Journal: Biomolecules

Article Title: yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated Proteolysis

doi: 10.3390/biom13060987

Figure Lengend Snippet: Chemicals used.

Article Snippet: Ethidium Bromide , Genesee Scientific , 20276.

Techniques:

Journal: Journal of Cancer

Article Title: Differences in Endothelin B Receptor Isoforms Expression and Function in Breast Cancer Cells

doi: 10.7150/jca.41004

Figure Lengend Snippet: EDNRB isoforms differentially regulate breast cancer invasion. Transfecting siRNA specific to EDNRB alongside control siRNA into breast cancer cell lines results in (A) significant reduction in mRNA expression (p=0.027) and a significant increase in in vitro invasion toward ET3 (p=0.02) and (B) significant reduction in mRNA expression (p=0.018) but a non-significant increase in invasion toward ET3 (p=0.18) in MDA-MB-231 cells. Over-expressing mammalian plasmids encoding for (C) EDNRB-442 significantly increased mRNA expression (p=.0002) and significantly decreased in vitro invasion toward ET3 (p=0.008); however, expressing EDNRB isoforms EDNRB-436 (D) and EDNRB-532 (E) significantly increased expression (p=0.0002; p=0.0026) but did not alter invasion toward ET3 (p=0.39; p=0.25, respectively). All graphs are from 3 biological replicates.

Article Snippet: Endothelin 3 was purchased from Sigma Aldrich and reconstituted to 1 mg/ml in water. siRNA specific for EDNRB (sc-39962), along with control siRNA (sc-37007) were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Control, Expressing, In Vitro

Journal: Journal of Cancer

Article Title: Differences in Endothelin B Receptor Isoforms Expression and Function in Breast Cancer Cells

doi: 10.7150/jca.41004

Figure Lengend Snippet: EDNRB isoforms differentially regulate cellular signaling. Western blotting of lysates extracted from (A) MDA-MB-231 cells transfected with EDNRB isoforms and (B) MCF-7 cells transfected EDNRB-specific siRNA were probed with antibodies specific for phosphorylated AKT1 and phosphorylated AKT2 at S473 (pAKT1, pAKT2), pan AKT, phosphorylated ERK (pERK), pan ERK, and GAPDH. Bands were quantitated using ImageJ Software and normalized to GAPDH intensity. (A) MDA-MB-231 cells transfected with EDNRB-442 had significantly higher levels of pAKT1 (ANOVA p=0.013) but not pAKT2 (p=0.28) or pERK (p=0.42); (B) MCF-7 cells transfected with EDNRB-siRNA had significantly reduced pAKT1/AKT levels (p=0.097). (C) Analysis of breast cancer cell lines from Cancer Cell Line Encyclopedia data (CCLE) shows a non-significant positive association between EDNRB and active AKT (left); this trend was not observed in non-transformed breast cell lines (right). (D) TCGA breast cancer exon expression data was separated by subtype and analyzed by median EDNRB expression using two isoform-specific probes that recognize EDNRB-442 or EDNRB-532; pAKT levels at both S473 and T308 sites from the RPPA dataset were compared between EDNRB-high and low groups (n=60 for both groups) in basal and (left) luminal A (right) breast cancer subtypes, and (E) normal breast tissue.

Article Snippet: Endothelin 3 was purchased from Sigma Aldrich and reconstituted to 1 mg/ml in water. siRNA specific for EDNRB (sc-39962), along with control siRNA (sc-37007) were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Western Blot, Transfection, Software, Transformation Assay, Expressing

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: ( A, B ) Highly expressed GPCR transcripts in mouse DRGs. RNAseq shows mRNA expression levels (FPKM) of the top 10 GPCR transcripts (A, n = 3) and the top 10 orphan GPCR transcripts (B, n = 3). Gpr37l1 expression is highlighted in red bars. Note that the Gpr37 expression is much lower than Gpr37l1 . ( C, D ) Highly expressed GPCR transcripts in human DRGs. Normalized microarray shows mRNA expression levels (intensity) of the top 10 GPCR transcripts (C, n = 214) and the top 10 orphan GPCR transcripts (D, n = 214). GRP37L1 expression is highlighted in red bars. GPR37 expression is included for comparison. Data are expressed as mean ± SEM.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Expressing, Microarray, Comparison

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: ( A ) Single-cell RNAseq showing expression of Gpr37l1 and Kcnj10 transcripts in SGCs of mouse DRG tissues . ( B, C ) Images of RNAscope in situ hybridization (ISH) of Gpr37l1 expression in trigeminal ganglion (TG, B) and nodose ganglion (NG, C). Small boxes are enlarged in the right panels. Neurons are lightly labeled with Nissl staining (white). Scales, 25 µm. ( D ) RNAscope images of Gpr37l1 expression in NG of Gpr37l1 +/+ mice (WT, top) and Gpr37l1 -/- mice (bottom). Small boxes are enlarged in the right panels. Scales, 25 µm. ( E ) Double staining of RNAscope ISH showing distinct expression of Gpr37 (red) and Gpr37l1 (white) in mouse NG. Scale = 25 µm.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Expressing, RNAscope, In Situ Hybridization, Labeling, Staining, Double Staining

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: ( A-C ) Flow cytometry data showing GPR37L1 expression in DRGs of WT and Gpr37l1 -/- mice. ( A ) Gating strategy. (B) GPR37L1 expression in DRGs of WT and Gpr37l1 -/- mice. Left, representative images of flow cytometry and histograms after gating by Glast-PE. Right, Quantification of GPR37L1+ cells in the Glast-PE+ population ( n = 6 for WT and n = 5 for KO mice). (C) Left, representative images of flow cytometry and histogram after gating by Glast-PE in DRGs of scRNA or siRNA treated mice (related to ). Right, Quantification of GPR37L1+ cells in the Glast-PE+ population ( n = 3 mice/group). GLAST was used as a marker to isolate SGCs in DRGs. ( D ) Genotyping for Gpr37l1 WT and mutant mice. Data are expressed as mean ± s.e.m. **** P <0.0001, ** P <0.01, unpaired t-test.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Flow Cytometry, Expressing, Marker, Mutagenesis

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: ( A ) Double staining of RNAscope in situ hybridization (ISH for Gpr37l1 ) and immunohistochemistry (IHC for Tuj1) show non-overlapping expression of Gpr37l1 mRNA (red) and Tuj1 (white) in mouse DRGs. Right, enlarged image from the box in the left panel. Yellow arrows indicate Gpr37l1 + cells surrounding DRG neurons. Scale = 25 µm. (B) Double IHC staining shows co-localization of GPR37L1 (red) with FABP7 (white). Scale bars, 25 µm. ( C ) Western blot showing GPR37L1 expression in mouse DRGs of Gpr37l1 +/+ , Gpr37l1 +/- , and Gpr37l1 -/- mice. GAPDH was included as a loading control. ( D ) Double staining of ISH ( GPR37L1, red) and IHC (glutamine synthetase, GS, green) shows co-localization of GPR37L1 mRNA and GS in human SGCs. (E) Double staining of IHC for GPR37L1 (red) and FABP7 (green) shows heavy co-localization of GPR37L1 and FABP7 in human SGCs. Note that GPR37L1 is enriched on the inner side of SGCs in close contact with neurons. * indicates a neuron and arrows indicate SGCs surrounding the neurons. Scale bars, 25 µm. (F) Top, western blots showing plasma membrane (PM) and cytosol fractions of GPR37L1 and GAPDH loading control in human DRGs of neuropathic pain patients and controls (Con, n = 3). Bottom, the ratio of PM/cytosol GPR37L1 expression in human DRGs of control and neuropathic pain patients. Data are expressed as mean ± SEM and analyzed by t-test. ** P <0.05, n = 3, unpaired student’s t-test.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Double Staining, RNAscope, In Situ Hybridization, Immunohistochemistry, Expressing, Western Blot, Control, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: ( A-C ) Baseline pain sensitivity in Gpr37l1 +/+ mice ( n = 12 ), Gpr37l1 +/- mice ( n = 12), and Gpr37l1 -/- mice ( n = 17), assessed in von Frey test (mechanical sensitivity, A), hot-plate test (heat sensitivity, B), and acetone test (cold sensitivity, C). ( D-F ) Mechanical and thermal hyperalgesia in mice treated with I.G. injection of Gpr37l1 siRNA ( n = 10) and control scRNA ( n = 10). ( D ) Paw withdrawal frequency (0.6 g) showing the siRNA-induced mechanical allodynia at 1d and 2d. ( E ) Hargreaves test showing siRNA-induced heat hyperalgesia at 1d. ( F ) Acetone test shows no cold allodynia following the siRNA treatment. Data are expressed as mean ± SEM and statistically analyzed by One-Way ANOVA with Turkey’s post-hoc test (A, B), Kruskal-Wallis test with Dunn’s post-hoc test (C), or Two-Way ANOVA with Bonferroni’s post-hoc test (scRNA vs siRNA, D-F). * P <0.05, *** P <0.001.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Hot Plate Test, Injection, Control

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: ( A, B ) Neuropathic pain (mechanical allodynia) induced by streptozotocin (75 mg/kg STZ, A) and paclitaxel (6 mg/kg, PTX, B) in wild-type and Gpr37l1 mutant mice. ( A ) Time course of STZ-induced mechanical allodynia in Gpr37l1 +/+ mice ( n = 5) , Gpr37l1 +/- mice ( n = 7) , and Gpr37l1 -/- mice ( n = 6). ( B ) Time course of PTX-induced mechanical allodynia in Gpr37l1 +/+ mice ( n = 7) , Gpr37l1 +/- mice ( n = 10) , and Gpr37l1 -/- mice ( n = 8). ( C, D ) GPR37L1 expression in the plasma membrane (PM) fraction of DRG tissues of control animals ( n = 3) and animals 4 weeks after STZ treatment ( n = 3). Transferrin (TfR) was used as a loading control for surface proteins. ( D ) Quantification of GPR37L1 as fold change from control. ( E-G ) Unilateral intraganglionic (I.G.) microinjection of Gpr37l1 -targeting siRNA reduces Gpr37l1 expression and induces persistent mechanical allodynia in naïve animals. (E ) Schematic of unilateral I.G. microinjection (2 μl) of Gpr37l1 -targeting siRNA (siRNA) and scrambled control RNA (scRNA) in the L4 and L5 DRGs, followed by von Frey testing on day 1 and day 2 and subsequent tissue collection for quantitative RT-PCR (qPCR) analysis. (F) Mechanical allodynia is induced by siRNA ( n = 10 mice) but not scRNA ( n = 10 mice). ( G ) qPCR analyses showing expression of Gpr37l1 in L4-L5 DRGs from the ipsilateral ( Ipsi ) and contralateral ( Contra ) sides 2 days after unilateral injection of siRNA ( n = 10 mice) and scRNA ( n = 10 mice). ( H-J ) Unilateral I.G. microinjection of Fabp7 - Gpr37l1 or Fabp7-mock AAV virus rescued Gpr37l1 expression and reduced persistent mechanical allodynia in CIPN mice (6 mg/kg PTX). (H ) Schematic of intraganglionic unilateral microinjection (2 μl) of Fabp7 promoter Gpr37l1 expression AAV9 virus (AAV-L1) and Mock control virus (AAV-Con) in the L4 and L5 DRGs, given one week after PTX, followed by von Frey testing and subsequent tissue collection for quantitative RT-PCR (qPCR) analysis. (I) PTX-mediated mechanical allodynia is reduced by AAV-L1 application ( n = 8 mice) but not AAV-Con ( n = 8 mice). Note that AAV-L1 injection in control mice without PTX treatment has no effects on mechanical pain. ( J ) qPCR analyses showing expression of Gpr37l1 in ipsilateral L4 DRGs ( Ipsi, n = 4 mice) 4 weeks after unilateral virus injection in naïve and PTX mice. Data are expressed as mean ± SEM and statistically analyzed by Two-Way ANOVA with Tukey’s post-hoc test (A, B, and I) or Bonferroni’s posthoc test (F), One-Way ANOVA with Tukey’s post-hoc test (G and J), and two-tailed t-test (D). * P <0.05, ** P <0.01, **** P <0.0001.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Mutagenesis, Expressing, Clinical Proteomics, Membrane, Control, Microinjection, Quantitative RT-PCR, Injection, Virus, Two Tailed Test

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: ( A, B ) Lipid overlay assay shows GPR37L1 binding to MaR1. ( A ) Schematic for detecting GPR37L1-binding lipid mediators. hGPR37L1 is FLAG-tagged and expressed in HEK293 cells after hGPR37L1 cDNA transfection. ( B ) Specific binding of hGPR37L1 to MaR1. The plate was coated with 10 different lipid mediators (1 μg/ml, #2-11), as well as vehicle control (0.1% ethanol, #1) and no coating control (NC, #12), and then incubated with cell lysates of hGPR37L1 or MOCK transfected cells. A yellow star indicates a positive response. (C) Up, a representative blot of MaR1-coated PVDF membrane. Down, quantification of dot intensity in DRG lysates from WT and Gpr37l1 -/- mice. n = 3 repeats. (D, E) The overall structure of hGPR37L1 (Green) in complex with MaR1 (Magenta). (E) 100 ns RMSD graph was obtained with a 1000 ns simulation of the GPR37L1-MaR1 complex (red) or GPR37L1(blue). Data are expressed as mean ± s.e.m. and analyzed by unpaired t-test (C) * P <0.05.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Overlay Assay, Binding Assay, Transfection, Control, Incubation, Membrane

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: Lipid pull-down assay showing MaR1 binding to GPR37L1 in GPR37L1-expressing HEK293 cells. (A) Schematic of the lipid pull-down assay using agarose bead-coated SPM (MaR1). (B) Top, Ponceau S staining; red arrowhead indicates the specific band at 50 kDa. Bottom: Anti-FLAG Western blot showing the GPR37L1 band, indicated by red arrowhead. One microgram of protein was loaded. ( C ) Quantification of the intensity of immune blots; DRG lysate and Mock were used as positive control (PC) and negative control (NC). n = 3 repeats. Data are expressed as mean ± s.e.m. and analyzed by One-Way ANOVA followed by Tukey’s post-hoc test (C). *** P <0.001.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Pull Down Assay, Binding Assay, Expressing, Staining, Western Blot, Positive Control, Negative Control

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: ( A ) Alignment of amino-acid sequences of a/the crystallized construct of human EDNRB (PDB: 6IGK) and human GPR37L1 (UniProt ID: O60883). Conservation of the residues is indicated as follows: red panels for completely conserved; red letters for partly conserved; and black letters for not conserved. Note there is a 30.23% homology between these two genes. (B) The overall structure of hGPR37L1 (Green) complex with NPD1 (Magenta). (C) 1000 ns molecular dynamics simulation of the GPR37L1-MaR1 complex (red) or GPR37L1(blue). (D) 1000 ns molecular dynamics simulation of the GPR37L1-NPD1 complex (red) or GPR37L1(blue). Note that MaR1, but not NPD1, has stable interaction with GPR37L1.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Construct

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: ( A, B ) Intrathecal MaR1 (100 ng) reduces mechanical allodynia in Gpr37l1 +/+ and Gpr37l1 +/- mice, induced by 75 mg/kg of STZ (A) and 6 mg/kg of PTX (B). ( A ) Paw withdrawal thresholds were assessed in Gpr37l1 +/+ mice (left, n = 7), Gpr37l1 +/- mice (middle, n = 10), and Gpr37l1 -/- mice ( n = 8) after intrathecal MaR1 injection. ( B ) Paw withdrawal thresholds in Gpr37l1 +/+ mice (left, n = 5), Gpr37l1 +/- mice (middle, n = 7), and Gpr37l1 -/- mice ( n = 6) after intrathecal MaR1 injection. Behavior was assessed 1 h after MaR1 injection on Post-PTX and Post-STZ day 3. Abbreviations: PTX, paclitaxel; STZ, streptozotocin. ( C-D ) Validation of knockdown effect in L4 DRGs after scRNA or siRNA treated in PTX + MaR1 application ( n = 10) (C) . I.G. injection of MaR1 (10 ng, 2 μl) reduces PTX-induced mechanical allodynia in control (scRNA-treated) animals but not in animals treated with Gpr37l1 -siRNA (D). (E) MaR1 inhibits paclitaxel-induced IL-1β release in SGC-neuron co-cultures via GPR37L1. IL-1β release in neuron-glia cultures from DRG of WT and Gpr37l1 -/- mice was analyzed by ELISA. The cultures were stimulated with 1 µM paclitaxel for 24 h in the absence or presence of MaR1 (100 nM). n = 6 mice for WT and n = 6 mice for Gpr37l1 -/- mice. Data are expressed as mean ± SEM and statistically analyzed by paired t-test (A, B), Two-Way ANOVA with Bonferroni’s post-hoc test (D, E), Tukey’s post-hoc test (D, E) or unpaired t-test (C). * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, n.s., not significant.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Injection, Biomarker Discovery, Knockdown, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: (A-C) Western blot analysis of surface GPR37L1 expression in whole-mount DRG preparations. (A) Schematic of whole-mount DRG treatment with PTX or MaR1, followed by plasma membrane (PM) preparation and western blot. (B ) Western blot showing the effects of PTX (1 μM) and MaR1 (100 ng/ml, 2 h) on PM and total fraction of GPR37L1. ( C ) Quantification of GRP37L1 expression ( n = 6 preps). ( D-J ) Patch-clamp recordings of K + currents in SGCs in whole-mount DRG preparations. ( D ) Micrograph showing patch-clamp recording in a SGC, indicated by a red arrow. ( E ) Representative trace for total K + currents in SGCs of WT-DRG treated with vehicle or PTX (1 μM, 1 h). (F) Average I/V curves in SGCs treated with vehicle ( n = 5 cells) and PTX (1 µM, n = 18 cells). (G) Quantification of the amplitude of K + currents (I k-160 ) for F, with the holding potential of -160 mV. (H) Representative traces for total K + currents in SGCs of Gpr37l1 +/+ DRG treated with PTX (1 μM, 1 h) or PTX + MaR1 (100 ng/ml, 1 h), or in Gpr37l1 +/- DRG treated with PTX (1 μM, 1 h) or PTX + MaR1 (100 ng/ml). (I) Average I/V curves in WT SGCs after treatment of PTX ( n = 18 cells) or PTX + MaR1 ( n = 15 cells). (J) The amplitude of κ + currents (I k-160 ) for H-I, with the holding potential of - 160 mV. In Gpr37l1 +/- DRG preps, n = 17 for PTX; n = 14 for PTX + MaR1. Note that the κ + currents are suppressed by PTX and MaR1 can increase the suppressed currents in WT mice but not in mutant mice. (K) Triple IHC staining showing heavy co-localization of GPR37L1 (green), GS (White), and KCNJ10 (red) in mouse DRG SGCs encircling a neuron. Scale bars, 25 µm. (L) Protein pull-down and co-immunoprecipitation (Co-IP) showing GPR37L1/KCNJ10 interaction in mouse DRGs. Upper labels, pull-down antibodies; left labels, detection antibodies. (M) Quantification of the pull-down results in L for GPR37L1 (left) and KCNJ10 (right). n = 5 mice. (N) Dose-response curve of Ti + influx after subtraction of background, as indicated by AUC (10 min, n = 6). Data are expressed as mean ± SEM and analyzed by Two-Way ANOVA with Bonferroni’s post-hoc test (F, I, and J) and One-Way ANOVA with Tukey’s post-hoc test (C, M), or two-tailed t-test (G). * P <0.05, ** P <0.01, *** , P <0.001. The EC 50 was calculated by Richard’s five-parameter dose-response curve (N).

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Western Blot, Expressing, Clinical Proteomics, Membrane, Patch Clamp, Mutagenesis, Immunohistochemistry, Immunoprecipitation, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: (A) Time course of PM expression of GPR37L1 and KCNJ10 following treatment of vehicle (left) and paclitaxel (PTX, 1 μM, right). (B) Quantification of PM fraction of GRP37L1 (left) and KCNJ10 (right) expression. n = 3 for vehicle and n = 4 for PTX. (C-F) Unilateral local injection of paclitaxel (PTX, 100 ng) via I.G. route (C) or I.T. route (E) is sufficient to induce acute mechanical pain. (C-D) MaR1 (I.G., 100 ng) prevented PTX (I.G.) induced acute mechanical allodynia. (E-F) MaR1 (I.T., 100 ng) attenuated PTX (I.T.) induced acute mechanical pain in WT mice but not Gpr37l1 -/- (KO) mice. n = 6 mice/group. Data are expressed as mean ± s.e.m. and analyzed by Two-Way ANOVA followed by Tukey’s post-hoc test (B, F) and Bonferroni’s post-hoc test (D). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Expressing, Injection

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: (A) Schematic diagram for whole-mount SGC recording. (B) K + current traces in SGCs at different times after PTX treatment (1 μM, 10 min 20 min, 60 min, and 120 min) were recorded using the step voltage injection protocol. (C) Voltage-current curves in SGCs at different times after PTX treatment. (D) Quantification of the amplitude of -160 mV currents in (B) and (C). (E) Quantification of the amplitude of -160 mV currents at different doses of PTX (vehicle, 0.03 n = 3, 0.1,1 µM, n = 7 cells) at 20 min. (F-G) Isolation of Barium sensitive currents in SGCs in whole-mount DRGs. (F) Example traces of Barium sensitive currents in SGCs. (G) Left, average voltage-current curves in SGCs for Barium sensitive or insensitive currents ( n = 6 cells, left). Right, quantification of -160 mV currents for Barium sensitive and total currents ( n = 6 cells, right). ( H ) Average I/V curves in Gpr37l1 +/- SGCs after treatment of PTX (n = 17 cells) or PTX + MaR1 ( n = 14 cells). (I-J) 96well plate Ti + influx assay in GPR37L1 and KCNJ10 expressed HEK293 cells after MaR1 incubation. (I) Flowchart of Ti + assay. (J) Representative traces of Ti + influx in cells treated with 100 nM MaR1 (red, n = 3) or control vehicle (Con, black, n = 3) for 1h incubation. Data are expressed as mean ± SEM and analyzed by Two-way ANOVA with Tukey’s post-hoc test (C, G left), One-way ANOVA with Tukey’s post-hoc (D, E), and unpaired t-test (H). * P <0.05, ** P <0.01, *** P <0.001, **** P < 0.0001, n.s., not significant.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Injection, Isolation, Incubation, Control

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: (A, B) Single-cell RNAseq meta-analysis of KCNJ transcripts expression in mouse (A) and human (B) trigeminal ganglia (TGs) from a published database . ( A) Mouse SGCs predominantly express Gpr37l1 and Kcnj10 , which are clustered together. (B) Human SGCs express GPR37L1, KCNJ3 , and KCNJ10 , which are clustered together. Note greater expression KCNJ3 than KCNJ10 . (C) Transcriptomic meta-analysis of human DRGs, from a published database , reveals downregulations of GPR37L1 and KCNJ3 , but not KCNJ10 , in patients with painful DPN. (D) Triple RNAscope in situ hybridization (ISH) showing the expression of KCNJ10 (red), KCNJ3 mRNA (white), and TUBB3 (Green ) in non-diseased human DRG. Note co-localization of KCNJ10 and KCNJ3 in SGCs surrounding the TUBB3 + neurons. Scale bar, 50 µm. (E) Western blots showing PM fractions of KCNJ3 levels in human DRGs of neuropathic pain patients and controls. (F) Quantification of the western blot results in E ( n = 4). ( G, H ) MaR1 increases K + levels in human SGCs. (G) Images of Ti + influx in human DRG co-cultures for SGCs and neurons treated with vehicle and MaR1. SGCs are indicated with white arrows. (H) Quantification of fluorescence intensity for G at 10 min after 500 µM Ti + simulation in human SGCs. n = 5 cultures from 2 donors. (I) Time course of Ti + influx in HEK293 cells expressing GPR37L1 with vehicle ( n = 16) or MaR1(100 nM, 30 min n = 16) and GPR37L1/KCNJ3 in the presence of vehicle( n = 13) and MaR1 (100 nM, 30 min, n = 12). (J) Quantification of Ti + influx in I after 10 min of 500 µM Ti + stimulation ( n = 6 culture). Data are expressed as mean ± SEM and analyzed by two-tailed t-test (C, F, H) or One-Way ANOVA with Tukey’s post-hoc test (J). * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Expressing, RNAscope, In Situ Hybridization, Western Blot, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: (A) Co-expression percentage of KCNJ3 and KCNJ10 in GPR37L1+ SGCs from a human TG database of RNAseq . (B) Representative traces for Ti + influx assay of cultured human SGCs treated with MaR1 (100 nM, n = 7) or vehicle ( n = 8). (C-D) Ti + influx assay in GPR37L1 and KCNJ3 expressing HEK293 cells. MaR1 increased Ti + influx, which was reduced by Gβγ inhibitor (Gallein, 1 µM) and KCNJ3 inhibitor (SCH 23390, 1 µM). The baseline value of the Ti + influx of the vehicle group was subtracted. (C) Traces of time-dependent Ti + influx and the effects of Gβγ or KCNJ3 inhibitors ( n = 14 at 30 min of drug incubation). (D) Quantification of Ti + influx at 10 min (n = 6 culture). ( E-F ) Thallium influx assay in KCNJ3-expressing HEK293 cells. (E) Time course of Ti + influx activity after vehicle, MaR1 (30 nM), and ML297 (300 nM). n = 4 cultures. (F) Quantification of Ti + influx at 10 min (n = 4 cultures). Data are expressed as mean ± SEM and analyzed by One-way ANOVA with Tukey’s post-hoc (D, F) * P <0.05.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Expressing, Cell Culture, Incubation, Activity Assay

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: (A) GPR37L1 stability changes in different GPR37L1 mutations were tested by protein stability prediction server using 9 different algorithms. (B) Realtime PCR shows the expression levels of GPR37L1 and KCNJ3 in mock, WT, or E296K mutant transfected human SGC cultures ( n = 3). (C) IL-1β secretion level in human SGC cultures after transfection of WT or mutant GPR37L1 and the effects of paclitaxel (PTX, 1 μg/ml, 24 hours). n = 8 cultures. ( D ) Ti + influx activity is reduced in E296K mutation in human SGCs in the presence of 30 nM MaR1. n = 12 cultures. Data are expressed as mean ± SEM, One sample t-test (A), Two-Way ANOVA with Tukey’s post-hoc test (B, C), and One-Way ANOVA with Tukey’s post-hoc test (D) * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Expressing, Mutagenesis, Transfection, Activity Assay

Journal: bioRxiv

Article Title: Satellite glial GPR37L1 regulates maresin and potassium channel signaling for pain control

doi: 10.1101/2023.12.03.569787

Figure Lengend Snippet: Schematic illustration of GPR37L1-mediated regulation of K + channels in SGCs of DRG. Top left, pain transduction and transmission in peripheral and central axons of DRG neurons. Bottom left, SGCs surrounding a DRG neuron. Right, control of potassium channels signaling by GPR37L1 in SGCs. Neuronal excitation causes K + efflux, and extracellular K + can be up-taken by KCNJ10 in mouse SGCs and KCNJ3/KCNJ10 in human SGCs. Loss of K + uptake activity after chemotherapy and diabetes, as a result of downregulations of GPR37L1/KCNJ3/KCNJ10, will lead to neuropathic pain (CIPN and DPN). Additionally, reduced intracellular K+ levels might initiate ROS-mediated inflammatory responses and contribute to IL-1β production. IL-1β production by both SGCs and neurons can subsequently elevate neuronal excitability . Activation of GPR37L1 by MaR1 has the potential to enhance GPR37L1 activity, augment K+ uptake in SGCs, and decrease IL-1β production. This cascade of events may ultimately alleviate pain.

Article Snippet: Flag-tagged human GPR37L1 was purchased from Origene (Cat No. RC208132).

Techniques: Transduction, Transmission Assay, Control, Activity Assay, Activation Assay

Journal: Scientific Reports

Article Title: Amygdalar Endothelin-1 Regulates Pyramidal Neuron Excitability and Affects Anxiety

doi: 10.1038/s41598-017-02583-6

Figure Lengend Snippet: ET1 mRNA levels in the amygdala of high-anxiety mice are lower than in low-anxiety mice, and infusing ET1 gene knock-down lentiviral vector (LV-ET1 shRNA) into the BLA enhanced anxiety-like behaviors. ( a ) Schematic illustration of the elevated plus maze test. The left trace is the path of a representative low-anxiety mouse, and the right trace is that of a high-anxiety mouse. This system is an acute selection of anxiety-like behavior and not a model of the trait anxiety. ( b ) The time spent in the open arms and the time spent in the closed arms in the elevated plus maze test. ( c ) The real-time quantitative fluorescence PCR analysis shows ET1 mRNA expression (n = 5/group). ( d ) The experimental protocol. ( e ) Infusion sites in the BLA. The infusion sites of the tips show that the virus was limited to the BLA. ( f ) The time spent in the open arms and the time spent in the closed arms in the elevated plus maze test. ( g ) The latency to biting the food and the food intake in the novelty-suppressed feeding test (n = 8/group). ( h ) The time spent in the center arena of the open field box. ( i,j ) The locomotor activity of mice in the open field test. Vertical bars represent the mean ± SEM. The asterisks indicate significant differences from the relevant control. *P < 0.05, **P < 0.01,***P < 0.001, two-way ANOVA test with one factor as repeated measure for 1b, 1c and 1f, Student’s t test for 1g–1j.

Article Snippet: ET1 shRNA Lentiviral Particles (sc-45395-v), ETAR Lentiviral Activation Particles (sc-420111-LAC), ETBR Lentiviral Activation Particles (sc-420112-LAC), ETBR shRNA (m) Lentiviral Particles (sc-39963-V) and ETAR shRNA (m) Lentiviral Particles (sc-39961-V) were bought from Santa Cruz Biotech.

Techniques: Knockdown, Plasmid Preparation, shRNA, Selection, Fluorescence, Expressing, Virus, Activity Assay, Control

Journal: Scientific Reports

Article Title: Amygdalar Endothelin-1 Regulates Pyramidal Neuron Excitability and Affects Anxiety

doi: 10.1038/s41598-017-02583-6

Figure Lengend Snippet: Up-regulating ETBR gene expression in the BLA with ETBR lentiviral activation particles (LV-ETBR) attenuated anxiety-related behaviors. Knocking down ETBR gene expression in the BLA increased anxiety-related behaviors, and down-regulating both ETBR and ET1 had no additional anxiogenic effect. ( a ) The time spent in the open arms and the time spent in the closed arms in the elevated plus maze test. ( b ) The latency time and the food intake of the mice in the novelty-suppressed feeding test. ( c ) The time spent in the center of the open field box. ( d , e ) The total path length and the speed in the open field test. ( f ) The time spent in the open arms and the time spent in the closed arms in the elevated plus maze test. ( g ) The latency time and the food intake of the mice in the novelty-suppressed feeding test. ( h ) The time spent in the central area in the open field test. ( i , j ) Locomotor activity in the open field test. (n = 16/group). *P < 0.05, **P < 0.01,***P < 0.001, two-way ANOVA test with one factor as repeated measure for 2a and 2f, one way ANOVA post hoc test for 2b–2e and 2g–2i.

Article Snippet: ET1 shRNA Lentiviral Particles (sc-45395-v), ETAR Lentiviral Activation Particles (sc-420111-LAC), ETBR Lentiviral Activation Particles (sc-420112-LAC), ETBR shRNA (m) Lentiviral Particles (sc-39963-V) and ETAR shRNA (m) Lentiviral Particles (sc-39961-V) were bought from Santa Cruz Biotech.

Techniques: Gene Expression, Activation Assay, Activity Assay

Journal: Scientific Reports

Article Title: Amygdalar Endothelin-1 Regulates Pyramidal Neuron Excitability and Affects Anxiety

doi: 10.1038/s41598-017-02583-6

Figure Lengend Snippet: ET1 and its receptors (ETARs and ETBRs) are mainly present at glutamatergic neurons in the BLA. ( a ) Coronal sections of the BLA of C57 mice were stained with anti-CamKII antibody, anti-ET1 antibody and DAPI (blue). Most the right image shows combinations of red (CamKII), green (ET1) and blue (DAPI) channels. Unfilled triangles, ET1-positive neurons without CamKII; arrows, ET1- and CamKII-positive neurons. ( b ) Coronal sections of the BLA were stained with anti-GAD antibody, anti-ET1 antibody and DAPI. Most the right image shows combinations of red (GAD), green (ET1) and blue (DAPI) channels. Unfilled triangles, ET1-positive neurons without GAD; arrows, ET1- and GAD-positive neurons.( c ) Quantitative analysis of pyramidal neurons and GABAergic neurons that are positive for ET1. ( d ) Coronal sections of the BLA were stained with anti-CamKII antibody, anti-ETAR antibody and DAPI. Most the right image shows combinations of red (CamKII), green (ETAR) and blue (DAPI) channels. Unfilled triangles, ETAR-positive neurons without CamKII; arrows, ETAR- and CamKII-positive neurons. ( e ) Coronal sections of the BLA were stained with anti-GAD antibody, anti-ETAR antibody and DAPI. Most the right image shows combinations of red (GAD), green (ET1) and blue (DAPI) channels. ( f ) Quantitative analysis of pyramidal neurons and GABAergic neurons that were positive for ETAR. Unfilled triangles, ETAR-positive neurons without GAD; arrows, ETAR- and GAD-positive neurons. ( g ) Coronal sections of the BLA were stained with anti-CamKII antibody, anti-ETBR antibody and DAPI. Most the right image shows combinations of red (CamKII), green (ETBR) and blue (DAPI) channels. Unfilled triangles, ETBR-positive neurons without CamKII; arrows, ETBR- and CamKII-positive neurons. ( h ) Coronal sections of the BLA were stained with anti-GAD antibody, anti-ETBR antibody and DAPI. Most the right image shows combinations of red (GAD), green (ETBR) and blue (DAPI) channels. Unfilled triangles, ETBR-positive neurons without GAD; arrows, ETBR- and GAD-positive neurons. ( i ) Quantitative analysis of pyramidal neurons and GABAergic neurons that were positive for ETBR. Scale Bar = 50 µm. Shown are means ± SE; n = 40 for CamKII-positive cells of 10 independent sections; n = 10 for GAD-positive cells of 10 independent sections. The 10 independent sections of each group were randomly selected from 5 mice. ***P < 0.001, Student’s t test for 3c, 3f, 3i.

Article Snippet: ET1 shRNA Lentiviral Particles (sc-45395-v), ETAR Lentiviral Activation Particles (sc-420111-LAC), ETBR Lentiviral Activation Particles (sc-420112-LAC), ETBR shRNA (m) Lentiviral Particles (sc-39963-V) and ETAR shRNA (m) Lentiviral Particles (sc-39961-V) were bought from Santa Cruz Biotech.

Techniques: Staining

Journal: Scientific Reports

Article Title: Amygdalar Endothelin-1 Regulates Pyramidal Neuron Excitability and Affects Anxiety

doi: 10.1038/s41598-017-02583-6

Figure Lengend Snippet: The firing frequency and threshold current of action potential generation are regulated by ET1 and ETBR antagonists. ( a ) Voltage responses of a representative pyramidal neuron in the BLA to current injections of, from bottom to top, 0, 100, 200, 300, 400, and 500 pA. Different concentrations of ET1 solution were applied. ( b ) Summary plot of the firing frequency before and after application of 200 pM ET1 (n = 8/group). ( c ) Summary histogram of the threshold current for action potential before and after application of 200 pM ET1 (n = 8/group). ( d ) Voltage response of a representative pyramidal neuron in the BLA to current injections of, from bottom to top, 0 to 500 pA, before and after BQ123 treatment. ( e ) Summary plot of the firing frequency before and after application of 1 nm BQ123 (n = 8/group). ( f ) Summary histogram of the threshold current for action potential before and after application of 1 nm BQ123 (n = 8/group). ( g ) Voltage response of a representative pyramidal neuron in the BLA to current injections of, from bottom to top, 0 to 500 pA, before and after BQ788 treatment. ( h ) Summary plot of the firing frequency before and after application of 2 nM BQ788 (n = 6/group). ( i ) Summary histogram of the threshold current before and after application of 2 nM BQ788 (n = 6/group). *P < 0.05, two-way ANOVA test with one factor as repeated measure for 4b, 4e and 4 h and Student’s t test for 4c, 4f and 4i.

Article Snippet: ET1 shRNA Lentiviral Particles (sc-45395-v), ETAR Lentiviral Activation Particles (sc-420111-LAC), ETBR Lentiviral Activation Particles (sc-420112-LAC), ETBR shRNA (m) Lentiviral Particles (sc-39963-V) and ETAR shRNA (m) Lentiviral Particles (sc-39961-V) were bought from Santa Cruz Biotech.

Techniques:

Journal: Scientific Reports

Article Title: Amygdalar Endothelin-1 Regulates Pyramidal Neuron Excitability and Affects Anxiety

doi: 10.1038/s41598-017-02583-6

Figure Lengend Snippet: ET1 directly regulates the excitability of BLA pyramidal neurons through ETBRs. Viruses were injected into the BLA two weeks before the patch-clamp recordings. ( a ) Representative action potentials of pyramidal neurons in the BLA from mice treated with the control lentivirus, LV-ET1 shRNA and LV-ETBR. ( b ) Summary histogram of the firing frequency of action potentials. ( c ) Summary histogram of the threshold current for action potential generation. ( d ) Representative action potentials of pyramidal neurons in the BLA from slices treated with the control lentivirus, LV-ETBR shRNA and LV-ETBR shRNA + ET1 (probing with 300 pA current). ( e ) Summary histogram of the firing frequency of action potentials. ( f ) Summary histogram of the threshold current for action potential generation (n = 8/group). ( g ) Schematic illustration of the injection sites for virus in the BLA and recording sites for the mEPSCs of BLA pyramidal neurons. ( h ) Representative mEPSC traces of BLA pyramidal neurons treated with control lentivirus or LV-ET1 shRNA. ( i ) Summary histogram of the effect of LV-ET1 shRNA on the mEPSC frequencies of BLA pyramidal neurons. ( j ) Summary histogram of the effect of LV-ET1 shRNA on mEPSC amplitudes of BLA pyramidal neurons (n = 6/group). ( k ) Schematic illustration of the injection sites for LV-ET1 shRNA in the BLA and the recording sites for the mIPSCs of BLA pyramidal neurons. ( l ) Representative mIPSC traces of BLA pyramidal neurons treated with control lentivirus or LV-ET1 shRNA. ( m ) Summary histogram of the effect of LV-ET1 shRNA on mIPSC frequencies of BLA pyramidal neurons. ( n ) Summary histogram of the effect of LV-ET1 shRNA on mIPSC amplitudes of BLA pyramidal neurons (n = 6/group). Triangles (▴) represent pyramidal neurons. *P < 0.05, two-way ANOVA test with one factor as repeated measure for 5b and 5e, one-way ANOVA with post hoc test for 5c and 5f, student’s t test for 5m-5i and 5i–5j.

Article Snippet: ET1 shRNA Lentiviral Particles (sc-45395-v), ETAR Lentiviral Activation Particles (sc-420111-LAC), ETBR Lentiviral Activation Particles (sc-420112-LAC), ETBR shRNA (m) Lentiviral Particles (sc-39963-V) and ETAR shRNA (m) Lentiviral Particles (sc-39961-V) were bought from Santa Cruz Biotech.

Techniques: Injection, Patch Clamp, Control, shRNA, Virus