Visual Overview
Abstract
Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdhQ7/Q7) or mutant huntingtin protein (STHdhQ111/Q111). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased—such as THC found at high levels in modern varieties of marijuana—may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced.
Introduction
Huntington Disease.
Expression of mutant huntingtin protein (mHtt) causes a myriad of molecular and cellular changes that ultimately cause progressive worsening of the symptoms of Huntington disease (HD). Early in HD progression, levels of type 1 cannabinoid receptor (CB1) mRNA and protein decrease in medium spiny projection neurons of the caudate and putamen (Denovan-Wright and Robertson, 2000; Glass et al., 2000; Van Laere et al., 2010). CB1 transcription is inhibited by mHtt (McCaw et al., 2004; Laprairie et al., 2013). The reduction in CB1 and loss of CB1 function have been shown to contribute to the cognitive, behavioral, and motor deficits of HD pathology in animal models of HD (Blázquez et al., 2011; 2015; Chiarlone et al., 2014). Furthermore, rescue of CB1 gene expression in the striatum using viral transduction prevents the loss of excitatory synaptic markers and reduces dendritic spine loss in animal models of HD (Naydenov et al., 2014). The benefit of adeno-associated viral CB1 delivery in HD provides strong proof for the concept of treating HD through enhancing CB1 function. However, gene-based therapies specifically for CB1 or other single alterations in gene expression, will probably not be used clinically for HD in the near future because of the invasive nature of delivery and because the potential adverse effects of gene therapy are still being investigated. The more effective gene-based therapies for HD will target the underlying cause of the disease, the mHtt gene and encoded protein, and not secondarily lost cellular components (Kumar et al., 2015). In contrast, pharmacological strategies aimed at elevating CB1 levels and/or signaling through remaining pool of CB1 receptors has significant therapeutic potential for the treatment and management of HD.
Pharmacological Targeting of CB1.
CB1 is activated by cannabinoids, which are a structurally diverse group of ligands that includes endogenously occurring cannabinoids (endocannabinoids) such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), phytocannabinoids from Cannabis sativa such as Δ9-tetrahydrocannabinol (THC), and synthetic cannabinoids such as CP55,940 (CP) and WIN55,212–2 (WIN) (Pertwee, 2008). Activation of CB1 in the brain results in inhibition of neurotransmitter release from presynaptic glutamatergic and GABAergic neurons and activation of prosurvival signaling cascades such as extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) (Fernández-Ruiz, 2009). We have reported that AEA, and structurally related compounds, increase the expression of CB1 via CB1 through Gαi/o and Gβγ signaling in a cell culture model expressing normal huntingtin (STHdhQ7/Q7) and cells expressing mHtt (STHdhQ111/Q111) (Laprairie et al., 2013). Importantly, this cell culture model endogenously expresses CB1 and other components of the endocannabinoid system. Increasing levels of CB1 improved neuronal viability in this cell culture model (Laprairie et al., 2013), lending further support to the strategy of enhancing signaling through the pool of CB1 that are retained in the presence of mHtt and elevating CB1 levels in these cells despite transcriptional repression via mHtt.
Not all cannabinoids increase CB1 levels. THC and CP treatment promote β-arrestin-dependent CB1 internalization and reduce CB1-dependent downstream signaling (Laprairie et al., 2014). Functional selectivity (i.e., signaling bias) describes the receptor- and ligand-dependent enhancement of certain signal transduction pathways and the simultaneous diminution of other signal transduction pathways at a single receptor (Luttrell et al., 2015). Functional selectivity occurs via a GPCR ligand that preferentially activates one effector (e.g., Gαi/o) more potently and efficaciously than another (e.g., β-arrestin) through ligand-specific changes in GPCR conformation or dimerization with other GPCRs (Christopoulos, 2014). Signaling bias could be exploited for enhancement of CB1 function in HD, at the same time limiting detrimental adverse on-target effects (Laprairie et al., 2014). Cannabinoids display signaling bias (Laprairie et al., 2014; Khajehali et al., 2015). Endocannabinoids acting at CB1 are Gαi/o-biased, whereas THC and CP are β-arrestin-biased in STHdhQ7/Q7 cells (Laprairie et al., 2014). In this study, we wanted to determine how the bias of different classes of cannabinoid affected neuronal viability. We hypothesized that Gαi/o-biased cannabinoids improve neuronal viability, whereas β-arrestin-biased cannabinoids reduce—or have no effect on—cell viability. The functional selectivity of six cannabinoids [AEA, 2-AG, THC, cannabidiol (CBD), WIN, and CP] between Gαi/o-, Gαs-, Gαq-, Gβγ-, and β-arrestin pathways was examined in STHdhQ7/Q7 and STHdhQ111/Q111 cells and compared with cannabinoid-dependent changes in ATP level, γ-aminobutyric acid (GABA) release, metabolic activity, and cell death.
Materials and Methods
Drugs.
Drugs were dissolved in ethanol (THC) or DMSO [2-AG, 8-OH-DPAT (5HT1A agonist), AEA, CP, CBD, gallein (Gβγ inhibitor), haloperidol (D2 antagonist), O-2050 (CB1 antagonist), quinpirole (D2 agonist), WAY-100,635 (5HT1A antagonist), WIN] and diluted to final solvent concentrations of 0.1%. 2-AG, AEA, CP, CBD, O-2050, and WIN were purchased from Tocris Bioscience (Bristol, UK). 8-OH-DPAT, haloperidol, quinpirole, THC, and WAY-100,635 were purchased from Sigma-Aldrich (Oakville, ON, CAN). The Gβγ modulator gallein was purchased from MilliporeSigma (Billerica, MA). Pertussis toxin (PTx) and Cholera toxin (CTx) (Sigma-Aldrich) were dissolved in dH2O (50 ng/ml) and added directly to the media 24 hours prior to cannabinoid treatment. Pretreatment of cells with PTx and CTx inhibits Gαi/o and Gαs, respectively (Milligan et al., 1989). In the case of CTx, this occurs via downregulation of Gαs following ADP-ribosylation (Milligan et al., 1989; McKenzie and Milligan, 1991). All experiments included a vehicle treatment control.
Cell Culture.
STHdhQ7/Q7 and STHdhQ111/Q111 cells are derived from the conditionally immortalized striatal progenitor cells of embryonic day 14 C57BlJ/6 mice (Coriell Institute, Camden, NJ) (Trettel et al., 2000). STHdhQ111/Q111 cells express exon 1 of the mutant human huntingtin gene containing 111 CAG repeats knocked into the mouse huntingtin locus (Trettel et al.., 2000). STHdhQ7/Q7 and STHdhQ111/Q111 cells endogenously express CB1 and dopamine D2 receptor (Paoletti et al., 2008; Laprairie et al., 2014). Cells were maintained at 33°C, 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 104 IU ml−1 penicillin/streptomycin, and 400 μg ml−1 geneticin. Cells were serum-deprived for 24 hours prior to experiments to promote differentiation (Trettel et al., 2000; Laprairie et al., 2014).
Plasmids and Transfection.
Human CB1-green fluorescent protein2 (GFP2) C-terminal fusion protein was generated using the pGFP2-N3 plasmid (PerkinElmer, Waltham, MA), as described previously (Bagher et al., 2013). Human arrestin2 (β-arrestin1)-Renilla luciferase II (Rluc) C-terminal fusion protein was generated using the pcDNA3.1 plasmid and provided by Dr. Denis J Dupré (Dalhousie University, NS). The GFP2-Rluc fusion construct, and Rluc plasmids have also been described (Bagher et al., 2013). The Gαq dominant negative mutant [Glu 209 Δ Leu, Asp 277 Δ Asn (Q209L,D277N)] pcDNA3.1 plasmid was obtained from the cDNA Resource Center (Missouri S&T, Rolla, MO) (Lauckner et al., 2005).
Cells were grown in six-well plates and transfected with 200 ng of the Rluc fusion plasmid and 400 ng of the GFP2 fusion plasmid according to previously described protocols (Laprairie et al., 2014) using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Burlington, Canada). Transfected cells were maintained for 48 hours prior to experimentation.
BRET2.
Interactions between CB1 and β-arrestin1 were quantified via BRET2 (Packard BioScience Company, Meriden, CT) according to previously described methods (James et al., 2006; Laprairie et al., 2014). Bioluminescence resonance energy transfer (BRET) efficiency (BRETEff) was determined such that Rluc alone was used to calculate BRETMIN and the Rluc-GFP2 fusion protein was used to calculate BRETMAX using previously described methods (James et al., 2006).
On- and In-Cell Western.
On-Cell Western (LI-COR Biosciences, Lincoln, NE) analyses were completed as described previously (Laprairie et al., 2014) using primary antibody directed against N-CB1 (1:500, cat. no. 101500; Cayman Chemical Company, Ann Arbor, MI). All experiments measuring CB1 included an N-CB1 blocking peptide (1:500) control, which was incubated with N-CB1 antibody (1:500). Immunofluorescence observed with the N-CB1 blocking peptide was subtracted from all experimental replicates. In-Cell Western (LI-COR Biosciences) analyses were conducted as described previously (Laprairie et al., 2014). Primary antibody solutions were directed against: the amino terminus of CB1 (N-CB1) (1:500), phosphorylated (p)ERK1/2(Tyr205/185) (1:500), ERK1/2 (1:500), pCREB(S133) (1:500), cAMP response element-binding protein (CREB) (1:500), pPLCβ3(S537) (1:500), PLCβ3 (1:1000), pAkt(S473) (1:500), protein kinase B (Akt) (1:1000), or β-actin (1:2000; Santa Cruz Biotechnology, Dallas, TX). Secondary antibody solutions were: IRCW700dye or IRCW800dye (1:500; Rockland Immunochemicals, Pottstown, PA).
ATP Quantification, γ-Aminobutyric Acid Enzyme-Linked Immunosorbent Assay, and Cell Viability Assays.
The CellTiter-Glo ATP quantification assay was used according to the manufacturer’s instructions (Promega, Madison, WI). The GABA enzyme-linked immunosorbent assay was conducted according to the manufacturer’s instructions for mouse cell culture media (Novatein Biosciences, Boston, MA). GABA levels were reported as ΔGABA relative to GABA in vehicle-treated cells. Viability assays [calcein-AM (cal-AM), ethidium homodimer-1 (EthD-1)] were conducted according to the manufacturer’s instructions (Live/Dead Cytotoxicity Assay, Life Technologies, Burlington, Canada). Cal-AM fluorescence is an indicator of cellular esterase activity and mitochondrial respiration. Cal-AM fluorescence (460/510 nm) is reported as % esterase activity relative to vehicle-treated STHdhQ7/Q7 cells (100%). EthD-1 fluorescence is an indicator of membrane permeability and cell death. EthD-1 fluorescence (530/620 nm) is reported as % membrane permeability relative to STHdhQ7/Q7 cells treated with 70% methanol for 30 minutes (100%). All measurements of viability (ATP, GABA, calcein-AM, EthD-1) were made 18 hours following cannabinoid treatment.
Statistical Analyses.
All experiments were conducted alongside WIN as a reference ligand. Although it is often considered ideal to choose the endogenous receptor agonist as a reference ligand (Kenakin and Christopoulos, 2013), WIN was chosen as a reference ligand for these studies because: 1) it is a widely used reference compound to study CB1-dependent signaling (Lauckner et al., 2005); 2) it acted as an agonist in all assays with nonsignificant differences in EC50 observed between assays; and 3) we wanted to determine whether the two endogenous cannabinoids, AEA and 2-AG, were inherently biased either in wild-type (STHdhQ7/Q7) or mHtt-expressing (STHdhQ111/Q111) cells. Concentration-response curves for ERK, BRET2 (CB1/β-arrestin1), CREB, phospholipase C (PLC)β3, and Akt are presented as % of WIN Emax in STHdhQ7/Q7 cells (Griffin et al., 2007).
Concentration-response curves were fit to nonlinear regression with variable slope (four-parameter) model to determine pEC50 and Emax (Table 1), or global nonlinear regression using the operational model (Black and Leff, 1983; Ehlert et al., 2011; Kenakin et al., 2012) (eq. 1) to estimate the transduction coefficient [logR (τ/KA)], change in transducer coefficient relative to the reference ligand (ΔlogR), and bias factor (ΔΔlogR) (Prism v. 5.0, GraphPad Software Inc., San Diego, CA), as indicated. In eq. (1) E is the response, Emax is the maximal response, [A] is agonist concentration, n is transducer slope, τ is agonist efficacy, and KA is the agonist’s affinity for the receptor (Kenakin et al., 2012). To obtain a global least-squares fit of the data to the operational model, n was constrained to 1 and logKA was shared between both STHdhQ7/Q7 and STHdhQ111/Q111 datasets and constrained to be greater than –15 (Griffin et al., 2007; Ehlert, 2015). Relative activity (ΔlogR) was calculated in Prism as the difference between transduction coefficients [logR (τ/KA)] values for two ligands, a “test” ligand, and a reference ligand (here WIN) as measured between sample-matched replicates (Kenakin et al., 2012) (eq. 2). In eq. (3) bias factor (i.e., log bias, ΔΔlogR) is the difference between response 1 (R1) and response 2 (R2) (Kenakin et al., 2012). All calculations of ΔΔlogR are reported using pERK response (Gαi/o) as R1. Statistical analyses were two-way analysis of variance (ANOVA) (Prism). Post-hoc analyses were performed using the Bonferroni test. Homogeneity of variance was confirmed using the Bartlett test. The level of significance was set to P < 0.01 where ANOVA was used or P < 0.05 where nonoverlapping confidence intervals (CI) were used to determine significance. Results are reported as the mean ± S.E.M. from at least four independent experiments.
Results
Cannabinoid-Dependent Signaling in the Presence of mHtt.
STHdhQ7/Q7 (Fig. 1, A–E) and STHdhQ111/Q111 (Fig. 1, F–J) cells were treated with 10 nM–10 μM WIN, CP, 2-AG, AEA, THC, CBD, or THC+CBD (1:1), and Gαi/o- (ERK1/2), β-arrestin1, Gαs- (CREB), Gαq- (PLCβ3), and Gβγ-dependent (Akt) signaling were measured. The coupling of each of these signaling pathways to CB1 and their respective G proteins or β-arrestin1 has been tested previously (Laprairie et al., 2014) and is presented in (Supplemental Fig. 1 for a subset of cannabinoids. The agonist effects of all cannabinoids tested were CB1-dependent, with the exception of CBD (see below).
For pERK1/2 (Gαi/o), the Emax observed for all cannabinoids was reduced by approximately 50% in STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells, with no change in pEC50 observed between STHdhQ7/Q7 and STHdhQ111/Q111 cells (Table 1; Fig. 1, A and F). This is consistent with our earlier finding that the Emax for pERK relative to total ERK (i.e., raw data without reference ligand) following arachidonoyl–2′-chloroethylamide treatment is 50% lower in STHdhQ111/Q111 cells expressing mHtt compared with STHdhQ7/Q7 cells (Laprairie et al., 2013). The pERK Emax values were greater in WIN- and AEA-treated STHdhQ7/Q7 cells compared with 2-AG-, CP-, THC-treated STHdhQ7/Q7 cells; CBD and THC+CBD displayed no agonist activity in STHdhQ7/Q7 cells (Table 1; Fig. 1A). In contrast, the pERK Emax values were not different in 2-AG-, AEA-, WIN-, and CP-treated STHdhQ111/Q111 cells, and the pERK Emax was lower in THC- and THC+CBD-treated STHdhQ111/Q111 cells compared with WIN; CBD did not elicit an agonist response (Table 1; Fig. 1F). THC+CBD-treated STHdhQ111/Q111 cells also displayed a lower pEC50 in the pERK assay (Table 1; Fig. 1F).
CB1 is known to interact with β-arrestin1, which mediates receptor internalization, recycling, and degradation (Sim-Selley and Martin, 2002; Laprairie et al., 2014). Unlike pERK, no differences in Emax and pEC50 were observed for β-arrestin1 assays. CP displayed higher pEC50 and Emax values than WIN, whereas no differences in pEC50 and Emax were observed between WIN, 2-AG, and THC, and AEA displayed lower Emax values for β-arrestin1 recruitment in both cell lines (Table 1; Fig. 1, B and G). CBD was not an agonist of β-arrestin1 recruitment. In the THC+CBD-treated cells, the Emax and pEC50 of BRETEff were both reduced compared with THC-treated cells (Table 1). These data are consistent with our previous finding that CBD is a negative allosteric modulator of THC-dependent effects at CB1 (Laprairie et al., 2015).
The observed Emax and pEC50 for pCREB (GαS) was not different in STHdhQ7/Q7 cells treated with WIN, CP, CBD, or THC+CBD, relative to STHdhQ111/Q111 cells (Table 1; Fig. 1, C and H). AEA and 2-AG did not evoke a pCREB response. CP, CBD, and THC+CBD treatment resulted in Emax values for pCREB higher than WIN treatment in both cell lines. pCREB pEC50 and Emax values were higher in CP- and CBD-treated cells compared with THC+CBD-treated cells (Table 1; Fig. 1, C and H). Because CB1-dependent GαS signaling is uncommon, this was examined further (see below).
CB1 can also couple Gαq to modulate Ca2+- and PLCβ3-dependent signaling (Lauckner et al., 2005). No differences were observed for PLCβ3 phosphorylation between STHdhQ7/Q7 and STHdhQ111/Q111 cells (Table 1; Fig. 1, D and I). pPLCβ3 Emax values were greater in WIN-, 2-AG-, and AEA-treated cells compared with CP- and THC-treated cells, with no change in pEC50 (Table 1; Fig. 1, D and I). CBD was not an agonist of PLCβ3 phosphorylation.
In the case of pAkt (Gβγ), no differences were observed between STHdhQ7/Q7 and STHdhQ111/Q111 cells (Table 1; Fig. 1, E and J). pAkt Emax values were greater in WIN-, 2-AG-, and AEA-treated cells compared with CP-treated cells, which were in turn greater compared with THC-treated cells (Table 1; Fig. 1, E and J). pAkt pEC50 values did not differ between agonists. CBD was not an agonist of Akt phosphorylation.
Operational Model Analysis of Cannabinoid Transduction Coefficients (logR) and Relative Activity (ΔlogR) in the Presence of mHtt.
The operational model global nonlinear regression (eq. 1) was used to analyze concentration-response data for cannabinoid signaling bias in STHdhQ7/Q7 and STHdhQ111/Q111 cells. CBD only displayed agonist activity for pCREB and these data were therefore omitted from global nonlinear regression analyses of pERK, β-arrestin1, pPLCβ3, and pAkt assays. The transduction coefficient [logR (τ/KA)] for the ERK response was lower in THC- and THC+CBD-treated cells compared with WIN-treated cells, and was lower in THC- and THC+CBD-treated STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells (Table 2). logR for β-arrestin1 was also lower in THC- (only STHdhQ111/Q111) and THC+CBD-treated cells compared with WIN-treated cells, was lower in THC- and THC+CBD-treated STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells, and was higher in THC- and THC+CBD-treated cells compared with the ERK response (Table 2). logR for the CREB response was higher in CP-treated cells, and lower in THC+CBD-treated cells, compared with WIN, was lower in WIN-treated STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells, and was lower in WIN-treated cells compared with the ERK response (Table 2, 3). logR for the PLCβ3 response was lower in CP- (only STHdhQ7/Q7), AEA-, THC-, and THC+CBD-treated cells, compared with WIN, was lower in CP-, AEA-, and THC-treated STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells, and was lower in AEA- and THC-treated cells compared with the ERK response (Table 2, 3). Finally, logR for the Akt response was lower in CP-, THC-, and THC+CBD-treated cells, was lower in THC- and THC+CBD-treated STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells, and was lower in THC-treated STHdhQ7/Q7 cells compared with the ERK response (Table 2, 4).
Relative activity (ΔlogR) was calculated using WIN as the reference ligand (eq. 2). WIN was chosen as a reference ligand, rather than the endocannabinoids 2-AG and AEA (Kenakin and Christopoulos, 2013), because it displayed activity in all assays, and we wanted to quantify the relative activity and bias of 2-AG and AEA in STHdhQ7/Q7 and STHdhQ111/Q111 cells. The ΔlogR for ERK response was lower in THC- and THC+CBD-treated cells compared with WIN (ΔlogR = 0) (Table 2). The ΔlogR for β-arrestin1 was lower in 2-AG-, AEA-, THC-, and THC+CBD-treated cells compared with WIN, and compared with the ERK response (Table 2). The ΔlogR for β-arrestin1 was lower in THC-treated STHdhQ111/Q111 cells, and higher in THC+CBD-treated STHdhQ111/Q111 cells, compared with STHdhQ7/Q7 cells (Table 2). The ΔlogR for the CREB response was higher in CP- (both cell types) and THC+CBD-treated STHdhQ111/Q111 cells, and lower in THC+CBD-treated STHdhQ7/Q7 cells, compared with WIN (Table 3). The ΔlogR for the CREB response was higher in CP- (both cell types) and THC+CBD-treated STHdhQ111/Q111 cells compared with the ERK response, and was greater in THC+CBD-treated STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells (Table 2, 3). The ΔlogR for the PLCβ3 response was lower in CP- (only STHdhQ7/Q7), 2-AG- (only STHdhQ7/Q7), AEA- (only STHdhQ111/Q111), THC- and THC+CBD-treated cells compared with WIN, and compared with the ERK response for CP, 2-AG, and AEA treatments (Table 2, 3). The ΔlogR for the PLCβ3 response was lower in THC- and THC+CBD-treated STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells (Table 2, 3). Finally, the ΔlogR for the Akt response was lower in CP- (only STHdhQ7/Q7), AEA- (only STHdhQ7/Q7), THC-, and THC+CBD-treated cells compared with WIN, and compared with the ERK response for CP and THC (Table 2, 4). ΔlogR values for the Akt response were lower and higher in THC- and THC+CBD-treated STHdhQ111/Q111 cells, respectively, compared with STHdhQ7/Q7 cells (Table 2, 4).
Summarizing the data in Table 2, we observed that the rank order of τ/KA and relative activity (ΔlogR) for pERK was AEA > WIN > CP (STHdhQ7/Q7) > 2-AG > CP (STHdhQ111/Q111) > THC ≥ THC+CBD. For β-arrestin1 this order was CP > THC ≥ WIN > 2-AG = AEA > THC (STHdhQ111/Q111) > THC+CBD. For pCREB this order was CP > WIN (STHdhQ7/Q7) > CBD (STHdhQ7/Q7) > THC+CBD (STHdhQ111/Q111) > CBD (STHdhQ111/Q111) > WIN (STHdhQ111/Q111) ≥ THC+CBD (STHdhQ7/Q7). For pPLCβ3 the order was WIN > CP (STHdhQ111/Q111) > AEA (STHdhQ7/Q7) > 2-AG (STHdhQ7/Q7) > CP (STHdhQ7/Q7) > 2-AG (STHdhQ7/Q7) > THC (STHdhQ7/Q7) > AEA (STHdhQ7/Q7) > THC (STHdhQ7/Q7) > THC+CBD. And for pAkt the order was AEA ≥ 2-AG = WIN > CP > THC > THC+CBD.
Operational Model Analysis of Cannabinoid-Dependent System Bias (ΔΔlogR) in the Presence of mHtt.
Bias values (ΔΔlogR) were calculated from the relative activity data (ΔlogR) to characterize functional selectivity in STHdhQ7/Q7 and STHdhQ111/Q111 cells (eq. 3) (Fig. 2, A–D). Because CB1 is classically considered a Gαi/o-coupled receptor (Kondo et al.., 1998; Lauckner et al., 2005), all comparisons were made using Gαi/o-dependent ERK1/2 signaling (pERK) as ΔlogR1. On the basis of these data, CP evoked GαS- and β-arrestin1-biased signaling compared with Gαi/o, and Gαi/o-biased signaling compared with Gαq or Gβγ in both cell types tested here (i.e., GαS > β-arrestin1 > Gαi/o > Gαq > Gβγ) (Fig. 2, A–D). 2-AG evoked Gαi/o-biased signaling compared with β-arrestin1 (in STHdhQ7/Q7 cells) and Gαq (more so in STHdhQ111/Q111 cells), and Gβγ-biased signaling compared with Gαi/o (in STHdhQ7/Q7 cells) (i.e., Gβγ > Gαi/o > β-arrestin1 > Gαq) (Fig. 2, A–D). Like 2-AG, AEA evoked Gαi/o-biased signaling compared with β-arrestin1 and Gαq (more so in STHdhQ111/Q111 cells), and Gβγ-biased signaling compared with Gαi/o (in STHdhQ7/Q7 cells) (i.e., Gβγ > Gαi/o > β-arrestin1 > Gαq) (Fig. 2, A–D). THC evoked β-arrestin1-, Gαq-, and Gβγ-biased signaling compared with Gαi/o, in both cell types (i.e., β-arrestin1 > Gαq = Gβγ > Gαi/o) (Fig. 2, A–D). CBD treatment only produced a significant activation of GαS-dependent CREB phosphorylation, and bias values could not be calculated for this ligand. The combination THC+CBD evoked GαS-biased signaling compared with Gαi/o- and Gαi/o-biased signaling compared with β-arrestin1, Gαq, or Gβγ (more so in STHdhQ7/Q7 cells) (i.e., GαS > Gαi/o > β-arrestin1 = Gαq = Gβγ) (Fig. 2, A–D).
Each cannabinoid analyzed here displayed unique functional selectivity for different signaling pathways. Overall, the bias factor of 2-AG and AEA was shifted toward Gαi/o-dependent ERK phosphorylation, and the bias factor of THC+CBD was shifted away from Gαi/o-dependent ERK phosphorylation, in STHdhQ111/Q111 cells. The reduced pERK Emax in mHtt-expressing STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells (Table 1) may result from lower CB1 levels (50%) (Laprairie et al., 2013). An important advantage of using the operational model to estimate the relative activity and ligand bias is that this model negates the effects of differences in receptor density (Kenakin et al., 2012). Therefore, differences in bias between STHdhQ7/Q7 and STHdhQ111/Q111 cells were probably mHtt-dependent and not the result of changes in agonist potency or efficacy.
Cannabinoid-Specific Changes in Cellular Function and Viability.
Treatment of STHdhQ7/Q7 cells with WIN, 2-AG, AEA, or THC resulted in a small increase in ATP, whereas treatment with CP, CBD, or THC+CBD resulted in a decrease in ATP (Fig. 3A). In STHdhQ111/Q111 cells, basal ATP levels were approximately 50% lower than basal ATP levels in STHdhQ7/Q7 cells. ATP levels increased in STHdhQ111/Q111 cells treated with WIN, 2-AG, AEA, or THC and decreased with CP or CBD (Fig. 3E). THC+CBD treatment resulted in higher ATP levels in STHdhQ111/Q111 cells. CP and CBD were the only cannabinoids tested that evoked GαS-biased (CREB) signaling in STHdh cells. The lower ATP levels observed in cells treated with CP or CBD may have resulted from cAMP production. However, given that cells expressing mHtt are deficient in ATP (Sadri-Vakili et al., 2006; Laprairie et al., 2013), cannabinoids that exaggerate this state may exacerbate cellular pathology.
Excessive glutamate release from cortical neurons and GABA release from striatal medium spiny projection neurons are both observed in HD (Benn et al., 2007; Botelho et al., 2014). Compounds that limit neurotransmitter release may, therefore, be beneficial in HD, whereas compounds that enhance neurotransmitter release may exacerbate HD pathophysiology. GABA release was inhibited by WIN, 2-AG, AEA, CP, and THC in STHdhQ7/Q7 and STHdhQ111/Q111 cells (Fig. 3, B and F). CBD treatment was associated with enhanced GABA release in STHdhQ7/Q7 and STHdhQ111/Q111 cells and the EC50 and Emax of this response were reduced in the presence of THC (THC+CBD) (Fig. 3, B and F). Therefore, CBD treatment may enhance excessive neurotransmitter release in HD, whereas other cannabinoids tested here limited neurotransmitter release.
Cell viability was measured by cal-AM fluorescence, which is an indicator of esterase activity and mitochondrial respiration that is positively correlated with viability, and EthD-1 fluorescence, which is an indicator of membrane permeability and cell death and therefore negatively correlated with viability (MacCoubrey et al., 1990). Basal cal-AM fluorescence (% esterase activity) was 60% less in STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells (Fig. 3, C and G). Cal-AM fluorescence was decreased by 40% in STHdhQ7/Q7 and STHdhQ111/Q111 cells treated with CP or THC and increased by 40% in STHdhQ111/Q111 cells treated with WIN, 2-AG, AEA, or CBD (Fig. 3, C and G). Basal EthD-1 fluorescence (% membrane permeable cells) was 40% greater in STHdhQ111/Q111 cells compared with STHdhQ7/Q7 cells (Fig. 3, D and H). EthD-1 fluorescence was increased by 30% in STHdhQ7/Q7 and STHdhQ111/Q111 cells treated with CP or THC (Fig. 3, D and H). EthD-1 fluorescence was decreased by 20% in AEA- and CBD-treated STHdhQ7/Q7 cells, and by 40% in WIN-, 2-AG-, AEA-, and CBD-treated STHdhQ111/Q111 cells (Fig. 3, D and H). The effect of CBD predominated over that of THC for both cal-AM and EthD-1 fluorescence in both cell lines. Therefore, in these viability assays, the CP and THC (which both displayed β-arrestin1 bias) appeared harmful, whereas other cannabinoids improved viability in STHdhQ111/Q111 cells.
Functional CB1 residing at the plasma membrane undergo internalization following ligand binding and β-arrestin recruitment (Blair et al., 2009). Total CB1 levels were higher in WIN-, 2-AG-, and AEA-treated STHdhQ7/Q7 and STHdhQ111/Q111 cells, compared with vehicle, whereas total CB1 levels were lower in CP- and THC-treated STHdhQ7/Q7 and STHdhQ111/Q111 cells (Fig. 4A). The fraction of CB1 at the plasma membrane and total CB1 was assayed in STHdhQ7/Q7 and STHdhQ111/Q111 cells treated with various cannabinoids for 12 hours (Fig. 4, A and B). The fraction of CB1 at the plasma membrane was lower in WIN-, 2-AG-, CP-, and THC-treated cells, and higher in CBD-treated cells (Fig. 4B). CP and THC—and to a lesser extent WIN and 2-AG—displayed greater β-arrestin1 bias than AEA or CBD. The mechanism of cannabinoid-dependent induction of CB1 expression has been described previously (Laprairie et al., 2013). Here, it is important to note that treatment with cannabinoids that evoked Gαi/o-and Gβγ-biased signaling (2-AG, AEA) was associated with higher CB1 levels, whereas treatment with CP and THC (β-arrestin1-biased cannabinoids) was associated with lower CB1 levels, suggesting that cannabinoids that are functionally selective for β-arrestin1 may reduce the available pool of CB1 receptors. The effects of THC and CBD were neutralized by one another (Fig. 4, A and B).
Mechanism of CP- and CBD-Dependent GαS Signaling.
CBD is known to modulate the activity of many cellular GPCRs, including CB1, the type 2 cannabinoid receptor (CB2) (Hayakawa et al., 2008), the serotonin 5HT1A receptor (Russo et al., 2005), G protein-coupled receptor 55(Ryberg et al., 2007), and the μ- and δ-opioid receptors (Kathmann et al., 2006). Here, CBD treatment resulted in CB1-independent CREB phosphorylation (Fig. 5). CREB phosphorylation was highest 30 minutes after CBD treatment and was sustained for the duration of the experiment (60 minutes) (Fig. 5A). Treatment of STHdhQ7/Q7 cells with the 5HT1A agonist 8-OH-DPAT resulted in a dose-dependent increase in CREB phosphorylation that was competitively inhibited by the 5HT1A antagonist WAY-100,635 and CBD (Fig. 5B). Treatment of STHdhQ7/Q7 cells with CBD alone also resulted in a dose-dependent increase in CREB phosphorylation, with less potency and efficacy that the full agonist 8-OH-DPAT (Fig. 5C). CBD-dependent CREB phosphorylation was not inhibited by the CB1 antagonist O-2050, but was inhibited by WAY-100,635 (Fig. 5C), indicating that CBD activated CREB via 5HT1A. It is not known whether the partial agonism of 5HT1A by CBD is functionally antagonistic of serotonergic signaling in vivo and whether this would play a role in CBD-based treatments of neurologic disorders.
Unexpectedly, we observed a switch in signaling following continued drug exposure for CP. At 10 minutes CP treatment produced Gαi/o-dependent ERK phosphorylation that returned to basal levels by 25 minutes; and at 30 minutes CP treatment produced Gαs-dependent CREB phosphorylation (Fig. 5A). STHdh cells endogenously express the type 2 dopamine receptor (D2) (Paoletti et al., 2008) and heterodimerization of CB1 and D2 is known to lead to a switch in coupling from Gαi/o to Gαs following treatment with CP (Glass and Felder, 1997; Kearn et al., 2005). Therefore, we hypothesized that CP could be functionally selective for CB1/D2 heterodimer signaling to explain the switch from Gαi/o to Gαs. Cotreatment of STHdhQ7/Q7 cells with CP and 1 μM quinpirole (a D2 agonist) shifted the concentration-response curve for CREB phosphorylation right, as did cotreatment with O-2050 (a competitive antagonist of CB1), whereas cotreatment with 10 μM haloperidol (a D2 antagonist) shifted the concentration-response curve left (Fig. 5D). Quinpirole and haloperidol did not effect CREB phosphorylation alone (Fig. 5D). From these data, we suggest that CP selectively enhanced either physical heterodimerization between CB1/D2 or functional signaling through these receptors with a subsequent switch from Gαi/o to Gαs (Kearn et al., 2005).
Discussion
Correlations between Functional Selectivity and Cellular Viability.
In this study, we described the biased signaling properties of six cannabinoids in the STHdh cell culture model of striatal medium spiny projection neurons. System bias shifted toward Gαi/o for 2-AG and AEA in STHdhQ111/Q111 (mHtt-expressing) cells compared with STHdhQ7/Q7 cells. Treatment of STHdhQ111/Q111 cells with cannabinoids that signaled via CB1 and were functionally selective for Gαi/o and Gβγ (2-AG, AEA) was associated with the greatest improvement in ATP production, inhibition of GABA release, cellular metabolic activity (esterase activity), and cell death (membrane permeability). In contrast, ligands that preferentially enhanced β-arrestin1-recruitment (THC and CP) reduced cellular viability in both STHdhQ7/Q7 and STHdhQ111/Q111 cells as determined by the same measures. We have previously observed that derivatives of AEA normalize CB1 levels in STHdhQ111/Q111 cells via Gαi/o, Gβγ, Akt, and nuclear factor (NF)-κB, and that normalization of CB1 was associated with improved cell function and viability (Laprairie et al., 2013, 2014). Recently, three studies have demonstrated that increasing CB1 levels in medium spiny projection neurons in the R6/2 mouse model of HD via adenovirus-mediated overexpression normalizes brain-derived neurotrophic factor levels, reduces striatal atrophy, and prevents decreases in dendritic spine density and levels of excitatory synaptic markers, such as synaptophysin and vesicular glutamate transporter, but does not improve deficits in motor coordination (Chiarlone et al., 2014; Naydenov et al., 2014; Blázquez et al., 2015). In accordance with this, knockdown or knockout of CB1 in medium spiny projection neurons of R6/2, N171-82Q, or HdhQ150/Q150 HD mice further reduces the pool of CB1 and exacerbates deficits in motor control, enhances striatal atrophy, and reduces survival (Blázquez et al., 2011; Mievis et al., 2011; Horne et al., 2013). Further, individuals with HD and a variant of the CB1 gene (CNR1 rs4707436) that is associated with lower levels of CB1 begin displaying motor-related symptoms of HD earlier than individuals with HD and normal CNR1 (Kloster et al., 2013). Together, these studies and our data provide support for Gαi/o- and Gβγ-selective activation of CB1 to maintain CB1 levels and the cellular function and viability of cells expressing mHtt (Blázquez et al., 2011, 2015; Mievis et al., 2011; Horne et al., 2013; Chiarlone et al., 2014; Naydenov et al., 2014).
Use of THC and CBD in HD.
Despite a lack of clinical evidence, patients suffering from HD may be seeking medical marijuana or acquiring it from other sources in an attempt to relieve some of the symptoms of their disease (Müller-Vahl et al., 1999; Meisel and Friedman, 2012; Koppel et al., 2014). Most medically available and tested illicit marijuana contains a high concentration of THC relative to other cannabinoids, such as CBD (De Backer et al., 2012). Here, we observed that THC reduced cellular function and viability in cells expressing mHtt whether THC was used alone or in a 1:1 combination with CBD. Likewise, treatment of R6/1 and R6/2 mouse models with 10 mg/kg THC is associated with worsening of HD signs and symptoms (Dowie et al., 2010). However, others have reported improvement in motor control and reduced striatal atrophy in R6/1 and R6/2 HD treated for 6 weeks with 2 mg/kg THC beginning at 4 weeks of age (Blázquez et al., 2011), suggesting that the deleterious effects of THC in HD are dose- and time course–dependent. CBD alone displayed mixed beneficial and negative effects in STHdhQ7/Q7 and STHdhQ111/Q111 cells. CBD is known to act through a number of effectors, including as a negative allosteric modulator at CB1 and a partial agonist at 5HT1A (Pazos et al.., 2013; Laprairie et al., 2015). It is unclear which effects of CBD predominate in vivo normally and in HD and how the combinations of any or all of the at least 65 cannabinoids found in marijuana (McPartland et al., 2015) influence one another’s pharmacokinetics and pharmacodynamics (Sagredo et al., 2011; Valdeolivas et al., 2012). Further, the utility of CBD in HD remains controversial, with some studies reporting no effects in animal models and human trials (Consroe et al., 1991; Valdeolivas et al., 2012), or positive effects in animal models (Sagredo et al., 2007, 2011). Overall, the use of THC or marijuana may exacerbate the signs and symptoms of HD via further downregulation of CB1 and reduced cellular viability.
Conclusions
Gαi/o- and Gβγ-selective CB1 ligands are probably the most therapeutically useful cannabinoids in the treatment of HD. However, highly potent synthetic cannabinoids, such as WIN, may produce unwanted psychoactive effects and their chronic use would probably result in receptor desensitization or downregulation (Sim-Selley and Martin, 2002; Blair et al., 2009). Endocannabinoids, which we observed to enhance Gαi/o- and Gβγ-dependent signaling in the STHdh cell culture system, are rapidly metabolized in vivo and consequently have limited efficacy when they are directly administered (Devane et al.., 1992; Kondo et al., 1998). The inhibitor of endocannabinoid catabolism URB597 has demonstrated limited efficacy at improving motor control deficits in R6/2 HD mice (Dowie et al., 2010), but additional studies are needed to understand how elevating endocannabinoid levels affects the signs and symptoms of HD in vivo. An alternative means of enhancing endogenous CB1 signaling is with the use of positive allosteric modulators (PAMs) of CB1. PAMs bind to a site on the receptor that is distinct from the site of endogenous ligand binding (i.e., the orthosteric site) and enhance the binding and efficacy of the endogenous ligands that are produced and regulated through intrinsic control mechanisms (Pamplona et al., 2012; Wootten et al., 2013). CB1 PAMs may increase Gαi/o-dependent pro-survival signaling occurring via endocannabinoids without producing the psychotropic effects associated with synthetic cannabinoid agonists, because they are unable to directly activate CB1. Our in vitro study of cannabinoid functional selectivity leads us to conclude that enhancement of endocannabinoid-dependent CB1 activation may be a means of treating the signs and symptoms of HD by targeting CB1.
Acknowledgments
The authors thank Drs. Laura Bohn and Edward Stahl for assistance and consultation in reviewing the data presented here.
Authorship Contributions
Participated in research design: Laprairie, Bagher, Kelly, and Denovan-Wright.
Conducted experiments: Laprairie.
Contributed new reagents or analytic tools: Kelly, Denovan-Wright.
Performed data analysis: Laprairie, Denovan-Wright.
Wrote or contributed to the writing of the manuscript: Laprairie, Bagher, Kelly, Denovan-Wright.
Footnotes
- Received October 9, 2015.
- Accepted December 22, 2015.
This work was supported by a partnership grant from the Canadian Institutes of Health Research, Nova Scotia Health Research Foundation, and the Huntington Society of Canada [ROP–97185] to E.M.D.-W., and a Canadian Institutes of Health Research operating grant [MOP–97768] to M.E.M.K. R.B.L. is supported by studentships from the Canadian Institutes of Health Research, the Huntington Society of Canada, Killam Trusts, and Nova Scotia Health Research Foundation. A.M.B. is supported by scholarships from Dalhousie University and King Abdul Aziz University, Jeddah, Saudi Arabia. Primary laboratory of origin: E.M.D.W.
This work has been included in the thesis: Laprairie RB (2016). Biased Agonists and Allosteric Modulators: Potential Treatments for Huntington Disease. Doctoral dissertation, Dalhousie University, Halifax NS CAN; and it was presented in the following conference abstract: Laprairie RB, Bagher AM, Kelly ME, Dupre DJ, and Denovan-Wright EM (2014) The therapeutic efficacy of cannabinoid receptor type 1 (CB1) ligands in Huntington’s disease may depend on their functional selectivity, at Experimental Biology and American Society for Pharmacology and Experimental Therapeutics Annual Meeting; 2014 April 25–May 1, San Diego CA.
↵This article has supplemental material available at molpharm.aspetjournals.org.
Abbreviations
- AEA
- anandamide
- Akt
- protein kinase B
- ANOVA
- analysis of variance
- BRET
- bioluminescence resonance energy transfer
- cal-AM
- calcein-AM
- CBD
- cannabidiol
- CB1
- type 1 cannabinoid receptor
- CP
- CP55,940
- CREB
- cAMP response element-binding protein
- CTx
- Cholera toxin
- 8-OH-DPAT
- 7-(dipropylamino)-5,6,7,8-tetrahydronaphthalen-1-ol
- D2
- type 2 dopamine receptor
- ERK
- extracellular signal-regulated kinase
- EthD-1
- ethidium homodimer-1
- GABA
- γ-aminobutyric acid
- GFP2
- green fluorescent protein 2
- HD
- Huntington disease
- mHtt
- mutant huntingtin protein
- N-CB1
- amino terminus of CB1
- O-2050
- (6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran
- p
- phosphorylated
- PAMs
- positive allosteric modulators
- PLC
- phospholipase C
- Rluc
- Renilla luciferase
- THC
- Δ9-tetrahydrocannabinol
- URB597
- [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate
- WAY-100,635
- N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide
- WIN
- WIN55,212–2
- Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics