Seizures: 2-AG dampens down seizure activity but increases post-seizure oxygen deprivation in the brain


A marijuana-like chemical in the brain, mirroring its plant-based counterpart, packs both ups and downs.

Epileptic seizures trigger the rapid synthesis and release of a substance mimicked by marijuana’s most psychoactive component, Stanford University School of Medicine investigators have learned.

This substance is called 2-arachidonoylglycerol, or 2-AG, and has the beneficial effect of damping down seizure intensity.

But there’s a dark side. The similarly rapid breakdown of 2-AG after its release, the researchers found, trips off a cascade of biochemical reactions culminating in blood-vessel constriction in the brain and, in turn, the disorientation and amnesia that typically follow an epileptic seizure.

The Stanford scientists’ findings, reached in collaboration with colleagues at other institutions in the United States, Canada and China, are described in a study to be published Aug. 4 in Neuron. Ivan Soltesz, PhD, professor of neurosurgery, shares senior authorship with G. Campbell Teskey, PhD, professor of cell biology and anatomy at the University of Calgary in Alberta, Canada. The study’s lead author is Jordan Farrell, PhD, a postdoctoral scholar in Soltesz’s group.

The researchers’ discoveries could guide the development of drugs that both curb seizures’ strength and reduce their aftereffects.

Electrical storm in the brain

About one in every hundred people has epilepsy. Epileptic seizures can be described as an electrical storm in the brain. These storms typically begin at a single spot where nerve cells begin repeatedly firing together in synchrony. The hyperactivity often spreads from that one spot to other areas throughout the brain, causing symptoms such as loss of consciousness and convulsions. It’s typical for the person experiencing a seizure to need tens of minutes before becoming clearheaded again.

The majority of epileptic seizures originate in the hippocampus, a brain structure buried in the temporal lobe, said Soltesz, the James R. Doty Professor of Neurosurgery and Neurosciences. The hippocampus plays an outsized role in short-term memory, learning and spatial orientation.

Its ability to quickly adopt new neuronal firing patterns renders it especially vulnerable to glitches that initiate seizures. (Most epileptic seizures in adults begin in or near the hippocampus, Soltesz noted.)

In the study, Soltesz and his associates monitored split-second changes in levels of 2-AG in the hippocampus of mice during periods of normal activity, like walking or running, and in experiments in which brief seizures were induced in the hippocampus.

2-AG is an endocannabinoid, a member of a family of short-lived signaling substances that are the brain’s internal versions of the psychoactive chemicals in marijuana. 2-AG and these plant-derived psychoactive chemicals share an affinity for a receptor, known as CB1, that’s extremely abundant on the surface of neurons throughout the brain.

“There have been lots of studies providing evidence for a connection between seizures and endocannabinoids,” Soltesz said. “What sets our study apart is that we could watch endocannabinoid production and action unfold in, basically, real time.”

A brake on excitement
Endocannabinoids are understood to play a role in inhibiting excessive excitement in the brain. When excitatory neurons, secreting chemical “go” signals, exceed a threshold, they induce the production and release of endocannabinoids, whose binding to CB1 on an excitatory neuron acts as a brake, ordering that neuron to chill out a little.

While smoking marijuana floods the entire brain with relatively long-lasting THC, endocannabinoids are released in precise spots in the brain under precise circumstances, and their rapid breakdown leaves them in place and active for extremely short periods of time, said Soltesz, who has been studying the connection between endocannabinoids and epilepsy for decades.

But because endocannabinoids are so fragile and break down so quickly, until recently there was no way to measure their fast-changing levels in animals’ brains. “Existing biochemical methods were far too slow,” he said.

The most recent study had its start when Soltesz learned of a new endocannabinoid-visualization method invented by study co-author Yulong Li, PhD, a professor of neuroscience at Peking University in Beijing.

The method involves the bioengineering of select neurons in mice so that these neurons express a modified version of CB1 that emits a fluorescent glow whenever a cannabinoid binds to the modified endocannabinoid receptor. The fluorescence can be detected by photosensitive instruments.

Using this new tool, the scientists could monitor and localize sub-second changes in fluorescence that correlate with endocannabinoid levels where that binding was occurring.

Zeroing in on 2-AG

By blocking enzymes critical to the production and breakdown of different endocannabinoids, the researchers proved that 2-AG alone is the endocannabinoid substance whose surges and rapid disappearance track neuronal activity in the mice. Several hundred times as much 2-AG was released when a mouse was having a seizure compared with when it was merely running in place.

The researchers were able to rule out the involvement of an alternative endocannabinoid, anandamide, that many neuroscientists and pharmacologists had assumed was the active substance. Anandamide’s name is derived from the Sanskrit word for “bliss.”

“This previously undetected activity-dependent surge in levels of 2-AG downregulates excitatory neurons’ excessive rhythmic firing during a seizure,” Soltesz said.

But 2-AG is almost immediately converted to arachidonic acid, a building block for inflammatory compounds called prostaglandins. The researchers showed that the ensuing increase in arachidonic acid levels resulted in the buildup of a particular variety of prostaglandin that causes constriction of tiny blood vessels in the brain where the seizure has induced thatprostaglandin’s production, cutting off oxygen supply to those brain areas.

Oxygen deprivation is known to produce the cognitive deficits — disorientation, memory loss — that occur after a seizure, Soltesz said.

“A drug that blocks 2-AG’s conversion to arachidonic acid would kill two birds with one stone,” Soltesz said. “It would increase 2-AG’s concentration, diminishing seizure severity, and decrease arachidonic acid levels, cutting off the production of blood-vessel-constricting prostaglandins.”

Soltesz is a member of Stanford Bio-X, the Wu Tsai Neurosciences Institute at Stanford, and the Stanford Maternal and Child Health Research Institute.

Another Stanford co-author of the study is postdoctoral scholar Barna Dudok, PhD.

Other researchers at the University of Calgary, as well as researchers at Vanderbilt University, contributed to the work.

Funding: The study was funded by the National Institutes of Health (grants K99NS117795, MH107435, 1S10OD017997-01A1, NS99457 and NS103558), the Canadian Institutes of Health Research, the Beijing Municipal Science & Technology Commission, the National Natural Science Foundation of China, and the Peking University School of Life Sciences.

Cannabinoids are a class of compounds that are plant-derived (phytocannabinoids, i.e. from Cannabis sativa), made naturally within the body, and chemically manufactured. For the purpose of this review, where the term “cannabinoids” is used, it will refer to the general class of compounds, unless specified otherwise. Endocannabinoids (endogenous cannabinoids) are unsaturated fatty acid derivatives with wide distribution in the human body [1,2,3].

The two most extensively studied endocannabinoids are N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG). AEA was the first endocannabinoid to be discovered [4] and is an important intermediate in lipid metabolism [5]. Its name was derived from the Sanskrit word “ananda” which means inner bliss, describing the euphoric effects of this ligand [5, 6].

Physiologically, it is produced ubiquitously [5] with the greatest tissue concentrations found in the brain [6]. Under most physiological conditions, 2-AG concentrations are much higher than that of AEA [5]. Interestingly, these endocannabinoids are not stored within cells, but are thought to be manufactured “on demand” from membrane phospholipid precursors [7, 8].

However, recent reports challenge this view suggesting that they may be stored intracellularly within lipid droplets referred to as adiposomes, thus allowing for intracellular accumulation [5, 9]. Others suggest that catabolic enzymes at the surface of adiposomes quickly lead to endocannabinoid degradation [7].

In light of this, sequestering of endocannabinoids in adiposomes may prolong their half-life (hours rather than minutes), allowing them time to trigger nuclear receptors [7].

AEA and 2-AG are known to bind to and activate two G-protein coupled receptors (GPCR): cannabinoid receptor 1 (CB1) and CB2. Both receptor isoforms are ubiquitously expressed, with CB1 found more predominantly in the central nervous system and CB2 found largely in cells of the immune system [5, 10, 11].

The receptor actions of AEA and 2-AG are mimicked by the exogenous cannabinoid Δ-9-tetrahydrocannabinol (THC), the primary psychoactive component of cannabis [4, 12]. In fact, it was the discovery in the 1980s that THC could bind to receptors in the brain that led researchers to discover AEA, the prototypical endocannabinoid [13].

AEA: Synthesis, transport, and degradation

AEA acts as a partial agonist at CB1 and as a weak/partial agonist at CB2 [5]. Interestingly, AEA is reported to demonstrate promiscuous binding activity [11], as it can trigger various signaling pathways via a number of different receptors, both extracellularly at CB1and CB2, intracellularly at the transient receptor potential vanilloid-1 (TRPV1) channel, and in the nucleus via the peroxisome proliferator-activated receptors (PPARs) [7]. More recently, studies have suggested that AEA may also act at the level of the mitochondria via CB1 receptors situated on the mitochondrial outer membrane [14].

The biosynthesis of AEA occurs in two steps. First, AEA is released from phospholipid precursors in the plasma membrane to a phosphatidylethanolamine, leading to the formation of N-acylphosphatidylethanolamine (NAPE). In the second step, a type D phospholipase (NAPE-PLD) catalyzes the formation of AEA from its NAPE precursor [5]. AEA is then rapidly taken up by cells [8, 15]. Movement of AEA across the phospholipid bilayer is thought to occur by simple diffusion or endocytosis [8, 15] since AEA is uncharged and lipid soluble. Other studies strongly suggest the involvement of a putative endocannabinoid membrane transporter (EMT) [8, 16, 17] which allows AEA to be rapidly shuttled to its intracellular targets [8, 15].

Some of the intracellular targets for AEA identified to date include AEA intracellular binding proteins (AIBPs), namely albumin, heat shock protein 70 (Hsp70) and fatty acid binding protein-5 and -7 (FABP-5 and -7) [8]. It has been proposed that FABPs are principally involved in AEA trafficking and breakdown, as the use of a novel reversible FABP inhibitor, BMS309403, partially reduced AEA uptake [15]. Ultimately, AEA is broken down by intracellular hydrolases into ethanolamine and arachidonic acid [5, 7]. A key regulator of AEA activity is the serine hydrolase fatty acid amide hydrolase (FAAH) which is bound to intracellular membranes, particularly the endoplasmic reticulum (ER) and nuclear membrane [7].

2-AG: Synthesis, transport, and degradation

Belonging to the monoacylglycerol (MAG) family of endocannabinoids, 2-AG acts equally at CB1 and CB2 as a full potent agonist, but it has not been shown to act at the TRPV1 receptor [5, 11]. Tissue levels of 2-AG are markedly higher than that of AEA within the same tissue [5]. Coupled with its full agonistic activity at cannabinoid receptors, it has been proposed as the primary endogenous agonist of both CB1 and CB2 [8].

The biosynthesis of 2-AG involves the combined action of two membrane-bound enzymes: phospholipase C (PLC) and diacylglycerol lipase (DAGL) [5]. Unlike AEA, only a few studies have investigated the mechanisms underlying the rapid cellular uptake of 2-AG, which is surprising given that it is more abundant than AEA [8]. While the mechanism(s) may be dependent upon the cell type [8, 16, 18], the most likely routes of entry for 2-AG may be via endocytosis, simple diffusion, the same EMT as AEA or other transporters [8, 16].

Once within the cytosol, 2-AG becomes a substrate for the chief catalytic enzyme monoacylglycerol lipase (MAGL), associated with the inner membrane, which degrades 2-AG to glycerol and arachidonic acid [8]. In addition to MAGL, two other 2-AG hydrolases have been identified: α,β-hydrolase-6 and -12 (ABHD-6 and -12), which are integral cell membrane proteins and thought to share the catalytic triad with MAGL [8]. In addition to the consensus regarding the putative degradative enzymes, these prototypical endocannabinoids may also be degraded via oxidation by cyclooxygenase (COX), lipoxygenase (LOX), or cytochrome P450 [5]. These and other biosynthetic and degradative pathways for AEA and 2-AG are discussed at length in Fezza et al [5].

THC and other ligands

THC was the first exogenous ligand of cannabinoid receptors to be discovered [13]. The physiological effects of THC are clinically concerning and need to be well delineated. Since this compound is highly lipophilic [5, 12, 19], it can be readily sequestered into adipose tissue, resulting in a rapid decrease in plasma concentrations [19], followed by a slow release into circulation over extended periods of time [12]. This tissue distribution permits longer-lasting stimulation of the cannabinoid receptors. This is unlike that of the locally released endocannabinoids AEA and 2-AG, which are rapidly inactivated by their transporters and hydrolases [12].

Endogenously, other fatty acid derived compounds, including N-arachidonoyldopamine (NADA), 2-arachidonoylglycerylether (noladin ether) and O-arachidonoyl ethanolamine (virodhamine) have also been identified as endocannabinoids, although information on their function and biological relevance is somewhat limited [4, 5, 10]. Interestingly, a novel group of ligands has been identified, referred to as retro-anandamides, which are characterized by a reversal in the positions of the carbonyl and the amido groups [20].

While these compounds demonstrate reduced affinity for CB1 and CB2 receptors as compared to AEA, they are resistant to FAAH catabolism resulting in increased stability relative to AEA [20]. These same authors, in an earlier report, identified the first metabolically stable AEA analogue, (R)-methanandamide, which exhibited significantly greater affinity for CB1 and resistance to FAAH degradation when compared to AEA [20]. Despite their presence, these and other ligands have received less scientific attention than AEA and 2-AG, perhaps due to the difficulty involved in isolating them from biological tissues [5].

Cannabinoid receptors

CB1 and CB2 belong to a large superfamily of seven-transmembrane spanning GPCRs [4]. AEA or 2-AG ligand binding to either CB1 or CB2 leads to multiple signal transduction mechanisms, including the inhibition of adenylate cyclase [4], a common target for activated G proteins, and consequent decrease in intracellular cAMP levels, increased potassium influx, and/or inhibition of certain calcium channels, thus reducing calcium influx [21, 22]. The intracellular signals arising from these cascades subsequently leads to the regulation of growth, proliferation, and/or differentiation [6].

The CB1 receptor

CB1 receptors are found mostly within the central nervous system [22]. Peripherally, CB1 receptors have been identified in the spleen, heart, adrenal gland, ovaries, endometrium, testes, among others [1, 22]. Furthermore CB1 receptors have also been localized intracellularly on the mitochondrial outer membrane [14]. Mitochondria regulate the energy demands of the cell, thus compromises in its function, from aberrant cannabinoid signaling [23, 24], will deregulate energy metabolism [25]. For example, THC induced mitochondrial dysfunction has been associated with pathologies such as stroke [26].

Mechanistically, mitochondrial CB1 receptors are thought to modulate complex I activity via a process involving soluble adenylyl cyclase [27]. Since mitochondrial function controls apoptosis, disruption of this role can impact the process of producing quality gametes, subsequently interfering with embryogenesis and lead to the production poor quality embryonic stem cells [28]. Furthermore, ovarian ageing is also associated with increased accumulation of mitochondrial DNA mutations which are likely to affect mitochondrial biogenesis and impact oocyte quality [29]. Additionally, placental oxidative stress is also linked to mitochondrial dysfunction [30] and may impact vascular remodeling in the placenta [31] which is important for tissue oxygenation and organ function.

Dysregulation of placental vascular development can result in a number of adverse pregnancy outcomes such as intrauterine growth restriction and preeclampsia [32, 33]. CB1 receptors are also found in the hypothalamus, the central regulator of energy homeostasis, further implicating a role for the ECS in energy balance. Moreover, the preoptic area of the hypothalamus contain CB1 receptors, from which secretory neurons for gonadotropin-releasing hormone (GnRH) are located [34].

The CB2 receptor

Like the CB1 receptor, CB2 is also a G protein coupled receptor demonstrating 44% sequence homology to CB1 [6, 22]. CB2 receptors are predominantly found peripherally within cells of the immune system, such as lymphocytes and macrophages [23]. El-Talatini et al confirmed the presence of CB2 receptors in the ovarian cortex, ovarian medulla, and ovarian follicles from human samples [1], which followed a similar staining pattern as CB1 in the same tissues [1]. However, Wang et al found that in murine oocytes, the action of endocannabinoids were mediated by CB1 receptor activation, not that of CB2. [35]. This interspecies difference highlights the importance of utilizing human reproductive tissues as a means to study the impact of the ECS on its function/dysregulation [1].

Other receptors

Endocannabinoids are thought to also target a number of other orphan receptors. These include the GPR55 (also known as CB3) and GPR119 receptors which have signaling mechanisms distinct from CB1/CB2 [3, 16, 36]. Other receptors targeted by endocannabinoids include TRPV1, cytosolic target for AEA; and nuclear PPAR. Pertwee et al provide a detailed review of these other receptors and their pharmacology [10].

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