Localization of the mechanotransducer channel and its accessory proteins during development of cochlear hair cells.

Our inner ear, comprised of the spiral shaped cochlea and the vestibular system, gives us our senses of hearing and balance. The molecular mechanisms involved in these senses are still the subject of much debate. Moreover, hearing loss is the commonest form of sensory deprivation suffered by humans as they age. In accordance with the BBSRC strategic priority on ageing, our project aims to understand basic mechanisms of hearing at the molecular level in order to provide information for strategies to protect our hearing and prevent damage with age. As well as the ageing process, there are still many questions about the development of hearing at the cellular and molecular level.

The stimulus in both hearing and balance is mechanical: in hearing, sound waves, which start as movements of air, are transformed into fluid movements in the cochlea; in balance, movements of the head cause fluid motion within the vestibular structures. In either case, the fluid motion directly, or indirectly via other structures, stimulates hair cells. These specialised sensory cells are characterised by a bundle of tiny hairs (stereocilia) sticking out of their top. Deflecting the stereocilia in one (excitatory) direction produces mechaonelectrocial transduction (MET) in the hair cell where the mechanical stimulius is turned into an electrical response. Key components of this MET apparatus are: the stereocilia themselves; tiny filaments that link the stereocilia together, called tip links and lateral links; and proteins associated with the links such as the MET ion channels (proteins embedded in the cell's outer layer or membrane that can form pores) and accessory proteins connecting the tip links to the MET channels. Mutations in candidates for all of these proteins are known to cause progressive hearing loss.

The prevailing view of the MET response is that bundle deflection stretches the tip links, allowing them to pull on the accessory proteins attached to the MET channels, and so cause the latter to open. Opening the channels allows positive ions (potassium and calcium) to enter the cell causing the electrical response. Studies of the channels in the mammalian cochlea also show that their properties vary along the length of this organ, but what underlies this variation is not known. Certain mutations that affect the tip links, and experimental destruction of them, cause this response to be reversed, a phenomanon so far unexplained. After damage in vitro the tip links are known to recover along with MET. It is important to explain the reversed responses and understand how the mechanism develops or recovers after damage.

The key objectives of this project are:

1 and 2. To localize candidate MET channels and accessory proteins that are thought to be required by the hair cell to detect the mechanical stimulus in hearing (and balance). Do these proteins occur at the end of the tip links?

3. To determine the distribution of these proteins in situations where hair cell responses are reversed. Are the proteins in abnormal locations, thus causing the reversal?

4. To follow changes in distribution of the proteins during mammalian hair cell development. When and how do these proteins become associated with the tip links?

5. To follow the protein distributions through the process of tip link recovery and remodeling. Does repair and restructuring of the tip link ensure they become correctly localised?

6. To quantify the amount of these proteins in different locations. Do they change in composition, and so confer the known changes in MET channel properties along the cochlea?

Determining the molecular organization of the apparatus that allows the hair cell to respond will enable us to fill in existing gaps in our knowledge of hearing and balance during development and ageing. It will furthermore give us new opportunities to find strategies that could ameliorate degenerative changes that occur with age.

Mechanosensory hair cells of vertebrates have a bundle of stereocilia arranged in rows of increasing height. Deflection towards the tallest row causes mechanoelectrical transduction (MET) channels to open, depolarising the cell. MET channels are located near the lower end of tip links, inter-stereociiary filaments composed of cadherin 23 and protocadherin 15 that join adjacent rows, and may be connected to them via lipoma HMGIC fusion partner-like 5 protein (LHFPL5). Recent reports suggest transmembrane channel like proteins 1 and 2 (TMC1, TMC2) form the MET channels, and mutations in TMC1 and LHFPL5 cause progressive hearing loss. However, the identity of the MET channels is controversial: transduction is reversed in tip link and TMC1/2 double mutants, confirming the latter's involvement, but implying that the other proteins may form the channel. Reversed transduction is also seen during recovery after BAPTA treatment and in development in zebrafish. In this project we will use confocal and quantitative immunoelectron microscopy to localise TMC1, TMC2 and LHFPL5 (or another isoform) during development and in adult mouse cochlea; determine whether their distribution is altered in mutants lacking tip link components with reversed transduction; determine how these proteins change in distribution with recovery of tip links in vitro; and evaluate whether there are quantitative changes in these proteins along the cochlea. The results of this study should (i) provide a better understanding of how TMC1 and TMC2 contribute to forming the MET; (ii) determine whether LHFPL5 is associated with TMC1 and TMC2 and the tip links; (iii) determine whether mislocalisation of the TMC1, TMC2 and LHFPL5 occurs with loss of tip links and underlies reverse transduction; and (iv) provide evidence for a stoichiometric change in MT/associated proteins that explains conductance changes along the cochlea. These findings will be relevant to development of hearing and hearing loss with age.

The research focuses on the basic mechanisms of transduction by cochlear hair cells and the proteins involved; how the transduction mechanism develops and goes wrong in mutations of the proteins; and how it recovers after damage. It addresses fundamental questions in hearing research. The beneficiaries are listed in order of increasingly broader cohorts of stakeholders:

1. Other researchers interested in the topic of hair-cell function: these researchers will benefit from the research outcomes because this project will confirm whether TMC proteins form the transduction channel of hair cells. Thus researchers will be able to develop new strategies to explore further into the mechanisms of transduction.

2. Researchers interested in basic biophysical mechanisms of sensory systems, such as the somatosensory system. The mechanisms of mechanosensitivity of different systems share some features and the result of this study may help to provide new avenues of research in studying mechanosensory systems elsewhere.

3. Researchers, clinicians and patients interested in the underlying causes and treatment of Usher syndrome and hearing disorders based on the TMC and LHFPL proteins. Usher syndrome has both retinal and auditory symptoms and thus has a major impact on the sensory function of sufferers. Several of the mice to be used in this study are also used as models of Usher syndrome. Mutations in TMC and LHFPL also are common causes of hearing loss.

4. Clinicians interested in hearing recovery and how to stimulate it. Recovery of the hair-cell transduction mechanism may be an underlying cause of hearing recovery in temporary loss of hearing caused, for instance, by noise damage.

5. Industrial stakeholders interested in pharmacological or other potential methods to treat hearing loss. Understanding how the transduction machinery recovers from damage gives a focus to developing new drugs and approaches to repair, thereby aiding recovery of hearing.

6. Industries interested more generally in cochlear function, such as the manufacturers of hearing aids and cochlear implants. Understanding hair cell function and signalling will inform the development of new hearing aids and approaches to compensating for hearing loss.

7. Developers of nanosensory and biomimetic materials, and engineering solutions. The transduction mechanism of the hair cell offer unparalleled sensitivity as a detector of sub-nanometre movements. The possibility of future developments for biological sensors or structural designs of nanomaterials based on this mechanism is speculative but nevertheless a potential source of engineering development and commercial activity.

8. Medical and general teaching. The results of this study will be integrated into teaching and learning activities worldwide that are associated with medical conditions and teaching of develomental, normal and ageing heairng function. E.g., the PI has recently revised the Inner Ear Chapter of Gray's Anatomy, a leading medical textbook.

9. Individual's health and well being through the life course. It is important to maintain good hearing through ageing because there is evidence that hearing loss causes not only impaired communication but decreased cognitive function. Since hearing loss affects more than half the population over the age of 65 (statistics from Action on Hearing Loss) this can have a significant social impact.

10. UK health and well being. 10 million people currently have hearing impairment, and this will rise to a predicted 14.5 million by 2031 (source of data: Action on Hearing loss).

11. Global health issues and economy. Understanding hearing mechanisms, hearing loss and recovery has a global impact: ~275 million people suffer from hearing impairment worldwide. One American study estimated that the economic cost of hearing loss was of the order of $300000 per person (as of 2000; Mohr et al., Int J Technol Assess Health Care. 16(4):1120-3