María L. de Ceballos

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

Alzheimers disease (AD) is characterized by enhanced β-amyloid peptide (βA) 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 βA 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 βA-induced microglial activation, cognitive impairment, and loss of neuronal markers. Cannabinoids (HU-210, WIN55,212-2, and JWH-133) block βA-induced activation of cultured microglial cells, as judged by mitochondrial activity, cell morphology, and tumor necrosis factor-α 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 βA 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.

The transcription factor Nrf2 is a therapeutic target against brain inflammation

Because chronic neuroinflammation is a hallmark of neurodegenerative diseases and compromises neuron viability, it is imperative to discover pharmacologic targets to modulate the activation of immune brain cells, the microglia. In this study, we identify the transcription factor Nrf2, guardian of redox homeostasis, as such target in a model of LPS-induced inflammation in mouse hippocampus. Nrf2 knockout mice were hypersensitive to the neuroinflammation induced by LPS, as determined by an increase in F4/80 mRNA and protein, indicative of an increase in microglial cells, and in the inflammation markers inducible NO synthase, IL-6, and TNF-α, compared with the hippocampi of wild-type littermates. The aliphatic isothiocyanate sulforaphane elicited an Nrf2-mediated antioxidant response in the BV2 microglial cell line, determined by flow cytometry of cells incubated with the redox sensitive probe dihydrodichlorofluorescein diacetate, and by the Nrf2-dependent induction of the phase II antioxidant enzyme heme oxygenase-1. Animals treated with sulforaphane displayed a 2–3-fold increase in heme oxygenase-1, a reduced abundance of microglial cells in the hippocampus and an attenuated production of inflammation markers (inducible NO synthase, IL-6, and TNF-α) in response to LPS. Considering that release of reactive oxygen species is a property of activated microglia, we propose a model in which late induction of Nrf2 intervenes in the down-regulation of microglia. This study opens the possibility of targeting Nrf2 in brain as a means to modulate neuroinflammation.

Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson's disease.

Neural injury leads to inflammation and activation of microglia that in turn may participate in progression of neurodegeneration. The mechanisms involved in changing microglial activity from beneficial to chronic detrimental neuroinflammation are not known but reactive oxygen species (ROS) may be involved. We have addressed this question in Nrf2‐knockout mice, with hypersensitivity to oxidative stress, submitted to daily inoculation of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) for 4 weeks. Basal ganglia of these mice exhibited a more severe dopaminergic dysfunction than wild type littermates in response to MPTP. The amount of CD11b‐positive/CD45‐highly‐stained cells, indicative of peripheral macrophage infiltration, did not increase significantly in response to MPTP. However, Nrf2‐deficient mice exhibited more astrogliosis and microgliosis as determined by an increase in messenger RNA and protein levels for GFAP and F4/80, respectively. Inflammation markers characteristic of classical microglial activation, COX‐2, iNOS, IL‐6, and TNF‐α were also increased and, at the same time, anti‐inflammatory markers attributable to alternative microglial activation, such as FIZZ‐1, YM‐1, Arginase‐1, and IL‐4 were decreased. These results were confirmed in microglial cultures stimulated with apoptotic conditioned medium from MPP+‐treated dopaminergic cells, further demonstrating a role of Nrf2 in tuning balance between classical and alternative microglial activation. This study demonstrates a crucial role of Nrf2 in modulation of microglial dynamics and identifies Nrf2 as molecular target to control microglial function in Parkinsons disease (PD) progression.

Cannabidiol and Other Cannabinoids Reduce Microglial Activation In Vitro and In Vivo: Relevance to Alzheimer's Disease

Microglial activation is an invariant feature of Alzheimers disease (AD). It is noteworthy that cannabinoids are neuroprotective by preventing β-amyloid (Aβ)-induced microglial activation both in vitro and in vivo. On the other hand, the phytocannabinoid cannabidiol (CBD) has shown anti-inflammatory properties in different paradigms. In the present study, we compared the effects of CBD with those of other cannabinoids on microglial cell functions in vitro and on learning behavior and cytokine expression after Aβ intraventricular administration to mice. CBD, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone [WIN 55,212-2 (WIN)], a mixed CB1/CB2 agonist, and 1,1-dimethylbutyl-1-deoxy-Δ9-tetrahydrocannabinol [JWH-133 (JWH)], a CB2-selective agonist, concentration-dependently decreased ATP-induced (400 μM) increase in intracellular calcium ([Ca2+]i) in cultured N13 microglial cells and in rat primary microglia. In contrast, 4-[4-(1,1-dimethylheptyl)-2,6-dimethoxyphenyl]-6,6-dimethyl-bicyclo[3.1.1]hept-2-ene-2-methanol [HU-308 (HU)], another CB2 agonist, was without effect. Cannabinoid and adenosine A2A receptors may be involved in the CBD action. CBD- and WIN-promoted primary microglia migration was blocked by CB1 and/or CB2 antagonists. JWH and HU-induced migration was blocked by a CB2 antagonist only. All of the cannabinoids decreased lipopolysaccharide-induced nitrite generation, which was insensitive to cannabinoid antagonism. Finally, both CBD and WIN, after subchronic administration for 3 weeks, were able to prevent learning of a spatial navigation task and cytokine gene expression in β-amyloid-injected mice. In summary, CBD is able to modulate microglial cell function in vitro and induce beneficial effects in an in vivo model of AD. Given that CBD lacks psychoactivity, it may represent a novel therapeutic approach for this neurological disease.

Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes

Glial cells perform critical functions that alter the metabolism and activity of neurons, and there is increasing interest in their role in appetite and energy balance. Leptin, a key regulator of appetite and metabolism, has previously been reported to influence glial structural proteins and morphology. Here, we demonstrate that metabolic status and leptin also modify astrocyte-specific glutamate and glucose transporters, indicating that metabolic signals influence synaptic efficacy and glucose uptake and, ultimately, neuronal function. We found that basal and glucose-stimulated electrical activity of hypothalamic proopiomelanocortin (POMC) neurons in mice were altered in the offspring of mothers fed a high-fat diet. In adulthood, increased body weight and fasting also altered the expression of glucose and glutamate transporters. These results demonstrate that whole-organism metabolism alters hypothalamic glial cell activity and suggest that these cells play an important role in the pathology of obesity.

Prolonged oral cannabinoid administration prevents neuroinflammation, lowers β-amyloid levels and improves cognitive performance in Tg APP 2576 mice.

BackgroundAlzheimers disease (AD) brain shows an ongoing inflammatory condition and non-steroidal anti-inflammatories diminish the risk of suffering the neurologic disease. Cannabinoids are neuroprotective and anti-inflammatory agents with therapeutic potential.MethodsWe have studied the effects of prolonged oral administration of transgenic amyloid precursor protein (APP) mice with two pharmacologically different cannabinoids (WIN 55,212-2 and JWH-133, 0.2 mg/kg/day in the drinking water during 4 months) on inflammatory and cognitive parameters, and on 18F-fluoro-deoxyglucose (18FDG) uptake by positron emission tomography (PET).ResultsNovel object recognition was significantly reduced in 11 month old Tg APP mice and 4 month administration of JWH was able to normalize this cognitive deficit, although WIN was ineffective. Wild type mice cognitive performance was unaltered by cannabinoid administration. Tg APP mice showed decreased 18FDG uptake in hippocampus and cortical regions, which was counteracted by oral JWH treatment. Hippocampal GFAP immunoreactivity and cortical protein expression was unaffected by genotype or treatment. In contrast, the density of Iba1 positive microglia was increased in Tg APP mice, and normalized following JWH chronic treatment. Both cannabinoids were effective at reducing the enhancement of COX-2 protein levels and TNF-α mRNA expression found in the AD model. Increased cortical β-amyloid (Aβ) levels were significantly reduced in the mouse model by both cannabinoids. Noteworthy both cannabinoids enhanced Aβ transport across choroid plexus cells in vitro.ConclusionsIn summary we have shown that chronically administered cannabinoid showed marked beneficial effects concomitant with inflammation reduction and increased Aβ clearance.

The AMP‐Activated Protein Kinase Is Involved in the Regulation of Ketone Body Production by Astrocytes

Abstract : The possible role of the AMP‐activated protein kinase (AMPK), a highly conserved stress‐activated kinase, in the regulation of ketone body production by astrocytes was studied. AMPK activity in rat cortical astrocytes was three times higher than in rat cortical neurons. AMPK in astrocytes was shown to be functionally active. Thus, incubation of astrocytes with 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR), a cellpermeable activator of AMPK, stimulated both ketogenesis from palmitate and carnitine palmitoyltransferase I. This was concomitant to a decrease of intracellular malonyl‐CoA levels and an inhibition of acetyl‐CoA carboxylase/fatty acid synthesis and 3‐hydroxy‐3‐methylglutaryl‐CoA reductase/cholesterol synthesis. Moreover, in microdialysis experiments AICAR was shown to stimulate brain ketogenesis markedly. The effect of chemical hypoxia on AMPK and the ketogenic pathway was studied subsequently. Incubation of astrocytes with azide led to a remarkable drop of fatty acid β‐oxidation. However, activation of AMPK during hypoxia compensated the depression of β‐oxidation, thereby sustaining ketone body production. This effect seemed to rely on the cascade hypoxia → increase of the AMP/ATP ratio → AMPK stimulation → acetyl‐CoA carboxylase inhibition → decrease of malonyl‐CoA concentration → carnitine palmitoyltransferase I deinhibition → enhanced ketogenesis. Furthermore, incubation of neurons with azide blunted lactate oxidation, but not 3‐hydroxybutyrate oxidation. Results show that (a) AMPK plays an active role in the regulation of ketone body production by astrocytes, and (b) ketone bodies produced by astrocytes during hypoxia might be a substrate for neuronal oxidative metabolism.

Molecular reorganization of endocannabinoid signalling in Alzheimer’s disease

Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB(1) cannabinoid receptors. Cognitive decline in Alzheimers disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimers disease. Comparative protein profiling and quantitative morphometry showed that overall CB(1) cannabinoid receptor protein levels in the hippocampi of patients with Alzheimers disease remain unchanged relative to age-matched controls, and CB(1) cannabinoid receptor-positive presynapses engulf amyloid-β-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase α and β isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimers (Braak stage VI), with ectopic sn-1-diacylglycerol lipase β expression found in microglia accumulating near senile plaques and apposing CB(1) cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimers disease (Braak stage III). However, Alzheimers pathology differentially impacts serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase α/β-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB(1) cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimers tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimers disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-β. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimers disease.

Hypoalgesia induced by antidepressants in mice: a case for opioids and serotonin

The tail flick assay was used to evaluate pain perception in mice treated acutely with either of the two classical antidepressant drugs (AD) imipramine or amitriptyline, or the atypical antidepressant iprindole. A sustained hypoalgesic effect, sensitive to the opiate antagonist naloxone, was detected for the AD used in this study. Administration of the aminopeptidase inhibitor bestatin or the enkephalinase blocker thiorphan made subeffective doses of AD hypoalgesic. This synergistic effect was reversed by naloxone. The antinociceptive action of the AD wore off in mice rendered tolerant to morphine by subcutaneous implantation of a pellet of the opiate. Subchronic treatment with p-chlorophenylalanine did not alter the effect of AD on pain perception, but in animals whose serotonin (5-HT) receptors were blocked by methysergide AD did not produce any change in pain threshold. It was concluded on the basis of these findings that short-chain opioids and 5-HT appear to have a role in the hypoalgesic effect of either classical or atypical AD.

Chronic antidepressant treatment increases enkephalin levels in n. accumbens and striatum of the rat.

Chronic (21 consecutive days) and not acute administration of typical (clomipramine, desipramine, amitriptyline) or atypical (iprindole, nomifensin) antidepressant drugs was found to provoke a selective increase in [Met5]enkephalin-like immunoreactivity ([Met5]ELI) in striatum and nucleus accumbens of rat brain. In parallel experiments, following chronic treatment with clomipramine, iprindole and nomifensin striatal [Leu5]enkephalin-like immunoreactivity ([Leu5]ELI) was also significantly enhanced. No variations in enzymatic activity of either enkephalinase or aminopeptidase were detected when assayed in several brain parts of animals chronically treated with antidepressants. Elevation of ELI in discrete regions of the brain might play a part in the mechanism of action of these centrally acting agents.