Assistant ProfessorMario Bortolozzi
Postdoctoral FellowsGiulia Crispino
Ph.D. studentFederico Ceriani
Mario Bortolozzi: VIMM Junior PI since September 2013
The Cochlea Webpage
Hearing and Deafness
Field of Interest
The sense of hearing relies on a sensitive mechanoelectrical transduction process in the cochlea of the inner ear, in which the organ of Corti encompasses highly specialized sensory hair cells that are embedded in a matrix of supporting and epithelial cells, henceforth designated as cochlear non–sensory cells. Fluid movements that deflect the hair bundles of hair cells activate mechanosensitive transducer channels at the tip of the hair cell stereocilia allowing K+ and Ca2+ to flow into cells. This unusual process relies on ionic gradients that are unique to the inner ear and whose maintenance depends heavily on cochlear non–sensory cells (Figure 1).
[click image to enlarge]
Indeed, the functional maturation of the cochlea relies not only on the differentiation of hair cells and the formation of coordinately polarized hair bundles, but also on the formation of non–sensory cell networks, which form vast functional syncytia (Figure 2) coupling transfer of ions, signaling molecules and nutrients through gap junction channels formed by connexin (Cx) subunits.
Most cell–cell channels in cochlear gap junction networks are composed of Cx26 and Cx30, which share 77% amino acid identity and may assemble to form heteromeric and heterotypic channels. While the exact function of connexins expressed by non–sensory cells of the inner ear remains unclear, it is important to mention that they also form unpaired connexons, i.e. non–junctional connexin hemichannels in the cell plasma membrane (Figure 3).
[click image to enlarge]
The genes encoding Cx26 (GJB2) and Cx30 (GJB6) are found within 50 kb of each other in the DFNB1 complex deafness locus. DFNB1 accounts for approximately 50% of congenital, severe-to-profound, autosomal recessive nonsyndromic hearing loss in the United States, France, Britain, and New Zealand/Australia. The incidence of congenital hereditary hearing impairment is 1:2000 neonates, of which 70% have nonsyndromic hearing loss. Seventy-five to 80% of cases of nonsyndromic hearing loss are autosomal recessive; of these, 50% result from GJB2 mutations. Thus, the approximate prevalence of DFNB1 in the general population is 5:10,000 x 0.7 x 0.8 x 0.5 = 14:100,000 (see http://www.ncbi.nlm.nih.gov/books/NBK1272/).
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.
Recent results and ongoing activities
Ca2+ signaling in the developing cochlea and hearing acquisition
[click image to enlarge]
Our work is shedding new light on the role of phosphoinositides in the crucial postnatal weeks that precede hearing acquisition in mice. We focused our attention on non-sensory cells of the developing sensory epithelium using organotypic cultures of P0–P5 cochlear tissue (Figure 5).
We reported that PIPKIγ deficiency in both homozygous and heterozygous transgenic mice results in a sizeable downregulation of IP3R Ca2+ signaling among cochlear non–sensory cells from pups, which appears unique in terms of anatomical sublocalization of the defect to a subset of these cells. Most importantly, this reduction of Ca2+ signaling correlates with the appearance of a specific deafness phenotype in adult PIPKIγ(+/-) mice. Hearing loss in these heterozygous mice, that breed normally and had no obvious phenotype, is characterized by normal sensitivity to low frequency sounds but a dramatic reduction of the sensitivity to frequencies in excess of 20 kHz (Figure 6). These are the first data that concern the hitherto unexplored function of PI(4,5)P2 in non-sensory (supporting and epithelial) cells of the mammalian cochlea, which appear of major relevance in terms of present models of inner ear development and sound perception. Furthermore, the data we presented contribute to solving a controversy present in the literature. Indeed, based on studies in cell lines or platelets it has been suggested that PIPKIγ is dispensable (platelets) or essential (HeLa cells) for the rapid synthesis of the pool of PI(4,5)P2 required for IP3 and diacylglycerol generation upon agonist stimulation. While the previous data have been collected in a cell line or cell remnants, the present data were obtained from primary cochlear cultures with intact anatomical structure. Our data suggest that the enzyme isoforms responsible of the synthesis of the key PI(4,5)P2 pool involved in IP3 synthesis may vary depending on the cell type, adding further complexity to the control of Ca2+ signaling.
- A representative movie of spontaneous Ca2+ transients recorded in our laboratory from a P5 cochlear organotypic culture is available at http://youtu.be/3u1l0LW4FB4
Further understanding how the Ca2+ signaling defects reported here link to hearing loss in these different mouse models may prove crucial to unravel how hearing acquisition proceeds under normal circumstances as well as to decipher the pathogenic processes underlying human hereditary deafness forms and, eventually, to test prospective therapies.
Restoration of gap junction coupling in cochlear organotypic cultures from DFNB1 mouse models by BAAV–mediated GJB2 gene transfer
Gene therapy offers an attractive method for modulating gene expression in the inner ear with the ultimate goal of treating cochlear disorders. Vectors based on recombinant bovine adeno associated virus (BAAV) have several attributes that make them well suited for gene transfer in the inner ear, including low toxicity and a unique serological identity. We optimized BAAV production in order to achieve the high viral titer required for efficient transduction of cochlear non-sensory cells. In parallel, we investigated the expression and function of Cx26 and Cx30 from P5 to adult age in double transgenic Cx26Sox10Cre mice, which we obtained by crossing Cx26 floxed mice (Cx26loxP/loxP) with a deleter Sox10–Cre line. Cx26Sox10Cre mice presented with complete Cx26 ablation in the epithelial gap junction network of the cochlea, whereas Cx30 expression was developmentally delayed. In vivo electrophysiological measurements in Cx26Sox10Cre mice revealed profound hearing loss accompanied by reduction of endocochlear potential (Figure 7).
[click image to enlarge]
We transduced these cultures with a BAAV vector encoding a Cx26CFP fusion protein (BAAVCx26CFP). Confocal fluorescence microscopy images obtained 48 hours post transduction showed recombinant Cx26CFP protein expressed in a large fraction of the non–sensory cells. The recombinant protein expression pattern resembled closely that of endogenous Cx26 in control cultures from Cx26loxP/loxP mice immunoassayed with a Cx26 specific antibody whereas no Cx26 immunoreactivity was detected in untreated Cx26Sox10Cre cultures (Figure 8a). In addition, we performed fluorescence recovery after photobleaching (FRAP) assays after loading P5 cochlear organotypic cultures with the acetoxymethyl ester of calcein, a fluorescent tracer that diffuses through gap junction channels in this preparation. Following the delivery of a 405 nm laser pulse to a restricted tissue area of wild type cultures, the intracellular calcein fluorescence was partially restored via diffusion of the indicator dye through gap junction channels from adjacent unbleached cell. Incomplete FRAP is ascribed to the fraction of the calcein pool which is not available for intercellular transfer (immobile fraction) due to trapping into subcellular organelles and/or binding to subcellular structures.
[click image to enlarge]
We did not observe any FRAP in Cx26Sox10Cre organotypic cultures, indicative of impaired gap junction coupling (not shown). By contrast, FRAP in Cx26Sox10Cre cultures transduced with BAAVCx26CFP was even faster than that of untreated Cx26loxP/loxP control cultures possibly due to a higher-than-normal level of recombinant Cx26 expression driven by the CMV promoter in the BAAV vector (Figure 8b).
- A representative movie of calcein FRAP recorded in our laboratory from a P5 cochlear organotypic culture is available at http://youtu.be/X5pP0FK4WAw.
Connexin structure and function: insight from Molecular Dynamics (MD)
The recently published X–ray crystallographic structure of a homomeric intercellular channel formed by human (h) Cx26 protomers at 3.5–Å resolution is the only connexin crystal structure available to date (Protein Data Bank accession code 2ZW3). We used this structure to construct MD models for the hCx26 connexon (Figure 9) and the hCx30 connexon embedded in a realistic environment comprising plasma membrane phospholipids, explicit solvent (water molecules) and a sufficient number of ions to simulate normal ionic strength. For both models we computed the free energy profile for the permeation of a K+ ion moving along the axis of the channel. By interpreting the results in light of Eyring transition state theory, we were able to compute the ratio between the unitary conductances of hCx26 and hCx30 channels which was found in good agreement with the experimental one (Zonta et al., 2012; http://www.ncbi.nlm.nih.gov/pubmed/22292956).
[click image to enlarge]
Next, we measured the unitary flux of calcein through hCx26wt gap junction channels, and compared the experimentally determined value to that predicted by MD simulations based on the 3.5 Å X–ray structural data. Term of comparison was the unitary transition rate, i.e. the number of calcein molecules that are able to transit trough a single channel per unit time. Simulations were performed with two different charge states for the calcein molecule. In the first case calcein had all the carboxylic groups unprotonated, as expected at physiological pH. In the other case, calcein was protonated and set to zero total charge. Our simulations indicate that a calcein molecule with a presumptive physiological charge is unable to traverse the channel due to the large energy barrier it faces (45.2 kBT). In contrast, the predicted transition rate for a calcein molecule with zero charge is compatible with the experimentally determined value. Based on this analysis we conclude that the structural model of the hCx26wt channel derived from the 3.5 Å X–ray data is not permeable to calcein and the blockade is essentially electrostatic. Our conclusion is in contrast with the proposal of Maeda et al. that the model represents an open channel. This proposal was based on the facts that: (i) there are no obvious obstructions along the pore of the hCx26wt channel; (ii) the crystallization conditions adopted by Maeda et al. are compatible with the formation of channels in the open state (neutral pH without aminosulphonate buffer or any divalent ions). We think that this can be explained as follows: (1) there is no way to guarantee that the open channel structure was preserved during the partial dehydration and crystallization procedures; (2) in a gap junction plaque, only 10% or less of the channels are in an open state and therefore the averaging procedure intrinsic in the generation of the crystal structure data more closely reflects that of a closed channel (Zonta et al., 2013; http://www.ncbi.nlm.nih.gov/pubmed/23445664).
- An animated MD model of the hCx26 hemichannel is available at: http://youtu.be/zBRYhZBxxPk
- An example of steered MD of a potassium ion moving along a hCx26 hemichannel is available at: http://youtu.be/kF3nehei3_A
- A MD simulation of calcein permeation in a hCx26 hemichannel is available at: http://youtu.be/_nnAIkueVAg
[click image to enlarge]
Advances in optical microscopy
The unique ability to transport light in a coherent way using graded index (GRIN) fiber optics allows the assembly of probes and objectives for microscopy applications requiring non-invasive and flexible operation in small and crowded spaces (in vivo microscopy, endoscopy, inspection). We used a GRIN sample produced by Grintech (Germany) based on a ¼ pitch objective with a 0.5 in-water numerical aperture (NA) coupled to a ¾ pitch relay lens with a 0.2 NA entrance. The probe (0.5 mm diameter) was inserted in an iron capillary tube (0.7 mm diameter and 10 mm length) acting as light shield and mechanical protection (Figure 10).
[click image to enlarge]
In the subsequent study, we coupled this compact GRIN fiber objective to VIMM’s two–photon microscope, which we retrofitted with a commercially available adaptive optics (AO) system (Figure 11). The application required the definition and set–up of:
- a calibration procedure and optical architecture to insert the AO components in the microscope optical path
- a computer algorithm to optimize the performance of the system
As a final test of our imaging apparatus, we imaged fluorescent micro–beads (1.0 µm diameter, peak emission around 515 nm) excited by the two–photon laser tuned at 830 nm with and without AO assist. The PSF was clearly sharpened and regularized independent of the position in the scanned field (about 80×80 µm). Notice that PSF sharpening and regularization allowed the detection of the minute spaces between adjacent spheres (Figure 12). Tests performed with smaller beads showed that the resolution limit of the system is close to 0.5 µm.
[click image to enlarge]
|2002–present||Associate Professor, University of Padua, Italy|
|2001–present||VIMM Principal Investigator|
|1995–2001||Assistant Professor, Biophysics Sector, SISSA|
|1992–1995||Research Associate, Dept of Physiology, University of Bristol, UK|
|1992||PhD in Biophysics, SISSA, Trieste, Italy|
|1990–1991||Ensign, Naval Academy, Livorno|
|1989||M.Phil. in Biophysics, SISSA, Trieste, Italy|
|1988||Visiting Scientist, MIT, Boston, MA, USA|
|1987||Degree in Physics, University of Parma, Italy|
|2013||Professor of Applied Physics and Principal Investigator, Department of Biomedical Science, University of Sheffield, UK|
|2010||Collegium Oto–Rhino–Laryngologicum Amicitiae Sacrum (CORLAS), Elected member since 15 September 2010; www.corlas.org|
|2005||Research and Development Prize
"The Roboscope Project", Start Cup 2005, 2nd Prize (12000 Euro) for the best business ideas of the Veneto Region|
Science Dissemination Award The Cochlea Webpage reviewed in the Web Watch section of Science (Vol 309, 26 August 2005, p. 1307), selected by Thomson ISI for inclusion in Current Web Contents, a value-added section of Current Contents Connect™ (CC Connect™)
Selected VIMM Publications
- Zonta F, Polles G, Sanasi MF, Bortolozzi M, Mammano F (2013) The 3.5 ångström X-ray structure of the human connexin26 gap junction channel is unlikely that of a fully open channel. Cell Commun. Signal 11:15.
- Mammano F (2013) ATP-dependent intercellular Ca2+ signaling in the developing cochlea: facts, fantasies and perspectives. Semin. Cell Dev. Biol. 24:31-9.
- Rodriguez L, Simeonato E, Scimemi P, Anselmi F, Calì B, Crispino G, Ciubotaru CD, Bortolozzi M, Ramirez FG, Majumder P, Arslan E, De Camilli P, Pozzan T, Mammano F (2012) Reduced phosphatidylinositol 4,5-bisphosphate synthesis impairs inner ear Ca2+ signaling and high-frequency hearing acquisition. Proc. Natl. Acad. Sci. U.S.A. 109:14013-8.
- Zonta F, Polles G, Zanotti G, Mammano F (2012) Permeation pathway of homomeric connexin 26 and connexin 30 channels investigated by molecular dynamics. J. Biomol. Struct. Dyn. 29:985-98.
- Crispino G, Di Pasquale G, Scimemi P, Rodriguez L, Galindo Ramirez F, De Siati RD, Santarelli RM, Arslan E, Bortolozzi M, Chiorini JA, Mammano F (2011) BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice. PLoS ONE 6:e23279.
- Blaauw B, Del Piccolo P, Rodriguez L, Hernandez Gonzalez VH, Agatea L, Solagna F, Mammano F, Pozzan T, Schiaffino S (2012) No evidence for inositol 1,4,5-trisphosphate-dependent Ca2+ release in isolated fibers of adult mouse skeletal muscle. J. Gen. Physiol. 140:235-41.
- Ceriani F, Mammano F (2012) Calcium signaling in the cochlea - Molecular mechanisms and physiopathological implications. Cell Commun. Signal 10:20.
- Decrock E, Krysko DV, Vinken M, Kaczmarek A, Crispino G, Bol M, Wang N, De Bock M, De Vuyst E, Naus CC, Rogiers V, Vandenabeele P, Erneux C, Mammano F, Bultynck G, Leybaert L (2012) Transfer of IP₃ through gap junctions is critical, but not sufficient, for the spread of apoptosis. Cell Death Differ. 19:947-57.
- Bortoletto F, Bonoli C, Panizzolo P, Ciubotaru CD, Mammano F (2011) Multiphoton fluorescence microscopy with GRIN objective aberration correction by low order adaptive optics. PLoS ONE 6:e22321.
- Mammano F (2011) Ca2+ homeostasis defects and hereditary hearing loss. Biofactors 37:182-8.
- Bortoletto F, Bonoli C, Panizzolo P, Mammano F (2011) Construction and test of a GRIN-based optical objective. J Microsc 242:100-3.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
|2013||Seminar, University College London, Ear Institure, 25 October|
International symposium on the role of intrinsic electrical activity in the maturation and refinement of the auditory and visual systems. IUPS, 21-26 July, Birmingham, UK
Seminar, Dept of Biomedical Sciences, University of Sheffield, UK, 20 May
|2012||International School for Advanced Studies, Trieste, Italy, 5 June|
Centre International des Rencontres Culturelles et Musicales de l'Abbaye de Sylvanès, France, 18 May
Annual Meeting of the CNR Institute of Neuroscience, Brixen, Italy, 2 March
|2011||Annual Meeting of the Collegium Oto–
Rhino–Laryngologicum Amicitiae Sacrum (CORLAS), Bruge, Belgium, 5 September1|
Med–El Electronics, Innsbruck, Austria, 17 June
Italian Institute of Technology (IIT), Genoa, Italy, 7 June
Annual Meeting of the Italian Society of Otoloryngology, Udine, Italy, 27 May
Symposium on the Development and Function of the Eye and the Ear, Sackler Faculty of Medicine, Tel Aviv University, Israel, 21 March
School on Convergent Science Network (CSN) of Biomimetic and Biohybrid Systems, a coordination action (CA) for the development of future real-world technologies supported by the European Commission under the Future and Emerging Technologies (FET) program (ICT, FP7), Padua, Italy, 14 March
|2006||"How hearing works", Innsbruck, Austria |
"Connexins and deafness", International Center for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
"ATP-induced calcium waves in cultured organs of Corti: long-range cochlear signalling", Advances in Microscopy, Ferrara, Italy
|2005||"Connexin and deafness: InsP3 permeability defects", One-day symposium on: Calcium signalling: from Physiology to Pathology. Sponsored by The Physiological Society and the Italian Physiological Society, Palermo, Italy|
"Effects of noise damage on cochlear hair cells", One-day symposium on: Noise in the workplace, in memory of Dr. Claudio Saulino, Naples, Italy
|2004||"A mechanism for sensing noise damage in the inner ear", NIH/NIDCD Intramural Seminar, 5 Research Court, Rockville, MD, USA|
Venetian Institute of Molecular Medicine
Via Orus 2
35129 Padua — Italy