Cannabinoid drugs have numerous therapeutic properties that can be exploited in the near future. However, the more we investigate the endocannabinoid system, the more complex it is becoming. It is thus necessary to fully dissect how cannabinoid receptors exert different functions in order to maximize their therapeutic potential and to avoid their possible side effects.

It is well known that CB1 receptors are mainly expressed in the brain but also in peripheral tissues. Moreover, it has been described how these receptors can be expressed in different cell types (reviewed in Busquets-Garcia et al. 2018). In this article, the localization of CB1 receptors in a subcellular localization, the mitochondrial membranes, is discussed.

Mitochondria in the brain: a hot topic in behavioral neuroscience research

Mitochondria is a double-membrane-bound organelle found in most eukaryotic organisms. The word mitochondria come from the Greek μίτος, mitos, "thread", and χονδρίον, chondrion, "grain-like". Mitochondria exert several functions for the cell, including energy production through adenosine triphosphate, oxidative stress modulation or the control of calcium metabolism inside the cell. In neuroscience, these organelles are gaining popularity and different studies demonstrate its importance for different brain functions, including the fine control of behavior (reviewed in Mattson et al. 2008, Kann et al. 2007 and Kovac et al. 2008).

Brain activity depends on the high energetic support provided by mitochondria. This ability to transform energy sources into usable molecular substrates represents the conditio sine qua non of life. However, there is something more than mere cell survival in the relationship between brain and mitochondria. The human brain weighs only 2% of the body, whereas it can consume up to 25% of body energy substrates. Taking into account that whereas other organs can undergo ischemia for relatively long periods (e.g. 20-30 minutes for heart and kidney, hours for skeletal muscles) and then rescue at least partially their functionality upon reperfusion, few seconds of blood flow interruption to the brain are lethal for the body. This extreme sensitivity is due to failure of brain functioning in terms of synaptic transmission, plasticity and circuit integrity. Thus, compared to other organs, the brain needs bioenergetic processes, likely controlled by mitochondria, not only to survive, but also for ongoing functional activity.

One of the main events taking place in the mitochondria is a complex chain of enzymatic reactions, which is called oxidative phosphorylation cascade, needed to supply energy for survival and ongoing activity of cells. Besides cell survival, energy is used in the brain for many different specific functions, ranging from enzymatic activities to neurotransmitter release and synaptic plasticity, among numerous processes. Production of energy is not the only task exerted by brain mitochondria, which are involved in (i) the production of reactive oxygen species, (ii) the metabolism of many neurotransmitters, gliotransmitters and other signaling molecules, (iii) the regulation of cellular ionic levels (e.g. of calcium) and (iv) the modulation of apoptosis, among others. Interestingly, recent data suggest that brain mitochondria can even be transferred between cells, thereby representing bona fide signaling elements of intercellular communication. Overall, it is not surprising that alterations of their properties have been involved in many neurological and neuropsychiatric disorders.

GPCR and mitochondria: an example of a broken dogma

In our pockets, we normally bring different keys to open different elements (e.g. home, work, lockers, ...). Each key will open only one of these elements in a specific manner. In biology, signals are transmitted through receptors and ligands that come in many forms, but they all have one thing in common: they come in closely matched pairs, with a receptor recognizing just one (or a few) specific ligands, and a ligand binding to just one (or a few) target receptors. The binding of a ligand to a receptor changes its shape or activity, allowing it to transmit a signal or directly produce a change inside of the cell. Among other receptors, G protein-coupled receptors, also known as seven-(pass)-transmembrane domain receptors, constitute a large protein family of receptors, which are involved in the regulation of several cellular responses. The ligands that bind and activate these receptors include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters.

In the last decades, the restricted view of G protein-coupled receptors present only in the plasma membrane has been challenged and the dogma that these receptors are found exclusively in this external membrane has been challenged. For example, these receptors are found on nuclear or endosomal membranes and important emerging studies are now pointing to their presence in the mitochondria (angiotensin receptor, purine receptors or beta-adrenergic receptors). In this article, a special emphasis will be given to the type-1cannabinoid (CB1) receptor, which is an interesting example of GPCR found in both the plasmatic and the mitochondria membranes, among other subcellular compartments.

Cannabinoids and mitochondria: a historical relationship

During the last century, different studies reported effects of cannabinoids on mitochondrial functions, including a decrease of the activity of different elements of the oxidative cascade and changes of mitochondrial ultrastructure. The scientific community did not fully understand these effects and they were kept aside without further explanation during some years. Moreover, with the identification of CB1 receptors as typical plasma membrane G-coupled receptors, they were ascribed to unspecific alterations of mitochondrial membrane properties by lipid molecules, to indirect CB1 receptor-dependent signaling or to CB1 receptor-independent events. However, this vision has changed with the use of novel approaches in the last decades.

The large majority of plant-derived, synthetic and endogenous arachidonic acid-derivatives endocannabinoids are lipids. 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. In line with the idea that they 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. 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. Even more recently, we and others found that CB1 receptors are functionally present at brain and peripheral mitochondrial membranes, where they can regulate cellular respiration and other bioenergetic processes, and mediate behavioral effects of cannabinoid drugs.

Mitochondrial CB1 receptors: knowledge and perspectives

In 2012, electron microscopic experiments accompanied by controlled functional assays revealed that a small but significant proportion of hippocampal CB1 receptors are localized at mitochondrial membranes, where they control mitochondrial respiration (Benard et al, 2012, Hebert-Chatelain et al, 2016). Interestingly, similar mitochondrial localization of CB1 receptors has been also shown in peripheral tissues, such as sperm cells and muscles, where the proportion of mtCB1 receptors appears to be higher than in the brain, and in specific brain cells such as astrocytes.

All these anatomical evidence lead to more laborious studies in order to dissect if mitochondrial CB1 receptors activate a specific signaling pathway. Pharmacological and genetic experiments showed that the effects of cannabinoids on mitochondrial respiration are involving a specific cascade formed by different enzymatic proteins that ultimately affect the phosphorylation oxidation cascade. Importantly, genetic approaches showed that this signaling cascade is necessary for specific effects of cannabinoids in vitro and in vivo (Benard et al, 2012, Hebert-Chatelain et al, 2016). However, the mitochondrial CB1-dependent intramitochondrial signaling cascade is far from being completely understood and presents surprising elements. For example, it is not clear yet how mitochondrial CB1 receptors could trigger the reduction of oxygen consumption by brain mitochondria via an interaction between G proteins and specific enzymatic proteins found in the mitochondria. Moreover, it is possible that cell-type specific intramitochondrial signaling complexes exist. Further studies are required to clarify this and other issues linked to the discovery of mitochondrial CB1 receptors and to identify the specific effects of cannabinoids and functions of the endocannabinoid system that are mediated by this novel direct interaction of a G-coupled receptors with mitochondrial functions. For example, we are still missing to understand how CB1 receptors decide to go to the mitochondrial membranes, whether this process is active and transient and which other mitochondrial functions can be regulated by CB1 receptors.

As mentioned above, by regulating innumerous cellular processes beyond energy production, mitochondria exert a plethora of functions that are particularly crucial for one of the most energy-demanding organs of the body, such as the brain. Thus, the elucidation of novel mitochondrial CB1-dependent functions can open great opportunities in the potential therapeutic aspect of cannabinoid drugs. Moreover, it will be very important to dissect the possible involvement of mitochondrial CB1 receptors in pathologic conditions were CB1 receptors exert a specific role. Overall, thanks to the use of new tools such as advanced imaging, genetic, viral and behavioral techniques, researchers in the cannabinoid field must fully understand how CB1 receptors, with or without the involvement of the mitochondria, are specifically exerting their physiological or pathological functions.

References:

Busquets-Garcia, A., Bains, J., and Marsicano, G. (2018). CB1 Receptor Signaling in the Brain: Extracting Specificity from Ubiquity. Neuropsychopharmacology 43, 4-20.

Mattson, M. P., Gleichmann, M. & Cheng, A. Mitochondria in neuroplasticity and neurological disorders. Neuron 60, 748-766 (2008).

Kann, O. & Kovacs, R. Mitochondria and neuronal activity. Am J Physiol Cell Physiol 292, C641-657 (2007).

Kovac, L. Bioenergetics: A key to brain and mind. Commun Integr Biol 1, 114-122 (2008).

Benard G, Massa F, Puente N, Lourenco J, Bellocchio L, Soria-Gomez E, et al (2012). Mitochondrial CB(1) receptors regulate neuronal energy metabolism. Nat Neurosci 15(4): 558-564.