Glaucoma is a prevalent blinding disease characterized by the progressive loss of retinal ganglion cells. Previously, we used Bax knockout mice to show that this proapoptotic gene was essential for ganglion cell death stimulated by optic nerve crush and in a mouse model of spontaneous glaucoma. Further study has now shown that variation in Bax expression plays a dramatic role in ganglion cell susceptibility to apoptotic stimuli. In different strains of mice, this variation is modulated by a single nucleotide change in the Bax gene promoter, which affects the binding affinity of an unidentified transcription factor. We propose to continue this investigation by identifying this factor and determining its role in regulating Bax expression in both in vitro (cell transfection) and in vivo (targeted deletion) studies. Since Bax expression is critical to the death of nearly all cells, a more complete characterization of the regulatory mechanisms controlling its transcription is a fundamental advance to our understanding of the cell death process. In addition to characterizing part of the transcriptional control of Bax gene transcription, we have also been investigating the early stages of apoptosis that occur in retinal ganglion cells before the critical BAX-dependent pathway. These studies were precipitated by our initial experiments to determine the regenerative potential of Bax- deficient ganglion cells that were part of the last funding period. We learned that cells arrested at the BAX-dependent step of apoptosis have undergone several molecular changes that render them incompetent to function normally, or mount a robust regenerative response. Two of these early events are the focus of this new proposal. The first of these is the apoptotic volume decrease (AVD), which occurs within the first two hours after optic nerve crush, suggesting that this may be one of the first changes occurring in the somas of damaged ganglion cells. We propose experiments to investigate the role of the Kv2.1 voltage-gated K+ channel in this process, including direct in vivo measurement of K+ efflux after optic nerve crush, an investigation of the signaling pathways that lead to Kv2.1 activation, and a direct in vivo test of Kv2.1 action in the cell death process using gene transfer of a dominant negative mutant of this channel. In addition to the AVD, retinal ganglion cells also exhibit an early stage of gene silencing. Work from the previous funding period showed that this was linked to epigenetic changes in the histones of active genes, which undergo deacetylation. We propose new experiments to characterize these changes further, including an investigation to identify accompanying histone methylation/demethylation events and the enzymes involved in these changes. Since we postulate that reversal of gene silencing is a critical component to reactivate ganglion cells, a complete understanding of the types of epigenetic changes associate with this phenomenon is essential.
This proposal is a continuation of studies of the process of retinal ganglion cell death. In the previous funding period, we examined the important role of the proapoptotic gene Bax in the process of ganglion cell death. Although cells do not die in the absence of Bax function, they have undergone important early molecular changes that disrupt their normal function and limit their ability to respond to neurotrophic treatments. This proposal will extend those studies to investigate the molecular basis of the early onset apoptotic volume decrease and the epigenetic basis of normal gene silencing. In addition to these aims, experiments are also proposed to investigate further a key regulatory element and binding protein complex in the Bax promoter that quantitatively affects normal gene expression.
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