Mark A. Rutherford, Ph.D. - US grants

? DESCRIPTION (provided by applicant): Glutamate-induced excitotoxicity is increasingly recognized as the trigger for swelling, retraction, and delayed degeneration of auditory nerve fibers (ANFs) following moderate overexposure to sound; however, little is known about the underlying mechanisms. This excitotoxicity seems to involve glutamate receptors, and research in other systems has indicated the crucial role of postsynaptic intracellular Ca2+ in mediating the excitotoxicity that produces slow neurodegeneration. Postsynaptic Ca2+ can also mediate homeostatic plasticity. It is still unknown if or how Ca2+ signals link excitotoxicity to neurodegeneration or protection in ANFs. Although all ANF terminals express glutamate receptors, they differ in susceptibility to noise-induced synaptopathy and degeneration. The roles of glutamate receptor subunits in the inner ear deserve attention because glutamate-induced Ca2+ influx through receptors depends upon subunit composition. The project encompasses studies of activity-dependent synaptic plasticity in the cochlea because our long- term goal is to identify mechanisms of synaptic damage and repair that can be manipulated to prevent or rapidly reverse damage before the onset of neurodegeneration. We have already demonstrated that ANF terminals differ from each other in their complements of AMPA-type glutamate receptor subunits. We hypothesize that heterogeneity of glutamate receptor subunit expression among ANF terminals is a crucial determinant of susceptibility to noise-induced damage. Thus, we are studying noise-activated changes in subunit composition. We are comparing receptor subunit composition with presynaptic molecular anatomy and reconstructing synapse position on the inner hair cell (IHC) to compare along the modiolar-pillar and orthogonal axes. We are using genetically modified mice to manipulate glutamatergic activity. We previously employed superresolution STED microscopy to measure synaptic structures at 50 nm resolution in 2D. We now implement, for the first time in the organ of Corti, 3D superresolution STORM microscopy at 20 nm resolution. We are now able to measure the intrasynaptic organization of AMPA receptor subunits with subunit-specific antibodies to GluA2, GluA3, and GluA4. Anatomical measurements will be complemented with functional recordings. In prior work with the patch- clamp technique we made the first measurements of ANF excitability with direct current injection into ANF terminals. Here, differences in firing behavior will be compared with synaptic structure by filling recorded neurons with dye, followed by fixation and immunohistochemistry. We are implementing Ca2+ imaging in ANFs for the first time, which allows for less invasive, simultaneous observation of activity across fibers. We will use Ca2+ imaging to test for functional routes of Ca2+ entry pharmacologically. Understanding how ANF diversity is shaped by glutamate receptor subunits and postsynaptic Ca2+ will deliver new perspectives on questions of clinical hearing loss as well as the basic mechanisms underlying this unique synapse.

Project Summary: Normally, glutamatergic synapses in the mature brain predominantly express GluA2-containing Ca2+- impermeable AMPARs. However, certain synapses, such as the synapses between hair cells and cochlear afferents, also express GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs). In the parent R01: EXCITATION AND EXCITOTOXICITY IN TYPE I COCHLEAR AFFERENTS: SYNAPTIC STRUCTURE AND FUNCTION, we published on the presence of GluA2-lacking AMPARs in rat cochlea and proposed that those receptors are Ca2+- permeable as is the case in hair cell organs from frog and zebrafish. We recently submitted a paper on GluA2- lacking CP-AMPARs in synaptopathic excitotoxicity in the mouse cochlea. Notably, certain neuropathologies are known to involve CP-AMPARs, including epilepsy, ischemia, traumatic brain injury, and addiction/withdrawal. Work on the parent R01 is focused on the hypothesis that CP-AMPARs contribute to induction of neurodegeneration. Other recent work on Alzheimer's Disease has implicated CP-AMPARs in progressive, selective neurodegeneration in the brain. The proposed studies will examine a possible link between cochlear synapses and neurodegeneration in Alzheimer's disease. The cellular mechanisms responsible for neuronal pathology in Alzheimer's disease (AD) remain poorly understood. Our project is motivated by the observation that two of the key genes and proteins in AD, amyloid precursor protein (APP) and Tau are highly expressed in the sensory tissues of the inner ears of mice. In addition, studies of human populations indicate that auditory and vestibular deficits are associated with the development of AD. Based on these observations, we hypothesize that AD may contribute to neurodegeneration in the inner ear through changes in synaptic molecular anatomy and Ca2+-permeability of AMPARs. A more complete understanding of how AD affects the inner ear may lead to novel diagnostics for the detection of early stage AD in humans. Moreover, understanding the basic and pathological functions of AD proteins in the inner ear may shed light on AD mechanisms in the brain. Specific Aim 1 will determine the expression of AMPAR subunits at inner ear synapses in mouse models of AD. Specific Aim 2 will measure inner ear function in mouse models of AD. Together, the proposed experiments will determine whether AD mouse models have molecular changes at inner ear synapses and if they associate with changes in hearing or vestibular function. If synaptic dysfunction and nerve degeneration in the ear involve processes similar to AD processes in the brain, then we should further study AD mechanisms in the ear. Insights and tools from AD research will be applied in the ear to prevent loss of hearing and balance function, and may also assist in a more complete understanding of AD neuropathology in the brain.