Astrocytes are the major cell type of the central nervous system, wherein they perform critical roles in its development, maintenance, and response to injury. Activities now attributed to astrocytes include participation in the development and maintenance of neurons and oligodendrocytes, formation of the blood-brain barrier, recycling of glutamate, and potassium homeostasis. Recent findings that astrocytes produce and/or have receptors for a large array of neurotransmitters, neuropeptides, cytokines, and growth factors have further stimulated speculation concerning the roles of these cells. Nearly all of the suggested functions for astrocytes are based on observed correlations, and many of these have been made on cultured cells whose properties may differ from those in vivo. As an alternative approach to understanding astrocytes, it is proposed to study their function by analyzing the transcriptional regulation of the gene encoding glial fibrillary acidic protein (GFAP), an astrocyte-specific protein. These studies will primarily be performed by analyzing expression patterns of reporter gene constructs in transgenic mice.
In specific aim 1, a detailed analysis will be performed of a small, 124 bp region of the GFAP promoter that previous experiments have shown to be sufficient for astrocyte specificity of transcription in cultured cells. The objective is to identify the precise DNA sequences required, and then to use this information to isolate and characterize the mediating transcription factors to parse out the signaling pathways involved.
In specific aim 2, a large segment of the GFAP promoter will be examined that is required for transcription in astrocytes in certain brain regions, but not in others.
This specific aim seeks to better define differences among astrocytes, with the ultimate goal of understanding the functional consequences of astrocyte heterogeneity.
In specific aim 3, more sophisticated tools will be developed for expressing transgenes in astrocytes. These tools include GFAP-based promoters that can be turned on and off, that are more potent, and that target specific subclasses of astrocytes. These tools will facilitate hypothesis-testing, creation of disease models, and gene therapy.
