David Poulsen - US grants

neuroregulation; glutamate transporter; technology /technique development; glutamates;

technology /technique development

DESCRIPTION (provided by applicant): Hair cells are the mechanosensory cells of the cochlea and may be lost as a consequence of aging or exposure to ototoxic agents and viral or bacterial pathogens. Lost hair cells spontaneously regenerate within the avian but not the mammalian cochlea. Thus, hair cell loss leads to permanent hearing deficits in humans. The lack of effective treatments for hair cell loss and many other forms of acquired and inherited hearing disorders has prompted interest in the potential application of gene transfer techniques to restore normal cochlear function. Recent gene transfer studies have examined the potential of driving hair differentiation in mature mammalian cochlea. It has been determined that the basic helix-loop-helix transcriptional activator Math1, is both required and sufficient to induce mature cochlear non-sensory epithelial cells to differentiate into hair cells. While extremely exciting and encouraging, a number of issues remain to be worked out before gene therapy approaches to treating hair cell loss can be successfully applied in humans. For example, natural hair cells are arrayed within a highly defined cytoarchitecture composed of a single row of inner hair cells and three rows of outer hair cells. In addition, the stereocilia of hair cells must come into contact with the tectorial membrane which come into direct contact. The inner and outer hair cells are associated with different subpopulations of support cells and make different types of synaptic contacts with spiral ganglion neurons. The inner and outer hair cells also perform different functions within the mature cochlea. Inner hair cells serve as the primary mechanosensory cells while outer hair cells serve more of an amplification capacity. Therefore, we hypothesize that nascent hair cells will need to be localized within the natural rows of inner and outer hair cells of the sensory epithelial ridge in order to make the appropriate contacts with support cells, the tectorial membrane and neurons to be functional. Hair cell regeneration studies published to date have relied on Adenoviral vectors to deliver, and the CMV promoter to drive constitutive expression of the Math1 gene. These conditions predominantly resulted in the development of ectopic hairs cells located outside of the normal sensory epithelial ridge. We have recently demonstrated that Adeno-associated virus (AAV) can efficiently transduce support cells immediately surrounding hair cells in the mature cochlea. We have also established that transgene expression can be limited to these support cell populations when expression is driven by the glial fibrillarv acidic protein (GFAP) promoter. We have further demonstrated that Math1 activity can be made inducible (in the presence of tamoxifen) by fusing the Math1 protein with the estrogen receptor protein. Therefore, we propose to use AAV to deliver a GFAP-Math1/ER construct and drive expression of the fusion protein specifically in support cells. Proposed studies will be carried out using both in vitro and in vivo models to study the effects of transient Math1 activity in specific support cell populations on the localization and development of nascent hair cells.

[unreadable] DESCRIPTION (provided by applicant): Under normal physiological conditions, glutamate-mediated activation of excitatory receptors on neurons is transient. However, under pathological conditions, extracellular glutamate levels can become elevated, resulting in prolonged stimulation of the excitatory receptors, and leading to excitotoxic death of neurons. Five excitatory amino acid transporters (EAATs 1-5) play a key role in insuring that excitatory transmission is transient by rapidly clearing glutamate from synaptic clefts, and the extracellular environment. However, under certain pathological conditions, such as ischemia, reversal of these transporters may actually contribute to pathology and neuronal damage. Thus, glutamate transporters have the potential to promote either neuroprotection or neurodegeneration. Contradictions exist in the current literature in regards to the contribution that individual transporter isotypes provide towards neuroprotection or neurodegeneration in the context of excitotoxic injury. The model systems used to study glutamate transporters have been primarily limited to transporter inhibition. Pharmacological studies have successfully identified a number of glutamate analogs that globally inhibit glutamate transporters. Very few pharmacological agents have been identified which enhance glutamate transporter activity and none have the ability to discriminate between selected transporters in a cell type specific manner. In contrast, our preliminary data strongly suggests that the expression of individual transporter isotypes can be either inhibited or enhanced in a cell type specific manner by using recombinant Adeno-associated virus (AAV) vectors to deliver selected glutamate transporter genes in either the sense or antisense orientation, under the control of cell type specific promoters. However, we have observed that gene expression is limited to focal regions within the CNS following stereotactic delivery of AAV vectors. Our goal in this proposal is to rigorously test this tool and determine to what extent functional transporter expression can be modulated and further develop this method to provide global, cell type specific transporter gene distribution and expression in the CNS through intracerebral ventricular injection of P0 rat pups with novel AAV serotypes. Relevance The development of this viral based gene delivery method will provide a novel tool which will permit the examination of glutamate transporter function in a way that has not been previously possible. This approach will allow us to directly test critical hypotheses regarding how individual glutamate transporter isotypes contribute to neuroprotection or neuropathology in diseases and disorders such as stroke, epilepsy and ALS. The development of these tools will also provide a solid scientific basis for future translational research involving the cell type specific modulation of selected transporters as potential treatments for disorders involving an excitotoxic component. [unreadable] [unreadable]

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