Glutamine synthetase (as) is a glial specific enzyme that constitutes in neural tissues an endogenous neuroprotection mechanism that amidates the neurotoxic neurotransmitter glutamate to the non-toxic amino acid glutamine. This mechanism fails to protect against glutamate neurotoxicity under pathological conditions. In the retina, glutamate neurotoxicity has been implicated in the process of neuronal degeneration that follows injury or focal ischemia and in the pathophysiology of degenerative disorders. These conditions are also accompanied by a decline in as expression. It has been suggested that glial cells cannot cope with glutamate neurotoxicity because the level of as is not sufficiently high to catalyze the excessive amounts of glutamate released by damaged neurons. Our long-term objective is to examine whether elevation of as expression has neuroprotective benefits.
The specific aim of this pilot study is to design an approach for the induction of the endogenous as gene in injured neural retina and to examine whether the resulting increase in as has neuroprotective benefits. We will exploit our recent finding of a NRSE site in the regulatory region of the as gene. A recombinant transcription factor, that contains a strong viral transactivation domain attached to the NRSF-DNA binding domain, will be constructed. Viral vectors that express the recombinant factor will be used to elevate as expression in explants of injured chick retina. The level of as expression will be monitored by enzyme activity, Western and Northern blotting and immunohistochemistry. Neuronal cell degeneration will be assessed in terms of LDH efflux, DNA fragmentation and histological appearance. Our results will indicate whether as is a limiting factor in the function of the endogenous neuroprotective mechanism in injured tissue. They are expected to contribute to the basic understanding of the mechanisms that underlie neuronal degeneration and to open new avenues for therapy.
