Journal: World Journal of Gastroenterology

Article Title: Inhibition of metabotropic glutamate receptor-5 alleviates hepatic steatosis by enhancing autophagy via activation of the AMPK signaling pathway

doi: 10.3748/wjg.v31.i7.98852

Figure Lengend Snippet: Inhibition of metabotropic glutamate receptor type 5 attenuates hepatocyte steatosis in vitro. A: Protein expression of metabotropic glutamate receptor type 5 (mGluR5) in HepG2 cells treated with different concentrations of free fatty acids (FFAs) for 24 hours; B: Protein expression of mGluR5 in HepG2 cells treated with 0.5 mmol/L FFAs for different Durations; C: CCK-8 assays revealed the effects of MPEP (2.5, 5, 10, 20, and 50 μM) on HepG2 cell viability; D: CCK-8 assays revealed the effects of CHPG (50, 100, 200, 500, and 1000 μM) on HepG2 cell viability; E and F: HepG2 cells were stimulated with 0.5 mmol/L FFAs for 24 hours and then treated with MPEP (10 μM) or CHPG (500 μM) for another 24 hours. The cells were stained with Oil Red O, and the intracellular triglyceride concentration was quantitatively determined. a P < 0.05 vs free fatty acids, b P < 0.01 vs free fatty acids, c P < 0.001 vs free fatty acids; d P < 0.001 vs bovine serum albumin. The data are presented as the mean ± SE. All cell experiments were repeated three times. mGluR5: Metabotropic glutamate receptor type 5; FFAs: Free fatty acids; BSA: Bovine serum albumin.

Article Snippet: Subsequently, cells were treated with FFAs supplemented with the mGluR5 antagonist MPEP (M5435, Sigma-Aldrich) (0, 2.5, 5, 10, 20, 50 μM) or the mGluR5 agonist CHPG (HY-101364, MedChemExpress) (0, 50, 100, 200, 500, 1000 μM) and incubated for an additional 24 hours.

Techniques: Inhibition, In Vitro, Expressing, CCK-8 Assay, Staining, Concentration Assay

Journal: World Journal of Gastroenterology

Article Title: Inhibition of metabotropic glutamate receptor-5 alleviates hepatic steatosis by enhancing autophagy via activation of the AMPK signaling pathway

doi: 10.3748/wjg.v31.i7.98852

Figure Lengend Snippet: Inhibition of metabotropic glutamate receptor type 5 improves lipid accumulation by increasing autophagy in free fatty acids-stimulated HepG2 cells. A and B: HepG2 cells were stimulated with 0.5 mmol/L free fatty acids (FFAs) for 24 hours and then treated with MPEP (10 μM) or CHPG (500 μM) for another 24 hours. Western blotting was used to detect the protein expression of LC3 and p62 in the cells; C and D: HepG2 cells were stimulated with 0.5 mmol/L FFAs for 24 hours and then treated with MPEP (10 μM) or MPEP (10 μM) +chloroquine (20 μM) for another 24 hours. The cells were stained with Oil Red O, and the intracellular triglyceride concentration was quantitatively determined. a P < 0.05 vs free fatty acids; b P < 0.01 vs free fatty acids; c P < 0.001 vs bovine serum albumin; d P < 0.001 vs MPEP; e P < 0.05 vs bovine serum albumin. The data are presented as the mean ± SE. All cell experiments were repeated three times. FFAs: Free fatty acids; BSA: Bovine serum albumin; CQ: Chloroquine.

Article Snippet: Subsequently, cells were treated with FFAs supplemented with the mGluR5 antagonist MPEP (M5435, Sigma-Aldrich) (0, 2.5, 5, 10, 20, 50 μM) or the mGluR5 agonist CHPG (HY-101364, MedChemExpress) (0, 50, 100, 200, 500, 1000 μM) and incubated for an additional 24 hours.

Techniques: Inhibition, Western Blot, Expressing, Staining, Concentration Assay

Journal: World Journal of Gastroenterology

Article Title: Inhibition of metabotropic glutamate receptor-5 alleviates hepatic steatosis by enhancing autophagy via activation of the AMPK signaling pathway

doi: 10.3748/wjg.v31.i7.98852

Figure Lengend Snippet: Inhibition of metabotropic glutamate receptor type 5-induced autophagy via activation of the AMPK signaling pathway. A and B: HepG2 cells were stimulated with 0.5 mmol/L free fatty acids (FFAs) for 24 hours and then treated with MPEP (10 μM) or CHPG (500 μM) for another 24 hours. Western blotting was used to detect the protein expression of P-AMPK, AMPK, P-ULK1 and ULK1 in the cells; C-E: HepG2 cells were stimulated with 0.5 mmol/L FFAs for 24 hours and then treated with MPEP (10 μM), CHPG or MPEP (10 μM) + Compound C (10 μM) for another 24 hours. The cells were stained with Oil Red O (C), and the level of intracellular triglyceride was quantitatively determined (D). Western blot was used to determine transcription factor EB expression in the cytoplasmic and nuclear fractions of HepG2 cells (E). Histone H3 was used as a reference control for nuclear expression. a P < 0.05 vs free fatty acids; b P < 0.01 vs free fatty acids; c P < 0.001 vs bovine serum albumin; d P < 0.01 vs bovine serum albumin; e P < 0.01 vs MPEP. The data are presented as the mean ± SE. All cell experiments were repeated three times. FFAs: Free fatty acids; BSA: Bovine serum albumin; TFEB: Transcription factor EB.

Article Snippet: Subsequently, cells were treated with FFAs supplemented with the mGluR5 antagonist MPEP (M5435, Sigma-Aldrich) (0, 2.5, 5, 10, 20, 50 μM) or the mGluR5 agonist CHPG (HY-101364, MedChemExpress) (0, 50, 100, 200, 500, 1000 μM) and incubated for an additional 24 hours.

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Staining, Control

Journal: World Journal of Gastroenterology

Article Title: Inhibition of metabotropic glutamate receptor-5 alleviates hepatic steatosis by enhancing autophagy via activation of the AMPK signaling pathway

doi: 10.3748/wjg.v31.i7.98852

Figure Lengend Snippet: Knockout of the metabotropic glutamate receptor type 5 gene improved glucose and lipid metabolism and induced hepatic autophagy in high-fat diet-fed mice. A: Weekly weights of male mice; B and C: Blood glucose curve and area under the curve of the Intraperitoneal glucose tolerance test in male mice; D: Serum insulin levels in male mice; E and F: Serum triglyceride and total cholesterol levels in male mice; G and H: Serum low-density lipoprotein and high-density lipoprotein levels in male mice; I: Western blot analysis was used to evaluate the protein expression levels of LC3 and p62 in mice livers. The data are presented as the mean ± SE. a P < 0.05 vs WT/NCD, b P < 0.01 vs WT/NCD, c P < 0.001 vs WT/NCD; d P < 0.05 vs WT/HFD, e P < 0.01 vs WT/HFD, f P < 0.001 vs WT/HFD. WT: Wild-type; KO: Knockout; HFD: High-fat diet; NCD: Normal chow diet; AUC: Area under the curve; TG: Triglyceride; TC: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol.

Article Snippet: Subsequently, cells were treated with FFAs supplemented with the mGluR5 antagonist MPEP (M5435, Sigma-Aldrich) (0, 2.5, 5, 10, 20, 50 μM) or the mGluR5 agonist CHPG (HY-101364, MedChemExpress) (0, 50, 100, 200, 500, 1000 μM) and incubated for an additional 24 hours.

Techniques: Knock-Out, Western Blot, Expressing

Journal: World Journal of Gastroenterology

Article Title: Inhibition of metabotropic glutamate receptor-5 alleviates hepatic steatosis by enhancing autophagy via activation of the AMPK signaling pathway

doi: 10.3748/wjg.v31.i7.98852

Figure Lengend Snippet: Schematic diagram summarizing the underlying mechanism by which metabotropic glutamate receptor type 5 alleviates hepatic steatosis. Metabotropic glutamate receptor type 5 inhibition promotes AMPK phosphorylation, which leads to increased LC3II expression and nuclear translocation of transcription factor EB and the induction of p62 transcription, thereby activating autophagy and reducing lipid accumulation in hepatocytes. mGluR5: Metabotropic glutamate receptor type 5; TFEB: Transcription factor EB.

Article Snippet: Subsequently, cells were treated with FFAs supplemented with the mGluR5 antagonist MPEP (M5435, Sigma-Aldrich) (0, 2.5, 5, 10, 20, 50 μM) or the mGluR5 agonist CHPG (HY-101364, MedChemExpress) (0, 50, 100, 200, 500, 1000 μM) and incubated for an additional 24 hours.

Techniques: Inhibition, Phospho-proteomics, Expressing, Translocation Assay

Journal: Neurotherapeutics

Article Title: PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson’s Disease

doi: 10.1007/s13311-019-00730-7

Figure Lengend Snippet: CAL modulates group I mGluRs-mediated AKT and ERK1/2 signaling in C6 astroglial cells. (A) Cells were treated with DHPG for 30 min followed by coimmunoprecipitation. The cell lysates were incubated with anti-CAL (left) or anti-mGluR5 (right) antibody coupled to protein A/G beads to immunoprecipitate, and the complexes were detected with anti-mGluR1 (left) or anti-CAL (right) antibody via Western blotting to test the interaction (up). Total lysates were probed with anti-mGluR1 or anti-mGluR5 antibody to visualize the equal expression (down). (B) Cells were transiently transfected with plasmids either for CAL knockdown or scramble control. At 48 h after transfection, cells were harvested to detect the level of CAL using Western blotting. The protein level was normalized to β-actin and expressed as the fold difference of scramble. (C) At 48 h after transfection, cells were stimulated with DHPG (100 μM, 5 min) followed by analysis of the activities of AKT and ERK1/2 by Western blotting (left). Protein levels were normalized to β-actin and represented as the percentage difference of the control shRNA group with DHPG stimulation in AKT (middle) or ERK1/2 (right) phosphorylation. (D) Cells after transfection were pretreated with AIDA (100 μM, 25 min) followed by DHPG stimulation (100 μM, 5 min), and cell lysates were detected for the activities of AKT and ERK1/2 by Western blotting (left). Protein levels were normalized to β-actin and represented as the percentage difference of the AKT (middle) or ERK1/2 (right) phosphorylation compared to control shRNA with DHPG stimulation. (E, F) After transfection, cells were pre-incubated with MPEP (10 μM, 25 min) or LY367385 (100 μM, 25 min) to block mGluR5 and mGluR1, respectively, followed by DHPG (100 μM, 5 min) treatment. Samples were analyzed for the activities of AKT and ERK1/2 via Western blotting (left). Protein levels were normalized to β-actin and represented as the percentage difference of p-AKT (middle) or p-ERK1/2 (right) compared with the control shRNA. Either the control shRNA or CAL shRNA group (in panels for quantification) was calculated as the fold difference relative to its corresponding phospho-AKT or phospho-ERK1/2 immunoreactivity by the presence of stimulation over the absence of stimulation. Data shown in this figure represent the means ± SEM of 3 independent experiments. The statistical significance was determined using Student’s t test. *p < 0.05 versus control shRNA group

Article Snippet: 1,4-Diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126, MEK inhibitor), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002, PI3K inhibitor), (S)-3, 5-dihydroxyphenylglycine (DHPG, group I mGluRs agonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA, group I antagonist), (s)-(+)-α-amine-4-carboxy-2-methylbenzene acetic acid ( {"type":"entrez-nucleotide","attrs":{"text":"LY367385","term_id":"1257996803","term_text":"LY367385"}} LY367385 , mGluR1 antagonist), (RS)-2-chloro-5-hidroxyphenylglycine (CHPG, mGluR5 agonist), and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP, mGluR5 antagonist) were all from Tocris Biosciences (Ellisville, MO, USA).

Techniques: Incubation, Western Blot, Expressing, Transfection, Knockdown, Control, shRNA, Phospho-proteomics, Blocking Assay

Journal: Neurotherapeutics

Article Title: PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson’s Disease

doi: 10.1007/s13311-019-00730-7

Figure Lengend Snippet: CAL attenuates rotenone-induced cell death by regulating mGluR5 expression in MN9D cells. (A, C) Cells were infected with LV-CAL or LV-NC followed by rotenone (0.2 μM) treatment for 24 h. The cells were then subjected to the cell viability assay (A) and cell apoptosis analysis via TUNEL staining (C). Scale bar = 100 μm. (B) Cells infected with LV-CAL or LV-NC were treated with 0.2 μM rotenone for different time periods and then cleaved PARP was determined by Western blotting (left). The protein level was normalized to GAPDH and represented as the fold difference of the control group (right). (D, E) Cells were treated with different doses of rotenone (0, 0.05, 0.1, 0.2, 0.5, 1 μM) for 24 h (D) or 0.2 μM rotenone for different times (0, 12, 24, 36, 48 h) (E), and cell lysates were obtained to detect mGluR5 and CAL protein levels using Western blotting (up). Protein levels were normalized to GAPDH and represented as the fold difference of the control group (down). (F) Cells were infected with LV-CAL or LV-NC followed by 0.2 μM rotenone treatment for 24 h, and lysates were obtained to detect mGluR5 expression (up). The protein level was normalized to GAPDH and represented as the fold difference of the control group (down). (G) After infection with LV-sh-CAL or LV-sh-NC, cells were exposed to rotenone (0.2 μM) for 24 h and analyzed for mGluR5 protein level (up). The protein level was normalized to GAPDH and represented as the fold difference of the control group (down). Untreated cells were used as controls (D, E). Cells infected with LV-NC without rotenone treatment were used as controls (A-C, F). Cells infected with LV-sh-NC without rotenone treatment were used as controls (G). Ctrl: control; Rot: Rotenone. Data shown in this figure represent the mean ± SEM of 3 separate experiments. The statistical significance was determined by 1-way ANOVA followed by Dunnett’s test (A, C–G). A comparison of different times of rotenone treatment between LV-CAL and LV-NC was carried out using 2-way ANOVA analysis (B). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control; #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the LV-NC/LV-sh-NC group subjected to corresponding rotenone treatment

Article Snippet: 1,4-Diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126, MEK inhibitor), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002, PI3K inhibitor), (S)-3, 5-dihydroxyphenylglycine (DHPG, group I mGluRs agonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA, group I antagonist), (s)-(+)-α-amine-4-carboxy-2-methylbenzene acetic acid ( {"type":"entrez-nucleotide","attrs":{"text":"LY367385","term_id":"1257996803","term_text":"LY367385"}} LY367385 , mGluR1 antagonist), (RS)-2-chloro-5-hidroxyphenylglycine (CHPG, mGluR5 agonist), and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP, mGluR5 antagonist) were all from Tocris Biosciences (Ellisville, MO, USA).

Techniques: Expressing, Infection, Viability Assay, TUNEL Assay, Staining, Western Blot, Control, Comparison

Journal: Neurotherapeutics

Article Title: PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson’s Disease

doi: 10.1007/s13311-019-00730-7

Figure Lengend Snippet: CAL inhibits ubiquitin-proteasome-dependent degradation of mGluR5 protein in rotenone-treated MN9D cells. (A) Cells were treated with 0.2 μM CHX for 1 h followed by rotenone exposure for 0–60 h and then Western blotting was performed to detect the alteration in mGluR5 protein level (up). The protein level was normalized to GAPDH and represented as the fold difference of the control group (down). Untreated cells were used as controls. **p < 0.01 versus the CHX group. (B, C) Cells with LV-CAL/LV-NC (B) or LV-sh-CAL/LV-sh-NC infection (C) were treated with 0.2 μM CHX for 1 h followed by rotenone exposure for 0–60 h and then Western blotting was performed to detect the alteration in mGluR5 protein levels (up). Protein levels were normalized to GAPDH and represented as the fold difference of the control group. Cells treated with the drug for 0 h in the LV-CAL/LV-NC or LV-sh-CAL/LV-sh-NC group were used as controls. #p < 0.05, ##p < 0.01 compared to the corresponding CHX+Rot group. (D) Cells were infected with LV-CAL or LV-NC and treated with 0.2 μM rotenone for 24 h. The cell lysates were then subjected to immunoprecipitation with anti-mGluR5 antibody coupled to beads, and the precipitated complexes were probed with anti-CAL antibody to examine the interaction between CAL and the anti-ubiquitin antibody to evaluate mGluR5 ubiquitination using Western blotting. The whole lysates were probed with anti-mGluR5 and anti-CAL antibodies to visualize the expression of mGluR5 and CAL (left). Protein levels were normalized to immunoprecipitated mGluR5 and represented as the fold difference of the control group (right). Cells infected with LV-NC without rotenone treatment were used as controls. *p < 0.05 and **p < 0.01 versus control group, #p < 0.05 and ###p < 0.01 compared to the LV-NC+rotenone group. (E) Cells were pretreated with MG132 (1, 2, 5 μM) for 30 min followed by rotenone (0.2 μM) treatment for 24 h and mGluR5 expression was detected by Western blotting (up). The protein level was normalized to GAPDH and represented as the fold difference of the control group (down). Untreated cells were used as controls. *p < 0.05 versus control group, #p < 0.05 compared to the rotenone group. Data shown in all panels of this figure represent the mean ± SEM of 3 independent experiments. The statistical significance was determined by 1-way ANOVA followed by Dunnett’s test (D, E) or 2-way ANOVA (A–C)

Article Snippet: 1,4-Diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126, MEK inhibitor), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002, PI3K inhibitor), (S)-3, 5-dihydroxyphenylglycine (DHPG, group I mGluRs agonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA, group I antagonist), (s)-(+)-α-amine-4-carboxy-2-methylbenzene acetic acid ( {"type":"entrez-nucleotide","attrs":{"text":"LY367385","term_id":"1257996803","term_text":"LY367385"}} LY367385 , mGluR1 antagonist), (RS)-2-chloro-5-hidroxyphenylglycine (CHPG, mGluR5 agonist), and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP, mGluR5 antagonist) were all from Tocris Biosciences (Ellisville, MO, USA).

Techniques: Ubiquitin Proteomics, Western Blot, Control, Infection, Immunoprecipitation, Expressing

Journal: Neurotherapeutics

Article Title: PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson’s Disease

doi: 10.1007/s13311-019-00730-7

Figure Lengend Snippet: CAL suppresses mGluR5-mediated JNK phosphorylation and ameliorates MN9D cell apoptosis induced by rotenone administration. (A) Cells with LV-CAL infection or SP600125 pretreatment were serum-starved for 16 h followed by rotenone treatment (0.2 μM) for 1 h, and then harvested for p-JNK analysis (up). The protein level was normalized to total JNK and represented as the fold difference of the control group (down). (B) Cells were pretreated with SP600125 (100 μM, 30 min) followed by rotenone (0.2 μM) for 24 h and then harvested for PARP detection by Western blotting (up). The protein level was normalized to GAPDH and represented as the fold difference of the control group (down). (C) Cells were infected with LV-CAL or pretreated with SP600125 (100 μM, 30 min) followed by rotenone (0.2 μM) treatment for 24 h, and then cell viability was estimated by the MTT assay. (D) Cells were pretreated with MPEP (100 μM, 30 min) or infected with LV-CAL followed by rotenone (0.2 μM) for 1 h, then cell lysates were collected for p-JNK assessment (up). The protein level was normalized to total JNK and represented as the fold difference of the control group (down). Untreated cells were used as controls. Data shown in all panels of this figure represent the mean ± SEM of 3 independent experiments. The statistical significance was determined by 1-way ANOVA followed by Dunnett’s test. *p < 0.05 and ***p < 0.001 versus control, #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the rotenone group

Article Snippet: 1,4-Diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126, MEK inhibitor), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002, PI3K inhibitor), (S)-3, 5-dihydroxyphenylglycine (DHPG, group I mGluRs agonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA, group I antagonist), (s)-(+)-α-amine-4-carboxy-2-methylbenzene acetic acid ( {"type":"entrez-nucleotide","attrs":{"text":"LY367385","term_id":"1257996803","term_text":"LY367385"}} LY367385 , mGluR1 antagonist), (RS)-2-chloro-5-hidroxyphenylglycine (CHPG, mGluR5 agonist), and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP, mGluR5 antagonist) were all from Tocris Biosciences (Ellisville, MO, USA).

Techniques: Phospho-proteomics, Infection, Control, Western Blot, MTT Assay

Journal: Neurotherapeutics

Article Title: PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson’s Disease

doi: 10.1007/s13311-019-00730-7

Figure Lengend Snippet: CAL inhibits rotenone-induced glutamate release to modulate mGluR5 activity. (A–D) Cells infected with LV-CAL or LV-NC were exposed to rotenone followed by DHPG treatment, and the alteration in protein levels of mGluR5 (A), p-JNK (B), TH (C), and cell viability (D) was evaluated. Protein levels shown in corresponding graphs were normalized to GAPDH or total JNK and represented as the fold difference of the control group. (E) Cells were transfected with CMV-hCAL-IRES-ZsGreen or vector CMV-IRES-ZsGreen followed by 0.2 μM rotenone treatment for 24 h and then the supernatant was assessed for glutamate concentration. (F, G) Cells transfected with shGFP or shCAL were pretreated with MPEP (100 μM, 30 min) followed by rotenone for 24 h, the supernatant was obtained for the glutamate assay (F), and the cell lysates were probed with anti-mGluR5 and anti-CAL antibodies by Western blotting (G). Protein levels were normalized to GAPDH and represented as the fold difference of the control group. LV-NC infected cells without rotenone treatment were used as controls (A–D). Cells transfected with vector without rotenone administration were used as controls (E–G). Data shown in all panels of this figure represent the mean ± SEM of 3 independent experiments. The statistical significance was determined by 1-way ANOVA followed by Dunnett’s test. **p < 0.01 and ***p < 0.001 versus control, #p < 0.05, ##p < 0.01, and ###p < 0.001 compared to the rotenone treatment group, &p < 0.05 and &&p < 0.01 compared to the rotenone-treated LV-CAL group or rotenone- and MPEP-treated shGFP group

Article Snippet: 1,4-Diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126, MEK inhibitor), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002, PI3K inhibitor), (S)-3, 5-dihydroxyphenylglycine (DHPG, group I mGluRs agonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA, group I antagonist), (s)-(+)-α-amine-4-carboxy-2-methylbenzene acetic acid ( {"type":"entrez-nucleotide","attrs":{"text":"LY367385","term_id":"1257996803","term_text":"LY367385"}} LY367385 , mGluR1 antagonist), (RS)-2-chloro-5-hidroxyphenylglycine (CHPG, mGluR5 agonist), and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP, mGluR5 antagonist) were all from Tocris Biosciences (Ellisville, MO, USA).

Techniques: Activity Assay, Infection, Control, Transfection, Plasmid Preparation, Concentration Assay, Glutamate Assay, Western Blot

Journal: Neurotherapeutics

Article Title: PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson’s Disease

doi: 10.1007/s13311-019-00730-7

Figure Lengend Snippet: CAL overexpression upregulates mGluR5 expression to protect against cell death in the rotenone-induced animal model of PD. (A) The treatment of rats is shown in the scheme. (B) At 4 weeks after AAV injection into the SN of animals, GFP and TH were immuno-labeled (red) and images were captured by confocal microscopy (up), and the expression of CAL in SN tissues was detected by Western blotting (down). Scale bar = 75 μm. The protein level was normalized to β-tubulin and represented as the fold difference of the AAV-GFP group (n = 3 or 4/group). (C) Following virus injection, animal behavioral asymmetry with the apomorphine-induced rotation test and the total hemilateral rotations within 30 min were evaluated (up) (n = 8/group). Body weight each week during surgery was measured (down) (n = 8/group). (D) TH-positive immunoreactive neurons in the SN and TH-IR fibers in the striatum were detected by immunohistochemistry. Scale bar = 500 μm (SN), 1 mm (striatum). Quantification of TH-positive immunoreactivity was represented by the ratio of lesion side to intact side (n = 3 or 4/group). (E) Brain lysates from the left (L) and right (R) striatum were analyzed by Western blotting to observe the alteration in mGluR5, CAL, and p-JNK protein levels. Protein levels were normalized to β-tubulin and represented as the ratio of lesion side to intact side and expressed as the fold difference of the control group (n = 3 or 4/group). (F) mGluR5 and CAL staining in SN sections were detected by immunohistochemistry (left) and the number of positive cells was analyzed (right). The quantification represented the percentage in the control group. The black arrows represented positive cells that were stained brown (scale bar = 50 μm). Data shown in all panels in this figure represent the mean ± SD. The statistical significance was determined using Student’s t test (B) or 1-way ANOVA followed by Dunnett’s test (C–F). Vehicle groups with AAV-GFP infection were used as controls. **p < 0.01, ***p < 0.001 versus control, #p < 0.05, ##p < 0.01 compared to the rotenone lesion with AAV-GFP delivery group

Article Snippet: 1,4-Diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126, MEK inhibitor), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002, PI3K inhibitor), (S)-3, 5-dihydroxyphenylglycine (DHPG, group I mGluRs agonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA, group I antagonist), (s)-(+)-α-amine-4-carboxy-2-methylbenzene acetic acid ( {"type":"entrez-nucleotide","attrs":{"text":"LY367385","term_id":"1257996803","term_text":"LY367385"}} LY367385 , mGluR1 antagonist), (RS)-2-chloro-5-hidroxyphenylglycine (CHPG, mGluR5 agonist), and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP, mGluR5 antagonist) were all from Tocris Biosciences (Ellisville, MO, USA).

Techniques: Over Expression, Expressing, Animal Model, Injection, Labeling, Confocal Microscopy, Western Blot, Virus, Immunohistochemistry, Control, Staining, Infection

Journal: Neurotherapeutics

Article Title: PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson’s Disease

doi: 10.1007/s13311-019-00730-7

Figure Lengend Snippet: Effect of CAL deficiency on rotenone-induced toxicity in vivo. (A) Following 4 weeks of AAV delivery, the SN sections were subjected to TH labeling (red) and the images with GFP immunofluorescence were obtained by confocal microscopy (up). Scale bar = 100 μm. The interference efficiency of AAV-shCAL was investigated by Western blotting (down, left) and quantification analysis was represented as the fold difference of the AAV-scramble group (down, right). (B) After virus injection, the animals underwent the apomorphine-induced rotational test to assess behavioral asymmetry. The total number of hemilateral rotational turns within 30 min was evaluated (up) (n = 7/ group) and the body weight during surgery was monitored (down) (n = 7/group). (C) TH-positive immunoreactivity was assessed by immunohistochemistry in the SN and striatum. Scale bar = 500 μm (SN), 1 mm (striatum). Quantification of TH-IR neurons in the SN or TH-IR fibers in the striatum represented the ratio of lesion side to intact side (n = 3 or 4/group). (D) The alterations in mGluR5, CAL, and p-JNK in the left (L) and right (R) striatum were assessed by Western blotting (up). Protein levels were normalized to GAPDH (TH, mGluR5, CAL) or total JNK (p-JNK) and represented as the ratio of lesion side to intact side by the expression of fold difference of the control group (n = 3/group). (E) mGluR5- and CAL-positive immunoreactivity in the SN was analyzed by immunohistochemistry (left) and the number of positive cells was analyzed (right). The quantification represented the percentage in the control group (n = 3 or 4/group). The black arrows represent positive cells that were stained brown (scale bar = 50 μm). Vehicle groups with AAV-scramble virus delivery were used as controls. Data shown in all panels of this figure represent the mean ± SD. Statistical significance was determined using Student’s t test (A) or 1-way ANOVA followed by Dunnett’s test (B–E). *p < 0.05, **p < 0.01, ***p < 0.001 versus control, #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the rotenone lesion with AAV-scramble delivery group

Article Snippet: 1,4-Diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126, MEK inhibitor), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002, PI3K inhibitor), (S)-3, 5-dihydroxyphenylglycine (DHPG, group I mGluRs agonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA, group I antagonist), (s)-(+)-α-amine-4-carboxy-2-methylbenzene acetic acid ( {"type":"entrez-nucleotide","attrs":{"text":"LY367385","term_id":"1257996803","term_text":"LY367385"}} LY367385 , mGluR1 antagonist), (RS)-2-chloro-5-hidroxyphenylglycine (CHPG, mGluR5 agonist), and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP, mGluR5 antagonist) were all from Tocris Biosciences (Ellisville, MO, USA).

Techniques: In Vivo, Labeling, Immunofluorescence, Confocal Microscopy, Western Blot, Virus, Injection, Immunohistochemistry, Expressing, Control, Staining

Journal: Neurotherapeutics

Article Title: PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson’s Disease

doi: 10.1007/s13311-019-00730-7

Figure Lengend Snippet: CAL regulated mGluR5 degradation through the interaction and deficiency of CAL abrogated the beneficial effect of MPEP in vivo. (A) The brain tissues were lysed for coimmunoprecipitation. Lysates were first incubated with mGluR5 antibody followed by protein-G beads to immunoprecipitate; the complex was detected with anti-ubiquitin or anti-CAL antibodies by Western blot to test mGluR5 ubiquitination and interaction of CAL (up). The whole lysates were detected with mGluR5 and CAL antibodies to visualize the expression (down). Protein levels were normalized to immunoprecipitated mGluR5 and represented as the fold different ratio (right/left) of the control group. (B) Immunofluorescence of mGluR5 (red) and CAL (green) in the SN section showing the protein expression and colocalization. Scale bar = 50 μm. (C) Rats were lesioned by rotenone for 2 weeks followed by CAL knockdown and MPEP administered for 3 weeks as the process shown. (D) The interference efficiency of AAV-shCAL was detected by Western blotting (n = 3, left) and quantification analysis was represented as the ratio of lesion side to intact side by the fold difference of the AAV-scramble group (right). (E) The body weight and apomorphine-induced rotational behavior were monitored (n = 6–8/group). (F) TH-positive immunoreactivity in SN and striatum was detected by immunohistochemistry and quantification represented as the ratio of lesion side to intact side (n = 3). Scale bar = 500 μm (SN), 1 mm (striatum). (G, H) The expression of mGluR5, CAL, TH, and p-JNK were detected by Western blot. Protein levels were normalized to β-tubulin or total JNK and represented as the ratio of lesion side to intact side by the expression of fold difference of the control group (n = 3). Data shown represent the mean ± SD. The statistical significance was determined by 1-way ANOVA followed by Dunnett’s test. *p < 0.05, **p < 0.01, ***p < 0.001 versus control, #p < 0.05, ##p < 0.01, ###p < 0.001 compared to rotenone group, &p < 0.05, &&p < 0.01, &&&p < 0.001 compared to rotenone group with MPEP treatment

Article Snippet: 1,4-Diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126, MEK inhibitor), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002, PI3K inhibitor), (S)-3, 5-dihydroxyphenylglycine (DHPG, group I mGluRs agonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA, group I antagonist), (s)-(+)-α-amine-4-carboxy-2-methylbenzene acetic acid ( {"type":"entrez-nucleotide","attrs":{"text":"LY367385","term_id":"1257996803","term_text":"LY367385"}} LY367385 , mGluR1 antagonist), (RS)-2-chloro-5-hidroxyphenylglycine (CHPG, mGluR5 agonist), and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP, mGluR5 antagonist) were all from Tocris Biosciences (Ellisville, MO, USA).

Techniques: In Vivo, Incubation, Ubiquitin Proteomics, Western Blot, Expressing, Immunoprecipitation, Control, Immunofluorescence, Knockdown, Immunohistochemistry

Journal: Neurotherapeutics

Article Title: PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson’s Disease

doi: 10.1007/s13311-019-00730-7

Figure Lengend Snippet: Schematic graph of the neuroprotective role of scaffold protein CAL via regulation of mGluR5. The association of CAL with mGluR5 controlled rotenone-induced receptor overactivation by decreasing the level of glutamate and u-regulating the protein level of mGluR5 through ubiquitination-proteasome-mediated mGluR5 degradation, and further mediation of the related signaling pathway to exert neuroprotection against cell apoptosis

Article Snippet: 1,4-Diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126, MEK inhibitor), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002, PI3K inhibitor), (S)-3, 5-dihydroxyphenylglycine (DHPG, group I mGluRs agonist), (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA, group I antagonist), (s)-(+)-α-amine-4-carboxy-2-methylbenzene acetic acid ( {"type":"entrez-nucleotide","attrs":{"text":"LY367385","term_id":"1257996803","term_text":"LY367385"}} LY367385 , mGluR1 antagonist), (RS)-2-chloro-5-hidroxyphenylglycine (CHPG, mGluR5 agonist), and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP, mGluR5 antagonist) were all from Tocris Biosciences (Ellisville, MO, USA).

Techniques: Ubiquitin Proteomics