Dissertation
The role of pendrin and cellular mechanisms in SLC26A4-related hearing loss
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2022
DOI: 10.25820/etd.006461
Abstract
In the USA, 50% of congenital sensorineural hearing loss (SNHL) is due to genetic factors, and the remaining 50% is due to environmental factors. Of the congenital SNHL caused by genetic factors, 15% are syndromic, and 35% are non-syndromic. Non-syndromic deafness only alters hearing function, whereas syndromic deafness includes other defects. Pendred syndrome (PDS) is the most common syndromic form of deafness, accounting for at least 10% of hereditary hearing loss in humans. Non-syndromic enlarged vestibular aqueduct (NSEVA) is the most common radiological malformation associated with childhood SNHL. Both disorders are caused by mutations in the coding sequence of SLC26A4. The disease phenotype is characterized by SNHL and structural malformation of the inner ear, such as enlarged vestibular aqueducts. Although digenic inheritance is very rare (< 1%), the PDS-NSEVA disease spectrum shows compound heterozygosity of mutations in SLC26A4, as well as mutations in other genes such as KCNJ10 (the potassium channel) and FOXI1 (the transcription factor), suggesting that there are more genes involved in the PDS-NSEVA disease spectrum. Furthermore, the SLC26A4 gene encodes a protein called pendrin, which is an anion exchanger. In the inner ear, pendrin is expressed in the epithelial cells of the cochlea, vestibular system, and endolymphatic sac. Pendrin transports bicarbonate (HCO3-) into the endolymph and maintains endolymph pH. However, little is known about the role of pendrin in maintaining endolymphatic potential (EP) and cochlear function. In mouse models, lacking pendrin leads to reduced EP and pH; however, underlying cellular and molecular mechanisms of the reduced EP and pH are still unknown.In this study, single-cell RNA-sequencing in the cochlear stria vascularis (SV) identified cells that express Slc26a4 and Kcnj10. Through transcriptome profiling and morphological analysis, we characterized intermediate cells (IMC), spindle cells (SC), and marginal cells (MC) in the cochlear SV. The pH-regulating genes identified in the Slc26a4-expressing cells (specifically SCs) work together with Slc26a4 (pendrin) to maintain endolymphatic pH homeostasis. Further, pendrin and carbonic anhydrase 13 co-localize and therefore may regulate intracellular pH via cAMP/PKA signaling pathway. To maintain EP, SCs express several other ion transporters. Illumina short-read RNA-sequencing identified an unrecognized transcription start site (TSS) of Slc26a4. Nanopore long-read RNA-sequencing identified a novel isoform in the Slc26a4-expressing cells. mRNA expression of the novel TSS in Slc26a4 was validated using RT-PCR. Protein expression of the novel short isoform was confirmed using western blotting and immunostaining experiments. The novel isoform has a strong Kozak sequence similar to another known isoform. Underlying cellular and molecular mechanisms of the reduced EP and pH in the Slc26a4-/- mouse model was due to 7 differentially regulated genes in SCs and 16 differentially regulated genes in IMCs. The cell-to-cell interaction between SCs, IMCs, and MCs in the SV is mediated, at least in part, by SC exosomes.
Taken together, findings from this study indicate novel marker genes, including deafness-causing genes in SV cells and novel isoforms resulting from previously unacknowledged isoform diversity. Our study has significant consequence on understanding the role of pendrin and the pathogenic mechanisms of SLC26A4-related hearing loss. Importantly, results from this study are expected to contribute to the advancement of novel approaches targeting for SLC26A4-related hearing loss prevention and therapy response.
Details
- Title: Subtitle
- The role of pendrin and cellular mechanisms in SLC26A4-related hearing loss
- Creators
- Jin-Young Koh
- Contributors
- Richard J.H. Smith (Advisor)Terry A. Braun (Committee Member)Michael J. Schnieders (Committee Member)Kai Wang (Committee Member)Bernd Fritzsch (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biomedical Engineering
- Date degree season
- Summer 2022
- DOI
- 10.25820/etd.006461
- Publisher
- University of Iowa
- Number of pages
- xx, 191 pages
- Copyright
- Copyright 2022 Jin-Young Koh
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 181-191).
- Public Abstract (ETD)
Hearing is an important function for communication, yet despite advanced therapy such as hearing aids for sound amplification or cochlear implants for profound hearing loss, the World Health Organization estimates that more than 466 million people (6.1% of the world’s population) suffer from hearing loss and 34 million (7% of people with hearing loss) are children. From this, hearing loss is the most common form of sensorial deficiency in humans. Therefore, a better understanding of the basic biology of hearing loss is necessary. In this study, we investigate the cellular and molecular mechanisms that drive the hearing loss.
Recent technological advances, such as single-cell RNA-sequencing (scRNA-seq), have enabled cost effective testing of individual cells. Using scRNA-seq of individual auditory cells to better characterize the rare and important cell types of the auditory system, we identify cells that express deafness-causing genes. Additionally, we identify a new transcript structure for hearing loss genes, indicating the expression of previously unreported isoforms. Therefore, this study leads to improve comprehensive genetic testing for deafness by identifying and characterizing novel exons and isoforms implicated in hearing loss.
To characterize the comprehensive impact of gene mutations in murine models of human hearing loss, we used the Slc26a4-/- mouse as a murine model. Our results show that Slc26a4 gene mutations regulate gene expression in individual cells. We identify that when cellular homeostasis is not maintained, the expression level of 23 genes changes in 2 different cell types. Results of this study support targeting the isoform level of single genes in the individual cells as a promising approach to preventing hearing loss progression and improving therapy outcomes.
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Craniofacial Anomalies Research Center
- Record Identifier
- 9984285248902771
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