Journal: bioRxiv

Article Title: Lupus auto-antibodies act as positive allosteric modulators at NMDA receptors and induce spatial memory deficits

doi: 10.1101/791715

Figure Lengend Snippet: (A) Bar graphs (mean ± SEM) (from left to right, n = 6, 6, 8, 8, 6, 6) showing normalized whole-cell peak current amplitudes for diheteromeric NMDARs (N1/N2A-N2A or N1/N2B-N2B) or triheteromeric receptors (N1/N2A-N2B) at 10 μg/mL of B1 or G11 antibody (** p < 0.01, Mann-Whitney U test ). (B) A single charge reversal in the GluN2A DWEYS motif (DW D YS) (D285K) disrupts G11 binding. Representative images from immunocytochemistry of HEK293T cells not transfected (no DNA) or transfected with GluN1/GluN2A or with GluN1/GluN2A(D285K). Cells were stained with either B1 or G11 (10 μg/mL) (green, Alexa-488) under non-permeabilizing conditions with DAPI (blue) counterstain. Scale (white bar): 40 μm. (C) Quantification of mean fluorescence intensity in (B) (mean ± SEM) (n = 5, all conditions) (** p < 0.01, one-way ANOVA with post-hoc Tukey’s test ). (D) Single charge reversal prevents DNRAb (10 μg/mL) potentiation of whole cell currents (mean ± SEM, n = 5, all conditions). (E) Left , Current records of triheteromeric (N1/N2A-N2A(D285K)) or diheteromeric (N1/N2A(D285K)-N2A(D285K)) NMDARs. Currents displayed as in . Right , Bar graphs (mean ± SEM) (from left to right, n = 5, 6, 5, 7, 5, 6) showing normalized current amplitude for diheteromeric NMDARs (N1/N2A-N2A or N1/N2A(D285K)-N2A(D285K)) or triheteromeric receptors (N1/N2A-N2A(D285K)) at 10 μg/mL of B1 or G11 (* p < 0.05, Mann-Whitney U test ). (F) GluN2A specific antagonists are neuro-protective at moderate pathophysiological levels of DNRAbs. Immunocytochemistry of primary hippocampal neuronal cultures (DIV14) incubated either in control antibody (B1+vehicle) or in DNRAb (G11) at 10 μg/mL. G11 was incubated either alone (+vehicle), with GluN2A antagonists TCN-201 (not shown) or MPX-004, or with ifenprodil, a GluN2B negative allosteric modulator at 3 μM. Upper panels, representative images of hippocampal neurons stained with antibodies against neuronal-specific β-tubulin, Tubb3 (green, Alexa-488), and activated caspase-3 (red, Alexa-647), with DAPI (blue) counterstain. Lower panel, only activated caspase-3 channel is shown. Scale (white bar): 40 μm. (G) Quantification of immunocytochemistry results shown in (F). Proportion of DAPI and activated-caspase-3-positive cells divided by total DAPI cells (mean ± SEM) (from left to right, n = 11, 11, 7, 7, 9, 6) per treatment condition (* p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with post-hoc Tukey’s test ).

Article Snippet: Final concentrations of antagonists were 3 μM: TCN-201 (AdooQ Bioscience, A11947), MPX-004 (Alomone Labs, M280), and Ifenprodil (Sigma, I2892).

Techniques: MANN-WHITNEY, Binding Assay, Immunocytochemistry, Transfection, Staining, Fluorescence, Incubation

Journal: eLife

Article Title: Tumor initiating cells induce Cxcr4-mediated infiltration of pro-tumoral macrophages into the brain

doi: 10.7554/elife.31918

Figure Lengend Snippet: Figure 5. Cxcr4b signaling is required for the increase in microglia numbers following AKT1 overexpression. (A) To achieve expression in neural cells in the cxcr4b-/- mutant zebrafish line, a driver plasmid containing the NBT promoter (NBT:DlexPR-lexOP-pA) was co-injected together with either a lexOP: AKT1-lexOP:tagRFP plasmid to induce AKT1 expression or together with a lexOP:tagRFP-pA plasmid to achieve control RFP expression. Immunohistochemistry expression of (B) the human AKT1 protein and (C) Synaptophysin in the AKT1-expressing cells in the cxcr4b-/- mutant fish at 8 Figure 5 continued on next page

Article Snippet: The authors are grateful to Graham Lieschke for sharing mpeg1:EGFP fish and to Darren Gilmour for sharing cxcr4b-/- zebrafish. pcDNA3 Myr HA Akt1 was a gift from William Sellers (Addgene plasmid # 9008).

Techniques: Over Expression, Expressing, Mutagenesis, Plasmid Preparation, Injection, Control, Immunohistochemistry

Journal: eLife

Article Title: Tumor initiating cells induce Cxcr4-mediated infiltration of pro-tumoral macrophages into the brain

doi: 10.7554/elife.31918

Figure Lengend Snippet: Figure 6. Cxcr4b signaling in macrophages is required for the increase in microglia numbers upon AKT1 transformation in the brain. (A) To rescue Cxcr4b expression in macrophages or neural cells in the cxcr4b-/- mutant fish, a cell-specific rescue construct was injected in addition to the NBT driver and lexOP:tagRFP-pA/lexOP:AKT1-lexOP:RFP constructs. Cxcr4b expression was recovered in macrophages through the mpeg1 promoter (‘macrophage’ rescue) via a mpeg1:cxcr4b-pA construct. The expression of cxcr4b in neural cells (‘Neural’ rescue) was rescued through the lexOP: cxcr4b-pA construct. (B) Immunohistochemistry using a Cxcr4b antibody showed that Cxcr4b expression is completely absent in microglia and neural cells in the cxcr4b-/- fish (upper panels). Cxcr4b expression is partially recovered following the respective rescue conditions (lower panels). (C) Quantification of the number of microglia in cxcr4b-/- mutants overexpressing AKT1 before, and after Cxcr4b rescue in macrophages (‘Macrophage’ rescue: 119.5 ± 4.59, n = 28 vs cxcr4b-/- Akt1: 91.4 ± 5.22, n = 17; p<0.001, N = 39) and in neural cells (‘Neural’ rescue: 92.2 ± 4.40, n = 27, p=1 (n.s.), N = 3). (D) Quantification of the level of proliferation of AKT1-expressing cells in cxcr4b-/- mutants overexpressing AKT1 before, and after Cxcr4b rescue in macrophages (‘Macrophage’ rescue: 37.8 ± 1.45%, n = 45 larvae, vs cxcr4b-/- AKT1: 22.1 ± 1.73%, n = 27 larvae p<0.001, N = 3) and in neural cells (‘Neural’ rescue: 23.8 ± 2.08%, n = 22 larvae, vs cxcr4b-/- AKT1: 22.1 ± 1.73%, n = 27 larvae, p=1.00 (n.s.), N = 3). Error bars represent mean ±SEM. Images were captured using a Zeiss LSM710 confocal microscope with a 20X/NA 0.8 objective. Scale bars represent 100 mm. DOI: https://doi.org/10.7554/eLife.31918.016

Article Snippet: The authors are grateful to Graham Lieschke for sharing mpeg1:EGFP fish and to Darren Gilmour for sharing cxcr4b-/- zebrafish. pcDNA3 Myr HA Akt1 was a gift from William Sellers (Addgene plasmid # 9008).

Techniques: Transformation Assay, Expressing, Mutagenesis, Construct, Injection, Immunohistochemistry, Microscopy

Journal: eLife

Article Title: Tumor initiating cells induce Cxcr4-mediated infiltration of pro-tumoral macrophages into the brain

doi: 10.7554/elife.31918

Figure Lengend Snippet: Figure 7. High levels of Sdf1b produced by AKT1-expressing cells lead to increased microglia numbers. (A) mRNA expression levels of mif, sdf1a, and sdf1b in AKT1-RFP+ cells determined by qPCR (N = 3 for each gene). Fold change was measured in relation to control-RFP+ cells using the comparative (DDCT) method. (B) Quantification of the number of microglia in control WT larvae, and following Sdf1b overexpression in both WT fish (WT Control: 85.8 ± 2.45, n = 35 larvae; WT Sdf1b: 111.0 ± 6.08, n = 25 larvae, p<0.001, N = 3) and cxcr4b-/- mutants (cxcr4b-/- Sdf1b: 82.8 ± 3.51, n = 12, p=0.225 (n. s.), N = 3). Error bars represent mean ±SEM. DOI: https://doi.org/10.7554/eLife.31918.017

Article Snippet: The authors are grateful to Graham Lieschke for sharing mpeg1:EGFP fish and to Darren Gilmour for sharing cxcr4b-/- zebrafish. pcDNA3 Myr HA Akt1 was a gift from William Sellers (Addgene plasmid # 9008).

Techniques: Produced, Expressing, Control, Over Expression