All auditory information received by the central nervous system is transferred across the inner hair cell - afferent fiber synapse. Dysfunction of this synapse due to aging, noise or genetic disorders reduces hearing dramatically. This remarkable synapse must transfer intensity and timing information faithfully for long periods of time. Ribbon synapses, like those in hair cells, are typically found in cells that use graded receptor potentials and need to have sustained release. The hair cell ribbon synapse is more robust than most, releasing at higher rates and sustaining release for longer periods of time. In order for synapses to operate in this manner they must have a robust means of recycling and replenishing vesicles to synaptic release sites. We have identified a nonlinear calcium dependent capacitance change that we hypothesize is a reflection of vesicle recruitment and replenishment. Our first major goal (Aim 1) is to determine whether this nonlinear change is in fact synaptically based and we will do that using paired recordings to identify a postsynaptic response, using a new glutamate sensor to determine if glutamate release mimics the capacitance response and by inactivating ribbons optically to see if we can ablate this nonlinear component. We have also identified a calcium induced calcium release process (CICR) associated with the nonlinear capacitance change. We are hypothesizing that vesicle trafficking is calcium dependent and that CICR is used as a means of ensuring continual vesicle supply while calcium at the synapse is tightly regulated. We will use optical, electrophysiological and immunocytochemical tools to investigate this possibility in aim 2. Otoferlin is a unique protein implicated to regulate vesicle fusion, trafficking and reuptake. We are postulating that biochemical modification of this protein via phosphorylation by CaM Kinase II or ubiquitination is responsible for targeting these different functions. We will investigate this hypothesis using electrophysiological, optical, molecular and immunocytochemical technologies.

Public Health Relevance

The auditory end organ, the cochlea, is a remarkable machine that converts mechanical vibration into an electrical signal that is conveyed to the central nervous system across a specialized synapse that can maintain high rates of release for long periods of time. To do this, these synapses need to replenish synaptic vesicles at rapid rates. The information needs to be transmitted with high fidelity as well which means the synapse needs to be able to turn on and off in a very synchronized manner. Likely this means that calcium, which controls synaptic transmission is tightly regulated. We have developed new technology to allow us to measure vesicle trafficking in real time. We have also developed the means to investigate calcium regulation with better spatial and temporal resolution than previously available. We will use these technologies to investigate how these synapses are specialized to operate. Given that many forms of hearing loss including age related loss, noise induced loss and a variety of genetic disorders cause synaptic dysfunction, understanding the underlying molecular mechanisms associated with these processes is critical to the human health condition.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
2R01DC009913-06A1
Application #
8885354
Study Section
Special Emphasis Panel (ZRG1-IFCN-B (02))
Program Officer
Platt, Christopher
Project Start
2008-12-08
Project End
2020-03-31
Budget Start
2015-04-01
Budget End
2016-03-31
Support Year
6
Fiscal Year
2015
Total Cost
$487,687
Indirect Cost
$182,588
Name
Stanford University
Department
Otolaryngology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Meese, Sandra; Cepeda, Andreia P; Gahlen, Felix et al. (2017) Activity-Dependent Phosphorylation by CaMKII? Alters the Ca2+ Affinity of the Multi-C2-Domain Protein Otoferlin. Front Synaptic Neurosci 9:13
Castellano-Muñoz, Manuel; Schnee, Michael E; Ricci, Anthony J (2016) Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells. J Neurophysiol 115:226-39
Sundaresan, Srividya; Kong, Jee-Hyun; Fang, Qing et al. (2016) Thyroid hormone is required for pruning, functioning and long-term maintenance of afferent inner hair cell synapses. Eur J Neurosci 43:148-61
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