Our long-term objective is to understand and counteract mechanisms that lead to cellular degeneration and impede axon regeneration after brain and spinal cord injury.
Our specific aims here are to generate new and innovative models to study the roles played by specific molecules that are expressed by reactive astrocytes, a principal cell type in the local tissue response to injury and disease in the central nervous system (CNS). These models will help to identify key molecules that determine the biology and function of reactive astrocytes. Numerous effector molecules produced by reactive astrocytes have been described, but the roles played by these cells are not well defined. Little mechanistic information is available about how the different functions of reactive astrocytes are triggered and implemented through specific molecules. We previously developed a model for the ablation of reactive astrocytes in transgenic mice and showed that these cells are essential for local neuroprotection, blood brat barrier repair and regulation of inflammation after CNS injury. Our next goal is to identify molecular mechanisms for these phenotypes. Here, we propose to develop transgenic models for the targeted and regulatable ablation of specific molecules molecules expressed by reactive astrocytes. These models use the Cre/loxP system, where bacterial Cre recombinase I excises DNA sequences between loxP sites inserted into genes targeted for inactivation.
Aim I is to generate and characterize transgenic mice that express Cre from the glial fibrillary protein (GFAP) promoter, thus targeting Cre to reactive astrocytes.
Aim 2 is to generate transgenic mice in which an additional level of regulation is achieved by using a tetracycline regulatable promoter system. In these mice, Cre expression in astrocytes will be suppressed during development and activated in adult mice. Transgenic lines will be validated for use by demonstrating the specificity of the targeting of Cre activity to reactive astrocytes at the single cell level.
Aim 3 is to demonstrate proof of principle by ablating a crucial regulatory molecule specifically from GFAP expressing astrocytes. STAT3 is an intracellular signal transducer of the JAK-STAT family involved in mediating the proliferation and differentiation responses to various cytokines and growth factors in may cell types. STAT3 signaling has been implicated in both astrocyte development and reactive astrocytosis. Mice with loxP sites inserted into the STAT3 gene are available and will be crossbred with Cre mice from Aims 1 and 2. STAT3-deficient astrocytes will be evaluated in vitro and after experimental CNS injury in vivo for their ability to become reactive. The consequences of the predicted failure of astrocytosis after CNS injury in mutant mice will be studied in vivo. The models from Aims 1 and 2 will allow the testing of many hypotheses about the roles that specific molecules expressed by reactive astrocytes play in the biology and function of reactive astrocytes after CNS injury. These models will be useful for many different future studies in our own and collaborating laboratories.
Aim 3 will begin this process. The findings obtained in both the short and long term will contribute fundamental information about cellular and molecular mechanisms that determine functional outcome after CNS injury. Mechanistic information of this kind is essential for the development of rationally based therapeutic strategies.
