Cannabinoids increase type 1 cannabinoid receptor expression in a cell culture model of striatal neurons: Implications for Huntington's disease

Highlights

• We asked: can cannabinoids increase CB₁ receptor expression in neuronal cells?

• CB₁ receptor expression was increased following treatment with cannabinoids.

• This increase was CB₁ receptor-, Akt, and NF-κB-dependent.

• CB₁ receptor expression was also increased in cell models of Huntington's disease.

• Cannabinoids increased BDNF and PGC1α levels in cells expressing mutant huntingtin.

Abstract

The type 1 cannabinoid receptor (CB₁) is a G protein-coupled receptor that is expressed at high levels in the striatum. Activation of CB₁ increases expression of neuronal trophic factors and inhibits neurotransmitter release from GABA-ergic striatal neurons. CB₁ mRNA levels can be elevated by treatment with cannabinoids in non-neuronal cells. We wanted to determine whether cannabinoid treatment could induce CB₁ expression in a cell culture model of striatal neurons and, if possible, determine the molecular mechanism by which this occurred. We found that treatment of STHdh^7/7 cells with the cannabinoids ACEA, mAEA, and AEA produced a CB₁receptor-dependent increase in CB₁ promoter activity, mRNA, and protein expression. This response was Akt- and NF-κB-dependent. Because decreased CB₁ expression is thought to contribute to the pathogenesis of Huntington's disease (HD), we wanted to determine whether cannabinoids could increase CB₁ expression in STHdh^7/111 and ^111/111 cells expressing the mutant huntingtin protein. We observed that cannabinoid treatment increased CB₁ mRNA levels approximately 10-fold in STHdh^7/111 and ^111/111 cells, compared to vehicle treatment. Importantly, cannabinoid treatment improved ATP production, increased the expression of the trophic factor BDNF-2, and the mitochondrial regulator PGC1α, and reduced spontaneous GABA release, in HD cells. Therefore, cannabinoid-mediated increases in CB₁ levels could reduce the severity of some molecular pathologies observed in HD.

Introduction

The type 1 cannabinoid receptor (CB₁) is a G protein-coupled receptor (GPCR) that is highly expressed in the cortex and medium spiny projection neurons of the striatum during adulthood in both rodents and humans (Vitalis et al., 2008; Fernandez-Ruiz et al., 2004). The level of CB₁ gene expression varies in different regions of the brain at different developmental stages. CB₁ mRNA expression is high in the cerebral white matter of the mouse, relative to other regions of the brain during embryonic development (Fernandez-Ruiz et al., 1999; Vitalis et al. 2008). For instance, CB₁ is expressed in the cerebral white matter during early development, and declines to undetectable levels by post-natal day 5 (Fernandez-Ruiz et al., 1999, 2004; Vitalis et al., 2008). CB₁ mRNA levels are also increased in the pre-frontal cortex and amygdala of non-human primates, relative to other regions of the brain, during adolescence (Eggan et al., 2010; reviewed in Laprairie et al., 2012). Because CB₁ levels are elevated in the pre-frontal cortex during adolescence, this period is thought to be a period of heightened sensitivity to exogenous cannabinoids (Eggan et al., 2010). Transcription factors such as AP-1, estrogen receptor, necrosis factor kappa B (NF-κB), nuclear factor activated in T cells (NFAT), Sp1, retinoic acid receptor (RAR), and repressive element silencing transcription factor (REST) are known to regulate the expression of CB₁ in T cells, hepatocytes, and cultured neurons (Blázquez et al., 2011; Borner et al., 2007a, 2007b, 2008; McCaw et al., 2004; Mukhopadhyay et al., 2010). However, the role these and other transcription factors play in regulating CB₁ gene expression throughout development in not well characterized.

Cortical and striatal CB₁ receptors are localized to the pre-synaptic boutons where they are activated by retrograde neurotransmission of endocannabinoids (Wilson and Nicoll, 2001). Activation of CB₁ is associated with inhibition of Ca²⁺-dependent neurotransmitter release (Kreitzer and Regehr, 2001), inhibition of adenylate cyclase, and activation of MAPK and Akt signalling pathways (Coutts et al., 2001; Pertwee, 2005). CB₁-mediated signal transduction is associated with increased pro-survival gene expression and synaptic plasticity at the pre-synaptic neuron (Howlett et al., 2002).

Recently, several authors have demonstrated that acute treatment of cultured rodent hepatocytes, human T cells, or human colorectal carcinoma cells with cannabinoids is associated with elevated CB₁ mRNA and protein expression (Borner et al., 2007a; Mukhopadhyay et al., 2010; Proto et al., 2011). Collectively, these data suggest that CB₁ expression in non-neuronal model systems is inducible via cannabinoid treatment. If cannabinoid-dependent CB₁ induction also occurs in neurons, then cannabinoid treatment may alter the functionality of the endocannabinoid system in the central nervous system.

Huntington's disease (HD) is a progressive, inherited, neurodegenerative disorder (reviewed in Walker, 2007). HD symptoms include choreic movements as well as cognitive impairments and psychiatric disturbances (Walker, 2007). During HD progression there is a pronounced, progressive destruction of striatal medium spiny projection neurons in addition to widespread neuronal atrophy (Sapp et al., 1995; Zuccato et al., 2008). HD is caused by inheritance of a single copy of the mutant huntingtin allele containing an expanded CAG repeat region in exon 1 (>36 CAG repeats; Walker, 2007). Translation of the mutant allele yields the mutant huntingtin protein (mHtt) containing an expanded polyglutamine (polyQ) region near the amino terminus. Expression of mHtt leads to several pathological changes, including impaired ATP production in mouse cell culture models of HD (Trettel et al., 2000), reduced neurotrophic support in HD patients and mouse models (Zuccato et al., 2008), increased spontaneous GABA release in mouse models of HD (Cepeda et al., 2004), and transcriptional dysregulation (Luthi-Carter et al., 2000). One of the earliest changes observed in HD patients and animal models of HD is transcriptional dysregulation of a subset of genes (Gomez et al., 2006). Among the genes dysregulated early in HD, there is a cell-specific decrease in CB₁ mRNA levels in the striatum. In particular, levels of CB₁ in the striatum are reduced by approximately 50% prior to motor symptom onset in human HD patients and in rodent models of HD, relative to age-matched, healthy controls (Denovan-Wright and Robertson, 2000; McCaw et al., 2004; Van Laere et al., 2010). Treatment of R6/2 HD mice with Δ⁹-tetrahydrocannabinol (THC) is associated with decreased striatal atrophy and improved motor coordination, and treatment of cell culture models of HD with THC is associated with increased viability relative to untreated controls (Blázquez et al., 2011). Furthermore, heterozygous CB₁ knockout mice that express the amino terminus of mHtt (CB₁^+/−/mHtt) exhibit more severe motor control deficits and striatal atrophy than HD mice with a full complement of CB₁ (Blázquez et al., 2011; Mievis et al., 2011). Therefore, decreased CB₁ likely represents a pathogenic feature of HD and not a cellular compensatory mechanism.

We wanted to determine if cannabinoid treatment could elevate CB₁ levels in a cell culture model of striatal neurons and characterize the mechanisms by which cannabinoids regulate CB₁ expression. We also wanted to learn whether cannabinoid treatment could increase CB₁ expression in cells expressing mHtt. Our final objective was to determine whether cannabinoid treatment could improve HD-specific pathogenic features, such as ATP deficit and transcriptional dysregulation, in a cell culture model of HD. To our knowledge, this is the first attempt to pharmacologically characterize the mechanism of a cannabinoid-mediated increase in CB₁ expression in a neuronal model and in cells expressing mHtt.