Biological insights from multi-omic analysis of 31 genomic risk loci for adult hearing difficulty.

Age-related hearing impairment (ARHI), one of the most common medical conditions, is strongly heritable, yet its genetic causes remain largely unknown. We conducted a meta-analysis of GWAS summary statistics from multiple hearing-related traits in the UK Biobank (n = up to 330,759) and identified 31 genome-wide significant risk loci for self-reported hearing difficulty (p < 5x10-8), of which eight have not been reported previously in the peer-reviewed literature. We investigated the regulatory and cell specific expression for these loci by generating mRNA-seq, ATAC-seq, and single-cell RNA-seq from cells in the mouse cochlea. Risk-associated genes were most strongly enriched for expression in cochlear epithelial cells, as well as for genes related to sensory perception and known Mendelian deafness genes, supporting their relevance to auditory function. Regions of the human genome homologous to open chromatin in epithelial cells from the mouse were strongly enriched for heritable risk for hearing difficulty, even after adjusting for baseline effects of evolutionary conservation and cell-type non-specific regulatory regions. Epigenomic and statistical fine-mapping most strongly supported 50 putative risk genes. Of these, 39 were expressed robustly in mouse cochlea and 16 were enriched specifically in sensory hair cells. These results reveal new risk loci and risk genes for hearing difficulty and suggest an important role for altered gene regulation in the cochlear sensory epithelium.

miR-143 Regulates Lysosomal Enzyme Transport across the Blood-Brain Barrier and Transforms CNS Treatment for Mucopolysaccharidosis Type I.

During brain maturation, cation-independent mannose-6-phosphate receptor (CI-MPR), a key transporter for lysosomal hydrolases, decreases significantly on the blood-brain barrier (BBB). Such a phenomenon leads to poor brain penetration of therapeutic enzymes and subsequent failure in reversing neurological complications in patients with neuropathic lysosomal storage diseases (nLSDs), such as Hurler syndrome (severe form of mucopolysaccharidosis type I [MPS I]). In this study, we discover that upregulation of microRNA-143 (miR-143) contributes to the decline of CI-MPR on the BBB during development. Gain- and loss-of-function studies showed that miR-143 inhibits CI-MPR expression and its transport function in human endothelial cells in vitro. Genetic removal of miR-143 in MPS I mice enhances CI-MPR expression and improves enzyme transport across the BBB, leading to brain metabolic correction, pathology normalization, and correction of neurological functional deficits 5 months after peripheral protein delivery at clinically relevant levels that derived from erythroid/megakaryocytic cells via hematopoietic stem cell-mediated gene therapy, when otherwise no improvement was observed in MPS I mice at a parallel setting. These studies not only uncover a novel role of miR-143 as an important modulator for the developmental decline of CI-MPR on the BBB, but they also demonstrate the functional significance of depleting miR-143 for "rescuing" BBB-anchored CI-MPR on advancing CNS treatment for nLSDs.

A cell type-specific approach to elucidate the role of miR-96 in inner ear hair cells.

Introduction: Mutations in microRNA-96 (miR-96), a microRNA expressed within the hair cells (HCs) of the inner ear, result in progressive hearing loss in both mouse models and humans. In this study, we present the first HC-specific RNA-sequencing (RNA-seq) dataset from newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Methods: Bulk RNA-seq was performed on HCs of newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Differentially expressed gene analysis was conducted on Mir96Dmdo homozygous mutant HCs compared to wildtype littermate controls, followed by GO term and protein-protein interaction analysis on these differentially expressed genes. Results: We identify 215 upregulated and 428 downregulated genes in the HCs of the Mir96Dmdo homozygous mutant mice compared to their wildtype littermate controls. Many of the significantly downregulated genes in Mir96Dmdo homozygous mutant HCs have established roles in HC development and/or known roles in deafness including Myo15a, Myo7a, Ush1c, Gfi1, and Ptprq and have enrichment in gene ontology (GO) terms with biological functions such as sensory perception of sound. Interestingly, upregulated genes in Mir96Dmdo homozygous mutants, including possible miR-96 direct targets, show higher wildtype expression in supporting cells compared to HCs. Conclusion: Our data further support a role for miR-96 in HC development, possibly as a repressor of supporting cell transcriptional programs in HCs. The HC-specific Mir96Dmdo RNA-seq data set generated from this manuscript are now publicly available in a dedicated profile in the gene expression analysis resource (gEAR-https://umgear.org/p?l=miR96).

Spatially distinct otic mesenchyme cells show molecular and functional heterogeneity patterns before hearing onset.

The cochlea consists of diverse cellular populations working in harmony to convert mechanical stimuli into electrical signals for the perception of sound. Otic mesenchyme cells (OMCs), often considered a homogeneous cell type, are essential for normal cochlear development and hearing. Despite being the most numerous cell type in the developing cochlea, OMCs are poorly understood. OMCs are known to differentiate into spatially and functionally distinct cell types, including fibrocytes of the lateral wall and spiral limbus, modiolar osteoblasts, and specialized tympanic border cells of the basilar membrane. Here, we show that OMCs are transcriptionally and functionally heterogeneous and can be divided into four distinct populations that spatially correspond to OMC-derived cochlear structures. We also show that this heterogeneity and complexity of OMCs commences during early phases of cochlear development. Finally, we describe the cell-cell communication network of the developing cochlea, inferring a major role for OMC in outgoing signaling.

Pou3f4-expressing otic mesenchyme cells promote spiral ganglion neuron survival in the postnatal mouse cochlea.

During inner ear development, primary auditory neurons named spiral ganglion neurons (SGNs) are surrounded by otic mesenchyme cells, which express the transcription factor Pou3f4. Mutations in Pou3f4 are associated with DFNX2, the most common form of X-linked deafness and typically include developmental malformations of the middle ear and inner ear. It is known that interactions between Pou3f4-expressing mesenchyme cells and SGNs are important for proper axon bundling during development. However, Pou3f4 continues to be expressed through later phases of development, and potential interactions between Pou3f4 and SGNs during this period had not been explored. To address this, we documented Pou3f4 protein expression in the early postnatal mouse cochlea and compared SGNs in Pou3f4 knockout mice and littermate controls. In Pou3f4y/- mice, SGN density begins to decline by the end of the first postnatal week, with approximately 25% of SGNs ultimately lost. This period of SGN loss in Pou3f4y/- cochleae coincides with significant elevations in SGN apoptosis. Interestingly, this period also coincides with the presence of a transient population of Pou3f4-expressing cells around and within the spiral ganglion. To determine if Pou3f4 is normally required for SGN peripheral axon extension into the sensory domain, we used a genetic sparse labeling approach to track SGNs and found no differences compared with controls. We also found that Pou3f4 loss did not lead to changes in the proportions of Type I SGN subtypes. Overall, these data suggest that otic mesenchyme cells may play a role in maintaining SGN populations during the early postnatal period.