TY - JOUR
AU - Bernal, Juan
AB - Abstract Astrocytes mediate the action of thyroid hormone in the brain on other neural cells through the production of the active hormone triiodothyronine (T3) from its precursor thyroxine. T3 has also many effects on the astrocytes in vivo and in culture, but whether these actions are directly mediated by transcriptional regulation is not clear. In this work, we have analyzed the genomic response to T3 of cultured astrocytes isolated from the postnatal mouse cerebral cortex using RNA sequencing. Cultured astrocytes express relevant genes of thyroid hormone metabolism and action encoding type 2 deiodinase (Dio2), Mct8 transporter (Slc16a2), T3 receptors (Thra1 and Thrb), and nuclear corepressor (Ncor1) and coactivator (Ncoa1). T3 changed the expression of 668 genes (4.5% of expressed genes), of which 117 were responsive to T3 in the presence of cycloheximide. The Wnt and Notch pathways were downregulated at the posttranscriptional level. Comparison with the effect of T3 on astrocyte-enriched genes in mixed cerebrocortical cultures isolated from fetal cortex revealed that the response to T3 is influenced by the degree of astrocyte maturation and that, in agreement with its physiological effects, T3 promotes the transition between the fetal and adult patterns of gene expression. Thyroid hormones thyroxine (T4) and triiodothyronine (T3) are important regulators of developmental and physiological homeostasis, and, in particular, they are crucial for proper brain maturation and function (1). Thyroid hormone action is exerted by the regulation of gene expression after T3 binding to its nuclear receptors (2). Accordingly, many genes are thyroid hormone–dependent in the developing brain. The majority of these genes are sensitive to thyroid hormone only during specific developmental windows, and only a minor fraction remains sensitive in the adult brain. This is consistent with the developmental role of thyroid hormones in promoting the progression from the fetal to adult states, reflected in their influence on the transition from embryonic to adult patterns of gene expression (3). Neurons and oligodendrocytes are relevant thyroid hormone cellular targets. T3 regulates the expression of genes expressed specifically in these cells (4) in a developmental window–dependent manner. Little is known about astrocytes as thyroid hormone target cells. Astrocytes express thyroid hormone receptors (5, 6) and have an important role in thyroid hormone metabolism, as they express type 2 deiodinase (Dio2), which converts T4 into the more active T3 (7). Regulation of this pathway during development is of crucial importance, as most T3 available to the brain during prenatal and early postnatal stages is generated through this mechanism (8). Thyroid hormones have many effects on astrocytes in vivo and in vitro (9–18). However, because thyroid hormones, especially T4, may have extragenomic actions (19), it is unclear to what extent thyroid hormones influence astrocyte maturation and function by the control of gene expression at a direct transcriptional level exerted by T3. Transcriptional responses to T3 in astrocytes have so far not been defined. Given the wide variety of functions performed by astrocytes and their involvement in brain disorders (20, 21), it is important to define whether astrocytes are transcriptional targets of T3, in addition to locally controlling the production of the active hormone. In a recent study, we used mouse embryo–derived primary cerebrocortical (CC) cells to analyze the effects of T3 on gene expression (3). Around one thousand genes were induced or repressed by T3, of which about one-third were regulated directly at the level of transcription. The culture was composed mainly of neurons, but astrocytes were also present. T3 addition resulted in the direct upregulation of 18 astrocyte-enriched genes and direct downregulation of 2 genes. These results indicated that astrocytes are primary genomic targets of T3, and a tentative list of genes regulated by T3 in astrocytes could be elaborated based on the relative enrichment of the target genes in specific neural cells (22). For example, Aqp4, encoding aquaporin, and Slc1a2, encoding the high-affinity glutamate transporter Glt1, were upregulated directly by T3. Because these genes are highly enriched in astrocytes over the rest of the neural cells, their regulation by T3 in the mixed CC culture most likely occurred in astrocytes. Another important gene, Kcnj10, encoding the K+ channel Kir4.1 (23), was also directly regulated by T3. Kcnj10 is enriched fourfold in astrocytes compared with the rest of neural cells and is also expressed by cells of oligodendroglial lineage. Therefore, it was uncertain whether it was regulated by an action of T3 on the astrocytes or in another cell type. In the present work, we have analyzed the effects of T3 on gene expression in astrocyte cultures derived from the early postnatal mouse cortex. Our goal was to analyze the action of T3 on astrocytes through changes of gene expression and compare with the effects of T3 on astrocyte-enriched genes in a mixed neural cell culture. Materials and Methods Materials Dulbecco’s modified Eagle medium (DMEM), glutamax, fungizone, penicillin, streptomycin, 4′,6-diamidino-2-phenylindole, and fetal calf serum were from Gibco (Thermo Fisher Scientific, Waltham, MA); 12-well culture plates were from Costar® (Corning, Corning, NY); the RNeasy Plus Micro Kit was from Qiagen (Valencia, CA); and poly-l-ornithine, cycloheximide (CHX), Triton X-100, and T3 (sodium salt) were from Sigma-Aldrich (St. Louis, MO). For antibodies, glial fibrillary protein (GFAP) antibody clone G-A-5 was from Sigma-Aldrich [Research Resource Identifier (RRID): AB_2314539]; rabbit polyclonal anti–neuronal-specific nuclear protein (NeuN) antibody was from Millipore (Burlington, MA; RRID: AB_10807945); and secondary antibodies and Alexa Fluor 555 (RRID: AB_10892947) and 488 (RRID: AB_2556542) were from Molecular Probes (Thermo Fisher Scientific). Astrocyte primary culture Establishment of astrocyte cultures Primary astrocyte (ASTRO) cultures (15) were established from the whole neocortex of P3 mice. Protocols for animal handling were approved by the local Institutional Animal Care and Use Committee, according to European Union rules. Mice of a hybrid genetic background of 129/OLa × 129/Sv+ and BALB/c × C57BL/6 were used (3). The pups were euthanized by decapitation; the brains were placed on a Petri dish in phosphate-buffered saline and dissected under a stereomicroscope. A midline cut was made to separate each hemibrain, the meninges and blood vessels were carefully removed with forceps, and the cerebral cortices isolated from other structures. The tissue was disaggregated in culture medium [DMEM containing 10% thyroid hormone–deprived fetal calf serum (24), 1% glutamax, 1% fungizone, and penicillin-streptomycin] by passing up and down through 1- and 0.2-mL pipette tips. After 5 minutes of centrifugation at 1200 rpm, the pellet was suspended in culture medium. The cells were established at low density in poly-l-ornithine–coated 12-well plates (130,000 cells per well) and incubated for 7 days, with medium changes every 72 hours. Cultures were established such that each 12-well plate was derived from a pool of five hemicortices. Three wells from each plate received the same treatment, and the RNA from each of these wells was pooled. Each experiment contained four replicas of the same treatment. Immunofluorescence Cells were fixed with acetone, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich), and stained with a mouse monoclonal anti-GFAP and donkey anti-mouse Alexa 488 (green). Neurons were identified with a rabbit polyclonal anti-NeuN and donkey anti-rabbit Alexa 555 (red). All antibodies were used at 1:500 dilution. 4′,6-Diamidino-2-phenylindole was used to stain the nuclei. T3 treatment The cells were incubated in DMEM containing 0.1% thyroid hormone–deprived serum 24 hours before adding T3. For RNA sequencing (RNA-Seq) and CHX experiments, 10 nM T3 was used, as we wanted to ensure that after 6-hour incubation, the occupancy of nuclear receptor was high. For the validation experiments involving incubation with T3 for 24 hours, we used 1 nM, as this is the concentration that in our experience results in a maximal effect. Cells were harvested 24 hours later. CHX was added to the cells at a final concentration of 8 µg/mL 30 minutes before adding T3 (10 nM), and the cells incubated for 6 hours after T3 addition before harvesting. RNA analysis RNA isolation and sequencing Total RNA was prepared using RNeasy Plus Micro Kit and quantified and purity checked using a NanoDrop ND-1000 (Thermo Fisher Scientific). RNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA-Seq was performed at the Genomics Unit of the Centro Nacional de Investigaciones Cardiovasculares (Madrid, Spain). The experimental design was as follows: (1) comparison at 24 hours in the absence (four samples) and presence of T3 (three samples), and (2) comparisons at 6 hours in the absence (four samples) and presence of T3 with or without CHX (four samples). Two hundred nanograms total RNA was used to construct index-tagged complementary DNA (cDNA) libraries using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA). Single reads of 50-base length using the TruSeq SBS Kit v5 (Illumina, San Diego, CA) were generated on the HiSeq 2500 following the standard RNA-Seq protocol. For the demultiplexing process, we used bcl2fastq with standard parameters and one mismatch allowed for the index identification. Read quality was determined by analyzing reads (∼30 million reads/sample; Supplemental Table 1) with the FastQC application (S. Andrews, FastQC, A Quality Control Tool for High Throughput Sequence Data, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We used the mouse sequenced genome GRCm38. The fasta file containing sequences of this genome was downloaded from Ensembl (http://www.ensembl.org/Mus_musculus/Info/Index). This genome was indexed from Bowtie (25), and sequence reads were aligned using TopHat (26). Mapping data quality (Supplemental Table 1) was determined with the application Qualimap (27). We quantified reads to specific genes using the Python module HT-SEQ (28). We explored gene expression data by principal component analysis and clustering methods. Before computing differential expression between samples, we also performed exploratory plots to evaluate saturation, count distribution, and type of detected features using the Bioconductor package NOISeq (29). The pattern was similar for all samples in the study. RNA-Seq data were normalized using trimmed mean of M values (30). Expression levels were estimated using the reads per kilobase of transcript per million mapped reads (RPKM) method. Group comparison and statistical significances were analyzed from the Bioconductor package edge (31). The multiple-testing P value correction procedure was used to derive adjusted P values (32). The RNA-Seq data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE110372 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE110372). Real-time polymerase chain reaction For the cDNA synthesis, 250 ng RNA was used with the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative polymerase chain reaction (qPCR) assays were performed using TaqMan® probes on a 7900HT fast real-time polymerase chain reaction (PCR) system (Thermo Fisher Scientific). The PCR program was 10 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. For analysis, we used the comparative CT method. Data were expressed relative to the values obtained for the corresponding untreated cells, which were given a value of 1.0 after correction for 18S RNA as internal control (33). As shown in Supplemental Fig. 1, 18S RNA abundance was not affected by T3 or CHX. The Mann-Whitney test was used to calculate significances. Calculations were done using Prism software version 7 (GraphPad Software, La Jolla, CA). Results Immunocytochemical and molecular characterization of the culture ASTRO culture was assessed by immunocytochemical and transcriptomic analysis. GFAP immunocytochemistry showed that >95% cells expressed GFAP (Supplemental Fig. 2a). We compared this culture with neuron-enriched CC cultures isolated from embryonic cortex as described (3). The CC culture contains a mixed population, with 75% NeuN+ neurons and 15% astrocytes GFAP+ astrocytes. The morphology of the astrocytes in each culture was clearly different. The ASTRO culture consisted of polygonal cells, whereas in the CC cultures, they presented slender ramifications with long and fine processes. The different morphology of each culture is what would be predicted from astrocytes incubated in the absence (ASTRO culture) or presence (CC culture) of neurons (34, 35). To further substantiate astrocyte enrichment of the ASTRO culture, we used a more sensitive and quantitative approach based on the transcriptomic data. To this end, we selected genes specifically expressed in native individual cell types from the data of Zhang et al. (22), which were obtained from acutely isolated cells from the P7 mouse cortex. These data provide the relative expression and cell-type enrichment of individual genes in native cells not subjected to further manipulations. We selected the top 50 genes with highest enrichment in each cell type (i.e., neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells) and compared their expression levels (in RPKM) with the RNA-Seq data from the ASTRO culture. Cluster analysis of the data showed that the ASTRO culture expressed non-astrocytic genes at background levels (Supplemental Fig. 2b and 2c). Thyroid hormone transporters and receptors in astrocytes Genes relevant to thyroid hormone transport, metabolism, and action (2, 36) were expressed in the culture (Supplemental Table 3). The major thyroid hormone transporter gene expressed was Slc16a2, encoding the monocarboxylate transporter 8, or MCT8, highly specific for T4 and T3. Other transporters were also expressed in lower amounts, especially Slc7a5 (the amino acid transporter LAT1) and the organic anion transporter polypeptides Slco1c1 (a specific T4 transporter), Slco3a1, and Slco1a4. These transporters are also expressed in native astrocytes from the neonatal mouse cortex, although in different proportions (36). Slc16a2 has similar expression levels in isolated native cells as in cultured cells (37), whereas Slco1c1 and Slc7a5 are more expressed in native cells than in cultured cells. Slc16a10 (MCT10) expressed in microglia was almost absent from the cultured astrocytes. The cultured astrocytes expressed Dio2 and much lower amounts of Dio3, the T3 receptors Thra1 and Thrb, and the non T3-binding isoform Thra2. The transcriptional coactivator Ncoa1 and corepressor Ncor1 were also expressed. Therefore, the astrocytes expressed genes that make a cell responsive to thyroid hormones. T3-induced transcriptomic changes Genes responsive to T3 after 24-hour incubation are listed in Supplemental Table 4. Of 14,838 genes expressed in the cultures, the expression of 668 genes [4.5%; false discovery rate (FDR) <0.05] was changed by 10 nM T3. Seventy-five of these genes show astrocyte enrichment (more than fivefold), and 260 are also regulated by thyroid hormones in vivo (38). A total of 430 genes were upregulated and 238 downregulated (Table 1). We refer to these genes as positive and negative genes, respectively. Table 1. Positive and Negative Regulation of Gene Expression by T3 in Mouse Astrocytes in Primary Culture Treatment  Number of Genes  Regulation    Positive  Negative  Total  T3 24 h  430  238  668  T3 6 h  194  58  252  T3 6 h + CHX  116  1  117  Treatment  Number of Genes  Regulation    Positive  Negative  Total  T3 24 h  430  238  668  T3 6 h  194  58  252  T3 6 h + CHX  116  1  117  RNA-Seq analysis (FDR <0.05) of primary astrocytes incubated with 10 nM T3 for 6 or 24 hours in the absence or presence of CHX. CHX was added to the culture 30 minutes before adding T3. View Large Table 1. Positive and Negative Regulation of Gene Expression by T3 in Mouse Astrocytes in Primary Culture Treatment  Number of Genes  Regulation    Positive  Negative  Total  T3 24 h  430  238  668  T3 6 h  194  58  252  T3 6 h + CHX  116  1  117  Treatment  Number of Genes  Regulation    Positive  Negative  Total  T3 24 h  430  238  668  T3 6 h  194  58  252  T3 6 h + CHX  116  1  117  RNA-Seq analysis (FDR <0.05) of primary astrocytes incubated with 10 nM T3 for 6 or 24 hours in the absence or presence of CHX. CHX was added to the culture 30 minutes before adding T3. View Large Gene ontology (GO) enrichment analysis was performed independently for each set of upregulated and downregulated genes. All of the significant GO terms (with Padjust < 0.05) and genes included in the analysis are detailed in Supplemental Table 5. A summary of the most relevant data is shown in Table 2. Within the set of positive genes, there was an overrepresentation of genes encoding integral membrane proteins, extracellular matrix proteins, and proteins involved in cell adhesion, receptor, and transporter activity. Conversely, there was a downrepresentation of genes encoding intracellular and nuclear proteins and involved in RNA metabolic process, DNA binding, and transcription factor activity. Within the negative genes, there was an overrepresentation of cytoskeletal and membrane receptor proteins involved in G-protein–coupled receptor activity, developmental processes, and cell movement. Table 2. GO Enrichment Analysis   Positive-Regulated Genes  Negative-Regulated Genes  Biological process  Cell adhesion  Cellular component movement    RNA metabolic process (−)  Nervous system development  Molecular function  Receptor activity  G protein–coupled receptor activity    Transporter activity    Nucleic acid binding (−)  Cellular component  Extracellular matrix  Cytoskeletal    Integral to membrane    Intracellular (−)    Nucleus (−)  Protein class  Receptors  Cytoskeletal protein receptors    Nucleic acid binding (−)    Pathways    Notch signaling pathway      Wnt signaling pathway    Positive-Regulated Genes  Negative-Regulated Genes  Biological process  Cell adhesion  Cellular component movement    RNA metabolic process (−)  Nervous system development  Molecular function  Receptor activity  G protein–coupled receptor activity    Transporter activity    Nucleic acid binding (−)  Cellular component  Extracellular matrix  Cytoskeletal    Integral to membrane    Intracellular (−)    Nucleus (−)  Protein class  Receptors  Cytoskeletal protein receptors    Nucleic acid binding (−)    Pathways    Notch signaling pathway      Wnt signaling pathway  GO enrichment analysis for the set of upregulated (positive genes) and downregulated genes (negative genes) after 24 hours of T3 treatment (PANTHER Classification System, http://pantherdb.org/; P < 0.05, Bonferroni correction for multiple testing). Abbreviation: (−), downrepresentation. View Large Table 2. GO Enrichment Analysis   Positive-Regulated Genes  Negative-Regulated Genes  Biological process  Cell adhesion  Cellular component movement    RNA metabolic process (−)  Nervous system development  Molecular function  Receptor activity  G protein–coupled receptor activity    Transporter activity    Nucleic acid binding (−)  Cellular component  Extracellular matrix  Cytoskeletal    Integral to membrane    Intracellular (−)    Nucleus (−)  Protein class  Receptors  Cytoskeletal protein receptors    Nucleic acid binding (−)    Pathways    Notch signaling pathway      Wnt signaling pathway    Positive-Regulated Genes  Negative-Regulated Genes  Biological process  Cell adhesion  Cellular component movement    RNA metabolic process (−)  Nervous system development  Molecular function  Receptor activity  G protein–coupled receptor activity    Transporter activity    Nucleic acid binding (−)  Cellular component  Extracellular matrix  Cytoskeletal    Integral to membrane    Intracellular (−)    Nucleus (−)  Protein class  Receptors  Cytoskeletal protein receptors    Nucleic acid binding (−)    Pathways    Notch signaling pathway      Wnt signaling pathway  GO enrichment analysis for the set of upregulated (positive genes) and downregulated genes (negative genes) after 24 hours of T3 treatment (PANTHER Classification System, http://pantherdb.org/; P < 0.05, Bonferroni correction for multiple testing). Abbreviation: (−), downrepresentation. View Large The most remarkable finding was that PANTHER pathway analysis identified the Notch and Wnt signaling pathways in the set of negative genes. Specifically, the Notch ligands Dll1 and Dll3, receptor Notch3, and target genes Hes5 and Hey2 were all downregulated by T3 (Supplemental Table 5), and Hey1 was transcriptionally upregulated (see following section). In the Wnt signaling pathway, genes involved in the canonical and noncanonical pathways were downregulated, including Wnt7b, Ccnd1, Pygo1, Frzb, Smarca1, Mycn, Mycl1, Map3k7cl, and Nfatc2, and Wnt7a was transcriptionally upregulated. Cadherins associated with the Wnt pathways such as Cdh4, Cdh5, and Cdh13 were also downregulated. In contrast, Daam1 and Daam2 involved in the Wnt/JNK pathway were transcriptionally upregulated by T3 (see following section). Direct transcriptional targets of T3 To confirm the RNA-Seq results in different astrocyte cultures, cells were incubated with 1 nM T3, and the expression of several genes was measured by qPCR. We selected some known T3 targets, such as Hr, Klf9, and Dbp, and some of the genes mentioned in the preceding section, such as Damm2, Hes5, and Wnt7a (Fig. 1a). There was an excellent correlation between the fold changes induced by T3 as calculated in the RNA-Seq and qPCR, respectively (Fig. 1b). Figure 1. View largeDownload slide Correlation between the effects of T3 by RNA-Seq and qPCR in replicate cultures. (a) Effect of 1 nM T3 on the expression of selected genes measured by qPCR using TaqMan probes. (b) Correlation between RNA-Seq and qPCR of the data from (a). Hr increased 25-fold by qPCR and 13-fold by RNA-Seq, and the values exceeded the axis limits. (c) Effect of 10 nM T3 on the expression of F3, Adamts5, Kcns1, and Hes5 at 6 and 24 hours in the absence and presence of CHX. All data are mean ± standard error of fold change (FC) after T3 addition of four samples for each condition. *P = 0.0286 by Mann-Whitney test. NS, not significant. Figure 1. View largeDownload slide Correlation between the effects of T3 by RNA-Seq and qPCR in replicate cultures. (a) Effect of 1 nM T3 on the expression of selected genes measured by qPCR using TaqMan probes. (b) Correlation between RNA-Seq and qPCR of the data from (a). Hr increased 25-fold by qPCR and 13-fold by RNA-Seq, and the values exceeded the axis limits. (c) Effect of 10 nM T3 on the expression of F3, Adamts5, Kcns1, and Hes5 at 6 and 24 hours in the absence and presence of CHX. All data are mean ± standard error of fold change (FC) after T3 addition of four samples for each condition. *P = 0.0286 by Mann-Whitney test. NS, not significant. To identify direct transcriptional targets of T3, the cells were incubated with 10 nM T3 for 6 hours in the presence and absence of the translational inhibitor CHX (39) to identify the fraction of the 24-hour responsive genes that were induced or repressed by T3 in the absence of protein synthesis (3, 33). Genes responsive to T3 in the presence of CHX represent direct transcriptional responses, not secondary to another primary effect of the hormone. Of the 668 genes with expression changes 24 hours after T3, 252 genes responded to T3 6 hours after addition (194 positives and 58 negatives) in the absence of CHX and 117 (116 positives and 1 negative) in its presence (Table 1; Supplemental Table 4). This result was confirmed by qPCR (Fig. 1c). F3 and Adamts5 are induced by T3 in the presence and absence of CHX. Kcns1 induction and Hes5 repression by T3 are blocked by CHX, confirming results by RNA-Seq. Primary gene responses by T3 should be expected to contain T3-responsive elements in regulatory regions. As an approximation to this question, we looked for T3 receptor binding sites (TRBS) identified by chromatin immunoprecipitation with antibodies against a GS tag (protein G and streptavidin) fused to overexpressed T3 receptors (40). Although proof that the TRBS are indeed T3-responsive elements requires functional characterization, their presence in genes regulated by T3 in the presence of CHX reinforces the idea of primary transcriptional regulation. We found that of the 117 genes under direct regulation by T3, 64 (63 positives and 1 negative) contain TRBS. A comparison with the CC cells showed that 27 of the directly regulated genes in the ASTRO culture were also directly regulated in the CC culture (Table 3). Twenty-one of these 27 genes are also regulated by thyroid hormones in vivo and include the universal T3-responsive genes Hr and Klf9 (38). Sixteen of the directly regulated genes contain TRBS, and therefore, the list of the 27 genes is a highly representative group of thyroid hormone action on gene expression. Table 3. A Partial List of the Direct Transcriptional Targets of T3 Associated Gene Name  log2 Fold Change 24-h T3  FDR  Astrocyte Enrichment  In Vivo Regulation  Contains TRBS  Dio3  5.09  2.83E-81    x    Hr  3.72  9.40E-259    x  x  Klf9  1.49  4.25E-27    x  x  Gpr30  1.11  4.91E-20        Gbp3  1.10  2.92E-15    x    Morc4  1.09  1.39E-17      x  Bcar3  1.02  3.61E-25    x  x  Daam2  0.92  2.30E-14  22×  x  x  F3  0.91  1.40E-17  19.3×  x  x  Dbp  0.79  3.28E-09    x  x  Kazn  0.77  1.32E-10        Trp53inp2  0.74  1.23E-12    x  x  Cldn12  0.73  8.88E-12    x  x  Sorl1  0.71  4.62E-05  9.6×  x    Mfsd2a  0.70  7.03E-08      x  Slc35g1  0.69  0.001118743    x    Elovl5  0.63  2.24E-10  5.5×  x  x  Nrarp  0.62  8.51E-08  6.7×  x    Ppargc1a  0.56  0.003203018    x    Gpr85  0.55  0.009420735    x    Mfap3l  0.54  0.000221799  7.9×  x  x  Hccs  0.54  4.50E-06    x  x  Kcnj10  0.48  0.00044663    x    Sgk1  0.44  0.003516857    x    Tbcel  0.44  0.000414875      x  Nudt18  0.44  0.023379335      x  Pmp22  0.31  0.016288763    x  x  Associated Gene Name  log2 Fold Change 24-h T3  FDR  Astrocyte Enrichment  In Vivo Regulation  Contains TRBS  Dio3  5.09  2.83E-81    x    Hr  3.72  9.40E-259    x  x  Klf9  1.49  4.25E-27    x  x  Gpr30  1.11  4.91E-20        Gbp3  1.10  2.92E-15    x    Morc4  1.09  1.39E-17      x  Bcar3  1.02  3.61E-25    x  x  Daam2  0.92  2.30E-14  22×  x  x  F3  0.91  1.40E-17  19.3×  x  x  Dbp  0.79  3.28E-09    x  x  Kazn  0.77  1.32E-10        Trp53inp2  0.74  1.23E-12    x  x  Cldn12  0.73  8.88E-12    x  x  Sorl1  0.71  4.62E-05  9.6×  x    Mfsd2a  0.70  7.03E-08      x  Slc35g1  0.69  0.001118743    x    Elovl5  0.63  2.24E-10  5.5×  x  x  Nrarp  0.62  8.51E-08  6.7×  x    Ppargc1a  0.56  0.003203018    x    Gpr85  0.55  0.009420735    x    Mfap3l  0.54  0.000221799  7.9×  x  x  Hccs  0.54  4.50E-06    x  x  Kcnj10  0.48  0.00044663    x    Sgk1  0.44  0.003516857    x    Tbcel  0.44  0.000414875      x  Nudt18  0.44  0.023379335      x  Pmp22  0.31  0.016288763    x  x  This table lists only the genes that are regulated directly by T3 in the ASTRO and CC cultures (39), correlated with astrocyte enrichment (22), in vivo regulation (37), and the presence of TRBS. TRBS were identified using antibodies against a GS tag (protein G and streptavidin) fused to the N terminus of overexpressed T3 receptors in C17 neuronlike cells (40). View Large Table 3. A Partial List of the Direct Transcriptional Targets of T3 Associated Gene Name  log2 Fold Change 24-h T3  FDR  Astrocyte Enrichment  In Vivo Regulation  Contains TRBS  Dio3  5.09  2.83E-81    x    Hr  3.72  9.40E-259    x  x  Klf9  1.49  4.25E-27    x  x  Gpr30  1.11  4.91E-20        Gbp3  1.10  2.92E-15    x    Morc4  1.09  1.39E-17      x  Bcar3  1.02  3.61E-25    x  x  Daam2  0.92  2.30E-14  22×  x  x  F3  0.91  1.40E-17  19.3×  x  x  Dbp  0.79  3.28E-09    x  x  Kazn  0.77  1.32E-10        Trp53inp2  0.74  1.23E-12    x  x  Cldn12  0.73  8.88E-12    x  x  Sorl1  0.71  4.62E-05  9.6×  x    Mfsd2a  0.70  7.03E-08      x  Slc35g1  0.69  0.001118743    x    Elovl5  0.63  2.24E-10  5.5×  x  x  Nrarp  0.62  8.51E-08  6.7×  x    Ppargc1a  0.56  0.003203018    x    Gpr85  0.55  0.009420735    x    Mfap3l  0.54  0.000221799  7.9×  x  x  Hccs  0.54  4.50E-06    x  x  Kcnj10  0.48  0.00044663    x    Sgk1  0.44  0.003516857    x    Tbcel  0.44  0.000414875      x  Nudt18  0.44  0.023379335      x  Pmp22  0.31  0.016288763    x  x  Associated Gene Name  log2 Fold Change 24-h T3  FDR  Astrocyte Enrichment  In Vivo Regulation  Contains TRBS  Dio3  5.09  2.83E-81    x    Hr  3.72  9.40E-259    x  x  Klf9  1.49  4.25E-27    x  x  Gpr30  1.11  4.91E-20        Gbp3  1.10  2.92E-15    x    Morc4  1.09  1.39E-17      x  Bcar3  1.02  3.61E-25    x  x  Daam2  0.92  2.30E-14  22×  x  x  F3  0.91  1.40E-17  19.3×  x  x  Dbp  0.79  3.28E-09    x  x  Kazn  0.77  1.32E-10        Trp53inp2  0.74  1.23E-12    x  x  Cldn12  0.73  8.88E-12    x  x  Sorl1  0.71  4.62E-05  9.6×  x    Mfsd2a  0.70  7.03E-08      x  Slc35g1  0.69  0.001118743    x    Elovl5  0.63  2.24E-10  5.5×  x  x  Nrarp  0.62  8.51E-08  6.7×  x    Ppargc1a  0.56  0.003203018    x    Gpr85  0.55  0.009420735    x    Mfap3l  0.54  0.000221799  7.9×  x  x  Hccs  0.54  4.50E-06    x  x  Kcnj10  0.48  0.00044663    x    Sgk1  0.44  0.003516857    x    Tbcel  0.44  0.000414875      x  Nudt18  0.44  0.023379335      x  Pmp22  0.31  0.016288763    x  x  This table lists only the genes that are regulated directly by T3 in the ASTRO and CC cultures (39), correlated with astrocyte enrichment (22), in vivo regulation (37), and the presence of TRBS. TRBS were identified using antibodies against a GS tag (protein G and streptavidin) fused to the N terminus of overexpressed T3 receptors in C17 neuronlike cells (40). View Large Factors influencing the response to T3 The comparison between the responses to T3 in the ASTRO culture with those on astrocytes present in the mixed CC culture (33) mentioned previously revealed some discrepancies. For example, Abcd2, a gene enriched 21.8-fold in astrocytes and subject of recent interest as a target of thyroid hormone (41), was induced by T3 in the CC culture and not in the ASTRO culture (Fig. 2a). Overall, there was only partial overlap in the T3 responses between each culture. This was evident not only in the total number of genes regulated, but also in the number of astrocyte-enriched genes responding to T3, including primary T3 responses. Thus, as shown in Fig. 2b, of 136 astrocyte-enriched genes regulated in any of the cultures, only 32 (6 regulated directly and 16 indirectly) were sensitive to T3 in both cultures. This observation prompted us to analyze possible causes for the discrepancy because they may shed light on the factors influencing the T3 responses. Figure 2. View largeDownload slide Comparison between the effects of T3 on ASTRO and CC cultures. (a) The astrocyte-enriched gene Abcd2 is regulated by T3 in the CC culture but not in the ASTRO culture. (b) Comparison between the numbers of astrocyte-enriched genes regulated by T3 in ASTRO cells and mixed CC cells. Shown is the number of genes with expression changes 24 hours after T3 in each culture and, common to both, the number of genes enriched at least fivefold in astrocytes within this set and the number of directly regulated astrocyte-enriched genes. (c) The astrocyte maturation marker Ranbp3l is highly expressed in the ASTRO but not the CC culture. Data are mean ± standard error of fold change after T3 addition of four samples for each condition. *P = 0.0286 by Mann-Whitney test. NS, not significant. Figure 2. View largeDownload slide Comparison between the effects of T3 on ASTRO and CC cultures. (a) The astrocyte-enriched gene Abcd2 is regulated by T3 in the CC culture but not in the ASTRO culture. (b) Comparison between the numbers of astrocyte-enriched genes regulated by T3 in ASTRO cells and mixed CC cells. Shown is the number of genes with expression changes 24 hours after T3 in each culture and, common to both, the number of genes enriched at least fivefold in astrocytes within this set and the number of directly regulated astrocyte-enriched genes. (c) The astrocyte maturation marker Ranbp3l is highly expressed in the ASTRO but not the CC culture. Data are mean ± standard error of fold change after T3 addition of four samples for each condition. *P = 0.0286 by Mann-Whitney test. NS, not significant. We first considered that different maturational degrees of the astrocytes might affect the T3 response, as the CC culture was derived from the embryonic cortex and the ASTRO culture from the postnatal cortex. From a set of 500 astrocyte-enriched genes, 392 genes were expressed in both cultures. Thirty-six genes were expressed in the ASTRO culture and not in the CC culture, and within this set, there were genes enriched >80-fold in mature over fetal astrocytes, such as Gjb6, Apoc1, and Ranbp3l (42). Ranbp3l was indeed confirmed by qPCR to be more abundantly expressed in the ASTRO culture (Fig. 2c) than in the CC culture. To analyze if the T3 response is influenced by the maturation stage, we looked at the relative enrichment of the T3-responsive genes in mature vs fetal astrocytes. In this analysis, we considered only genes enriched in astrocytes (more than fivefold enrichment over the rest of the neural cell types) that were regulated by T3 in the presence of CHX and therefore likely directly regulated genes. Table 4 shows the list of astrocyte genes from Fig. 2b regulated directly by T3 only in the ASTRO cells (7 genes) or in the CC cells (18 genes) and in both (6 genes). Four of the six genes responding to T3 in both cultures (Sorl1, Mfap3l, Daam2, and F3) were enriched in the mature vs the fetal brain, which might indicate that T3 is needed under physiological conditions for the mature-type expression of these genes. Accordingly, of the 18 genes regulated only in the CC culture, 8 also showed higher expression in the mature brain (Slc1a2, Gpam, Aqp4, Prss35, Dio2, Dtna, Abcd2, and Gabrg1). Table 4. Regulation of Astrocyte Genes by T3 in Primary Cells in Relation to Astrocyte Maturity and the Presence of Neurons in the Culture Postnatal ASTRO Cells  Embryonic CC Cells  Astrocyte Enrichment  Fold Change Mature/Fetal  Fold Change Monoculture/Coculture  Sorl1  Sorl1  9.6  13    Mfap3l  Mfap3l  7.9  17    Nrarp  Nrarp  6.7    −9.51  Daam2  Daam2  22.0  7    F3  F3  19.3  5    Elovl5  Elovl5  5.5        Wwc1  10.1        Rgma  8.2        Megf10  16.0        Slc1a2  17.3  58  −10.8    Gpam  14.6  5      Aqp4  70.4  32      Prss35  7.8  9  −5.9    Dio2  31.3  10  −6.5    Dtna  12.4  6      Abcd2  21.8  9      Gabbr2  6.5  −7      Rasl11b  6.8        Btbd17  14.9        Arhgef26  14.0        Gpc6  9.0  −25      Gabrg1  12.0  27      Sybu  5.3        Grm5  8.2      Baalc    8.3  31    Adamts5    6.5      Wnt7a    8.5      Bdnf    10.4      Gli2    67.8  −6    Fndc5    6.3      Stk17b    5.1      Postnatal ASTRO Cells  Embryonic CC Cells  Astrocyte Enrichment  Fold Change Mature/Fetal  Fold Change Monoculture/Coculture  Sorl1  Sorl1  9.6  13    Mfap3l  Mfap3l  7.9  17    Nrarp  Nrarp  6.7    −9.51  Daam2  Daam2  22.0  7    F3  F3  19.3  5    Elovl5  Elovl5  5.5        Wwc1  10.1        Rgma  8.2        Megf10  16.0        Slc1a2  17.3  58  −10.8    Gpam  14.6  5      Aqp4  70.4  32      Prss35  7.8  9  −5.9    Dio2  31.3  10  −6.5    Dtna  12.4  6      Abcd2  21.8  9      Gabbr2  6.5  −7      Rasl11b  6.8        Btbd17  14.9        Arhgef26  14.0        Gpc6  9.0  −25      Gabrg1  12.0  27      Sybu  5.3        Grm5  8.2      Baalc    8.3  31    Adamts5    6.5      Wnt7a    8.5      Bdnf    10.4      Gli2    67.8  −6    Fndc5    6.3      Stk17b    5.1      This table shows that there is only partial overlap in the astrocyte-enriched genes (enrichment factor more than fivefold) regulated directly by T3 in astrocytes from the postnatal cortex (ASTRO cells) and in a mixed neuron-astrocyte culture from the embryonic cortex. Data on enrichment in mature cortex compared with fetal cortex (fold change mature/fetal) are from Zhang et al. (42). Data on differences between the astrocyte monoculture and coculture with neurons are from Gil-Ibáñez et al. (33). More genes are regulated transcriptionally by T3 in the CC culture than in the ASTRO culture, and lack of sensitivity to T3 in the ASTRO culture appears to be related to gene enrichment in the mature brain. View Large Table 4. Regulation of Astrocyte Genes by T3 in Primary Cells in Relation to Astrocyte Maturity and the Presence of Neurons in the Culture Postnatal ASTRO Cells  Embryonic CC Cells  Astrocyte Enrichment  Fold Change Mature/Fetal  Fold Change Monoculture/Coculture  Sorl1  Sorl1  9.6  13    Mfap3l  Mfap3l  7.9  17    Nrarp  Nrarp  6.7    −9.51  Daam2  Daam2  22.0  7    F3  F3  19.3  5    Elovl5  Elovl5  5.5        Wwc1  10.1        Rgma  8.2        Megf10  16.0        Slc1a2  17.3  58  −10.8    Gpam  14.6  5      Aqp4  70.4  32      Prss35  7.8  9  −5.9    Dio2  31.3  10  −6.5    Dtna  12.4  6      Abcd2  21.8  9      Gabbr2  6.5  −7      Rasl11b  6.8        Btbd17  14.9        Arhgef26  14.0        Gpc6  9.0  −25      Gabrg1  12.0  27      Sybu  5.3        Grm5  8.2      Baalc    8.3  31    Adamts5    6.5      Wnt7a    8.5      Bdnf    10.4      Gli2    67.8  −6    Fndc5    6.3      Stk17b    5.1      Postnatal ASTRO Cells  Embryonic CC Cells  Astrocyte Enrichment  Fold Change Mature/Fetal  Fold Change Monoculture/Coculture  Sorl1  Sorl1  9.6  13    Mfap3l  Mfap3l  7.9  17    Nrarp  Nrarp  6.7    −9.51  Daam2  Daam2  22.0  7    F3  F3  19.3  5    Elovl5  Elovl5  5.5        Wwc1  10.1        Rgma  8.2        Megf10  16.0        Slc1a2  17.3  58  −10.8    Gpam  14.6  5      Aqp4  70.4  32      Prss35  7.8  9  −5.9    Dio2  31.3  10  −6.5    Dtna  12.4  6      Abcd2  21.8  9      Gabbr2  6.5  −7      Rasl11b  6.8        Btbd17  14.9        Arhgef26  14.0        Gpc6  9.0  −25      Gabrg1  12.0  27      Sybu  5.3        Grm5  8.2      Baalc    8.3  31    Adamts5    6.5      Wnt7a    8.5      Bdnf    10.4      Gli2    67.8  −6    Fndc5    6.3      Stk17b    5.1      This table shows that there is only partial overlap in the astrocyte-enriched genes (enrichment factor more than fivefold) regulated directly by T3 in astrocytes from the postnatal cortex (ASTRO cells) and in a mixed neuron-astrocyte culture from the embryonic cortex. Data on enrichment in mature cortex compared with fetal cortex (fold change mature/fetal) are from Zhang et al. (42). Data on differences between the astrocyte monoculture and coculture with neurons are from Gil-Ibáñez et al. (33). More genes are regulated transcriptionally by T3 in the CC culture than in the ASTRO culture, and lack of sensitivity to T3 in the ASTRO culture appears to be related to gene enrichment in the mature brain. View Large Differences between the response of astrocytes to T3 in the ASTRO culture and in the CC cells might be also due to the absence or presence of neurons. It is known that gene expression in astrocytes is influenced by the presence of neurons, and the morphology of astrocytes in the ASTRO culture was as described for astrocytes cultured in the absence of neurons (34, 35). To explore the possibility that the absence of neurons might have modified the astrocyte response to T3, we looked at how the astrocyte-enriched genes regulated directly by T3 (Fig. 2b; Table 4) were influenced by coculture with neurons using the datasets from Hasel et al. (34). Out of the six genes regulated by T3 in CC and ASTRO cells, only Nrarp showed changes of expression between the astrocyte monoculture and coculture. From the 18 genes regulated by T3 only in CC cells, 3 genes, Slc1a2, Prss35, and Dio2, showed similar changes. The seven genes regulated only in the ASTRO culture were not influenced by the culture conditions. These results indicated that the culture conditions might have had influence on some genes, but differences in the response between the two types of astrocytes appeared to correlate better with gene enrichment in the mature brain. The meaning of this is that T3 facilitates the transition between the immature to the mature state, but the T3-responsive genes in the transition become refractory to the hormone once maturity is reached, similar to the regulation of myelin genes (43, 44). Discussion Astrocytes carry out important functions in the central nervous system and mediate the actions of thyroid hormones in the control of their metabolism. Dio2, the enzyme responsible for the conversion of T4 to T3 through 5′ deiodination, is highly enriched in astrocytes relative to the rest of neural cells. During maturation, most T3 available to the developing brain is generated locally in the astrocytes from circulating T4, and this pathway compensates for the decrease of T4 availability taking place in hypothyroidism, iodine deficiency, and other conditions (8, 45). Astrocytes are also cellular targets of T3. As shown in this study, they express relevant genes involved in thyroid hormone transport, metabolism, and action, and the addition of T3 to primary astrocytes results in changes of expression of 4.5% of the astrocyte transcriptome. Using the protein synthesis inhibitor CHX to block secondary T3 responses, we conclude that in at least 17.5% of all of the genes with expression changes 24 hours after T3 addition, the action of the hormone did not require protein synthesis and therefore was likely at the transcriptional level. Unexpectedly, all directly regulated genes except one, Trim47, were upregulated by T3. In contrast, using the same criteria in primary CC cells (3), the number of downregulated genes was 52% of the set of directly regulated genes. Trim47 contains TRBS (40), supporting a direct transcriptional effect of T3 through the thyroid hormone receptor. According to pathways analysis, there was an overrepresentation of the Notch and Wnt pathways in the set of genes downregulated by T3. The significance of this finding is not clear to us. Hypothyroidism delays developmental changes of Notch expression in the cerebellum (9). Two Notch ligands (Dll1 and Dll3), one receptor (Notch3), and two target genes (Hes5 and Hey2) were downregulated, though indirectly by T3. In contrast, another target, Hey1, was directly upregulated. Nine genes related to the Wnt pathways were also indirectly downregulated by T3, whereas two were directly upregulated. Despite the positive transcriptional effect of T3 on some discrete components of these pathways, the overall effect of T3 was inhibitory. As these pathways are intricately interrelated, it is difficult to formulate a hypothesis on the primary effect of T3 leading to the secondary changes in expression of Notch and Wnt pathway genes. Pathways analysis in CC cells (3) did not disclose a significant effect of T3 on these pathways, although again, T3 had a transcriptional effect on isolated components of these pathways such as Pygo1, Smarca1, Mycn, Map3k5, and the Notch effector Nrp1. The latter is downregulated by T3 and also by T4 in Thra1-expressing N2a neuroblastoma cells (46). The presence or absence of neurons in the culture greatly modifies astrocyte phenotype and gene expression (34, 35). In particular, neurons maintain a mature astrocyte phenotype through the activation of Notch pathways (34). Coculture of astrocytes with neurons upregulates Hes5 and Hey2, two genes that in the present work are downregulated by T3. Hasel et al. (34) concluded that stimulation of Notch signaling by neurons was necessary for the induction of Slc1a2 in astrocytes and thus glutamate uptake. We found that Slc1a2 expression was not influenced by T3 in the astrocytes, despite the fact that it contains a TRBS and was transcriptionally upregulated by T3 in CC culture (3, 40). The lack of effect of T3 on this gene in the ASTRO culture could, therefore, be due to the absence of neurons. However, Slc1a2 is highly expressed in mature astrocytes over fetal astrocytes (Table 4), in agreement with the proposed effect of T3 on maturation-dependent genes (3). The astrocyte culture of the present work was prepared from the postnatal cortex and appears to be composed of cells at a higher maturity stage than in the CC culture, as indicated by expression of maturity markers such as Ranbp3l. Differences in expression between the CC and ASTRO culture in what concerns astrocyte-enriched genes most probably reflect the developmental effect of thyroid hormone driving the expression of genes that will be enriched in mature cells and then become refractory, as it is well known for the myelin genes, as noted previously. However, we do not discard the possibility that the response of astrocytes to T3, including the Notch pathway, is influenced by the presence or absence of neurons. Actually, within the set of genes induced by astrocyte-neuron coculture are the thyroid hormone transporter Slco1c1 and Dio2 (34), indicating that the effects of neurons and T3 on the astrocytes are interrelated. Finally, another factor that may modify the response to T3 is the astrocyte type. Astrocytes are classified as types 1 and 2 based on expression of specific markers (47). Type 1 astrocytes predominate in the upper layers of the cortex in contact with the pial surface and express Gfap and Aqp4, whereas type 2 astrocytes are present in the rest of the layers and express Mfge8. In CC culture, T3 upregulates Gfap and Aqp4, the latter at the transcriptional level, whereas in the ASTRO culture, T3 downregulates Mfge8. These results suggest that type 1 astrocytes are responsive to T3 in the CC culture, whereas in the ASTRO culture, the response of the type 2 astrocytes is more relevant. Conclusion In addition to playing a fundamental role in thyroid hormone metabolism with the generation of local T3 from T4 in the brain, astrocytes are sensitive genomic targets of T3. One hundred seventeen genes, ∼0.8% of the astrocytic transcriptome, were transcriptionally regulated by T3. The response to T3 appears to be influenced by the degree of astrocyte maturation, and, in general, T3 facilitates the transition between fetal and adult patterns of gene expression. Abbreviations: Abbreviations: ASTRO primary astrocyte CC cerebrocortical cDNA complementary DNA CHX cycloheximide Dio2 type 2 deiodinase DMEM Dulbecco’s modified Eagle medium FDR false discovery rate GFAP glial fibrillary protein GO gene ontology NeuN neuronal-specific nuclear protein PCR polymerase chain reaction qPCR quantitative polymerase chain reaction RNA-Seq RNA sequencing RPKM reads per kilobase of transcript per million mapped reads RRID Research Resource Identifier T3 triiodothyronine T4 thyroxine TRBS triiodothyronine receptor binding sites Acknowledgments Financial Support: This work was supported by the Center for Biomedical Research on Rare Diseases under the framework of E-Rare-2, the ERA-Net for Research on Rare Diseases (project acronym THYRONERVE); Grant SAF2014-54919-R from the Plan Nacional de I+D+I of Ministry of Economy and Competitivity of Spain; and by the European Regional Funds. Author Contributions: B.M. performed the analysis of the RNA-Seq data. 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TI - Regulation of Gene Expression by Thyroid Hormone in Primary Astrocytes: Factors Influencing the Genomic Response
JO - Endocrinology
DO - 10.1210/en.2017-03084
DA - 2018-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/regulation-of-gene-expression-by-thyroid-hormone-in-primary-astrocytes-W8eshvbBBS
SP - 2083
EP - 2092
VL - 159
IS - 5
DP - DeepDyve
ER -