Journal: The Journal of Neuroscience

Article Title: Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue

doi: 10.1523/JNEUROSCI.0218-19.2019

Figure Lengend Snippet: Expression of P2Y12 and A2A receptors by human microglia. A, Immunostaining against P2Y12 and A2A receptors in an IBA1+, ramified PTC microglia. B, P2Y12 and A2A receptor immunostaining for an amoeboid IBA1+ cell from MTLE tissue. A, B, From left, P2Y12 immunostaining (green), IBA1 (white), A2A (red), and merged P2Y12 and A2A staining. C, Proportion of ramified (clear; n = 33) and amoeboid (hatched; n = 25) microglia immunopositive for P2Y12 (gray) and A2A receptors (red). D, Color-coded staining intensity (green represents low; red represents high) shows that P2Y12 staining was highest toward the tips of processes of a ramified microglia (above) in the resting state. Intensity was highest in a somatic region of a nonstimulated amoeboid cell (below). E, Color-coded intensity (green represents low; red represents high) for P2Y12 immunopositivity for initially amoeboid microglia fixed at full extension induced by 10 μm ADP. Staining intensity was low in perisomatic regions (top) and highest at bulbous endings of processes (bottom).

Article Snippet: We used primary antibodies directed against the following: Iba1 (Abcam, ab5076; 1/500), P2Y12 (Novus, NBP2–33870; 1/200), A2AR (Santa Cruz Biotechnology, sc-32261; 1/100), P2Y1 (Abcam, ab168918; 1/200), and P2Y13 (LSBio, LS-A1622; 1/200).

Techniques: Expressing, Immunostaining, Staining

Journal: The Journal of Neuroscience

Article Title: Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue

doi: 10.1523/JNEUROSCI.0218-19.2019

Figure Lengend Snippet: P2Y12 receptors and microglial motilities. A, The nonhydrolyzable ADP analog 2MeSADP (1 nm) increased mean microglial cross-sectional area (n = 4 PTC cells). B, The P2Y12 receptor antagonist PSB0739 (1 μm) reduced the mean cross-sectional area of ramified microglia (3 PTC and 2 MTLE microglia). It prevented the process extension induced by 10 μm ADP. C, PSB0739 suppressed surveillance motility of processes of ramified microglia. Right, Control. Left, Process positions at 5 min intervals during control period shown by colors (green, red, yellow, dark blue, light blue). D, Right, After PSB0739. Middle, Processes in the presence of PSB0739 (1 μm) and ADP (10 μm). Left, Process positions at 5 min intervals (colors as in C). Movie 4 shows that PSB0739 suppresses surveillance motility and blocks ADP-induced process extension.

Article Snippet: We used primary antibodies directed against the following: Iba1 (Abcam, ab5076; 1/500), P2Y12 (Novus, NBP2–33870; 1/200), A2AR (Santa Cruz Biotechnology, sc-32261; 1/100), P2Y1 (Abcam, ab168918; 1/200), and P2Y13 (LSBio, LS-A1622; 1/200).

Techniques: Control

Journal: The Journal of Neuroscience

Article Title: Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue

doi: 10.1523/JNEUROSCI.0218-19.2019

Figure Lengend Snippet: Role of P2Y receptors in microglial motility induced by tissue damage. A, The effects of local laser stimulation. Control images of an initially amoeboid cell from PTC before (CTRL), immediately after laser damage (yellow arrow), and at 10 and 15 min later. Merge is the difference between control and 15 min images. Red represents new membrane signal. Green represents lost signal. B, The same cell after a second laser stimulus (yellow arrow) applied 5 min after the P2Y12 antagonist PSB0739 (1 μm). Images are shown at 5 and 15 min after stimulation. Merge is the difference between PSB0739 and 15 min images. Red represents lost membrane signal due to process retraction. Blue represents new membrane signal from ruffling. Movie 6 shows that local laser damage induces process extension and is blocked by P2Y12R antagonist. Movie 7 shows that process extension is restored in the absence of P2Y12R antagonist.

Article Snippet: We used primary antibodies directed against the following: Iba1 (Abcam, ab5076; 1/500), P2Y12 (Novus, NBP2–33870; 1/200), A2AR (Santa Cruz Biotechnology, sc-32261; 1/100), P2Y1 (Abcam, ab168918; 1/200), and P2Y13 (LSBio, LS-A1622; 1/200).

Techniques: Control, Membrane

Journal: Blood Advances

Article Title: The Na/K-ATPase α1 subunit fine-tunes platelet P2Y 12 function and mediates sex dimorphism–associated thrombosis

doi: 10.1182/bloodadvances.2025016605

Figure Lengend Snippet: α1 binds to P2Y 12 , and α1 deficiency attenuates ADP-induced AKT activation in platelets. (A) WT platelets pooled from 6 male WT mice were lysed and subjected to co-IP to assess the interaction between NKA α1 and P2Y 12 or P2Y 1 . (B) Platelet lysates from α1 +/− and α1 +/+ mice were analyzed using blue-native polyacrylamide gel electrophoresis (PAGE). The membrane was first probed for α1, then stripped and reprobed for P2Y 12 . The image represents 2 independent experiments. (C) COS-7 cells were transfected with plasmids encoding human P2Y 12 (p-hP2Y 12 ; Addgene number 66471) or the A2B receptor (p-hA2BR; Addgene number 37202) for 36 hours. Cell lysates were then used for co-IP assays to examine α1 binding to P2Y 12 and A2B receptors. (D) PRP from α1 +/− and α1 +/+ mice was pooled from 5 males, adjusted to a final concentration of 2.5 × 10 8 cells per mL using platelet-poor plasma, and divided into 4 aliquots. PRP was supplemented with MgCl 2 /CaCl 2 (1mM final concentration) and stimulated with ADP (2.5μM final concentration) for different time points. The reaction was stopped using an EDTA/PGE1 cocktail, and platelets were lysed for western blot analysis. The blot represents 2 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation.

Article Snippet: The image represents 2 independent experiments. (C) COS-7 cells were transfected with plasmids encoding human P2Y 12 (p-hP2Y 12 ; Addgene number 66471) or the A2B receptor (p-hA2BR; Addgene number 37202) for 36 hours.

Techniques: Activation Assay, Co-Immunoprecipitation Assay, Polyacrylamide Gel Electrophoresis, Membrane, Transfection, Binding Assay, Concentration Assay, Clinical Proteomics, Western Blot, Immunoprecipitation

Journal: Blood Advances

Article Title: The Na/K-ATPase α1 subunit fine-tunes platelet P2Y 12 function and mediates sex dimorphism–associated thrombosis

doi: 10.1182/bloodadvances.2025016605

Figure Lengend Snippet: LGL mediates the interaction between NKA α1 and P2Y 12 . (A) COS-7 cells were transfected with plasmids encoding mouse WT P2Y 12 along with either WT or LGL→SFT mutant α1. (B-C) COS-7 cells were transfected with plasmids encoding mouse WT α1 along with either WT or LGL→SFT mutant P2Y 12 , and cell lysates were used for co-IP assays (B); α1 band intensity was quantified and expressed as the ratio relative to the average of WT P2Y 12 cotransfected with WT α1 (C). (D) COS-7 cells were transfected with different plasmid combinations and treated with ouabain or digoxin for 36 hours before being lysed for co-IP assays. α1 band intensity was quantified with ImageJ and normalized to lane 1. (E) COS-7 cells were transfected with plasmids encoding human P2Y 12 for 24 hours, then treated with LGL peptide at the indicated dosages for 16 hours. Cells were harvested for co-IP analysis of α1 and P2Y 12 . α1 band intensity was quantified with ImageJ and normalized to the non-LGL condition. GAPDH was blotted as a loading control for input. (F) Washed platelets from male α1 +/− or α1 +/+ mice were resuspended in PBS at 2.5 × 10 8 /mL, and 400 μL was used per aggregation assay. LGL peptide or a leucine-glycine mixture (control) was added to a final concentration of 50μM and incubated for 3 minutes before initiating aggregation. Data were expressed as (LGL – control)/control. An unpaired t test was used. IgG, immunoglobulin G; SB, 2× Laemmli sample buffer.

Article Snippet: The image represents 2 independent experiments. (C) COS-7 cells were transfected with plasmids encoding human P2Y 12 (p-hP2Y 12 ; Addgene number 66471) or the A2B receptor (p-hA2BR; Addgene number 37202) for 36 hours.

Techniques: Transfection, Mutagenesis, Co-Immunoprecipitation Assay, Plasmid Preparation, Control, Concentration Assay, Incubation

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Microglia alter the threshold of spreading depolarization and related potassium uptake in the mouse brain

doi: 10.1177/0271678x19900097

Figure Lengend Snippet: Figure 1. Spreading depolarization (SD) attracts microglial processes to neuronal somata and selective elimination of microglia alters the electrical threshold of SD elicitation. (a) Schematic illustration of the experimental setting in series 1, and areas of quantification for the immunofluorescent analysis shown in Panels (b) and (c). Area size (420 mm 320 mm). (b) Changes in microglial cell body area and the number of branches as obtained from 2D morphological analysis of Iba1-positive microglia in the cerebral cortex (the region used for analysis is identical to Area 1 shown in panel (a)). Mann–Whitney U test (p < 0.05*). Scale bar, 10 lm. (c) Representative images demonstrating microglial (P2Y12R, red) process recruitment to Kv2.1 labeled neuronal soma (green) in the neocortex, contralateral and ipsilateral to SD elicitation. Microglial process density is increased around neurons in the ipsilateral cortex relative to the contralateral cortex in Area 1 (near SD induction) depicted in Panel A. Data are expressed as meanstdev. Control vs. ipsi p < 0.01, two-way ANOVA followed by Sidak’s multiple comparison test (p < 0.05 for Area 1). Scale bar, 10 lm. (d) Representative images and quantitative analysis confirming elimination of microglia (P2Y12 receptors, green) after feeding mice a PLX5622 (1200 ppm)-containing diet for three weeks. Data are expressed as meanstdev. Unpaired t-test (p < 0.01**). Scale bar, 50 lm. (e) Microglia depletion increased the electric threshold of SD elicitation for each consecutive SD in a train (Series 1). Data are shown as median and interquartile ranges. Friedman ANOVA for time (p < 0.05* vs. 1st SD, in the depleted group), and a Mann–Whitney U test for group comparison (no significant differences between groups). (f) Microglia depletion altered the electric threshold of SD elic- itation (series 1). First SDs (circles) are shown apart from pooled, recurrent SDs (triangles). Black circle and error bars stand for mean 95% CI. Mann–Whitney U test (p < 0.05* and p < 0.01** vs. respective Control). (g) The induction of SD required a KCl concentration higher than 1 M in some of the microglia-depleted and P2Y12R KO animals (series 2). Each colored sphere in the graph stands for the induction of an individual SD.

Article Snippet: Experiments were carried out in 12–14-weeks-old adult male C57BL/6J (n1⁄4 28) and P2Y12R KO (#TF1881, Taconic) (n1⁄4 7) mice (male, C57BL/6J background), bred in the SPF unit of the Institute of Experimental Medicine (Budapest, Hungary).

Techniques: MANN-WHITNEY, Labeling, Control, Comparison, Concentration Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Microglia alter the threshold of spreading depolarization and related potassium uptake in the mouse brain

doi: 10.1177/0271678x19900097

Figure Lengend Snippet: Figure 2. Selective elimination of microglia or absence of P2Y12R curbs SD and supports hyperpolarization after SD. (a) Confocal analysis revealed increased microglial P2Y12R mean fluorescence intensity (MFI) in response to a series of SDs (assessed in Area 1 according to Figure 1(a)). Unpaired t-test, n ¼ 40 randomly selected microglia from the contralateral hemisphere and n ¼ 38 microglia from the ipsilateral hemisphere from seven mice per group. (b) Representative images depict the enrichment of P2Y12R (STORM, cyan) on microglial processes (P2Y12R, confocal, red) recruited to the Kv2.1 (green) labeled neurons in the neocortex, contralateral and ipsilateral to SD elicitation. Scale bar, 1000 nm. STORM super-resolution microscopy reveals increased P2Y12R densities on microglial processes recruited to neurons, in response to SD. Meanstdev values of the number of localization points (NLP) are shown normalized to P2Y12R clusters determined by the density-based spatial clustering of applications with noise (DBSCAN) algorithm (Local density filter: 10 neighbours within 150 nm Z-filter: 300 nm from focal plane). Unpaired t test, p < 0.05*, n ¼ 20 neurons randomly selected from seven mice per group. (c) Changes in microglial cell body area and the number of branches in P2Y12R KO mice as obtained from 2D morphological analysis of Iba1-positive microglia in the cerebral cortex (the region used for analysis is identical to Area 1 shown on Figure 1 Panel (a)). Values normalized to the contralateral side are shown as median interquartile range. Mann–Whitney U test, (p < 0.05*). Scale bar, 10 lm. (d) The direct current (DC) potential signature of recurrent SDs (rSDs) (each trace is the mean of rSDs in each experimental group, series 2). Capital letters indicate variables quantitated in the respective Panels. (e) Amplitude of the negative DC potential shift of rSDs (series 1 and 2). (f) Duration of the negative DC potential shift of rSDs (series 1 and 2). (g) Area under the curve of the hyperpolarization after rSDs (series 1 and 2). In Panels (d)–(f), data are given as meanstdev. Sample size is indicated in each bar. Statistical analysis of data in series 1 relied on an independent t-test or a Welch-t test. Data in series 2 were evaluated by a one-way ANOVA paradigm followed by a multiple comparison of Tukey for equal variances, or Games-Howell for unequal variances (p < 0.01** depleted vs. respective control, p < 0.05# and p < 0.01## P2Y12R KO vs. respective control).

Article Snippet: Experiments were carried out in 12–14-weeks-old adult male C57BL/6J (n1⁄4 28) and P2Y12R KO (#TF1881, Taconic) (n1⁄4 7) mice (male, C57BL/6J background), bred in the SPF unit of the Institute of Experimental Medicine (Budapest, Hungary).

Techniques: Fluorescence, Labeling, Super-Resolution Microscopy, MANN-WHITNEY, Comparison, Control

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Microglia alter the threshold of spreading depolarization and related potassium uptake in the mouse brain

doi: 10.1177/0271678x19900097

Figure Lengend Snippet: Figure 4. The absence of microglia is associated with augmented neuronal activation after spreading depolarization (SD). (a) Schematic showing the areas used for quantitative analysis relative to the site of SD induction. (b) Representative images showing glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) immunofluorescence in the ipsilateral hemisphere 1.5 h after SD induction (Area 1 is displayed). Graphs showing integrated density values of GFAP and GS signal normalized to the corresponding areas of the contralateral hemisphere as meanstdev. Two-way ANOVA followed by Sidak’s multiple comparison. (c) Representative images demonstrate activated, c-fos labeled (green) neurons (Kv2.1, a voltage-dependent Kþ channel; magenta) in the cerebral cortex ipsilateral to SD elicitation (meanstdev). Mice were sacrificed 1.5 h after the induction of the first SD in a train of four events, to allow the assessment of c-fos protein expression. Scale bar, 10 mm. Quantification of c-fos protein expression was performed in areas indicated in the schematic coronal brain section in Panel (a) (ROI: 300 mm 200 mm). p < 0.01**, control vs. depleted (ipsilaterally), two-way ANOVA followed by Sidak’s multiple comparison. (d) Representative images demonstrate activated, c-fos labeled (green) neurons (Kv2.1, magenta) in the cerebral cortex ipsilateral to SD elicitation in control and P2Y12R KO mice (Area 1 on panel A is shown). Quantification of c-fos protein expression was performed in areas indicated in the schematic coronal brain section in Panel (a) (ROI: 300 mm 200 mm, only Area 1 and Area 2 assessed based on the results of microglia depletion studies). n ¼ 4–7, data are expressed meanstdev.

Article Snippet: Experiments were carried out in 12–14-weeks-old adult male C57BL/6J (n1⁄4 28) and P2Y12R KO (#TF1881, Taconic) (n1⁄4 7) mice (male, C57BL/6J background), bred in the SPF unit of the Institute of Experimental Medicine (Budapest, Hungary).

Techniques: Activation Assay, Immunofluorescence, Comparison, Labeling, Expressing, Control