RESUMO
The role astrocytes play in brain development and function has garnered greater attention as the diversity of roles they are involved in has become apparent. We have previously shown that ethanol-exposed astrocytes alter neuronal neurite outgrowth in an in vitro co-culture system and that ethanol alters the astrocyte-produced extracellular matrix (ECM) in vitro, with similar alterations in vivo. In this study, we utilized the translating ribosome affinity purification (TRAP) procedure in Aldh1l1-EGFP/Rpl10a transgenic mouse primary cortical astrocyte cultures to transcriptionally and translationally profile the astrocyte response to ethanol. We found a large number of differences between the total RNA pool and the translating RNA pool, indicating that the transcriptional state of astrocytes may not always reflect the translational state of astrocytes. In addition, there was a considerable overlap between ethanol-dysregulated genes in the total RNA pool and the translating RNA pool. Comparisons to published datasets indicate the in vitro model used here is most similar to PD1 or PD7 in vivo cortical astrocytes, and the ethanol-regulated genes showed a significant overlap with models of chronic ethanol exposure in astrocytes, a model of third-trimester ethanol exposure in the hippocampus and cerebellum, and an acute model of ethanol exposure in the hippocampus. These findings will further our understanding of the effects of ethanol on astrocyte gene expression and protein translation and how these changes may alter brain development and support the use of in vitro astrocyte cultures as models of neonatal astrocytes.
RESUMO
Neurotoxicology is hampered by the inability to predict regional and cellular targets of toxicant-induced damage. Evaluating astrogliosis overcomes this problem because reactive astrocytes highlight the location of toxicant-induced damage. While enhanced expression of glial fibrillary acidic protein is a hallmark of astrogliosis, few other biomarkers have been identified. However, bacterial artificial chromosome - translating ribosome affinity purification (bacTRAP) technology allows for characterization of the actively translating transcriptome of a particular cell type; use of this technology in aldehyde dehydrogenase 1 family member L1 (ALDH1L1) bacTRAP mice can identify genes selectively expressed in astrocytes. The aim of this study was to characterize additional biomarkers of neurotoxicity-induced astrogliosis using ALDH1L1 bacTRAP mice. The known dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 12.5 mg/kg s.c.) was used to induce astrogliosis. Striatal tissue was obtained 12, 24, and 48 h following exposure for the isolation of actively translating RNA. Subsequently, MPTP-induced changes in this RNA pool were analyzed by microarray and 184 statistically significant, differentially expressed genes were identified. The dataset was interrogated by gene ontology, pathway, and co-expression network analyses, which identified novel genes, as well as those with known immune and inflammatory functions. Using these analyses, we were directed to several genes associated with reactive astrocytes. Of these, TIMP1 and miR-147 were identified as candidate biomarkers because of their robust increased expression following both MPTP and trimethyl tin exposures. Thus, we have demonstrated that bacTRAP can be used to identify new biomarkers of astrogliosis and aid in the characterization of astrocyte phenotypes induced by toxicant exposures. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14518.