BAC-TRAP technology in neurotoxicology: the ALDH1L1BAC-TRAP mouse as a tool to assess astrocyte specific responses to neural injury.
O'Callaghan-JP; Kelly-KA; Miller-DB
Toxicologist 2014 Mar; 138(1):95
A central problem in neurotoxicology is detecting the selective and unpredictable damage to specific cells produced by toxic agents and mixtures. Evaluating astrogliosis overcomes this problem because reactive astrocytes show the location of toxicant-induced damage occurring anywhere in the CNS. Enhanced expression of GFAP is a hallmark of reactive astrocytes; however, few other astrogliosis biomarkers are known. Thus, determining the specific in vivo transcriptomic profile of astrocytes under control and reactive conditions will allow for the identification of additional astrogliosis biomarkers. Heintz and Greengard (2008) introduced BAC-TRAP (translating ribosome affinity purification) technology as an approach for identification of cell-type-specific responses in vivo. Here, we implemented this approach for the assessment of mRNA translation specific to astrocytes responding to damage caused by known neurotoxicants. ALDH1L1 is an enzyme thought to serve a housekeeping function in astrocytes. Using hippocampal and striatal damage due to TMT and MPTP, respectively, we evaluated the localization and response of ALDH1L1 compared to astrogliosis seen by GFAP immunohistochemistry and ELISA. Staining of ALDH1L1 revealed localization to astrocytes after TMT and MPTP, while immunoblots of ALDH1L1 revealed basal expression of this protein but little enhanced expression after MPTP and TMT, confirming it to be an astrocytic "housekeeping" gene/protein. We then used the ALDH1L1 BAC-TRAP mouse to evaluate astrocytic-specific mRNA with expression analyses by gene array in control conditions. Tissue was subjected to TRAP utilizing an eGFP antibody that only binds to actively translating RNA in astrocytes. This revealed numerous genes in "resting" astrocytes, including genes previously localized to astrocytes (e.g., GFAP), as well as novel genes to this cell type (e.g. PHOX2A). MPTP expression data are reported in accompanying poster. The ALDH1L1 BAC-TRAP mouse represents a promising tool to investigate astrocytic responses to neural injury.
Toxicology; Neurotoxicity; Models; Cell-function; Cellular-function; Toxic-effects; Biomarkers; In-vivo-study; Neurotoxins; Enzymes; Laboratory-animals; Animals; Genes; Neurological-system; Neurological-reactions
The Toxicologist. Society of Toxicology 53rd Annual Meeting and ToxExpo, March 23-27, 2014, Phonex, Arizona