Journal: Nature Communications

Article Title: Astroblastomas exhibit radial glia stem cell lineages and differential expression of imprinted and X-inactivation escape genes

doi: 10.1038/s41467-022-29302-8

Figure Lengend Snippet: a Classic astroblastic pseudorossette. b Neural stem cell morphologies. NEC, neuroepithelial cell; vRG, ventricular zone radial glia cell; oRG, outer radial glia cell; tRG, truncated radial glia cell; VZ, ventricular zone; SVZ, subventricular zone; OSVZ, outer subventricular zone. c Unsupervised hierarchical clustering of RNAseq expression data separates AB-like tumors into MN1- rearranged and MAPK/PI3K pathway transcriptomic groups. Genes overexpressed in MN1-BEND2 tumors are highlighted in red, those overexpressed in MAPK-ABC tumors in blue and PTPR in bronze. Some genes highly expressed in both MN1-BEND2 tumors and PTPR are depicted in red. Abbreviations: AFL, adult frontal lobe; AAM, adult amygdala; ACP, adult choroid plexus; ATH, adult thalamus; FCE, fetal cerebrum; FCM, fetal cerebellum; FCG, fetal germinal matrix; FHP, fetal hippocampus; PCM, pediatric cerebellum, PHP, pediatric hippocampus; PTH, pediatric thalamus. d tSNE analysis of RNAseq expression data. e Hierarchical clustering and f tSNE analysis of Affymetrix HTA2 RNA expression data. Classical PXA tumors cluster separately from MAPK-ABC AB-like tumors demonstrating that they are separate biologic entities. Source data: Supplementary Fig. , Gene Expression Omnibus (GEO) accession numbers GSE165351 , GSE165813 .

Article Snippet: Immunohistochemical stains for GFAP clone EP672Y (Cell Marque 258R-16, lot 32653, 1:25), MN1 (Proteintech 24697-1-AP, lot 00021048, 1:40) and IGF2 (Abcam; ab9574, lots GR31975-61, and GR31975-64, 1:500) were performed on the Leica Bond III using Leica Epitope Retrieval 1 (Leica; AR9961) for GFAP, and Epitope Retrieval 2 (Leica; AR9640) for MN1 and IGF2.

Techniques: Expressing, RNA Expression, Gene Expression

Journal: Nature Communications

Article Title: Astroblastomas exhibit radial glia stem cell lineages and differential expression of imprinted and X-inactivation escape genes

doi: 10.1038/s41467-022-29302-8

Figure Lengend Snippet: Patient demographic and tumor pathology data.

Article Snippet: Immunohistochemical stains for GFAP clone EP672Y (Cell Marque 258R-16, lot 32653, 1:25), MN1 (Proteintech 24697-1-AP, lot 00021048, 1:40) and IGF2 (Abcam; ab9574, lots GR31975-61, and GR31975-64, 1:500) were performed on the Leica Bond III using Leica Epitope Retrieval 1 (Leica; AR9961) for GFAP, and Epitope Retrieval 2 (Leica; AR9640) for MN1 and IGF2.

Techniques:

Journal: Nature Communications

Article Title: Astroblastomas exhibit radial glia stem cell lineages and differential expression of imprinted and X-inactivation escape genes

doi: 10.1038/s41467-022-29302-8

Figure Lengend Snippet: a AB tumor histology is characterized by astroblastic pseudorosettes , and oRG-like morphology in Group C tumors. a – i H&E-stained AB-like tumor cases. Top row MN1-BEND2 cases; Middle row BRAF V600E - mutant MAPK-A cases ; Bottom row Group C methylation class tumor cases. Subpial accumulations of tumor cells in C22 ( g ) demonstrated long basal processes extending to the pial surface and inconspicuous apical processes, morphologically resembling outer radial glial . Similar cells extend basal processes to blood vessels in C27 and C19 ( h – i ). b Astroblastoma tumors cells within astroblastic pseudorosettes from Bailey and Bucy’s original cohort. a , b Tumor cells with long basal processes resembling those in Group C tumors (Plate III, Figs. . and ). c Tumor cells from a case more similar in appearance to those in MAPK-AB tumors (Plate IV, Fig. ). Mallory-Davidoff stain ( a , b 850× and c , 600×) . Reproduced with the publisher’s permission. c MN1-BEND2 and MAPK-ABC AB-like tumors show characteristic GFAP immunoreactivity. a – c Only focal staining for GFAP is observed in MN1-BEND2 tumors. d – f MAPK-ABC tumors are strongly and diffusely GFAP immunoreactive. d MN1 diffusely highlights tumor nuclei in all MN1-BEND2 tumors tested ( a – c ). Significant nuclear MN1 immunoreactivity is absent in non- MN1-BEND2 AB cases ( d – f ). e MN1-BEND2 tumors showed strong granular cytoplasmic IGF2 immunostaining ( a – c ), not seen in non- MN1-BEND2 AB-like tumors ( d – f ). Immunohistochemical staining for all antibodies and samples was performed at least twice with equivalent results. f , g Mutations in AB-like tumor related genes occur in a patient age dependent manner. ABCC1 mutations and fusions all occurred in patients 16 years old or less. Its highest expression in the Allen Human Developmental Transcriptome (AHDT) was before 25 pcw. VEZT and VEZF mutations and CTD-2152M20.2-GOLPH3 fusions were all found in tumors from patients aged 25 and younger (Supplementary Data ). The highest GOLPH3 , VEZT and VEZF1 expression in the AHDT was under 25 pcw. MEG8 fusions occurred only in females 30 years of age and younger, most below 16 years, and only in MN1-BEND2 and MAPK-A tumors. GRIA2 mutations were present in MAPK-ABC cases. ANK3 , PARP8 and PTEN alterations were mostly present in non- BRAF V600E MAPK-AB patients. A PARP8 fusion was present in a BRAF V600E tumor. Conversely, high GRIA2 expression spans the fetal and postnatal AHDT, but not below approximately 12 pcw. GRIA2 mutations are absent in MN1 -rearranged tumors. Some genetic lesions were relatively specific to AB-like tumor genetic types: MN1-BEND2 fusions and ABCC1 mutation or fusion in MN1 -rearranged tumors, and TRIO and KALRN fusions in BRAF V600E tumors. SON mutations span the gamut of patient ages, as does its expression in the AHDT. These presumed somatic mutations were found in tumors clinically presenting at the indicated ages, however this data only indicates that the mutation was acquired in a patient sometime prior to that age, and some could have indeed been germline. Colors are arbitrary in f . In g MN1-BEND2 tumor mutated genes are in red and MAPK-ABC tumor mutated genes are in blue. Source data: Supplementary Data and .

Article Snippet: Immunohistochemical stains for GFAP clone EP672Y (Cell Marque 258R-16, lot 32653, 1:25), MN1 (Proteintech 24697-1-AP, lot 00021048, 1:40) and IGF2 (Abcam; ab9574, lots GR31975-61, and GR31975-64, 1:500) were performed on the Leica Bond III using Leica Epitope Retrieval 1 (Leica; AR9961) for GFAP, and Epitope Retrieval 2 (Leica; AR9640) for MN1 and IGF2.

Techniques: Staining, Mutagenesis, Methylation, Immunostaining, Immunohistochemical staining, Expressing

Journal: Nature Communications

Article Title: Astroblastomas exhibit radial glia stem cell lineages and differential expression of imprinted and X-inactivation escape genes

doi: 10.1038/s41467-022-29302-8

Figure Lengend Snippet: The developmental expression time course of NSC markers and select genes overexpressed or mutated in MN1-BEND2 and MAPK-ABC AB-like tumors was obtained from the Allen Human Developmental Transcriptome database and subjected to unsupervised hierarchical clustering. MN1-BEND2 tumor-associated genes (relatively overexpressed or mutated) are depicted in red and MAPK-ABC tumor associated genes are in blue. Genes associated with NEC, vRG, tRG and oRG are indicated. The x-axis age bar progressive color scheme is arbitrary. Transcript expression is normalized by reads per kilobase of transcript per million mapped reads (RPKM) to compensate for RNAseq generation of more sequencing reads from longer RNA molecules. Data is from up to 16 brain regions from 42 specimens (Allen Human Brain Atlas Developmental Transcriptome, Institute for Brain Science, available from: human.brain-map.org). The figure source data is available in the Source Data File.

Article Snippet: Immunohistochemical stains for GFAP clone EP672Y (Cell Marque 258R-16, lot 32653, 1:25), MN1 (Proteintech 24697-1-AP, lot 00021048, 1:40) and IGF2 (Abcam; ab9574, lots GR31975-61, and GR31975-64, 1:500) were performed on the Leica Bond III using Leica Epitope Retrieval 1 (Leica; AR9961) for GFAP, and Epitope Retrieval 2 (Leica; AR9640) for MN1 and IGF2.

Techniques: Expressing, Sequencing

Journal: Nature Communications

Article Title: Astroblastomas exhibit radial glia stem cell lineages and differential expression of imprinted and X-inactivation escape genes

doi: 10.1038/s41467-022-29302-8

Figure Lengend Snippet: a – g Immunofluorescence stained embryonic (E16), newborn (P0) and postnatal day 10 (P10) mouse brain coronal sections are shown. a , c , d and f In E16 and P10 coronal sections MN1 and IGF2 staining is not apparent. b IGF2 staining is seen in the ventricular zone of the medial and lateral ventricular walls in P0 animal brains (arrows). e MN1 labels immature ependymal cells in the P0 lateral ventricular wall covering the striatum on the right (arrows). The medial wall ependyma are mature and demonstrate multiple centrioles (red) and motile cilia (blue). MN1 staining is not evident in these cells. g High power image of P0 lateral ventricular wall showing green MN1 staining of immature ependymal cell nuclei and cytoplasm (white arrows) and more basally located striatal cells (arrowheads), which could explain the cortical or juxtacortical location of MN1-BEND2 tumors. The pink arrow indicates a more mature differentiating ependymal cell demonstrating multiple centrioles (pink fluorescence). h , i P0 whole mounts of the lateral ventricular wall show IGF2-positive microglia situated on the ependymal surface (arrows). An IGF2-positive, late differentiating ependymal cell with short multiple cilia is indicated by the arrowhead. Earlier stage differentiating ependymal cells with focal IGF2 staining are indicated by yellow arrows. Red circles highlight very early “halo stage” differentiating ependymal cells ( i , l ), which are IGF2-negative ( i ). j , k In P6 whole mount lateral wall, IGF2 again stains microglia (arrows) and immature ependyma with short cilia (arrowheads). l , m Mostly unciliated differentiating ependymal cells show MN1 staining in the P0 lateral wall (arrows). Very early halo stage nascent ependyma are MN1-negative ( l ). n , o In P6 lateral wall, differentiating ependymal cells with few cilia are MN1-positive (arrows). A later differentiating multiciliated ependymal cell is MN1-negative (arrowhead). Experiments were performed twice with equivalent results.

Article Snippet: Immunohistochemical stains for GFAP clone EP672Y (Cell Marque 258R-16, lot 32653, 1:25), MN1 (Proteintech 24697-1-AP, lot 00021048, 1:40) and IGF2 (Abcam; ab9574, lots GR31975-61, and GR31975-64, 1:500) were performed on the Leica Bond III using Leica Epitope Retrieval 1 (Leica; AR9961) for GFAP, and Epitope Retrieval 2 (Leica; AR9640) for MN1 and IGF2.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Nature Communications

Article Title: Astroblastomas exhibit radial glia stem cell lineages and differential expression of imprinted and X-inactivation escape genes

doi: 10.1038/s41467-022-29302-8

Figure Lengend Snippet: a Heatmap of unsupervised hierarchical clustering of RNAseq expression data for a subset of imprinted genes. b Similar data from the Affymetrix analysis including controls and PXA. c Heatmaps for expression of subsets of XIE genes from the Affymetrix data. IGF2 is absent in the NCBI annotation for the HTA 2.0 array as of April 2021. IGF2-AS (arrow) may serve as a surrogate marker for IGF2 and was upregulated in MN1 -rearranged tumors and normal choroid plexus (choroid plxs). The latter develops as an out-pouching of the ependyma and is known to express IGF2 . The XIST gene message is indicated by the arrowhead. MN1 -rearranged tumors are highlighted in red, MAPK-AB in blue, and MAPK-C in purple. Complete source data for heatmaps are available as Supplementary Fig. - and . d GSEA plot showing enrichment of imprinted gene expression in MN1-BEND2 versus MAPK-ABC tumors. e GSEA plot depicting enrichment of XIE gene expression in MAPK-ABC versus MN1-BEND2 tumors. GSEA is two-sided with adjustment for multiple comparisons. Tumors or control tissues comparatively enriched for neural stem cell gene sets are depicted in larger and bold font. Colors are arbitrary.

Article Snippet: Immunohistochemical stains for GFAP clone EP672Y (Cell Marque 258R-16, lot 32653, 1:25), MN1 (Proteintech 24697-1-AP, lot 00021048, 1:40) and IGF2 (Abcam; ab9574, lots GR31975-61, and GR31975-64, 1:500) were performed on the Leica Bond III using Leica Epitope Retrieval 1 (Leica; AR9961) for GFAP, and Epitope Retrieval 2 (Leica; AR9640) for MN1 and IGF2.

Techniques: Expressing, Marker, Gene Expression, Control

Journal: Nature Communications

Article Title: Astroblastomas exhibit radial glia stem cell lineages and differential expression of imprinted and X-inactivation escape genes

doi: 10.1038/s41467-022-29302-8

Figure Lengend Snippet: a Summary of anatomic locations and patient age groups of molecular subtypes of AB-like tumors. b Hypothetical model for EET MN1-BEND2 tumorigenesis. c Hypothetical model for MAPK-ABC astroblastoma tumorigenesis. GSEA and RNA expression patterns of FOXJ1 and IGF2 suggest a temporal progression of early to late vRG to tRG and oRG genes and development of related tumor types: EET MN1-BEND2, FOXJ1 +/ IGF2 +; RELA STE, FOXJ1 +/ IGF2 +; PTPR, FOXJ1 +/ IGF2 −; MAPK-ABC and PXA, FOXJ1 −/ IGF2 .

Article Snippet: Immunohistochemical stains for GFAP clone EP672Y (Cell Marque 258R-16, lot 32653, 1:25), MN1 (Proteintech 24697-1-AP, lot 00021048, 1:40) and IGF2 (Abcam; ab9574, lots GR31975-61, and GR31975-64, 1:500) were performed on the Leica Bond III using Leica Epitope Retrieval 1 (Leica; AR9961) for GFAP, and Epitope Retrieval 2 (Leica; AR9640) for MN1 and IGF2.

Techniques: RNA Expression

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Partial loss of endothelial nitric oxide leads to increased cerebrovascular beta amyloid

doi: 10.1177/0271678x18822474

Figure Lengend Snippet: Figure 4. PS1, a major g-secretase component, was significantly increased in microvascular tissue of eNOSþ/ mice. (a) Microvascular tissue from 18-month-old eNOSþ/ and wild type animals was analyzed by Western blot analyses for g-secretase components: PS1, PS2, nicastrin, Pen2, and Aph1. Representative image is shown. Densitometric analysis was performed for (b) PS1 (n ¼ 6 animals; *P < 0.05), (c) PS2 (n ¼ 4 animals), (d) nicastrin (n ¼ 6 animals), (e) Pen2 (n ¼ 6 animals), and (f) Aph1 (n ¼ 4 animals). All data are presented as meanSD with individual data points shown.

Article Snippet: Presenilin enhancer 2 (Pen2) was purchased from ProSCi (Poway, CA.).

Techniques: Western Blot