Gene expression in astrocytes and astrocytosis: In vitro and in vivo regulation of novel genes identified by differential cloning methods.

摘要

To define the molecular basis of gene expression in astrocytes and astrogliosis, we used differential cloning methods. Initially, we identified genes expressed in activated astrocytes involved in brain injury or disease using differential display. Additionally, we identified genes expressed in regionally-defined astrocytes cultured under basal conditions using subtractive hybridization. Using these techniques, we identified both known and novel genes that are involved in astrocyte function in the activated state, as well as under basal conditions.; In the first part of our studies, we developed an in vitro model of astrogliosis using cultures of cortical type I astrocytes stimulated with basic fibroblast growth factor (bFGF). bFGF, which is elevated in brain injury, induced a process-bearing morphology, comparable to that observed in cortical disease. Treatment for 24 hours increased glial fibrillary acidic protein (GFAP), measured by western blot analysis. We used this model to investigate activation-associated expression. At 4 and 24 hours, activated astrocytes exhibited unique patterns of gene expression. Seven cDNA bands were isolated, PCR re-amplified, and cloned; slot blot analysis confirmed differential expression. Using partial sequence analysis, we assigned the cDNAs to three categories: known genes with known functions, previously cloned genes of unknown function and novel genes of unknown brain function. Finally, northern analysis confirmed that the novel clone 4.1 was temporally regulated in vitro as well as in vivo, when bFGF was given subcutaneously.; In the second part of our studies, we addressed issues of astrocyte heterogeneity by characterizing regional-specific gene expression in the substantia nigra (SN). Using PCR-based subtractive hybridization, two astrocyte cDNAs of interest were identified, one known and one novel. The known cDNA was peroxisomal membrane protein, PMP70, a peroxisome-specific protein differentially expressed in astrocytes from different brain regions. The novel cDNA, AT1-46, was preferentially expressed by the olfactory-limbic system of the adult rat brain, regulated in the brain in a developmentally- and cell-specific manner. These findings indicated that astrocytes from different brain regions are different, expressing both known and novel genes potentially involved in the development of the nervous system.; This work showed that differential cloning methods can be used to identify both known and novel genes that may define activated astrocyte function, as well as basal astrocyte gene expression. An understanding of molecular gene expression involved in normal and reactive states will establish fundamental knowledge of astrocyte function and define their complex interactions in the brain.