cGMP survival signalling for prevention of hearing loss

Lukas Rüttiger and Marlies Knipper, Tübingen

The potential capacity of cGMP activation for protection of sensory organs in the inner ear is not fully understood. Our previous findings suggested that the phosphodiesterase 5 (PDE5) inhibitor vardenafil may provide protection against trauma-induced hearing loss and sensory hair cell death potentially by signalling via a common cGMP/cGMP-dependent protein kinase type I (cGKI) pathway in the inner ear. At the same time we have identified a higher vulnerability and a reduced recovery from noise-induced auditory injury in mouse mutants globally lacking cGKI. Hence, it is the overall aim of this project to elucidate the role of the cGMP generators and effectors involved in hair cell survival in the injured or challenged auditory system, and to monitor spatiotemporal cGMP dynamics during both sudden and progressive damage. In order to validate this pathway for early identification and treatment of hearing loss, we will monitor the auditory responses in the presence and absence of various cGMP elevating agents in different animal models. To date, we have subjected membrane-bound guanylyl cyclase GC-A and GC-B mutants as well as global knockout mice lacking either the nitric oxide (NO)-stimulated GC subunits a1 and a2 (NO-GC1-KO and NO-GC2-KO, respectively) or the β1 subunit deficient NO-GC animals to a combination of physiological and molecular settings for spontaneous or induced auditory signal processing disorders (in collaboration with other groups of this research unit). Interestingly, we find that GC-A rather than NO-GC mediates protection from noise-induced hearing loss, whereas NO-GC1 and NO-GC2 seem to have autonomous functions in inner and outer hair cells, which may involve changes in central brainstem adaptation and a distinct efferent feedback control. Further studies need to dissect the individual functions of the NO-GC isoforms in sensory cells and central nuclei for spontaneous and trauma-induced hearing loss and their potential use as pharmacological targets in the development of auditory pathologies. Moreover, we will explore the molecular and cellular aspects of degeneration and regeneration during auditory injury, which should validate the therapeutic potential of GC-A and its ligands for the protection of the inner ear during auditory stress. GC-B deletion leads to failure of auditory nerve bifurcation in the auditory brainstem. Our preliminary findings suggest that this may lead to a moderate hearing loss possibly linked to disturbed efferent feedback loops. We will therefore also explore the role of GC-B/cGMP for the central inhibitory neuronal circuits in the cochlear nucleus complex and for damage-induced changes in binaural hearing, adaptation, and plasticity in more detail. These functional experiments shall be corroborated by exploring the cGMP effector proteins downstream of the distinct cGMP generating molecules and by directly visualizing cGMP in different cell types of the organ of corti under pathophysiological conditions using cGi500 sensor transgenic mice with inducible expression of the cGMP sensor specifically in e.g. sensory hair cells, auditory neurons, and supporting cells. Together, our comprehensive approach using cellular, functional and perceptual assays should decode the cGMP cascade in the hearing organ and thereby contribute to the general state of knowledge for cell protection and survival by cGMP and cGMP elevating compounds.

Fig. 1. Scheme of afferent and efferent neuronal connections in the cochlea and signalling during noise exposure. (A) Afferent signals are transmitted in the auditory nerve from the cochlea to the brainstem. The afferent connections, type-I afferents from the IHCs and type-II afferents from the OHCs, can be modulated in activity by inhibitory axo-dentritic (LOC-efferents to the IHCs) or axo-somatic (MOC-efferents to the OHCs) contacts. Components of the cGMP/cGKI signalling cascade have been identified in the IHCs, the neurons of the SGN, and the brainstem nuclei. In subproject 1, the role of NO-GC for efferent feedback to cochlear hair cells and auditory responses will be studied. Subproject 2 will focus on GC-A deficient mice, that exhibit a hearing phenotype strikingly similar to the cGKI deficient mice (Jaumann et al 2012 and preliminary data). Subproject 3 will analyse the involvement of other PDEs for hearing protection and cell survival. The significance of GC-B mediated bifurcation of auditory fibres in the cochlear nucleus will be examined in subproject 4. (B) Noise exposure and age-related hearing loss seem to be influenced by several cGMP signalling cascades. NO-GC stimulation may support central efferent pathways. GC-A seems to protect hair cells and afferent connections in the inner ear. GC-B enables central axonal bifurcation to disperse the auditory information into the sensory channels required for temporal and spatial recognition and detection. While we found no evidence for the involvement of IRAG in the protective cascades, stimulation of NO-GC preserves function after damage, as does inhibition of PDE5 (modified after Layman & Zuo 2012). (C) L. Rüttiger, M. Knipper
Fig. 2. cGKI and PDE5 localization in the cochlea. Immunohistochemistry on cochlear sections demonstrates PDE5 (c,d,e) and cGKI (f,g,h) expression in outer hair cells (OHCs, c,f), inner hair cells (IHCs, d,g), spiral ganglion neurons (SGN, e,h, solid arrowheads) and supporting cells (satellite cells, Sat, e,h, open arrows). Whirlin, a protein that is expressed in the stereocilia of sensory IHC and OHC was used to localize PDE5 and cGKI proteins to the region under the cuticular plate. No signals were detected with the cGKI antibody in cGKI-deficient mice (inserts). Scale bars, c,d,f,g, 10 µm; e,h, 20 µm. From Jaumann et al. 2012.

Project-related publications

PIs of this project; PIs of other FOR 2060 projects are in bold.

1.    Schubert T, Gleiser C, Heiduschka P, Franz C, Nagel-Wolfrum K, Sahaboglu A, Weisschuh N, Eske G, Rohbock K, Rieger N, Paquet-Durand F, Wissinger B, Wolfrum U, Hirt B, Singer W, Rüttiger L, Zimmermann U, Knipper M. Deletion of myosin VI causes slow retinal optic neuropathy and age-related macular degeneration (AMD)-relevant retinal phenotype. Cell Mol Life Sci. 2015;72:3953-69 [Project 8 and Project 9] [pubmed]

2.    Knipper M, Panford-Walsh R, Singer W, Rüttiger L, Zimmermann U. Specific synaptopathies diversify brain responses and hearing disorders: you lose the gain from early life. Cell Tissue Res. 2015;361:77-93. [Project 8] [pubmed]

3.    Chen JT, Guo D, Campanelli D, Frattini F, Mayer F, Zhou L, Kuner R, Heppenstall PA, Knipper M, Hu J. Presynaptic GABAergic inhibition regulated by BDNF contributes to neuropathic pain induction. Nat Commun. 2014;5:5331. [Project 8] [pubmed]

4.    Dettling J, Franz C, Zimmermann U, Lee SC, Bress A, Brandt N, Feil R, Pfister M, Engel J, Flamant F, Rüttiger L, Knipper M. Autonomous functions of murine thyroid hormone receptor TRα and TRβ in cochlear hair cells. Mol Cell Endocrinol. 2014;382:26-37. [Project 1 and Project 8[pubmed]

5.    Typlt M, Mirkowski M, Azzopardi E, Rüttiger L, Ruth P, Schmid S. Mice with deficient BK channel function show impaired prepulse inhibition and spatial learning, but normal working and spatial reference memory. PLoS One. 2013;8:e81270. [Project 5 and Project 8] [pubmed]

6.    Duncker SV, Franz C, Kuhn S, Schulte U, Campanelli D, Brandt N, Hirt B, Fakler B, Blin N, Ruth P, Engel J, Marcotti W, Zimmermann U, Knipper M. Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells. J Neurosci. 2013;33:9508-19. [Project 5 and Project 8] [pubmed]

7.    Jaumann M, Dettling J, Gubelt M, Zimmermann U, Gerling A, Paquet-Durand F, Feil S, Wolpert S, Franz C, Varakina K, Xiong H, Brandt N, Kuhn S, Geisler H, Rohbock K, Ruth P, Schlossmann J, Hütter J, Sandner P, Feil R, Engel J, Knipper M, Rüttiger L. cGMP-Prkg1 signaling and PDE5 inhibition shelter cochlear hair cells and hearing function. Nat Med. 2012;18:252-9. [Project 1Project 5Project 8, and Project 9] [pubmed]

8.    Zuccotti A, Kuhn S, Johnson SL, Franz C, Singer W, Hecker D, Geisler HS, Köpschall I, Rohbock K, Gutsche K, Dlugaiczyk J, Schick B, Marcotti W, Rüttiger L, Schimmang T, Knipper M. Lack of brain-derived neurotrophic factor hampers inner hair cell synapse physiology, but protects against noise-induced hearing loss. J Neurosci. 2012;32:8545-53. [Project 8[pubmed]