The Alamar Blue assay indicated that 20 μ m FCCP (overnight) caused a mean reduction in cell viability of 70 ± 2%. When CBD (100 n m and 1 μ m ) was coapplied with FCCP it was neuroprotective at both concentrations [percentage protection: 10% and 15%, respectively, n = 12 for both (p < 0.01)] ( Fig. 9 A), in line with the evidence from previous acute imaging experiments. This experiment was next repeated with the cells exposed to CBD for 1 h before FCCP application. A markedly enhanced protection was observed (100 n m CBD yielding 35 ± 3% protection and 1 μ m conferring 53 ± 2% protection). This level of protection was significantly greater than that seen without CBD preexposure in each case (p values <0.05). CsA was also found to be protective in this model in a dose-dependent manner, reaching maximal protection (43 ± 2%, n = 24, p < 0.001) at 20 μ m (shown in Fig. 9 A). As for oligomycin, when the FCCP toxicity experiment was repeated in cultured hippocampal neurons (with 1 h CBD preincubation), CBD proved to be protective by 27 ± 3% (n = 6, p < 0.01) ( Fig. 9 A).
The highly lipophilic nature of cannabinoids grants them access to intracellular sites of action, and a number of studies have suggested mitochondria as targets for cannabinoids (Bartova and Birmingham, 1976; Sarafian et al., 2003; Athanasiou et al., 2007). Modulation of [Ca 2+ ]i by CBD has also been observed in a variety of cell types (Ligresti et al., 2006; Giudice et al., 2007), including our previous work which demonstrated a CBD-induced non-CB1/TRPV1-receptor-mediated increase in [Ca 2+ ]i in hippocampal neurons (Drysdale et al., 2006). Subsequent studies showed CBD effects to be negatively modulated by the endocannabinoid system (Ryan et al., 2007), but the exact mechanisms remained to be fully characterized. Therefore, the present study investigated CBD actions upon mitochondria and Ca 2+ homeostasis as a potential basis for CBD’s neuroprotective properties.
Determination of cell death in hippocampal cultured neurons (live–dead cell staining kit) by multichannel image capture in cells treated with 20 μ m oligomycin. A, Transmission image. B, Cells with compromised cell membranes (rhodamine filter). C, Healthy cells (FITC filter). D, Merged image. A dead sample neuron is circled in each image. For further details, see Materials and Methods.
In an alternative approach, we induced seizure-like Ca 2+ oscillations by applying the K + channel blocker 4AP, thus also probing previously reported anti-convulsant actions of CBD. Here, 4AP (50 μ m ) applied to primary hippocampal cultures ( Fig. 3 A) induced a sustained rise in [Ca 2+ ]i that continued to cause Ca 2+ oscillations a few minutes after wash. When the 4AP application was immediately followed by 1 μ m CBD ( Fig. 3 B), oscillations were silenced (n = 29; 5 glia, 24 neurons). Alternating the order of application robustly demonstrated that CBD could also prevent the initiation of epileptiform activity by 4AP. This was proven to be the case in all neurons (n = 10) and almost all glia (n = 30/31) investigated ( Fig. 3 C).
Protection by CBD against mitochondrial toxins
Previous studies from our group have strongly suggested a link between CBD signaling and [Ca 2+ ]i regulation via intracellular Ca 2+ stores (Drysdale et al., 2006; Ryan et al., 2007). To explore and characterize the underlying mechanisms, experimental conditions were used which enhance excitability and increase the degree of loading of intracellular Ca 2+ stores. It was predicted that such conditions should increase the CBD response compared with responses in standard HBS ( Fig. 2 A), as reported for other store-operated signaling cascades (Irving and Collingridge, 1998). Thus, CBD was applied (1 μ m ; 5 min) in HBS with doubled K + concentration (10.8 m m ). Surprisingly, under these conditions the effect of CBD application was to reduce [Ca 2+ ]i in both neurons and glia ( Fig. 1 B). The neuronal response was −26 ± 2% ΔF/F (n = 19), with almost identical responses in glia [−26 ± 4% ΔF/F (n = 19)], p values <0.001 compared with CBD controls ( Fig. 1 C), suggesting that [Ca 2+ ]i regulation by CBD is bidirectional and depends on excitability.
SH-SY5Y cells were grown on 96-well plates and treated with FCCP overnight as described above. MitoCapture reagent (Calbiochem) was diluted 1:1000 in PBS (at room temperature) before use. The medium was removed from the wells of the plate and replaced with 50 μl of reagent solution and placed in an incubator (37°C at 5% CO2) for 15–20 min. The cells were then washed twice with PBS and run through the plate reader (Synergy HT, Bio-Tek) with two fluorescence channels measured (green monomers: excitation 488 nm and emission 530 nm; red aggregates: excitation 488 nm and emission 590 nm). Control and toxin groups were run in 6 samples (wells) per experiment and performed three times.
For calcium imaging experiments (see also Ryan et al., 2006), hippocampal cultures were washed with HBS (as above) at room temperature and loaded with the cell-permeable fluorescent calcium indicator fura-2 AM (10 μ m , Invitrogen) for 1 h in the dark. To allow the monitoring of postsynaptic events uncontaminated by spontaneous activity and transmitter release, the sodium channel blocker tetrodotoxin (TTX, 0.5 μ m , Alomone Labs) was added to all perfusion media (except in experiments with 4AP). Cultures were perfused with HBS or low-Mg 2+ (0.1 m m ) HBS, using a gravity perfusion system at a flow rate of 1–2 ml/min.
Materials and Methods
CBD effects on epileptiform activity in cultured hippocampal neurons. A, Application of the K + channel antagonist 4AP to naive cultures induces spontaneous Ca 2+ oscillations. B, C, The presence of CBD following (B), or preceding (C), 4AP application dampened Ca 2+ oscillations. Data are presented as %ΔF/F.
The reversal of Ca 2+ responses in hippocampal cultures in the presence of an already elevated [Ca 2+ ]i (as a result of increased K + in the perfusion media) ruled out the ER receptors as the primary source of Ca 2+ in CBD responses, but instead echoed the theory of the mitochondrial Ca 2+ “set point” (Nicholls, 2005), i.e., the cytosolic concentration of Ca 2+ at which mitochondrial uptake and efflux of Ca 2+ are equal: interactions between Ca 2+ influx and efflux mechanisms in the mitochondria maintain extramitochondrial Ca 2+ concentrations at a fixed value (Nicholls, 1978; Thayer and Miller, 1990). Therefore, the opposing CBD responses may be achieved via reversal of one and the same Ca 2+ transport mechanism (see also Poburko et al., 2006). Additionally, the ER as the principal source of Ca 2+ released by CBD was effectively discounted by the combined application of dantrolene and 2-APB, which did not prevent CBD responses. 2-APB blocks sites other than IP3 receptors, including TRP channels (for review, see Bootman et al., 2002), a subset of which can act as Ca 2+ release channels from the ER. This is an important consideration, as phytocannabinoids can raise Ca 2+ via these channels (De Petrocellis et al., 2008), yet a contribution to the CBD response in our experiments is unlikely as suggested by our data obtained with 2-APB ( Fig. 6 ) (see also Tsuzuki et al., 2004). Instead, the trend of increased CBD responses in the presence of 2-APB is in agreement with similarly enhancing actions observed with TRPV1 and CB1 antagonists (Ryan et al., 2007).
In another immunological study using murine thymocytes, treatment with 8 μM CBD led to an
CBD treatment range of all in vitro studies.
In addition to studying brain mitochondria directly, the effects of CBD on respiration have also been tested in a rat primary hippocampal cell culture . In this system, which contained approximately 2:1 neurons to glia, CBD protected against the death of cells subjected to two different mitochondrial toxins that inhibit cellular respiration . First, oligomycin was used to inhibit the phosphorylation of ADP by complex V, which decreased the viability of the cells by 35% . The administration of 1 μM CBD diminished the effects of oligomycin, significantly increasing the number of viable cells by 15 ± 4% . Similar effects were observed following addition of the mitochondrial oxidative phosphorylation uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). While FCCP had a greater negative effect, reducing cell viability by 70%, the co-administration of 1 μM CBD with FCCP once again improved the viability of the cells by 15% .
Although these pathways and processes are often investigated separately, they demonstrate notable convergence on mitochondrial function and, indeed, growing evidence has demonstrated a role for CBD in the modulation of mitochondrial processes. Recently, CBD has been found to regulate intracellular and mitochondrial calcium concentrations , mitochondria-mediated intrinsic apoptosis , mitochondrial DNA epigenetics , mitochondrial ferritin concentrations , electron transport chain activity , mitochondrial biogenesis , and mitochondrial network dynamics . Since abnormal mitochondrial function has been closely linked to the development of a wide variety of disorders, ranging from age-related neurodegenerative diseases  to cardiovascular diseases , diabetes mellitus , and multiple cancers , development of a more complete understanding of the effects of CBD on mitochondrial function may identify novel therapeutic applications. Here, we review recent advances in the understanding of the mechanisms of CBD-mediated regulation of mitochondrial function, with an emphasis on neural health and translational relevancy based on current knowledge of CBD pharmacokinetics.
4. CBD and Epigenetic Modifications of Mitochondrial DNA
Effects of CBD on mitochondrial activity.
In a study of THP monocytes, cellular respiration was also determined following treatment with 10.68 and 21.64 μM CBD, concentrations found to inhibit cell viability by 10% and 50%, respectively . While 10.68 μM CBD had no significant effect on mitochondrial bioenergetics, 21.64 μM CBD inhibited the maximal respiration and ATP production of THP monocytes by 58% and 60%, respectively . Current evidence, therefore, indicates that CBD exposure exceeding 10 μM has a detrimental effect on the mitochondrial respiration of immune cells. Additional research is therefore clearly warranted, examining the effects of CBD on mitochondrial respiration of other immune cell types, such as macrophages, B and T cells, dendritic cells, basophils, and others, and also examining CBD doses in a pharmacologically relevant range.
Apoptosis is a normal and critical regulatory process for immune system functioning , and CBD has been found to alter the intrinsic apoptosis of immune cells. In a study by Wu and colleagues, treatment of human CD14 + monocytes with 16 μM CBD induced a greater than 25-fold increase in apoptosis after 1 h, with an approximate doubling evident at 30 min . This was accompanied by a doubling in intracellular ROS at 1 h, but was preceded by a significant increase in the percent of depolarized cells already at 5 min. Cytochrome c release into the cytosol and cardiolipin oxidation were also increased by CBD treatment at this level . To determine the primary mechanism behind the effects of CBD on monocyte apoptosis, the researchers co-administered CBD with a mitochondrial permeability transition pore (mPTP) inhibitor, cyclosporin A (CsA). This treatment markedly attenuated the apoptotic effects of CBD , indicating that opening of the mPTP is a mechanism through which CBD induced apoptosis in monocytes in this study.
Cannabidiol (CBD) is part of a group of phytocannabinoids derived from Cannabis sativa. Initial work on CBD presumed the compound was inactive, but it was later found to exhibit antipsychotic, anti-depressive, anxiolytic, and antiepileptic effects. In recent decades, evidence has indicated a role for CBD in the modulation of mitochondrial processes, including respiration and bioenergetics, mitochondrial DNA epigenetics, intrinsic apoptosis, the regulation of mitochondrial and intracellular calcium concentrations, mitochondrial fission, fusion and biogenesis, and mitochondrial ferritin concentration and mitochondrial monoamine oxidase activity regulation. Despite these advances, current data demonstrate contradictory findings with regard to not only the magnitude of effects mediated by CBD, but also to the direction of effects. For example, there are data indicating that CBD treatment can increase, decrease, or have no significant effect on intrinsic apoptosis. Differences between studies in cell type, cell-specific response to CBD, and, in some cases, dose of CBD may help to explain differences in outcomes. Most studies on CBD and mitochondria have utilized treatment concentrations that exceed the highest recorded plasma concentrations in humans, suggesting that future studies should focus on CBD treatments within a range observed in pharmacokinetic studies. This review focuses on understanding the mechanisms of CBD-mediated regulation of mitochondrial functions, with an emphasis on findings in neural cells and tissues and therapeutic relevance based on human pharmacokinetics.
Following the first study, McKallip and colleagues (2006) conducted a series of experiments examining CBD and apoptosis in leukemia and lymphoma cell lines . The first experiment showed a dramatic increase in apoptosis of murine EL-4 lymphoma cells grown in vitro following treatment with 2.5 μM CBD . This effect was recapitulated in vivo. C57BL/6 mice were injected intraperitoneally with EL-4 tumor cells and then with 25 mg/kg body weight CBD 10 days later. Twenty-four hours later, cells were collected from the peritoneal cavity, and cancer cell apoptosis was found to be almost 10% greater in mice given the single-dose CBD treatment. In the final experiments, human Jurkat and MOLT-4 leukemia cells were studied. Treatment of cells with 2.5 μM CBD significantly decreased viable cell numbers and increased apoptosis in both lines, with the largest effects evident in MOLT-4 cells. This included a seven-fold decrease in cell viability and an almost 50% increase in the percentage undergoing apoptosis, although effects in Jurkat cells were also significant, with an approximate 33% reduction in cell viability and 15% increase in apoptosis. Further analysis of Jurkat cells revealed greater activation of caspase 9, decreased full-length BID, and increased release of cytochrome c into the cytosol as mechanistic regulators of the observed effects on viability and apoptosis . It was also suggested that the apoptotic effects of CBD on Jurkat cells was due, at least in part, to an increase in oxidative stress mediated by the greater expression of ROS-producing NAD(P)H oxidases . Notably, the concentrations used in this study were aligned with circulating concentrations reported following intravenous infusion of CBD and, therefore, are therapeutically relevant .