ABSTRACT
OBJECTIVES: Diabetes is closely linked to hearing loss, yet the exact mechanisms remain unclear. Cochlear stria vascularis and pericytes (PCs) are crucial for hearing. This study investigates whether high glucose induces apoptosis in the cochlear stria vascularis and pericytes via elevated ROS levels due to oxidative stress, impacting hearing loss. METHODS: We established a type II diabetes model in C57BL/6J mice and used auditory brainstem response (ABR), Evans blue staining, HE staining, immunohistochemistry, and immunofluorescence to observe changes in hearing, blood-labyrinth barrier (BLB) permeability, stria vascularis morphology, and apoptosis protein expression. Primary cultured stria vascularis pericytes were subjected to high glucose, and apoptosis levels were assessed using flow cytometry, Annexin V-FITC, Hoechst 33342 staining, Western blot, Mitosox, and JC-1 probes. RESULTS: Diabetic mice showed decreased hearing thresholds, reduced stria vascularis density, increased oxidative stress, cell apoptosis, and decreased antioxidant levels. High glucose exposure increased apoptosis and ROS content in pericytes, while mitochondrial membrane potential decreased, with AIF and cytochrome C (CytC) released from mitochondria to the cytoplasm. Adding oxidative scavengers reduced AIF and CytC release, decreasing pericyte apoptosis. DISCUSSION: Hyperglycemia may induce mitochondrial apoptosis of cochlear stria vascularis pericytes through oxidative stress.
Subject(s)
Apoptosis Inducing Factor , Apoptosis , Cytochromes c , Hyperglycemia , Mice, Inbred C57BL , Mitochondria , Oxidative Stress , Pericytes , Proto-Oncogene Proteins c-bcl-2 , Reactive Oxygen Species , Stria Vascularis , Animals , Pericytes/metabolism , Pericytes/drug effects , Pericytes/pathology , Stria Vascularis/metabolism , Stria Vascularis/pathology , Mice , Reactive Oxygen Species/metabolism , Mitochondria/metabolism , Cytochromes c/metabolism , Apoptosis Inducing Factor/metabolism , Hyperglycemia/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Male , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Cochlea/metabolism , Cochlea/pathologyABSTRACT
Sgms1 encodes sphingomyelin synthase 1, an enzyme in the sphingosine-1-phosphate signalling pathway, and was previously reported to underlie hearing impairment in the mouse. A new mouse allele, Sgms1tm1a, unexpectedly showed normal Auditory Brainstem Response thresholds. We found that the Sgms1tm1a mutation led to incomplete knockdown of transcript to 20 % of normal values, which was enough to support normal hearing. The Sgms1tm1b allele was generated by knocking out exon 7, leading to a complete lack of detectable transcript in the inner ear. Sgms1tm1b homozygotes showed largely normal auditory brainstem response thresholds at first, followed by progressive loss of sensitivity until they showed severe impairment at 6 months old. The endocochlear potential was consistently reduced in Sgms1tm1b mutants at 3, 4 and 8 weeks old, to around 80 mV compared with around 120 mV in control littermates. The stria vascularis showed a characteristic irregularity of marginal cell surfaces and patchy loss of Kcnq1 expression at their apical membrane, and expression analysis of the lateral wall suggested that marginal cells were the most likely initial site of dysfunction in the mutants. Finally, significant association of auditory thresholds with DNA markers within and close to the human SGMS1 gene were found in the 1958 Birth Cohort, suggesting that SGMS1 variants may play a role in the range of hearing abilities in the human population.
Subject(s)
Evoked Potentials, Auditory, Brain Stem , Hearing Loss , Stria Vascularis , Transferases (Other Substituted Phosphate Groups) , Animals , Female , Male , Mice , Auditory Threshold , Cochlea/physiopathology , Cochlea/metabolism , Disease Models, Animal , Genetic Predisposition to Disease , Hearing/genetics , Hearing Loss/genetics , Hearing Loss/physiopathology , Homozygote , Mice, Inbred C57BL , Mice, Knockout , Mutation , Phenotype , Stria Vascularis/metabolism , Transferases (Other Substituted Phosphate Groups)/geneticsABSTRACT
Hearing crucially depends on cochlear ion homeostasis as evident from deafness elicited by mutations in various genes encoding cation or anion channels and transporters. Ablation of ClCK/barttin chloride channels causes deafness by interfering with the positive electrical potential of the endolymph, but roles of other anion channels in the inner ear have not been studied. Here we report the intracochlear distribution of all five LRRC8 subunits of VRAC, a volume-regulated anion channel that transports chloride, metabolites, and drugs such as the ototoxic anti-cancer drug cisplatin, and explore its physiological role by ablating its subunits. Sensory hair cells express all LRRC8 isoforms, whereas only LRRC8A, D and E were found in the potassium-secreting epithelium of the stria vascularis. Cochlear disruption of the essential LRRC8A subunit, or combined ablation of LRRC8D and E, resulted in cochlear degeneration and congenital deafness of Lrrc8a-/- mice. It was associated with a progressive degeneration of the organ of Corti and its innervating spiral ganglion. Like disruption of ClC-K/barttin, loss of VRAC severely reduced the endocochlear potential. However, the mechanism underlying this reduction seems different. Disruption of VRAC, but not ClC-K/barttin, led to an almost complete loss of Kir4.1 (KCNJ10), a strial K+ channel crucial for the generation of the endocochlear potential. The strong downregulation of Kir4.1 might be secondary to a loss of VRAC-mediated transport of metabolites regulating inner ear redox potential such as glutathione. Our study extends the knowledge of the role of cochlear ion transport in hearing and ototoxicity.
Subject(s)
Membrane Proteins , Mice, Knockout , Animals , Mice , Membrane Proteins/metabolism , Membrane Proteins/genetics , Hearing , Cochlea/metabolism , Cochlea/pathology , Hair Cells, Auditory/metabolism , Hair Cells, Auditory/pathology , Stria Vascularis/metabolism , Stria Vascularis/pathology , Deafness/metabolism , Deafness/pathology , Deafness/genetics , Voltage-Dependent Anion Channels/metabolism , Voltage-Dependent Anion Channels/geneticsABSTRACT
Age-related hearing loss (HL), or presbycusis, is a complex and heterogeneous condition, affecting a significant portion of older adults and involving various interacting mechanisms. Metabolic presbycusis, a type of age-related HL, is characterized by the dysfunction of the stria vascularis, which is crucial for maintaining the endocochlear potential necessary for hearing. Although attention on metabolic presbycusis has waned in recent years, research continues to identify strial pathology as a key factor in age-related HL. This narrative review integrates past and recent research, bridging findings from animal models and human studies, to examine the contributions of the stria vascularis to age-related HL. It provides a brief overview of the structure and function of the stria vascularis and then examines mechanisms contributing to age-related strial dysfunction, including altered ion transport, changes in pigmentation, inflammatory responses, and vascular atrophy. Importantly, this review outlines the contribution of metabolic mechanisms to age-related HL, highlighting areas for future research. It emphasizes the complex interdependence of metabolic and sensorineural mechanisms in the pathology of age-related HL and highlights the importance of animal models in understanding the underlying mechanisms. The comprehensive and mechanistic investigation of all factors contributing to age-related HL, including cochlear metabolic dysfunction, remains crucial to identifying the underlying mechanisms and developing personalized, protective, and restorative treatments.
Subject(s)
Aging , Presbycusis , Stria Vascularis , Humans , Stria Vascularis/metabolism , Stria Vascularis/pathology , Animals , Presbycusis/metabolism , Presbycusis/pathology , Presbycusis/physiopathology , Aging/metabolism , Aging/physiology , Cochlea/metabolism , Cochlea/pathology , Hearing Loss/metabolism , Hearing Loss/pathologyABSTRACT
Studies have highlighted oxidative damage in the inner ear as a critical pathological basis for sensorineural hearing loss, especially the presbycusis. Poly(ADP-ribose) polymerase-1 (PARP1) activation responds to oxidative stress-induced DNA damage with pro-repair and pro-death effects resembling two sides of the same coin. PARP1-related cell death, known as parthanatos, whose underlying mechanisms are attractive research hotspots but remain to be clarified. In this study, we observed that aged rats showed stria vascularis degeneration and oxidative damage, and PARP1-dependent cell death was prominent in age-related cochlear disorganization and dysfunction. Based on oxidative stress model of primary cultured stria marginal cells (MCs), we revealed that upregulated PARP1 and PAR (Poly(ADP-ribose)) polymers are responsible for MCs oxidative death with high mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse, while inhibition of PARP1 ameliorated the adverse outcomes. Importantly, the PARylation of apoptosis-inducing factor (AIF) is essential for its conformational change and translocation, which subsequently causes DNA break and cell death. Concretely, the interaction of PAR and truncated AIF (tAIF) is the mainstream in the parthanatos pathway. We also found that the effects of AIF cleavage and release were achieved through calpain activity and mPTP opening, both of which could be regulated by PARP1 via mediation of mitochondria Ca2+ concentration. In conclusion, the PAR-Ca2+-tAIF signaling pathway in parthanatos contributes to the oxidative stress damage observed in MCs. Targeting PAR-Ca2+-tAIF might be a potential therapeutic strategy for the early intervention of presbycusis and other oxidative stress-associated sensorineural deafness.
Subject(s)
Apoptosis Inducing Factor , Calcium , Oxidative Stress , Poly (ADP-Ribose) Polymerase-1 , Presbycusis , Animals , Apoptosis Inducing Factor/metabolism , Apoptosis Inducing Factor/genetics , Rats , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , Calcium/metabolism , Presbycusis/metabolism , Presbycusis/pathology , Presbycusis/genetics , Parthanatos/genetics , Membrane Potential, Mitochondrial , Stria Vascularis/metabolism , Stria Vascularis/pathology , Apoptosis , Mitochondrial Permeability Transition Pore/metabolism , Mitochondria/metabolism , Mitochondria/pathology , Rats, Sprague-Dawley , DNA Damage , Aging/metabolism , Aging/pathology , Cochlea/metabolism , Cochlea/pathology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Male , Humans , Cells, CulturedABSTRACT
A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.
Subject(s)
Cell Differentiation , Cochlea , Patched-1 Receptor , Stria Vascularis , Patched-1 Receptor/metabolism , Patched-1 Receptor/genetics , Animals , Mice , Stria Vascularis/metabolism , Stria Vascularis/cytology , Cochlea/metabolism , Cochlea/embryology , Cochlea/cytology , Signal Transduction , Zinc Finger Protein Gli2/metabolism , Zinc Finger Protein Gli2/genetics , Hedgehog Proteins/metabolism , Hedgehog Proteins/geneticsABSTRACT
BACKGROUND: Water homeostasis is essential for inner ear function. Several aquaporins (AQPs), which are water transport proteins in the cell or plasma membrane, have been reported in the lateral wall of the rat inner ear (cochlea). However, the presence of AQP-10, -11 and -12 has not been reported in the rat stria vascularis (SV) to date. AIMS/OBJECTIVES: We have aimed to clarify the expression of AQP-10, -11 and -12 in the cochlea lateral wall. MATERIALS AND METHODS: Using Wistar rats, we examined the expression of AQP-10, -11 and -12 in the cochlea lateral wall using molecular approaches and immunohistochemistry. RESULTS: AQP-11 was molecular biologically expressed, but the expression of AQP-10 and -12 was not observed. Immunohistochemically, AQP-11 was diffusely localized in the basal cells and marginal cells of the rat SV but was not expressed at the apical site of marginal cells with double staining. The expression of AQP-10 and -12 was not observed. CONCLUSIONS AND SIGNIFICANCE: Only AQP-11 was expressed in the basal cells and marginal cells, but it was not expressed at the apical site of marginal cells. Based on this study, AQP-11 may not have an important role in water flux between the perilymph and endolymph.
Subject(s)
Aquaporins , Rats, Wistar , Stria Vascularis , Animals , Rats , Aquaporins/metabolism , Immunohistochemistry , Stria Vascularis/metabolismABSTRACT
BACKGROUND: The stria vascularis (SV), located in the lateral wall of the cochlea, maintains cochlear fluid homeostasis and mechanoelectrical transduction (MET) activity required for sound wave conduction. The pathogenesis of a number of human inheritable deafness syndromes, age related hearing loss, drug-induced ototoxicity and noise-induced hearing loss results from the morphological changes and functional impairments in the development of the SV. In this study, we investigate the implications of intercellular communication within the SV in the pathogenesis of sensorineural hearing loss (SNHL). We aim to identify commonly regulated signaling pathways using publicly available single-cell transcriptomic sequencing (scRNA-seq) datasets. METHODS: We analyzed scRNA-seq data, which was derived from studying the cochlear SV in mice with SNHL compared to normal adult mice. After quality control and filtering, we obtained the major cellular components of the mouse cochlear SV and integrated the data. Using Seurat's FindAllMarkers and FindMarkers packages, we searched for novel conservative genes and differential genes. We employed KEGG and GSEA to identify molecular pathways that are commonly altered among different types of SNHL. We utilized pySCENIC to discover new specific regulatory factors in SV subpopulation cells. With the help of CellChat, we identified changes in subpopulation cells showing similar trends across different SNHL types and their alterations in intercellular communication pathways. RESULTS: Through the analysis of the integrated data, we discovered new conserved genes to SV specific cells and identified common downregulated pathways in three types of SNHL. The enriched genes for these pathways showing similar trends are primarily associated with the Electron Transport Chain, related to mitochondrial energy metabolism. Using the CellChat package, we further found that there are shared pathways in the incoming signaling of specific intermediate cells in SNHL, and these pathways have common upstream regulatory transcription factor of Nfe2l2. Combining the results from pySCENIC and CellChat, we predicted the transcription factor Nfe2l2 as an upstream regulatory factor for multiple shared cellular pathways in IC. Additionally, it serves as an upstream factor for several genes within the Electron Transport Chain. CONCLUSION: Our bioinformatics analysis has revealed that downregulation of the mitochondrial electron transport chain have been observed in various conditions of SNHL. E2f1, Esrrb, Runx1, Yy1, and Gata2 could serve as novel important common TFs regulating the electron transport chain. Adm has emerged as a potential new marker gene for intermediate cells, while Itgb5 and Tesc show promise as potential new marker genes for marginal cells in the SV. These findings offer a new perspective on SV lesions in SNHL and provide additional theoretical evidence for the same drug treatment and prevention of different pathologies of SNHL.
Subject(s)
Hearing Loss, Sensorineural , Stria Vascularis , Adult , Humans , Animals , Mice , Stria Vascularis/metabolism , Stria Vascularis/pathology , Single-Cell Gene Expression Analysis , Hearing Loss, Sensorineural/genetics , Hearing Loss, Sensorineural/pathology , Cochlea , Transcription Factors/metabolismABSTRACT
The stria vascularis (SV) is a stratified epithelium in the lateral wall of the mammalian cochlea, responsible for both endolymphatic ion homeostasis and generation of the endocochlear potential (EP) critical for normal hearing. The SV has three layers consisting predominantly of basal, intermediate, and marginal cells. Intermediate and marginal cells form an intricate interdigitated network of cell projections making discrimination of the cells challenging. To enable intermediate cell visualization, we engineered by BAC transgenesis, reporter mouse lines expressing ZsGreen fluorescent protein under the control of Kcnj10 promoter and regulatory sequences. Kcnj10 encodes KCNJ10 protein (also known as Kir4.1 or Kir1.2), an ATP-sensitive inwardly-rectifying potassium channel critical to EP generation, highly expressed in SV intermediate cells. In these transgenic mice, ZsGreen fluorescence mimics Kcnj10 endogenous expression in the cochlea and was detected in the intermediate cells of the SV, in the inner phalangeal cells, Hensen's, Deiters' and pillar cells, in a subset of spiral ganglion neurons, and in glial cells. We show that expression of the transgene in hemizygous mice does not alter auditory function, nor EP. These transgenic Tg(Kcnj10-ZsGreen) mice allow live and fixed tissue visualization of ZsGreen-expressing intermediate cells and will facilitate future studies of stria vascularis cell function.
Subject(s)
Ear, Inner , Potassium Channels, Inwardly Rectifying , Animals , Mice , Stria Vascularis/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Cochlea/metabolism , Ear, Inner/metabolism , Mice, Transgenic , Mammals/metabolismABSTRACT
TRPC channels are critical players in cochlear hair cells and sensory neurons, as demonstrated in animal experiments. However, evidence for TRPC expression in the human cochlea is still lacking. This reflects the logistic and practical difficulties in obtaining human cochleae. The purpose of this study was to detect TRPC6, TRPC5 and TRPC3 in the human cochlea. Temporal bone pairs were excised from ten body donors, and the inner ear was first assessed based on computed tomography scans. Decalcification was then performed using 20% EDTA solutions. Immunohistochemistry with knockout-tested antibodies followed. The organ of Corti, the stria vascularis, the spiral lamina, spiral ganglion neurons and cochlear nerves were specifically stained. This unique report of TRPC channels in the human cochlea supports the hypothesis of the potentially critical role of TRPC channels in human cochlear health and disease which has been suggested in previous rodent experiments.
Subject(s)
Cochlea , Ear, Inner , Animals , Humans , Immunohistochemistry , Cochlea/metabolism , Ear, Inner/metabolism , Stria Vascularis/metabolism , HearingABSTRACT
BACKGROUND: The primary pathological alterations of Pendred syndrome are endolymphatic pH acidification and luminal enlargement of the inner ear. However, the molecular contributions of specific cell types remain poorly characterized. Therefore, we aimed to identify pH regulators in pendrin-expressing cells that may contribute to the homeostasis of endolymph pH and define the cellular pathogenic mechanisms that contribute to the dysregulation of cochlear endolymph pH in Slc26a4-/- mice. METHODS: We used single-cell RNA sequencing to identify both Slc26a4-expressing cells and Kcnj10-expressing cells in wild-type (WT, Slc26a4+/+) and Slc26a4-/- mice. Bioinformatic analysis of expression data confirmed marker genes defining the different cell types of the stria vascularis. In addition, specific findings were confirmed at the protein level by immunofluorescence. RESULTS: We found that spindle cells, which express pendrin, contain extrinsic cellular components, a factor that enables cell-to-cell communication. In addition, the gene expression profile informed the pH of the spindle cells. Compared to WT, the transcriptional profiles in Slc26a4-/- mice showed downregulation of extracellular exosome-related genes in spindle cells. Immunofluorescence studies in spindle cells of Slc26a4-/- mice validated the increased expression of the exosome-related protein, annexin A1, and the clathrin-mediated endocytosis-related protein, adaptor protein 2. CONCLUSION: Overall, cell isolation of stria vascularis from WT and Slc26a4-/- samples combined with cell type-specific transcriptomic analyses revealed pH-dependent alternations in spindle cells and intermediate cells, inspiring further studies into the dysfunctional role of stria vascularis cells in SLC26A4-related hearing loss.
Subject(s)
Deafness , Stria Vascularis , Mice , Animals , Stria Vascularis/metabolism , Stria Vascularis/pathology , Anion Transport Proteins/genetics , Anion Transport Proteins/metabolism , Cochlea/metabolism , Cochlea/pathology , Deafness/genetics , Sulfate Transporters/genetics , RNA/metabolismABSTRACT
The stria vascularis (SV) contributes to cochlear homeostasis and consists of three layers, one of which contains the blood-labyrinthic barrier (BLB), with a large number of bovine cochlear pericytes (BCPs). Cisplatin is a chemotherapeutic drug that can damage the SV and cause hearing loss. In this study, cell viability, proliferation rate, cytotoxicity and reactive oxygen species production were evaluated. The protein content of phospho-extracellular signal-regulated kinases (ERK) 1/2, total ERK 1/2, phospho-cytosolic phospholipase A2 (cPLA2), total cPLA2 and cyclooxygenase 2 (COX-2) and the release of prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) from BCPs were analyzed. Finally, the protective effect of platelet-derived growth factor (PDGF-BB) on BCPs treated with cisplatin was investigated. Cisplatin reduced viability and proliferation, activated ERK 1/2, cPLA2 and COX-2 expression and increased PGE2 and VEGF release; these effects were reversed by Dexamethasone. The presence of PDGF-BB during the treatment with cisplatin significantly increased the proliferation rate. No studies on cell regeneration in ear tissue evaluated the effect of the PDGF/Dex combination. The aim of this study was to investigate the effects of cisplatin on cochlear pericytes and propose new otoprotective agents aimed at preventing the reduction of their vitality and thus maintaining the BLB structure.
Subject(s)
Pericytes , Stria Vascularis , Animals , Cattle , Stria Vascularis/metabolism , Cisplatin/toxicity , Cisplatin/metabolism , Vascular Endothelial Growth Factor A/metabolism , Becaplermin/metabolism , Cyclooxygenase 2/metabolism , Dinoprostone/metabolism , Platelet-Derived Growth Factor/metabolismABSTRACT
Aminoglycosides (AGs) are commonly used antibiotics that cause deafness through the irreversible loss of cochlear sensory hair cells (HCs). How AGs enter the cochlea and then target HCs remains unresolved. Here, we performed time-lapse multicellular imaging of cochlea in live adult hearing mice via a chemo-mechanical cochleostomy. The in vivo tracking revealed that systemically administered Texas Red-labeled gentamicin (GTTR) enters the cochlea via the stria vascularis and then HCs selectively. GTTR uptake into HCs was completely abolished in transmembrane channel-like protein 1 (TMC1) knockout mice, indicating mechanotransducer channel-dependent AG uptake. Blockage of megalin, the candidate AG transporter in the stria vascularis, by binding competitor cilastatin prevented GTTR accumulation in HCs. Furthermore, cilastatin treatment markedly reduced AG-induced HC degeneration and hearing loss in vivo. Together, our in vivo real-time tracking of megalin-dependent AG transport across the blood-labyrinth barrier identifies new therapeutic targets for preventing AG-induced ototoxicity.
Subject(s)
Anti-Bacterial Agents/metabolism , Gentamicins/metabolism , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , Animals , Anti-Bacterial Agents/toxicity , Biological Transport , Cilastatin/pharmacology , Endolymph/metabolism , Gentamicins/toxicity , Hair Cells, Auditory/drug effects , Hair Cells, Auditory/metabolism , Hearing/drug effects , Low Density Lipoprotein Receptor-Related Protein-2/antagonists & inhibitors , Mice , Stria Vascularis/metabolismABSTRACT
Mitochondrial dysfunction is associated with aging and age-related hearing loss (AHL). However, the precise mechanisms underlying the pathophysiology of hearing loss remain unclear. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) enables efficient intramitochondrial translation by catalyzing the deposition of 2-methylthio modifications on mitochondrial tRNAs. Here we investigated the effect of defective mitochondrial protein translation on hearing and AHL in a Cdk5rap1 deficiency C57BL/6 mouse model. Compared to control C57BL/6 mice, Cdk5rap1-knockout female mice displayed hearing loss phenotypically similar to AHL from an early age. The premature hearing loss in Cdk5rap1-knockout mice was associated with the degeneration of the spiral ligament and reduction of endocochlear potentials following the loss of auditory sensory cells. Furthermore, cultured primary mouse embryonic fibroblasts displayed early onset of cellular senescence associated with high oxidative stress and cell death. These results indicate that the CDK5RAP1 deficiency-induced defective mitochondrial translation might cause early hearing loss through the induction of cellular senescence and cochlear dysfunction in the inner ear. Our results suggest that the accumulation of dysfunctional mitochondria might promote AHL progression. Furthermore, our findings suggest that mitochondrial dysfunction and dysregulated mitochondrial tRNA modifications mechanistically cause AHL. Understanding the mechanisms underlying AHL will guide future clinical investigations and interventions in the attempt to mitigate the consequences of AHL.
Subject(s)
Aging/pathology , Cell Cycle Proteins/deficiency , Mitochondrial Proteins/genetics , Presbycusis/genetics , Sulfur Group Transferases/genetics , Action Potentials , Animals , Apoptosis , Cell Cycle Proteins/metabolism , Female , Fibroblasts/metabolism , Hair Cells, Auditory/metabolism , Metabolome , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/metabolism , Mitochondria/ultrastructure , Phenotype , Reactive Oxygen Species/metabolism , Spiral Ganglion/metabolism , Spiral Ligament of Cochlea/metabolism , Stress, Physiological , Stria Vascularis/metabolismABSTRACT
Purpose: Slc26a4-/- mice exhibit severer defects in the development of the cochlea and develop deafness, while the underlying mechanisms responsible for these effects remain unclear. Our study was to investigate the potential mechanism linking SLC26A4 deficiency to hearing loss. Materials and Methods: RNA sequencing was applied to analyze the differential gene expression of the stria vascularis (SV) from wildtype and Slc26a4-/- mice. GO and KEGG pathway analysis were performed. Quantitative RT-PCR was applied to validate the expression of candidate genes affected by Slc26a4. ELISA and immunofluorescence technique were used to detect the homocysteine (Hcy) level in serum, brain, and SV, respectively. Results: 183 upregulated genes and 63 downregulated genes were identified in the SV associated with Slc26a4 depletion. Transcriptomic profiling revealed that Slc26a4 deficiency significantly affected the expression of genes associated with cell adhesion, transmembrane transport, and the biogenesis of multicellular organisms. The SV from Slc26a4-/- mice exhibited a higher expression of Bhmt mRNAs, as well as altered homocysteine (Hcy) metabolism. Conclusions: The altered expression of Bhmt results in a dramatic change in multiple biochemical reactions and a disruption of nutrient homeostasis in the endolymph which may contribute to hearing loss of Slc26a4 knockout mouse.
Subject(s)
Goiter, Nodular/genetics , Hearing Loss, Sensorineural/genetics , Homocysteine/metabolism , Stria Vascularis/metabolism , Animals , Betaine-Homocysteine S-Methyltransferase/genetics , Cell Adhesion , Disease Models, Animal , Gene Expression Regulation , Goiter, Nodular/pathology , Hearing Loss, Sensorineural/pathology , Mice , Mice, Knockout , RNA/genetics , Signal Transduction/genetics , Stria Vascularis/pathology , Sulfate Transporters/genetics , TranscriptomeABSTRACT
In the inner ear, the neural crest gives rise to the glia of the VIII ganglion and two types of melanocytic cells: The pigmented cells of the vestibular system and intermediate cells of the stria vascularis. We analyzed the transcriptome of neonatal intermediate cells in an effort to better understand the development of the stria vascularis. We found that the expression of endothelin receptor B, which is essential for melanocyte development, persists in intermediate cells long after birth. In contrast, skin melanocytes rapidly downregulate the expression of EdnrB. Our findings suggest that endothelins might have co-opted new functions in the inner ear during evolution of the auditory organ.
Subject(s)
Cochlea/metabolism , Ear, Inner/metabolism , Melanocytes/metabolism , Receptor, Endothelin B/metabolism , Skin/metabolism , Transcriptome , Animals , Cochlea/cytology , Ear, Inner/cytology , Gene Expression Regulation, Developmental , Melanocytes/cytology , Mice , Mice, Inbred C57BL , Receptor, Endothelin B/genetics , Skin/cytology , Stria Vascularis/cytology , Stria Vascularis/metabolism , Vestibular System/cytology , Vestibular System/metabolismABSTRACT
Na+, K+-ATPase (Na,K-ATPase) is an ubiquitous enzyme in the inner ear and a key factor in the maintenance of the osmotic gradient of the endolymph. This study uses Na,K-ATPase α1 subunit immunoreactivity (IR) to identify cellular structures in the normal and disease human cochlea. Formalin-fixed celloidin-embedded (FFCE) human temporal bone sections were immunoreacted with mouse monoclonal antibodies against Na,K-ATPase α1 subunit. Na,K-ATPase α1 IR was examined in the cochlea of 30 patients: four with normal hearing, 5 with Meniere's disease, and 21 with other inner ear diseases: 11 male, 19 female; ages 42 to 96 years-old (yo), average age of 77 yo. Na,K-ATPase α1 IR area was quantified using the ImageJ software program. Na,K-ATPase α1 IR was located in the stria vascularis, and in type I, II and IV fibrocytes of the spiral ligament in the cochlea from patients with normal hearing. Na,K-ATPase α1 IR was seen in Deiters's cells and inner phalangeal cells of the organ of Corti. Na,K-ATPase α1 IR was present in satellite cells that surround the neurons of the spiral ganglia. In the inner ear of pathological specimens, Na,K-ATPase IR area was decreased (compared to the normal) in the stria vascularis, supporting cells in the organ of Corti and satellite cells of the spiral ganglia. These results show that Na,K-ATPase α1 IR is a good marker to identify cellular structures of the human inner ear and may be used to study cellular changes in the cochlea associated with aging and disease. The ubiquitous localization of Na,K-ATPase α1 in the human cochlea is consistent with the Na,K-ATPase role in ionic homeostasis and osmolarity, similar to that seen in animal models.
Subject(s)
Ear, Inner , Adult , Aged , Aged, 80 and over , Animals , Cochlea/metabolism , Ear, Inner/metabolism , Endolymph/metabolism , Female , Humans , Male , Mice , Middle Aged , Sodium-Potassium-Exchanging ATPase/metabolism , Stria Vascularis/metabolismABSTRACT
The stria vascularis (SV) in the cochlea generates and maintains the endocochlear potential, thereby playing a pivotal role in normal hearing. Knowing transcriptional profiles and gene regulatory networks of SV cell types establishes a basis for studying the mechanism underlying SV-related hearing loss. While we have previously characterized the expression profiles of major SV cell types in the adult mouse, transcriptional profiles of rare SV cell types remained elusive due to the limitation of cell capture in single-cell RNA-Seq. The role of these rare cell types in the homeostatic function of the adult SV remain largely undefined. In this study, we performed single-nucleus RNA-Seq on the adult mouse SV in conjunction with sample preservation treatments during the isolation steps. We distinguish rare SV cell types, including spindle cells and root cells, from other cell types, and characterize their transcriptional profiles. Furthermore, we also identify and validate novel specific markers for these rare SV cell types. Finally, we identify homeostatic gene regulatory networks within spindle and root cells, establishing a basis for understanding the functional roles of these cells in hearing. These novel findings will provide new insights for future work in SV-related hearing loss and hearing fluctuation.
Subject(s)
Biomarkers/analysis , Cochlea/metabolism , Gene Expression Regulation , RNA-Seq/methods , Stria Vascularis/metabolism , Transcriptome , Animals , Female , Male , MiceABSTRACT
Previous work demonstrates that the hearing loss in Alport mice is caused by defects in the stria vascularis. As the animals age, progressive thickening of strial capillary basement membranes (SCBMs) occurs associated with elevated levels of extracellular matrix expression and hypoxia-related gene and protein expression. These conditions render the animals susceptible to noise-induced hearing loss. In an effort to develop a more comprehensive understanding of how the underlying mutation in the COL4A3 gene influences homeostasis in the stria vascularis, we performed vascular permeability studies combined with RNA-seq analysis using isolated stria vascularis from 7-week old wild-type and Alport mice on the 129 Sv background. Alport SCBMs were found to be less permeable than wild-type littermates. RNA-seq and bioinformatics analysis revealed 68 genes were induced and 61 genes suppressed in the stria from Alport mice relative to wild-type using a cut-off of 2-fold. These included pathways involving transcription factors associated with the regulation of pro-inflammatory responses as well as cytokines, chemokines, and chemokine receptors that are up- or down-regulated. Canonical pathways included modulation of genes associated with glucose and glucose-1-PO4 degradation, NAD biosynthesis, histidine degradation, calcium signaling, and glutamate receptor signaling (among others). In all, the data point to the Alport stria being in an inflammatory state with disruption in numerous metabolic pathways indicative of metabolic stress, a likely cause for the susceptibility of Alport mice to noise-induced hearing loss under conditions that do not cause permanent hearing loss in age/strain-matched wild-type mice. The work lays the foundation for studies aimed at understanding the nature of strial pathology in Alport mice. The modulation of these genes under conditions of therapeutic intervention may provide important pre-clinical data to justify trials in humans afflicted with the disease.
Subject(s)
Gene Expression Regulation/genetics , Hearing Loss, Noise-Induced/metabolism , Nephritis, Hereditary/metabolism , Stria Vascularis/metabolism , Animals , Autoantigens/genetics , Autoantigens/metabolism , Basement Membrane/metabolism , Chemokines/genetics , Chemokines/metabolism , Collagen Type IV/genetics , Collagen Type IV/metabolism , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Down-Regulation , Extracellular Matrix/metabolism , Female , Glucose/genetics , Glucose/metabolism , Hearing Loss, Noise-Induced/genetics , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Male , Mice , Nephritis, Hereditary/genetics , Nephritis, Hereditary/pathology , RNA-Seq , Signal Transduction/genetics , Stria Vascularis/pathology , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome/genetics , Up-RegulationABSTRACT
Background: Waardenburg syndrome is an uncommon genetic condition characterized by at least some degree of congenital hearing loss and pigmentation deficiencies. However, the genetic pathway affecting the development of stria vascularis is not fully illustrated. Methods: The transcript profile of stria vascularis of Waardenburg syndrome was studied using Mitf-M mutant pig and mice models. Therefore, GO analysis was performed to identify the differential gene expression caused by Mitf-M mutation. Results: There were 113 genes in tyrosine metabolism, melanin formation, and ion transportations showed significant changes in pig models and 191 genes in mice models. In addition, there were some spice's specific gene changes in the stria vascularis in the mouse and porcine models. The expression of tight junction-associated genes, including Cadm1, Cldn11, Pcdh1, Pcdh19, and Cdh24 genes, were significantly higher in porcine models compared to mouse models. Vascular-related and ion channel-related genes in the stria vascularis were also shown significantly difference between the two species. The expression of Col2a1, Col3a1, Col11a1, and Col11a2 genes were higher, and the expression of Col8a2, Cd34, and Ncam genes were lower in the porcine models compared to mouse models. Conclusions: Our data suggests that there is a significant difference on the gene expression and function between these two models.