Hearing and Deafness

Sensory transduction in the cochlea and vestibular labyrinth depends on fluid movements that deflect the hair bundles of mechanosensitive hair cells. Mechanosensitive transducer channels at the tip of the hair cell stereocilia allow K²⁺ and Ca²⁺ to flow into cells. This unusual process relies on ionic gradients unique to the inner ear. Linking genes to deafness in humans and mice has been instrumental in identifying critical aspects of the molecular machinery important for hearing and balance. Functional analysis is difficult if not impossible in patients, but mouse models have helped to investigate phenotypes at different developmental time points, as well as to set blueprints for future therapeutic interventions.

Inner ear connexin expression and function under pathophysiological conditions

Figure 1. (A) Spontaneous intracellular Ca²⁺ concentration changes in a cochlear colture averaged during 7 minutes of recording and encoded in false-colors by the Fluo-4 fluorescence changes (Δf/f0). Six regions of interest are shown superimposed on different Ca²⁺ hot spots; GER, greater epithelial ridge; LER, lesser epithelial ridge; scale bar, 100µm; (B) Fluo-4 traces obtained as pixel averages from the six regions in (A).

Figure 1. (A) Spontaneous intracellular Ca²⁺ concentration changes in a cochlear colture averaged during 7 minutes of recording and encoded in false-colors by the Fluo-4 fluorescence changes (Δf/f0). Six regions of interest are shown superimposed on different Ca²⁺ hot spots; GER, greater epithelial ridge; LER, lesser epithelial ridge; scale bar, 100µm; (B) Fluo-4 traces obtained as pixel averages from the six regions in (A).
[click image to enlarge]

In Europe profound deafness affects at least 3 million individuals and at least 6.7 million people are severely deaf. We have recently offered a mechanistic explanation for the pathogenesis of deafness due to connexin mutations. Abnormal or impaired connexin function has been linked to several other diseases, including skin disease, peripheral neuropathies, and cataracts, thus our data may have a more general impact. With help of funds from the Italian Telethon Foundation, we are contributing to create and use mouse models of deafness to advance our understanding of inner ear physiology and pathology and to open the way to genetic and pharmacologic approaches. We investigated the connexin network of cochlear inner supporting cells, in which ATP acts as an IP₃-generating agonist and evokes Ca²⁺ responses that have been linked to noise-induced hearing loss and development of hair cell-afferent synapses (Figure 1). Our results demonstrate that these connexins play a dual crucial role in inner ear Ca²⁺ signaling.
The recent publication of the structure of the connexin 26 (Cx26) gap junction channel at 3.5 Å resolution opens many new perspectives also to the study of the single channel properties. Efforts are ongoing in our lab to simulate Cx26 and Cx30 channels in a realistic environment, including solvent and membrane phospholipids. We can simulate the diffusion through Cx26 and Cx30 channel pores of selected ions and signaling molecules such as Ca²⁺, IP₃, cAMP, cGMP and ATP in the presence of an electrochemical gradient, by applying steered dynamics. This will allow us to compare transit time and investigate specific interactions with the pore lining residues of wild-type (WT) and mutant channels. This work is combined with unitary permeability measurements in WT and mutant channels by patch-clamp.

Applying a gene therapy approach to the inner ear

Figure 2. Cochlear outer supporting cells from Cx30 KO mice transduced with BAAVCx30GFP (green; red is actin) show recombinant Cx30GFP protein expression in 90% of the cells; images were acquired 48h after adding the viral vectors to the culture medium. Scale bar: 30µm.

Figure 2. Cochlear outer supporting cells from Cx30 KO mice transduced with BAAVCx30GFP (green; red is actin) show recombinant Cx30GFP protein expression in 90% of the cells; images were acquired 48h after adding the viral vectors to the culture medium. Scale bar: 30µm.
[click image to enlarge]

The molecular diagnosis of hereditary hearing impairment, especially of the highly prevalent form due to a defect in Cx26, is now available in both developed and some developing countries. But so far, aside from hearing aids, there is no remedy for this hereditary disease. At the moment we have good expectations for a new therapeutic tool based on gene transfer by a novel bovine virus (BAAV) [Di Pasquale G et al., Mol Ther. (2005) 11:849-55]. Immunocytochemistry and quantitative PCR analysis of Cx30 KO mouse cochlea cultures revealed that Cx26 is downregulated at the protein level and at the mRNA level in nonsensory cells located between outer hair cells and the stria vascularis. The manipulation of Cx30 gene expression in the Cx30 KO mouse by transduction with BAAV restored Cx26 expression, permitted the formation of functional gap junction channels, and rescued propagating Ca²⁺ signals (Figure 2). Based on in vitro results and in vivo preliminary results, we have reasons to believe that injection of the BAAV adenoviral vector via a canalostomy in the posterior semicircular canal of the mouse can preserve hearing and is particularly attractive due to the simplicity of the surgical approach.

Mechanoelectrical transduction, Ca²⁺ homeostasis and congenital hearing loss

(collaboration with E. Carafoli)

Ca²⁺ acts as a fundamental signal transduction element in the inner ear, delivering information about sound acceleration and gravity through a small number of mechano-transduction channels in the hair cell stereocilia as far as to the ribbon synapse, where it drives neurotransmission. In particular, ablation or missense mutations of the unusual splicing isoform (w/a) of the PMCA2 Ca²⁺-pump of stereocilia cause deafness and loss of balance. To investigate the physiological significance of these genetic defects, we used a combination of confocal fluorescence microscopy and cytosolic Ca²⁺ photoliberation in hair cells of organotypic cultures of the inner ear utricle from neonatal mice . Ca²⁺ extrusion was found to be compromised by various degrees in PMCA2 knockout mice as well as in the mutant deafwaddler, Oblivion and Tommy mice, thus providing an explanation for the observed reduced endolymphatic Ca²⁺ concentration which can trouble the finely tuned control mechanisms of signal transduction, eventually resulting in hair cell death.

Technological advances in the field of optical microscopy

In 2005, our group received a 12000 Euro prize in a regional competition for new business ideas. The Foundation for Advanced Biomedical Research subsequently created a spin-off company called Optical and Electronic Solutions (OES) S.r.l. that is now out on the market, trying to attract investors or venture capitalists. To study signal transduction in vivo, we have also developed an imaging system based on adaptive optics and graded index (GRIN) fibers. The basic idea is to retro-fit the VIMM Biorad 2000 two-photon confocal microscope by intercepting its laser beam and reflecting it off a deformable mirror . The necessary hardware and software have been developed in collaboration with a team of engineers at the Padua branch of the National Institute of Astrophysics lead Dr. Favio Bortoletto (former Director of the European Observatory "Galileo" in the Canary Islands).

Synoptic CV

Honours

Selected VIMM Publications

• Schütz M, Scimemi P, Majumder P, De Siati RD, Crispino G, Rodriguez L, Bortolozzi M, Santarelli R, Seydel A, Sonntag S, Ingham N, Steel KP, Willecke K, Mammano F (2010) The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice. Hum. Mol. Genet. 19:4759-73.
• Ortolano S, Di Pasquale G, Crispino G, Anselmi F, Mammano F, Chiorini JA (2008) Coordinated control of connexin 26 and connexin 30 at the regulatory and functional level in the inner ear. Proc. Natl. Acad. Sci. U.S.A. 105:18776-81.
• Anselmi F, Hernandez VH, Crispino G, Seydel A, Ortolano S, Roper SD, Kessaris N, Richardson W, Rickheit G, Filippov MA, Monyer H, Mammano F (2008) ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear. Proc. Natl. Acad. Sci. U.S.A. 105:18770-5.
• Spiden SL, Bortolozzi M, Di Leva F, de Angelis MH, Fuchs H, Lim D, Ortolano S, Ingham NJ, Brini M, Carafoli E, Mammano F, Steel KP (2008) The novel mouse mutation Oblivion inactivates the PMCA2 pump and causes progressive hearing loss. PLoS Genet. 4:e1000238.
• Hernandez VH, Bortolozzi M, Pertegato V, Beltramello M, Giarin M, Zaccolo M, Pantano S, Mammano F (2007) Unitary permeability of gap junction channels to second messengers measured by FRET microscopy. Nat. Methods 4:353-8.

VIMM Publications

• Bortolozzi M, Brini M, Parkinson N, Crispino G, Scimemi P, De Siati RD, Di Leva F, Parker A, Ortolano S, Arslan E, Brown SD, Carafoli E, Mammano F (2010) The Novel PMCA2 Pump Mutation Tommy Impairs Cytosolic Calcium Clearance in Hair Cells and Links to Deafness in Mice. J. Biol. Chem. 285:37693-703.
• Majumder P, Crispino G, Rodriguez L, Ciubotaru CD, Anselmi F, Piazza V, Bortolozzi M, Mammano F (2010) ATP-mediated cell-cell signaling in the organ of Corti: the role of connexin channels. Purinergic Signal. 6:167-87.
• Pantano S, Zonta F, Mammano F (2008) A fully atomistic model of the Cx32 connexon. PLoS ONE 3:e2614.
• Bortolozzi M, Lelli A, Mammano F (2008) Calcium microdomains at presynaptic active zones of vertebrate hair cells unmasked by stochastic deconvolution. Cell Calcium 44:158-68.
• Mammano F, Bortolozzi M, Ortolano S, Anselmi F (2007) Ca2+ signaling in the inner ear. Physiology (Bethesda) 22:131-44.
• Ficarella R, Di Leva F, Bortolozzi M, Ortolano S, Donaudy F, Petrillo M, Melchionda S, Lelli A, Domi T, Fedrizzi L, Lim D, Shull GE, Gasparini P, Brini M, Mammano F, Carafoli E (2007) A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness. Proc. Natl. Acad. Sci. U.S.A. 104:1516-21.

Selected Seminars

Contact

Fabio Mammano Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Tel.:  (+39) 049 7923 231 Fax:  (+39) 049 7923 250