4 aminopyridine  (Alomone Labs)


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    Structured Review

    Alomone Labs 4 aminopyridine
    Influences of voltage-gated potassium (K v ) channel inhibition on progenitor c ell proliferation . Proliferation of hNPCs was analyzed via BrdU incorporation assay. (A): Progenitor cell proliferation was measured colorimetrically after 72 h of K v channel inhibition and normalized to control values without addition of inhibitor. Electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of <t>4-aminopyridine</t> (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) were applied. Progenitor cell proliferation was significantly reduced by inhibition of I A with 4-AP, PTX, NH 4 Cl as well as by unspecific blockers like TEA and higher doses of QND. In contrast, the I K antagonist DTX increased proliferation of hNPCs (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, *p
    4 Aminopyridine, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells"

    Article Title: Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006168

    Influences of voltage-gated potassium (K v ) channel inhibition on progenitor c ell proliferation . Proliferation of hNPCs was analyzed via BrdU incorporation assay. (A): Progenitor cell proliferation was measured colorimetrically after 72 h of K v channel inhibition and normalized to control values without addition of inhibitor. Electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) were applied. Progenitor cell proliferation was significantly reduced by inhibition of I A with 4-AP, PTX, NH 4 Cl as well as by unspecific blockers like TEA and higher doses of QND. In contrast, the I K antagonist DTX increased proliferation of hNPCs (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, *p
    Figure Legend Snippet: Influences of voltage-gated potassium (K v ) channel inhibition on progenitor c ell proliferation . Proliferation of hNPCs was analyzed via BrdU incorporation assay. (A): Progenitor cell proliferation was measured colorimetrically after 72 h of K v channel inhibition and normalized to control values without addition of inhibitor. Electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) were applied. Progenitor cell proliferation was significantly reduced by inhibition of I A with 4-AP, PTX, NH 4 Cl as well as by unspecific blockers like TEA and higher doses of QND. In contrast, the I K antagonist DTX increased proliferation of hNPCs (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, *p

    Techniques Used: Inhibition, BrdU Incorporation Assay

    Cell viability after inhibition of voltage-gated potassium (K v ) channels. Determination of cell viability in proliferating hNPCs via 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt (MTT) assay. (A): Cell viability was measured colorimetrically after 72 h of K v channel inhibition with different concentrations of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) and normalized to control values without addition of inhibitor. (B): Viability of hNPCs was significantly reduced by electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-AP, PTX and NH 4 Cl, which specifically blocked I A , as well as by TEA and higher doses of QND, which inhibited both current components (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, ***p
    Figure Legend Snippet: Cell viability after inhibition of voltage-gated potassium (K v ) channels. Determination of cell viability in proliferating hNPCs via 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt (MTT) assay. (A): Cell viability was measured colorimetrically after 72 h of K v channel inhibition with different concentrations of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH 4 Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) and normalized to control values without addition of inhibitor. (B): Viability of hNPCs was significantly reduced by electrophysiologically determined inhibitory doses (IC 50 /IC 80 ) of 4-AP, PTX and NH 4 Cl, which specifically blocked I A , as well as by TEA and higher doses of QND, which inhibited both current components (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, ***p

    Techniques Used: Inhibition, MTT Assay

    Pharmacological inhibition of K v currents in hNPCs. Biophysically separated A-type (I A ) and delayed-rectifying (I K ) K v currents in proliferating hNPCs were differentially inhibited by the 4-aminopyridine (4-AP, i), phrixotoxin-1 (PTX, ii), ammonium chloride (NH 4 Cl, iii), tetraethylammonium chloride (TEA, iv), quinidine (QND, v) and α-dendrotoxin (DTX, vi). (A): Peak amplitudes of I A were measured during a depolarizing voltage step from −130 mV to 0 mV between 0 and 20 ms (inset). (B): I K was determined between 280 and 300 ms of a 100 mV depolarization step following a −40 mV prepulse during the application of different antagonist concentrations (insets). (C): Both current values were normalized to the non-inhibited peak amplitudes. Dose-response relationships were fitted with the Hill equation and IC 50 values were determined (see Tab. 2 ). Note that PTX selectively and 4-AP preferentially inhibited I A , while DTX selectively blocked I K .
    Figure Legend Snippet: Pharmacological inhibition of K v currents in hNPCs. Biophysically separated A-type (I A ) and delayed-rectifying (I K ) K v currents in proliferating hNPCs were differentially inhibited by the 4-aminopyridine (4-AP, i), phrixotoxin-1 (PTX, ii), ammonium chloride (NH 4 Cl, iii), tetraethylammonium chloride (TEA, iv), quinidine (QND, v) and α-dendrotoxin (DTX, vi). (A): Peak amplitudes of I A were measured during a depolarizing voltage step from −130 mV to 0 mV between 0 and 20 ms (inset). (B): I K was determined between 280 and 300 ms of a 100 mV depolarization step following a −40 mV prepulse during the application of different antagonist concentrations (insets). (C): Both current values were normalized to the non-inhibited peak amplitudes. Dose-response relationships were fitted with the Hill equation and IC 50 values were determined (see Tab. 2 ). Note that PTX selectively and 4-AP preferentially inhibited I A , while DTX selectively blocked I K .

    Techniques Used: Inhibition, Mass Spectrometry

    Voltage-activated potassium (K v ) outward currents in hNPCs. (A): In whole-cell patch-clamp recordings human neural progenitor cells (hNPCs) expressed inactivating A-type (I A ) and non-inactivating delayed-rectifier-like potassium currents in activation (i) and inactivation protocols (ii, insets). (B): Pharmacological separation of current components was performed by application of 10 mM 4-aminopyridine (4-AP). I K was defined as 4-AP-insensitive component and I A as 4-AP-sensitive component. (C): Biophysical separation of I K was observed in activation protocols by a depolarizing prepulse to −40 mV (500 ms), which caused inactivation of I A . In inactivation protocols I A was revealed by a test pulse to 0 mV only since it activated at slightly more negative potentials than I K . During each voltage step peak values of the transient component were measured between 0 and 20 ms and sustained currents were determined between 280 and 300 ms. Chord conductances and current values respectively were normalized to their peak amplitudes and fitted to a Boltzmann distribution and current-voltage-relationships of control currents (A), pharmacologically (B) as well as biophysically (C) separated currents were calculated (iii, see Tab. 1 ). Note the similar I–V relations for both separation procedures.
    Figure Legend Snippet: Voltage-activated potassium (K v ) outward currents in hNPCs. (A): In whole-cell patch-clamp recordings human neural progenitor cells (hNPCs) expressed inactivating A-type (I A ) and non-inactivating delayed-rectifier-like potassium currents in activation (i) and inactivation protocols (ii, insets). (B): Pharmacological separation of current components was performed by application of 10 mM 4-aminopyridine (4-AP). I K was defined as 4-AP-insensitive component and I A as 4-AP-sensitive component. (C): Biophysical separation of I K was observed in activation protocols by a depolarizing prepulse to −40 mV (500 ms), which caused inactivation of I A . In inactivation protocols I A was revealed by a test pulse to 0 mV only since it activated at slightly more negative potentials than I K . During each voltage step peak values of the transient component were measured between 0 and 20 ms and sustained currents were determined between 280 and 300 ms. Chord conductances and current values respectively were normalized to their peak amplitudes and fitted to a Boltzmann distribution and current-voltage-relationships of control currents (A), pharmacologically (B) as well as biophysically (C) separated currents were calculated (iii, see Tab. 1 ). Note the similar I–V relations for both separation procedures.

    Techniques Used: Patch Clamp, Activation Assay, Mass Spectrometry

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    Alomone Labs ns8593
    TRPM7 activity is dispensable for phagocytosis but indispensable for reactive oxygen species (ROS) production of human neutrophils. (A) Phagocytic activity of neutrophils was measured using fluorescent Escherichia coli particles together with human whole blood pre-incubated with <t>NS8593</t> (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min or vehicle (Ctrl, black) and analyzed by flow cytometry (n = 5). Representative dot plot analysis (left panel) and quantification of phagocytic activity (right panel). Data are shown as mean ± s.e.m., two-way repeated measurements ANOVA, Sidak’s multiple comparison. (B) Effects of TRPM7 channel and kinase blockade on lipopolysaccharides (LPS)-triggered ROS production. Human neutrophils were pretreated with or without (Ctrl, black), NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min and then incubated with LPS (10 ng/ml) for 0, 15, 30, 60 and 90 min. Intracellular ROS levels over time (left panel) and quantification at 60 min (middle panel) and 90 min (right panel). Data are normalized to t 0 and represented as mean ± s.e.m.; n=5. Statistics: one-way ANOVA *p
    Ns8593, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rjr 2403 oxalate
    Schematic model. A , impact of Aβ oligomers. In the hippocampus, α7- and α4β2-nAChRs are prominently expressed on inhibitory interneurons; thus, selective binding of soluble Aβ42 oligomers (oAβ42) to α7- and α4β2-nAChRs but not α3β4-nAChRs, reduces neuronal activity in inhibitory cells, leading to a decrease in the release of GABA onto hippocampal excitatory neurons. Consequently, excitatory cells have increased frequency of Ca 2+ transients, resulting in elevated calcineurin (CaN) activity. Calcineurin then dephosphorylates the AMPA receptor (AMPAR) subunit, GluA1, promoting AMPAR endocytosis and resulting in an overall decrease of AMPAR surface expression. This ultimately contributes to disruptions of long-term potentiation. B , reversal of Aβ-induced synaptic and neuronal dysfunction by costimulation with α7- and α4β2-nAChRs agonists. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, costimulation of α7- and α4β2-nAChRs by selective agonists, PNU-282987 (PNU) and <t>RJR-2403</t> <t>Oxalate</t> (RJR), can restore normal activity of both hippocampal inhibitory and excitatory cells, reversing Aβ-induced synaptic dysfunction. This restoration of normal Ca 2+ activity prompts a decrease in calcineurin activity, leading to a decrease in AMPAR dephosphorylation and AMPAR endocytosis, ultimately restoring normal long-term potentiation. However, an agonist for α3β4-nAChRs, NS-3861 (NS), does not appear to have neuroprotective effects. Moreover, nonspecific stimulation of nAChRs by using three agonists together or carbachol is unable to reverse the Aβ effects on neuronal activity and synaptic function, emphasizing the importance of selective costimulation of nAChRs as potential therapeutic approaches.
    Rjr 2403 Oxalate, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TRPM7 activity is dispensable for phagocytosis but indispensable for reactive oxygen species (ROS) production of human neutrophils. (A) Phagocytic activity of neutrophils was measured using fluorescent Escherichia coli particles together with human whole blood pre-incubated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min or vehicle (Ctrl, black) and analyzed by flow cytometry (n = 5). Representative dot plot analysis (left panel) and quantification of phagocytic activity (right panel). Data are shown as mean ± s.e.m., two-way repeated measurements ANOVA, Sidak’s multiple comparison. (B) Effects of TRPM7 channel and kinase blockade on lipopolysaccharides (LPS)-triggered ROS production. Human neutrophils were pretreated with or without (Ctrl, black), NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min and then incubated with LPS (10 ng/ml) for 0, 15, 30, 60 and 90 min. Intracellular ROS levels over time (left panel) and quantification at 60 min (middle panel) and 90 min (right panel). Data are normalized to t 0 and represented as mean ± s.e.m.; n=5. Statistics: one-way ANOVA *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 activity is dispensable for phagocytosis but indispensable for reactive oxygen species (ROS) production of human neutrophils. (A) Phagocytic activity of neutrophils was measured using fluorescent Escherichia coli particles together with human whole blood pre-incubated with NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min or vehicle (Ctrl, black) and analyzed by flow cytometry (n = 5). Representative dot plot analysis (left panel) and quantification of phagocytic activity (right panel). Data are shown as mean ± s.e.m., two-way repeated measurements ANOVA, Sidak’s multiple comparison. (B) Effects of TRPM7 channel and kinase blockade on lipopolysaccharides (LPS)-triggered ROS production. Human neutrophils were pretreated with or without (Ctrl, black), NS8593 (30 µM, red), TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) for 30 min and then incubated with LPS (10 ng/ml) for 0, 15, 30, 60 and 90 min. Intracellular ROS levels over time (left panel) and quantification at 60 min (middle panel) and 90 min (right panel). Data are normalized to t 0 and represented as mean ± s.e.m.; n=5. Statistics: one-way ANOVA *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Activity Assay, Incubation, Flow Cytometry

    TRPM7 is essential for human neutrophil transmigration. (A) Representative purity of primary human neutrophils isolated from whole blood using magnetic cell sorting. (B) Representative human neutrophils stained with Alexa Fluor-488 conjugated anti-CD16 antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. TRPM7 current densities in human neutrophils treated with 30 µM NS8593 (NS8593, red circles, n = 6), 20 µM TG100-115 (TG, blue circles, n = 5), or without (Ctrl, black circles, n = 6) were plotted versus time of the experiment in seconds (s). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of human neutrophils treated with NS8593 (red), TG100-115 (blue) or without (black) (middle panel). Quantification of the current density extracted at +80 mV and displayed as pA/pF at 250 s of human neutrophils treated with NS8593 (NS8593, red, n = 6), 20 µM TG100-115 (TG, blue, n = 5), or without (Ctrl, black, n = 7) (right panel). Data are shown as mean ± s.e.m. *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 is essential for human neutrophil transmigration. (A) Representative purity of primary human neutrophils isolated from whole blood using magnetic cell sorting. (B) Representative human neutrophils stained with Alexa Fluor-488 conjugated anti-CD16 antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. TRPM7 current densities in human neutrophils treated with 30 µM NS8593 (NS8593, red circles, n = 6), 20 µM TG100-115 (TG, blue circles, n = 5), or without (Ctrl, black circles, n = 6) were plotted versus time of the experiment in seconds (s). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of human neutrophils treated with NS8593 (red), TG100-115 (blue) or without (black) (middle panel). Quantification of the current density extracted at +80 mV and displayed as pA/pF at 250 s of human neutrophils treated with NS8593 (NS8593, red, n = 6), 20 µM TG100-115 (TG, blue, n = 5), or without (Ctrl, black, n = 7) (right panel). Data are shown as mean ± s.e.m. *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Transmigration Assay, Isolation, FACS, Staining, Patch Clamp, Activity Assay

    TRPM7 kinase is essential for neutrophil chemotaxis and infiltration in an in vivo murine peritonitis model. (A) Representative purity of primary bone marrow derived murine neutrophils isolated from Trpm7 +/+ and Trpm7 R/R mice using magnetic cell sorting. (B) Representative murine neutrophil stained with PE conjugated anti-Ly6G antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. Primary murine neutrophils were treated with or without NS8593 (30 µM). TRPM7 current densities in neutrophils isolated from Trpm7 +/+ mice (circles) without (black, n = 8) and with NS8593 treatment (red, n = 5) as well as from Trpm7 R/R mice (triangles) without (green, n = 9) or with NS8593 (red, n = 3) were averaged and plotted versus time of the experiment in seconds (s) (left panel). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of murine neutrophils (middle panel). Quantification of the current density extracted at +80 mV and displayed as average current density (pA/pF) at 250 s (right panels). Data are shown as mean ± s.e.m. Statistics: one-way ANOVA *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 kinase is essential for neutrophil chemotaxis and infiltration in an in vivo murine peritonitis model. (A) Representative purity of primary bone marrow derived murine neutrophils isolated from Trpm7 +/+ and Trpm7 R/R mice using magnetic cell sorting. (B) Representative murine neutrophil stained with PE conjugated anti-Ly6G antibody (left panel). Whole-cell patch clamp analysis of TRPM7 ion channel activity. Primary murine neutrophils were treated with or without NS8593 (30 µM). TRPM7 current densities in neutrophils isolated from Trpm7 +/+ mice (circles) without (black, n = 8) and with NS8593 treatment (red, n = 5) as well as from Trpm7 R/R mice (triangles) without (green, n = 9) or with NS8593 (red, n = 3) were averaged and plotted versus time of the experiment in seconds (s) (left panel). Error bars indicate s.e.m. Representative current-voltage relationships extracted at 250 s of murine neutrophils (middle panel). Quantification of the current density extracted at +80 mV and displayed as average current density (pA/pF) at 250 s (right panels). Data are shown as mean ± s.e.m. Statistics: one-way ANOVA *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Chemotaxis Assay, In Vivo, Derivative Assay, Isolation, Mouse Assay, FACS, Staining, Patch Clamp, Activity Assay

    TRPM7 regulates neutrophil function via NFκB and Akt/mTOR signaling pathways. Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR utilizing a Bio-Plex assay and phospho-specific antibodies on lysates of bone marrow derived murine neutrophils of Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Trpm7 +/+ and Trpm7 R/R neutrophils were pre-incubated with or without (control, black) the TRPM7 inhibitor NS8593 (30 µM, red), the TRPM7 kinase blocker TG100-115 (20 µM, blue), or a combination of IPI and NEM (160 and 100 nM, gray), respectively, for 30 min. Presented data depict the phosphorylation status upon stimulation with 10 ng/ml LPS for 30 min of (A, E) NFκB p65 (Ser536), (B, F) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C, G) Akt (Ser473), and (D, H) mTOR (Ser2448). For comparison results from control Trpm7 R/R neutrophils (open green triangle) were taken from the respective panels above. Data are normalized to protein content and represented as mean ± s.e.m.; n = 3, measured in duplicates; a total number of 5–6 mice were used for each genotype. Statistics: one-way ANOVA *p

    Journal: Frontiers in Immunology

    Article Title: TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

    doi: 10.3389/fimmu.2020.606893

    Figure Lengend Snippet: TRPM7 regulates neutrophil function via NFκB and Akt/mTOR signaling pathways. Assessment of the activity of the cell signaling molecules NFκB, Erk1/2, Akt1, and mTOR utilizing a Bio-Plex assay and phospho-specific antibodies on lysates of bone marrow derived murine neutrophils of Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Trpm7 +/+ and Trpm7 R/R neutrophils were pre-incubated with or without (control, black) the TRPM7 inhibitor NS8593 (30 µM, red), the TRPM7 kinase blocker TG100-115 (20 µM, blue), or a combination of IPI and NEM (160 and 100 nM, gray), respectively, for 30 min. Presented data depict the phosphorylation status upon stimulation with 10 ng/ml LPS for 30 min of (A, E) NFκB p65 (Ser536), (B, F) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C, G) Akt (Ser473), and (D, H) mTOR (Ser2448). For comparison results from control Trpm7 R/R neutrophils (open green triangle) were taken from the respective panels above. Data are normalized to protein content and represented as mean ± s.e.m.; n = 3, measured in duplicates; a total number of 5–6 mice were used for each genotype. Statistics: one-way ANOVA *p

    Article Snippet: Bio-Plex Assay For Bio-Plex Pro™ Cell Signaling Assay (Bio-Rad) human and murine neutrophils were pre-incubated with DMSO, NS8593 (30 µM, Alomone Labs), TG100-115 (20 µM, Selleckchem) or a combination of IPI-549 (160 nM, Selleckchem) and nemiralisib (100 nM, Selleckchem) for 30 min and then treated with LPS (10 ng/ml, Sigma-Aldrich) for 30 min at 37°C.

    Techniques: Activity Assay, Plex Assay, Derivative Assay, Mouse Assay, Incubation

    Schematic model. A , impact of Aβ oligomers. In the hippocampus, α7- and α4β2-nAChRs are prominently expressed on inhibitory interneurons; thus, selective binding of soluble Aβ42 oligomers (oAβ42) to α7- and α4β2-nAChRs but not α3β4-nAChRs, reduces neuronal activity in inhibitory cells, leading to a decrease in the release of GABA onto hippocampal excitatory neurons. Consequently, excitatory cells have increased frequency of Ca 2+ transients, resulting in elevated calcineurin (CaN) activity. Calcineurin then dephosphorylates the AMPA receptor (AMPAR) subunit, GluA1, promoting AMPAR endocytosis and resulting in an overall decrease of AMPAR surface expression. This ultimately contributes to disruptions of long-term potentiation. B , reversal of Aβ-induced synaptic and neuronal dysfunction by costimulation with α7- and α4β2-nAChRs agonists. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, costimulation of α7- and α4β2-nAChRs by selective agonists, PNU-282987 (PNU) and RJR-2403 Oxalate (RJR), can restore normal activity of both hippocampal inhibitory and excitatory cells, reversing Aβ-induced synaptic dysfunction. This restoration of normal Ca 2+ activity prompts a decrease in calcineurin activity, leading to a decrease in AMPAR dephosphorylation and AMPAR endocytosis, ultimately restoring normal long-term potentiation. However, an agonist for α3β4-nAChRs, NS-3861 (NS), does not appear to have neuroprotective effects. Moreover, nonspecific stimulation of nAChRs by using three agonists together or carbachol is unable to reverse the Aβ effects on neuronal activity and synaptic function, emphasizing the importance of selective costimulation of nAChRs as potential therapeutic approaches.

    Journal: The Journal of Biological Chemistry

    Article Title: Selective coactivation of α7- and α4β2-nicotinic acetylcholine receptors reverses beta-amyloid–induced synaptic dysfunction

    doi: 10.1016/j.jbc.2021.100402

    Figure Lengend Snippet: Schematic model. A , impact of Aβ oligomers. In the hippocampus, α7- and α4β2-nAChRs are prominently expressed on inhibitory interneurons; thus, selective binding of soluble Aβ42 oligomers (oAβ42) to α7- and α4β2-nAChRs but not α3β4-nAChRs, reduces neuronal activity in inhibitory cells, leading to a decrease in the release of GABA onto hippocampal excitatory neurons. Consequently, excitatory cells have increased frequency of Ca 2+ transients, resulting in elevated calcineurin (CaN) activity. Calcineurin then dephosphorylates the AMPA receptor (AMPAR) subunit, GluA1, promoting AMPAR endocytosis and resulting in an overall decrease of AMPAR surface expression. This ultimately contributes to disruptions of long-term potentiation. B , reversal of Aβ-induced synaptic and neuronal dysfunction by costimulation with α7- and α4β2-nAChRs agonists. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, costimulation of α7- and α4β2-nAChRs by selective agonists, PNU-282987 (PNU) and RJR-2403 Oxalate (RJR), can restore normal activity of both hippocampal inhibitory and excitatory cells, reversing Aβ-induced synaptic dysfunction. This restoration of normal Ca 2+ activity prompts a decrease in calcineurin activity, leading to a decrease in AMPAR dephosphorylation and AMPAR endocytosis, ultimately restoring normal long-term potentiation. However, an agonist for α3β4-nAChRs, NS-3861 (NS), does not appear to have neuroprotective effects. Moreover, nonspecific stimulation of nAChRs by using three agonists together or carbachol is unable to reverse the Aβ effects on neuronal activity and synaptic function, emphasizing the importance of selective costimulation of nAChRs as potential therapeutic approaches.

    Article Snippet: The following agonists were used in this study: 1 μM PNU-120596 (Alomone Labs), 2 μM RJR-2403 Oxalate (Alomone labs), 1 μM NS-3861 (Tocris Bioscience), and 1 μM Carbamoylcholine chloride (carbachol) (Tocris Bioscience).

    Techniques: Binding Assay, Activity Assay, Expressing, De-Phosphorylation Assay

    Schematic model. A ) Impact of Aβ oligomers. In the hippocampus, nAChRs are prominently expressed on inhibitory interneurons, thus, selective binding of soluble Aβ42 oligomers (oAβ42) to α7- and α4β2-nAChRs but not α3β4-nAChRs, reduces neuronal activity in inhibitory cells, leading to a decrease in the release of GABA onto hippocampal excitatory neurons. Consequently, excitatory cells have increased frequency of Ca 2+ transients, resulting in elevated calcineurin (CaN) activity. CaN dephosphorylates the AMPAR subunit, GluA1, promoting AMPAR endocytosis and resulting in an overall decrease of AMPAR surface expression. This ultimately contributes to disruptions of LTP. B) Reversal of Aβ-induced synaptic and neuronal dysfunction by co-stimulation with α7- and α4β2-nAChRs agonists. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, co-stimulation of α7- and α4β2-nAChRs by selective agonists, PNU-282987 (PNU) and RJR-2403 Oxalate (RJR), can restore normal activity of both hippocampal inhibitory and excitatory cells, reversing Aβ-induced synaptic dysfunction. This restoration of normal Ca 2+ activity prompts a decrease in calcineurin activity, leading to a decrease in AMPAR dephosphorylation and AMPAR endocytosis, ultimately restoring normal LTP. However, an agonist for α3β4-nAChRs, NS-3861 (NS), does not appear to have neuroprotective effects. Moreover, non-specific stimulation of nAChRs by using three agonists together or carbachol is unable to reverse the Aβ effects on neuronal activity and synaptic function, emphasizing the importance of selective co-stimulation of nAChRs as potential therapeutic approaches.

    Journal: bioRxiv

    Article Title: Selective co-activation of α7- and α4β2-nicotinic acetylcholine receptors reverses beta-amyloid-induced synaptic dysfunction

    doi: 10.1101/2020.11.05.370080

    Figure Lengend Snippet: Schematic model. A ) Impact of Aβ oligomers. In the hippocampus, nAChRs are prominently expressed on inhibitory interneurons, thus, selective binding of soluble Aβ42 oligomers (oAβ42) to α7- and α4β2-nAChRs but not α3β4-nAChRs, reduces neuronal activity in inhibitory cells, leading to a decrease in the release of GABA onto hippocampal excitatory neurons. Consequently, excitatory cells have increased frequency of Ca 2+ transients, resulting in elevated calcineurin (CaN) activity. CaN dephosphorylates the AMPAR subunit, GluA1, promoting AMPAR endocytosis and resulting in an overall decrease of AMPAR surface expression. This ultimately contributes to disruptions of LTP. B) Reversal of Aβ-induced synaptic and neuronal dysfunction by co-stimulation with α7- and α4β2-nAChRs agonists. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, co-stimulation of α7- and α4β2-nAChRs by selective agonists, PNU-282987 (PNU) and RJR-2403 Oxalate (RJR), can restore normal activity of both hippocampal inhibitory and excitatory cells, reversing Aβ-induced synaptic dysfunction. This restoration of normal Ca 2+ activity prompts a decrease in calcineurin activity, leading to a decrease in AMPAR dephosphorylation and AMPAR endocytosis, ultimately restoring normal LTP. However, an agonist for α3β4-nAChRs, NS-3861 (NS), does not appear to have neuroprotective effects. Moreover, non-specific stimulation of nAChRs by using three agonists together or carbachol is unable to reverse the Aβ effects on neuronal activity and synaptic function, emphasizing the importance of selective co-stimulation of nAChRs as potential therapeutic approaches.

    Article Snippet: The following agonists were used in this study: 1μM PNU-120596 (Alomone labs), 2μM RJR-2403 Oxalate (Alomone labs), 1μM NS-3861 (Tocris Bioscience), and 1μM Carbamoylcholine chloride (carbachol) (Tocris Bioscience).

    Techniques: Binding Assay, Activity Assay, Expressing, De-Phosphorylation Assay