Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 25 (8), 1904-13

Prevention of Alzheimer's Disease Pathology by Cannabinoids: Neuroprotection Mediated by Blockade of Microglial Activation

Affiliations

Prevention of Alzheimer's Disease Pathology by Cannabinoids: Neuroprotection Mediated by Blockade of Microglial Activation

Belén G Ramírez et al. J Neurosci.

Abstract

Alzheimer's disease (AD) is characterized by enhanced beta-amyloid peptide (betaA) 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. This background prompted us to study the localization, expression, and function of cannabinoid receptors in AD and the possible protective role of cannabinoids after betaA treatment, both in vivo and in vitro. Here, we show that senile plaques in AD patients express cannabinoid receptors CB1 and CB2, together with markers of microglial activation, and that CB1-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 CB1 receptor protein expression are markedly decreased in AD brains. Additionally, in AD brains, protein nitration is increased, and, more specifically, CB1 and CB2 proteins show enhanced nitration. Intracerebroventricular administration of the synthetic cannabinoid WIN55,212-2 to rats prevent betaA-induced microglial activation, cognitive impairment, and loss of neuronal markers. Cannabinoids (HU-210, WIN55,212-2, and JWH-133) block betaA-induced activation of cultured microglial cells, as judged by mitochondrial activity, cell morphology, and tumor necrosis factor-alpha release; these effects are independent of the antioxidant action of cannabinoid compounds and are also exerted by a CB2-selective agonist. Moreover, cannabinoids abrogate microglia-mediated neurotoxicity after betaA 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.

Figures

Figure 1.
Figure 1.
Cannabinoid receptor localization in AD brain. a, CB1 and CB2 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 CB1 (brown, asterisks). CB1-positive neurons in controls (top); CB1-positive neurons are still present (middle) or completely lost (bottom) in areas of intense microglial activation in AD. c, CB1-positive and CB2-positive neurons and dystrophic neurites in AD. Insets, Absence of labeling by preabsorption of the antibodies with the antigenic peptide (pep). Scale bars, 25 μm.
Figure 2.
Figure 2.
Nitration of CB1 and CB2 is increased in AD brain. a, N-Tyr-immunoreactive astrocytes in control (top); nuclear N-Tyr expression in control (middle); cytoplasmic N-Tyr expression in AD (arrows, bottom). Scale bar, 25 μm. b, Total protein nitration (as detected by Western 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-CB1 or CB2 antibodies. 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); representative blots are shown. Error bars represent SEM.
Figure 3.
Figure 3.
CB1 receptor binding is unaltered and G-protein coupling is reduced in AD frontal cortex. a, Specific 3H-WIN55,212-2 (WIN) binding (n = 18 in each group). b, Representative 3H-WIN55212-2 binding saturation curves (n = 3 in each group). c, Basal 35S-GTPγS binding (n = 18 in each group). d, WIN55,212-2-stimulated 35S-GTPγS binding (n = 18; *p < 0.05 compared with controls; Student's t test). e, f, CB1 (e) and CB2 (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.
Figure 4.
Figure 4.
Cannabinoid treatment prevents βA-induced microglial activation in rats. Tomato lectin binding to microglial cells in frontal cortex of rats 24 h after treatment completion was increased by βA compared with SCR peptide and prevented by WIN55,212-2 (WIN) cotreatment; pictures of one representative animal of three per group are shown. Initial magnification was 200×.
Figure 5.
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 clarity and were always <12% of the mean; *p < 0.05 and **p < 0.01 compared with SCR-treated rats at the same training day; #p < 0.05 and ##p < 0.01 compared with βA-treated rats (ANOVA with Bonferroni's post hoc test). WIN, WIN55,212-2. b, c, Expression of calbindin (b) and α-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 CB1 (d), calbindin (e), and α-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 compared with SCR-treated rats (ANOVA with Bonferroni's post hoc test); representative blots are shown. Error bars represent SEM.
Figure 6.
Figure 6.
Cannabinoids prevent βA-induced microglial activation in vitro. a, Immunostaining of cultured microglia with anti-OX42 (top), CB1 (middle), and CB2 (bottom) antibodies. b, Fibrillar βA1-40 (fib), but not soluble βA1-40 (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 nm for 4 h] prevented TNF-α release and mitochondrial activity, as induced by fibrillar βA1-40 (500 nm). TNF-α 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 βA1-40-treated control cultures; #p < 0.05, ##p < 0.01 compared with fibrillar βA1-40-treated cultures (ANOVA with Bonferroni's post hoc test).
Figure 7.
Figure 7.
Cannabinoids prevent βA-induced microglia-mediated neurotoxicity in vitro. a, Neurons (neu) were treated with fibrillar βA1-40 (fib) or soluble βA1-40 (sol) alone (500 nm) or in combination with cannabinoid agonists [WIN55,212-2 (WIN) or JWH-133 (JWH), 100nm] and antagonists [SR141716 (SR1) or SR144528 (SR2), 100 nm] for 20 h (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 βA1-40-treated control cultures; #p < 0.05 compared with fibrillar βA1-40-treated cultures (ANOVA with Bonferroni's post hoc test). Bottom, The set of three columns on the left represents single treatments (fibrillar βA1-40, SR141716, or SR144528); *p < 0.05 compared with cultures treated with fibrillar βA1-40 alone; #p < 0.05 compared with fibrillar βA1-40 plus the cannabinoid agonist. b, Microglia-neuron cocultures showing neurotoxicity after exposure to fibrillar βA1-40-activated microglia and prevention by cannabinoids.

Similar articles

See all similar articles

Cited by 184 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback