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Primed to Die: an Investigation of the Genetic Mechanisms Underlying Noise-Induced

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bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435183; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Title. 2 Primed to Die: An Investigation of the Genetic Mechanisms Underlying Noise-Induced 3 Hearing Loss and Cochlear Damage in Homozygous Foxo3-knockout Mice 4 5 Running title. 6 Noise induced outer hair cell loss in Foxo3 mutant mice 7 8 Authors. 9 Holly J. Beaulac1, Felicia Gilels1†, Jingyuan Zhang1††, Sarah Jeoung2, and Patricia M. White1* 10 11 Institutional Affiliations. 12 1Department of Neuroscience, Ernest J. Del Monte Institute for Neuroscience, University of 13 Rochester School of Medicine and Dentistry, Rochester, NY, USA. 14 15 2University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. 16 17 †Now at Department of Pathology, University of Rochester School of Medicine and Dentistry, 18 Rochester, NY, USA. 19 20 ††Now at Department of Otolaryngology, Otolaryngology-Head and Neck Surgery, Harvard 21 Medical School, Boston Children’s Hospital Center for Life Science, Boston, MA, USA. 22 23 (*) Corresponding Author. Department of Neuroscience, Ernest J. Del Monte Institute for 24 Neuroscience, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, 25 Rochester, NY, 14642, USA. Email: [email protected]; Phone: 585-273-2340 26 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435183; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 27 Abbreviations. 28 NIHL, noise-induced hearing loss; OHC, outer hair cell; IHC, inner hair cell; SC, supporting cell; 29 P, postnatal day; WT, wild-type; ABR, auditory brainstem response; DPOAE, distortion product 30 otoacoustic emission; DPN, days post noise; HPN, hours post noise; kHz, kilohertz; dB, decibel; 31 DEGs, differentially expressed genes; GDPD3, Glycerophosphodiester Phosphodiesterase 32 Domain Containing 3; LPA, lysophosphatidic acid. 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435183; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 33 Abstract. 34 The prevalence of noise-induced hearing loss (NIHL) continues to increase, with limited therapies 35 available for individuals with cochlear damage. We have previously established that the 36 transcription factor FOXO3 is necessary to preserve outer hair cells (OHCs) and hearing 37 thresholds up to two weeks following a mild noise exposure in mice. The mechanisms by which 38 FOXO3 preserves cochlear cells and function are unknown. In this study, we analyzed the 39 immediate effects of mild noise exposure on wild-type, Foxo3 heterozygous (Foxo3+/KO), and 40 Foxo3 knock-out (Foxo3KO/KO) mice to better understand FOXO3’s role(s) in the mammalian 41 cochlea. We used confocal and multiphoton microscopy to examine well-characterized 42 components of noise-induced damage including calcium regulators, oxidative stress, necrosis, 43 and caspase-dependent and -independent apoptosis. Lower immunoreactivity of the calcium 44 buffer oncomodulin in Foxo3KO/KO OHCs correlated with cell loss beginning 4 hours post-noise 45 exposure. Using immunohistochemistry, we identified parthanatos as the cell death pathway for 46 OHCs. Oxidative stress response pathways were not significantly altered in FOXO3’s absence. 47 We used RNA sequencing to identify and RT-qPCR to confirm differentially expressed genes. We 48 further investigated a gene downregulated in the unexposed Foxo3KO/KO mice that may contribute 49 to OHC noise susceptibility. Glycerophosphodiester Phosphodiesterase Domain Containing 3 50 (GDPD3), a possible endogenous source of lysophosphatidic acid (LPA), has not previously been 51 described in the cochlea. As LPA reduces OHC loss after severe noise exposure, we treated 52 noise exposed Foxo3KO/KO mice with exogenous LPA. LPA treatment delayed immediate damage 53 to OHCs but was insufficient to ultimately prevent their death or prevent hearing loss. These 54 results suggest that FOXO3 acts prior to acoustic insult to maintain cochlear resilience, possibly 55 through sustaining endogenous LPA levels. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435183; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 56 Introduction. 57 Noise induced hearing loss (NIHL) is a pervasive health threat. In 2017, the NIDCD estimated that 58 despite increased awareness and access to protective equipment, nearly 40 million American adults 59 had signs of NIHL1. Therapies for NIHL remain limited, in many cases due to the permanent loss of 60 cochlear sensory cells. Individuals have varying susceptibilities to noise damage, partially due to 61 differences in gene expression crucial for hearing recovery and cochlear preservation2. By studying 62 the underlying genetic mechanisms of NIHL susceptibility, our goal is to better prevent or mitigate 63 NIHL using biological approaches. 64 65 Transcriptional regulators comprise a class of NIHL susceptibility genes, including forkhead box 66 O-3 (FOXO3)3. FOXO3 is a winged helix/forkhead class transcription factor involved in cellular 67 processes including autophagy4, survival5, stress resistance6–9, apoptosis10–12, and longevity13–16. 68 Individuals may harbor genetic variants that can lead to varying levels of FOXO3 transcription, 69 with its spatiotemporal regulatory roles susceptible to manipulation in autoimmune diseases17–19 70 and cancers20,21. FOXO3 is expressed from birth into adulthood throughout the mammalian 71 cochlea including inner and outer hair cells (IHCs and OHCs), supporting cells (SCs), and spiral 72 ganglion neurons (SGNs)22. FOXO3 is capable of translocating from the cytoplasm to nucleus in 73 response to a stressor, as SGNs respond to non-traumatic noise22. This suggests a role in driving 74 gene transcription, which could prevent additional damage or promote death in dysfunctional 75 cells. We have previously shown that FOXO3 is required to preserve OHCs and hearing 76 thresholds following mild noise exposure23. In the hematopoietic system, loss of FOXO3 leads to 77 the overaccumulation of reactive oxygen species (ROS), leading to the death of hematopoietic 78 stem cells24 and developing erythrocytes25. Accumulation of ROS in cochlear cells after noise 79 exposure also drives cell death26–28. We hypothesize that in the absence of FOXO3 following 80 noise exposure, higher levels of reactive oxygen species would accumulate in Foxo3KO/KO OHCs 81 versus WT OHCs, triggering an apoptotic cascade. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435183; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 82 83 To better understand FOXO3’s role in NIHL susceptibility, we analyzed the immediate effects of 84 mild noise exposure on wild-type (WT, Foxo3+/+), Foxo3 heterozygous (Foxo3+/KO), and Foxo3 85 knock-out (Foxo3KO/KO) mice. Previous studies have shown that after traumatic noise exposure, 86 OHC damage drives apoptosis, necroptosis, and a third death pathway29–31. Depending on the 87 severity of the traumatic exposure, OHC caspase-3 activation may be immediately evident29, or it 88 may develop over the following 24 hours post noise (HPN)32. OHC loss continues over a period 89 of days to weeks32,33. The degree of OHC loss seen in the Foxo3KO/KO cochlea after a mild noise 90 exposure could reflect either changes in cochlear homeostasis prior to damage or a compromised 91 damage response. Determining the time course of OHC death could shed light on when FOXO3 92 acts to protect the cochlea. 93 94 To this end, we characterized the time course of OHC loss from noise in the Foxo3KO/KO mouse 95 and identified a likely mode of cell death. We compared activation of known damage pathways 96 in the Foxo3KO/KO cochlea to that of the WT cochlea to assess their involvement. We performed 97 RNA-SEQ, identified a candidate downstream effector enzyme, and tested its involvement by 98 exogenous supplementation of its product. Our goals were to determine what pathways were 99 immediately active after noise and when therapeutic intervention would be most efficacious to 100 mitigate the effects of FOXO3’s absence. 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.12.435183; this version posted March 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 101 Results. 102 Establishing the time course of OHC loss in the Foxo3KO/KO noise-exposed cochlea. 103 Foxo3KO/KO mice acquire severe permanent threshold shifts throughout the cochlea after mild 104 noise exposure23. By two weeks, about one third of Foxo3KO/KO OHCs were lost from the basal 105 cochlea without any IHC loss23. WT littermates experienced only temporary threshold shifts and 106 no cell loss from the same exposure23. These observations did not establish when or how 107 Foxo3KO/KO OHCs are lost. Here we characterize cellular pathology in Foxo3KO/KO and WT 108 cochleae immediately after noise exposure. We used cochlear cryosectioning to obtain cross- 109 sections for detailed cellular analysis (Fig. S1A), and whole mounts preparations for cellular 110 quantification according to tonotopic frequency (Fig. S1B). The latter is important as high 111 frequency (basal) OHCs are more susceptible to trauma34–36. To test for haploinsufficiency, we 112 analyzed the FOXO3 heterozygote (Foxo3+/KO). We measured Foxo3+/KO auditory brainstem 113 responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) at baseline, 1-day 114 post-noise (1 DPN), and 14 DPN (Fig.
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