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Cellular/Molecular
Cannabidiol Targets Mitochondria to Regulate Intracellular
Ca
2⫹
Levels
Duncan Ryan, Alison J. Drysdale, Carlos Lafourcade, Roger G. Pertwee, and Bettina Platt
School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom
Cannabinoids and the endocannabinoid system have attracted considerable interest for therapeutic applications. Nevertheless, the
mechanism of action of one of the main nonpsychoactive phytocannabinoids, cannabidiol (CBD), remains elusive despite potentially
beneficial properties as an anti-convulsant and neuroprotectant. Here, we characterize the mechanisms by which CBD regulates Ca
2⫹
homeostasis and mediates neuroprotection in neuronal preparations. Imaging studies in hippocampal cultures using fura-2 AM sug-
gested that CBD-mediated Ca
2⫹
regulation is bidirectional, depending on the excitability of cells. Under physiological K
⫹
/Ca
2⫹
levels,
CBD caused a subtle rise in [Ca
2⫹
]
i
, whereas CBD reduced [Ca
2⫹
]
i
and prevented Ca
2⫹
oscillations under high-excitability conditions
(high K
⫹
or exposure to the K
⫹
channel antagonist 4AP). Regulation of [Ca
2⫹
]
i
was not primarily mediated by interactions with
ryanodine or IP
3
receptors of the endoplasmic reticulum. Instead, dual-calcium imaging experiments with a cytosolic (fura-2 AM) and a
mitochondrial (Rhod-FF, AM) fluorophore implied that mitochondria act as sinks and sources for CBD’s [Ca
2⫹
]
i
regulation. Application
of carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the mitochondrial Na
⫹
/Ca
2⫹
exchange inhibitor, CGP 37157, but
not the mitochondrial permeability transition pore inhibitor cyclosporin A, prevented subsequent CBD-induced Ca
2⫹
responses. In
established human neuroblastoma cell lines (SH-SY5Y) treated with mitochondrial toxins, CBD (0.1 and 1
M) was neuroprotective
against the uncoupler FCCP (53% protection), and modestly protective against hydrogen peroxide- (16%) and oligomycin- (15%) medi-
ated cell death, a pattern also confirmed in cultured hippocampal neurons. Thus, under pathological conditions involving mitochondrial
dysfunction and Ca
2⫹
dysregulation, CBD may prove beneficial in preventing apoptotic signaling via a restoration of Ca
2⫹
homeostasis.
Key words: excitotoxicity; hippocampus; cannabinoids; ATP synthase; Na
⫹
/Ca
2⫹
exchanger; neuroprotection
Introduction
Two fundamental determinants of neuronal survival and viabil-
ity under pathological conditions are Ca
2⫹
homeostasis and met-
abolic activity, both reliant on mitochondrial function. Neurons
have a particularly high energy demand and correspondingly
high metabolic activity, alongside large fluctuations in [Ca
2⫹
]
i
;
thus, mitochondria play a particularly important role in this cell
type. Even subtle mitochondrial deficits can have deleterious ef-
fects that can ultimately result in degenerative processes (for re-
view, see Kajta, 2004). Energy deficiencies are also associated with
aging (Bowling et al., 1993) (for review, see Wiesner et al., 2006)
and age-related disorders, e.g., Alzheimer’s disease (de la Monte
and Wands, 2006), indicating a correlation with mitochondrial
dysfunction, as also recently suggested by a corresponding treat-
ment success in Alzheimer’s patients (Doody et al., 2008). Mito-
chondria are preferentially located in areas of highest [Ca
2⫹
]
i
adjacent to the endoplasmic reticulum, essential for the func-
tional coupling of these two organelles (Robb-Gaspers et al.,
1998; Szabadkai et al., 2003; Saris and Carafoli, 2005). Moreover,
mitochondria determine cellular survival by generation of reac-
tive oxygen species (Lafon-Cazal et al., 1993) and apoptotic fac-
tors (Hong et al., 2004). This process involves an increased per-
meability of mitochondrial membranes [including opening of
the mitochondrial permeability transition pore (mPTP) (Hunter
et al., 1976)]. Therefore, identification of agents that can restore
normal mitochondrial function is highly desirable.
The plant Cannabis sativa has for many centuries been re-
puted to possess therapeutically relevant properties. Its most
widely studied and characterized component, ⌬
9
-
tetrahydrocannabinol (THC), is one of 60⫹compounds from
Cannabis sativa, collectively known as phytocannabinoids. How-
ever, THC may have a limited usefulness due to psychoactivity,
dependence, and tolerance (Sim-Selley and Martin, 2002); there-
fore, attention has turned to some of the nonpsychoactive phy-
tocannabinoids, most notably cannabidiol (CBD). CBD has little
agonistic activity at the known cannabinoid receptors (CB
1
and
CB
2
) (Pertwee, 2004), and may possess therapeutic potential, e.g.,
anti-epileptic (Cunha et al., 1980), anxiolytic (Guimara˜es et al.,
1994), anti-inflammatory (Carrier et al., 2006), and even anti-
psychotic properties (Leweke et al., 2000) [for review, see Pertwee
(2004) and Drysdale and Platt (2003)]. In addition, CBD has
shown neuroprotection in a range of in vivo (Lastres-Becker et al.,
2005) and in vitro models (Esposito et al., 2006), some in associ-
ation with a reduction in [Ca
2⫹
]
i
(Iuvone et al., 2004).
The highly lipophilic nature of cannabinoids grants them ac-
Received Sept. 4, 2008; revised Jan. 13, 2009; accepted Jan. 13, 2009.
We thank GW Pharmaceuticals for the provision of CBD.
Correspondence should be addressed to Bettina Platt at the above address. E-mail: b.platt@abdn.ac.uk.
C. Lafourcade’s present address: Departments of Physiology and Psychiatry, University of Maryland School of
Medicine, 655 West Baltimore Street, BRB 5-025, Baltimore, MD 21201.
DOI:10.1523/JNEUROSCI.4212-08.2009
Copyright © 2009 Society for Neuroscience 0270-6474/09/292053-11$15.00/0
The Journal of Neuroscience, February 18, 2009 •29(7):2053–2063 • 2053
cess 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 vari-
ety of cell types (Ligresti et al., 2006; Giudice et al., 2007), includ-
ing our previous work which demonstrated a CBD-induced non-
CB
1
/TRPV
1
-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 endocan-
nabinoid 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⫹
ho-
meostasis as a potential basis for CBD’s neuroprotective
properties.
Materials and Methods
Hippocampal culture preparation. Preparation of standard primary hip-
pocampal cultures from Lister–Hooded rat pups (1–3 d old) was con-
ducted as described previously (Drysdale et al., 2006; Ryan et al., 2006),
conforming to Home Office and institute regulations. Briefly, pups were
killed by cervical dislocation and the brain removed, and the hippocampi
were dissected out and placed in filtered ice-cold HEPES-buffered solu-
tion (HBS, composition in mM: NaCl, 130; KCl, 5.4; CaCl
2
, 1.8; MgCl
2
,1;
HEPES, 10; glucose, 25; compounds from Sigma-Aldrich). Hippocampal
tissue was finely chopped and placed in a 1 mg/ml protease solution (type
X and XIV, Sigma-Aldrich) for 40 min. Graded fire-polished glass Pas-
teur pipettes were used to triturate the tissue a number of times. Follow-
ing centrifugations, the tissue pellet was resuspended in tissue culture
medium [90% minimum essential medium (MEM; Invitrogen), 10%
fetal bovine serum (FBS) (Helena Biosciences), and 2 mML-glutamine
(Sigma-Aldrich)], kept in a humidified incubator at 37°C and in 5% CO
2
,
and plated in 35 mm culture dishes (Invitrogen, coated with poly-L-
lysine, Sigma-Aldrich). After 1 h, an additional 2 ml of tissue culture
medium was gently added to each dish and stored in a humidified incu-
bator (37°C; 5% CO
2
). After2dofmaturation, the MEM was replaced
with Neurobasal medium (Invitrogen) to reduce glial growth [composi-
tion of culture by cell-type (2:1, neurons:glia) was in keeping with that
outlined in previous publications (Platt et al., 2007)], containing 2% B27,
2m
ML-glutamine, and 25
ML-glutamate (Sigma-Aldrich). Culture
dishes were checked for uniform density and deemed suitable for imag-
ing experiments from 5 to 10 d in vitro based on fully reproducible
NMDA responses (variability: ⬍5%), with control experiments con-
ducted at regular intervals.
Fura-2 AM Ca
2⫹
imaging. 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 fluores-
cent calcium indicator fura-2 AM (10
M, Invitrogen) for1hinthedark.
To allow the monitoring of postsynaptic events uncontaminated by
spontaneous activity and transmitter release, the sodium channel blocker
tetrodotoxin (TTX, 0.5
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 mM) HBS, using a gravity perfusion system at a
flow rate of 1–2 ml/min.
The imaging system, fitted onto an Olympus BX51WI fixed stage mi-
croscope, used the Improvision software package Openlab (version 4.03,
Improvision) with a DG-4 illumination system (Sutter Instruments) and
a Hamamatsu Orca-ER CCD camera for ratiometric imaging. After an
appropriate field of cells was identified, a gray-scale transmission image
was visualized and captured. Cells were excited with wavelengths of 340
and 380 nm, and the ratio of fluorescence emitted at 510 nm analyzed
after subtraction of background fluorescence levels. As described in our
previous publications, fields of cells and regions of interest (ROIs) were
chosen based on homogenous and equal cell densities, with a neuronal
population of 15– 40 cells per field of view. ROIs were placed on all fura-2
AM-loaded neuronal cell bodies and large, star-shaped glia, confirmed to
be astrocytes by GFAP staining, and based on an overlay of a transmission
image (Koss et al., 2007). Following this, time courses were created for all
cells (neurons and glia), with frames captured every 5 s.
Mitochondrial Ca
2⫹
imaging. The mitochondrial and cytosolic Ca
2⫹
compartments were visualized simultaneously by preloading cultures
with the mitochondrion-specific Ca
2⫹
sensor Rhod-FF, AM (Invitro-
gen). Culture dishes were incubated with Rhod-FF, AM (5
M, in stan-
dard HBS) for 15 min on the day before experimentation to allow com-
partmentalization of the marker (specificity of this marker was
confirmed by the abolition of compartmentalization by FCCP applica-
tion) (see Fig. 4Ci,Cii). HBS was replaced with fresh Neurobasal medium
and returned to the incubator overnight. The following day, cells were
loaded with fura-2 AM as described above. Dual imaging was performed
with alternating wavelengths relevant to Rhod-FF (excitation: 550 nm;
emission: 580 nm) and fura-2 AM (as above) delivered at intervals of 3 s.
Both images were background subtracted, and separate graphs were plot-
ted on-line (see Fig. 4). For off-line analysis of mitochondrial responses,
data were imported into the Volocity analysis program (version 4.02,
Improvision). Areas of most intense Rhod-FF mitochondrial fluores-
cence within a single neuron were allocated ROIs.
SH-SY5Y cell preparation. The established human neuroblastoma cell
line, SHSY-5Y (SH), was grown in 30 ml flasks in MEM-based medium
supplemented with growth factor F12, 10% fetal bovine serum, 2 mM
L-glutamine, and 50
g/ml antibiotic. Cells were maintained at 37°C at
5% CO
2
. Medium was replaced every 2–4 d, after washing with PBS (1
mMphosphate). Once cells proliferated to ⱖ80% confluency they were
passaged or transferred to a 96-well plate (Greiner) for experimental
treatment (final volume in medium: 150
l of cell suspension per well).
Plates were used for experimentation when ⱖ80% confluency was
achieved (typically taking 4 d).
Each treatment and relevant vehicle controls were run in six samples
(wells) per experiment and repeated at least twice, viability was com-
pared using the nontoxic cell viability marker Alamar Blue (Serotec).
This marker was made up as a 10% solution in MEM and applied to all
wells (following the removal of treatment medium) for 2 h at 37°C, 5%
CO
2
. The plates were then run in a plate reader (either Victor
2
1420,
Wallac, Perkin-Elmer or Synergy HT, Bio-Tek) and the fluorescence
(excitation: 530 nm and emission: 590 nm) measured.
Cell death in hippocampal cultures. Hippocampal cultures were prein-
cubated with CBD for 1 h before coapplication of CBD with
mitochondrion-acting toxins overnight, following which cell death was
quantified using a Live-Dead staining kit (Sigma) (modified from our
previous publications) (Platt et al., 2007). Briefly, 10
l of solution A and
4
l of solution B were diluted in 5 ml of HBS (at room temperature).
Each dish was washed in HBS three times and 500
l of the staining
solution added and incubated for 20 min (in the dark, at room temper-
ature). After a further wash with HBS, live images were capture in HBS
with a 40⫻phase-contrast water-immersion objective [brightfield, FITC
(live cells) and rhodamine filters (dead cells)] using an Axioskop 2 plus
microscope (Carl Zeiss) fitted with an AxioCam HRc camera, with Ax-
ioVision software (version 3.1). Three images were taken from each dish
and each experiment performed on at least two dishes from three differ-
ent cultures.
MitoCapture. SH-SY5Y cells were grown on 96-well plates and treated
with FCCP overnight as described above. MitoCapture reagent (Calbio-
chem) 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
l
of reagent solution and placed in an incubator (37°C at 5% CO
2
) 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 per-
formed three times.
Drugs and stock solutions. CBD, obtained from GW Pharmaceuticals,
and AM281 (Tocris Bioscience) were stored in ethanol (1 mg/ml) at
⫺20°C. For use in experiments, the ethanol was evaporated and the
cannabinoid resuspended in dimethyl sulfoxide (DMSO) at 1 mM(con-
trol experiments confirmed that 0.1% DMSO did not alter basal Ca
2⫹
levels or NMDA-induced Ca
2⫹
responses, data not shown). The toxins
tested in the SH-SY5Y model were as follows: hydrogen peroxide (H
2
O
2
,
Sigma-Aldrich) at 0.1 and 0.5 mM, 3 h application; oligomycin (20
M,
2054 •J. Neurosci., February 18, 2009 •29(7):2053–2063 Ryan et al. •CBD and Mitochondria
Sigma-Aldrich) applied overnight (used in the same manner in hip-
pocampal culture cell death models also); FCCP (Sigma-Aldrich) was
also applied overnight at 20
M(also applied in the same concentration
and duration in hippocampal culture cell death models). In each case,
pilot experiments were performed to determine suitable concentrations
resulting in a degree of cell death that leaves capacity for either a reduc-
tion or increase in cell viability (targeted reduction in cell viability: 40 –
70%). The sites of action of these toxins can be seen in Figure 1. Other
compounds tested to elucidate CBD’s mechanisms of action were (with
final concentrations and stock solvents listed) as follows: catalase (500
and 1000 U/ml; MEM), cyclosporin A (CsA; 1–20
M; DMSO), butylated
hydroxytoluene (BHT; 3 and 10
M; DMSO), and dantrolene (10
M;
H
2
O), all from Sigma-Aldrich. Additionally, 4-aminopyridine (4AP; 50
M; DMSO), 2-aminoethoxydiphenyl borate (2-APB; 100
M; DMSO),
and CGP 37157 (10
M; DMSO) were obtained from Tocris Bioscience.
For all compounds tested, drug-only controls were performed.
Data analysis. All fura-2 AM fluorescence values were converted into
%⌬F/F, where ⌬Fis the change in fluorescence, calculated as a percent-
age of baseline fluorescence (F), which was defined as an average of five
baseline values before drug application (Drysdale et al., 2006; Ryan et al.,
2007). Each group of experiments consisted of at least three independent
replications from different cultures. A change in fluorescence of ⱖ10% of
baseline fluorescence was deemed a genuine response to drug applica-
tions (with intrinsic Ca
2⫹
fluctuations ⫾5%). Data were exported to
Excel and GraphPad Prism (version 4, Graph Pad Software) for prepara-
tion of graphs and statistical analysis. Due to the absence of normal
distribution, Kruskal–Wallis nonparametric tests with Dunn’s post hoc
test were used for multiple-group comparisons, and a Mann–Whitney U
test applied for paired comparisons.
For work with SH cells, data generated as units of fluorescence inten-
sity were transferred to Excel and converted into percentage of within-
plate controls for graphical presentation only. Statistical analysis was
performed on raw data using Prism, with an overall one-way ANOVA
performed for multiple-group comparison. For overall pvalues ⬍0.05,
Tukey’s posttest was used for paired comparison. Comparison of two
relevant groups was conducted using an unpaired ttest.
Significance for all statistical analyses performed was set at p⬍0.05 ⫽
significant; p⬍0.01 ⫽highly significant; p⬍0.001 ⫽very highly significant.
Results
CBD regulates Ca
2ⴙ
homeostasis in hippocampal tissue
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 con-
ditions 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 com-
pared with responses in standard HBS (Fig. 2A), as reported for
other store-operated signaling cascades (Irving and Collingridge,
1998). Thus, CBD was applied (1
M; 5 min) in HBS with dou-
Figure 1. Mitochondrial components of Ca
2⫹
regulation and targets for drug action used to
assess the action of CBD. Drugs/enzymes used to induce cell death in SH-SY5Y cells are italic and
underlined,andthoseapplied to identify possible mechanisms ofprotectionareoutlined. SOD2,
Superoxide dismutase 2.
Figure 2. Bidirectional Ca
2⫹
responses to CBD in hippocampal cultures. A,B, Sample traces
for CBD-mediated Ca
2⫹
responses in neurons (black traces) and glia (gray traces) in normal (A)
andhigh-excitability(B)HBS(doubleK
⫹
concentration).NMDAapplicationsattheendofeach
experiment were used to confirm intact signaling in neurons. C, Mean responses of CBD in
normal (ctrl) and high (high ex)-excitability HBS. Data are presented as %⌬F/F⫹SEM.
***p⬍0.001.
Ryan et al. •CBD and Mitochondria J. Neurosci., February 18, 2009 •29(7):2053–2063 • 2055
bled K
⫹
concentration (10.8 mM). Surprisingly, under these con-
ditions the effect of CBD application was to reduce [Ca
2⫹
]
i
in
both neurons and glia (Fig. 1B). The neuronal response was
⫺26 ⫾2% ⌬F/F(n⫽19), with almost identical responses in glia
[⫺26 ⫾4% ⌬F/F(n⫽19)], pvalues ⬍0.001 compared with CBD
controls (Fig. 1C), suggesting that [Ca
2⫹
]
i
regulation by CBD is
bidirectional and depends on excitability.
In an alternative approach, we induced seizure-like Ca
2⫹
os-
cillations by applying the K
⫹
channel blocker 4AP, thus also
probing previously reported anti-convulsant actions of CBD.
Here, 4AP (50
M) applied to primary hippocampal cultures
(Fig. 3A) 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
MCBD (Fig. 3B),
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. 3C).
CBD, mitochondria, and [Ca
2ⴙ
]
i
levels
Previous work from our laboratory indicated a link between
CBD-induced Ca
2⫹
responses and intracellular Ca
2⫹
stores
(Drysdale et al., 2006), rather than extracellular Ca
2⫹
sources.
Thus, we next investigated a potential role of mitochondria, fun-
damental players in cellular Ca
2⫹
homeostasis, in CBD’s action.
To simultaneously study mitochondrial signaling together with
cytosolic Ca
2⫹
responses, cultures were preloaded with the
mitochondrion-specific Ca
2⫹
-sensitive fluorescent marker,
Rhod-FF, AM, followed by fura-2 AM loading (Fig. 4). The fluo-
rescence pattern and responses to FCCP (10
M), an uncoupler of
ATP synthesis due to its action as a protonophore, confirmed the
specificity of this protocol, causing leakage of mitochondrial
Ca
2⫹
from mitochondria accompanied by an increased cytosolic
Ca
2⫹
concentration (Fig. 4). Application of CBD (1
M) resulted
in an increase in cytosolic Ca
2⫹
, preceded by a response in the
Rhod-FF fluorescence (Fig. 5). Two Rhod-FF response patterns
were observed, biphasic (an initial rise followed by a decrease) or
a continuous decline (see sample traces given in Fig. 5A,B). Sub-
sequently, we confirmed that the pattern observed with CBD in
this dual-fluorescence model genuinely represents a release from
mitochondrial Ca
2⫹
stores by preapplication of FCCP (1
M),
applied to dual-loaded cultures (see Fig. 1 for the sites of action
for this and other mitochondrion-acting compounds). At this
concentration, FCCP induced an immediate reduction in
Rhod-FF fluorescence in the mitochondrial compartment, and
somewhat delayed in onset and progression, an increase in cyto-
solic Ca
2⫹
levels was observed. More importantly, no further
responses to CBD could be induced in mitochondria (Fig. 5C),
while raised cytosolic Ca
2⫹
levels recovered partially, in agree-
ment with our previous experiments in high-K
⫹
HBS and 4AP.
Overall, FCCP eradicated CBD responses in neurons [mean:
⫺1⫾6% ⌬F/F(n⫽25), p⬍0.001 compared with controls] and
significantly reduced responses in glia [reduced by 61 ⫾5% (n⫽
8), p⬍0.001]. As these data strongly suggested a mitochondrial
site of action, we aimed to exclude the ER as the primary source of
Ca
2⫹
for CBD responses by applying CBD in the presence of
specific antagonists to the receptors linked to Ca
2⫹
release path-
ways from the ER (dantrolene and 2-APB, acting as ryanodine
and IP3 receptor antagonists, respectively). The blockade of one
of these receptors has been shown to upregulate the activity of the
other, implying that both release mechanisms share a common
pool of Ca
2⫹
(White and McGeown, 2003). Thus, both antago-
nists were coapplied to fully block ER receptor-mediated release.
Such a blockade transiently altered baseline Ca
2⫹
levels, but
longer duration of antagonist treatment (10 min) allowed a set-
tled baseline to be established before CBD application. When
CBD was coapplied with 2-APB and dantrolene, responses did
not significantly differ from control values ( p⬎0.05), with glial
responses increased compared with controls ( p⬍0.001) (Fig. 6),
further confirming that ER receptors are somewhat modulating,
but not mediating CBD-induced responses.
Thus, our data strongly suggested that [Ca
2⫹
]
i
regulation via
CBD is achieved via mitochondrial uptake and release, which
could potentially be achieved via either the mPTP or the mito-
Figure 3. CBD effects on epileptiform activity in cultured hippocampal neurons. A, Applica-
tion of the K
⫹
channel antagonist 4AP to naive cultures induces spontaneous Ca
2⫹
oscilla-
tions. B,C, The presence of CBD following (B), or preceding (C), 4AP application dampened
Ca
2⫹
oscillations. Data are presented as %⌬F/F.
2056 •J. Neurosci., February 18, 2009 •29(7):2053–2063 Ryan et al. •CBD and Mitochondria
chondrial Na
⫹
/Ca
2⫹
-exchanger (NCX) (Griffiths, 1999). Exper-
iments with the mPTP inhibitor CsA showed no difference to
control CBD responses, implying that the mPTP is not the prin-
cipal mechanism of CBD’s actions (Fig. 6). When the role of the
NCX in CBD-mediated responses was investigated using the spe-
cific antagonist CGP 37157 (CGP) (Chiesi et al., 1988; Med-
vedeva et al., 2008), preapplied and coapplied (10
M), CBD (1
M) responses were abolished [remaining response: neurons:
10 ⫾10% ⌬F/F(n⫽8), glia: 3 ⫾7% ⌬F/F(n⫽14), pvalues
⬍0.001] (Fig. 6). To confirm that NCX was also fundamental to
[Ca
2⫹
]
i
reducing CBD responses, the experiment was repeated in
the presence of elevated [K
⫹
]
e
(as above). The reversal of neuro-
nal CBD responses normally seen under these conditions was no
longer observed. Accordingly, the CBD response in CGP no
longer differed between high-K
⫹
and standard HBS in both neu-
rons and glia ( pvalues ⬎0.05) (Fig. 6). Therefore, we conclude
that CBD is acting via the mitochondrial
NCX to elevate or decrease cytosolic Ca
2⫹
levels, dependent on resting [Ca
2⫹
]
i
.
Protection by CBD against
mitochondrial toxins
The apparent mitochondrial site of action
of CBD led to the hypothesis that CBD
may act as a neuroprotectant against mito-
chondrially acting toxins, acting either di-
rectly on mitochondrial sites or down-
stream thereof (Fig. 1). Initial tests used
the mitochondria-reliant viability assay
Alamar Blue in SH-SY5Y cells, with pro-
tective actions of CBD confirmed in hip-
pocampal cultures using a live– dead stain
(Fig. 7).
Application of hydrogen peroxide
(H
2
O
2
), produced in response to cell stress
and metabolic impairment as a byproduct
of the dismutation of the superoxide (O
2
⫺
)
free radical, to SH cells at 100
Mfor3h
reduced cell viability by ⬃50% (range: 40 –
60%). As a positive control for the mode of
cell death, the peroxide-specific catalyzing
enzyme catalase was coapplied. With and
without 1 h preapplication, catalase (at
both 500 and 1000 U/ml) fully protected
against peroxide-induced cell death. Next,
CBD (0.1 and 1
M) was assessed as a po-
tential neuroprotectant and was initially
coapplied with H
2
O
2
. The lower concen-
tration of CBD proved to be marginally,
though significantly, protective (by 16 ⫾
5% (n⫽18); p⬍0.05), whereas the higher
concentration had no significant effect
(Fig. 8A). This experiment was repeated
with cells preexposed to CBD (concentra-
tions as above) for 1 h before H
2
O
2
expo-
sure. The neuroprotective effects of 100
nMCBD were no longer evident, while 1
MCBD worsened the fate of cells ( p⬍
0.05, compared with peroxide controls).
Overall, this pattern argues against a sim-
ple antioxidant action of CBD.
Next, ATP production was targeted
with oligomycin, an inhibitor of ATP syn-
thase (Fig. 1) that blocks the phosphorylation of ADP at this
complex of the electron transport chain (Penefsky, 1985;
Duchen, 2004). Following overnight dose–response experiments,
a concentration of 20
Mwas selected for further experimenta-
tion (average reduction in cell viability: 35%). A modest, though
significant, protection was conferred by coapplication of the
toxin with 1
MCBD [improved by 15 ⫾4% (n⫽23), p⬍0.05]
(Fig. 8B), but not with the lower CBD concentration (100 nM,
data not shown). As a confirmation of mPTP involvement in this
toxicity assay, the inhibitor of mPTP formation, CsA (1
M), was
applied and proved to be protective [increase in cell viability:
53 ⫾10% (n⫽24), p⬍0.001 compared with oligomycin con-
trols]. In comparison, catalase conferred no protection against
oligomycin toxicity, coapplication of CsA and CBD also proved
not to be additive (data not shown). Notably, CsA alone (in the
absence of any toxin) improved cell viability (CsA control being
Figure 4. Dual-loading of hippocampal cultures with fura-2 AM and Rhod-FF, AM. A, Typical transmission image shows clearly
defined neuronal appearance. B,C, Rhod-FF fluorescence (B) demonstrates a clear compartmentalization into mitochondria, a
pattern disrupted by FCCP application (Ci,Cii). The corresponding cytosolic Ca
2⫹
alterations are monitored using fura-2 AM (D)
with responses shown in both compartments to the mitochondrial uncoupler FCCP and NMDA (B,Di–Div). E, The raw values (OD,
optical density) for each channel are plotted.
Ryan et al. •CBD and Mitochondria J. Neurosci., February 18, 2009 •29(7):2053–2063 • 2057
110 ⫾3% of control value, p⬍0.05), implying under resting
conditions there may be some activation of mPTP (data not
shown). Neuroprotection of CBD was also confirmed in hip-
pocampal cultures, where CBD (with 1 h preincubation) again
proved to be neuroprotective by 31 ⫾3% (n⫽9, p⬍0.01) (Fig.
8B).
Finally, as a continuation of our imaging data, CBD was again
tested in combination with the uncoupler of ATP synthesis,
FCCP (see above and Fig. 1). To further confirm the mitochon-
drial site of action of FCCP, SH-SY5Y cells were loaded with the
MitoCapture fluorescent dye, also used as a marker for apoptosis.
In healthy cells, the reagent congregates in the mitochondria and
is detected as a red fluorescence signal. Conversely, in apoptotic
cells, MitoCapture remains in the cell cytosol (due to the dis-
rupted mitochondrial membrane potential) and can be moni-
tored as a green fluorescent signal. Following FCCP incuba-
tion (20
M, overnight) green fluorescence was increased by
45 ⫾8%, while red fluorescence was decreased by 51 ⫾6% (in
both cases n⫽30 and p⬍0.001 compared with controls) (Fig.
9C). This indicates that FCCP is acting to primarily depolarize
mitochondria.
Figure 5. Mitochondrial and cytosolic CBD (1
M) responses in naive cultures loaded with
Rhod-FF,AMandfura-2AM.A,B,Sampletracesfromneuronsshowingdelayedcytosolic (black
trace) and early biphasic mitochondrial (gray trace) Ca
2⫹
responses. NMDA application was
used as an indicator of neuronal viability and to make a clear distinction between neurons and
glia. C, Application of FCCP (1
M) led to a drop in mitochondrial Ca
2⫹
levels and prevented a
further Ca
2⫹
rise by CBD. Vertical lines have been added to visualize the order of responses. All
Rhod-FF data are raw fluorescence values (OD, optic density), and fura-2 AM responses are
presented as ratio values.
Figure 6. Effects of ER- and mitochondrion-acting drugs on CBD responses. A,B, The role of
mitochondria in CBD responses were confirmed in neurons (A) and glia (B). The uncoupler FCCP
prevented neuronal CBD response and largely reduce glial responses while blockade of IP3 and
ryanodine receptors [by 2-APB and dantrolene (Dant.), respectively] did not significantly alter
CBD responses in neurons, a sample trace of which is also shown (C). In the presence of CGP
37157 (CGP), but not in the presence of the mPTP inhibitor cyclosporin A (CsA), CBD responses
were also blocked in normal and high-excitability HBS; CBD responses under high-excitability
conditions no longer differed from standard HBS responses. Data are presented as %⌬F/F⫹
SEM; n.s., not statistically significant; **p⬍0.01, ***p⬍0.001.
2058 •J. Neurosci., February 18, 2009 •29(7):2053–2063 Ryan et al. •CBD and Mitochondria
The Alamar Blue assay indicated that 20
MFCCP (overnight)
caused a mean reduction in cell viability of 70 ⫾2%. When CBD
(100 nMand 1
M) was coapplied with FCCP it was neuroprotec-
tive at both concentrations [percentage protection: 10% and
15%, respectively, n⫽12 for both ( p⬍0.01)] (Fig. 9A), 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 nMCBD yielding 35 ⫾3% protection and 1
Mconferring 53 ⫾2% protection). This level of protection was
significantly greater than that seen without CBD preexposure in
each case ( pvalues ⬍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
M(shown in Fig.
9A). As for oligomycin, when the FCCP toxicity experiment was
repeated in cultured hippocampal neurons (with 1 h CBD prein-
cubation), CBD proved to be protective by 27 ⫾3% (n⫽6, p⬍
0.01) (Fig. 9A).
Since an antioxidant capacity has been widely reported for
CBD (Hampson et al., 1998; Chen and Buck, 2000), its neuropro-
tective properties were subsequently compared with the protec-
tive capabilities of the free radical scavenger butylated hydroxy-
toluene (BHT). Interestingly, coapplication of FCCP with BHT
(at 3 and 10
M;nvalues ⫽11 and 12, respectively) conferred no
significant protection ( pvalues ⬎0.05), yet joint application of
CBD (1
M) applied with the higher concentration of BHT (10
M) provided a complete prevention of FCCP’s toxic effects
[100 ⫾7% protection (n⫽12), p⬍0.001 compared with FCCP
controls], significantly more potent than CBD alone ( p⬍0.001)
(Fig. 9B). The superadditive nature of this protection strongly
suggests independent but synergistic modes of action. Overall,
our data suggest that CBD directly acts on
mitochondria, and this action offers pro-
tection against toxins that directly target
mitochondria.
Discussion
Enhanced excitability and
epileptiform activity
We here report bidirectional regulation of
[Ca
2⫹
]
i
and protection provided by CBD.
This was evident acutely as CBD reduced
cytosolic Ca
2⫹
levels in high-K
⫹
solution,
and also silenced and prevented
epileptiform-like activity induced by 4AP.
The latter experiments were performed in
the absence of TTX (as sustained sponta-
neous firing and neurotransmitter release
is fundamental for epileptiform activity),
hence one possibility is that anticonvul-
sant activity could be mediated by actions
on transmitter release, a property already
identified for a number of cannabinoids
with respect to glutamate (Szabo and
Schlicker, 2005; Shen et al., 1996) and
GABA (Katona et al., 1999; Ko¨falvi et al.,
2005). Such actions can potentially alter
excitability but would require CBD to act
on the endocannabinoid system. While
modulatory interactions between CBD
and endocannabinoids were demon-
strated in our previous work (Ryan et al.,
2007), this did not involve agonism on
known CB receptors, although an indirect
action on these receptors via inhibition of endocannabinoid re-
uptake and hydrolysis remains a possibility (Bisogno et al., 2001;
Ligresti et al., 2006). A number of other studies have identified
CBD as an anti-epileptic agent both in vitro and in vivo (for re-
view, see Pertwee, 2004), and our data imply that this can be
achieved by a mitochondrial regulation of [Ca
2⫹
]
i
. We also pro-
pose that this action would offer beneficial protection in disease
states that involve hyperexcitability, as CBD’s mode of action
may allow it to functioning as a Ca
2⫹
sensor and regulator.
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 ex-
tramitochondrial 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). Addition-
ally, 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 IP
3
receptors, including TRP channels (for re-
view, 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 Pet-
rocellis et al., 2008), yet a contribution to the CBD response in
our experiments is unlikely as suggested by our data obtained
Figure 7. Determination of cell death in hippocampal cultured neurons (live– dead cell staining kit) by multichannel image
capture in cells treated with 20
Moligomycin. 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.
Ryan et al. •CBD and Mitochondria J. Neurosci., February 18, 2009 •29(7):2053–2063 • 2059
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).
NCX as a target for CBD
The ER is a fundamental player in Ca
2⫹
homeostasis with per-
turbations of this organelle’s functioning associated with excito-
toxicity (e.g., in Alzheimer’s disease) (for review, see Mattson and
Chan, 2003). Moreover, the close interaction between ER and
mitochondrial Ca
2⫹
signaling is an important factor in apoptotic
signaling (for review, see Szabadkai and Rizzuto, 2004), although
a recent study has suggested mitochondrial regulation of cytoso-
lic Ca
2⫹
independent of the ER, with the NCX as the rate-limiting
factor for temporal decoding (Young et al., 2008).
A direct role for mitochondria in CBD signaling was con-
firmed here by the use of dual-loaded hippocampal neurons with
Ca
2⫹
-sensitive probes for mitochondrial and cytosolic compart-
ments, with changes in mitochondrial Ca
2⫹
levels preceding the
rise of cytosolic Ca
2⫹
. Moreover, the depletion of Ca
2⫹
from
mitochondria (using FCCP) resulted in the inability of CBD to
yield any subsequent response. CBD’s point of action upon mi-
tochondria was identified to be the NCX (and not the mPTP), as
CBD responses were no longer present when this exchanger was
blocked by CGP, yet unaffected by CsA. The NCX functions nor-
mally to remove Ca
2⫹
from the mitochondria but can reverse
when the ionic gradients are sufficiently altered, especially in dis-
ease states (Jung et al., 1995; Griffiths, 1999; Poburko et al., 2006).
A fundamental role for the mitochondrial NCX in Ca
2⫹
signaling
associated with ischemia and excitotoxic events, where the influx
of Na
⫹
into the cell causes release of Ca
2⫹
from mitochondria,
has previously been identified in hippocampal tissue (Zhang and
Lipton, 1999).
CGP may act not only upon mitochondrial NCX, but also as
an inhibitor of VGCCs in dorsal root ganglion neurons (Baron
and Thayer, 1997). However, our previous data with VGCC
blockers (Drysdale et al., 2006) are not consistent with an effect of
CBD on this target. Others have found CGP to inhibit the NCX in
the plasma membrane of cerebellar granule cells (Czyz and Kied-
rowski, 2003), although with an IC
50
of 13
M, a concentration
higher than that used here, and higher than CGP’s IC
50
(4
M) for
the mitochondrial NCX in cultured rat DRGs (Baron and Thayer,
1997). Indeed, the concentration of CGP used here is in keeping
with recent work by others in cultured neurons (Medvedeva et
al., 2008).
CBD as a neuroprotectant
Our study uncovers a new intracellular, and potentially direct,
target for CBD, which has so far been largely elusive despite the
wide-ranging use of this phytocannabinoid in diverse prepara-
tions and applications. Evidence for actions of CBD on mito-
chondria was strongly supported by cell death models with mito-
chondrial toxins. The most potent CBD protection was seen
against FCCP toxicity (in SH cells and reproduced in cultured
hippocampal neurons), a mitochondrial uncoupler, causing the
collapse of the mitochondrial membrane potential and the re-
lease of Ca
2⫹
into the cytosol. A more modest protection by CBD
was observed against other oxidative stress related agents, hydro-
gen peroxide and oligomycin. FCCP causes the accumulation of
protons into mitochondria leading to uncoupling of the mito-
chondrial potential (⌬⌿
m
), ultimately causing a loss of ATP pro-
duction (for review, see Wallace and Starkov, 2000), and has
previously been demonstrated to cause apoptosis in PC12 cells
(Dispersyn et al., 1999) and primary neurons (Moon et al., 2005).
The loss of ⌬⌿
m
(confirmed by MitoCapture) and resultant cell
death is also assumed to involve mPTP formation (Marques-
Santos et al., 2006), in keeping with our finding that CsA can
protect against FCCP toxicity in a dose-dependent manner.
Exogenous application of hydrogen peroxide (as well as the
cellular generation of this oxidative agent) has been shown to
induce apoptosis in association with MAP kinase activation
(Guyton et al., 1996). Thus, CBD’s neuroprotective action
against H
2
O
2
toxicity in SH cells is in line with CBD’s reported
inhibition of p38 MAP kinase, although proposed to be second-
ary to CBD’s antioxidant capacity (El-Remessy et al., 2006),
which was not apparent here. Ligresti et al. (2006) also showed
protection in breast cancer cells against H
2
O
2
toxicity at low (nM)
but not higher (
M) concentrations of CBD. The latter effect was
suggested to involve the generation of ROS, also reported in gli-
oma cells (Massi et al., 2006). Therefore, the possibility has been
raised that CBD might have potential as an anticancer treatment
Figure 8. Cannabinoids as potential neuroprotectants against mitochondrial stressors in
SH-SY5Y cells. Data are expressed as percentage protection (⫹SEM) relative to within-
experiment controls and shown for peroxide (0.1 mM)(A) and oligomycin (20
M)(B) toxicity.
CBD conferred protection against oligomycin toxicity in both SH-SY5Y cells and hippocampal
cultures (HIPP.). Pre-Inc., Following 1 h preincubation; *p⬍0.05, ***p⬍0.001.
2060 •J. Neurosci., February 18, 2009 •29(7):2053–2063 Ryan et al. •CBD and Mitochondria
(for review, see Mechoulam et al., 2007). A comparison between
the antioxidant properties of CBD and BHT has been performed
previously (Hamelink et al., 2005), with approximately equiva-
lent antioxidant capacities reported for both compounds. This is
surprising in the light of the data generated here where CBD, but
not BHT, alone was neuroprotective against FCCP toxicity. In-
terestingly, the combination of CBD and BHT caused complete
protection against FCCP. Previous work conducted in our lab has
shown an interaction between this phytocannabinoid and BHT,
with BHT preexposure (saturating antioxidant pathways) to pri-
mary rat hippocampal cultures facilitating [Ca
2⫹
]
i
responses to a
subsequent CBD application (our unpublished observations).
This implies a synergy between CBD and antioxidant pathways,
with the latter facilitating CBD’s effects, rather than mediating
them. Since CBD showed only little protection in the peroxide
model, it seems that its anti-oxidant properties are not of major
relevance for its protective action. While similar protection was
seen for CsA and CBD in the FCCP model, differences in efficacy
between CsA and CBD and the lack of additivity between CBD
and CsA in the oligomycin model (Co-
melli et al., 2003) suggest that the mPTP is
not a major target in CBD’s action.
Overall, the apparent capacity for CBD
to reduce [Ca
2⫹
]
i
when it is abnormally
elevated via interactions with
mitochondria-dependent Ca
2⫹
regulation
may be a valuable property for many dis-
ease states associated with Ca
2⫹
dysregu-
lation. Moreover, neurodegenerative dis-
eases linked directly to mitochondrial
malfunction, such as Huntington’s disease
and Friedreich’s ataxia, may benefit greatly
from CBD-based medicines.
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