Sha Lab

Sensory Hair Cell Survival & Death in the Inner Ear

The cochlea is the sensory organ responsible for hearing and plays a role in verbal communication. The sensory cells, or ‘hair cells’, of the organ of Corti are responsible for the transduction of acoustic input into nerve impulses that are then transmitted to the brain. Damage to or loss of hair cells leads to permanent hearing loss, as mammalian hair cells lack the ability to regenerate. According to the Center for Hearing and Communication, an estimated 38 million people in the United States—one of every 10 Americans—have hearing loss, which imposes a huge detriment on the quality of life of the affected individuals and a formidable economic burden on society.

Our research focuses on acquired hearing loss, which is hearing loss that develops during one's lifetime from exposure to noxious insults to the inner ear. Exposure to excessive noise or ototoxic drugs (such as aminoglycoside antibiotics or cisplatin, an anti-neoplastic), and aging are the three main causes of acquired hearing loss. The histopathological hallmark of acquired hearing loss is the destruction of sensory hair cells in the inner ear, leading to permanent hearing loss.

Research Program

Noise-induced hearing loss (NIHL)

Research into NIHL using animal models has borne out two leading theories for the cause of hearing loss. One is mechanical damage from vibration of the organ of Corti beyond the tolerance of its physical structure. Another is so called 'metabolic damage', wherein metabolic overstimulation and stress trigger cell death pathways. A variety of biochemical and molecular responses to noise trauma have been identified. For example, noise exposure elevates intracellular calcium levels in hair cells, likely through influx via calcium channels, and activation of calcium/calmodulin-dependent protein kinases or calcium-dependent phosphatase. Another well-documented response to noise trauma is the generation of reactive oxygen species (ROS). It appears that excessive noise decreases cochlear blood flow, which causes vasoactive lipid peroxidation and leads to generation of ROS. In addition, release of the neurotransmitter glutamate at inner hair cell synapses in response to traumatic noise has been implicated in excitotoxicity (neuronal damage and cell death from overstimulation by neurotransmitters) at this synapse.

The traditional method for prevention of NIHL by use of ear protection to reduce noise levels reaching the ear has proven insufficient, primarily due to non-compliance. Based on animal experimentation, pharmacological prevention using a ‘hearing pill’ is a promising and appealing route. Antioxidants such as glutathione, D-methionine, ascorbic acid, and water-soluble coenzyme Q10 have been reported to attenuate NIHL to some extent, while other antioxidants have failed to prevent NIHL. These conflicting results underscore the challenge still faced by the field in elucidating detailed molecular mechanisms and preventing NIHL.

Figure 1: Noise-induced permanent hearing loss.

(A) Auditory brainstem response (ABR) threshold shifts: The bar graph represents the permanent NIHL in 3-month-old male CBA/J mice resulting two weeks after exposure to 2–20 kHz broadband noise (BBN) at 106 dB SPL for two hours. Permanent threshold shifts (PTS) were greatest at high frequencies and decreased toward lower frequencies. Data are presented as means + SD; n = 5 for each condition.

(B) The images were taken of the cochlear epithelium with a 63× lens. The representative images illustrate hair cell losses in the middle region (3 mm from the apex) two weeks after exposure to 106 dB SPL 2–20 kHz BBN. Preparations from control mice illustrate the normal structure of the sensory epithelium with three rows of outer hair cells (OHC1–3) and one row of inner hair cells (IHC). Scale bar = 10 µm.

(C) PTS-noise exposure results in apoptotic and necrotic OHC nuclei. Both swollen necrotic (arrowheads) and condensed apoptotic nuclei (arrows) were present one hour post PTS-noise exposure. Red: propidium iodide staining of nuclei.

(D) Noise-trauma-induced translocation of the mitochondrial protein endonuclease G (Endo G) into the nucleus indicates activation of caspase-independent cell death pathways. Immunofluorescent labeling of surface preparations from control mice showed Endo G in OHCs, but not in their nuclei. Three hours (3 h) post PTS-noise exposure, Endo G was translocated into condensed nuclei of some OHCs (arrows). Green: Endo G; blue: Hoechst 33342 staining for nuclei.

Figure 2: Application of shCaMKKβ, short-hairpin RNA specific to inhibition of CaMKKβ, via adeno-associated virus (AAV) vector transfection significantly reduces CaMKKβ expression in the inner ear. Representative images show AAV-infected sensory hair cell expression of eGFP (green) in the apical, middle, and basal turns co-localized by immunolabeling for myosin VIIa (Myo7a, red). Cochleae were harvested at p21 after microinjection of 2 µL of AAV-shControl (shCtrl) or AAV-shCaMKKβ stock solutions (2.5 × 1013 GCs/mL) into the left ear of FVB/NJ mice at p1–2. eGFP: enhanced green fluorescent protein, Myo7a: myosin VIIa antibody used as a specific marker for sensory hair cells, shCtrl: scrambled shRNA, AAV-shCtrl: AAV2.7m8-U6-shControl-CMV-eGFP, and AAV-shCaMKKβ: AAV2.7m8-U6-shCaMKKβ-CMV-eGFP. Scale bar = 10 µm.

Figure 3: Fluorescence in-situ hybridization (FISH) of Myosin7a mRNA. Confocal images of 30 compressed Z-sections of the middle turn shows Myosin7a mRNA (probe #462771, red puncta) of control mice. Enlarged images of OHCs and IHCs are for better visualization of FISH of Myosin7a mRNA. Green: counter-labeling with a myosin VIIa antibody to localize sensory hair cells, blue: DAPI stains for nuclei. Scar bar: 10 µm.

Figure 4: ATP-depletion agent oligomycin induces Endo G nuclear translocation in HEI-OC1 cells.

(A) Endo G staining appeared in the nuclei of some HEI-OC1 cells one hour after 1-µM oligomycin treatment (arrows). Endo G is stained green, and nuclei red with propidium iodide (PI).

(B) Oligomycin induces HEI-OC1 cell death. Following incubation with 1 µM oligomycin for 24 hours, cell death of HEI-OC1 cells was detected by a flow cytometry assay. Oligomycin treatment significantly increased the amount of cell death compared to the control treatment. PI: propidium iodide.

Aminoglycoside-Induced & Age-Related Hearing Loss

Interestingly, noise- and aminoglycoside-induced hearing loss and age-related hearing loss share similar pathologies and, at the very least, share oxidative stress as a pathological feature. Both in-vivo and in-vitro studies from our laboratories have convincingly shown that oxidative stress is a causative factor in aminoglycoside-induced hearing loss and that this ototoxicity can be attenuated by the administration of antioxidants. We are, therefore, extending our studies to aminoglycoside-induced hearing loss in collaboration with Dr. Dev Arya at Clemson and Dr. David Crich at UGA to screen aminoglycoside-derivatives, collaboration with Dr. Samson Jamesdaniel at Wayne State University (WSU) to investigate cisplatin-induced hearing loss, and collaboration with Dr. Kumar Sambamuriti to study AD-induced and age-related hearing loss.

Selected Publications

1. Lai, R., Fang, QJ., Wu, F., Pan, S., Haque, K., Sha, S.-H. (2023). Prevention of noise-induced hearing loss by calpain inhibitor MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathway. Frontiers in Cellular Neuroscience, 17:1199656.

2. Wu, F., Sambamurti, K., & Sha, S. (2022). Current Advances in Adeno-Associated Virus-Mediated Gene Therapy to Prevent Acquired Hearing Loss. Journal of the Association for Research in Otolaryngology: JARO, 23, 569–578.

3. Wu, F., Hill, K., Fang, Q., He, Z., Zheng, H., Wang, X., Xiong, H., & Sha, S.-H. (2022). Traumatic-noise-induced hair cell death and hearing loss is mediated by activation of CaMKKβ. Cellular and Molecular Life Sciences: CMLS, 79, 249.

4. He, Z.-H., Pan, S., Zheng, H.-W., Fang, Q.-J., Hill, K., & Sha, S.-H. (2021). Treatment With Calcineurin Inhibitor FK506 Attenuates Noise-Induced Hearing Loss. Frontiers in Cell and Developmental Biology, 9, 648461.

5. Wu, F., Xiong, H., & Sha, S. (2020). Noise-induced loss of sensory hair cells is mediated by ROS/AMPKα pathway. Redox Biology, 29, 101406.

6. Xiong, H., Long, H., Pan, S., Lai, R., Wang, X., Zhu, Y., Hill, K., Fang, Q., Zheng, Y., & Sha, S.-H. (2019). Inhibition of Histone Methyltransferase G9a Attenuates Noise-Induced Cochlear Synaptopathy and Hearing Loss. Journal of the Association for Research in Otolaryngology: JARO, 20, 217–232.

7. Fang, Q.-J., Wu, F., Chai, R., & Sha, S.-H. (2019). Cochlear Surface Preparation in the Adult Mouse. Journal of Visualized Experiments: JoVE, Article 153.

8. Wang, X., Zhu, Y., Long, H., Pan, S., Xiong, H., Fang, Q., Hill, K., Lai, R., Yuan, H., & Sha, S.-H. (2018). Mitochondrial Calcium Transporters Mediate Sensitivity to Noise-Induced Losses of Hair Cells and Cochlear Synapses. Frontiers in Molecular Neuroscience, 11, 469.

9. Yang, C. H., Liu, Z., Dong, D., Schacht, J., Arya, D., & Sha, S.-H. (2017). Histone Deacetylase Inhibitors Are Protective in Acute but Not in Chronic Models of Ototoxicity. Frontiers in Cellular Neuroscience, 11, 315.

10. Chen, J., Hill, K., & Sha, S.-H. (2016). Inhibitors of Histone Deacetylases Attenuate Noise-Induced Hearing Loss. Journal of the Association for Research in Otolaryngology: JARO, 17, 289–302.

11. Hill, K., Yuan, H., Wang, X., & Sha, S.-H. (2016). Noise-Induced Loss of Hair Cells and Cochlear Synaptopathy Are Mediated by the Activation of AMPK. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 36, 7497–7510.

12. Chen, J., Yuan, H., Talaska, A. E., Hill, K., & Sha, S.-H. (2015). Increased Sensitivity to Noise-Induced Hearing Loss by Blockade of Endogenous PI3K/Akt Signaling. Journal of the Association for Research in Otolaryngology: JARO, 16, 347–356.

13. Han, Y., Wang, X., Chen, J., & Sha, S.-H. (2015). Noise-induced cochlear F-actin depolymerization is mediated via ROCK2/p-ERM signaling. Journal of Neurochemistry, 133, 617–628.

14. Yuan, H., Wang, X., Hill, K., Chen, J., Lemasters, J., Yang, S.-M., & Sha, S.-H. (2015). Autophagy attenuates noise-induced hearing loss by reducing oxidative stress. Antioxidants & Redox Signaling, 22, 1308–1324.

15. Zheng, H.-W., Chen, J., & Sha, S.-H. (2014). Receptor-interacting protein kinases modulate noise-induced sensory hair cell death. Cell Death & Disease, 5, e1262.

16. Oishi, N., Chen, J., Zheng, H.-W., Hill, K., Schacht, J., & Sha, S.-H. (2013). Tumor necrosis factor-alpha-mutant mice exhibit high frequency hearing loss. Journal of the Association for Research in Otolaryngology: JARO, 14, 801–811.

17. Chen, F.-Q., Zheng, H.-W., Schacht, J., & Sha, S.-H. (2013). Mitochondrial peroxiredoxin 3 regulates sensory cell survival in the cochlea. PloS One, 8, e61999.

18. Oishi, N., Chen, F.-Q., Zheng, H.-W., & Sha, S.-H. (2013). Intra-tympanic delivery of short interfering RNA into the adult mouse cochlea. Hearing Research, 296, 36–41.

19. Chen, F.-Q., Zheng, H.-W., Hill, K., & Sha, S.-H. (2012). Traumatic noise activates Rho-family GTPases through transient cellular energy depletion. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 32, 12421–12430.

20. Chen, F.-Q., Hill, K., Guan, Y.-J., Schacht, J., & Sha, S.-H. (2012). Activation of apoptotic pathways in the absence of cell death in an inner-ear immortomouse cell line. Hearing Research, 284, 33–41.

21. Sha, S.-H., Chen, F.-Q., & Schacht, J. (2010). PTEN attenuates PIP3/Akt signaling in the cochlea of the aging CBA/J mouse. Hearing Research, 264, 86–92.

22. Chen, F.-Q., Schacht, J., & Sha, S.-H. (2009). Aminoglycoside-induced histone deacetylation and hair cell death in the mouse cochlea. Journal of Neurochemistry, 108, 1226–1236.

23. Sha, S.-H., Kanicki, A., Dootz, G., Talaska, A. E., Halsey, K., Dolan, D., Altschuler, R., & Schacht, J. (2008). Age-related auditory pathology in the CBA/J mouse. Hearing Research, 243, 87–94.

24. Jiang, H., Talaska, A. E., Schacht, J., & Sha, S.-H. (2007). Oxidative imbalance in the aging inner ear. Neurobiology of Aging, 28, 1605–1612.

25. Sha, S.-H., Qiu, J.-H., & Schacht, J. (2006). Aspirin to prevent gentamicin-induced hearing loss. The New England Journal of Medicine, 354, 1856–1857.

26. Sha, S. H., Zajic, G., Epstein, C. J., & Schacht, J. (2001). Overexpression of copper/zinc-superoxide dismutase protects from kanamycin-induced hearing loss. Audiology & Neuro-Otology, 6, 117–123.