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Hearing Loss: Molecular Biological Insights

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: 20 April 2025 | Viewed by 4304

Special Issue Editor


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Guest Editor
Department of Biochemistry and Molecular Biology and Physioloy, Faculty of Medicine, Avda. Ramón y Cajal, 7, 47005 Valladolid, Spain
Interests: cochlea; inner ear; molecular mechanisms; neurodegeneration; regeneration; neuroprotection; aging; senescence

Special Issue Information

Dear Colleagues,

Disabling hearing loss is the most common sensory disorder, affecting over 5% of the world’s population and presenting alarmingly rising incidence rates. Obstacles to the development of therapeutics to treat these patients arise from the location of the cochlear tissue and the complexity that underlies the auditory function, which is regulated by a large number of genes and molecular pathways and is under the influence of environmental factors. These all act in concert to modify the severity and onset of disease; hearing problems may become apparent long after the harmful events have taken place, which further hinders the identification of the causing agents and prevents any efficacious treatment. Recent advancements in high-throughput screening approaches and single-cell transcriptomics, the development of novel tools to achieve the delivery of molecules into the inner ear, and the generation of new animal and human models of disease will all contribute to the identification of the molecular mechanisms leading to auditory disability and the elucidation of molecular targets for therapeutic strategies.

I am delighted to invite you to contribute to this Special Issue launched by the International Journal of Molecular Sciences on “Hearing Loss: Molecular Biological Insights”. The aim of this Special Issue is to provide a meeting point where scientists in the field of hearing may bring together their most recent discoveries on the molecular mechanisms leading to hearing disability as well as report on the latest advancements in molecular biology-based diagnostics and therapeutics for hearing loss. This Special Issue should help us gain an overview of the work that is currently being conducted to decipher the molecular basis of hearing and the impressive achievements that are already being reached, as well as spark the debate on the most promising leads for the development of novel therapies against this devastating disorder.

In this Special Issue, original research articles and reviews are welcome. Research areas may include (but are not limited to) the following:

  • Genetic and molecular bases of hearing loss;
  • Epigenetics of hearing loss;
  • Molecular biological techniques to study the structure and function of the auditory organ;
  • Multi-omic approaches for the elucidation of the molecular mechanisms underlying hearing loss;
  • Molecular biology of inner ear development;
  • Animal models and human organoid-based models to study the molecular biology of inner ear development and function;
  • Molecular mechanisms involved in age-related or drug-induced hearing loss;
  • Application of CRISPR/Cas and other molecular biology techniques to analyze the effect of altering molecular processes on inner ear development and function;
  • Diagnostic markers of hearing dysfunction;
  • Molecular pathways associated with otoprotection or inner ear cell regeneration;
  • Intracochlear delivery of molecular agents as a possible therapy against hearing loss.

I am looking forward to receiving your valuable contributions.

Dr. María Beatriz Durán-Alonso
Guest Editor

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Keywords

  • genetics
  • microRNA
  • epigenetics
  • CRISPR
  • multi-omics
  • presbycusis
  • ototoxicity
  • development
 

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Published Papers (4 papers)

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Research

17 pages, 4363 KiB  
Article
Molecular Characterization of Subdomain Specification of Cochlear Duct Based on Foxg1 and Gata3
by Yongjin Gil, Jiho Ryu, Hayoung Yang, Yechan Ma, Ki-Hoan Nam, Sung-Wuk Jang and Sungbo Shim
Int. J. Mol. Sci. 2024, 25(23), 12700; https://doi.org/10.3390/ijms252312700 - 26 Nov 2024
Viewed by 302
Abstract
The inner ear is one of the sensory organs of vertebrates and is largely composed of the vestibule, which controls balance, and the cochlea, which is responsible for hearing. In particular, a problem in cochlear development can lead to hearing loss. Although numerous [...] Read more.
The inner ear is one of the sensory organs of vertebrates and is largely composed of the vestibule, which controls balance, and the cochlea, which is responsible for hearing. In particular, a problem in cochlear development can lead to hearing loss. Although numerous studies have been conducted on genes involved in the development of the cochlea, many areas still need to be discovered regarding factors that control the patterning of the early cochlear duct. Herein, based on the dynamic expression pattern of FOXG1 in the apical and basal regions of the E13.5 cochlear duct, we identified detailed expression regions through an open-source analysis of single-cell RNA analysis data and demonstrated a clinical correlation with hearing loss. The distinct expression patterns of FOXG1 and GATA3 during the patterning process of the cochlear duct provide important clues to understanding how the fates of the apical and basal regions are divided. These results are expected to be extremely important not only for understanding the molecular mechanisms involved in the early development of the cochlear duct, but also for identifying potential genes that cause hearing loss. Full article
(This article belongs to the Special Issue Hearing Loss: Molecular Biological Insights)
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Figure 1

Figure 1
<p>Expressions of FOXG1 and GATA3 in the developing inner ear. At E9.5–E10.5, the otic vesicle begins to extend, and from E11.5, the cochlear duct begins to extend; the cross section of the cochlear duct is observed at E12.5 and E13.5. The section plane is indicated in the embryo image on the left. (<b>A</b>,<b>B</b>) At E9.5–E10.5, FOXG1 and GATA3 are expressed throughout the otic vesicle. (<b>C</b>) In the cochlea at E11.5, <span class="html-italic">Gata3</span> shows a change in expression toward the medial–lateral part, whereas <span class="html-italic">Foxg1</span> maintains global expression. (<b>D</b>) In the cochlea at E12.5, <span class="html-italic">Gata3</span> is expressed only in the dorsal region, whereas <span class="html-italic">Foxg1</span> continues to be expressed in both the dorsal and ventral regions. (<b>E</b>) At E13.5, three cochlear sections and nearby ganglia are visible. In all sections, <span class="html-italic">Gata3</span> is expressed at the base, <span class="html-italic">Foxg1</span> is expressed throughout the apical duct (asterisk in <b>E</b>), and only <span class="html-italic">Gata3</span> is expressed at the base. Scale bar = 100 μm. CD, cochlear duct; fac, facio-acoustic (VII-VIII) neural crest complex; GER, the greater epithelial ridge; LER, the lesser epithelial ridge; OV, otic vesicle; PHV, primary head vein; PIN, Pinna; SC, Spinal cord. (<b>F</b>) Quantitative analysis of changes in <span class="html-italic">Foxg1</span> expression at the base and apex of E13.5 cochlea. The cochlea was divided into the roof and floor, and the proportion of cells expressing <span class="html-italic">Foxg1</span> and <span class="html-italic">Gata3</span> in the corresponding region was shown (n = 5). At the apex, <span class="html-italic">Foxg1</span> expression was high at the roof and floor, but was decreased at the floor of the base compared to the apex. **** <span class="html-italic">p</span> ≤ 0.0001.</p>
Full article ">Figure 2
<p>Identification of inner-ear cell type. (<b>A</b>–<b>C</b>) UMAP plots for cell type (<b>A</b>), reference (<b>B</b>), and stage (<b>C</b>). (<b>D</b>) UMAP plots showing marker gene expression in individual clusters. (<b>E</b>) Dot plots showing the expressions of the top 5 marker genes and their proportion in individual cell clusters. Color gradient represents the average expression, and dot size indicates the percentage of expressed cells.</p>
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<p>hdWGCNA of cochlear epithelial cell subtype. (<b>A</b>) UMAP plots showing modules identified by hdWGCNA and the top 10 hub genes for each module. Colors represent the following modules: CD−M1 (roof) in orange, CD−M2 (LER and PD) in green, CD−M3 (GER and PD) in red, CD−M4 (cell cycle) in cyan, CD−M5 (epithelial patterning) in dark green, CD−M6 (GER and Roof) in light pink, CD−M7 (cartilage) in dark blue, CD−M8 (protein and RNA metabolism) in medium blue, CD−M9 (ribosome) in pink, and CD−M10 (mitochondria) in dark purple. (<b>B</b>) Radar plot showing the kME values of hub genes. Colors represent the following genes: <span class="html-italic">Foxg1</span> in light pink, <span class="html-italic">Gata3</span> in green, <span class="html-italic">Otx2</span> in orange, <span class="html-italic">Bmp4</span> in red, <span class="html-italic">Sox2</span> in cyan, and <span class="html-italic">Fgf10</span> in medium blue. (<b>C</b>) UMAP plot showing the hub gene network.</p>
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<p>Network analysis of <span class="html-italic">Foxg1</span> and <span class="html-italic">Gata3</span>. (<b>A</b>) UMAP plot showing the expression of <span class="html-italic">Foxg1</span> and <span class="html-italic">Gata3</span>. Colors represent the following genes: <span class="html-italic">Foxg1</span> in red, <span class="html-italic">Gata3</span> in green, and cells with blended expression of both genes in yellow. (<b>B</b>) Gene network of the top 20 genes highly correlated with <span class="html-italic">Foxg1</span>. Colors represent the following modules: CD−M3 (GER and PD) in red and CD−M6 (GER and roof) in light pink. (<b>C</b>) Gene network of the top 20 genes highly correlated with <span class="html-italic">Gata3</span>. Colors represent the following modules: CD−M2 (LER and PD) in green. (<b>D</b>) Dot plot showing the Gene Ontology (GO) terms related to the biological process for genes included in the <span class="html-italic">Foxg1</span> network. Dot size represents the gene count, whereas color represents the adjusted <span class="html-italic">p</span>-value. (<b>E</b>) Dot plot showing the GO terms related to the biological process for genes included in the <span class="html-italic">Gata3</span> network. Dot size represents gene count, and color represents the adjusted <span class="html-italic">p</span>-value.</p>
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<p>Expression of various marker genes in the E13.5 cochlear duct. (<b>A</b>) OTX2, a cochlear roof marker, is specifically expressed in the roof of the apical and primary ducts. FOXG1 is expressed throughout the basal, roof, and GER. In vertex ducts, they are visible on both the roof and the floor. (<b>B</b>) SOX2, a sensory domain marker, is co-expressed in the basal region with <span class="html-italic">Gata3</span>, and <span class="html-italic">Gata3</span> is expressed more broadly throughout the LER. (<b>C</b>) Unlike GATA3, SOX9 is expressed throughout the cochlear epithelium. (<b>D</b>) The expression of JAG1 can be observed in the prosensory domain and GER, together with that of FOXG1. Scale bar = 100 μm. GER, greater epithelial ridge; LER, lesser epithelial ridge; PSD, prosensory domain; SGN, spiral ganglion neurons.</p>
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<p>The association between hearing loss-associated disease and modules. (<b>A</b>) Heatmap showing genes and hearing loss-related diseases divided by modules. (<b>B</b>) Heatmap showing genes and the Z-scored kME values for each module. (<b>C</b>) Scheme summarizing the structure of the cochlear duct and genes related to hearing loss diseases within each module.</p>
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13 pages, 2128 KiB  
Article
Neutrophil Extracellular Traps Affect Human Inner Ear Vascular Permeability
by Marijana Sekulic, Stavros Giaglis, Nina Chatelain, Daniel Bodmer and Vesna Petkovic
Int. J. Mol. Sci. 2024, 25(18), 9766; https://doi.org/10.3390/ijms25189766 - 10 Sep 2024
Viewed by 939
Abstract
The integrity of the blood–labyrinth barrier (BLB) is essential for inner ear homeostasis, regulating the ionic composition of endolymph and perilymph and preventing harmful substance entry. Endothelial hyperpermeability, central in inflammatory and immune responses, is managed through complex intercellular communication and molecular signaling [...] Read more.
The integrity of the blood–labyrinth barrier (BLB) is essential for inner ear homeostasis, regulating the ionic composition of endolymph and perilymph and preventing harmful substance entry. Endothelial hyperpermeability, central in inflammatory and immune responses, is managed through complex intercellular communication and molecular signaling pathways. Recent studies link BLB permeability dysregulation to auditory pathologies like acoustic trauma, autoimmune inner ear diseases, and presbycusis. Polymorphonuclear granulocytes (PMNs), or neutrophils, significantly modulate vascular permeability, impacting endothelial barrier properties. Neutrophil extracellular traps (NETs) are involved in diseases with autoimmune and autoinflammatory bases. The present study evaluated the impact of NETs on a BLB cellular model using a Transwell® setup. Our findings revealed a concentration-dependent impact of NETs on human inner ear-derived endothelial cells. In particular, endothelial permeability markers increased, as indicated by reduced transepithelial electrical resistance, enhanced dextran permeability, and downregulated junctional gene expression (ZO1, OCL, and CDH5). Changes in cytoskeletal architecture were also observed. These preliminary results pave the way for further research into the potential involvement of NETs in BLB impairment and implications for auditory disorders. Full article
(This article belongs to the Special Issue Hearing Loss: Molecular Biological Insights)
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Figure 1

Figure 1
<p>NET generation from healthy donors’ PMNs. (<b>A</b>) SytoxGreen and DAPI fluorescence captions depicting extracellular DNA and cell nuclei, respectively. (<b>B</b>) Spectro-photometrical determination of DNA quantities (NETs) in cell supernatants. Magnification: 10×; scale bars: 100 μm; ns: not significant; ***: <span class="html-italic">p</span> &lt; 0.001; data are presented as the mean ± SD.</p>
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<p>NETs induce endothelial toxicity in a dose-dependent manner. An LDH test was performed after 96 h of exposure to 50 ng/mL, 100 ng/mL, and 200 ng/mL of untreated PMN supernatant (UT PMN Sup) and NETs, with TNFα as a positive control. All three treatments showed increased luminescence intensity, indicating increased cellular toxicity compared with the control, with TNFα causing the most significant impact and the UT PMN supernatant having the lowest impact. At 100 ng/mL, NETs showed a significant increase in luminescence compared with both the EC medium-only control and the 100 ng/mL UT PMN supernatant. With the 200 ng/mL treatment, the cytotoxic luminescence signal increased significantly for NETs and TNFα, whereas UT PMN supernatant treatment values remained similar to values obtained with a 100 ng/mL concentration. Asterisks above the 100 ng/mL and 200 ng/mL bars indicate comparisons to the respective 50 ng/mL treatments. *: <span class="html-italic">p</span> &lt; 0.05, **: <span class="html-italic">p</span> &lt; 0.01, ***: <span class="html-italic">p</span> &lt; 0.001, ****: <span class="html-italic">p</span> &lt; 0.0001. Total number of individual treatment experiments (<span class="html-italic">n</span> = 3); data are presented as the mean ± SD.</p>
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<p>TEER values are altered in the endothelial monolayer upon NET exposure. (<b>A</b>) TEER values after seeding human ECs on a Transwell<sup>®</sup> insert with a polyester membrane. Treatment with 100 ng/mL non-PMA-treated PMN supernatant (UT PMN Sup) started on day 10 and continued over the next 10 days. TEER measurements were performed once per day, with the first measurement starting 24 h after seeding and labeled as day 1. TEER measurement was performed in three consecutive repetitions for each individual well. Control cells were treated with media only. No significant difference between the UT PMN supernatant and control conditions was observed during the treatment period. (<b>B</b>) TEER values after treatment with NETs (100 ng/mL) vs. control showed a significant drop, starting from day 14 and continuing until the end of the treatment period. **: <span class="html-italic">p</span> &lt; 0.01; ****: <span class="html-italic">p</span> &lt; 0.0001; number of individual experiments with all treatment conditions (<span class="html-italic">n</span> = 6). Data are presented as the mean ± SD.</p>
Full article ">Figure 4
<p>Expression of junctional genes is altered under the influence of NETs. Treatment with NETs (100 ng/mL) for 96 h resulted in a significant decrease in <span class="html-italic">ZO1</span>, <span class="html-italic">OCL</span>, and <span class="html-italic">CDH5</span> expression, whereas JAM1 and CLDN levels remained unchanged compared with the control. Data were obtained from six donor-derived cell cultures and performed in triplicate (technical); number of individual experiments (<span class="html-italic">n</span> = 3); ns: not significant; ****: <span class="html-italic">p</span> &lt; 0.0001; data are presented as the mean ± SD.</p>
Full article ">Figure 5
<p>Increased EC permeability in the presence of NETs. Incubation of the endothelial cells with NETs (100 ng/mL) for 96 h produced a significant increase in dextran influx compared with the untreated cells, whereas the non-PMA-treated PMN supernatant (UT PMN Sup) did not result in any significant changes between control and treated cells. MFI: mean fluorescence intensity; ns: not significant; *: <span class="html-italic">p</span> &lt; 0.05, **: <span class="html-italic">p</span> &lt; 0.01; number of individual experiments with all treatment conditions (<span class="html-italic">n</span> = 6). Data are presented as the mean ± SD.</p>
Full article ">Figure 6
<p>Decreased actin signal and minor rearrangement were observed in EC samples exposed to NETs. (<b>A</b>) Cells were stained with phalloidin conjugated with Alexa-488 (green) and DAPI (blue). In the NET-treated cells (100 ng/mL; right), changes in F-actin arrangement and a decrease in phalloidin intensity compared with control (left) were observed. Scale bar: 100 µm. (<b>B</b>) The fluorescence signals were weaker in NET-treated cells, as confirmed by signal quantification. *: <span class="html-italic">p</span> &lt; 0.05, number of individual experiments (<span class="html-italic">n</span> = 3). Data are presented as the mean ± SD.</p>
Full article ">
21 pages, 8172 KiB  
Article
Differentiation of Spiral Ganglion Neurons from Human Dental Pulp Stem Cells: A Further Step towards Autologous Auditory Nerve Recovery
by Yassine Messat, Marta Martin-Fernandez, Said Assou, Keshi Chung, Frederic Guérin, Csilla Gergely, Frederic Cuisinier and Azel Zine
Int. J. Mol. Sci. 2024, 25(16), 9115; https://doi.org/10.3390/ijms25169115 - 22 Aug 2024
Viewed by 1081
Abstract
The degeneration of spiral ganglion neurons (SGNs), which convey auditory signals from hair cells to the brain, can be a primary cause of sensorineural hearing loss (SNHL) or can occur secondary to hair cell loss. Emerging therapies for SNHL include the replacement of [...] Read more.
The degeneration of spiral ganglion neurons (SGNs), which convey auditory signals from hair cells to the brain, can be a primary cause of sensorineural hearing loss (SNHL) or can occur secondary to hair cell loss. Emerging therapies for SNHL include the replacement of damaged SGNs using stem cell-derived otic neuronal progenitors (ONPs). However, the availability of renewable, accessible, and patient-matched sources of human stem cells is a prerequisite for successful replacement of the auditory nerve. In this study, we derived ONP and SGN-like cells by a reliable and reproducible stepwise guidance differentiation procedure of self-renewing human dental pulp stem cells (hDPSCs). This in vitro differentiation protocol relies on the modulation of BMP and TGFβ pathways using a free-floating 3D neurosphere method, followed by differentiation on a Geltrex-coated surface using two culture paradigms to modulate the major factors and pathways involved in early otic neurogenesis. Gene and protein expression analyses revealed efficient induction of a comprehensive panel of known ONP and SGN-like cell markers during the time course of hDPSCs differentiation. Atomic force microscopy revealed that hDPSC-derived SGN-like cells exhibit similar nanomechanical properties as their in vivo SGN counterparts. Furthermore, spiral ganglion neurons from newborn rats come in close contact with hDPSC-derived ONPs 5 days after co-culturing. Our data demonstrate the capability of hDPSCs to generate SGN-like neurons with specific lineage marker expression, bipolar morphology, and the nanomechanical characteristics of SGNs, suggesting that the neurons could be used for next-generation cochlear implants and/or inner ear cell-based strategies for SNHL. Full article
(This article belongs to the Special Issue Hearing Loss: Molecular Biological Insights)
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Figure 1

Figure 1
<p>Schematic illustration outlining the timeline and conditions of the newly established protocol for the generation of hDPSC-derived SGN-like cells. The hDPSCs were exposed to SB/LDN for 4 days in vitro, followed by treatment with SB/BMP4 for an additional 3 days in vitro to generate neurospheres in ultra-low attachment (ULA) plates. The generated neurospheres were then plated on a Geltrex-coated surface and differentiated following differentiation paradigms 1 or 2. For paradigm 1, neurospheres were exposed to ATRA/SHH/CHIR until 13 DIV and then to BDNF/NT3 until 21 DIV. For paradigm 2, neurospheres were only exposed to BDNF/NT3 for the same period in vitro. The culture period was extended to 32 DIV in paradigm 2 to assess the effect of a prolonged in vitro maturation. Abbreviations: DIV: day in vitro; SB: SB431542: TGFb inhibitor; LDN: LDN-193189: bone morphogenetic protein (BMP) pathway inhibitor; BMP4: bone morphogenetic protein 4, ATRA: all trans retinoic acid; SHH: Sonic hedgehog; BDNF: brain-derived neurotrophic factor; NT3: neurotrophin 3.</p>
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<p>Induction of early neuronal and otic/placodal markers expression in hDPSC-derived neurospheres. (<b>A</b>) Bar chart showing relative gene expression levels in logarithmic (Ln) scale, obtained by qPCR analyses for three distinct panels of genes featuring the neural crest, otic placode, and otic vesicle, respectively. Cells were collected at 3 and 7 DIV of differentiation and analyzed to assess the effects of the dual inhibition/activation of BMP-signaling under continuous TGFb inhibition on otic induction. Noticeably, the results demonstrate a significant upregulation of otic/placode (<span class="html-italic">Dlx5</span>) and otic/vesicle markers (<span class="html-italic">Pax2</span>, <span class="html-italic">Sox2</span>). Bars represent SD. Statistically significant differences are indicated by * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01 (one-way ANOVA), <span class="html-italic">n</span> = 3 independent experiments. Abbreviations: NC: neural crest; OP: otic placodal; OV: otic vesicle. The dashed line represents gene expression of undifferentiated hDPSCs. (<b>B</b>) Immunocytochemistry shows the expression of NESTIN and PAX2 during the time course of in vitro differentiation. A large proportion of cells within the neurospheres are NESTIN immunopositive at 3 and 7 DIV (shown in green), while PAX2 expression (shown in red) was principally observed at 7 DIV, and PAX2 immunopositive cells co-expressed SOX2. Cell nuclei were counterstained with DAPI (blue). Scale bars = 100 µm.</p>
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<p>Analyses of otic vesicle and SGN gene markers in differentiated cells at 21 DIV. Bar charts showing relative gene expression levels were obtained by qPCR analyses of genes featuring OV and SGN lineages in cells differentiated under paradigms 1 and 2, as compared to control culture conditions. The differentiated cells were collected at 21 DIV from 3 independent culture experiments. A significant upregulation of OV and SGN markers was observed in both paradigms when compared to the control culture condition. Moreover, paradigm 2 showed the most significant upregulation of a subset (<span class="html-italic">Prph</span>, <span class="html-italic">TrkB</span>) related to both OV and SGN lineage. Statistical differences were determined with one way ANOVA. <span class="html-italic">p</span> values are indicated with * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> ≤ 0.001, ns: not significant.</p>
Full article ">Figure 4
<p>Representative images of immunocytochemical analyses of the expression of neural markers at 21 DIV in paradigm 1 and 2 cell cultures. (<b>A</b>) Phase-contrast image showing hDPSC-derived otic neuronal progenitors in paradigm 1 cultures. Differentiated cells have a bipolar morphology, with a round soma (asterisks), in a dense network. (<b>B</b>) The immunostaining shows the expression of MAP2/TUJ1/GFAP in the cytoplasm (shown in green) and SOX2/NEUN at nuclear level (shown in red) in paradigm 1 cultures. (<b>C</b>) Phase-contrast image showing hDPSC-derived otic neuronal progenitors in paradigm 2 cultures. Among differentiated cells, some have a bipolar morphology (asterisks), while others display a phenotype close to glial cells (arrows). (<b>D</b>) The immunostaining shows expression of MAP2/TUJ1/GFAP in the cytoplasm (shown in green) and SOX2/NEUN in the nuclei (shown in red) in paradigm 2 cultures. Under this paradigm, more double immunopositive SOX2/GFAP cells differentiated were detected, as compared to paradigm 1. DAPI staining is shown in blue. Scale bars = 100 μm in all panels.</p>
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<p>Characterization of the expression of SGN markers at 32 DIV in paradigm 2. (<b>A</b>) The differentiated SGN-like cells displayed a bipolar morphology and expressed a panel of SGN known markers such as SOX2 (green), BRN3A (red), TUJ1 (green), PRPH (red), and TRKC (red). A subset of these differentiated cells was double immunopositive for SOX2/BRN3A, characteristic of SGN phenotype. In addition, they express TRKC and PRPH which are also SGN-related markers. Scale bar = 100 µm. (<b>B</b>) The table represents the percentage of cells expressing SOX2, BRN3A, and SOX2/BNR3A. Cell count indicates about 40% of the differentiated cells at 32 DIV are double immunopositive for BRN3A/SOX2 from n = 2709 counted cells.</p>
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<p>AFM-based nanomechanical characterization of SGN-like cells at 32 DIV. (<b>A</b>) Violin plot of measured Young’s modulus (<b>E</b>) of fixed SGN in vivo, SGN in vitro, and undifferentiated hDPSCs. A significant difference was observed between hDPSCs and SGN samples. However, both SGN in vivo and in vitro were similar related to Young’s modulus measurements. (<b>B</b>) Violin plot of measured Young’s modulus of unfixed SGN in vitro, SGN in vivo, and undifferentiated hDPSCs. A significant difference was also observed between hDPSCs and SGN samples. Young’s modulus measurements of SGN in vivo and in vitro were similar. These measurements indicate strong similarities between SGN in vivo and SGN in vitro differentiated from hDPSCs at the nanomechanical level. (<b>C</b>) Three-dimensional reconstruction of analyzed SGNs in vivo, showing a bipolar morphology. (<b>D</b>) Three-dimensional reconstruction of analyzed SGNs in vitro, showing their bipolar morphology acquired after the in vitro differentiation process, which is different form the morphology of hDPSCs shown in (<b>E</b>). (<b>E</b>) Three-dimensional reconstruction of analyzed hDPSCs showing the characteristic elongated morphology of these cells. Statistical differences were determined with one-way ANOVA. <span class="html-italic">p</span> values are indicated with **** <span class="html-italic">p</span> ≤ 0.0001. n = 10 measurements. Scale bar = 20 µm.</p>
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<p>Characterization of the co-cultures between human ONP cells and rat SG explants. (<b>A</b>) Schematic representation of different steps of the co-culture procedure. Neurospheres were generated from hDPSCs and differentiated to ONP cells until 13 DIV. In parallel, cochlear explants were dissected from the inner ear of postnatal day P3 rats, followed by SG isolation and culture on Geltrex-coated plates for 48 h. Then, the ONP cells were detached from the substrate and co-cultured with SG explants for 5 additional days. (<b>B</b>) Representative image of neurite outgrowths from SG explant (asterisk) immunostained with anti-TUJ1 (shown in magenta), projected towards the ONP cells, immunostained with anti-human nuclei (shown in green). DAPI was used to counterstain the nuclei. Scale bar = 1000 µm. (<b>C</b>) Magnification of the area indicated by the white rectangle in (B, showing outgrowth neurites (magenta) emanating from the SG explant towards ONP cells (green), Scale bar = 500 µm. (<b>D</b>) Magnification of the area indicated by the dashed rectangle in (<b>B</b>) highlights contacts between neurites from SG explant and the membrane of ONP cells. Scale bar = 50 µm. Abbreviations: ONP: otic neuronal progenitors; SG: spiral ganglion.</p>
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<p>Three-dimensional reconstruction of SG explant-emanating neurites and ONP contacts using IMARIS. <a href="#app1-ijms-25-09115" class="html-app">Figure S8B</a> was used for the reconstruction with two look-up tables for TUJ1 staining. (<b>A</b>) Image represents the 3D reconstruction of neurite contacts between SGNs from the SG explant and ONP cells using IMARIS 10 software. The neurites (magenta = TUJ1 immunostaining) establish direct contacts with the membrane of ONP derived from hDPSCs (green: human nuclei, and purple: TUJ1). Scale bar = 30 µm. (<b>B</b>) Magnification of the area indicated by the square in (A) representing the image reconstruction by IMARIS of the neurites which established contacts during the co-culture. Scale bar = 10 µm.</p>
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22 pages, 3720 KiB  
Article
ERK1/2 Inhibition via the Oral Administration of Tizaterkib Alleviates Noise-Induced Hearing Loss While Tempering down the Immune Response
by Richard D. Lutze, Matthew A. Ingersoll, Alena Thotam, Anjali Joseph, Joshua Fernandes and Tal Teitz
Int. J. Mol. Sci. 2024, 25(12), 6305; https://doi.org/10.3390/ijms25126305 - 7 Jun 2024
Cited by 3 | Viewed by 1418
Abstract
Noise-induced hearing loss (NIHL) is a major cause of hearing impairment and is linked to dementia and mental health conditions, yet no FDA-approved drugs exist to prevent it. Downregulating the mitogen-activated protein kinase (MAPK) cellular pathway has emerged as a promising approach to [...] Read more.
Noise-induced hearing loss (NIHL) is a major cause of hearing impairment and is linked to dementia and mental health conditions, yet no FDA-approved drugs exist to prevent it. Downregulating the mitogen-activated protein kinase (MAPK) cellular pathway has emerged as a promising approach to attenuate NIHL, but the molecular targets and the mechanism of protection are not fully understood. Here, we tested specifically the role of the kinases ERK1/2 in noise otoprotection using a newly developed, highly specific ERK1/2 inhibitor, tizaterkib, in preclinical animal models. Tizaterkib is currently being tested in phase 1 clinical trials for cancer treatment and has high oral bioavailability and low predicted systemic toxicity in mice and humans. In this study, we performed dose–response measurements of tizaterkib’s efficacy against permanent NIHL in adult FVB/NJ mice, and its minimum effective dose (0.5 mg/kg/bw), therapeutic index (>50), and window of opportunity (<48 h) were determined. The drug, administered orally twice daily for 3 days, 24 h after 2 h of 100 dB or 106 dB SPL noise exposure, at a dose equivalent to what is prescribed currently for humans in clinical trials, conferred an average protection of 20–25 dB SPL in both female and male mice. The drug shielded mice from the noise-induced synaptic damage which occurs following loud noise exposure. Equally interesting, tizaterkib was shown to decrease the number of CD45- and CD68-positive immune cells in the mouse cochlea following noise exposure. This study suggests that repurposing tizaterkib and the ERK1/2 kinases’ inhibition could be a promising strategy for the treatment of NIHL. Full article
(This article belongs to the Special Issue Hearing Loss: Molecular Biological Insights)
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Figure 1

Figure 1
<p>AZD0364 protects mice from noise-induced hearing loss when administered 45 min before noise exposure. (<b>A</b>) Molecular structure of tizaterkib. (<b>B</b>) Schedule of administration of noise exposure and tizaterkib treatments in FVB mice. Mice were given their first treatment of tizaterkib via an oral gavage 45 min before noise exposure. Mice were treated with the drug for a total of 3 days, twice a day, and exposed to noise once. (<b>C</b>) ABR threshold shifts following procedure in (<b>B</b>). Shaded region is the frequency range of the noise exposure. (<b>D</b>) Representative post-noise-exposure ABRs of noise-alone- and noise + tizaterkib-treated mice. (<b>E</b>) Percent weight change of different experimental cohorts throughout the 14-day protocol shown in (<b>B</b>). Noise + Carrier (red), noise + tizaterkib (green), tizaterkib alone (blue), and carrier (black). Data shown as means ± SEM; ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 compared to noise alone by two-way ANOVA with a Bonferroni post hoc test. The color of the asterisks indicates the statistical significance of the treatment group with that same color compared to noise + carrier treated mice. <span class="html-italic">n</span> = 9–10 mice.</p>
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<p>Tizaterkib protects mice from noise-induced hearing loss when administered 24 h after noise exposure. (<b>A</b>) Schedule of noise exposure and tizaterkib, which began 24 h after noise exposure. Mice were treated with varying concentrations of tizaterkib twice a day for 3 whole days. (<b>B</b>) ABR threshold shifts following procedure in (<b>A</b>), with 25 and 5 mg/kg tizaterkib given to separate groups. Shaded region is the frequency range of the noise exposure. (<b>C</b>) ABR threshold shifts following the procedure in (<b>A</b>) with 0.5 mg/kg administered to mice. (<b>D</b>) ABR threshold shifts following the procedure in (<b>A</b>) with the 0.1 mg/kg tizaterkib treatment. (<b>E</b>) Dose–response curve of tizaterkib protection from noise-induced hearing loss at 16 kHz, with 100% protection as a 0 dB SPL threshold shift. (<b>F</b>) ABR threshold shifts of males and females, graphed separately, that were treated with tizaterkib or carrier following the procedure in (<b>A</b>). (<b>G</b>) Representative ABR traces of noise-alone- and noise + tizaterkib-treated mice. Noise + carrier (red), noise + tizaterkib (green), noise + 5 mg/kg tizaterkib (purple), tizaterkib alone (blue), and carrier (black). Data shown as means ± SEM; * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001 compared to noise alone by two-way ANOVA with a Bonferroni post hoc test. The color of the asterisks indicates the statistical significance of the treatment group with that same color compared to noise + carrier treated mice. <span class="html-italic">n</span> = 9–10 mice.</p>
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<p>Tizaterkib protects mice from noise-induced synaptopathy in the 8 and 16 kHz regions. (<b>A</b>) Representative images of whole-mount cochlear sections stained with myosin VI (green) and Ctbp2 (red) in the 8 kHz region. (<b>B</b>) Number of Ctbp2 puncta per IHC in the 8 kHz region. (<b>C</b>) Representative images of whole-mount cochlear sections in the 16 kHz region. (<b>D</b>) Number of Ctbp2 puncta per IHC in the 16 kHz region. (<b>E</b>) ABR wave 1 amplitude for 16 kHz from the post experimental ABR recordings shown in <a href="#ijms-25-06305-f002" class="html-fig">Figure 2</a>C. The wave 1 amplitude was measured from 60–90 dB. Data shown as means ± SEM; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared to noise alone by one-way ANOVA with a Bonferroni post hoc test. Tizaterkib alone (blue), carrier (black), noise alone (red), noise + tizaterkib (green). <span class="html-italic">n</span> = 9 mice.</p>
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<p>Tizaterkib protects from noise-induced hearing loss when mice are exposed to 106 dB. (<b>A</b>) Schedule of noise exposure and tizaterkib treatment. Mice were exposed to 106 dB SPL for 2 h and tizaterkib treatment started 24 h after noise exposure. Mice were treated for 3 whole days, twice a day. (<b>B</b>) ABR threshold shifts following the protocol in (<b>A</b>). Shaded region is the frequency range of the noise exposure. (<b>C</b>) Representative ABR traces of noise-alone- and noise + tizaterkib-treated mice following the 106 dB SPL noise exposure. (<b>D</b>) DPOAE threshold shifts following the protocol in (<b>A</b>). Noise + carrier (red), noise + tizaterkib (green), tizaterkib alone (blue), and carrier (black). Data shown as means ± SEM; * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001 compared to noise alone by two-way ANOVA with a Bonferroni post hoc test. The color of the asterisks indicates the statistical significance of the treatment group with that same color compared to noise + carrier treated mice. <span class="html-italic">n</span> = 13 mice.</p>
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<p>Tizaterkib treatment phenocopies the resistance to noise-induced hearing loss measured in the KSR1 KO mouse model. (<b>A</b>) KSR1 is a scaffolding protein for RAF, MEK, and ERK which enables the efficient transmission of MAPK signals. (<b>B</b>) Schedule of noise exposure and 5 mg/kg tizaterkib treatment in KSR1 WT and KO mice. Mice were exposed to 100 dB SPL for 2 h and tizaterkib treatment began 24 h after noise exposure. Mice were treated with tizaterkib or carrier twice a day for 3 whole days. (<b>C</b>) ABR threshold shifts following the protocol in (<b>B</b>). Shaded region is the frequency range of the noise exposure. WT + noise (red), KO + noise + tizaterkib (blue), WT + noise + tizaterkib (green), KO + noise (purple), and WT + carrier alone (black). Data shown as means ± SEM; * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001 compared to noise alone by two-way ANOVA with a Bonferroni post hoc test. The color of the asterisks indicates the statistical significance of the treatment group with that same color compared to noise + carrier treated mice. <span class="html-italic">n</span> = 5–6 mice.</p>
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<p>Three days of treatment beginning 24 h after noise exposure produces the optimal protection in terms of tizaterkib administration. (<b>A</b>) Schedule of administration for noise exposure and tizaterkib treatment. Treatment with tizaterkib began 48 h after noise exposure and mice were treated for 3 days, twice a day. (<b>B</b>) ABR threshold shifts following the protocol in (<b>A</b>). Shaded region is the frequency range of the noise exposure. Noise alone (red), noise + tizaterkib (green), carrier (black), and tizaterkib alone (blue). (<b>C</b>) Schedule of administration of noise exposure and tizaterkib treatment. Treatment began 24 h after noise exposure and one cohort was treated for 1 day, one cohort was treated for 2 days, and another cohort was treated for 3 days. (<b>D</b>) ABR threshold shifts following the protocol in (<b>C</b>). Noise alone (red), 1-day treatment + noise (purple), 2-day treatment + noise (blue), 3-day treatment + noise (green), carrier alone (black). Data shown as means ± SEM; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 compared to noise alone by two-way ANOVA with a Bonferroni post hoc test. The color of the asterisks indicates statistical significance of the treatment group with respect to that same color compared to noise + carrier-treated mice. <span class="html-italic">n</span> = 6–10 mice.</p>
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<p>Tizaterkib treatment lowers the number of CD45-positive cells in the cochlea 4 days post noise exposure. (<b>A</b>) Representative low-magnification images of cochlear cryosections stained with CD45 (red) and DAPI (blue). The treatment protocol shown in <a href="#ijms-25-06305-f002" class="html-fig">Figure 2</a>A was utilized and mice were sacrificed 4 days after noise exposure, 1 h after the final tizaterkib treatment. (<b>B</b>) Quantification of the CD45-positive cells in the cochlear sections in (<b>A</b>). (<b>C</b>) Higher magnification of the images shown in (<b>A</b>) of the scala tympani. (<b>D</b>) Quantification of CD45-positive cells in the walls of the scala tympani as presented in (<b>C</b>). (<b>E</b>) Representative images of cochlear cryosections of the stria vascularis following noise and tizaterkib treatment. (<b>F</b>) Quantification of CD45-positive cells per experimental group from the images in (<b>E</b>). Carrier (black), tizaterkib alone (blue), noise alone (red), noise + tizaterkib (green). Data shown as means ± SEM, * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001 compared to noise alone by one-way ANOVA with a Bonferroni post hoc test. <span class="html-italic">n</span> = 3–6 mice, with 3 sections each per mouse.</p>
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<p>Tizaterkib treatment lowers the amount of CD45 and CD68 in the cochlea 6 days post noise exposure. (<b>A</b>) Western blots showing the amount of CD45 and CD68 in the cochlea following noise exposure and tizaterkib treatment. The same treatment protocol shown in <a href="#ijms-25-06305-f002" class="html-fig">Figure 2</a>A was utilized and mice were sacrificed 6 days after noise exposure. (<b>B</b>) CD45/GAPDH ratio, normalized to the carrier-alone lane. Band intensities were measured using ImageJ software (version 1.54g). (<b>C</b>) CD68/GAPDH ratio, normalized to the carrier-alone lane. Data shown as means ± SEM, * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 compared to noise alone by one-way ANOVA with a Bonferroni post hoc test. The experimental groups, from left to right, are as follows: carrier alone, tizaterkib alone, noise alone, and noise + tizaterkib. Each group had the cochleae from 5 mice (10 cochleae) pooled together to make the tissue lysate. <span class="html-italic">n</span> = 5.</p>
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