pathways for therapeutic targeting.

Altered Fhod3 Expression Involved in Progressive High-Frequency Hearing Loss via Dysregulation of Actin Polymerization Stoichiometry in The Cuticular Plate.

Age-related hearing loss (ARHL) is a common sensory impairment with comlex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide association studies (GWAS) in mice and identified a novel locus on chromosome 18 associated with ARHL specifically linked to a 32 kHz tone burst stimulus. Consequently, we investigated the role of Formin Homology 2 Domain Containing 3 (Fhod3), a newly discovered candidate gene for ARHL based on the GWAS results. We observed Fhod3 expression in auditory hair cells (HCs) and primarily localized at the cuticular plate (CP). To understand the functional implications of Fhod3 in the cochlea, we generated Fhod3 overexpression mice (Pax2-Cre+/-; Fhod3Tg/+) (TG) and HC-specific conditional knockout mice (Atoh1-Cre+/-; Fhod3fl/fl) (KO). Audiological assessments in TG mice demonstrated progressive high-frequency hearing loss, characterized by predominant loss of outer HCs and decrease phalloidin intensities of CP. Ultrastructural analysis revealed shortened stereocilia in the basal turn cochlea. Importantly, the hearing and HC phenotype in TG mice were replicated in KO mice. These findings indicate that Fhod3 plays a critical role in regulating actin dynamics in CP and stereocilia. Further investigation of Fhod3-related hearing impairment mechanisms may facilitate the development of therapeutic strategies for ARHL in humans.

Attenuation of Age-Related Hearing Impairment in Senescence-Accelerated Mouse Prone 8 (SAMP8) Mice Treated with Fatty Acid Synthase Inhibitor CMS121.

In the senescence-accelerated mouse prone 8 (SAMP8) mouse model, oxidative stress leads to premature senescence and age-related hearing impairment (ARHI). CMS121 inhibits oxytosis/ferroptosis by targeting fatty acid synthase. The aim of our study was to determine whether CMS121 is protective against ARHI in SAMP8 mice. Auditory brainstem responses (ABRs) were used to assess baseline hearing in sixteen 4-week-old female SAMP8 mice, which were divided into two cohorts. The control group was fed a vehicle diet, while the experimental group was fed a diet containing CMS121. ABRs were measured until 13 weeks of age. Cochlear immunohistochemistry was performed to analyze the number of paired ribbon-receptor synapses per inner hair cell (IHC). Descriptive statistics are provided with mean ± SEM. Two-sample t-tests were performed to compare hearing thresholds and paired synapse count across the two groups, with alpha = 0.05. Baseline hearing thresholds in the control group were statistically similar to those of the CMS121 group. At 13 weeks of age, the control group had significantly worse hearing thresholds at 12 kHz (56.5 vs. 39.8, p = 0.044) and 16 kHz (64.8 vs. 43.8, p = 0.040) compared to the CMS121 group. Immunohistochemistry showed a significantly lower synapse count per IHC in the control group (15.7) compared to the CMS121 group (18.4), p = 0.014. Our study shows a significant reduction in ABR threshold shifts and increased preservation of IHC ribbon synapses in the mid-range frequencies among mice treated with CMS121 compared to untreated mice.

Cochlear transcriptome analysis of an outbred mouse population (CFW).

Age-related hearing loss (ARHL) is the most common cause of hearing loss and one of the most prevalent conditions affecting the elderly worldwide. Despite evidence from our lab and others about its polygenic nature, little is known about the specific genes, cell types and pathways involved in ARHL, impeding the development of therapeutic interventions. In this manuscript, we describe, for the first time, the complete cell-type specific transcriptome of the aging mouse cochlea using snRNA-seq in an outbred mouse model in relation to auditory threshold variation. Cochlear cell types were identified using unsupervised clustering and annotated via a three-tiered approach - first by linking to expression of known marker genes, then using the NS-Forest algorithm to select minimum cluster-specific marker genes and reduce dimensional feature space for statistical comparison of our clusters with existing publicly-available data sets on the gEAR website (https://umgear.org/), and finally, by validating and refining the annotations using Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH) and the cluster-specific marker genes as probes. We report on 60 unique cell-types expanding the number of defined cochlear cell types by more than two times. Importantly, we show significant specific cell type increases and decreases associated with loss of hearing acuity implicating specific subsets of hair cell subtypes, ganglion cell subtypes, and cell subtypes withing the stria vascularis in this model of ARHL. These results provide a view into the cellular and molecular mechanisms responsible for age-related hearing loss and pathways for therapeutic targeting.

Hearing loss and tinnitus: association studies for complex-hearing disorders in mouse and man.

Genome-wide association studies (GWAS) provide an unbiased first look at genetic loci involved in aging and noise-induced sensorineural hearing loss and tinnitus. The hearing phenotype, whether audiogram-based or self-report, is regressed against genotyped information at representative single nucleotide polymorphisms (SNPs) across the genome. Findings include the fact that both hearing loss and tinnitus are polygenic disorders, with up to thousands of genes, each of effect size of < 0.02. Smaller human GWAS' were able to use objective measures and identified a few loci; however, hundreds of thousands of participants have been required for the statistical power to identify significant variants, and GWAS is unable to assess rare variants with mean allele frequency < 1%. Animal studies are required as well because of inability to access the human cochlea. Mouse GWAS builds on linkage techniques and the known phenotypic differences in auditory function between inbred strains. With the advantage that the laboratory environment can be controlled for noise and aging, the Hybrid Mouse Diversity Panel (HDMP) combines 100 strains sequenced at high resolution. Lift-over regions between mice and humans have identified over 17,000 homologous genes. Since most significant SNPs are either intergenic or in introns, and binding sites between species are poorly preserved between species, expression quantitative trait locus information is required to bring humans and mice into agreement. Transcriptome-wide analysis studies (TWAS) can prioritize putative causal genes and tissues. Diverse species, each making a distinct contribution, carry a synergistic advantage in the quest for treatment and ultimate cure of sensorineural hearing difficulties.

Large-scale phenotyping of ABR P1-N1 amplitudes before and after exposure to noise in 69 strains of mice.

ABR wave I amplitude represents the synapse of auditory nerve fibers with the inner hair cell and is highly correlated with synapse counts. Cochlear synaptopathy, the loss of synaptic connections between inner hair cells and auditory nerve fibers, has been well-demonstrated in animal models of noise-induced hearing loss. The peak-to-peak wave I amplitude was determined at baseline and 2 weeks after noise exposure. We determined the ABR wave I amplitude at 80 dB SPL at the frequencies of 8, 12, 16, 24, and 32 kHz. A total of 69 strains (1-8 mice/strain) were analyzed. A statistically significant post-noise reduction in wave I amplitude was observed in all the tested frequencies (p < 0.00001). We identify distinct patterns of noise susceptibility and make this complete phenotypic dataset available for general use. This data establishes a new resource for the study of NIHL in mice and we hope this database will be a useful tool to expand the research in this field.

The Genetics of Variation of the Wave 1 Amplitude of the Mouse Auditory Brainstem Response.

This is the first genome-wide association study with the Hybrid Mouse Diversity Panel (HDMP) to define the genetic landscape of the variation in the suprathreshold wave 1 amplitude of the auditory brainstem response (ABR) both pre- and post-noise exposure. This measure is correlated with the density of the auditory neurons (AN) and/or the compliment of synaptic ribbons within the inner hair cells of the mouse cochlea. We analyzed suprathreshold ABR for 635 mice from 102 HMDP strains pre- and post-noise exposure (108 dB 10 kHz octave band noise exposure for 2 h) using auditory brainstem response (ABR) wave 1 suprathreshold amplitudes as part of a large survey (Myint et al., Hear Res 332:113-120, 2016). Genome-wide significance levels for pre- and post-exposure wave 1 amplitude across the HMDP were performed using FaST-LMM. Synaptic ribbon counts (Ctbp2 and mGluR2) were analyzed for the extreme strains within the HMDP. ABR wave 1 amplitude varied across all strains of the HMDP with differences ranging between 2.42 and 3.82-fold pre-exposure and between 2.43 and 7.5-fold post-exposure with several tone burst stimuli (4 kHz, 8 kHz, 12 kHz, 16 kHz, 24 kHz, and 32 kHz). Immunolabeling of paired synaptic ribbons and glutamate receptors of strains with the highest and lowest wave 1 values pre- and post-exposure revealed significant differences in functional synaptic ribbon counts. Genome-wide association analysis identified genome-wide significant threshold associations on chromosome 3 (24 kHz; JAX00105429; p < 1.12E-06) and chromosome 16 (16 kHz; JAX00424604; p < 9.02E-07) prior to noise exposure and significant associations on chromosomes 2 (32 kHz; JAX00497967; p < 3.68E-08) and 13 (8 kHz; JAX00049416; 1.07E-06) after noise exposure. In order to prioritize candidate genes, we generated cis-eQTLs from microarray profiling of RNA isolated from whole cochleae in 64 of the tested strains.This is the first report of a genome-wide association analysis, controlled for population structure, to explore the genetic landscape of suprathreshold wave 1 amplitude measurements of the mouse ABR. We have defined two genomic regions associated with wave 1 amplitude variation prior to noise exposure and an additional two associated with variation after noise exposure.