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Meniere's disease (MD) is an inner ear disorder, characterized by episodes of vertigo associated with sensorineural hearing loss, tinnitus with a multifactorial origin including genetic mutations. Familial MD is associated with several genes, OTOG being the most commonly found with a compound recessive inheritance pattern. This research proposal plans to design and phenotype a mutant otogelin mouse model to validate the causal role of OTOG in Meniere disease. The PhD project goal is to perform a mouse phenotyping by audiological and vestibular testing during mouse development. The PhD work will also include histological studies including scanning electron microscopy and confocal microscopy.
A/Professor Jose Lopez Escamez.
North Shore - Kolling Institute of Medical Research
Background
Meniere's disease (MD) is an inflammatory inner ear disorder with a multifactorial origin, including a genetic contribution (1-3). MD is associated with endolymph accumulation in the inner ear; however, this considered a late event in MD pathophysiology and is associated with hearing loss and duration of the disease (4-6).
Familial aggregation studies have shown that MD has a genetic contribution in European descendent population (7), however there are few studies supporting a familial clustering in East Asian or other ethnicities. Nine genes including FAM136A, DTNA, PRKCB, COCH, DPT, SEMA3D, TECTA, GUSB and SLC6A7 have been associated with autosomal dominant familial MD, whilst 4 genes that have been reported in AR familial MD, including HMX2, LSAMP, OTOG and STRC (8). However, few genes have been reported in multiple families and they encode proteins that links the hair cell stereocilia in the sensory epithelia with the tectorial and otolithic membranes. In this sense, OTOG, MYO7A or TECTA are the main genes associated with familial MD in European population (9-11), but molecular epidemiology studies in other ethnicities are missing.
Our research team has recently elucidated the relevance of two mutations in the OTOG that encodes otogelin, an extracellular protein that couples the hair cell stereocilia to the tectorial membrane (TM) of the sensory organs in the inner ear, (OTOGV269I and OTOGA2037V) in multiple families with MD. The hypothesis is that these mutations will affect the interaction of otogelin with other TM proteins leading to morphological changes in the TM structure and eventually detachment of the TM, with hearing loss or vestibular loss. To better understand the pathophysiology of these mutations in the inner ear we plan to generate transgenic mice harboring these mutations and perform hearing testing and histopathological studies.
Aim of the study
To generate a transgenic mouse and to perform mouse phenotyping by audiological and vestibular testing during mouse development. The PhD work will also include histological studies including scanning electron microscopy and confocal microscopy.
Research Methods
1. Testing baseline hearing function of the OTOGV269I and OTOGA2037V mouse models
Otog knockout mice exhibit hearing deficits and have an impaired outer hair cell (OHC) function (12). OHC are the sensory cells in the cochlea responsible for amplifying sound signals. We hypothesis that OTOG knock in mice (OTOGV269I and OTOGA2037V) would have loss of function and would exhibit similar hearing deficits. OTOG knock in mice will be tested for hearing function using Auditory Brainstem response (ABR) using ‘click’ and tone-pip testing (8kHz32kHz). When comparing patients with familiar MD, hearing loss is typically seen at lower frequencies, but this needs to be established in the mouse model. Having that function of OHC impaired in OTOG deficient mice, we hypothesis that OTOGV269I and OTOGA2037V heterozygous and/or double mutant will also exhibit this phenotype. ABR testing of auditory neurotransmission will be complemented with the use of cubic Distortion product otoacoustic emission (DPOAE) (8-24 kHz). Baseline hearing test will be performed at (6-8) weeks of age and again in 12 – 14 weeks to ascertain if the OTOG transgenic mice exhibit hearing impairment and if it progresses with age which would be tested at later time point (12-14) week [GTT1] [PN2] which can support if the mutation leads to progressive hearing loss. This assessment will be run against wildtype littermates by breeding heterozygous transgenic mice (C57Bl/6J background).
2. Testing susceptibility of OTOGV269I and OTOGA2037V mouse to noise.
We plan to test if noise leads to temporary hearing loss or permanent hearing loss in OTOGV269I and OTOGA2037V heterozygous and double mutants as compared to wild type littermates. To test this hypothesis, we would measure baseline hearing for ABR spanning (8-32kHz) and DPOAE across (8-24 kHz) for 12-14 week old mice followed by white noise exposure for 1 hour at 102 dB (13) [GTT3] [PN4] (8-32KHZ). To test temporary hearing loss ABR and DPOAE measurement will be undertaken before and then immediately after noise exposure while the mice remain anaesthetized (isoflurane).
3. Testing vestibular function of the OTOGV269I and OTOGA2037V mouse.
Otog knockout mice exhibited vestibular dysfunction (12), hence we hypothesis that vestibular function will likely be degraded in OTOGV269I and OTOGA2037V transgenic knock-in mice. The heterozygous, double mutant and wild type littermates will be tested for its vestibular function and motor coordination at two time points (6-8) weeks and (12-14) weeks by gait test and rotarod.
4. To study the structural morphology of the Organ of Corti and vestibular end organs (saccule and utricule) in OTOGV269I and OTOGA2037V mice.
Mice lacking Otog gene show defects in the morphology of the organ of Corti (12). The OHC hair bundles (stereocilia) lack horizontal connectors and are not anchored to the tectorial membrane. We hypothesis that a similar phenotype will be observed in the knock in mice. To study structural defects in OTOG knock in mice, cochlea from Postnatal P0-P20 of heterozygous and double mutants will be extracted after euthanizing the mice. Structural morphology of Inner and outer hair cells will be analyzed using a combination of techniques such as scanning electron microscopy, Transmission electron microscopy, whole mount Immunostaining and cryosection staining of the cochlear and vestibular tissues.
The Meniere disease Neuroscience Laboratory is located at the Kolling Institute.
Prof. Antonio Lopez Escamez profile
References
1. Lopez-Escamez JA, Carey J, Chung WH, Goebel JA, Magnusson M, Mandalà M, et al. J Vestib Res. (2015) 25:1–7.
2. Espinosa-Sanchez JM, Lopez-Escamez JA. Menière’s disease. In: Furman J, Lempert T, editors. Handbook of Clinical Neurology. Amsterdam: Elsevier B.V. (2016). p. 257–77.
3. Stahle J, Friberg U, Svedberg A. Long-term progression of Meniere's disease. Acta Oto-laryngol Suppl. 1991, 485
4. Rauch SD, Merchant SN, Thedinger BA. Ménière’s syndrome and endolymphatic hydrops: a double-blind temporal bone study. Ann Otol Rhinol Laryngol 1989;98: 873–83.
5. Attyé A, Eliezer M, Medici M, Tropres I, Dumas G, Krainik A, Schmerber S. In vivo imaging of saccular hydrops in humans reflects sensorineural hearing loss rather than Meniere's disease symptoms. Eur Radiol. 2018 Jul;28(7):2916-2922.
6. Perez-Carpena P, Lopez-Escamez JA. Current Understanding and Clinical Management of Meniere’s Disease: A Systematic Review. Semin Neurol. 2020;40(1):138–50.
7. Morrison AW. Anticipation in Menière’s disease. J Laryngol Otol. 1995 Jun 29;109(6):499–502.
8.Parra-Perez AM, Lopez-Escamez JA. Types of Inheritance and Genes Associated with Familial Meniere Disease. J Assoc Res Otolaryngol. 2023 Apr 6. doi: 10.1007/s10162-023-00896-0.
9. Roman-Naranjo P, Gallego-Martinez A, Soto-Varela A, Aran I, Moleon MDC, Espinosa-Sanchez JM, Amor-Dorado JC, Batuecas-Caletrio A, Perez-Vazquez P, Lopez-Escamez JA. Burden of Rare Variants in the OTOG Gene in Familial Meniere's Disease. Ear Hear. 2020 Nov/Dec;41(6):1598-1605.
10. Roman-Naranjo P, Moleon MDC, Aran I, Escalera-Balsera A, Soto-Varela A, Bächinger D, Gomez-Fiñana M, Eckhard AH, Lopez-Escamez JA. Rare coding variants involving MYO7A and other genes encoding stereocilia link proteins in familial meniere disease. Hear Res. 2021 Sep 15;409:108329.
11. Roman-Naranjo P, Parra-Perez AM, Escalera-Balsera A, Soto-Varela A, Gallego-Martinez A, Aran I, Perez-Fernandez N, Bächinger D, Eckhard AH, Gonzalez-Aguado R, Frejo L, Lopez-Escamez JA. Defective α-tectorin may involve tectorial membrane in familial Meniere disease. Clin Transl Med. 2022 Jun;12(6):e829.
12. Avan P, Le Gal S, Michel V, Dupont T, Hardelin JP, Petit C, Verpy E. Otogelin, otogelin-like, and stereocilin form links connecting outer hair cell stereocilia to each other and the tectorial membrane. Proc Natl Acad Sci U S A. 2019 Dec 17;116(51):25948-25957. doi: 10.1073/pnas.1902781116.
13. Milon B, Shulman ED, So KS, Cederroth CR, Lipford EL, Sperber M, Sellon JB, Sarlus H, Pregernig G, Shuster B, Song Y, Mitra S, Orvis J, Margulies Z, Ogawa Y, Shults C, Depireux DA, Palermo AT, Canlon B, Burns J, Elkon R, Hertzano R. A cell-type-specific atlas of the inner ear transcriptional response to acoustic trauma. Cell Rep. 2021 Sep 28;36(13):109758.
The opportunity ID for this research opportunity is 3396