mt7  (Alomone Labs)


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    Alomone Labs mt7
    <t>MT7</t> 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 ( ∗∗∗∗
    Mt7, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    mt7 - by Bioz Stars, 2022-01
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    Images

    1) Product Images from "Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons"

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

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2018.00402

    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 ( ∗∗∗∗
    Figure Legend 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 ( ∗∗∗∗

    Techniques Used: SDS Page

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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) .
    Figure Legend 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) .

    Techniques Used: Over Expression, Staining, Whisker Assay, Expressing

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Over Expression, Activity Assay

    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) .
    Figure Legend 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) .

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

    2) Product Images from "Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase β to Augment Mitochondrial Function and Prevent Neurodegeneration"

    Article Title: Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase β to Augment Mitochondrial Function and Prevent Neurodegeneration

    Journal: Molecular Neurobiology

    doi: 10.1007/s12035-020-01900-x

    Topical MT7 to the eye restored AMPK phosphorylation in the ipsilateral trigeminal ganglia (TG) in CIPN mice (oxaliplatin-treated; CIPN). Control or CIPN mice were treated topically to the left eye (ipsilateral) with MT7 using a stock of 25 ng/ml, and 30 μl of this solution was delivered to the surface of one eye daily, Monday–Friday, for 2 weeks. The right (contralateral) eye received vehicle. At the end of the study, the ipsilateral and contralateral TG from control (Ctrl) or CIPN mice were isolated and subjected to a Western blotting. b Western blot band intensities of phosphorylated AMPK (P-AMPK) and total AMPK (T-AMPK) were normalized to T-AMPK or T-ERK, respectively. The +MT7 is the treated (ipsilateral) eye, and without MT7 reflects the untreated (contralateral) eye. Data are mean ± SEM of N = 4 (ganglia were combined from two mice; 8 mice dissected to give n = 4); ** p
    Figure Legend Snippet: Topical MT7 to the eye restored AMPK phosphorylation in the ipsilateral trigeminal ganglia (TG) in CIPN mice (oxaliplatin-treated; CIPN). Control or CIPN mice were treated topically to the left eye (ipsilateral) with MT7 using a stock of 25 ng/ml, and 30 μl of this solution was delivered to the surface of one eye daily, Monday–Friday, for 2 weeks. The right (contralateral) eye received vehicle. At the end of the study, the ipsilateral and contralateral TG from control (Ctrl) or CIPN mice were isolated and subjected to a Western blotting. b Western blot band intensities of phosphorylated AMPK (P-AMPK) and total AMPK (T-AMPK) were normalized to T-AMPK or T-ERK, respectively. The +MT7 is the treated (ipsilateral) eye, and without MT7 reflects the untreated (contralateral) eye. Data are mean ± SEM of N = 4 (ganglia were combined from two mice; 8 mice dissected to give n = 4); ** p

    Techniques Used: Mouse Assay, Isolation, Western Blot

    MT7 improves mitochondrial function in sensory neurons cultured from STZ-induced diabetic rats. a Oxygen consumption rate (OCR) was measured in the DRG from a 6-week-old diabetic rat cultured overnight and in the presence or absence of 100 nM MT7 for 1 h and in the presence or absence of a 3-h STO-609 (3 μM) pretreatment. OCR in pmol/min was normalized to mg of protein. b Maximal respiration and c spare respiratory capacity were determined after subtracting the non-mitochondrial OCR. Values are mean ± SEM, n = 6–7 replicate cultures, and adjusted to total protein levels. * p
    Figure Legend Snippet: MT7 improves mitochondrial function in sensory neurons cultured from STZ-induced diabetic rats. a Oxygen consumption rate (OCR) was measured in the DRG from a 6-week-old diabetic rat cultured overnight and in the presence or absence of 100 nM MT7 for 1 h and in the presence or absence of a 3-h STO-609 (3 μM) pretreatment. OCR in pmol/min was normalized to mg of protein. b Maximal respiration and c spare respiratory capacity were determined after subtracting the non-mitochondrial OCR. Values are mean ± SEM, n = 6–7 replicate cultures, and adjusted to total protein levels. * p

    Techniques Used: Cell Culture

    MT7 enhanced transcriptional activity of PGC-1α through a CaMKKβ-dependent pathway. a Neurons from a diabetic rat were transfected with reporter plasmid for PGC-1α and, then after 48 h, were treated with varying doses of MT7 for 1 h. Cell lysates underwent dual-luciferase reporter assay for PGC-1α and data presented relative to PGL3 internal control plasmid (indicated by black bar). Values are means ± SEM, n = 3 replicate cultures; * p
    Figure Legend Snippet: MT7 enhanced transcriptional activity of PGC-1α through a CaMKKβ-dependent pathway. a Neurons from a diabetic rat were transfected with reporter plasmid for PGC-1α and, then after 48 h, were treated with varying doses of MT7 for 1 h. Cell lysates underwent dual-luciferase reporter assay for PGC-1α and data presented relative to PGL3 internal control plasmid (indicated by black bar). Values are means ± SEM, n = 3 replicate cultures; * p

    Techniques Used: Activity Assay, Pyrolysis Gas Chromatography, Transfection, Plasmid Preparation, Luciferase, Reporter Assay

    Topical MT7 promotes increased corneal nerve density in diabetic and CIPN mice. a Corneal nerve density measured by confocal microscopy in female Swiss Webster mice before induction of diabetes (day 0) and 28 days later. The same mice underwent further corneal nerve measurements after 42 days of diabetes before receiving daily delivery of MT7 to one eye (30 μl of 25 ng/ml solution), Monday–Friday, for 2 weeks. Sub-basal nerve plexus (SBNP) occupancy was calculated by tracing nerves and using a grid overlay system [ 56 ]. b Corneal nerve density (SBNP) was measured by confocal microscopy in control or oxaliplatin-treated (CIPN) female Swiss Webster mice before (pre) and 2 weeks after (+MT7) daily delivery of MT7 to one eye (30 μl of 25 ng/ml solution), Monday–Friday, for 2 weeks. In this study, the SBNP was measured by tracing all nerves and recording total pixels/image area [ 58 ]. Data are group mean + SEM of N = 7–9/group. Within-group statistical comparisons before and after treatment with STZ or MT7 by paired Student’s t test; * p
    Figure Legend Snippet: Topical MT7 promotes increased corneal nerve density in diabetic and CIPN mice. a Corneal nerve density measured by confocal microscopy in female Swiss Webster mice before induction of diabetes (day 0) and 28 days later. The same mice underwent further corneal nerve measurements after 42 days of diabetes before receiving daily delivery of MT7 to one eye (30 μl of 25 ng/ml solution), Monday–Friday, for 2 weeks. Sub-basal nerve plexus (SBNP) occupancy was calculated by tracing nerves and using a grid overlay system [ 56 ]. b Corneal nerve density (SBNP) was measured by confocal microscopy in control or oxaliplatin-treated (CIPN) female Swiss Webster mice before (pre) and 2 weeks after (+MT7) daily delivery of MT7 to one eye (30 μl of 25 ng/ml solution), Monday–Friday, for 2 weeks. In this study, the SBNP was measured by tracing all nerves and recording total pixels/image area [ 58 ]. Data are group mean + SEM of N = 7–9/group. Within-group statistical comparisons before and after treatment with STZ or MT7 by paired Student’s t test; * p

    Techniques Used: Mouse Assay, Confocal Microscopy

    Neurite outgrowth was raised by pirenzepine and MT7 and blocked by the CaMKKβ inhibitor, STO-609. a Cultured sensory neurons from a normal rat were exposed to 1 μM pirenzepine and a range of STO-609 concentrations for 24 h (pretreated with STO-609 for 1 h). Cells were fixed and immunostained for β-tubulin III, and neurite outgrowth was assessed. Values are means ± SEM, n = 8–10 replicate cultures; * p
    Figure Legend Snippet: Neurite outgrowth was raised by pirenzepine and MT7 and blocked by the CaMKKβ inhibitor, STO-609. a Cultured sensory neurons from a normal rat were exposed to 1 μM pirenzepine and a range of STO-609 concentrations for 24 h (pretreated with STO-609 for 1 h). Cells were fixed and immunostained for β-tubulin III, and neurite outgrowth was assessed. Values are means ± SEM, n = 8–10 replicate cultures; * p

    Techniques Used: Cell Culture

    MT7 elevated phosphorylation of AMPK in a dose and time–dependent manner. a Sensory neurons derived from a 3–5-month STZ-diabetic rat were cultured overnight and then treated with varying doses of MT7 for 1 h. Western blots are shown for P-AMPK, T-AMPK, and T-ERK. b Levels of expression of these proteins presented relative to T-ERK or T-AMPK. c Samples from the experiment in b were probed for P-ACC. d Neurons from a diabetic rat were cultured overnight and then exposed to 100 nM MT7 for varying times. e Levels of P-AMPK presented relative to T-ERK and T-AMPK. For b , c , and e , values are means ± SEM, n = 3 replicate cultures; * p
    Figure Legend Snippet: MT7 elevated phosphorylation of AMPK in a dose and time–dependent manner. a Sensory neurons derived from a 3–5-month STZ-diabetic rat were cultured overnight and then treated with varying doses of MT7 for 1 h. Western blots are shown for P-AMPK, T-AMPK, and T-ERK. b Levels of expression of these proteins presented relative to T-ERK or T-AMPK. c Samples from the experiment in b were probed for P-ACC. d Neurons from a diabetic rat were cultured overnight and then exposed to 100 nM MT7 for varying times. e Levels of P-AMPK presented relative to T-ERK and T-AMPK. For b , c , and e , values are means ± SEM, n = 3 replicate cultures; * p

    Techniques Used: Derivative Assay, Cell Culture, Western Blot, Expressing

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    Alomone Labs mt7
    <t>MT7</t> 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 ( ∗∗∗∗
    Mt7, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 ( ∗∗∗∗

    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

    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.

    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

    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.

    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

    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) .

    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

    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.

    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

    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.

    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

    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) .

    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

    Topical MT7 to the eye restored AMPK phosphorylation in the ipsilateral trigeminal ganglia (TG) in CIPN mice (oxaliplatin-treated; CIPN). Control or CIPN mice were treated topically to the left eye (ipsilateral) with MT7 using a stock of 25 ng/ml, and 30 μl of this solution was delivered to the surface of one eye daily, Monday–Friday, for 2 weeks. The right (contralateral) eye received vehicle. At the end of the study, the ipsilateral and contralateral TG from control (Ctrl) or CIPN mice were isolated and subjected to a Western blotting. b Western blot band intensities of phosphorylated AMPK (P-AMPK) and total AMPK (T-AMPK) were normalized to T-AMPK or T-ERK, respectively. The +MT7 is the treated (ipsilateral) eye, and without MT7 reflects the untreated (contralateral) eye. Data are mean ± SEM of N = 4 (ganglia were combined from two mice; 8 mice dissected to give n = 4); ** p

    Journal: Molecular Neurobiology

    Article Title: Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase β to Augment Mitochondrial Function and Prevent Neurodegeneration

    doi: 10.1007/s12035-020-01900-x

    Figure Lengend Snippet: Topical MT7 to the eye restored AMPK phosphorylation in the ipsilateral trigeminal ganglia (TG) in CIPN mice (oxaliplatin-treated; CIPN). Control or CIPN mice were treated topically to the left eye (ipsilateral) with MT7 using a stock of 25 ng/ml, and 30 μl of this solution was delivered to the surface of one eye daily, Monday–Friday, for 2 weeks. The right (contralateral) eye received vehicle. At the end of the study, the ipsilateral and contralateral TG from control (Ctrl) or CIPN mice were isolated and subjected to a Western blotting. b Western blot band intensities of phosphorylated AMPK (P-AMPK) and total AMPK (T-AMPK) were normalized to T-AMPK or T-ERK, respectively. The +MT7 is the treated (ipsilateral) eye, and without MT7 reflects the untreated (contralateral) eye. Data are mean ± SEM of N = 4 (ganglia were combined from two mice; 8 mice dissected to give n = 4); ** p

    Article Snippet: Cultures were treated with MT7 (Alomone Labs, Jerusalem, Israel), STO-609 (Tocris, Minneapolis, MN, USA), or pirenzepine (Sigma-Aldrich, Oakville, ON, Canada).

    Techniques: Mouse Assay, Isolation, Western Blot

    MT7 improves mitochondrial function in sensory neurons cultured from STZ-induced diabetic rats. a Oxygen consumption rate (OCR) was measured in the DRG from a 6-week-old diabetic rat cultured overnight and in the presence or absence of 100 nM MT7 for 1 h and in the presence or absence of a 3-h STO-609 (3 μM) pretreatment. OCR in pmol/min was normalized to mg of protein. b Maximal respiration and c spare respiratory capacity were determined after subtracting the non-mitochondrial OCR. Values are mean ± SEM, n = 6–7 replicate cultures, and adjusted to total protein levels. * p

    Journal: Molecular Neurobiology

    Article Title: Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase β to Augment Mitochondrial Function and Prevent Neurodegeneration

    doi: 10.1007/s12035-020-01900-x

    Figure Lengend Snippet: MT7 improves mitochondrial function in sensory neurons cultured from STZ-induced diabetic rats. a Oxygen consumption rate (OCR) was measured in the DRG from a 6-week-old diabetic rat cultured overnight and in the presence or absence of 100 nM MT7 for 1 h and in the presence or absence of a 3-h STO-609 (3 μM) pretreatment. OCR in pmol/min was normalized to mg of protein. b Maximal respiration and c spare respiratory capacity were determined after subtracting the non-mitochondrial OCR. Values are mean ± SEM, n = 6–7 replicate cultures, and adjusted to total protein levels. * p

    Article Snippet: Cultures were treated with MT7 (Alomone Labs, Jerusalem, Israel), STO-609 (Tocris, Minneapolis, MN, USA), or pirenzepine (Sigma-Aldrich, Oakville, ON, Canada).

    Techniques: Cell Culture

    MT7 enhanced transcriptional activity of PGC-1α through a CaMKKβ-dependent pathway. a Neurons from a diabetic rat were transfected with reporter plasmid for PGC-1α and, then after 48 h, were treated with varying doses of MT7 for 1 h. Cell lysates underwent dual-luciferase reporter assay for PGC-1α and data presented relative to PGL3 internal control plasmid (indicated by black bar). Values are means ± SEM, n = 3 replicate cultures; * p

    Journal: Molecular Neurobiology

    Article Title: Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase β to Augment Mitochondrial Function and Prevent Neurodegeneration

    doi: 10.1007/s12035-020-01900-x

    Figure Lengend Snippet: MT7 enhanced transcriptional activity of PGC-1α through a CaMKKβ-dependent pathway. a Neurons from a diabetic rat were transfected with reporter plasmid for PGC-1α and, then after 48 h, were treated with varying doses of MT7 for 1 h. Cell lysates underwent dual-luciferase reporter assay for PGC-1α and data presented relative to PGL3 internal control plasmid (indicated by black bar). Values are means ± SEM, n = 3 replicate cultures; * p

    Article Snippet: Cultures were treated with MT7 (Alomone Labs, Jerusalem, Israel), STO-609 (Tocris, Minneapolis, MN, USA), or pirenzepine (Sigma-Aldrich, Oakville, ON, Canada).

    Techniques: Activity Assay, Pyrolysis Gas Chromatography, Transfection, Plasmid Preparation, Luciferase, Reporter Assay

    Topical MT7 promotes increased corneal nerve density in diabetic and CIPN mice. a Corneal nerve density measured by confocal microscopy in female Swiss Webster mice before induction of diabetes (day 0) and 28 days later. The same mice underwent further corneal nerve measurements after 42 days of diabetes before receiving daily delivery of MT7 to one eye (30 μl of 25 ng/ml solution), Monday–Friday, for 2 weeks. Sub-basal nerve plexus (SBNP) occupancy was calculated by tracing nerves and using a grid overlay system [ 56 ]. b Corneal nerve density (SBNP) was measured by confocal microscopy in control or oxaliplatin-treated (CIPN) female Swiss Webster mice before (pre) and 2 weeks after (+MT7) daily delivery of MT7 to one eye (30 μl of 25 ng/ml solution), Monday–Friday, for 2 weeks. In this study, the SBNP was measured by tracing all nerves and recording total pixels/image area [ 58 ]. Data are group mean + SEM of N = 7–9/group. Within-group statistical comparisons before and after treatment with STZ or MT7 by paired Student’s t test; * p

    Journal: Molecular Neurobiology

    Article Title: Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase β to Augment Mitochondrial Function and Prevent Neurodegeneration

    doi: 10.1007/s12035-020-01900-x

    Figure Lengend Snippet: Topical MT7 promotes increased corneal nerve density in diabetic and CIPN mice. a Corneal nerve density measured by confocal microscopy in female Swiss Webster mice before induction of diabetes (day 0) and 28 days later. The same mice underwent further corneal nerve measurements after 42 days of diabetes before receiving daily delivery of MT7 to one eye (30 μl of 25 ng/ml solution), Monday–Friday, for 2 weeks. Sub-basal nerve plexus (SBNP) occupancy was calculated by tracing nerves and using a grid overlay system [ 56 ]. b Corneal nerve density (SBNP) was measured by confocal microscopy in control or oxaliplatin-treated (CIPN) female Swiss Webster mice before (pre) and 2 weeks after (+MT7) daily delivery of MT7 to one eye (30 μl of 25 ng/ml solution), Monday–Friday, for 2 weeks. In this study, the SBNP was measured by tracing all nerves and recording total pixels/image area [ 58 ]. Data are group mean + SEM of N = 7–9/group. Within-group statistical comparisons before and after treatment with STZ or MT7 by paired Student’s t test; * p

    Article Snippet: Cultures were treated with MT7 (Alomone Labs, Jerusalem, Israel), STO-609 (Tocris, Minneapolis, MN, USA), or pirenzepine (Sigma-Aldrich, Oakville, ON, Canada).

    Techniques: Mouse Assay, Confocal Microscopy

    Neurite outgrowth was raised by pirenzepine and MT7 and blocked by the CaMKKβ inhibitor, STO-609. a Cultured sensory neurons from a normal rat were exposed to 1 μM pirenzepine and a range of STO-609 concentrations for 24 h (pretreated with STO-609 for 1 h). Cells were fixed and immunostained for β-tubulin III, and neurite outgrowth was assessed. Values are means ± SEM, n = 8–10 replicate cultures; * p

    Journal: Molecular Neurobiology

    Article Title: Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase β to Augment Mitochondrial Function and Prevent Neurodegeneration

    doi: 10.1007/s12035-020-01900-x

    Figure Lengend Snippet: Neurite outgrowth was raised by pirenzepine and MT7 and blocked by the CaMKKβ inhibitor, STO-609. a Cultured sensory neurons from a normal rat were exposed to 1 μM pirenzepine and a range of STO-609 concentrations for 24 h (pretreated with STO-609 for 1 h). Cells were fixed and immunostained for β-tubulin III, and neurite outgrowth was assessed. Values are means ± SEM, n = 8–10 replicate cultures; * p

    Article Snippet: Cultures were treated with MT7 (Alomone Labs, Jerusalem, Israel), STO-609 (Tocris, Minneapolis, MN, USA), or pirenzepine (Sigma-Aldrich, Oakville, ON, Canada).

    Techniques: Cell Culture

    MT7 elevated phosphorylation of AMPK in a dose and time–dependent manner. a Sensory neurons derived from a 3–5-month STZ-diabetic rat were cultured overnight and then treated with varying doses of MT7 for 1 h. Western blots are shown for P-AMPK, T-AMPK, and T-ERK. b Levels of expression of these proteins presented relative to T-ERK or T-AMPK. c Samples from the experiment in b were probed for P-ACC. d Neurons from a diabetic rat were cultured overnight and then exposed to 100 nM MT7 for varying times. e Levels of P-AMPK presented relative to T-ERK and T-AMPK. For b , c , and e , values are means ± SEM, n = 3 replicate cultures; * p

    Journal: Molecular Neurobiology

    Article Title: Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase β to Augment Mitochondrial Function and Prevent Neurodegeneration

    doi: 10.1007/s12035-020-01900-x

    Figure Lengend Snippet: MT7 elevated phosphorylation of AMPK in a dose and time–dependent manner. a Sensory neurons derived from a 3–5-month STZ-diabetic rat were cultured overnight and then treated with varying doses of MT7 for 1 h. Western blots are shown for P-AMPK, T-AMPK, and T-ERK. b Levels of expression of these proteins presented relative to T-ERK or T-AMPK. c Samples from the experiment in b were probed for P-ACC. d Neurons from a diabetic rat were cultured overnight and then exposed to 100 nM MT7 for varying times. e Levels of P-AMPK presented relative to T-ERK and T-AMPK. For b , c , and e , values are means ± SEM, n = 3 replicate cultures; * p

    Article Snippet: Cultures were treated with MT7 (Alomone Labs, Jerusalem, Israel), STO-609 (Tocris, Minneapolis, MN, USA), or pirenzepine (Sigma-Aldrich, Oakville, ON, Canada).

    Techniques: Derivative Assay, Cell Culture, Western Blot, Expressing