Functional Analysis of Mitochondrial CB1 Cannabinoid Receptors (mtCB1) in the Brain

Abstract

Recent evidence indicates that, besides its canonical localization at cell plasma membranes, the type-1 cannabinoid receptor, CB1 is functionally present at brain and muscle mitochondrial membranes (mtCB1). Through mtCB1 receptors, cannabinoids can directly regulate intramitochondrial signaling and respiration. This new and surprising discovery paves the way to new potential fields of research, dealing with the direct impact of G protein-coupled receptors on bioenergetic processes and its functional implications. In this chapter, we summarize some key experimental approaches established in our laboratories to identify anatomical, biochemical, and functional features of mtCB1 receptors in the brain. In particular, we describe the procedures to obtain reliable and controlled detection of mtCB1 receptors by immunogold electromicroscopy and by immunoblotting methods. Then, we address the study of direct cannabinoid effects on the electron transport system and oxidative phosphorylation. Finally, we present a functional example of the impact of mtCB1 receptors on mitochondrial mobility in cultured neurons. Considering the youth of the field, these methodological approaches will very likely be improved and refined in the future, but this chapter aims at presenting the methods that are currently used and, in particular, at underlining the need of rigorous controls to obtain reliable results. We hope that this chapter might help scientists becoming interested in this new and exciting field of research.

Introduction

Humans have recognized the properties of the plant Cannabis sativa (marijuana) for almost 5000 years (Russo et al., 2008). The identification of its specific molecular lipid compounds (cannabinoids) in 1942 and 1964 (Adams, 1942, Gaoni and Mechoulam, 1964), the characterization and cloning of cannabinoid receptors (CB1 and CB2) in the late 1980s and early 1990s (Howlett, 1987, Matsuda et al., 1990, Munro et al., 1993), and the description of endogenous ligands for these receptors (endocannabinoids) (Devane et al., 1992, Di Marzo et al., 1994, Mechoulam et al., 1995, Sugiura et al., 1995) represent the keystones of one of the most fascinating discoveries of the last decades in modern biology. Indeed, the endocannabinoid system (ECS) is nowadays considered one of the most important regulators of brain and peripheral functions and its study has attracted an exponential and still rising attention of scientists interested in physiology, pathology, pharmacology, and toxicology (Pacher et al., 2006, Piomelli, 2003).

In the 25 years between the complete characterization of the most psychoactive compound of the plant, Δ⁹-tetrahydrocannabinol (THC) (Gaoni & Mechoulam, 1964) and the cloning of the first cannabinoid receptor CB1 in 1990 (Matsuda et al., 1990), a large body of studies have tried to address the question of how THC could modify cellular information processing. Some of these studies pointed to the possibility that mitochondrial activity could be altered by cannabinoids (Bartova and Birmingham, 1976, Bino et al., 1972, Chari-Bitron and Bino, 1971, Mahoney and Harris, 1972, Schurr and Livne, 1975). However, with the later identification of CB1 receptors (Matsuda et al., 1990), these effects were ascribed to unspecific alterations of mitochondrial membrane properties by lipid molecules (Howlett et al., 2002) or to indirect CB1 receptor-dependent signaling (Campbell, 2001) and, as such, were long forgotten in favor of mechanisms involving classical pathways of plasma membrane G protein-coupled receptors (GPCRs).

Indeed, CB1 receptors are GPCRs, which “detect molecules outside the cell and activate internal signal transduction pathways and, ultimately, cellular responses” (Wikipedia, 2017) and are mainly localized at plasma membrane, in the optimal position to “detect molecules outside the cell.” However, most of our knowledge about GPCRs is based on seminal studies on beta-adrenergic receptors (Nobelprize, 2014), which detect adrenalin, a water-soluble hormone. However, apart from the novel class of potential peptides recently described to modulate cannabinoid receptors (Bauer et al., 2012), the large majority of plant-derived, synthetic, or endogenous cannabinoids are lipid compounds (Piomelli, 2003). Despite the clear differences between water-soluble and -insoluble ligands, very little is known about the specific features of GPCRs targeted by lipids. For instance, lipid cannabinoids likely reach their cellular receptors by lateral diffusion within lipid bilayers (Kimura, Cheng, Rice, & Gawrisch, 2009), a notion that is compatible with the recent analyses of the CB1 receptor's crystal structure (Hua et al., 2016, Shao et al., 2016). Thus, lipid (endo)cannabinoids can easily move within cellular membranes and can likely reach intracellular compartments more easily than water-soluble ligands. Early anatomical studies revealed that a large proportion of CB1 receptors in brain cells are intracellular (Freund, Katona, & Piomelli, 2003). In line with the idea that GPCRs are functional only at plasma membranes, these intracellular pools of the protein were exclusively considered as nonfunctional receptors, caught in the process of transport or recycling from or to their functional “natural” location, the plasma membrane (Freund et al., 2003).

Recent data challenged this idea, suggesting that part of intracellular CB1 receptors are functional and respond to (endo)cannabinoid activation. For instance, cannabinoids can activate CB1 receptors localized in late endosomal/lysosomal compartments, where they can trigger G protein-dependent signaling (Rozenfeld & Devi, 2008). Even more recently, we and others found that CB1 receptors are functionally present at brain and peripheral mitochondrial membranes (mtCB1), where they can regulate cellular respiration and other bioenergetic processes (Aquila et al., 2010, Benard et al., 2012, Hebert-Chatelain et al., 2014a, Koch et al., 2015, Mendizabal-Zubiaga et al., 2016, Vallee et al., 2014), and mediate behavioral effects of cannabinoid drugs (Hebert-Chatelain et al., 2016).

Mitochondria are very important organelles, able to transform nutrients and oxygen into adenosine triphosphate (ATP) molecules, the central molecule utilized by cells as a source of energy for all their functions. As such, mitochondria are key elements of cell survival, but their roles go beyond mere “housekeeping” activity to regulate a plethora of functions involved in most biological processes. This is particularly evident in the brain, which is one of the most energy-consuming organs in the body; with a weight of 2% of the whole body, the human brain consumes up to 25% of its energy (Mattson, Gleichmann, & Cheng, 2008). Importantly, this large amount of energy needed for brain functions need to be very constant, and recent data showed that even a very short interruption of ATP supply to neurons is enough to disrupt synaptic functions (Rangaraju, Calloway, & Ryan, 2014). Therefore, regulation of mitochondrial activity is crucial for brain functions. Considering the large impact of CB1 receptors on brain tasks, from synaptic activity to perception, memory, and regulation of vital processes, the possibility that part of these functions might be exerted through a direct control of mitochondrial activity represents a novel paradigm in neuroscience.

In the brain, only a minor part of CB1 receptors (about 10%–15%) appear to be present at mitochondrial membranes (Benard et al., 2012, Hebert-Chatelain et al., 2014a). These low levels imply particularly strict methodological constraints for the study of mtCB1 receptors, in order to avoid the possibility of false-positive or false-negative results. Indeed, the existence of mtCB1 receptors have been recently questioned (Morozov et al., 2013). Discrepancies in the findings obtained by different groups were recently analyzed and methodological issues were identified as their cause (Hebert-Chatelain et al., 2014a, Hebert-Chatelain et al., 2014b, Koch et al., 2015). Within the large interest in the ECS, the biology of mtCB1 receptors remains a very young field of research, and thus it is important to reach early consensus on the methodological approaches to address the issue. Due to the low levels of mtCB1 receptors and the sensitivity of mitochondria to experimental manipulation, the danger of “false-positive” results derived from artifacts is particularly strong (Morozov et al., 2013). Therefore, the need of negative controls is a key element of any experiment aimed at investigating the functions of mtCB1 receptors. The use of tissues, cells, and organelles derived from genetically modified mice lacking the full CB1 protein (CB1-KO) (Marsicano et al., 2002) together with samples from wild-type littermates is a key and necessary precaution to obtain rigorous and trustable results.

In this chapter, we will describe the methods currently used in our laboratories to study the functional localization of CB1 receptors in mice. As mentioned earlier, all these experiments must be conducted with the use of reliable negative controls and only significant difference between samples from wild-type and CB1-KO mice should be considered as evidence for mitochondrial effects of cannabinoids. Of course, the youth of this research field leaves margins for improvements and for additional methods to study mtCB1 receptors. Some of these are currently tested in our laboratories, but only validated and confirmed methods will be described in this chapter. We hope that this work will be useful for scientists who might be interested by the novel field of the direct control of mitochondrial activity by CB1 receptors.