Receptor Heteromerization Expands the Repertoire of Cannabinoid Signaling in Rodent Neurons

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB1R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB1R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB1R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB1R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB1R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB1R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB1R desensitization. Additionally, presence of DOR facilitates signaling via a new CB1R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB1R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling

Sex Differences in the Neuroadaptations of Reward-related Circuits in Response to Subchronic Variable Stress

Women are twice as likely to be diagnosed with major depressive disorder. However, fewer studies in rodent models of depression have used female animals, leading to a relative lack of understanding of the female brain's response to stress, especially at a neural circuit level. In this study, we utilized a 6-day subchronic variable stress (SCVS) mouse model and measured novelty suppressed feeding as behavioral criteria to evaluate susceptibility to SCVS in male and female mice. First, we showed that SCVS induced a decrease in latency to eat (susceptible phenotype) in female mice, but not in males (resilient phenotype). After determining behavioral phenotypes, we investigated the firing activities of dopamine (DA) neurons in the ventral tegmental area (VTA), as well as the neurons that project from lateral habenula (LHb) to the VTA and from locus coeruleus (LC) to the VTA. Utilizing retrograding lumafluor fluorescent tracers and electrophysiology techniques, we performed cell type- and circuit-specific measures of neuronal firing rates. Our data show that SCVS significantly increased the firing rate of LHb-VTA circuit neurons in female mice when compared to that of their female controls, an effect that was absent in SCVS-exposed males. Interestingly, SCVS did not induce significant firing alterations in VTA DA neurons and LC-VTA circuit neurons in either female mice or male mice when compared to their stress-naïve controls. Overall, our data show sex differences in the LHb-VTA circuit responses to SCVS, and implicates a potential role of this projection in mediating vulnerability of female mice to stress-induced depression

Association between CB₁R and DOR alters CB₁R localization.

<p><b>A</b>, Lysates (100–200 µg) from N2A^CB1R and N2A^CB1RDOR cells were subjected to immunoprecipitation with 1 µg of anti-CB₁R (C-terminal) antibody, the immunoprecipitates were resolved on 10% SDS-PAGE and probed for the presence of myc-DOR using mouse monoclonal anti-myc antibody (1∶1000) and for CB₁R using rabbit polyclonal anti-CB₁R (C-terminal) antibody (1∶500) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Representative of 3 independent experiments shown. <b>B</b>, CB₁R-DOR complexes exhibit greater interaction with AP-2 than AP-3. Lysates (100–200 µg) from N2A^CB1R and N2A^CB1RDOR cells were subjected to immunoprecipitation using 1 µg of an anti-CB₁R (C-terminal) antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. The immunoprecipitates were resolved on 10% SDS-PAGE and probed for the presence of AP-3 (1∶1000), AP-2 (1∶1000) and CB₁R (C-term) (1∶500) using specific antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Representative of 3 independent experiments shown. <b>C</b>, Localization of endogenous CB₁R in N2A^CB1R and of CB₁R and DOR in N2A^CB1RDOR cells. Cells fixed with 4%PFA in PBS and permeablized with 0.1% Triton, were stained with the rabbit polyclonal anti-CB₁R (C-terminal) antibody (1∶500; green) and the mouse monoclonal anti-myc antibody (1∶1000; red) and visualized using Alexa 488-coupled anti-rabbit or Alexa 594-coupled anti-mouse secondary antibodies (1∶1000) using confocal microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Representative of 3 independent experiments shown. <b>D</b>, Cell surface staining of endogenous CB₁R and stably expressed DOR in N2A^CB1RDOR cells. N2A^CB1R and N2A^CB1RDOR cells were stained with a goat polyclonal anti-CB1R (N-terminal) antibody (1∶500) and mouse monoclonal anti-myc antibodies (1∶1000) prior to fixation of the cells to label cell surface receptors, as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#pone.0029239-Rozenfeld1" target="_blank">[28]</a>. After fixation, cells were visualized with Alexa 594-coupled anti-goat and Alexa 488-coupled anti-mouse secondary antibodies (1∶1,000) using confocal microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Representative of 3 independent experiments shown.</p

Engagement of arrestin3-dependent signaling in N2A^CB1RDOR.

<p>Time course of Hu210-mediated ERK phosphorylation in <b>A</b>, N2A^CB1R; <b>B</b>, N2A^CB1RDOR; and <b>C</b>, N2A^CB1RDORΔ15 cells transfected with control or arrestin3-targeting siRNA. N2A^CB1R alone or stably expressing either DOR or DORΔ15, transfected with a control or arrestin3-targeting siRNA, were starved for 4 hours, then stimulated with 100 nM Hu210 for the indicated times. Cell lysates (30 µg protein) were subjected to Western blotting for the levels of ERK (1∶1000), pERK (1∶1000), and arrestin3 (1∶500) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Data represent Mean ± SEM (n = 3); *p<0.05; **p<0.01; ***p<0.001, for control vs Arr3 siRNA (t test).</p

PLC-dependent arrestin3 association with CB1R-DOR complex.

<p><b>A</b>, [³⁵S]GTPγS binding assay in membranes from N2A^CB1R and N2A^CB1RDOR cells. Membranes (10 µg) were treated with indicated concentrations of the CB₁R agonist Hu210. [³⁵S]GTPγS binding was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. EC₅₀ and E_max values were calculated using GraphPad Prism software. Data represent Mean ± SEM (n = 3 independent experiments in triplicate). <b>B</b>, Dose-response of Hu210-mediated ERK phosphorylation in N2A^CB1R and N2A^CB1RDOR cells. Starved N2A^CB1R and N2A^CB1RDOR cells seeded in 24 well-plates were treated with indicated concentrations of Hu210 for 5 minutes. Cell lysates (30 µg protein) were analyzed by Western blotting and probed for the levels of pERK (1∶1000) and ERK (1∶1000) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). EC₅₀ and E_max values were calculated using GraphPad Prism software. Data represent Mean ± SEM (n = 3 independent experiments). *p<0.05 for N2A^CB1RDOR vs N2A^CB1R (t test). <b>C</b>, Effect of DOR down-regulation on ERK phosphorylation. F11 cells transduced with the DOR shRNA expressing lentivirus were starved for 4–6 h and treated with Hu210 (100 nM) for 5 min. Cell lysates (30 µg protein) were analyzed by Western blotting and probed for the levels of pERK (1∶1000) and ERK (1∶1000) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Data from 3 independent experiments is shown. *p<0.05 (t test). <b>D</b>, Examination of arrestin3 interaction with CB₁R after Hu210 treatment. N2A^CB1R and N2A^CB1RDOR, starved for 4 hours were stimulated with 100 nM Hu210 for 5 minutes and cell lysates prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Lysates (30 µg protein) were subjected to either Western blotting using rabbit anti-CB1R (C-terminal 1∶500) and mouse anti-arrestin 3 antibodies (1∶500) or to immunoprecipitation using 1 µg of agarose-coupled anti-CB₁R (C-terminal) antibody. Immunoprecipitates were probed for arrestin3 levels by Western blot using the mouse anti-arrestin 3 antibody. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). Representative of 3 independent experiments shown. <b>E</b>, Effect of Hu210 on arrestin recruitment. U2OS cells co-expressing ProLink/Enzyme Donor (PK)-tagged DOR and the Enzyme Activator (EA)-tagged arrestin3 fusion protein without or with CB₁R were treated with indicated concentrations of Hu-210. Arrestin3 recruitment was determined using the PathHunter Detection Kit as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Data represent Mean ± SEM (n = 4). <b>F</b>, Effect of PLC inhibitor (U73122) on DOR phosphorylation at serine 363 after Hu210 treatment. N2A^CB1RDOR cells were starved for 4–6 hours, and incubated with vehicle (DMSO) or U73122 (1 µM) for 30 minutes, then stimulated with 100 nM Hu210 for 5 minutes. Cell lysates (30 µg protein) were subjected to Western blotting using rabbit polyclonal phosphoDOR Ser 363 (1∶1000), mouse monoclonal anti-myc (1∶1000) antibodies and IR Dye 680 anti-rabbit and IR Dye 800 anti-mouse secondary antibodies (1∶10,000) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Data represent Mean ± SEM (n = 3).</p

Role of arrestin3 and ERK substrates in cannabinoid signaling by the CB₁R-DOR heteromer.

<p><b>A–B</b>, Effect of CB1R-DOR heteromerization on subcellular localization of pERK. <b>A</b>, N2A^CB1R and N2A^CB1RDOR cells were treated with Hu210 (100 nM; 0, 5 or 10 min), cytoplasmic and nuclear extracts were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a> and analyzed (30 µg protein) by Western blotting with pERK (1∶1000), ERK (1∶1000), lamin A/C (1∶2000), and GAPDH (1∶2000) antibodies. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). <b>B</b>, N2A^CB1R and N2A^CB1RDOR cells treated with Hu210 (100 nM; 5 min) were immunostained with pERK (1∶1000, red) and pericentrin (1∶1000, green) antibodies and visualized using Alexa 488-conjugated anti-rabbit (1∶1000) and Alexa 594-conjugated anti-mouse (1∶1000) secondary antibodies using confocal microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Representative of 3 independent experiments shown. <b>C</b>, Time course of phosphorylation of pERK substrates. Lysates (30 µg protein) from N2A^CB1R and N2A^CB1RDOR cells treated with Hu210 (100 nM; 0, 5, 10 or 30 min) were analyzed by Western blotting with STAT3 (1∶1000), phosphoSTAT3 (1∶1000), phospho-p70S6K (1∶1000), phospho-p90rsk (1∶1000) and phosphoBAD (1∶1000) antibodies. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). ERK (1∶1000) is used as a loading control. Representative of 3 independent experiments shown. <b>D</b>, Involvement of PLC and MEK in the phosphorylation of STAT3 and BAD. Lysates (30 µg protein) from N2A^CB1R and N2A^CB1RDOR cells treated with 100 nM Hu210 for 5 min in the absence or presence of U73122 (1 µM) or PD98059 (PD, 10 µM) were analyzed by Western blotting with STAT3 (1∶1000), phosphoSTAT3 (1∶1000), and phosphoBAD (1∶1000) antibodies. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). ERK (1∶1000) is used as a loading control. Representative of 3 independent experiments shown. <b>E</b>, Involvement of arrestin3 in BAD phosphorylation. Arrestin3 was down-regulated in N2A^CB1RDOR cells by transfection with a siRNA. These cells were stimulated with Hu210 (100 nM) for 5 min, in the absence or presence of PD (10 µM). Lysates (30 µg protein) were analyzed by Western blotting with phospho-p90rsk (1∶1000) and phosphoBAD (1∶1000) antibodies. IRDye 680 anti-rabbit and IRDye 800 anti-mouse were used as secondary antibodies (1∶10,000). ERK (1∶1000) is used as a loading control. Representative of 3 independent experiments shown.</p

CB₁R-DOR heteromerization promotes cell survival.

<p><b>A</b>, Hu210-treated N2A^CB1RDOR cells exhibit increased survival compared to N2A^CB1R cells. N2A^CB1R or N2A^CB1RDOR cells were treated with 1 µM Hu210 for the indicated days and survival measured by trypan blue exclusion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Data represent Mean ± SEM (n = 4 in triplicate). <b>B</b>, Hu210-treated N2A^CB1RDOR cells exhibit lower apoptosis as compared to N2A^CB1R cells. Apoptosis of N2A^CB1R or N2A^CB1RDOR treated for 3 or 8 days with 1 µM Hu210 was measured using caspase-3 activity assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Data represent Mean ± SEM (n = 4 in triplicate); ***p<0.001 N2A^CB1RDOR vs N2A^CB1R (t test). <b>C</b>, CB₁R antagonist treatment decreases neuronal survival of striatal neurons from wild-type but not DOR−/− mice. Primary striatal neurons from wild-type or from DOR−/− mice were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. The CB₁R antagonist AM251 (10 µM) was added to the growth media at DIV7 and cellular viability assessed at DIV10 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029239#s2" target="_blank">Methods</a>. Data represent Mean ± SEM (n = 2–4) <b>D–E</b>, A schematic of the signaling pathways emanating from CB₁R in N2A^CB1R (<b>D</b>) and N2A^CB1RDOR (<b>E</b>) cells. Activation of CB₁R in N2A^CB1RDOR cells leads to differential activation of signaling molecules and phosphorylation of ERK substrates.</p

Schwellen-Erfahrungen Berichte und Impressionen vom Besuch der Triestiner Psychiatrie

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