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Manuscript Text (Sequence of Content: Main Text, Methods, Legends [For Figures/Tables], and References) bioRxiv preprint doi: https://doi.org/10.1101/764670; this version posted September 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Manuscript text (sequence of content: main text, methods, legends [for 2 figures/tables], and references) 3 Homeostatic maintenance and age-related functional decline in the 4 Drosophila ear 5 Alyona Keder1, Camille Tardieu1, Liza Malong1, Anastasia Filia2, Assel Kashkenbayeva1, 6 Jonathan E. Gale1, Mike Lovett2, Andrew P. Jarman3 & Joerg T. Albert1,4,5,6,7* 7 Affiliations: 8 1Ear Institute, University College London, 332 Gray’s Inn Road, London WC1X 8EE, UK 9 2National Heart and Lung Institute, Imperial College London, Guy Scadding Building, 10 Dovehouse 9 Street, London, SW3 6LY, UK 11 3Centre for Discovery Brain Sciences, Edinburgh Medical School, University of Edinburgh, 12 Edinburgh EH8 9XD, Scotland, UK 13 4Centre for Mathematics and Physics in the Life Sciences and Experimental Biology 14 (CoMPLEX), University College London, Gower Street, London WC1E 6BT, UK 15 5The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK 16 6Department of Cell and Developmental Biology, University College London, Gower Street, 17 London WC1E 6DE, UK 18 7Lead Contact 19 *Correspondence to: [email protected] 20 21 Abstract (150 words): 22 The widespread loss of hearing is one of the major threats to future wellbeing in ageing human 23 societies. Amongst its various forms, age-related hearing loss (ARHL) carries the vast bulk of 24 the global disease burden. The causes for the terminal decline of auditory function, however, 25 are as unknown as the mechanisms that maintain sensitive hearing before its breakdown. We 26 here present an in-depth analysis of maintenance and ageing in the auditory system of the 27 fruit fly Drosophila melanogaster. We show that Drosophila, just like humans, display ARHL 28 and that their auditory life span is homeostatically supported by a set of evolutionarily 29 conserved transcription factors. The transcription factors Onecut (closest human orthologues: 30 ONECUT2, ONECUT3), Optix (SIX3, SIX6), Worniu (SNAI2) and Amos (ATOH1, ATOH7, 31 NEUROD1) emerged as key regulators acting upstream of core sensory genes, including 32 components of the fly’s molecular machinery for auditory transduction and amplification. 1 bioRxiv preprint doi: https://doi.org/10.1101/764670; this version posted September 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 33 Introduction 34 A surface calm can be misleading. All living things, from unicellular amoeba to neurons in the 35 human brain, require continual maintenance and the constant flow of their seemingly equable 36 physiological operations is in fact the product of complex homeostatic networks. All life, it has 37 been said, needs to run to stand still. As with many things, the underlying homeostasis 38 machinery remains mostly unappreciated until it breaks down. A most pertinent example of 39 such a breakdown are the hearing impairments that affect about 1.23 billion people worldwide, 40 corresponding to one sixth of the world’s total population 1. The aetiology of hearing loss is 41 diverse, including infections, ototoxic drugs, noise trauma, as well as genetic factors. The 42 arguably single most important cause, however, is age. Age-related hearing loss (ARHL) 43 carries the vast bulk of the global disease burden, but its scientific understanding is poor and 44 no treatments, neither preventive nor curative, are currently in sight 2. 45 Over the past few decades, gene discovery studies using mouse models have identified 46 numerous candidate genes for human deafness 3-9. Three recent larger scale screens have 47 brought the total number of candidate hearing loss genes for humans to 111 10-12. Yet, the 48 underlying mechanisms and molecular networks of ARHL, and particularly the maintenance 49 of hearing throughout the lifespan, have remained elusive. We here use the auditory system 50 of the fruit fly to shed some light on these issues. 51 The auditory systems of vertebrates and Drosophila share marked similarities; these include 52 (i) the fundamental mechanisms of auditory transduction 13 and amplification 14,15, (ii) the 53 general architecture of neuronal pathways from the ear to higher-order centres in the brain 16 54 and, finally, (iii) conserved families of proneural genes that control hearing organ development, 55 such as e.g. ato in flies and Atoh1 in mice (or ATOH1 in humans) 17,18. The high degree of 56 similarity, and partially near identity, between the ears of Drosophila and vertebrates (including 57 mammals) has recommended the fly as a powerful model to study fundamental aspects of 58 human hearing and deafness 19. 59 Many Drosophila hearing genes have been identified 19-21, but so far no study has explored 60 the flies’ hearing across their life course. We found that the ears of fruit flies also display ARHL. 61 Going one step further we set out to identify those homeostatic regulators that maintain the 62 fly’s sensitive hearing before the onset of ARHL. We combined RNA-Seq-based 63 transcriptomics with bioinformatical, biophysical and behavioural tools to explore the 64 landscape of age-variable genes of Johnston’s Organ (JO) - the flies’ inner ear’. Our data 65 suggests that the thereby identified transcriptional regulators are not restricted to Drosophila 66 - or the sense of hearing - but represent key players of homeostasis across taxa and probably 67 across sensory modalities. 2 bioRxiv preprint doi: https://doi.org/10.1101/764670; this version posted September 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 68 Results 69 Drosophila is prone to age-related hearing loss (ARHL) 70 Functionally, the Drosophila antennal ear (Fig. 1a) is composed of two components: (i) the 71 external sound receiver (jointly formed by the third antennal segment, A3, and its lateral 72 appendage, the arista) and (ii) the actual ‘inner ear’, which is formed by Johnston’s Organ 73 (JO), a chordotonal organ 22 located in the second antennal segment, A2. JO harbours ~500 74 mechanosensory neurons 23. 75 To assess hearing across the Drosophila life course we first measured the locomotor activities 76 of flies in response to a playback of courtship song components at different ages. Drosophila 77 melanogaster males increase locomotor activity in response to courtship song 24. While 10- 78 and 50-day-old flies increased their locomotor activity in response to a 15 min long train of 79 courtship song pulses (inter-pulse-interval, IPI: 40ms), sound-induced responses were absent 80 in 60-day-old flies (Fig. 1b, left). Baseline locomotor activities of 60-day-old flies, however, 81 remained the same as in 10-day-old flies (Fig. 1b, right), suggesting an auditory - rather than 82 a more generalised neurological - deficit as the underlying cause for the non-responsiveness 83 to sound. 84 A simple, but quantitatively powerful, test of auditory performance was then conducted by 85 recording the vibrations of unstimulated sound receivers (free fluctuations) 15. A receiver’s free 86 fluctuations reveal three principal parameters of auditory function: (1) the ear’s best frequency, 87 f0 (measured in Hz), (2) its frequency selectivity or quality factor, Q (dimensionless), and (iii) 88 its energy - or power - gain (measured in KBT). Much like hair cells in the vertebrate inner ear, 89 the antennal ears of Drosophila are active sensors, which inject energy into sound-induced 90 receiver motion 25. 91 Our data show that the ears of flies, much like those of humans, show age-related hearing 92 loss (ARHL) (Fig. 1c). At 25 C, the antennal receivers of 70-day-old flies show (i) best 93 frequency shifts towards the passive system, where no energy injection is observed, (ii) a 94 greatly reduced tuning sharpness and (iii) a ~90% loss of their energy gains (Fig. 1c and 95 Supplementary Table 1), indicating a near-complete breakdown of the active process - which 96 supports hearing - at day 70. The time course of this auditory decline was broadly similar 97 between males and females (Supplementary Table 1). 98 To probe auditory function in more detail, we also quantified the mechanical and 99 electrophysiological signatures of auditory mechanotransduction in response to force-step 100 actuation of the fly’s antennal ear at different ages (Fig. 1d). Direct mechanotransducer gating 101 introduces characteristic nonlinearities - namely drops in stiffness - into the mechanics of the 3 bioRxiv preprint doi: https://doi.org/10.1101/764670; this version posted September 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 102 sound receiver. These so-called ‘gating compliances’ can be modelled with a simple gating 103 spring model 13,26 thereby allowing for calculating the number - and molecular properties - of 104 different populations of mechanosensory ion channels present in the fly’s JO 27. Two distinct 105 mechanotransducer populations have previously been described: a sensitive population, 106 linked to hearing, and an insensitive population, linked to the sensation of wind and gravity 16.
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