Brief career history

• 2012 - present: Professor of Sensory Neuroscience
• 2006 - present: Royal Society University Research Fellow, University of Sheffield, UK.
• 2004 - 2005: Royal Society University Research Fellow, University of Sussex, UK.
• 2001 - 2004: Postdoctoral Fellow, University of Sussex, UK.
• 1997 - 2000: Postdoctoral Fellow, University of Bristol, UK.
• 1994 - 1997: PhD, University of Pavia, Italy.
• 1989 - 1992: University Degree in Biological Science, University of Pavia, Pavia, Italy.

Research interests

• Molecular and physiological mechanisms controlling the functional maturation of the auditory system.
• Mechanoelectrical transduction at the hair cell stereocilia
• Signal processing at ribbon synapses
• Age-related hearing loss
• Mechanisms underlying different forms of hearing loss and deafness

Professional activities

• Reviewing editor for Journal of Physiology
• BBSRC Core panel member – Panel A
• Grant reviewer for UK/EU Research Councils and Charities
• Reviewer for many leading scientific journals

Awards and prizes

• Wellcome Trust Senior Investigator (2014)
• Sharpey-Schafer Lecture and Prize (2011) – Physiological Society, Oxford.
• Royal Society University Research Fellowship (2004), The Royal Society, UK.

Full publications

Auditory neuroscience and Deafness Sensory transduction Synaptic transmission

Sensory organs and the neural networks responsible for processing sensory information are supremely well adapted for detecting input from the external environment. Their challenge is to maximize sensitivity and fidelity over a wide dynamic range. The sensory receptors of the mammalian auditory system, the inner hair cells (IHCs), do this with unparalleled temporal precision (kHz range). We know little about the molecular and physiological mechanisms controlling the functional maturation of the auditory system or signal processing at the primary auditory synapses, the IHC ribbon synapses. Crucial to this work, is the need of near-physiological in vitro and the development of in vivo experimental models.

My laboratory is uniquely suited for this task because it is the only one in the world that routinely uses near-physiological conditions for in vitro mammalian cochlear physiology and performs in-vivo electrophysiology from the zebrafish.  How biological systems orchestrate their development and how complex signals are processed by mature neuronal networks are major challenges in the quest to understand human biology and disease.

The auditory system provides an ideal model with which to address these questions, primarily because it involves a highly ordered array of a very small number of sensory cells with well-defined neuronal circuitry. It is also a key priority for human health because hearing loss affects more than 360 million people worldwide (WHO 2013), a number that will increase with the aging population.

Undergraduate and postgraduate taught modules

Level 3:

• BMS355 Sensory Neuroscience
• BMS349 Extended Library Project
• BMS369 Laboratory Research Project

Masters (MSc):

• BMS6355 Sensory Neuroscience

Dystroglycan function in mammalian auditory hair cells and neurons - Action on Hearing Loss Studentship

Action on Hearing Loss PhD Studentship (3 year fully funded PhD studentship (Uk and EU only)

Supervisors: Professor Steve Winder and Professor Walter Marcotti, Department of Biomedical Science

Overview

This PhD offers an exciting opportunity for a talented student to work on a multidisciplinary project in the areas of auditory physiology and molecular cell biology investigating the role of dystroglycan in mammalian hearing.

This proposal focuses on the function of a transmembrane protein called b-dystroglycan, which forms the core of dystroglycan complexes that enable cells to establish mechanical connections and communication pathways between their internal structures and the extracellular environment.

The inner ear interprets mechanical vibrations induced by sound. This requires the precise transduction of incoming auditory stimuli into an electrical signal that can be perceived by the brain. This sophisticated biological process occurs at the sensory hair cells. The hair cell mechanotransducer apparatus projects into a unique extracellular fluid compartment that is rich in potassium ions and that provides the ‘battery’ for the inner ear. Furthermore, hair cells transfer electrical information rapidly to their sensory nerves via specialised ‘ribbon’ synapses. All of these processes are expected to involve dystroglycan complexes, although published evidence suggests that b-dystroglycan is not present in the sensory organ. This is puzzling because there is clear evidence for its partner, a-dystroglycan, which is encoded by the same gene. We also know that defects in a number of other proteins linked to dystroglycan complexes are associated with hearing loss. One of these proteins is dystrophin, mutations of which is normally linked to muscular dystrophy.

Recently, we found that tyrosine phosphorylation of b-dystroglycan occurs at unusually high levels in the ear and that this explains why the protein was not previously detected. It changes the binding properties to other proteins, which is of general biological interest because dystroglycans are ubiquitously expressed yet they serve a wide range of different functions. In muscle, dystrophin binds to dystroglycan complexes and dissipates contraction forces to the extracellular connective tissue, thus protecting muscle membrane integrity. Mutations in dystrophin weaken these connections so that muscle cells become more easily damaged and degenerate, leading to muscular dystrophy. The strength of the attachment can be altered through the modification to b-dystroglycan that we observe in the ear and the mechanism has been proposed as a potential therapy to stabilise the muscle. Similar modifications to b-dystroglycan act as switches to alter association with other proteins in non-muscle cells, including nerves, and they are important for the organisation of signalling molecules at synaptic junctions and of molecules that regulate the flow of water and inorganic ions across fluid compartments. Finally, longer term changes in the function of dystroglycan complexes have been associated with aging. Thus phosphorylation of b-dystroglycan in the ear is likely to be critical for auditory function and for greater understanding of noise-induced and age-related hearing loss.

Outline of the research methods

Animal models: We have made a knock-in mouse (Dag1Y890F) with a phenylalanine substitution that prevents tyrosine phosphorylation of β-dystroglycan [1]. This will allow us to focus on the specific function of this modification in the ear. We will characterise the model, using the structural, physiological and biochemical techniques listed below. To identify potential hearing loss linked to the inability to phosphorylate Y890 in b-dystroglycan  we will perform in vivo electrophysiological recording (auditory brainstem responses, ABRs) from normal and Dag1Y890F mice at 1, 4, 8 and 12 months old at frequencies of 4, 8, 16 and 32kHz.

Cell lines and antibodies: We have a conditionally immortal, mouse otic neuronal cell line, US/VOT-N33 [2] that expresses β-dystroglycan and eps8, which has been assayed both by oligonucleotide gene expression arrays and by immunolabeling. The cells also react strongly with antibodies to pY890 b-dystroglycan. Eps8 is known to be expressed in neurons and it is associated with synaptic plasticity and the morphology of dendritic spines [3]. We have the hybridoma cell line that produces antibodies to non-phosphorylated β-dystroglycan (Mandag2) and our own polyclonal antibody to pY890 b-dystroglycan (Ab1709) with which we will be able to estimate relative expression of the two forms in tissue sections, organotypic whole-mounts of the organ of Corti and in western blots and immunoprecipitations. We will use antibodies to ribeye (ribbon synapse), eps8 and eps8l2 (hair bundles and neurons), and Kir4.1 and aqp4 (fluid regulation) to study the distribution of these proteins in Dag1Y890F mice.

Single cell electrophysiology - Patch clamp recording will carried out on hair cells as described previously [4]. We will use acutely dissected tissue to record mechanoelectrical transducer current from hair cells in response to displacements of hair bundles with a fluid jet and to assess basolateral membrane properties such as ion currents and vesicle release at the ribbon synapses.

Microscopy – Confocal fluorescence microscopy will be used to localise proteins in cells, cryostat sections and organotypic cultures. Scanning and transmission electron microscopy will be used to study the hair bundles and the localisation and structure of ribbon synapses.

Biochemistry – We will validate the Eps8 SH3 domain interaction with β-dystroglycan using peptide SPOT arrays to define the precise binding site on dystroglycan and using reciprocal fusion protein pull down assays from otic epithelial cell lysates. The interaction will be further validated by immunoprecipitation, immunofluorescence localisation and proximity ligation in otic epithelial cells in vitro. We have used similar approaches to validate SH3 domain interactions between β-dystroglycan and other cytoskeletal proteins [5, 6].

References

[1] Miller G, Moore CJ, Terry R, et al. Preventing phosphorylation of dystroglycan ameliorates the dystrophic phenotype in mdx mouse. Hum Mol Genet  2012;21:4508-20.
[2] Nicholl AJ, Kneebone A, Davies D, et al. Differentiation of an auditory neuronal cell line suitable for cell transplantation. Eur J Neurosci  2005;22:343-53.
[3] Winder SJ, Lipscomb L, Angela Parkin C, Juusola M. The proteasomal inhibitor MG132 prevents muscular dystrophy in zebrafish. PLoS Curr;3:RRN1286.
[4] Zampini V, Ruttiger L, Johnson SL, et al. Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells. PLoS Biol  2011;9:e1001048.
[5] Thompson O, Kleino I, Crimaldi L, Gimona M, Saksela K, Winder SJ. Dystroglycan, Tks5 and Src mediated assembly of podosomes in myoblasts. PLoS One  2008;3:e3638.
[6] Thompson O, Moore CJ, Hussain SA, et al. Modulation of cell spreading and cell-substrate adhesion dynamics by dystroglycan. J Cell Sci  2010;123:118-27.

Supervisors

For informal enquiries about this project, please contact:

Professor Steve Winder

Web: http://www.shef.ac.uk/bms/research/winder
Email: s.winder@shef.ac.uk

Professor Walter Marcotti

Web: http://www.shef.ac.uk/bms/research/marcotti
Email: w.marcotti@shef.ac.uk