Homeostatic maintenance of the auditory system and its relation to age-dependent hearing loss: A Drosophila model organ study

Lead Research Organisation: University College London
Department Name: Ear Institute

Abstract

In the UK, some 70% of people over 70 suffer from decline of hearing. Yet, we have acquiesced into this age-related decay of our senses almost as tacitly as we take their faultless operation for granted for most of our lives. Scientifically, both of these phenomena are equally remarkable. Our eyes and ears are parts of the living world and as such they are in constant need of metabolic energy and permanently exposed to countless environmental challenges. In contrast to a granite rock, a biological system that retains constant properties over periods of days, months, or even years, needs an explanation. If then at some point, and without any obvious external cause, system performance eventually declines, a second explanation is required. This research project will study both of these phenomena and test the hypothesis that they are mechanistically interlinked. In other words, what are the cellular mechanisms that maintain our sense of hearing and how do these mechanisms deteriorate over time? Using the antennal ears of the fruit fly Drosophila melanogaster as a model sensory organ, we will (i) dissect the molecular network that maintains sensitive hearing throughout the fly's life course and (ii) study how this maintenance, or homeostasis, changes during ageing. We will specifically test the hypothesis that the same molecular networks that direct the construction of the ear in the growing fly (about which we already know quite a lot) also contribute to its homeostatic control during adult life (about which we know very little). Importantly, the molecular networks of ear construction are known to have much in common between flies and humans, and we expect our research will provide a relevant model to understand decline of hearing in humans.

Technical Summary

Knowledge about the homeostatic equilibria that maintain sensory systems throughout their life courses is scarce, contrasting starkly with the wealth of information available for the cellular, and molecular, processes that bring about their development. This project will test sets of candidate genes for their contributions to the homeostasis of Johnston's Organ (JO) in the antennal ears of adult fruit flies (Drosophila melanogaster). Specific focus will be given to the post-developmental roles of classic 'regulatory' genes, such as proneural transcription factors, developmental assembly genes, or genes contributing to transport or chaperone pathways. Preliminary evidence suggests that vast parts of the adult JO transcriptome fall into such regulatory categories, but their specific non-developmental functions in the adult ear have remained unknown. We will probe the genes' homeostatic roles in three different ways. First, we will check the expression of candidate genes in JO. Second, we will use the constant turnover of auditory transducer modules as background paradigm. JO mechanotransducer function can be measured in vivo and we will thus use RNAi-mediated gene knockdown strategies to quantify the effects of adult-specific gene knockdowns on the integrity and specific properties of JO transducer modules. We will also use photo-bleaching of fluorescent transducer modules to directly compare turnover rates in between ears expressing knockdown constructs and their respective controls. Third, we will probe the homeostasis, and possible breakdowns thereof, in the ageing auditory system. In biophysical assays (e.g. gating compliance analyses, nerve recordings), we will quantify auditory transducer function in flies of different ages; in molecular assays (e.g. by RNASeq) we will explore age-related changes in the JO transcriptome. Based on these data, we will finally test individual genes for their ability to accelerate, or slow down, age-related hearing loss in flies.

Planned Impact

Commercial benefits

Biomedical drug companies may be interested in the targets identified in this research and in the in vivo screening skills we develop during the project. For instance, our screening strategy can be more generally applied to identify genes required for mechanosensory function, and therefore may be new therapeutic targets for deafness.

Insects are major pests of agriculture and are vectors of disease. Our research has potential benefits for agrochemical and insect control research. Chordotonal auditory and proprioceptive neurons (the subject of this proposal) are the reported targets of action for some well-known insecticides whose molecular mode of action is uncharacterised. It is highly possible that functional homeostasis is itself the insecticide target. The identification of genes required for chordotonal neuron homeostasis will provide excellent targets for understanding the modes of action of such pesticides, and may allow more specific pesticide intervention. Syngenta?

Charity interest

Although this is a basic science proposal, there are several medical charities that are likely to be interested in the research in this proposal for its long term potential to help understand deafness. Implications for hearing research should be of interest to Action on Hearing Loss. Potential impacts would be the identification of molecular targets for possible therapeutic intervention to prevent age-related inner ear hair cell degeneration or to promote regeneration.

Skills/training

The PDRAs to be recruited to this programme will benefit from training in the wide range of molecular and biophysical techniques involved. This will increase their long-term employability in laboratory research.

Public engagement

The wider public is likely to be interested in this research from several points of view. The use of an insect to understand hearing is a fascinating and unexpected concept to many, and it is essential to communicate this as an important aspect of BBSRC's remit.
 
Description Hearing loss affects ~10% of the general population and ~70% of the over 70s. In this project, we have prepared the ground for using the hearing organs of the fruit fly Drosophila as a model to explore ageing - and auditory homeostasis - in an adult ear. The fly's 'inner ear' is formed by the Johnston's Organ (JO). JO harbours ~500 ciliated neurons, which mediate the sensation of various mechanosensory stimuli, including sound, wind and gravity. Although some more subtle noise-induced impairments have been reported, we have found that the fly's ear presents itself as remarkably resistant to both stimulus-induced and age-related functional decline. Up to the age of ~50 days fly hearing fluctuates around normal (healthy) baseline values. A marked age -related hearing loss (ARHL), however, is evident in 70 days old flies. The ARHL is characterised by (i) reduced auditory amplification, (ii) loss of frequency tuning, and (iii) reduced frequency selectivity.
We dissected JOs, and profiled the auditory transcriptome, that means the changes in gene expression, across different ages. The regulatory landscape of the 1,000 most age-variable genes was explored using specialised software package, which can predict genes that act as regulators (upstream) of the identified auditory transcriptomes and genes that are themselves regulated by the identified genes (downstream). Four master regulators emerged from our analyses, which show expression in the fly ear and are conserved in humans (orthologues in brackets): onecut (ONECUT1-3), optix (SIX1-6), wor (SNAI2), amos (ATOH7). We probed the genes' auditory function in vivo by means of Laser Doppler Vibrometry (LDV) combined with auditory nerve recordings. Gene-specific knockdown of onecut and amos caused dramatic declines in auditory function resembling those seen during ARHL; knockdown of optix and wor, in contrast, improved hearing. The consequences of adult -specific RNAi knockdowns were further investigated in quantitative detail revealing a homeostatic regulation of core components of the flies' auditory transducer machinery, namely the transient receptor potential (TRP) channel genes, nompC, nanchung (nan) and inactive (iav). The results show that knockdown of onecut, e.g., leads to downregulation of iav and nan; knockdown of Optix, in turn, leads to upregulation of nompC expression.
The main findings of this project are:
(i) fly ears display age - related hearing loss;
(ii) a homeostatic transcriptional program maintains hearing during large parts of the flies' life course, its eventual breakdown results in auditory decline;
(iii) four transcription factors could be identified as core components of the underlying homeostatic network, corresponding knockdowns either accelerated auditory ageing or protected hearing in older flies.
(iv) onecut emerged as positive regulator of both nan and iav whereas Optix acts as negative regulator of NompC.
(v) NompC itself undergoes a permanent turnover, i.e. old molecules are constantly replaced by new ones. We estimate that this turnover replaces every NompC molecule within ~ 1 or 1 ½ days. The permanent renewal of transducer proteins might be a key mechanism of auditory maintenance.

A second, potentially very impactful, finding of our analyses relates to a set of master genes for auditory (and generally sensory) development, the atonal family of transcription factors. The atonal gene has received widespread academic and clinical attention as it provides currently one of the most promising lines of approach for restorative gene therapy of deafness. While current attention largely concentrates on the atonal gene itself and leaves other family members (paralogs) out of the equation, we found that in Drosophila, the closest paralog to atonal, a gene called amos, can rescue atonal null mutant flies virtually completely.
Exploitation Route The molecular inventory we provided of the ageing auditory transcriptome will be of great use for researchers beyond Drosophila and beyond audition.
The master regulators we identified all have human homologue and several of them have already been linked to sensory function or disease. The overlap between the auditory and visual system we unveiled will also be of particular clinical relevance as it not only provides an explanation for observed sensory co-morbidities (between auditory and visual decline in syndromic and non-syndromic disease clusters) but also a potential route for dual strategies (to counteract age-related visual and auditory disease in parallel).
Finally, our finding that the amos gene when driven in the pattern of the atonal gene can also take over the function of the atonal gene might suggest a slight shift in ongoing gene-therapeutical approaches. Under natural conditions, the flies' ears, e.g., do no longer express the master gene atonal but only its paralog amos. This could indicate that gene-therapeutical approaches in humans might benefit from also looking more closely into the roles, and use of, atonal paralogs (such as ATOH7).
Our findings have already been met with great interest at scientific meetings and are currently being prepared for publication. We ourselves have already initiated the necessary preparations to take these findings forward by securing two world leading experts, one for the biology of ageing (Prof Dame Linda Partridge) one for and state-of-the-art neurotranscriptomics (Dr Tony Southall) to apply for a follow-up project, which aims to take this approach to the next level.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The findings of this grant have directly led to an attempt to explore routes for commercialization in order to identify novel drugs to fight human age-related hearing loss. this effort uses three avenues: 1) Exploiting synergies with an ongoing ERC consolidator grant on circadian function, we have submitted an application for a Proof-of-Concept (PoC) grant (decision pending). 2) We have made contact with various players from pharma (Boehringer Ingelheim, Decibel Therapeutics, Selleck Chemicals, and others) to drive on this effort. 3) We have identified collaborators from human hearing research (and clinical domains), such as Prof Anne Schilder and Prof Karen Steel to team up on this effort. Formalizations of these interactions (such as joint follow-up grant applications) are in place (or process). 4) Selleck Chemicals have agreed to support our activities by providing free-of-charge compounds for testing. 5) Decibel Therapeutics currently support an ongoing project of our collaborator prof Karen Steel (King's College London), which we will join in and team up on. This project will use the fruit fly model that we established in the current award to explore the cross-over between mammalian (human) and insect (Drosophila) signalling networks of auditory homeostasis and age-related hearing loss. The ambition is to bring this research rapidly to the biomedical market. The corresponding activities - and considerations around IP-strategy related questions - have led to a minor delay of the project's key publication but the respective paper is currently in submission. We expect that it will have a huge impact on the current landscape, and future pathways, of research into age-related hearing loss and healthy ageing in general. In a further step we are right now preparing the translation of our findings into two directions: 1) testing the effective compounds identified in the ERC PoC grant in a mouse model of age-related hearing loss and 2) applying for a follow-up funding to (i) explore the genes identified in the BBSRC grant BB/M008533/1 in a mouse model and (ii) explore the downstream landscape of the identified regulatory genes in Drosophila.
First Year Of Impact 2018
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description (Clockstop) - A Fast Throughput Drosophila Model to Identify Drug Treatments for Age-related Hearing Loss (ARHL)
Amount € 150,000 (EUR)
Funding ID 862216 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 06/2019 
End 11/2020
 
Description EMBO Short Term Fellowship (Host Lab)
Amount € 7,500 (EUR)
Organisation European Molecular Biology Organisation 
Sector Charity/Non Profit
Country Germany
Start 01/2017 
End 04/2017
 
Description Homeostatic maintenance of the auditory system and its relation to age-dependent hearing loss: A Drosophila model organ study
Amount £711,952 (GBP)
Funding ID BB/M008533/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2015 
End 12/2018
 
Description Research Grant, International Partnering Award (Taiwan Partnering Award: Mosquito Research - From Sensory Biology to Vector Control)
Amount £50,000 (GBP)
Funding ID BB/R021007/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2020
 
Description Collaboration with Bassem Hassan Lab (Leuven/Paris) 
Organisation University of Leuven
Department VIB Switch Laboratory
Country Belgium 
Sector Academic/University 
PI Contribution Together with the Hassan Lab we explored how a coding sequence exchange of atonal, one of the major proneural (and prosensory) genes, affects the function of mechanosensory submodalities within the fly's Johnston's Organ (JO). We analysed the response behaviours of JOs in flies carrying different atonal knock-in (KI) constructs (from sponges, over annelids to mice). Our analyses covered electrophysiological responses and biomechanical signatures of transduction.
Collaborator Contribution The Hassan lab generated the various atonal knockin constructs and analysed other mechanosensory and visual organs neuroanatomically.
Impact A first publication is currently pending (resubmission of revised manuscript to eLife, currently under review), publication is expected to occur within the next two months.
Start Year 2014
 
Description Collaboration with Dame Linda Partridge (UCL Institute of Healthy Ageing and Max Planck Institute Biology of Ageing, Cologne, Germany) 
Organisation University College London
Department Institute of Healthy Ageing
Country United Kingdom 
Sector Academic/University 
PI Contribution From the AoHL funded PhD studentship on noise and stimulation effects on the Drosophila auditory system we have developed a wider framework of sensory homeostasis during ageing, which has not only led to a grant award in the joint the AoHL/BBSRC highlight notice but also to establishing a longer term collaboration between our group and Prof Dame Linda Partridge, one of the global leaders in ageing research. A better understanding of auditory homeostasis is a crucial requirement for fighting both age-related and activity (noise) dependent hearing deficits. Together with Prof Partridge our fly lab has embarked on that journey.
Collaborator Contribution 1) We have tested (and continue to do so) life span extending compounds, which were provided by the Partridge lab for their effects on auditory function and homeostasis. The Partridge lab has longstanding experience in Drosophila longevity and life span extending strategies.
Impact Joint expression of interest sent to BBSRC for responsive mode round in January (highlight notice on new approaches to ageing)
Start Year 2017
 
Description Collaboration with Dr Chun-Hong Chen International Partnering Award (Taiwan Partnering Award: Mosquito Research - From Sensory Biology to Vector Control) 
Organisation National Health Research Institutes (NHRI) Taiwan
Country Taiwan, Province of China 
Sector Charity/Non Profit 
PI Contribution Together with Dr Chun-Hong Chen we will conduct joint UK/Taiwan workshops with international leaders in the field of mosquito sensory and circadian biology. We will also start proof-of principle experiments with novel mosquito mutants and we will conduct skill/knowledge transfer workshops between our labs. We hold the expertise in mosquito auditory and circadian biology.
Collaborator Contribution Together with Dr Chun-Hong Chen we will conduct joint UK/Taiwan workshops with international leaders in the field of mosquito sensory and circadian biology. We will also start proof-of principle experiments with novel mosquito mutants and we will conduct skill/knowledge transfer workshops between our labs. Dr Chen's lab holds the expertise in mosquito mutagenesis.
Impact none yet (still to start)
Start Year 2017
 
Description Collaboration with Dr Tony Southall (Imperial College London) 
Organisation Imperial College London
Department Department of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Ryan Kavlie, the postdoctoral research associate has visited the Southall lab to learn and conduct targeted DamID (TaDa) on neuronal submodailities of the fly's auditory organ.
Collaborator Contribution The Albert lab has prepared the necessary biological samples for the analyses and Ryan Kavlie, Albert lab member, is conducting the necessary experiments. The Southall lab is providing the tools and lab space as well as the technical knowledge in order to perform TaDa.
Impact The collaboration is still ongoing and sample processing and analyses are underway.
Start Year 2016
 
Description Collaboration with Professor Karen Steel: "Building a translational platform for the study of age-related hearing loss (ARHL)" 
Organisation King's College London
Department Wolfson Centre for Age-Related Diseases
Country United Kingdom 
Sector Academic/University 
PI Contribution This collaboration project will aim to cross-calibrate - and translate in between - two models of human age-related hearing loss (ARHL): the mouse and fly model. The molecular networks known to be involved in age-related hearing loss in flies and mice will be probed bioinformatically, experimentally and genetically, with the aim to reduce mouse experiments in large parts of the gene- and drug discovery process around human ARHL. Experiments will take place at the UCL Ear Institute (lab of Prof Joerg Albert) and King's College (lab of Prof Karen Steel).
Collaborator Contribution Professor Karen Stell shared with us the gene profiling data from different studies, including the patients data with progressive hearing loss, and new genes involved in progressive hearing loss in mice. We then analysed this data and compared it with the RNA sequencing data from the flies antennaes at different ages obtained during the BBSRC project. The results of these bioinformatic analyses revealed over 65% of the genes were age-variable in Drosophila ears, and, specifically, 50 genes showed very high sequence similarity (DIOPT score higher than 10). We further analysed hearing of the three mutant fly lines that previously showed deafness phenotype in mice and/or humans, and all three candidate genes showed profound deafness in flies. We used these preliminary results in order to co-write the NC3Rs research grant application that is due on the 16th of April. The main idea of the research proposal is to analyse the genes that were prevously linked to hearing loss in mammals in flies, and to recommend the genes that showed greater deafness phenotype in flies, to study them in mice hearing loss model.
Impact Main outcome of this collaboration was the outline application for the research grant NC3Rs with the following title:"Building a translational platform for the study of age-related hearing loss (ARHL)". The results of this outline application will be communicated soon and we are planning to apply for the full application in mid-April of 2019.
Start Year 2017
 
Description 3rd Interventions in Aging Conference, 3rd-6th of MArch 2019, Nassau, Bahamas 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact I gave a talk on new finidings in age-related hearing loss, and particular use of the Drosphila model in the hearing research and auditory ageing. The talk was received good by the community and new ideas and collaborations were discussed.
Top-researchers from the ageing research field attended the conference, presented their projects and participated in the active discussion.
Industrial partners also attended the conference, presented their work and discussed the possibilities of new collaborations.
Year(s) Of Engagement Activity 2019
URL https://www.fusion-conferences.com/conference79.php
 
Description H3 symposium / Physiological Society / Sensory Transduction in Insects / 8th December 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact This symposium was conceived by Dr Ben Warren and Prof Joerg Albert. It was motivated by work which bridged sensory modalities and highlighted the common principles of operation of superficially very different sensory neurons. The main aim is to bring together researchers studying sensory transduction in different modalities to promote new interactions, and new insights, in insect sensory biology. The whole day meeting continued until long after the last talks with vivid discussions about the interactions, differences and similarities between the senses and how those could be exploited scientifically. The Physiological Society funding made it possible to assemble a stellar line up of internationally renowned speakers.
Year(s) Of Engagement Activity 2017
 
Description Molecular Biology of Hearnig and Deafness (MBHD) Conference, Hinxton, 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The oral preseantation at the conference leaded to a new collabration with the group of Dr. Jose Antonio Lopez Escamez from Otology & Neurotology Group CTS495
Genomic Medicine Area
Pfizer - Universidad de Granada - Junta de Andalucía Centre for Genomics and Oncological Research (GENYO)
Year(s) Of Engagement Activity 2016
 
Description Public seminar 23/11/2017: What one can hear when listening to the ear of a fly University of Leicester (Sponsor: The Physiological Society) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact Lecture to University of Leicester staff and students led to multiple new engagements and also fostered an ongoing interaction and collaboration with Drs Kyriacou and Matheson. Abstract: All sensation starts with the elementary act of sensory transduction. For the sense of hearing this involves ion channels that are directly (mechanically) gated by the forces of sound. Multiple properties of the entire auditory systems arise from, and have evolved around, these transducer channels; investigating their function, and the constraints that govern their operation, thus can reveal fundamental properties of sound sensation.
Year(s) Of Engagement Activity 2017
URL https://www2.le.ac.uk/departments/npb/news-1/invspk/seminar-abstracts/albert-231117
 
Description Royal Society Theo Murphy International Scientific Meeting: From sender to receiver: physics and sensory ecology of hearing in insects and vertebrates, 4th / 5th December 2017, Kavli Royal Society Centre, Chicheley Hall, Newport Pagnell, Buckinghamshire, MK16 9JJ 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The meeting was a prestigious Royal Society Theo Murphy international scientific meeting organised by Dr Andrei Kozlov and Dr Joerg Albert. It assembled colleagues and scientists from across the globe and from various fields (visual/auditory + insect/vertebrate) to hold a 2-day scientific exchange, which challenged and tried to change, some of the currently held views in order to establish a novel, more interdisciplinary approach to sensory biology. As judged by the following collaborations and the feedback from the audience, the meeting was a great success!
Year(s) Of Engagement Activity 2017
URL https://royalsociety.org/science-events-and-lectures/2017/12/physics-ecology-hearing/
 
Description Symposium International Symposium on Inner Ear Therapies (ISIET) 2017, Marrakesh 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Poster presentation of novel Drosophila model for sensory homeostasis and ageing.
Year(s) Of Engagement Activity 2017
URL http://www.isiet2017.org/
 
Description UK Clock Club Leicester, 12/1/2018, University of Leicester 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact This the regular workshop meeting of all UK researchers with interest in circadian biology, across all taxa (from single cells, over plants to animals). It is the most relevant domestic meeting in the field. We attended as a whole group to interact with the community over the day of the meeting, consolidating existing contacts, establishing new ones, and discussing our work and the current trends in the field.
Year(s) Of Engagement Activity 2018
URL https://www.eventbrite.com/e/uk-clock-club-leicester-2018-tickets-38653586919?utm_source=eb_email&ut...
 
Description collaborative, cross-project visit at the Ludwig Maximilian University (LMU) of Munich 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact During a 4-day visit at the Ludwig Maximilian University of Munich, Prof Albert explored the collaborative potential across three ongoing (or recently ceased) grants:
Award References: BB/L02084X/1 + BB/M008533/1 + BB/R021007/1 + ERC-consolidator grant Clock mechanics (648709), S34, BB/R000549/1, 1206383, 1336457

The visit included a central seminar with the LMU's neurolunch series and meetings with various group leaders and internationally leading PIs in animal evolution, neuroscience and health (e.g. Prof Benedikt Grothe, Prof Axel Borst, Prof Nicolas Gompel, Prof Peter Becker, Prof Till Roenneberg and Prof Martha Merrow). The aim of this visit was to present the recent data of the involved projects just prior to submission for publication and to explore how the overlaps between the different projects can be harvested by new collabrations with experts in the respective fields. Examples include the exploitation of circadian clock function and auditory homeostasis (Profs Becker, Grothe and Merrow), or the evolution of sensory modality and submodality (Profs Borst + Gompel). The short-term goal will be to apply for larger national (e.g. Wellcome Trust collaborative awards) or international (e.g. European Research Council) follow-up grants! The LMU is a centre of German academic excellence, which is of strategic interest for UCL.
Year(s) Of Engagement Activity 2018