p2x7 receptors  (Alomone Labs)

Journal: The Journal of Biological Chemistry

Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

doi: 10.1074/jbc.M109.062364

Figure Lengend Snippet: Differential binding of pro-BDNF and neurotension to sortilin and pro-sortilin. A , SPR analysis showing that unprocessed pro-BDNF (50 n m ) binds nearly as efficiently to the receptor in the presence (pro-sortilin) as in the absence (sortilin) of the receptor

Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

Techniques: Binding Assay, SPR Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

doi: 10.1074/jbc.M109.062364

Figure Lengend Snippet: Selective competition of ligands by sortilin-derived peptide antagonist. SPR binding analysis of 50 n m unprocessed pro-BDNF ( A ), 50 n m unprocessed pro-NGF ( B ), and 90 n m RAP ( C ) to immobilized sortilin in the absence and presence of the sort166–181

Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

Techniques: Derivative Assay, SPR Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *

doi: 10.1074/jbc.M109.062364

Figure Lengend Snippet: Mutation of the linear binding site specifically impairs binding of both the NGF and the BDNF pro-domains. SPR analysis showing reduced binding of equal amounts (analyte concentration: 200 n m ) of the soluble extracellular domains of sortilin-4A compared

Article Snippet: Internalization of bound ligands was studied by the incubation of cells for 30 min at 37 °C with anti-sortilin polyclonal antibodies (10 μg/ml), GST-NGFpro (1 μg/ml), or 100 n m BDNF pro-domain (Alomone) diluted in culture medium.

Techniques: Mutagenesis, Binding Assay, SPR Assay, Concentration Assay

Journal: Frontiers in Neuroscience

Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

doi: 10.3389/fnins.2018.00402

Figure Lengend Snippet: MT7 and pirenzepine elevated sequestration of trimeric G proteins associated with M 1 R in SH-SY5Y cells. (A) Diagrammatic representation of the experimental strategy. (B–D) Halo-tagged M 1 R was expressed transiently in sensory neurons and then treated with 100 nM MT7 or 1 μM PZ for 1 h. Cells were then lysed and Halo-M 1 R was pulled down using halo-linked resin. The halo tag was cleaved by TEV protease and the cleaved M 1 R associated multiprotein complex (MPC) was resolved in denaturing SDS-PAGE and immunoblotted using (B) anti-M 1 R, (C) anti-Gγ2/3/4/7, and (D) anti-Gα12/13 antibodies. (E,F) Scatter plot showing the relative amount of G proteins (Gα and Gγ, respectively) associated with M 1 R following drug treatment. The data represent mean ± SEM of three independent experiments. p -values ( ∗∗∗∗

Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

Techniques: SDS Page

Journal: Frontiers in Neuroscience

Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

doi: 10.3389/fnins.2018.00402

Figure Lengend Snippet: MT7 and pirenzepine elevated sequestration of trimeric G proteins in a protein complex associated with M 1 R in SH-SY5Y cells. (A,B) BN-PAGE analysis showing recruitment and sequestration of G proteins to M 1 R following MT7 treatment. GFP-M 1 R transfected cells were treated with 100 nM MT7 and incubated for 1 h. Cell lysates were then separated on 1D BN PAGE followed by 2D SDS-PAGE, (A) Control, (B) 100 nM MT7 treatment. Top panel: Coomassie stained gel piece showing 1D BN-PAGE separation of native page protein molecular weight marker. The red horizontal and vertical arrows indicate the direction of the 1D BN-PAGE and 2D SDS-PAGE, respectively. The blue rectangle shows PTMs of GFP-M 1 R associated with 1000 kDa and > 1200 kDa MPCs. The red circle indicates native form of the GFP-M 1 R associated with the MPCs. The red arrow connecting the red circles indicates shift of molecular weight in MPC due to recruitment of G proteins following drug treatment. The red rectangle in the bottom panel shows the co-migration of possible interacting G proteins with the MPCs.

Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Incubation, SDS Page, Staining, Clear Native PAGE, Molecular Weight, Marker, Migration

Journal: Frontiers in Neuroscience

Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

doi: 10.3389/fnins.2018.00402

Figure Lengend Snippet: Altered mitochondrial trafficking in M 1 R overexpressing neurons was rescued by antagonist treatment. (A) Representative images (top panels) of the first frame of a series of live cell time-lapse images showing the expression of DsRed2Mito7 in the mitochondria of GFP/M 1 R–GFP expressing neurons. The circles represent mitochondria identified by MTrackJ plugin, which then tracked migration through time in a series of time lapse images and calculated velocity of specific mitochondria. White/blue line represents neurite trace. The bottom panel represents Kymographs generated from live cell time-lapse images. The Kymograph was generated using ImageJ Kymograph plugin. The X -axis represents the physical location of mitochondria on the neurite, and the Y -axis represents the location of mitochondria in time. Streak of particles traversing the kymograph from left to right in angular lines indicates retrograde/anterograde mitochondrial motion. (B) Whisker plot showing mitochondrial velocity. DRG neurons were cultured in LGF media supplemented with 100 nM MT7 or 1 μM pirenzepine for 48 h following transfection. N = 40 from three independent experiments, p -value by one-way ANOVA followed by Dunnett’s multiple comparisons test. (C) Binning of the entire data set presented in (B) . (D,E) Immunofluorescence images showing mito7-RFP and β-tubulin-III staining in the GFP/GFP-M 1 R expressing neurites. White arrows indicate continuous/discontinuous tubulin cytoskeleton in GFP/GFP-M 1 R expressing neurites, respectively. Scale bar: 5 μm.

Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

Techniques: Expressing, Migration, Generated, Whisker Assay, Cell Culture, Transfection, Immunofluorescence, Staining

Journal: Frontiers in Neuroscience

Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

doi: 10.3389/fnins.2018.00402

Figure Lengend Snippet: Restoration of cytoskeleton, mitochondrial abundance and neurite outgrowth by M 1 R antagonists MT7 and pirenzepine treatment. (A) Time-lapse live confocal images of GFP-M 1 R over-expression in sensory neurons showing MT7 induced re-localization of M 1 R from perikaryon to neurites. The overexpressed neurons were grown in defined media for 48 h and imaged (left panel). Neurons were then treated with 100 nM MT7 for 24 h and imaged (middle panel). Right panel: The same neuron depicted left was fixed and stained for β-tubulin III to show continuity of cytoskeleton. Scale bars: 50 μm. (B,C) Whiskers box (Tukey) showing total neurite outgrowth per neuron (B) and average neurite width per neuron (C) . p -value calculated by one-way ANOVA, N = 224, 230 and 198 cells, respectively, for (A) and N = 1250, 1268, 1280, and 1306 cells, respectively, for (B) . Asterisks indicate p -value calculated by unpaired t -test. (D) Whisker box plot showing amount of mitochondria in the M 1 R expressing neurons treated with 100 nM MT7 or 1 μM pirenzepine. N = 101, p -values were calculated by one-way ANOVA followed by Dunnett’s multiple comparisons tests. (E) Binning of the entire data set presented in (D) .

Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

Techniques: Over Expression, Staining, Whisker Assay, Expressing

Journal: Frontiers in Neuroscience

Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

doi: 10.3389/fnins.2018.00402

Figure Lengend Snippet: M 1 R antagonists, MT7 and pirenzepine, augment neurite outgrowth in primary sensory neurons and M 1 R overexpression inhibits neurite outgrowth. (A,B) Whiskers box (Tukey) showing total neurite outgrowth per neuron. Neurons were grown for 48 h in defined media containing LGF (A) or HGF (B) condition and treated with 100 nM MT7 or 1 μM pirenzepine (PZ), respectively. N = 1249 (LGF) and 1517 (HGF), respectively. P -values were calculated by one-way ANOVA followed by post hoc multiple comparison tests. Dunnett’s multiple comparisons test was used to compare the MT7 and PZ treatment groups with the control group and Sidak’s multiple comparisons test was used to compare between the MT7 and PZ treatment groups; ∗ indicates the p -value obtained by Sidak’s multiple comparisons test. (C,D) Binning of the entire data set presented in (A,B) . (E) Immunoblot showing GFP-tagged muscarinic receptors (M 1 R to M 5 R) and GFP expression in transfected adult rat DRG neurons. pEGFP-C1-(M 1 R-M 5 R) plasmids were transfected in to DRG neurons and the lysate was resolved in SDS page and subsequently immunoblotted with anti-M 1 R (bottom panel) and anti-GFP (top panel) antibodies. (F) Time lapse confocal images showing increasing internalization (white arrows) of the GFP-M 1 R following treatment with carbachol (10 μM). Scale bar: 10 μm. (G) Immunofluorescence images showing colocalization of 24h CCh treated GFP-M1R with endosomal marker Rab5. Scale bar: 10 μm. (H) Whiskers box (Tukey) showing total neurite outgrowth per neuron, N = 634 (GFP), and N = 553 (GFP-M 1 R), neurons, respectively. P -value was calculated by t -test (unpaired). (I) Immunofluorescence images showing β-tubulin III staining and corresponding neurite trace (red lines) images in GFP and GFP-M 1 R overexpressed neurons. The total neurite outgrowth measurement was performed in Cellomics ArrayScan HCS Reader using neuronal profiling software. Scale bar: 10 μm.

Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

Techniques: Over Expression, Expressing, Transfection, SDS Page, Immunofluorescence, Marker, Staining, Software

Journal: Frontiers in Neuroscience

Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

doi: 10.3389/fnins.2018.00402

Figure Lengend Snippet: Model explaining the effect of M 1 R overexpression and antagonism on tubulin associated cytoskeleton and mitochondrial trafficking. (Left) M 1 R overexpressing neurons have increased basal activity in response to secreted acetylcholine signaling through overexpressed receptor. This is turn causes recruitment of trimeric G proteins that destabilize tubulin polymers by increasing intrinsic GTPase activity of tubulin. The lack of tubulin cytoskeleton in M 1 R overexpressing neuron leads to decreased mitochondrial trafficking and stagnation of mitochondria in the neurites that impairs outgrowth. (Right) M 1 R overexpression in the presence of antagonists MT7 and pirenzepine (PZ) stabilizes tubulin polymerization. The antagonists bind to the M 1 R and may stabilize a specific structural ensemble, which in turn recruit trimeric G proteins. However, the antagonist mediated M 1 R structural ensemble may sequester the bound G proteins and makes them unavailable for exerting their effect on tubulin polymerization which in turn stabilizes microtubule cytoskeleton and promotes mitochondrial trafficking and neurite outgrowth.

Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

Techniques: Over Expression, Activity Assay

Journal: Frontiers in Neuroscience

Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

doi: 10.3389/fnins.2018.00402

Figure Lengend Snippet: Knockdown of Gα13 reversed M 1 R overexpression-induced inhibition of neurite outgrowth. (A) Immunoblots showing relative expression of Gα12 and Gα13 proteins in cultured DRG neurons. (B) Scatter plot showing relative amount of Gα12 and Gα13 proteins in cultured sensory neurons. N = 5 independent experiments. p -value was calculated by unpaired t -test. (C) Immunoblots showing siRNA (cocktail of 3 siRNAs targeted to rat Gα) based knockdown of Gα13 protein in cultured adult rat DRG neurons. (D,F) Whisker box (Tukey) showing total neurite outgrowth per neuron. DRG neurons were transfected with a pEFGP-C1-M 1 R plasmid and siRNA using Amaxa nucleofection reagent and allowed to grow for 48 h. Scrambled siRNAs were used for control. In drug treatment groups, neurons were cultured in media supplemented with 100 nM MT7 or 1 μM pirenzepine following transfection. The neurons were fixed after 48 h of culture, stained with β-tubulin III and imaged using Cellomics ArrayScan HCS Reader. p -value by unpaired t -test or one-way ANOVA test followed by Dunnett’s multiple comparisons tests. N = 432/452 (in D ) and 406/694 (in F ). (E,G) Binning of the entire data set presented in (D,F) .

Article Snippet: Cultures were treated with 100 nM MT7 (M-200, Alomone Labs, Jerusalem, Israel) or 1 μM pirenzepine (P7412, Sigma).

Techniques: Over Expression, Inhibition, Western Blot, Expressing, Cell Culture, Whisker Assay, Transfection, Plasmid Preparation, Staining