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Hearing requires otoferlin-dependent efficient replenishment of synaptic vesicles in hair cells

Abstract

Inner hair cell ribbon synapses indefatigably transmit acoustic information. The proteins mediating their fast vesicle replenishment (hundreds of vesicles per s) are unknown. We found that an aspartate to glycine substitution in the C2F domain of the synaptic vesicle protein otoferlin impaired hearing by reducing vesicle replenishment in the pachanga mouse model of human deafness DFNB9. In vitro estimates of vesicle docking, the readily releasable vesicle pool (RRP), Ca2+ signaling and vesicle fusion were normal. Moreover, we observed postsynaptic excitatory currents of variable size and spike generation. However, mutant active zones replenished vesicles at lower rates than wild-type ones and sound-evoked spiking in auditory neurons was sparse and only partially improved during longer interstimulus intervals. We conclude that replenishment does not match the release of vesicles at mutant active zones in vivo and a sufficient standing RRP therefore cannot be maintained. We propose that otoferlin is involved in replenishing synaptic vesicles.

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Figure 1: OtofPga/Pga mice have normal hair cell transduction and receptor potentials, but severe hearing impairment.
Figure 2: Normal vesicle fusion and impaired vesicle replenishment in OtofPga/Pga IHCs.
Figure 3: Fatigue of exocytosis, slowed RRP refilling are seen in OtofPga/Pga IHCs, but docking and endocytic membrane retrieval are unaltered.
Figure 4: Synchronized multivesicular release and spike generation in otoferlin mutants.
Figure 5: Reduced and fatiguing sound encoding in OtofPga/Pga mice.
Figure 6: The pachanga mutation causes a reduction in otoferlin protein levels and a change in secondary structure.

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References

  1. Kiang, N.Y.-S., Watanabe, T., Thomas, E.C. & Clark, L.F. Discharge Pattern of Single Fibers in the Cat's Auditory Nerve (MIT Press, Cambridge, Massachusetts, 1965).

  2. Geisler, C.D. From Sound to Synapse (Oxford University Press, New York, 1998).

  3. Parsons, T.D., Lenzi, D., Almers, W. & Roberts, W.M. Calcium-triggered exocytosis and endocytosis in an isolated presynaptic cell: capacitance measurements in saccular hair cells. Neuron 13, 875–883 (1994).

    Article  CAS  Google Scholar 

  4. Moser, T. & Beutner, D. Kinetics of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse of the mouse. Proc. Natl. Acad. Sci. USA 97, 883–888 (2000).

    Article  CAS  Google Scholar 

  5. Schnee, M.E., Lawton, D.M., Furness, D.N., Benke, T.A. & Ricci, A.J. Auditory hair cell-afferent fiber synapses are specialized to operate at their best frequencies. Neuron 47, 243–254 (2005).

    Article  CAS  Google Scholar 

  6. Griesinger, C.B., Richards, C.D. & Ashmore, J.F. Fast vesicle replenishment allows indefatigable signaling at the first auditory synapse. Nature 435, 212–215 (2005).

    Article  CAS  Google Scholar 

  7. Keen, E.C. & Hudspeth, A.J. Transfer characteristics of the hair cell's afferent synapse. Proc. Natl. Acad. Sci. USA 103, 5537–5542 (2006).

    Article  CAS  Google Scholar 

  8. Goutman, J.D. & Glowatzki, E. Time course and calcium dependence of transmitter release at a single ribbon synapse. Proc. Natl. Acad. Sci. USA 104, 16341–16346 (2007).

    Article  CAS  Google Scholar 

  9. von Gersdorff, H. & Matthews, G. Depletion and replenishment of vesicle pools at a ribbon-type synaptic terminal. J. Neurosci. 17, 1919–1927 (1997).

    Article  CAS  Google Scholar 

  10. Gomis, A., Burrone, J. & Lagnado, L. Two actions of calcium regulate the supply of releasable vesicles at the ribbon synapse of retinal bipolar cells. J. Neurosci. 19, 6309–6317 (1999).

    Article  CAS  Google Scholar 

  11. Zenisek, D., Steyer, J.A. & Almers, W. Transport, capture and exocytosis of single synaptic vesicles at active zones. Nature 406, 849–854 (2000).

    Article  CAS  Google Scholar 

  12. Thoreson, W.B., Rabl, K., Townes-Anderson, E. & Heidelberger, R. A highly Ca2+-sensitive pool of vesicles contributes to linearity at the rod photoreceptor ribbon synapse. Neuron 42, 595–605 (2004).

    Article  CAS  Google Scholar 

  13. Rabl, K., Cadetti, L. & Thoreson, W.B. Kinetics of exocytosis is faster in cones than in rods. J. Neurosci. 25, 4633–4640 (2005).

    Article  CAS  Google Scholar 

  14. Singer, J.H. & Diamond, J.S. Vesicle depletion and synaptic depression at a mammalian ribbon synapse. J. Neurophysiol. 95, 3191–3198 (2006).

    Article  CAS  Google Scholar 

  15. Jackman, S.L. et al. Role of the synaptic ribbon in transmitting the cone light response. Nat. Neurosci. 12, 303–310 (2009).

    Article  CAS  Google Scholar 

  16. Hosoi, N., Sakaba, T. & Neher, E. Quantitative analysis of calcium-dependent vesicle recruitment and its functional role at the calyx of Held synapse. J. Neurosci. 27, 14286–14298 (2007).

    Article  CAS  Google Scholar 

  17. Saviane, C. & Silver, R.A. Fast vesicle reloading and a large pool sustain high bandwidth transmission at a central synapse. Nature 439, 983–987 (2006).

    Article  CAS  Google Scholar 

  18. Khimich, D. et al. Hair cell synaptic ribbons are essential for synchronous auditory signaling. Nature 434, 889–894 (2005).

    Article  CAS  Google Scholar 

  19. Moser, T., Neef, A. & Khimich, D. Mechanisms underlying the temporal precision of sound coding at the inner hair cell ribbon synapse. J. Physiol. (Lond.) 576, 55–62 (2006).

    Article  CAS  Google Scholar 

  20. Wittig, J.H. Jr. & Parsons, T.D. Synaptic ribbon enables temporal precision of hair cell afferent synapse by increasing the number of readily releasable vesicles: a modeling study. J. Neurophysiol. 100, 1724–1739 (2008).

    Article  Google Scholar 

  21. Eisen, M.D., Spassova, M. & Parsons, T.D. Large releasable pool of synaptic vesicles in chick cochlear hair cells. J. Neurophysiol. 91, 2422–2428 (2004).

    Article  Google Scholar 

  22. Johnson, S.L., Marcotti, W. & Kros, C.J. Increase in efficiency and reduction in Ca2+ dependence of exocytosis during development of mouse inner hair cells. J. Physiol. (Lond.) 563, 177–191 (2005).

    Article  CAS  Google Scholar 

  23. Roux, I. et al. Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse. Cell 127, 277–289 (2006).

    Article  CAS  Google Scholar 

  24. Roux, I. et al. Myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses. Hum. Mol. Genet. 18, 4615–4628 (2009).

    Article  CAS  Google Scholar 

  25. Heidrych, P. et al. Otoferlin interacts with myosin VI: implications for maintenance of the basolateral synaptic structure of the inner hair cell. Hum. Mol. Genet. 18, 2779–2790 (2009).

    Article  CAS  Google Scholar 

  26. Schwander, M. et al. A forward genetics screen in mice identifies recessive deafness traits and reveals that pejvakin is essential for outer hair cell function. J. Neurosci. 27, 2163–2175 (2007).

    Article  CAS  Google Scholar 

  27. Brandt, A., Khimich, D. & Moser, T. Few CaV1.3 channels regulate the exocytosis of a synaptic vesicle at the hair cell ribbon synapse. J. Neurosci. 25, 11577–11585 (2005).

    Article  CAS  Google Scholar 

  28. Frank, T., Khimich, D., Neef, A. & Moser, T. Mechanisms contributing to synaptic Ca2+ signals and their heterogeneity in hair cells. Proc. Natl. Acad. Sci. USA 106, 4483–4488 (2009).

    Article  CAS  Google Scholar 

  29. Spassova, M.A. et al. Evidence that rapid vesicle replenishment of the synaptic ribbon mediates recovery from short-term adaptation at the hair cell afferent synapse. J. Assoc. Res. Otolaryngol. 5, 376–390 (2004).

    Article  Google Scholar 

  30. Meyer, A.C. et al. Tuning of synapse number, structure and function in the cochlea. Nat. Neurosci. 12, 444–453 (2009).

    Article  CAS  Google Scholar 

  31. Li, G.L., Keen, E., Andor-Ardo, D., Hudspeth, A.J. & von Gersdorff, H. The unitary event underlying multiquantal EPSCs at a hair cell's ribbon synapse. J. Neurosci. 29, 7558–7568 (2009).

    Article  CAS  Google Scholar 

  32. Glowatzki, E. & Fuchs, P.A. Transmitter release at the hair cell ribbon synapse. Nat. Neurosci. 5, 147–154 (2002).

    Article  CAS  Google Scholar 

  33. Grant, L., Yi, E. & Glowatzki, E. Two modes of release shape the postsynaptic response at the inner hair cell ribbon synapse. J. Neurosci. 30, 4210–4220 (2010).

    Article  CAS  Google Scholar 

  34. Taberner, A.M. & Liberman, M.C. Response properties of single auditory nerve fibers in the mouse. J. Neurophysiol. 93, 557–569 (2005).

    Article  Google Scholar 

  35. Strenzke, N. et al. Complexin-I is required for high-fidelity transmission at the endbulb of held auditory synapse. J. Neurosci. 29, 7991–8004 (2009).

    Article  CAS  Google Scholar 

  36. Starr, A., Michalewski, H.J., Feng, G. & Moser, T. Perspectives on auditory neuropathy: disorders of inner hair cell, auditory nerve and their synapse. in The Senses: a Comprehensive Reference (eds. Dallos, P. & Oertel, D.) 397–412 (Elsevier, Amsterdam, 2008).

  37. Obholzer, N. et al. Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells. J. Neurosci. 28, 2110–2118 (2008).

    Article  CAS  Google Scholar 

  38. Seal, R.P. et al. Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3. Neuron 57, 263–275 (2008).

    Article  CAS  Google Scholar 

  39. Ruel, J. et al. Impairment of SLC17A8 encoding vesicular glutamate transporter-3, VGLUT3, underlies nonsyndromic deafness DFNA25 and inner hair cell dysfunction in null mice. Am. J. Hum. Genet. 83, 278–292 (2008).

    Article  CAS  Google Scholar 

  40. Hosoi, N., Holt, M. & Sakaba, T. Calcium dependence of exo- and endocytotic coupling at a glutamatergic synapse. Neuron 63, 216–229 (2009).

    Article  CAS  Google Scholar 

  41. Wadel, K., Neher, E. & Sakaba, T. The coupling between synaptic vesicles and Ca2+ channels determines fast neurotransmitter release. Neuron 53, 563–575 (2007).

    Article  CAS  Google Scholar 

  42. Wölfel, M., Lou, X. & Schneggenburger, R. A mechanism intrinsic to the vesicle fusion machinery determines fast and slow transmitter release at a large CNS synapse. J. Neurosci. 27, 3198–3210 (2007).

    Article  Google Scholar 

  43. Neef, J. et al. The Ca2+ channel subunit beta2 regulates Ca2+ channel abundance and function in inner hair cells and is required for hearing. J. Neurosci. 29, 10730–10740 (2009).

    Article  CAS  Google Scholar 

  44. de Wit, H. et al. Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes. Cell 138, 935–946 (2009).

    Article  CAS  Google Scholar 

  45. Sterling, P. & Matthews, G. Structure and function of ribbon synapses. Trends Neurosci. 28, 20–29 (2005).

    Article  CAS  Google Scholar 

  46. Dulon, D., Safieddine, S., Jones, S.M. & Petit, C. Otoferlin is critical for a highly sensitive and linear calcium-dependent exocytosis at vestibular hair cell ribbon synapses. J. Neurosci. 29, 10474–10487 (2009).

    Article  CAS  Google Scholar 

  47. Neef, A. et al. Probing the mechanism of exocytosis at the hair cell ribbon synapse. J. Neurosci. 27, 12933–12944 (2007).

    Article  CAS  Google Scholar 

  48. Beutner, D., Voets, T., Neher, E. & Moser, T. Calcium dependence of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse. Neuron 29, 681–690 (2001).

    Article  CAS  Google Scholar 

  49. Goodman, M.B. & Lockery, S.R. Pressure polishing: a method for re-shaping patch pipettes during fire polishing. J. Neurosci. Methods 100, 13–15 (2000).

    Article  CAS  Google Scholar 

  50. McCue, M.P. & Guinan, J. Jr. Acoustically responsive fibers in the vestibular nerve of the cat. J. Neurosci. 14, 6058–6070 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to thank E. Glowatzki and D. Khimich for teaching us the postsynaptic patch clamp, K. Tittman, D. Fasshauer and R. Jahn for advice and support for protein biochemistry, C.P. Richter and M.A. Cheatham for advice on the electrocochleography, A. Leonov and C. Griesinger for the DM-nitrophen, P. Jonas for the parvalbumin taqman probe, members of the InnerEarLab for discussion, M. Rutherford, J. Singer, E. Neher, J. Siegel, P. Heil, T. Sakaba, R. Nouvian, A. Lysakowski and A. Lee for comments on the manuscript and C. Rüdiger, S. Blume, N. Dankenbrink-Werder and M. Köppler for expert technical assistance. This work was supported by a fellowship of the Alexander von Humboldt Foundation to T.P., a fellowship of the Boehringer Ingelheim Fonds to K.R., a fellowship of MED-EL company to H.T., grants from the German Research Foundation (Center for Molecular Physiology of the Brain, T.M. and N.B.; Fellowship to N.S.), the European Commission (Eurohear, T.M.), the Max-Planck-Society (Tandemproject, T.M. and N.B.), the German Ministry for Education and Science via the Bernstein Focus Neurotechnology Goettingen (grant no. 01GQ0810 to T.M.), the State of Lower Saxony ('VW-Vorab' to T.M. and Christoph Matthias), and the US National Institutes of Health (DC007704, U.M.).

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Contributions

The study was designed by T.M., T.P., U.M., N.S., E.R. and N.B. T.P. carried out the IHC patch clamp and flash photolysis, extracellular postsynaptic recordings and immunohistochemistry and contributed to the electron microscopy. L.L. performed in vivo single-unit recordings, electrocochleography and auditory brainstem responses. K.R. carried out real-time PCR, protein purification, circular dichroism spectroscopy, fluorimetry and the floatation assay. H.T. performed postsynaptic recordings. M.S. carried out the ENU screen and initial auditory testing. D.R. performed electron microscopy. T.F. carried out Ca2+ imaging. N.S. performed in vivo physiology. E.R. generated the knockout mice. J.S.B. and L.M.T. started the ENU screen. T.M. and T.P. prepared the manuscript.

Corresponding authors

Correspondence to Ellen Reisinger or Tobias Moser.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–4 and Tables 1–3 (PDF 6382 kb)

Supplementary Movie 1

Tomogram of the ribbon in OtofPga/Pga IHC fixed in stimulatory condition from Figure 3d. Gold particles are visible on both surfaces of the sections. (MOV 3212 kb)

Supplementary Movie 2

Model of the same ribbon synapse in OtofPga/Pga IHC (as in Supplementary Movie 1). (PDF 737 kb)

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Pangršič, T., Lasarow, L., Reuter, K. et al. Hearing requires otoferlin-dependent efficient replenishment of synaptic vesicles in hair cells. Nat Neurosci 13, 869–876 (2010). https://doi.org/10.1038/nn.2578

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