Homeostatic maintenance and age-related functional decline in the Drosophila ear

Abstract

Age-related hearing loss (ARHL) is a threat to future human wellbeing. Multiple factors contributing to the terminal auditory decline have been identified; but a unified understanding of ARHL - or the homeostatic maintenance of hearing before its breakdown - is missing. We here present an in-depth analysis of homeostasis and ageing in the antennal ears of the fruit fly Drosophila melanogaster. We show that Drosophila, just like humans, display ARHL. By focusing on the phase of dynamic stability prior to the eventual hearing loss we discovered a set of evolutionarily conserved homeostasis genes. The transcription factors Onecut (closest human orthologues: ONECUT2, ONECUT3), Optix (SIX3, SIX6), Worniu (SNAI2) and Amos (ATOH1, ATOH7, ATOH8, NEUROD1) emerged as key regulators, acting upstream of core components of the fly's molecular machinery for auditory transduction and amplification. Adult-specific manipulation of homeostatic regulators in the fly's auditory neurons accelerated - or protected against - ARHL.

Figure 1

Drosophila Hearing across the life course. (a) Schematic representation of Johnston’s Organ (JO), a chordotonal organ located in the 2^nd antennal segment. JO harbours the mechanosensory units (scolopidia) that mediate the sensation of sound in Drosophila. Sound waves act on the feathery arista, forcing the 3^rd antennal segment to rotate about its longitudinal axis, thereby stretch-activating specialised mechanosensory ion channels (Nan, Iav, NompC) in the scolopidial neurons. (b) Sound-evoked activity (shown in light blue, male locomotor responses to courtship song, seen in 10-day and 50-day old flies (p < 0.001 in both, paired t-test) are abolished in 60-day old flies. Baseline activity levels (shown in grey, male locomotor activity when not stimulated) are not significantly different between 10 and 60 day old flies. [p = 0.487, t-test; sample sizes: n(day 10) = 12, n(day 50) = 10, n(day 60) = 14]. (c) Power Spectral Densities of unstimulated antennal sound receivers betray age-related decline of hearing in both males (left, shades of blue) and females (right, shades of red). Preceded by homeostatic oscillations around their baseline values, all principal parameters of hearing (shown in right-hand panels for both sexes) indicate a loss of hearing from day ~50 onwards: the receiver’s best frequency starts rising towards the level of the passive system, the auditory energy gain drops to near zero and tuning sharpness falls to values around ~1. [sample sizes males: n(day 1) = 18, n(day 5) = 13, n(day 10) = 6; n(day 25) = 19, n(day 50) = 16), n(day 60) = 11, n(day 70) = 18; sample sizes females: n(day 1) = 17, n(day 5) = 8, n(day 10) = 4; n(day 25) = 17, n(day 50) = 20), n(day 60) = 12, n(day 70) = 17]. (d) Mechanical and electrophysiological responses to force steps allowed for probing JO mechanotransducer function across the auditory life course in male (left, blue) and female (right, red) flies. Mechanical integrity of auditory transducers was quantified by fitting gating spring models to the antennal receiver’s dynamic stiffness (slope stiffness) as a function of its peak displacement (see ref. for details). Electrophysiological function was assessed by recording compound action potential (CAP) responses from the antennal nerve. CAP responses showed an identical pattern across the life course in both males and females: CAP response magnitudes substantially increased from day 1 to day 25, then monotonously declined from day 25 to day 70. The largest drop in CAP magnitudes occurred between day 50 and day 70, with responses of 70-day-old flies even falling below those of 1-day-old flies. Transducer mechanics, in contrast, remained more intact throughout. However, at day 70 the four principal parameters of transducer function, i.e. the number of sensitive transducer channels N_s), the number of insensitive transducer channels (N_i), the sensitive single channel gating force (z_s) and the insensitive single channel gating force (z_i) were all significantly different from their values at day 1, in both males and females (Mann-Whitney U test, p < 0.01 for all). Interestingly, no such change was observed for the stiffness of the antennal joint (K_steady), which is a transducer-independent measure of antennal mechanics. Next to these properties shared between males and females, our analyses also revealed some sexually dimorphic phenomena: K_GS was significantly different only in males (Mann-Whitney U test, p < 0.01). Whereas in females N_s, N_i, z_s and z_i, remain at constant values until the age of 50 days, the respective values of male flies change monotonously throughout the life course, with continually falling numbers of transducer channels being compensated by increasing single channel gating forces (thereby homeostatically balancing the male antenna’s nonlinear stiffness). [all error bars are SEM; sample sizes males: n(day 1) = 8, n(day 25) = 10, n(day 50) = 10), n(day 70) = 8; sample sizes females: n(day 1) = 11, n(day 25) = 10, n(day 50) = 10), n(day 70) = 7].

Figure 2

Gene-Ontology and Bioinformatics of the Drosophila age-variable JO transcriptome. (a) Gene Ontology (GO) based summary of age-variable genes in JO as derived from RNAseq data taken across different age points (days 1, 5, 10, 25 and 50). Down-regulated (blue) and up-regulated (red) genes from multiple pairwise comparisons between all age points are shown on the left and right side of the graph, respectively. The bubble diameter is proportional to the gene number (the larger the diameter the more genes were down -or up-regulated. The y-axis shows numbers of the genes and the x-axis the Log₂Fold-Change (Log₂FC) of gene expression. GO terms correspond to the up-regulated (right) or down-regulated (left) genes, a selection of which is shown in the respective neighbouring boxes; enrichment scores are shown in brackets. Selection of the most age variable genes are shown in individual boxes corresponding to each GO term next to it. Individual bubbles denote the number of genes (y-position of bubble centre) and their corresponding range of Log₂FC values; negative ranges like ‘−1 < x < −0.59’ mean that genes were downregulated between 2 and 2^0.59 times, whereas positive ranges like ‘1 < x < 0.59’ denote an upregulation between 2¹ and 2^0.59 times. (b) Prediction of upstream transcriptional master regulators for age-variable JO target genes (based on motif-binding analysis in the iRegulon software package). Identified master regulators wor, amos, Optix and onecut are shown in yellow with arrows leading to their predicted targets. Targets are grouped, up and down-regulated genes shown in blue and red, respectively. Mechanosensory ion channels, previously linked to fly hearing, are shown in green: iav and nompC are predicted to be downstream of onecut, amos and Optix, whereas nan is predicted to be downstream of wor, Optix and onecut.

Figure 3

Expression validation of homeostatic master regulators in JO. All four predicted regulators (Wor, Amos, Optix and Onecut) are expressed in JO (expression analysis was done at the age of day 10 for all genotypes). Expression of Wor was detected by expressing EGFP under the control of a wor-Gal4 driver; expression of Amos, Optix and Onecut was detected by using GFP-tagged flyFos gene expression constructs. Co-labelling with antibodies against two pan-neuronal markers (the nuclear marker Elav, red; and the membrane marker HRP, blue) confirmed neuronal expression for all four regulators. Arrowheads indicate examples of clear co-localization between the three signals.

Figure 4

Functional validation of homeostatic master regulators. (a) Average vibration velocities of female unstimulated sound receivers (‘free fluctuations’) after adult-specific, RNAi-mediated knockdown (KD; red solid lines) for all four master regulators alongside their respective controls (grey dashed lines). KDs of amos and onecut show a loss of sound receiver function, as evident from (i) reduced energy content (‘power gain’), (ii) reduced frequency selectivities, and - in the case of onecut - also (iv) best frequency shifts towards higher values. KDs of wor and Optix, in contrast, show enhanced sound receiver function, as evident from (i) increased energy content and (ii) increased frequency selectivity (wor) or best frequency shifts to lower values (Optix). [Supplementary Table 6 for numerical details and statistics]. All flies were assessed 15 days after eclosion. (b) Line plot summaries comparing the KD sound receiver phenotypes [as from (a)] to the sound receiver phenotypes occurring naturally during ageing (reference for comparison: Canton-S day 1 to day 70). Arrows indicate significant changes in parameters. Black arrows indicate that KD phenotypes (relative to their corresponding controls) phenocopy the age-related hearing loss (ARHL) phenotypes seen in wildtype flies. White arrows indicate a reversal of the specific ARHL phenotype. (c) Gating compliances (average fits, top) and CAP responses (medians plus standard errors, bottom) to force step actuation across adult-specific KDs of four master regulators (red) and their corresponding controls (grey). CAP responses are plotted against both stimulus force and antennal displacements. KD of onecut leads to a dramatic loss of auditory transducer function, as evident from the near complete loss of the gating compliance for the most sensitive transducers and the loss of nerve responses to small stimulus forces/displacements. KDs of wor, amos and Optix have subtler effects on transducer mechanics but all reduce nerve responses to larger stimulus forces/displacements. (d) Line plot summaries of transducer mechanics [from (C)] in four regulator KDs (red) relative to controls (green). Dashed lines indicate respective control values. Significant changes are asterisked (*). (e) Sound-induced behavioural responses in males after wor, amos, Optix and onecut KD (red) compared to control flies (grey). wor KD mutants show hypersensitivity to sound and show significant reduction of locomotor activity to sound compared to the baseline (n = 14,p = 0.029, Mann-Whitney Rank Sum Test), amos KD mutants do not respond to sound (n = 12, p = 0.503, t-test), Optix KD mutants show hypersensitivity to sound and show significant reduction of locomotor activity to sound compared to the baseline (n = 17,p = 0.001, Mann-Whitney Rank Sum Test), onecut KD mutants do not respond to sound (n = 10, p = 0.277, t-test), while their respective controls show an increase in locomotor activities in response to sound (n = 36, p = <0.001, Mann-Whitney Rank Sum Test).

Figure 5

Key molecular targets validation and gene therapeutic approach to ARHL. (a) Gene expression changes after regulator KDs as quantified by RT-qPCR. wor and amos KDs show significant reduction of the dynein motor Dhc98D, while KD of Optix leads to overexpression of NompC; onecut KD reduces expression of both nan and iav. (n indicates the biological replicates, error bars show standard deviations, *p > 0.05, **p > 0.01). (b) Vibration velocity of the sound-receiver and the sharpness of the tuning Q are significantly reduced in Dhc98D knockdown flies (shown in red) compared to the controls (shown in dotted grey). See also Supplementary Table 6. (c) Power Spectral Densities of unstimulated antennal sound receivers betray accelerated age-related hearing loss (aARHL) in flies kept at 30 °C (left), with a near complete loss of receiver activity already at ~day 25 (light blue area: 1 day old flies; dark blue area: 25 day old flies). A 30-day-long amos (middle) overexpression (OE) or wor (right) knockdown (at 30 °C) protects receivers from the age-related loss of activity (dark blue: KD or OE, respectively; light blue: controls). Box plots show energy contents (power gains) for each transgenic intervention (dark blue) relative to controls (light blue).

Figure 6

Comparison of lifespan and auditory healthspan. The flies’ auditory health span (here depicted as median auditory gain in % of its maximum value) and survival rates (three independent cohorts shown) are closely aligned. Both show sharp drops from ~50 days on (stocks kept at 25 °C and 60% relative humidity).