igfbp2 blocking antibody  (R&D Systems)

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a,c,e. Example images from Figure 5a,​,cc,​,ee prior to processing and analysis. Neurons cultured for 48 hours in RTT (a), FXS (c) or DS (e) ACM and immunostained with MAP2 (dendrites, green) and tau (axon, red). b,d,f. Relative frequency distribution plot of total neurite outgrowth, pooled data from 3 (b), 4 (d), 5 (f) experiments per graph, same data as Figure 5b,​,dd,​,f.f. Number of neurons: RTT (b): alone=439, WT ACM=549, RTT ACM=633, RTT ACM + Igfbp2-Ab=621; FXS (d): alone=492, WT ACM=608, FXS ACM=621, FXS ACM + Igfbp2-Ab=647; DS (f): alone=716, WT ACM=992, DS ACM=765, DS ACM + Igfbp2-Ab=858. g. Relative frequency distribution plot of neuronal cell body size, pooled data from 3 experiments, same data as Figure 5g. h-l Cell body size of upper layer cortical neurons is not different between WT and RTT mice, and unaffected by the Igfbp2 neutralizing antibody. h. Schematic of the experiment: P2 mice were injected in the visual cortex with AAV synapsin-GFP +/− antibody, and tissue collected at P7 and GFP-expressing neurons imaged, with the region to be imaged identified by the presence of the fluorescently labeled antibody. i,j. Cell body area in upper layer neurons is unaltered in RTT compared to WT mice. i. Analysis by mice, graph average ± s.e.m., individual data points represent mice, N = 4 WT and 4 RTT mice, statistics by 2-sided T-test. j. Analysis by cells, graph individual data points represent cells, dashed line represents the mean, n = 162 WT and 177 RTT cells, statistics by 2-sided T-test. k,l. Cell body area in upper layer neurons in RTT mice is unaffected by an Igfbp2 neutralizing antibody. k. Analysis by mice, graph average ± s.e.m., individual data points represent mice, N = 5 control-Ab and 5 Igfbp2-Ab mice, statistics by 2-sided T-test. l. Analysis by cells, graph individual data points represent cells, dashed line represents the mean, n = 183 control-Ab and 195 Igfbp2-Ab cells, statistics by 2-sided T-test.

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Cell Culture, Injection, Expressing, Labeling

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a-c. BMP6-treated WT astrocytes show upregulated protein secretion (a) and gene expression (b) that overlaps with ND astrocytes. c. Heatmap of proteins increased in all ND and BMP6-treated astrocytes vs. WT, ranked by protein abundance in BMP6-treated ACM. N=6 cultures each WT, FXS, RTT, DS, plus 6 cultures WT +/− BMP6. d,e. ACM from WT astrocytes treated with BMP6 inhibits WT neurite outgrowth. d. Example images of WT neurons cultured for 48 hours, conditions as marked (merged images of MAP2 + Tau). e. Quantification of total neurite outgrowth. Data from 3 experiments, number of neurons: alone=467, WT ACM=733, BMP6 WT ACM=610. f. Blocking Igfbp2 overcomes the inhibitory effect of BMP6 WT ACM on neurite outgrowth. Data from 3 experiments, number of neurons: alone=378, WT ACM=380, BMP6 WT ACM=506, BMP6 WT ACM + Igfbp2 blocking Ab=335. g-j. Blocking BMP signaling in ND astrocytes rescues deficits in WT neurite outgrowth. g. Experimental schematic for noggin treatment of FXS or RTT astrocytes. h. Blocking BMP signaling in FXS astrocytes rescues deficits in WT neurite outgrowth. Example images of WT neurons cultured for 48 hours, conditions as marked (merged images of MAP2 + Tau). i. Quantification of total neurite outgrowth. Data from 3 experiments, number of neurons: alone=923, WT ACM=1164, FXS ACM=1132, Noggin FXS ACM=1099. j. Blocking BMP signaling in RTT astrocytes rescues deficits in WT neurite outgrowth. Quantification of total neurite outgrowth. Data from 3 experiments, number of neurons: alone=238, WT ACM=279, RTT ACM=387, Noggin RTT ACM=365. Violin plot: dashed line median, dotted lines 25th and 75th percentile. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. See also Extended Data Figure 7; Tables S5, S7.

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Expressing, Cell Culture, Blocking Assay

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a. Schematic of IGF signaling via the PI3K/Akt pathway. b,c. Protein secretion (b) and gene expression (c) profiles of IGF family members in WT and ND astrocytes. Proteomics, N=6 cultures/genotype, *p<0.05, abundance >0.01%, fold change between WT and ND ≥1.5 calculated with T-fold test in Patternlab. RNASeq, N=6 cultures WT, RTT, FXS; 4 cultures DS, *adjusted p<0.05, FPKM>1, fold change between ND and WT ≥1.5 calculated with DESeq2. d,e. Excess Igfbp2 in ACM inhibits WT neurite outgrowth. d. Example images WT neurons grown 48 hours, conditions as marked (image merge of MAP2 + Tau). e. Quantification total neurite outgrowth normalized to control alone condition. Violin plot: dashed line median, dotted lines 25th and 75th percentile. Data from 3 separate experiments, number of neurons: control alone=447, control ACM=626, Igfbp2 alone=438, Igfbp2 ACM=596, Igfbp2 + Ab alone=376, Igfbp2 + Ab + ACM=562. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. f, g. Astrocytes express Igfbp2 mRNA in the P7 mouse visual cortex. f. smFISH against Igfbp2 mRNA in Aldh1l1-GFP mice to mark astrocytes, combined with probe for neurons (Tubb3). Left panels overview of cortex across all layers, right panel high power image from L2/3. g. Analysis of images in f, expression of Igfbp2 mRNA within astrocytes, neurons and OPCs. See Figure S4d for OPC image. N=3 WT mice. Bar graph mean±s.e.m., individual data points mice; statistics by one-way ANOVA with Tukey’s test for multiple comparisons. h-j. Immunostaining for Igfbp2 in each ND and littermate WT visual cortex at P7 (RTT - Mecp2 KO; FXS – Fmr1 KO; DS - Ts65Dn TG) reveals an increase in extracellular Igfbp2 in RTT and intracellular Igfbp2 in DS. h. Example images of L2/3 astrocytes (cyan, Aldh1l1-GFP) immunostained for Igfbp2 (magenta). i. Quantification of extracellular Igfbp2. j. Quantification of intracellular Igfbp2. N=6 littermate pairs RTT and FXS; 7 littermate pairs DS. Bar graph mean±s.e.m., individual points mice, same mouse in i and j for each genotype denoted with same shape; statistics two-sided T-test. See also Extended Data Figure 4.

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Expressing, Immunostaining

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a-f. Application of an Igfbp2-neutralizing antibody reduces WT neurite outgrowth inhibition induced by RTT ACM. a,c,e. Example images neurons cultured for 48 hours in RTT (a), FXS (c) or DS (e) ACM (merged images of MAP2 + Tau). b,d,f. Quantification total neurite outgrowth, normalized to alone condition. Violin plot: dashed line median, dotted lines 25th and 75th percentile. Data from 3 (b), 4 (d), 5 (f) separate experiments. Number of neurons: RTT (b): alone=439, WT ACM=549, RTT ACM=633, RTT ACM + Igfbp2-Ab=621; FXS (d): alone=492, WT ACM=608, FXS ACM=621, FXS ACM + Igfbp2-Ab=647; DS (f): alone=716, WT ACM=992, DS ACM=765, DS ACM + Igfbp2-Ab=858. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. g. Application of an Igfbp2 neutralizing antibody reduces WT neuronal cell body size deficits induced by RTT ACM. Graph mean ± s.e.m., individual data points represent average per experiment, data from 3 experiments. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. h-l. Delivery of an Igfbp2-neutralizing antibody increases cell body size of deep layer neurons in P7 visual cortex of RTT mice. h. Schematic: P2 mice were injected in the visual cortex with AAV synapsin-GFP +/− antibody, tissue collected at P7 and GFP-expressing neurons imaged. i,j. Cell body area in deep layer neurons is decreased in RTT compared to WT mice. i. Analysis by mice, graph average ± s.e.m., individual data points mice, N=4 WT, 4 RTT mice, statistics by 2-sided T-test. j. Analysis by cells, graph individual data points represent cells, dashed line at mean, n=133 WT, 189 RTT cells, statistics by 2-sided T-test. k,l. Cell body area in deep layer neurons in RTT mice is increased by an Igfbp2-Ab. k. Analysis by mice, graph average ± s.e.m., individual data points mice, N=5 control-Ab, 5 Igfbp2-Ab mice, statistics by 2-sided T-test. l. Analysis by cells, graph individual data points represent cells, dashed line at mean, n=219 control-Ab, 274 Igfbp2-Ab cells, statistics by 2-sided T-test. See also Extended Data Figure 5.

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Inhibition, Cell Culture, Injection, Expressing

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a,b. BMP6-treated WT astrocytes show protein secretion (a) and gene expression (b) downregulations that overlap with ND astrocytes. N=6 cultures WT, FXS, RTT, DS, plus 6 cultures WT +/− BMP6 proteomics; N=6 cultures WT, FXS, RTT; 4 DS, plus 6 cultures WT +/− BMP6 RNA sequencing. c. Example images from Figure 7d prior to processing and analysis. Neurons immunostained with MAP2 (dendrites, green) and tau (axon, red). d. Relative frequency distribution plot of total neurite outgrowth length, same data as Figure 7e. Data from 3 experiments, number of neurons: alone=467, WT ACM=733, BMP6 WT ACM=610.e. Relative frequency distribution plot of total neurite outgrowth length, same data as Figure 7f. Data from 3 experiments, number of neurons: alone=378, WT ACM=380, BMP6 WT ACM=506, BMP6 WT ACM + Igfbp2 blocking Ab=335. f. Example images from Figure 7h prior to processing and analysis. Neurons immunostained with MAP2 (dendrites, green) and tau (axon, red). g. Relative frequency distribution plot of total neurite outgrowth, same data as Figure 7i. Data from 3 experiments, number of neurons: alone=923, WT ACM=1164, FXS ACM=1132, Noggin FXS ACM=1099. h. Relative frequency distribution plot of total neurite outgrowth, same data as Figure 7j. Data from 3 experiments, number of neurons: alone=238, WT ACM=279, RTT ACM=387, Noggin RTT ACM=365. i. Example images of cortical neurons treated with noggin at the time of plating, ± WT ACM or ± FXS ACM (image merge of MAP2 + Tau). j. Quantification of total neurite outgrowth, data from 3 experiments. Number of neurons: alone=2197, alone + noggin=1981, WT ACM=2167, WT ACM + noggin=2421, FXS ACM=2060, FXS ACM + noggin=2523. Violin plots dashed line marks median, dotted lines 25th and 75th percentile. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons.

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Expressing, RNA Sequencing Assay, Blocking Assay

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a. Expression of IGF family members in cortical cell types (data from Zhang et al., 2014). b,c. Addition of Igfbp2 protein to WT ACM inhibits WT neurite outgrowth, which is reduced by adding IGF1. Addition of CPE protein to WT ACM does not inhibit WT neurite outgrowth. b. Example images of WT neurons cultured for 48 hours, conditions as marked (image merge of MAP2 + Tau). c. Quantification of total neurite outgrowth. Example experiment shown, repeated 2 times with same result, number of neurons: control alone=49, control ACM=50, CPE alone=36, CPE ACM=46, Igfbp2 alone=44, Igfbp2 ACM=48, Igfbp2 ACM + Igf1=39. d. smFISH against Igfbp2 mRNA in the P7 visual cortex in Aldh1l1-GFP mice to mark astrocytes, combined with probe for OPCs (Cspg4). See Figure 4g for quantification. N=3 WT mice. e. Example images from Figure 4d prior to processing and analysis. Neurons immunostained with MAP2 (dendrites, green) and tau (axon, red). f. Relative frequency distribution plot of total neurite outgrowth length, pooled data from 3 experiments, same data as Figure 4e. g. Adding the IgG control antibody to WT ACM does not alter neurite outgrowth. Example experiment shown, repeated twice with same result. Number of neurons: control alone=216, control ACM=333, Igfbp2-Ab alone=257, Igfbp2-Ab ACM=267, IgG con-Ab alone=277, IgG con-Ab ACM=266. Violin plots (c,g), dashed line marks median, dotted lines 25th and 75th percentile. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons, p values compared to control alone condition (c).

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Expressing, Cell Culture

Journal: JCI Insight

Article Title: CD44 expression in endothelial colony-forming cells regulates neurovascular trophic effect

doi: 10.1172/jci.insight.89906

Figure Lengend Snippet: (A–H) OIR eyes after injection of CD44 knockdown ECFCs did not show apparent morphological differences in samples of flat-mount stained retinas (A–D) or eye sections (E–H). Green, GFP; red, human-VE-cadherin; blue, Hoechst33342. (A–D) Low-magnification (A and C) or high-magnification (B and D) images of flat-mount staining for posterior lens capsule and retina harvested at P14 after injection at P12 of control ECFCs (ECFCs-scrRNA) (A and B) or CD44 knockdown ECFCs (ECFCs-shCD44) (C and D). (E–H) Low-magnification (E and G) or high-magnification images (F and H) of immunohistochemistry for eyeball sections harvested at P14 after injection at P12 of ECFCs-scrRNA (E and F) or ECFCs-shCD44 (G and H). Scale bar: 500 μm (A and C); 50 μm (B and D); 100 μm (E–H). (I) Experimental schema for isolation of P12 injected ECFCs using FAC sorting at P13. (J) Flow cytometry analysis for GFP-positive ECFCs-scrRNA or ECFCs-shCD44 from OIR eyes. (K) qPCR-based gene profile analysis for angiogenic growth factors expressed on injected ECFCs-scrRNA or ECFCs-shCD44. Genes dysregulated by >1.5-fold with P values of less than 0.05 were plotted. (L) Mesoscale discovery (MSD) analysis based on electrochemiluminescence detection technology for human IGFBP2 and IGFBP3 protein levels in a whole OIR eyes at P14 injected ECFCs at P12. Error bars represent Min to Max. n = 4. *P < 0.05, Mann-Whitney test.

Article Snippet: Briefly, we incubated the IGFBP2 and IGFBP3 blocking antibody or goat IgG (R&D) with Dynabeads.

Techniques: Injection, Staining, Immunohistochemistry, Isolation, Flow Cytometry, Electrochemiluminescence, MANN-WHITNEY

Journal: JCI Insight

Article Title: CD44 expression in endothelial colony-forming cells regulates neurovascular trophic effect

doi: 10.1172/jci.insight.89906

Figure Lengend Snippet: (A and B) Culture supernatant from 3D gel culture repairs the oxygen-induced retinopathy (OIR) model’s phenotype, and its rescue effect is regulated by CD44. (A) Confocal image of GFP-ECFC in collagen gel culture. (B) qPCR array gene profile analysis for human angiogenic growth factors expressed on ECFCs-scrRNA or ECFCs-shCD44 cultured in 3D gel for 48 hours. n = 3. Genes dysregulated by >1.5-fold with P values of less than 0.05 were plotted. (C) Angiogenic protein arrays of conditioned media from 3D gel culture of control ECFCs (ECFCs-scrRNA) (A and B) or CD44 knockdown ECFCs (ECFCs-shCD44). Boxes outlined with various colors indicate ECFC-secreted angiocrine factors. (D) ELISA analysis for human IL-8, IGFBP2, and IGFBP3 in conditioned media from 3D gel culture of ECFCs-scrRNA or ECFCs-shCD44. n = 7. Student’s t test. (E) Representative GS lectin–stained flat-mount retinas and (F) quantification for eyes harvested at P17 after injection at P12 with cell culture supernatant from 3D gel culture of control ECFCs (ECFCs-scrRNA), CD44 knockdown ECFCs (ECFCs-shCD44), or gel without ECFCs in OIR. *P < 0.05, Kruskal-Wallis test with Dunn’s multiple comparison test. n = 16–18. Scale bar: 500 μm. Error bars represent SD in D and SEM in F.

Article Snippet: Briefly, we incubated the IGFBP2 and IGFBP3 blocking antibody or goat IgG (R&D) with Dynabeads.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Injection

Journal: JCI Insight

Article Title: CD44 expression in endothelial colony-forming cells regulates neurovascular trophic effect

doi: 10.1172/jci.insight.89906

Figure Lengend Snippet: (A–D) Treatment with 1 ng rhIGFBP2 (A and B) or rhIGFBP3 (C and D) efficiently decreased neovascular tufts (NV) and vaso-obliterated regions (VO) at P17 when compared with vehicle-injected eyes. Results are expressed as a percentage of NV or VO area, normalized to vehicle-injected eyes. n = 9–16. *P < 0.05, Mann-Whitney test. Scale bar: 500 μm. (E–G) Depletion of IGFBPs in cell culture supernatant from 3D gel culture of ECFCs partially cancelled the rescue effect in oxygen-induced retinopathy (OIR). (E) ELISA analysis after depletion of IGFBP2 and IGFBP3 using Protein G magnetic beads with each neutralizing antibody. (F) Representative GS lectin–stained flat-mount retinas and (G) quantification for eyes harvested at P17 after injection at P12 with cell culture supernatant treated with Protein G magnetic beads with neutralizing antibody either of IGFBP2 or IGFBP3 and both (combo-Ab) from 3D gel culture of ECFCs in OIR. n > 21. *P < 0.05, **P < 0.01, Kruskal-Wallis test with Dunn’s multiple comparison test. Error bars represent SEM. Scale bar: 500 μm.

Article Snippet: Briefly, we incubated the IGFBP2 and IGFBP3 blocking antibody or goat IgG (R&D) with Dynabeads.

Techniques: Injection, MANN-WHITNEY, Cell Culture, Enzyme-linked Immunosorbent Assay, Magnetic Beads, Staining