Anne E. Luebke
- Biomedical Engineering
- Neurobiology & Anatomy
- Center for Navigation and Communication Sciences
The long-term goal of my laboratory is to understand the role of cochlear outer hair cells in hearing and hearing loss both at the molecular and the systems level. Specifically, we are interested in agents that block or enhance the action of receptors on outer hair cells of the cochlea, which enhance hearing or decrease hearing loss. To design and discover agents that block or enhance receptor action on cochlear outer hair cells, we are cloning neurotransmitter receptors expressed in the hair cells, and modulating expression of these receptors by viral-mediated gene transfer into the cochlea.
Using the guinea pig as an experimental animal model, we have isolated clones for neuronal nicotinic acetylcholine receptors (nAChRs) present on cochlear outer hair cells. We have made antibodies to the alpha 9 and alpha 10 subunits of the nAChR, and utilized these antibodies in Western blot and immunohistochemistry on vestibular and cochlear tissues. The efferent system is believed to be responsible for (1) protecting the cochlea from acoustic overstimulation and (2) enhancing sound recognition in the presence of background noise. We have also developed a method to assess the strength of an animal's efferent system across a multi-frequency extent based on measuring distortion product otoacoustic emissions (DPOAE). We determined that the efferent strength of an animal correlated with the amount of alpha 9 nAChR present in that animal's cochlea, and strong efferent strength correlated with decreased hearing loss associated with exposure to intense noise. In collaboration with Drs. Liberman, Zuo, and Maison, we have also determined that mice over-expressing the alpha 9 nAChR subunit are less susceptible to moderate and intense noise exposures. These studies suggest that acetylcholine is an important neurotransmitter in regulating hearing function, and that the cochlear nAChR is a candidate for therapeutic intervention to prevent hearing damage.
One drawback to studies based on the loss or overexpression of a gene in transgenic mouse models is that it is difficult to separate the acute effects from developmental effects of gain or loss of a gene of interest. To overcome this drawback, we have used virally-mediated gene transfer to deliver mRNAs to the ipsilateral cochleas of adult guinea pigs in vivo. As a first step, we determined which viral vector could infect hair cells, and which promoters could drive expression of transgenes in the infected hair cells. We found that adeno-associated virus (AAV) was able to deliver mRNAs to blood vessels and certain nerve fibers of the cochlea, but was not able to infect cochlear hair cells using a variety of promoter constructs. We found that first-generation adenovirus [E1-, E3-] could infect cochlear hair cells, yet was ototoxic, similar to the toxicity observed in cultured hair cells. However, we determined that a modified adenovirus [E1-,E3-, E2b-] could infect cochlear hair cells both in culture and in vivo with no loss of either transduction currents or cochlear function. Furthermore, we determined that the cytomegalovirus (CMV) promoter could drive expression of either lacZ or green fluorescent protein (GFP) in infected hair cells, indicating that we have developed a delivery system to express transgenes in cochlear hair cells without causing damage to the infected cells.