Superoxide produced in the cochlea of inner ears causes acquired hearing loss

(Press-News.org) Professor UEYAMA Takehiko (Biosignal Research Center, Kobe University) and the inner ear research group (Kyoto Prefectural University of Medicine) have identified the cell types in the inner ear cochlea (*1) responsible for the production of superoxide (Nox3*2-expressing cells). They achieved this by using genetically modified mice that they developed. The researchers discovered that these superoxide-producing cells increase in number in response to aging, noise damage, and ototoxic drugs, thus causing age-related, noise-induced and drug-induced hearing loss. In addition, they were able to suppress the onset of these three types of acquired hearing loss in genetically modified mice with no Nox3 expression (Nox3 knockout).

These results are expected to contribute towards the development of the world's first drug-based treatment for hearing loss. Future treatments could involve removing reactive oxygen species from the cochlea, suppressing their production, or the development of Nox3 inhibitors.

These research results were published in the official journal of the Society for Neuroscience 'Journal of Neuroscience' on April 12, 2021.

Main Points

It is known that NADPH oxidase 3 (Nox3) is only expressed in the inner ear; however, it was not known which cells are involved in this, nor when or how Nox3 is expressed. In a world first, this research team has revealed the cells in the inner ear responsible for Nox3 expression and its mode of expression by using the genetically modified mice that they developed. It was discovered that Nox3-expressing cells increase gradually with age, and suddenly in response to noise damage and ototoxic drugs. The researchers were able to control the onset of age-related, noise-induced, and drug-induced hearing loss in Nox3 knockout mice, in which Nox3 is unable to be expressed. In particular, they were able to significantly suppress the onset of age-related and drug-induced hearing loss. If a drug to suppress the expression and/or activity of Nox3 could be developed, this would aid the development of effective treatment methods for the many patients with the abovementioned major types of acquired hearing loss. Research Background

Sensorineural hearing loss (*3) is the most common type of sensorineural impairment. Around 5% of the global population have some kind of hearing impairment; however, there are still no established treatments, particularly pharmacological interventions, for these conditions.

It is said that hearing loss related to aging occurs in between 25%-40% of adults over 65, and there are around 10 million people with this condition in Japan alone. Noise-induced hearing loss is still one of the most common aftereffects experienced by former soldiers; however, they are not the only people who develop this impairment. According to a statement by the World Health Organization (WHO) in 2019, around 50% of young people aged 12-35 (1.1 billion people) are at risk of suffering from noise-induced hearing loss in the future due to overexposure to high noise levels resulting from the popularization of portable media players, such as smartphones. There is also drug-induced hearing loss, which can occur as a side effect of taking medications such as antibiotics, anticancer drugs, diuretics, painkillers and anti-inflammatory drugs. Consequently, the number of people with hearing loss is increasing in modern society, and developing treatments for this has become a pressing issue.

Superoxide, a kind of reactive oxygen species, produced by NADPH oxidases (Noxs) contribute towards the development of different types of sensorineural hearing loss. Among the seven types of Noxs found in humans, Nox3, which is specifically expressed in the inner ear, has been reported to be the origin of superoxide. However, the cell types in the inner ear cochlea that express Nox3 was not known, and it was not understood when nor how Nox3 is expressed. The mechanism by which it causes hearing loss was also unclear.

Research Methodology and Findings

This research group has created many mouse models using gene manipulation in order to investigate the causes of hearing loss in patients and develop treatments for them. For this experiment, they developed a mouse model (Nox3-Cre;tdTomato), in which red fluorescent light-emitting proteins (tdTomato) would be expressed in the Nox3-expressing cells, and a Nox3 knockout mouse line (Nox3-KO), in which Nox-3 is unable to be expressed. Using these mice, they were able to identify the Nox3-expressing cells in the inner ear cochlea (which is responsible for hearing), in other words the cells responsible for producing superoxide (Figures 1 and 2). Furthermore, they also revealed that Nox3 plays an important role in the onset of all of the following major types of acquired hearing loss (*4): age-related hearing loss, noise-induced hearing loss and drug-induced hearing loss (Figure 3).

First, the researchers used the mice with red fluorescent tdTomato proteins in Nox3-expressing cells, and a fluorescence microscope to track the temporal expression of red fluorescence in the cells of the mice after birth. They discovered increased red fluorescence in the following cells inside the cochlea (which is responsible for hearing): hair cells (*5), various types of supporting cells (that provide anatomical support to hair cells) and spiral ganglion neurons (the first afferent neuron for hearing connected to hair cells) (Figures 1 and 2).

Of the two types of hair cell inside the inner ear; outer hair cells are more vulnerable to various outside stimuli than the inner hair cells. A loss of outer hair cells is a known manifestation in many cases of hearing impairment. The research team investigated this in detail using two different types of genetically modified mice: mice that retained the Nox3-mediated ability to produce superoxide (heterozygous Nox3-Cre END