Astrocyte‐specific transcriptome analysis using the ALDH1L1 bacTRAP mouse reveals novel biomarkers of astrogliosis in response to neurotoxicity

Abstract

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: .

Meta-analysis of mouse transcriptomic studies supports a context-dependent astrocyte reaction in acute CNS injury versus neurodegeneration

“…The MPTP dataset [ 15 ] was classified as acute injury rather than chronic neurodegeneration because the authors examined the changes in astrocyte transcriptome after an acute neurotoxic injury to the substantia nigra dopaminergic neurons (i.e., 12 h, 24 h, and 48 h after a single MPTP intra-peritoneal injection), rather than after chronic MPTP administration through a subcutaneous osmotic pump [ 39 ]. Since the three acute MPTP time points are not independent datasets, only the 24 h dataset was used for the meta-analysis [ 15 ]. Mouse neurodegenerative datasets ( n = 9) included ALS (various SOD1 mutants, n = 3) [ 16 – 18 ] and AD models of both brain β-amyloidosis ( n = 5) (APPswe/PSEN1deltaE9 n = 3 and PS2APP n = 2) [ 19 – 23 ], and tauopathy (P301S-MAPT, n = 1) [ 23 ].…”

“…inj.) 3/3 3F/3F 4–7 months str TRAP of Aldh1L1-eGFP-L10a Microarray 10.17632/ktgcp4mtk2.1 [ 15 ] SOD1-G93A-1 Chronic G93A SOD1 vs wt 4/3 NA 90 days Spinal cord FACS Aldh1L1-eGFP/G93A SOD1 Microarray GSE111031 [ 16 ] SOD1-G93A-2 Chronic G93A SOD1 vs wt 3/3 3M/3M 120 days Spinal cord LCM of Aldh1L1+ cells Microarray GSE69166 [ 17 ] SOD1-G37R Chronic G37R SOD1 vs wt 4/6 4M/6M 8 months Spinal cord TRAP of Aldh1L1-eGFP-L10a RNA-seq GSE74724 [ 18 ] APPPS1-1 Chronic APPswePS1dE9 vs wt 4/4 NA 15–18 months Whole ctx FACS with Glt-1 Ab Microarray GSE74615 [ 19 ] APPPS1-2 Chronic APPswePS1dE9 vs wt 4/4 NA 15–18 months Whole ctx FACS of GFAP-GFP cells Microarray GSE74614 [ 20 ] APPPS1-3 Chronic APPswePS1dE9-GFP vs wt-GFP 4/7 4M/7M 9 months CA1 hipp FACS of eGFP+ cells RNA-seq GSE108520 [ 21 ] PS2APP-1 Chronic PS2APP vs wt 4/5 …”

“…Dataset category (acute injury versus neurodegeneration), mouse model, mouse age and sex, CNS region, method of astrocyte isolation, GEO accession number, and literature reference for these datasets can be found in Table 1. Mouse acute injury datasets (n = 6) included LPS-induced sepsis (n = 1) [1], MCAO stroke (n = 2) [1,12], SCI (n = 2) [13,14], and acute toxic parkinsonism (MPTP, n = 1) [15]. For one of the stroke studies [1], only the dataset from 3 days after MCAO was analyzed, because gene expression changes were maximum 3 days and attenuated by 7 days after MCAO surgery.…”

“…For one of the stroke studies [1], only the dataset from 3 days after MCAO was analyzed, because gene expression changes were maximum 3 days and attenuated by 7 days after MCAO surgery. The MPTP dataset [15] was classified as acute injury rather than chronic neurodegeneration because the authors examined the changes in astrocyte transcriptome after an acute neurotoxic injury to the substantia nigra dopaminergic neurons (i.e., 12 h, 24 h, and 48 h after a single MPTP intra-peritoneal injection), rather than after chronic MPTP administration through a subcutaneous osmotic pump [39]. Since the three acute MPTP time points are not independent datasets, only the 24 h dataset was used for the meta-analysis [15].…”

Cell-type specificin vitrogene expression profiling of stem-cell derived neural models

“…RiboTag and TRAP rely on the tight interaction between the ribosome and mRNA to indirectly immunoprecipitate translated RNA via RPL22 or RPL10a, respectively. They do not require specialized equipment, dissociation to single cells, and these methods have been used extensively for studies of murine neurons 27,28 , microglia 29 , astrocytes 30 , and macrophages 31 . We reasoned that the same strategy could be used to purify cell-type specific mRNA from heterogeneous in vitro co-cultures.…”