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Identification of a unique TGF-β-dependent molecular and functional signature in microglia.

Butovsky O, Jedrychowski MP, Moore CS, Cialic R, Lanser AJ, Gabriely G, Koeglsperger T, Dake B, Wu PM, Doykan CE, Fanek Z, Liu L, Chen Z, Rothstein JD, Ransohoff RM, Gygi SP, Antel JP, Weiner HL - Nat. Neurosci. (2013)

Bottom Line: Of the 239 genes, 106 were enriched in microglia as compared with astrocytes, oligodendrocytes and neurons.We found that TGF-β was required for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia and that microglia were absent in the CNS of TGF-β1-deficient mice.Our results identify a unique microglial signature that is dependent on TGF-β signaling and provide insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Microglia are myeloid cells of the CNS that participate both in normal CNS function and in disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia versus myeloid and other immune cells. Of the 239 genes, 106 were enriched in microglia as compared with astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS, and was also observed in human microglia. We found that TGF-β was required for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia and that microglia were absent in the CNS of TGF-β1-deficient mice. Our results identify a unique microglial signature that is dependent on TGF-β signaling and provide insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.

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MG400 profile in microglia vs. astrocytes, oligodendrocytes and neurons(a) Dendogram of unsupervised hierarchical clustering (Pearson correlation; average linkage) of biological duplicates for FCRLS+ adult microglia (n = 5 mice), Glt-EGFP+ adult astrocytes (n = 9 mice), adult oligodendrocytes (n = 5 mice) and primary postnatal hippocampal and cortical neurons (see Source data – Figure 2). Individual cell types are identified as follows: green, oligodendrocytes; orange, astrocytes; cyan, cortical neurons; dark-blue, hippocampal neurons and red, microglia. (b) Heatmap of top 25 enriched genes in each cell type based on hierarchical clustering. Each lane represents the average expression value of two biological duplicates per cell type. (c) Principle component analysis based on MG400 expression for CNS cells. (d) MG400 profile of detected genes (>100 mRNA transcripts) in microglia, astrocytes, oligodendrocytes and neurons. Venn diagram displays unique and intersecting genes among cell types. (e) Correspondence analysis of samples (large spheres) and genes (small spheres). (f) qPCR analysis of CNS cell type specific genes for each population. (g) qPCR analysis of microglial unique genes (P2ry12, Fcrls, Tmem119, Olfml3, Hexb and Tgfbr1) as compared to spleen red pulp macrophages and CNS cell types. Expression levels were normalized to Gapdh (n = 3). Bars show mean ± s.e.m. Shown is one of two individual experiments.
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Figure 2: MG400 profile in microglia vs. astrocytes, oligodendrocytes and neurons(a) Dendogram of unsupervised hierarchical clustering (Pearson correlation; average linkage) of biological duplicates for FCRLS+ adult microglia (n = 5 mice), Glt-EGFP+ adult astrocytes (n = 9 mice), adult oligodendrocytes (n = 5 mice) and primary postnatal hippocampal and cortical neurons (see Source data – Figure 2). Individual cell types are identified as follows: green, oligodendrocytes; orange, astrocytes; cyan, cortical neurons; dark-blue, hippocampal neurons and red, microglia. (b) Heatmap of top 25 enriched genes in each cell type based on hierarchical clustering. Each lane represents the average expression value of two biological duplicates per cell type. (c) Principle component analysis based on MG400 expression for CNS cells. (d) MG400 profile of detected genes (>100 mRNA transcripts) in microglia, astrocytes, oligodendrocytes and neurons. Venn diagram displays unique and intersecting genes among cell types. (e) Correspondence analysis of samples (large spheres) and genes (small spheres). (f) qPCR analysis of CNS cell type specific genes for each population. (g) qPCR analysis of microglial unique genes (P2ry12, Fcrls, Tmem119, Olfml3, Hexb and Tgfbr1) as compared to spleen red pulp macrophages and CNS cell types. Expression levels were normalized to Gapdh (n = 3). Bars show mean ± s.e.m. Shown is one of two individual experiments.

Mentions: Next, we investigated whether the microglial genes we identified were expressed in CNS cells including astrocytes, oligodendrocytes and neurons by profiling these CNS cells with the MG400 microglial chip (Source data Fig. 2). A dendogram based on unsupervised hierarchical clustering of CNS cell types demonstrates that microglia are different from CNS cells (Fig. 2a). Fig. 2b shows a heatmap of the 25-top specific genes in microglia and the other CNS cells (Supplementary Table 1). Principal component analysis (PCA) based on the MG400 gene expression profile shows that microglia are different than other CNS cells (Fig. 2c). We compared the identified molecular signature of detected genes across all four populations and found 106 genes which were specifically expressed in microglia (Fig. 2d). Correspondence analysis (COA) shows the 106 microglial specific genes (Source data Fig. 2). We highlight six highly expressed microglial genes in the COA plot: Fcrls, Olfml3, Tmeme119, P2ry12, Hexb and Tgfbr1 (Fig. 2e). The purity of CNS cells types was established by qPCR analysis using their known unique markers (Fig. 2f). qPCR analysis demonstrated that the microglia unique genes Fcrls, Olfml3, Tmeme119, P2ry12, Hexb and Tgfbr1 were absent in oligodendrocytes, astrocytes and neurons (Fig. 2g).


Identification of a unique TGF-β-dependent molecular and functional signature in microglia.

Butovsky O, Jedrychowski MP, Moore CS, Cialic R, Lanser AJ, Gabriely G, Koeglsperger T, Dake B, Wu PM, Doykan CE, Fanek Z, Liu L, Chen Z, Rothstein JD, Ransohoff RM, Gygi SP, Antel JP, Weiner HL - Nat. Neurosci. (2013)

MG400 profile in microglia vs. astrocytes, oligodendrocytes and neurons(a) Dendogram of unsupervised hierarchical clustering (Pearson correlation; average linkage) of biological duplicates for FCRLS+ adult microglia (n = 5 mice), Glt-EGFP+ adult astrocytes (n = 9 mice), adult oligodendrocytes (n = 5 mice) and primary postnatal hippocampal and cortical neurons (see Source data – Figure 2). Individual cell types are identified as follows: green, oligodendrocytes; orange, astrocytes; cyan, cortical neurons; dark-blue, hippocampal neurons and red, microglia. (b) Heatmap of top 25 enriched genes in each cell type based on hierarchical clustering. Each lane represents the average expression value of two biological duplicates per cell type. (c) Principle component analysis based on MG400 expression for CNS cells. (d) MG400 profile of detected genes (>100 mRNA transcripts) in microglia, astrocytes, oligodendrocytes and neurons. Venn diagram displays unique and intersecting genes among cell types. (e) Correspondence analysis of samples (large spheres) and genes (small spheres). (f) qPCR analysis of CNS cell type specific genes for each population. (g) qPCR analysis of microglial unique genes (P2ry12, Fcrls, Tmem119, Olfml3, Hexb and Tgfbr1) as compared to spleen red pulp macrophages and CNS cell types. Expression levels were normalized to Gapdh (n = 3). Bars show mean ± s.e.m. Shown is one of two individual experiments.
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Figure 2: MG400 profile in microglia vs. astrocytes, oligodendrocytes and neurons(a) Dendogram of unsupervised hierarchical clustering (Pearson correlation; average linkage) of biological duplicates for FCRLS+ adult microglia (n = 5 mice), Glt-EGFP+ adult astrocytes (n = 9 mice), adult oligodendrocytes (n = 5 mice) and primary postnatal hippocampal and cortical neurons (see Source data – Figure 2). Individual cell types are identified as follows: green, oligodendrocytes; orange, astrocytes; cyan, cortical neurons; dark-blue, hippocampal neurons and red, microglia. (b) Heatmap of top 25 enriched genes in each cell type based on hierarchical clustering. Each lane represents the average expression value of two biological duplicates per cell type. (c) Principle component analysis based on MG400 expression for CNS cells. (d) MG400 profile of detected genes (>100 mRNA transcripts) in microglia, astrocytes, oligodendrocytes and neurons. Venn diagram displays unique and intersecting genes among cell types. (e) Correspondence analysis of samples (large spheres) and genes (small spheres). (f) qPCR analysis of CNS cell type specific genes for each population. (g) qPCR analysis of microglial unique genes (P2ry12, Fcrls, Tmem119, Olfml3, Hexb and Tgfbr1) as compared to spleen red pulp macrophages and CNS cell types. Expression levels were normalized to Gapdh (n = 3). Bars show mean ± s.e.m. Shown is one of two individual experiments.
Mentions: Next, we investigated whether the microglial genes we identified were expressed in CNS cells including astrocytes, oligodendrocytes and neurons by profiling these CNS cells with the MG400 microglial chip (Source data Fig. 2). A dendogram based on unsupervised hierarchical clustering of CNS cell types demonstrates that microglia are different from CNS cells (Fig. 2a). Fig. 2b shows a heatmap of the 25-top specific genes in microglia and the other CNS cells (Supplementary Table 1). Principal component analysis (PCA) based on the MG400 gene expression profile shows that microglia are different than other CNS cells (Fig. 2c). We compared the identified molecular signature of detected genes across all four populations and found 106 genes which were specifically expressed in microglia (Fig. 2d). Correspondence analysis (COA) shows the 106 microglial specific genes (Source data Fig. 2). We highlight six highly expressed microglial genes in the COA plot: Fcrls, Olfml3, Tmeme119, P2ry12, Hexb and Tgfbr1 (Fig. 2e). The purity of CNS cells types was established by qPCR analysis using their known unique markers (Fig. 2f). qPCR analysis demonstrated that the microglia unique genes Fcrls, Olfml3, Tmeme119, P2ry12, Hexb and Tgfbr1 were absent in oligodendrocytes, astrocytes and neurons (Fig. 2g).

Bottom Line: Of the 239 genes, 106 were enriched in microglia as compared with astrocytes, oligodendrocytes and neurons.We found that TGF-β was required for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia and that microglia were absent in the CNS of TGF-β1-deficient mice.Our results identify a unique microglial signature that is dependent on TGF-β signaling and provide insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Microglia are myeloid cells of the CNS that participate both in normal CNS function and in disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia versus myeloid and other immune cells. Of the 239 genes, 106 were enriched in microglia as compared with astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS, and was also observed in human microglia. We found that TGF-β was required for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia and that microglia were absent in the CNS of TGF-β1-deficient mice. Our results identify a unique microglial signature that is dependent on TGF-β signaling and provide insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.

Show MeSH
Related in: MedlinePlus