Limitless Nexus
Search site

exhibition Info
Cannabinoids in Study Prevent Alzheimers Disease

Type: Literature (pdf)

Submitter: [anonymous]

Category:

Exhibition Date: 2014-05-30 10:26:51 MST

Views: 1104

Score: 5.00 / 5.00
(Based on 3 votes.)

Rate this exhibition

Actions

Sponsor
 
Gifts Copper Mugs Moscow Mules

Exhibition

Submitter's Comment
Neurobiology of Disease Prevention of Alzheimer’s Disease Pathology by Cannabinoids: Neuroprotection Mediated by Blockade of Microglial Activation
Bele
́n G. Ramı
́rez,
1
Cristina Bla
́zquez,
2
Teresa Go
́mez del Pulgar,
2
Manuel Guzma
́n,
2
and Marı
́a L. de Ceballos
1
1
Neurodegeneration Group, Cajal Institute, Consejo Superior de Investigaciones Cientı
́ficas, 28002 Madrid, Spain, and
2
Department of Biochemistry and
Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain
Alzheimer’s disease (AD) is characterized by enhanced
-amyloid peptide (
A) deposition along with glial activation in senile plaques,
selective neuronal loss, and cognitive deficits. Cannabinoids are neuroprotective agents against excitotoxicity
in vitro
and acute brain
damage
in vivo
.Thisbackgroundpromptedustostudythelocalization,expression,andfunctionofcannabinoidreceptorsinADandthe
possible protective role of cannabinoids after
A treatment, both
in vivo
and
in vitro
. Here, we show that senile plaques in AD patients
express cannabinoid receptors CB
1
and CB
2
, together with markers of microglial activation, and that CB
1
-positive neurons, present in
high numbers in control cases, are greatly reduced in areas of microglial activation. In pharmacological experiments, we found that
G-protein coupling and CB
1
receptor protein expression are markedly decreased in AD brains. Additionally, in AD brains, protein
nitration is increased, and, more specifically, CB
1
and CB
2
proteins show enhanced nitration. Intracerebroventricular administration of
the synthetic cannabinoid WIN55,212-2 to rats prevent
A-induced microglial activation, cognitive impairment, and loss of neuronal
markers. Cannabinoids (HU-210, WIN55,212-2, and JWH-133) block
A-induced activation of cultured microglial cells, as judged by
mitochondrial activity, cell morphology, and tumor necrosis factor-
release; these effects are independent of the antioxidant action of
cannabinoid compounds and are also exerted by a CB
2
-selective agonist. Moreover, cannabinoids abrogate microglia-mediated neuro
-
toxicity after
A addition to rat cortical cocultures. Our results indicate that cannabinoid receptors are important in the pathology of AD
and that cannabinoids succeed in preventing the neurodegenerative process occurring in the disease.
Key words:
Alzheimer’s disease;
-amyloid; cannabinoids; microglia; neurotoxicity; neuroprotection
Introduction
Alzheimer’s disease (AD), the most common form of dementia, is
characterized by the deposition of
-amyloid peptide (
A)
within one of its pathological hallmarks: the senile plaque. Acti-
vated microglia cluster at senile plaques (McGeer et al., 1987;
Dickson et al., 1988), and this seems to be responsible for the
ongoing inflammatory process in the disease. Transgenic mouse
models of AD also develop plaques in which
A deposits and
activated microglia exist (Masliah et al., 1996; Frautschy et al.,
1998; Jantzen et al., 2002). Furthermore, microglial activation
results in neurodegeneration both
in vitro
(Meda et al., 1995; Gao
et al., 2002; Xie et al., 2002) and
in vivo
(Weldon et al., 1998;
Herrera et al., 2000; Iravani et al., 2002) paradigms. In this con-
text, recent studies have focused on the therapeutic interest of
limiting microglial activation and inflammation in AD and other
neurological disorders.
Cannabinoids, the active components of marijuana and their
analogs, exert a wide spectrum of central and peripheral effects by
activating specific cannabinoid receptors, two of which have been
well characterized to date: CB
1
and CB
2
(Howlett et al., 2002;
Piomelli, 2003). CB
1
receptors are found in high density in the
nervous system (Herkenham et al., 1990), in which they mediate
cannabinoid psychoactivity, and all types of neural cells express
them. Thus, in addition to being present in neurons, CB
1
recep
-
tors exist in astrocytes (Bouaboula et al., 1995; Sa
́ nchez et al.,
1998), microglia (Waksman et al., 1999; Walter et al., 2003), and
oligodendrocytes (Molina-Holgado et al., 2002). In contrast, the
CB
2
receptor is considered to be expressed solely in cells and
organs of the immune system and is unrelated to cannabinoid
psychoactivity. There are also recent reports on the existence of
CB
2
receptors in microglia (Walter et al., 2003) and on cannabi
-
noids affecting migration (Walter et al., 2003), as well as nitric
oxide (NO) and cytokine production (Waksman et al., 1999;
Puffenbarger et al., 2000; Facchinetti et al., 2003) in microglial
cell cultures
in vitro
.
Cannabinoids exert neuroprotection under different experi-
Received Sept. 9, 2004; revised Dec. 28, 2004; accepted Dec. 30, 2004.
This work was supported by Grants SAF 2002-01566 (M.L.C) and SAF 2003-00745 (M.G.) from the Spanish
Ministry of Science and Technology, Grant CAM 08.1/0079 from the Community of Madrid (M.G., M.L.C.), Red de
Investigacio
́n de Enfermedades Neurolo
́gicas (M.L.C.), and Fundacio
́n Cientı ́fica de la Asociacio
́n Espan
̃ola contra el
Ca
́ncer (M.G.). B.G.R. is the recipient of a fellowship from the Community of Madrid. We thank Dr. K. Mackie for
anti-CB
1
antibodyandCB
1
andCB
2
antigenicpeptides,Dr.J.Rodrigoforanti-N-Tyrantibody,andSanofi-Synthelabo
for SR141716 and SR144528. Dr. I. Ferrer is acknowledged for helpful discussions, Dr. L. Lo
́pez-Mascaraque for
assistance in microscopic imaging, and M. E. Ferna
́ndez de Molina for excellent technical assistance.
Correspondence should be addressed to Dr. Marı ́a L. de Ceballos, Neurodegeneration Group, Cajal Institute,
Consejo Superior de Investigaciones Cientı ́ficas, Avenida Doctor Arce, 37, 28002 Madrid, Spain. E-mail:
mceballos@cajal.csic.es.
DOI:10.1523/JNEUROSCI.4540-04.2005
Copyright © 2005 Society for Neuroscience 0270-6474/05/251904-10$15.00/0
1904

The Journal of Neuroscience, February 23, 2005

25(8):1904–1913
mental conditions. Thus, cannabinoid receptor activation pro-
tects hippocampal or granule cerebellar neurons from excitotox-
icity (Skaper et al., 1996; Shen and Thayer, 1998; Hampson and
Grimaldi, 2001) and from hypoxia and glucose deprivation (Na-
gayama et al., 1999).
In vivo
, cannabinoids decrease hippocampal
neuronal loss and infarct volume after cerebral ischemia (Na-
gayama et al., 1999), acute brain trauma (Panikashvili et al.,
2001), and ouabain-induced excitotoxicity (van der Stelt et al.,
2001). These effects have been ascribed to inhibition of glutamate
transmission, reduction of calcium influx, and subsequent inhi-
bition of noxious cascades, such as tumor necrosis factor-
(TNF-
) generation and oxidative stress.
This background prompted us to study the characteristics and
localization of cannabinoid receptors in AD brain, with particu-
lar emphasis on any relationship with microglial activation. Fur-
thermore, the effects of cannabinoid receptor activation were
studied in an animal model of AD
in vivo
and in a model of
A-induced microglial activation
in vitro
.
Materials and Methods
Materials.
A
25–35
and a peptide containing the same 11 amino acids but
with a scrambled (SCR) sequence (NeoMPS, Strasbourg, France) were
used throughout.
A
25–35
and the scrambled peptide were dissolved in
distilled water at a concentration of 2.5 mg/ml to allow self-aggregation
of the peptide and stored at
80°C until used.
A
1– 40
(NeoMPS) was
dissolved in PBS (1.72 mg/ml), aged at 37°C for 24 h (“fibrillar” peptide),
and was vortexed several times during that period, and aliquots were stored
at
80°C until used. The control (C) peptide was not subjected to aging
(“soluble” peptide). Aggregation of all of the peptides was confirmed by
microscopy after staining with Coomassie brilliant blue (Ferna
́ ndez-Tome
́et
al., 2004). WIN55,212-2 [
R
-(
)-(2,3-dihydro-5-methyl-3-[(4-morpholi-
nyl)methyl]1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone
sulfonate] was obtained from Sigma (St. Louis, MO), JWH-133
[(6a
R
,10a
R
),-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-tri-
methyl-6H-dibenzo[b,d]pyran] was a generous gift from Dr. J. W. Huffman
(Clemson University, Clemson, SC), HU-210 [(6a
R
,10a
R
)-3-(1,1
-
dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-
dibenzo[b,d]pyran-9-methanol] was kindly given by Dr. R. Mechoulam
(The Hebrew University of Jerusalem,
Jerusalem, Israel), AM251 [
N
-1-
(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-
N
-1-piperidinyl-1H-
pyrazole-3-carboxamide] was from Tocris Cookson (Bristol, UK), and
SR141716 [
N
-piperidino-5-(4-chloro-
phenyl)-1-(2,4-dichlorophenyl)-
4-methyl-3-pyrazole carboxamide] and
SR144528 [
N
-[(1
S
)-endo-
1,3,3-trimethyl bicyclo[2.2.1]heptan-2-
yl]-5-(4-chloro-3-methylphenyl)-
1-(4-methylbenzyl)-pyrazole-3-carbo-xamide] were kindly donated by
Sanofi-Synthelabo (Montpellier, France). Each of these compounds was dis-
solved in DMSO at 10 m
M
concentration, and aliquots were stored at
80°C,
with the exception of WIN55,212-2, which was initially dissolved in chloro-
form (on ice), quickly aliquoted to prevent evaporation, and dried under a
stream of N
2
, and aliquots were stored desiccated. Before their use, drugs
were diluted in the appropriate solvent (e.g., PBS or cell culture medium),
and DMSO never exceeded 0.1% in pharmacological or cell culture experi-
ments. Cell culture reagents were from Sigma unless otherwise stated. Salts
and other reactives were analytical grade from Merck (Darmstadt,
Germany).
Patient samples.
For immunocytochemistry, cryoprotected and fixed
frozen samples from frontal cortex were obtained from the Neurologic
Tissue Bank (Hospital Clinic, Barcelona, Spain). Controls consisted of
three males and two females (mean
SEM; 62.8
7.2 years of age; 9.5
2.2 h of postmortem interval), and clinically diagnosed and neuropatho-
logically defined AD patients consisted of three females and three males
(76.0
3.3 years of age; 12.5
3.1 h of postmortem interval). For
biochemical studies, frozen frontal cortex samples (control,
n
18; AD,
n
18) were obtained from the London Brain Bank for Neurodegenera-
tive Diseases (Institute of Psychiatry, London, UK). Control subjects and
AD patients were matched for age (C, 71.8
2.6; AD, 77.4
2.2 years),
sex (13 females and 5 males per group), and postmortem delay (C, 38.7
4.7; AD, 33.3
5.3 h). Samples were maintained at
80°C until assayed.
Immunocytochemistry.
Immunostaining was performed on floating
sections (30
m) as described previously (Go
́ mez del Pulgar et al., 2002).
For CB
2
and N-Tyr immunostaining, antigen retrieval was achieved by
boiling sections in 1 m
M
EDTA, pH 8.0, for 5 min before the standard
labeling technique. Sections were incubated with the different antibodies
overnight at 4°C. Dilutions of antibodies were as follows: polyclonal
anti-CB
1
(1:900; from Dr. K. Mackie, University of Washington, Seattle,
WA), polyclonal anti-CB
2
(1:900; Affinity BioReagents, Golden, CO),
polyclonal anti-N-Tyr [1:000; Dr. J. Rodrigo, Cajal Institute, Consejo
Superior de Investigaciones Cientı
́ficas (CSIC), Madrid, Spain] (Uttenthal et
al., 1998), monoclonal anti-GFAP (1:1000; Sigma), monoclonal anti-human
leukocyte antigen-D region related (HLA-DR) (1:15; MP Biomedicals,
Irvine, CA), and biotinylated tomato lectin (TL) (1:150; Sigma). Develop-
ment was conducted by the ABC method (Pierce, Rockford, IL), and immu-
noreactivity was visualized by 3,3
-diaminobenzidine oxidation as chromo-
gen, with or without nickel enhancement.
Immunostaining of cell cultures, either neuronal or microglial, were
similarly performed after fixation with paraformaldehyde (4% in 0.1
M
phosphate buffer) for 30 min, followed by rinses with PBS. In addition,
rat microglia was immunostained with monoclonal anti-rat CD11
(OX42; 1:100; Serotec, Oxford, UK).
Omission of primary or secondary antibodies resulted in no immuno-
staining. Specificity of anti-CB
1
and anti-CB
2
staining was performed by
preabsorption of the antibodies with the antigenic peptides (kindly given
by Dr. K. Mackie), which completely abolished labeling.
3
H-WIN55,212-2 and
35
S-GTP
S binding.
3
H-WIN55,212-2 binding
was conducted as described previously (Breivogel et al., 2001). Briefly, P
2
membrane fractions (80
g of protein) were incubated in assay buffer
(Tris 50 m
M
, pH 7.4, containing 3 m
M
MgCl
2
,1m
M
EDTA, and 1 mg/ml
fatty acid-free BSA) with 3 n
M
3
H-WIN55,212-2 (specific activity, 41.0
Ci/mmol; PerkinElmer Life Sciences, Boston, MA) at 30°C for 60 min.
Nonspecific binding was determined in the presence of 1
M
SR141716.
Saturation curves were constructed with increasing concentrations of
3
H-WIN55,212-2 (0.25–10 n
M
), and, in competition curves, several con
-
centrations of the agonists or antagonists (10
10
to 10
4
M
) were as
-
sayed. The binding was terminated by filtering through GF/C Whatman
(Maidstone, UK) filters (0.45
m), incubated previously with 0.5% poly-
ethyleneimine for at least 2 h, using a Brandel (Gaithersburg, MD)
harvester, washed three times with ice-cold 5 m
M
Tris HCl, pH 7.4,
containing 1 mg/ml BSA, and the radioactivity was counted using an
LKB-Wallac (Gaithersburg, MD) scintillation counter.
35
S-GTP
S bind
-
ing was performed as described previously (Breivogel et al., 2001). P
2
membrane fractions (30
g of protein) were incubated in assay buffer
(50 m
M
Tris HCl, pH 7.4, containing 3 m
M
MgCl
2
, 100 m
M
Na Cl, 0.2 m
M
EGTA, and 0.5 mg/ml fatty acid-free BSA) with 0.04 n
M
35
S-GTP
S
(specific activity, 1250 Ci/mmol; PerkinElmer Life Sciences), 30
M
GDP, and 5
M
WIN55,212-2 at 30°C for 60 min. Binding was termi-
nated by rapid filtration as above. Basal activity was determined in the
absence of agonist and nonspecific binding in the presence of 10
M
of
unlabeled GTP
S. Net stimulation was the difference of the binding in
the presence and in the absence (basal binding) of WIN55,212-2. Data
were expressed in femtomoles per milligram of protein. All of the proce-
dures for both assays were conducted in plasticware and dilutions of
radioligands and cold reagents in plastic tubes coated with 50 m
M
Tris
HCl, pH 7.4, containing 1 mg/ml BSA.
A and cannabinoid administration to rats.
All of the experiments were
performed according to ethical regulations on the use and welfare of
experimental animals of the European Union and the Spanish Ministry
of Agriculture, and the procedures were approved by the bioethical com-
mittee of the CSIC.
A administration to male Wistar rats was performed
essentially as described previously (Pavı
́a et al., 2000).
A
25–35
or a pep
-
tide with a scrambled sequence (NeoMPS), which was used as control,
were injected intracerebroventricularly daily fo
r7d(20
gin10
lof
saline per day), and the animals were tested at different times after the
first injection. Other animals received a cannabinoid (WIN55,212-2, 10
gin10
l of 20% DMSO/80% saline per day) together with the pep-
tides. The Hamilton syringe used for intracerebroventricular injections
Ramı ́rez et al.

Cannabinoid Receptors in AD J. Neurosci., February 23, 2005

25(8):1904–1913
• 1905
was repeatedly washed with distilled water, followed by flushing with 1
mg/ml BSA solution, which reduces drastically binding to glass. This
procedure was performed before every injection.
Behavioraltests.
All of the behavioral procedures were conducted at the
same time of the day (9:00 A.M. to 2:00 P.M.). Motor activity was mon-
itored in four activity cages (Digiscan; AccuScan Instruments, Colum-
bus, OH) in an isolated room for 30 min, immediately after injections at
days 1 or 7 of treatment, or at 2 months after treatment initiation. Hor-
izontal motor activity and number of stereotypies and rearings were
recorded. To determine spatial learning, rats were trained to find a hid-
den platform in a water tank of 150 cm of diameter. Four trials per day
with different start positions, each 30 min apart, were conducted for 5 d
(Mu
̈ ller et al., 1994), and latency to reach the platform was recorded.
Cutoff time to find the platform was 120 s, and rats failing to find the
platform were placed on it and left there for 15 s. Data acquisition was
performed with a video camera (Noldus Information Technology,
Wageningen, The Netherlands).
Cell cultures and treatments.
Primary mixed glial cultures were pre-
pared from neonatal rat cortex as described previously (McCarthy and de
Vellis, 1980). Mechanically dissociated cortices were seeded onto 75 cm
2
flasks in DMEM/Ham’s F-12, supplemented with 10% fetal calf serum
(FCS) and 40
g/ml gentamicine. Cells were cultured in a humified
atmosphere of 5% CO
2
/95% air at 37°C, and the medium was changed
every 2 or 3 d. After being cultured for 3 weeks, flasks were shaken for 2–3
h at 230 rpm, and floating cells were pelleted, seeded onto a plastic dish,
and incubated at 37°C for 4 h, and loosely bound cells were aspirated.
Adherent cells were detached (PBS plus 1 m
M
EDTA) and 3
10
4
cells
were seeded onto poly-Orn-coated 96-well plates in DMEM/Ham’s F-12
supplemented with 0.5% FCS. The cultures were at least 99% pure, as
judged by immunocytochemical criteria. Mitochondrial activity (redox
state) was assessed by the MTT assay. Neuron cultures from rat cortices
were prepared as described previously (Ferna
́ ndez-Tome
́ et al., 2004)
with some modifications. Briefly, cerebral cortices were mechanically
dissociated in trypsin (1 mg/ml; Worthington, Freehold, NJ) at 37°C for
10 min, followed by DNaseI addition (50
l; Roche Products, Welwyn
Garden City, UK). Cells were collected after centrifugation and seeded
further in Neurobasal (Invitrogen, San Diego, CA), containing B27 ad-
ditives (Invitrogen), onto gelatin/poly-Lys-coated P24 plates (Falcon,
Franklin Lakes, NJ) at a density of 25
10
4
cells. Afte
r4din
culture, they
were switched to MEM with N2 additives (Sigma), and treatments (6 d
in
vitro
) were performed in MEM. Neuron cultures were completely devoid
of microglia as determined by TL or OX42 immunostaining (data not
shown). For cocultures, 15
10
4
microglial cells were seeded in inserts
(membrane pore size, 0.4
m; diameter, 9 mm; Costar, Cambridge, MA)
in DMEM containing 0.5% FCS and, at 24 h, were treated with
A
1– 40
for
4 h to avoid direct toxicity of the peptide on neurons, and the medium
was aspirated and placed over neuron cultures (25
10
4
; 24-well plates,
Costar) for an additional 20 h. Drugs were added in
1

10
of the final
volume to maintain aggregation of peptides. Cell counts were performed
by Coomassie brilliant blue (0.2% in 10% acetic acid/40% methanol)
staining under phase-contrast microscopy by an observer unaware of the
treatments (four fields per condition in triplicate) in a Zeiss
(Oberkochen, Germany) Axiovert microscope. Neurons showing intact
neurites with uniform diameter and soma with a smooth round appear-
ance were considered viable, whereas neurons with fragmented neurites
and a shrunken cell body were considered nonviable.
TNF-
analysis.
Cell-free supernatants from microglial cultures were
collected, microfuged, stored at
80°C, and assayed by a TNF-
com-
mercial sandwich ELISA (Biosource, Camarillo, CA) in strict accordance
with instructions of the manufacturer. The sensitivity was 10 pg/ml.
Western blot analysis.
Western blot was performed as described previ-
ously (Molina-Holgado et al., 2002). Tissues were sonicated in lysis
buffer, samples were centrifuged at high speed for 10 min, and superna-
tants were collected. Total protein was assessed by the Bio-Rad (Hercules,
CA) protein assay. An aliquot of each sample (30
g of protein) was
separated by SDS-PAGE (10%), and proteins were transferred from the
gels onto nitrocellulose membranes. The blots were blocked with 1%
defatted dry milk fo
r1hat
room temperature and incubated overnight at
4°C with the following antibodies: anti-CB
1
(1:5000), anti-CB
2
(1:2000),
anti-N-Tyr (1:3000), polyclonal anti-calbindin D-28K (1:5000; Swant,
Bellinzona, Switzerland), and monoclonal anti-
-tubulin (1:20,000;
Sigma). Finally, samples were subjected to enhanced chemiluminescence
and densitometric analysis. Densitometric analysis of bands was per-
formed by Quantity One quantitation software (version 4.2; Bio-Rad)
from film exposures; the background was always subtracted, and the
percentage of optical density was obtained considering 100% of that of
control samples within the same film. Immunoprecipitations were per-
formed by incubating overnight at 4°C lysate aliquots (100
g of protein)
with the anti-N-Tyr antibody (1
g/mg protein lysate) prebound to pro-
tein A-agarose (Sigma). After washing, the immunoprecipitates were
resolved by 10% SDS-PAGE and Western blotted with anti-CB
1
and
anti-CB
2
antibodies as above.
Statistical analysis.
Statistical significance analysis was assessed by us-
ing two-way or one-way ANOVA, followed by Bonferroni’s
post hoc
test
or by unpaired Student’s
t
test (Prism software, version 4.0; GraphPad
Software, San Diego, CA). A value of
p
0.05 was considered significant.
Binding saturation and inhibition curves were plotted by nonlinear re-
gression, and IC
50
values were determined using the one-site competi
-
tion model (Prism software).
Results
Cannabinoid receptors in AD brain
We studied by immunolabeling the localization of cannabinoid
receptors in relation to senile plaques and microglial activation.
All of the AD cases studied (six of six) showed CB
1
and CB
2
immunoreactivity in frontal cortical senile plaques (Fig. 1
a
),
along with markers of microglial activation, such as the histo-
compatibility glycoprotein HLA-DR (McGeer et al., 1987)
(Fig. 1
a
) and protein nitration (Fig. 1
a
, N-Tyr). Double-
immunolabeling studies addressed the question of whether CB
1
neurons are vulnerable in AD, and microglial activation ac-
counted for it. In agreement with previous immunohistochemi-
cal studies in nonhuman primates (Ong and Mackie, 1999), high
numbers of CB
1
neurons and fibers, surrounded by resting mi
-
croglia, were observed in all layers of the frontal cortex in controls
(Fig. 1
b
). CB
1
-positive neuron density was greatly reduced in AD.
Thus, although in some AD cases (two of six) CB
1
-positive neu
-
rons were still present in areas of microglial activation (Fig. 1
b
),
they were completely absent in others (four of six) (Fig. 1
b
).
Likewise, no coexistence of CB
1
-positive plaques and neurons
was evident in these cases. In addition to CB
2
expression in AD
plaques, we also found labeling in tangle-like neurons and dys-
trophic neurites (four of six cases) (Fig. 1
c
), whereas normal
brain was devoid of any signal (five of five cases). Labeling spec-
ificity was demonstrated by antigenic peptide preabsorption of
the anti-CB antibodies used (Fig. 1
c
), which completely blocked
immunoreactivity.
AD brain protein nitration (Smith et al., 1996) is thought to be
a consequence of the reaction of NO and superoxide to form the
toxic peroxynitrite radical. In control brain, N-Tyr immunoreac-
tivity was present in astrocytes and in neuronal nuclei (Fig. 2
a
).
N-Tyr-positive astrocytes were present in AD brain as well (data
not shown), but now many pyramidal neurons showed cytoplas-
mic labeling in all of the cases studied (six of six) (Fig. 2
a
). Protein
nitration was significantly increased in AD compared with con-
trol brain (Fig. 2
b
). Moreover, nitration of CB
1
and CB
2
protein
was markedly increased in AD, as shown by immunoprecipita-
tion studies (Fig. 2
c
).
Subsequently, we examined the pharmacological characteris-
tics of cannabinoid receptors by using the synthetic cannabinoid
agonist WIN55,212-2.
3
H-WIN55,212-2-specific binding, as de
-
fined by the selective CB
1
antagonist SR141716 (Rinaldi-
Carmona et al., 1994) at 1
M
, was similar in control and AD cases
(Fig. 3
a
), as were the saturation curves for the binding process
1906

J. Neurosci., February 23, 2005

25(8):1904–1913 Ramı ́rez et al.

Cannabinoid Receptors in AD
(Fig. 3
b
). Indeed, the density of receptors (
B
max
; control, 318.1
87.5 fmol/mg protein; AD, 304.7
62.2 fmol/mg protein;
n
3)
and the apparent affinity of the binding (
K
d
; control, 3.72
0.83
n
M
; AD, 5.36
0.62 n
M
;
n
3) were unaltered in AD. Indeed,
there was considerable overlap of data points among controls and
AD patients. Although basal
35
S-GTP
S binding was unchanged
in AD cortex (Fig. 3
c
), WIN55,212-2-stimulated
35
S-GTP
S
binding, which measures G-protein coupling and therefore can-
nabinoid receptor activation, was dramatically decreased
(
63%) in AD (Fig. 3
d
). There was no correlation between age,
sex, or postmortem parameters and the values of binding, in
neither controls nor AD patients. Analysis of cannabinoid recep-
tor levels by Western blot showed that CB
1
protein expression
was reduced in AD (Fig. 3
e
), whereas no changes in CB
2
expres
-
sion were observed (Fig. 3
f
).
WIN55,212-2 is a mixed CB
1
/CB
2
cannabinoid receptor ago
-
nist that activates, with similar affinities, CB
1
and CB
2
receptors
(Howlett et al., 2002) and may also interact with other, as yet
uncharacterized, receptor subtypes (Breivogel et al., 2001).
Therefore, its pharmacological selectivity was assessed in normal
human brain.
3
H-WIN55,212-2 binding was completely dis
-
placed by the CB
1
-selective antagonists SR141716 and AM251
(Lan et al., 1999) (pEC
50
, 6.88
0.14 and 6.22
0.12, respec
-
tively) but was not affected by the selective CB
2
antagonist
SR144528 (Rinaldi-Carmona et al., 1998) and the CB
2
-selective
agonist JWH-133 (Huffman et al., 1999).
35
S-GTP
S binding
was stimulated by WIN55,212-2 (maximal effect over basal,
87.9
20.2%; pEC
50
, 6.31
0.05), not affected by JWH-133 (up
to 5
M
), and completely blocked by
SR141716 and AM251 (pEC
50
, 7.87
0.13
and 7.87
0.10, respectively). Together,
these results indicate that, in human brain,
WIN55,212-2 interacts with CB
1
or CB
1
-
like receptors.
Cannabinoid treatment prevents
A-
induced toxic effects
in vivo
A overexpression or administration to
rodents models AD by activating micro-
glia (Masliah et al., 1996; Frautschy et al.,
1998; Netland et al., 1998) and inducing
cognitive impairment (Mu
̈ ller et al., 1994;
Delobette et al., 1997). We administered
A (20
g/d, i.c.v) or the control peptide
(SCR) alone or in combination with
WIN55,212-2 (10
g/d) and studied mi-
croglia staining at the end of the treatment
(8 d). Resting microglia were observed in
rats injected with SCR and/or the canna-
binoid (Fig. 4). Interestingly, there was an
intense microglial activation in the cortex
of
A-treated rats, which was prevented
by WIN55,212-2 treatment (Fig. 4).
We next examined whether cannabi-
noid administration affected cognitive
function. In contrast to control rats that
learned the spatial navigation task over 5 d
of training,
A-treated rats failed to do so.
More important, WIN55,212-2 treatment
prevented
A-induced cognitive impair-
ment (Fig. 5
a
), whereas it did not alter the
learning process when combined with
SCR. This cannabinoid effect was not at-
tributable to changes in the locomotor activity of the animals.
Thus, WIN55,212-2 produced its typical hypolocomotor action
on the first day of treatment, but this effect was not evident at the
seventh day of administration and 2 months later (data not
shown), when the navigation task was performed. As expected,
intracerebroventricular administration of the cannabinoid ago-
nist did not induce adverse effects. Indeed, just after the cessation
of the treatment, the general hematological profiles of the
WIN55,212-2-treated rats were normal. Likewise, neither the
biochemical parameters nor markers for tissue damage changed
at the end of the 7 d administration period.
Interestingly, the changes in neuronal protein expression ob-
served in AD patients were mimicked by repeated administration
of
A to rats. Thus, the expression of the neuronal markers cal-
bindin (Fig. 5
b
,
e
) and
-tubulin (Fig. 5
c
,
f
) was significantly re-
duced in both situations. Of importance, this reduction was at-
tenuated by cannabinoid administration to rats (Fig. 5
e
,
f
). CB
1
expression was also decreased in
A-treated rats (Fig. 5
d
), as was
the case in AD brain (Fig. 3
e
).
Cannabinoid treatment prevents
A-induced microglial
activation and neurotoxicity
in vitro
First, we examined the effects of cannabinoids on
A-induced
alterations in pure microglial cell cultures. As expected, microglia
in culture expressed both CB
1
and CB
2
receptors (Walter et al.,
2003) (Fig. 6
a
). Microglial activation after fibrillar
A challenge
(0.5
M
) included morphological changes at 24 h (Fig. 6
b
), as well
as increased mitochondrial activity (in the absence of cell prolif-
Figure 1.
Cannabinoid receptor localization in AD brain.
a
,CB
1
and CB
2
immunostaining in senile plaques, along with the
markers of microglial activation HLA-DR and N-Tyr.
b
, Double immunostaining of HLA-DR (black, arrows) and CB
1
(brown, aster
-
isks).CB
1
-positiveneuronsincontrols(top);CB
1
-positiveneuronsarestillpresent(middle)orcompletelylost(bottom)inareasof
intense microglial activation in AD.
c
,CB
1
-positive and CB
2
-positive neurons and dystrophic neurites in AD. Insets, Absence of
labeling by preabsorption of the antibodies with the antigenic peptide (pep). Scale bars, 25
m.
Ramı ́rez et al.

Cannabinoid Receptors in AD J. Neurosci., February 23, 2005

25(8):1904–1913
• 1907
eration; data not shown) and TNF-
release (Meda et al., 1995;
Casal et al., 2002) a
t 4 h (Fig. 6
c
) and 24 h (data not shown). The
cannabinoid agonist HU-210 (100 n
M
), which alone had no sig-
nificant effect on these parameters, counteracted fibrillar
A-
induced microglial activation. Indeed, the rod-like morphology
with lamellipodia acquired by fibrillar
A-treated cells (Casal et
al., 2002) turned to the resting oval morphology by HU-210 co-
treatment (Fig. 6
b
). HU-210 also prevented the enhancement in
TNF-
release observed after fibrillar
A addition. This effect was
mimicked by WIN55,212-2 (100 n
M
), a cannabinoid devoid of
antioxidant properties (Marsicano et al., 2002), and JWH-133
(100 n
M
),aCB
2
-selective agonist (Huffman et al., 1999) devoid of
psychoactive effects when administered
in vivo
(Sa
́ nchez et al.,
2001). In summary, cannabinoids counteract
A-mediated acti-
vation of microglia in culture.
A exerts direct toxicity on neurons, but indirect neurotoxic-
ity through microglia-mediated activation has been observed as
well (Meda et al., 1995; Tan et al., 2000; Xie et al., 2002). Canna-
binoids were unable to prevent direct toxicity of high concentra-
tions (1–10
M
)of
A in primary cortical neurons in culture
(data not shown). Subsequently, we assessed indirect
A neuro-
toxicity through microglia activation in cocultures. To avoid any
direct toxicity to neurons, microglia seeded in inserts was treated
with the peptides or the cannabinoids for 4 h, a time sufficient for
microglia activation. Indeed, there was significant neurotoxicity
when neurons were exposed for 20 h to microglial cells pretreated
with 0.5
M
fibrillar
A for 4 h (Fig. 7
a
,
b
), whereas, at this time
point and concentration, the peptide did not significantly alter
neuron survival in the absence of microglial cells (Fig. 7
a
) (Barger
et al., 1995; Ferna
́ ndez-Tome
́ et al., 2004). Both WIN55,212-2
and JWH-133 (100 n
M
) prevented microglia-mediated neuro-
toxicity after
A treatment (Fig. 7
a
,
b
). The neuroprotective effect
of WIN55,212-2 on
A neurotoxicity in cocultures was pre-
vented by the selective antagonists SR141716 and SR144528,
whereas the effect of JWH-133 was only counteracted by the latter
(Fig. 7
a
). These results support that the neuroprotective effect of
cannabinoids relies on the prevention of
A-induced microglial
activation.
Discussion
One of the key features of AD is microglial activation, a process
that may serve dual functions. For a long time, it was thought to
be responsible for the ongoing inflammatory process occurring
in the neurological condition (for review, see Akiyama et al.,
2000). However, it has been recognized recently that removal of
A by activated microglia may be beneficial (Bard et al., 2000;
Wyss-Coray et al., 2001; Jantzen et al., 2002). These cells are part
of the senile plaques in cerebral cortex and hippocampus of the
afflicted patients. We have shown that CB
1
and CB
2
receptors are
colocalized in plaques, in line with a recent report (Benito et al.,
2003). Upregulation of inducible nitric oxide synthase occurs in
senile plaques in AD (Lee et al., 1999) and in microglia after
A
administration
invivo
(Weldon et al., 1998). Furthermore, N-Tyr
immunoreactivity has been observed in plaques in AD (Vodovotz
et al., 1996) and in mice carrying amyloid precursor protein plus
presenilin-1 mutations (Matsuoka et al., 2001). Protein nitration
therefore appears to be a marker of the effects of the peroxynitrite
radical and a consequence of microglial activation. In normal
human brain, we observed a high density of CB
1
-positive neurons
(Ong and Mackie, 1999; Benito et al., 2003), mostly pyramidal
cells, which are lost in AD brain, in particular in areas bearing
plaques, pointing to the vulnerability of such neurons to the toxic
species generated by microglia (e.g., NO and cytokines). Given
that CB-positive neurons were segregated from plaques, their
possible contribution to CB receptors in plaques may be ruled
out, and this points to microglial cells as the unique source.
Previous works in human brain have mapped CB receptors by
autoradiographic techniques (Westlake et al., 1994; Glass et al.,
1997, 2000), but their pharmacological characteristics were not
determined. Furthermore, it is widely accepted that CB
2
recep
-
tors are not present in normal brain, but we wondered whether,
by using an appropriate selection of cold ligands to define specific
binding, we would be able to show CB
2
receptors, at least in
pathological brain, such as AD. In human cortical membranes,
3
H-WIN55,212-2 binding to CB
1
receptors, as defined by the
CB
1
-selective antagonist SR141716, was saturable and of high
affinity. The apparent affinity is similar to that observed in rat
brain (Breivogel et al., 1997), albeit the density of binding sites
(
B
max
) is much lower. Rather than a species trait, aging (Mailleux
and Vanderhaeghen, 1992; Romero et al., 1998; Mato and Pazos,
2004) may account for the difference in density, given that our
controls were nonpathological aged subjects. WIN55,212-2 is a
mixed CB
1
/CB
2
cannabinoid receptor agonist that activates, with
similar affinities, CB
1
and CB
2
receptors (Howlett et al., 2002)
and may also interact with other, as yet uncharacterized, receptor
subtypes (Breivogel et al., 2001). The relative potencies of the
drugs in our pharmacological studies are in general agreement
with previous studies (Rinaldi-Carmona et al., 1994, 1998;
Figure 2.
Nitration of CB
1
and CB
2
is increased in AD brain.
a
, N-Tyr-immunoreactive astro
-
cytes in control (top); nuclear N-Tyr expression in control (middle); cytoplasmic N-Tyr expres-
sion in AD (arrows, bottom). Scale bar, 25
m.
b
, Total protein nitration (as detected by West-
ern blot) in control (C) and AD brain. OD, Optical density.
c
, Lysates from control and AD brains
were immunoprecipitated with anti-N-Tyr antibody and blotted with anti-CB
1
or CB
2
antibod
-
ies. The percentage of nitration of total CBs is shown.
b
,
c
, Results are mean
SEM of
n
18
in each group; **
p
0.01 and ***
p
0.001 compared with controls (Student’s
t
test); repre-
sentative blots are shown. Error bars represent SEM.
1908

J. Neurosci., February 23, 2005

25(8):1904–1913 Ramı ́rez et al.

Cannabinoid Receptors in AD
Breivogel et al., 1997), including those in cells transfected with
the cloned human cannabinoid receptors (Felder et al., 1995;
MacLennan et al., 1998). Together, these results indicate that, in
human brain, WIN55,212-2 interacts with CB
1
or CB
1
-like recep
-
tors. CB
1
receptors outnumber other characterized cannabinoid
receptors, and such a high density may have masked CB
2
recep
-
tors, which were observed by immunohistochemistry, with a very
restricted localization in AD brain.
In a previous work, cannabinoid receptor binding in cortical
areas of AD brain was unchanged, and the reduction in hip-
pocampal subfields was unrelated to the pathological changes
(Westlake et al., 1994). However, in agreement with our histolog-
ical findings, in biochemical studies, we have shown that CB
1
protein expression and function are markedly reduced in AD.
Indeed G-protein coupling, as judged by
35
S-GTP
S binding,
was greatly diminished in samples from
AD patients. Given that enhanced protein
nitration was observed along CB receptors
in plaques, we examined whether they
may be a target for such alteration. In AD,
total protein nitration is increased com-
pared with controls (Aoyama et al., 2000).
More specifically, both CB
1
and CB
2
re
-
ceptor proteins show enhanced protein
nitration in AD than in controls. It should
be noted that protein nitration, known to
inactivate other proteins in AD (Aoyama
et al., 2000), might interfere with CB re-
ceptor function as well. In summary, sig-
nificant alterations in the localization, ex-
pression, and function of cannabinoid
receptors occur in AD and may play a role
in its physiopathology.
Our present results confirm and ex-
tend those of previous works, showing
marked alterations in cannabinoid re-
ceptors in AD brain. Benito et al. (2003)
reported that CB
1
-positive neurons ap
-
peared to be preserved in AD parahip-
pocampal cortex, but frontal cortex was
not studied. Therefore, the region of study
may account for the difference between their and our results.
Autoradiographic studies, used by Westlake et al. (1994), lack
cellular resolution, which may explain why decreased binding did
not correlate with areas showing pathology (senile plaques and
tangles) in hippocampal subfields in AD. In this work, we com-
bined histological, pharmacological, and biochemical techniques
to show loss of CB
1
-positive neurons in frontal cortex of AD
brain, as well as decreased CB
1
protein expression and G-protein
coupling, despite preserved binding.
In the present study, we used rats repeatedly injected intrac-
erebroventricularly with
A to model AD. Indeed, we reported
decreased muscarinic receptors in cortex (Pavı
́a et al., 2000) and
increased monomeric G
1
acetylcholinesterase isoform (Sa
́ ez-
Valero et al., 2002), similar to those in AD. These animals show
increased microglial reaction at the end of
A administration
(Netland et al., 1998; Weldon et al., 1998) and cognitive impair-
ment at 2 months (Delobette et al., 1997) compared with control
animals. Interestingly,
A-treated rats reproduced the same re-
duction in neuronal markers, including CB
1
protein, occurring in
AD, validating further the experimental model. Cannabinoids
induce neuroprotection against excitotoxicity (Shen and Thayer,
1998), ischemia (Nagayama et al., 1999), and glucose deprivation
(Nagayama et al., 1999)
in vitro
. These compounds are also effec-
tive against acute brain damage
in vivo
, including excitotoxic
insult (van der Stelt et al., 2001), ischemia (Nagayama et al.,
1999), and acute brain trauma (Panikashvili et al., 2001). Fur-
thermore, the involvement of CB
1
receptors and their endoge
-
nous ligands as neuroprotectants has been demonstrated recently
in CB
1
-null mutants, which are more susceptible to neurodegen
-
eration (Parmentier-Batteur et al., 2002; Marsicano et al., 2003).
In this work, we report that microglial activation induced
in vivo
by
A was completely prevented by cannabinoid administration.
We have shown that the loss of neuronal markers induced by
A
is attenuated by cannabinoid administration. More importantly,
the cognitive deficits occurring in
A-treated rats are also pre-
vented by the cannabinoid. Together, these results constitute the
Figure3.
CB
1
receptorbindingisunalteredandG-proteincouplingisreducedinADfrontalcortex.
a
,Specific
3
H-WIN55,212-2
(WIN)binding(
n
18ineachgroup).
b
,Representative
3
H-WIN55212-2bindingsaturationcurves(
n
3ineachgroup).
c
,Basal
35
S-GTP
S binding (
n
18 in each group).
d
, WIN55,212-2-stimulated
35
S-GTP
S binding (
n
18; *
p
0.05 compared with
controls; Student’s
t
test).
e
,
f
,CB
1
(
e
) and CB
2
(
f
) expression (as detected by Western blot) in control and AD brain. OD, Optical
density. Results are mean
SEM of
n
18 in each group; *
p
0.05 compared with controls (Student’s
t
test); representative
blots are shown. Error bars represent SEM.
Figure4.
Cannabinoidtreatmentprevents
A-inducedmicroglialactivationinrats.Tomato
lectin binding to microglial cells in frontal cortex of rats 24 h after treatment completion was
increased by
A compared with SCR peptide and prevented by WIN55,212-2 (WIN) cotreat-
ment;picturesofonerepresentativeanimalofthreepergroupareshown.Initialmagnification
was 200
.
Ramı ́rez et al.

Cannabinoid Receptors in AD J. Neurosci., February 23, 2005

25(8):1904–1913
• 1909
first evidence that cannabinoids exert
neuroprotection in a model of AD.
Cannabinoids prevent microglial acti-
vation and decrease NO production
(Waksman et al., 1999) and TNF-
ex-
pression and release (Puffenbarger et al.,
2000; Facchinetti et al., 2003) evoked by
different agents. In this work, cannabi-
noids with different pharmacological pro-
files effectively counteracted the well
known
A-induced microglial activation,
in particular TNF-
release, which is po-
tentially neurotoxic. Milton (2002) re-
ported that the toxicity of high concentra-
tions (25
M
)of
A peptides was
prevented by endocannabinoids in NT-2
cells, a human teratocarcinoma cell line
that can be differentiated into neuronal
phenotype, and in myeloma cells. Thus,
anandamide and noladin ether protected
the cells by a CB
1
-oraCB
1
/CB
2
-mediated
effect, respectively, depending on the cell
line used. These results were not repro-
duced in our neuronal cultures. Thus, dif-
ferent synthetic cannabinoids did not pre-
vent direct toxicity of
A on primary
neurons in culture. However, a neuropro-
tective effect of cannabinoids was dis-
closed using a
A microglia-mediated
neurotoxicity paradigm. The selective CB
2
agonist JWH-133 was as effective as
WIN55,212-2, the mixed CB
1
/CB
2
ago
-
nist. As shown in microglia–neuron co-
cultures and in
A-treated rats, these ben-
eficial effects of cannabinoids may rely on
their ability to block
A-induced micro-
glial activation. In line with this notion,
cannabinoids are unable to prevent
A-
induced death of primary neurons in cul-
ture. This may be of particular importance
for the possible endorsement of cannabi-
noids to therapeutic applications because
of their psychotropic side effects, which
may cast clinical concern. CB
2
receptors
appear to be exclusively expressed by mi-
croglia, and blockade of its activation may
be attained by CB
2
agonists with no overt
psychoactivity.
The search for new and effective treat-
ments for AD is of crucial importance, and
limiting ongoing inflammatory responses
secondary to microglial activation has
been proposed (Akiyama et al., 2000).
Furthermore, recent studies on therapeu-
tic strategies for neurodegenerative dis-
eases such as Parkinson’s disease and AD
have focused on the neuroprotective
properties (e.g., slowing the ongoing neu-
rodegeneration) rather than just on palli-
ating symptoms of the diseases (Dawson
and Dawson, 2002). Because cannabi-
noids combine both anti-inflammatory
and neuroprotective actions, our findings
Figure 6.
Cannabinoids prevent
A-induced microglial activation
in vitro
.
a
, Immunostaining of cultured microglia with
anti-OX42 (top), CB
1
(middle), and CB
2
(bottom) antibodies.
b
, Fibrillar
A
1–40
(fib), but not soluble
A
1–40
(sol), induced a
rod-like morphology, which was prevented by HU-210 (HU).
c
, Cannabinoids [HU-210 (HU), WIN55,212-2 (WIN), and JWH-133
(JWH), at 100 n
M
for 4 h] prevented TNF-
release and mitochondrial activity, as induced by fibrillar
A
1–40
(500 n
M
). TNF-
release in controls was 26.1
4.5 pg/ml. Results are mean
SEM of
n
4–6;*
p
0.05 and **
p
0.01 compared with
soluble
A
1–40
-treated control cultures;
#
p
0.05,
##
p
0.01 compared with fibrillar
A
1–40
-treated cultures (ANOVA with
Bonferroni’s
post hoc
test).
Figure 5.
Cannabinoid treatment prevents cognitive impairment and loss of neuronal markers in rats.
a
, Latency (in seconds)
to find a hidden platform in the water maze during training. Results are mean of
n
5 in each group; SEM have been omitted for
clarityandwerealways
12%ofthemean;*
p
0.05and**
p
0.01comparedwithSCR-treatedratsatthesametrainingday;
#
p
0.05 and
##
p
0.01 compared with
A-treated rats (ANOVA with Bonferroni’s
post hoc
test). WIN, WIN55,212-2.
b
,
c
,
Expression of calbindin (
b
) and
-tubulin (
c
) in control (C) and AD frontal cortex; results are mean
SEM of
n
18 control and
AD; ***
p
0.001 compared with controls.
d

f
, Expression of CB
1
(
d
), calbindin (
e
), and
-tubulin (
f
) in frontal cortex of rats at
2 months after treatment. OD, Optical density. Results are mean
SEM of
n
5 in each group; *
p
0.05 and **
p
0.01
comparedwithSCR-treatedrats(ANOVAwithBonferroni’s
post hoc
test);representativeblotsareshown.ErrorbarsrepresentSEM.
1910

J. Neurosci., February 23, 2005

25(8):1904–1913 Ramı ́rez et al.

Cannabinoid Receptors in AD
may set the basis for the use of these compounds as a therapeutic
approach for AD.
References
Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, Cooper NR,
Eikelenboom P, Emmerling M, Fiebich BL, Finch CE, Frautschy SA, Grif-
fin WS, Hampel H, Hull M, Landreth G, Lue L, Mrak R, Mackenzie IR,
McGeer PL, et al. (2000) Inflammation and Alzheimer’s disease. Neuro-
biol Aging 21:383– 421.
Aoyama K, Matsubara K, Fujikawa Y, Nagahiro Y, Shimizu K, Umegae N,
Hayase N, Shiono H, Kobayashi S (2000) Nitration of manganese super-
oxide dismutase in cerebrospinal fluids is a marker for peroxynitrite-
mediated oxidative stress in neurodegenerative diseases. Ann Neurol
47:524 –527.
Bard F, Cannon C, Barbour R, Burke R-L, Games D, Grajeda H, Guido T, Hu
K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Lieberbug
I, Motter R, Nguyen M, Soriano F, Vasquez N, Weiss K, Welch B, et al.
(2000) Peripherally administered antibodies against amyloid
-peptide
enter the central nervous system and reduce pathology in a mouse model
of Alzheimer disease. Nat Med 6:916 –919.
Barger SW, Ho
̈rster D, Furukawa K, Goodman Y, Krieglstein J, Mattson MP
(1995) Tumor necrosis factors
and
protect neurons against amyloid
-peptide toxicity: evidence for involvement of
B-binding factor and
attenuation of peroxide and Ca
2
accumulation. Proc Natl Acad Sci USA
92:9328 –9332.
Benito C, Nu
́n
̃ez E, Tolo
́ n RM, Carrier EJ, Rabano A, Hillard CJ, Romero J
(2003) Cannabinoid CB
2
receptors and fatty acid amide hydrolase are
selectively overexpressed in neuritic plaque-associated glia in Alzheimer’s
disease brains. J Neurosci 23:11136 –11141.
Bouaboula M, Bourrie B, Rinaldi-Carmona M, Shire D, Le Fur G, Casellas P
(1995) Stimulation of cannabinoid receptor CB1 induces krox-24 ex-
pression in human astrocytoma cells. J Biol Chem 270:13973–13980.
Breivogel CS, Sim LJ, Childers SR (1997) Regional differences in cannabi-
noid receptor/G-protein coupling in rat brain. J Pharmacol Exp Ther
282:1632–1642.
Breivogel CS, Griffin G, Di Marzo V, Martin BR (2001) Evidence for a new
G protein-coupled cannabinoid receptor in mouse brain. Mol Pharmacol
60:155–163.
Casal C, Serratosa J, Tusell JM (2002) Relationship between
-AP peptide
aggregation and microglial activation. Brain Res 928:76 – 84.
Dawson TM, Dawson VL (2002) Neuroprotective and neurorestorative
strategies for Parkinson’s disease. Nat Neurosci 5:1058 –1061.
Delobette S, Privat A, Maurice T (1997) In vitro aggregation facilitates
-amyloid peptide-(25–35)-induced amnesia in the rat. Eur J Pharmacol
319:1– 4.
Dickson DW, Farlo J, Davies P, Crystal H, Fuld P, Yen SH (1988) Alzhei-
mer’s disease. A double-labeling immunohistochemical study of senile
plaques. Am J Pathol 132:86 –101.
Facchinetti F, Del Giudice E, Furegato S, Passarotto M, Leon A (2003) Can-
nabinoids ablate release of TNF
in rat microglial cells stimulated with
lypopolysaccharide. Glia 41:161–168.
Felder CC, Joyce KE, Briley EM, Mansouri J, Mackie K, Blond O, Lai Y, Ma
AL, Mitchell RL (1995) Comparison of the pharmacology and signal
transduction of the human cannabinoid CB
1
and CB
2
receptors. Mol
Pharmacol 48:443– 450.
Ferna
́ ndez-Tome
́ P, Brera B, Are
́valo M-A, de Ceballos ML (2004)
-Amy-
loid
25–35
inhibits glutamate uptake in cultured neurons and astrocytes:
modulation of uptake as a survival mechanism. Neurobiol Dis
15:580 –589.
Frautschy SA, Yang F, Irrizarry M, Hyman B, Saido TC, Hsiao K, Cole GM
(1998) Microglial response to amyloid plaques in APPsw transgenic
mice. Am J Pathol 152:307–317.
Gao H-M, Hong J-S, Zhang W, Liu B (2002) Distinct role for microglia in
rotenone-induced degeneration of dopaminergic neurons. J Neurosci
22:782–790.
Glass M, Dragunow M, Faull RLM (1997) Cannabinoid receptors in the
human brain: a detailed anatomical and quantitative autoradiographic
study in the fetal, neonatal and adult human brain. Neuroscience
77:299 –318.
Glass M, Dragunow M, Faull RLM (2000) The pattern of neurodegenera-
tion in Huntington’s disease: a comparative study of cannabinoid, dopa-
Figure 7.
Cannabinoids prevent
A-induced microglia-mediated neurotoxicity
in vitro
.
a
,
Neurons(neu)weretreatedwithfibrillar
A
1–40
(fib)orsoluble
A
1–40
(sol)alone(500n
M
)or
incombinationwithcannabinoidagonists[WIN55,212-2(WIN)orJWH-133(JWH),100n
M
]and
antagonists[SR141716(SR1)orSR144528(SR2),100n
M
]for20h(top);alternatively,microglia
(mg) seeded in inserts were treated with the same compounds for 4 h, media were removed,
and inserts were placed on neurons for 20 h (middle and bottom). Neurons were fixed, stained
with Coomassie brilliant blue, and counted. Results are mean
SEM of
n
3. Middle, *
p
0.05 compared with soluble
A
1–40
-treated control cultures;
#
p
0.05 compared with fibril
-
lar
A
1–40
-treated cultures (ANOVA with Bonferroni’s
post hoc
test). Bottom, The set of three
columns on the left represents single treatments (fibrillar
A
1–40
, SR141716, or SR144528);
*
p
0.05 compared with cultures treated with fibrillar
A
1–40
alone;
#
p
0.05 compared
with fibrillar
A
1–40
plus the cannabinoid agonist.
b
, Microglia–neuron cocultures showing
neurotoxicity after exposure to fibrillar
A
1–40
-activated microglia and prevention by
cannabinoids.
Ramı ́rez et al.

Cannabinoid Receptors in AD J. Neurosci., February 23, 2005

25(8):1904–1913
• 1911
mine, adenosine, and GABA
A
receptor alterations in the human basal
ganglia in Huntington’s disease. Neuroscience 97:505–519.
Go
́ mez del Pulgar T, de Ceballos ML, Guzma
́ n M, Velasco G (2002) Canna-
binoids protect astrocytes from ceramide-induced apoptosis through
phosphatidylinositol 3-kinase/protein kinase B. J Biol Chem
277:36527–36533.
Hampson AJ, Grimaldi M (2001) Cannabinoid receptor activation and ele-
vated cyclic AMP reduce glutamate neurotoxicity. Eur J Neurosci
13:1529 –1536.
Herkenham M, Lynn AB, Little DM, Johnson MR, Melvin LS, De Costa BR,
Rice KC (1990) Cannabinoid receptor localization in brain. Proc Natl
Acad Sci USA 87:1932–1936.
Herrera AJ, Castan
̃o A, Venero JL, Cano J, Machado A (2000) The single
intranigral injection of LPS as a new model for studying the selective
effects of inflammatory reactions on dopaminergic systems. Neurobiol
Dis 7:429 – 447.
Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane W, Herkenham
M, Mackie K, Martin BR, Mechoulan R, Pertwee RG (2002) Interna-
tional Union of Pharmacology. XXVII. Classification of cannabinoid re-
ceptors. Pharmacol Rev 54:161–202.
Huffman JW, Liddle J, Yu S, Aung MM, Abood ME, Wiley JL, Martin BR
(1999) 3-(1
,1
-Dimethylbutyl)-1-deoxy-delta8-THC and related com-
pounds: synthesis and selective ligands for the CB2 receptor. Bioorg Med
Chem 7:2905–2914.
Iravani MM, Kashefi K, Mander P, Rose S, Jenner P (2002) Involvement of
inducible nitric oxide synthase in inflammation-induced dopaminergic
neurodegeneration. Neuroscience 110:49 –58.
Jantzen PT, Connor KE, DiCarlo G, Wenk GL, Wallace JL, Rojiani AM,
Coppola D, Morgan D, Gordon MN (2002) Microglial activation and
-amyloid deposit reduction caused by nitric oxide-releasing nonsteroi-
dal anti-inflammatory drug in amyloid precursor protein plus
presenilin-1 transgenic mice. J Neurosci 22:2246 –2254.
Lan R, Liu Q, Fan P, Lin S, Fernando SR, McCallion D, Pertwee R, Makriy-
annis A (1999) Structure-activity relationships of pyrazole derivatives as
cannabinoid receptor antagonists. J Med Chem 42:769 –774.
Lee SC, Zhao M-L, Hirano A, Dickson DW (1999) Inducible nitric oxide
synthase immunoreactivity in the Alzheimer disease hippocampus: asso-
ciation with Hirano bodies, neurofibrillary tangles and senile plaques.
J Neuropathol Exp Neurol 58:1163–1169.
MacLennan SJ, Reynen PH, Kwan J, Bonhaus DW (1998) Evidence for in-
verse agonism of SR141716A at human recombinant cannabinoid CB1
and CB2 receptors. Br J Pharmacol 124:619 – 622.
Mailleux P, Vanderhaeghen JJ (1992) Age-related loss of cannabinoid re-
ceptor binding sites and mRNA in the rat striatum. Neurosci Lett
147:179 –181.
Marsicano G, Moosmann B, Hermann HM, Lutz B, Behl C (2002) Neuro-
protective properties of cannabinoids against oxidative stress: role of the
cannabinoid receptor CB1. J Neurochem 80:448 – 456.
Marsicano G, Goodenough S, Monory K, Hermann H, Eder M, Cannich A,
Azad SC, Cascio MG, Gutierrez SO, van der Stelt M, Lo
́ pez-Rodrı
́guez
ML, Casanova E, Schu
̈ tz G, Zieglga
̈nsberger W, Di Marzo V, Behl C, Lutz
B (2003) CB1 cannabinoid receptors and on-demand defense against
excitotoxicity. Science 302:84 – 88.
Masliah E, Sisk A, Mallory M, Mucke L, Schenk D, Games D (1996) Com-
parison of neurodegenerative pathology in transgenic mice overexpress-
ing V717F
-amyloid precursor protein and Alzheimer’s disease. J Neu-
rosci 16:5795–5811.
Mato S, Pazos A (2004) Influence of age, postmortem delay and freezing
storage period on cannabinoid receptor density and functionality in hu-
man brain. Neuropharmacology 46:716 –726.
Matsuoka Y, Picciano M, La Francois, Duff K (2001) Fibrillar
-amyloid
evokes oxidative damage in a transgenic mouse model of Alzheimer’s
disease. Neuroscience 104:609 – 613.
McCarthy KD, de Vellis J (1980) Preparation of separate astroglial and oli-
godendroglial cell cultures from rat cerebral tissue. J Cell Biol
263:17205–17208.
McGeer PL, Itagaki S, Tago H, McGeer EG (1987) Reactive microglia in
patients with senile dementia of the Alzheimer type are positive for the
histocompatibility glycoprotein HLA-DR. Neurosci Lett 79:195–200.
Meda L, Cassatella MA, Szendrei GI, Otvos Jr L, Baron P, Villalba M, Ferrari
D, Rossi F (1995) Activation of microglial cells by
-amyloid protein
and interferon-
. Nature 374:647– 650.
Milton NG (2002) Anandamide and noladin ether prevent neurotoxicity of
the human amyloid-
peptide. Neurosci Lett 332:127–130.
Molina-Holgado E, Vela JM, Are
́valo-Martı
́n A, Almaza
́ n G, Molina-
Holgado F, Borrell J, Guaza C (2002) Cannabinoids promote oligoden-
drocyte progenitor survival: involvement of cannabinoid receptors and
phosphatylinositol-3 kinase/Akt signaling. J Neurosci 22:9742–9753.
Mu
̈ ller U, Cristina N, Li Z-W, Wolfer DP, Lipp H-P, Rulicke T, Brandner S,
Aguzzi T, Weissmann C (1994) Behavioral and anatomical deficits in
mice homozygous for a modified
-amyloid precursor protein. Cell
79:755–765.
Nagayama T, Sinor AD, Simon RP, Chen J, Graham SH, Jin K, Greenberg DA
(1999) Cannabinoids and neuroprotection in global and focal cerebral
ischemia and in neuronal cultures. J Neurosci 19:2987–2995.
Netland EE, Newton JL, Majocha RE, Tate BA (1998) Indomethacin re-
verses the microglial response to amyloid
-protein. Neurobiol Aging
19:201–204.
Ong WY, Mackie K (1999) A light and electron microscopic study of the
CB1 cannabinoid receptor in primate brain. Neuroscience 92:1177–1191.
Panikashvili D, Simeonidou C, Ben-Shabat S, Hanus L, Breuer A, Mechoulam
R, Shohami E (2001) An endogenous cannabinoid (2-AG) is neuropro-
tective after brain injury. Nature 413:527–531.
Parmentier-Batteur S, Jin K, Mao XO, Xie L, Greenberg DA (2002) In-
creased severity of stroke in CB
1
cannabinoid receptor knock-out mice.
J Neurosci 22:9771–9775.
Pavı
́a J, Alberch J, Alvarez I, Toledano A, de Ceballos ML (2000) Repeated
intracerebroventricular administration of
-amyloid(25–35) to rats de-
creases muscarinic receptors in cerebral cortex. Neurosci Lett 278:69 –72.
Piomelli D (2003) The molecular logic of endocannabinoid signalling. Nat
Rev Neurosci 4:873– 884.
Puffenbarger RA, Boothe AC, Cabral GA (2000) Cannabinoids inhibit LPS-
inducible cytokine mRNA expression in rat microglial cells. Glia
29:58 – 69.
Rinaldi-Carmona M, Barth F, He
́aulme M, Shire D, Calandra B, Congy C,
Martinez S, Maruani J, Ne
́liat G, Caput D, Ferrara P, Soubrie
́ P, Brelie
`re
JC, Le Fur G (1994) SR141716A, a potent and selective antagonist of the
brain cannabinoid receptor. FEBS Lett 350:240 –244.
Rinaldi-Carmona M, Barth F, Millan J, Derocq JM, Casellas P, Congy C,
Oustric D, Sarran M, Bouaboula M, Calandra B, Portier M, Shire D,
Brelie
́re JC, Le Fur GL (1998) SR144528, the first potent and selective
antagonist of the CB2 cannabinoid receptor. J Pharmacol Exp Ther
284:644 – 650.
Romero J, Berrendero F, Garcia-Gil L, de la Cruz P, Ramos JA, Fernandez-
Ruiz JJ (1998) Loss of cannabinoid receptor binding and messenger
RNA and cannabinoid agonist-stimulated [
35
S]guanylyl-5
O-(thio)-
triphosphate binding in the basal ganglia of aged rats. Neuroscience
84:1075–1083.
Sa
́ ez-Valero J, de Ceballos ML, Small DH, de Felipe C (2002) Changes in
molecular isoform distribution of acetylcholinesterase in rat cortex and
cerebrospinal fluid after intracerebroventricular administration of amy-
loid
-peptide. Neurosci Lett 325:199 –202.
Sa
́ nchez C, Galve-Roperh I, Canova C, Brachet P, Guzma
́ n M (1998)
9
-
tetrahydrocannabinol induces apoptosis in C6 glioma cells. FEBS Lett
436:6 –10.
Sa
́ nchez C, de Ceballos ML, Go
́ mez del Pulgar T, Rueda D, Corbacho C,
Velasco G, Galve-Roperh I, Huffman JW, Ramo
́ n y Cajal S, Guzma
́n M
(2001) Inhibition of glioma growth
in vivo
by selective activation of the
CB
2
cannabinoid receptor. Cancer Res 61:5784 –5789.
Shen M, Thayer SA (1998) Cannabinoid receptor agonists protect cultured
rat hippocampal neurons from excitotoxicity. Mol Pharmacol
54:459 – 462.
Skaper SD, Buriani A, Dal Toso R, Petrelli L, Romanello S, Facci L, Leon A
(1996) The ALIAmide palmitoylethanolamide and cannabinoids, but
not anadamide, are protective in a delayed postglutamate paradigm of
excitotoxic death in cerebellar granule neurons. Proc Natl Acad USA
93:3984 –3989.
Smith MA, Harris PLR, Sayre LM, Beckman JS, Perry G (1996) Widespread
peroxynitrite-mediated damage in Alzheimer’s disease. J Neurosci
17:2653–2657.
Tan J, Town T, Mori T, Saxe Y, Crawford F, Mullan M (2000) CD45 opposes
-amyloid peptide-induced microglial activation via inhibition of p44/42
mitogen-activated protein kinase. J Neurosci 20:7587–7594.
1912

J. Neurosci., February 23, 2005

25(8):1904–1913 Ramı ́rez et al.

Cannabinoid Receptors in AD


Keywords: study shows cannabinoids useful in prevention of alzheimers marijuana legalize

Most Recent User Comments
There are no comments for this exhibition yet.

Leave a comment
Please log in or create an account to post a comment.
Related sponsor

Related Literature

Other related exhibitions

Related links